# Patent

## Title
Antibody that binds zinc transporter protein 108P5H8

## Abstract

A novel gene (designated 108P5H8) and its encoded protein, and variants thereof, are described wherein 108P5H8 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 108P5H8 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 108P5H8 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 191P1E1B can be used in active or passive immunization.

## Background
RELATED APPLICATIONS
This application claims priority from provisional application U.S. Ser. No 60/256,210 filed 15 Dec. 2000, the contents of which are incorporated herein by reference.
SUBMISSION ON COMPACT DISC
The contents of the following submission on compact discs are incorporated herein by reference in its entirety: A compact disc copy of the Sequence Listing (COPY 1) (file name: 511582002500, date recorded: 2 Jun. 2002, size: 434 KB); a duplicate compact disc copy of Sequence Listing (COPY 2) (file name: 511582002500, date recorded: 2 Jun. 2002, size: 434 KB); a computer readable form copy of the Sequence Listing (CRF COPY) (file name: 511582002500, date recorded: 2 Jun. 2002, size: 434 KB).


FIELD OF THE INVENTION
The invention described herein relates to a gene and its encoded protein, termed 108P5H8, expressed in certain cancers such as those listed in Table I, and to diagnostic, prognostic, prophylactic and/or therapeutic methods and compositions useful in the management of cancers that express 108P5H8.
BACKGROUND OF THE INVENTION
Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.
Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.
Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die annually of this disease—second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.
On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.
Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 September 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci USA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).
While previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and treat prostate cancer, there is need for the identification of additional markers and therapeutic targets for prostate and related cancers in order to further improve diagnosis and therapy.
Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States, and more than 11,600 patients died of this disease in 1998. Transitional cell carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.
Surgery has been the primary therapy for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been refractory to any systemic therapy. With recent developments in systemic therapies, particularly immunotherapies, metastatic renal cell carcinoma may be approached aggressively in appropriate patients with a possibility of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.
Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and 8 per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking patterns in women. There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an increasing problem as the population becomes more elderly.
Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points out the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.
An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women. Incidence rates declined significantly during 1992-1996 (−2.1% per year).
Research suggests that these declines have been due to increased screening and polyp removal, preventing progression of polyps to invasive cancers. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, accounting for about 11% of all U.S. cancer deaths.
At present, surgery is the most common form of therapy for colorectal cancer, and for cancers that have not spread, it is frequently curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery to most patients whose cancer has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (creation of an abdominal opening for elimination of body wastes) is occasionally needed for colon cancer and is infrequently required for rectal cancer. There continues to be a need for effective diagnostic and treatment modalities for colorectal cancer.
There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, accounting for 14% of all U.S. cancer diagnoses. The incidence rate of lung and bronchial cancer is declining significantly in men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to slow. In 1996, the incidence rate in women was 42.3 per 100,000.
Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. During 1992-1996, mortality from lung cancer declined significantly among men (−1.7% per year) while rates for women were still significantly increasing (0.9% per year). Since 1987, more women have died each year of lung cancer than breast cancer, which, for over 40 years, was the major cause of cancer death in women. Decreasing lung cancer incidence and mortality rates most likely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking patterns among women lag behind those of men. Of concern, although the declines in adult tobacco use have slowed, tobacco use in youth is increasing again.
Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.
An estimated 182,800 new invasive cases of breast cancer were expected to occur among women in the United States during 2000. Additionally, about 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000. After increasing about 4% per year in the 1980s, breast cancer incidence rates in women have leveled off in the 1990s to about 110.6 cases per 100,000.
In the U.S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black. These decreases were probably the result of earlier detection and improved treatment.
Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.
Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast and/or tamoxifen may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop into invasive breast cancer. Nevertheless, there are serious side effects or sequelae to these treatments. There is, therefore, a need for efficacious breast cancer treatments.
There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. It accounts for 4% of all cancers among women and ranks second among gynecologic cancers. During 1992-1996, ovarian cancer incidence rates were significantly declining. Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.
Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgery usually includes the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy). In some very early tumors, only the involved ovary will be removed, especially in young women who wish to have children. In advanced disease, an attempt is made to remove all intra-abdominal disease to enhance the effect of chemotherapy. There continues to be an important need for effective treatment options for ovarian cancer.
There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, rates of pancreatic cancer have declined in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant decrease in mortality rates among men (about −0.9% per year) while rates have increased slightly among women.
Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival and/or relieve symptoms in many patients but are not likely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer.
SUMMARY OF THE INVENTION
The present invention relates to a gene, designated 108P5H8, that has now been found to be over-expressed in the cancer(s) listed in Table I. Northern blot expression analysis of 108P5H8 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 108P5H8 are provided. The tissue-related profile of 108P5H8 in normal adult tissues, combined with the over-expression observed in the tumors listed in Table I, shows that 108P5H8 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 108P5H8 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 108P5H8-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 108P5H8-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 108P5H8 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 108P5H8 genes, mRNAs, or to 108P5H8-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 108P5H8. Recombinant DNA molecules containing 108P5H8 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 108P5H8 gene products are also provided. The invention further provides antibodies that bind to 108P5H8 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 108P5H8 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 108P5H8. A typical embodiment of this invention provides methods for monitoring 108P5H8 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 108P5H8 such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 108P5H8 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 108P5H8 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 108P5H8. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 108P5H8 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 108P5H8 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 108P5H8 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 108P5H8. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 108P5H8 (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 108P5H8 production) or a ribozyme effective to lyse 108P5H8 mRNA.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. The 108P5H8 sequence of 448 nucleotides (SEQ ID NO: 2568).
FIG. 2. The cDNA (SEQ ID. NO. : 2569) and amino acid sequence (SEQ ID. NO. : 2570) of 108P5H8 v.1 is shown in FIG. 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 253-1542 including the stop codon. The nucleic acid (SEQ ID. NO. : 2571) and amino acid sequence of 108P5H8 variant 2 (SEQ ID. NO. : 2572) is shown in FIG. 2B, the codon for the start methionine is underlined. The open reading frame for variant 2 extends from nucleic acid 1 to 1290 including the stop codon. The nucleic acid (SEQ ID. NO.: 2573) and amino acid sequence of 108P5F18 variant 3 (SEQ ID. NO. : 2574) is shown in FIG. 2C, the codon for the start methionine is underlined. The open reading frame for variant 3 extends from nucleic acid 1-1290 including the stop codon.
FIG. 3. Amino acid sequence of 108P5H8 variant 1 and of 108P5H8 variant 2 (SEQ ID. NO. : 2572) is shown in FIG. 3A. The proteins encoded by the variant 1 and variant 2 nucleic acid sequences are identical and each have 429 amino acids. The amino acid sequence of 1 08P5H8 variant 3 (SEQ ID. NO. : 2574) is shown in FIG. 3B, the 108P5H8 v.3 protein has 429 amino acids.
FIG. 4. 4A shows nucleotide sequence alignments of 108P5H8 variants 1-3 and 4B shows amino acid alignments of 108P5H8 variant 1 (SEQ ID. NO. : 2570), variant 2 (SEQ ID. NO. : 2572) and variant 3 (SEQ ID. NO. : 2574).
FIG. 5. Hydrophilicity amino acid profile of 108P5H8 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 through the ExPasy molecular biology server.
FIG. 6. Hydropathicity amino acid profile of 108P5H8 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 through the ExPasy molecular biology server.
FIG. 7. Percent accessible residues amino acid profile of 108P5H8 determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website through the ExPasy molecular biology server.
FIG. 8. Average flexibility amino acid profile of 108P5H8 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 through the ExPasy molecular biology server.
FIG. 9. Beta-turn amino acid profile of 108P5H8 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 through the ExPasy molecular biology server.
FIG. 10. Expression of 108P5H8 by RT-PCR. First strand cDNA was prepared from vital pool 1 (VP1: liver, lung and kidney), vital pool 2 (VP2, pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI), normal thymus, prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, pancreas cancer pool, and from prostate cancer metastasis to lymph node from 2 different patients. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR using primers to 108P5H8 was performed at 26 and 30 cycles of amplification. Strong expression of 108P5H8 was observed in prostate cancer xenograft pool, prostate cancer pool and in the 2 different prostate cancer metastasis samples. Lower expression was detected in bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis pool, pancreas cancer pool, VP1 and VP2.
FIG. 11. Expression of 108P5H8 in normal human tissues and in prostate cancer xenografts. (A and B) Two multiple tissue Northern blots, with 2 mg of mRNA/lane, were probed with 108P5H8 sequence. Size standards in kilobases (kb) are indicated on the side. The results show strong expression of an approximately 7 kb 108P5H8 transcript in prostate and lower expression in other tissues. (C) RNA was extracted from normal prostate, and from prostate cancer xenografts, LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI. Northern blot with 10 mg of total RNA/lane was probed with 108P5H8 sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 108P5H8 in all 4 xenografts and in normal prostate.
FIG. 12. Expression of 108P5H8 in prostate cancer xenografts. RNA was extracted from prostate cancer xenografts, LAPC-4AD, and LAPC-9AD, injected either subcutaneously (sc) or intratibially (it) within the mouse bone. LAPC-4 was also grown within a human bone implant in SCID mice (LAPC-4 AD2). Northern blots with 10 μg of total RNA/lane were probed with the 108P5H8 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Results show expression of 108P5H8 in all prostate cancer xenograft tissues tested.
FIG. 13. Expression of 108P5H8 in human cancer cell lines. RNA was extracted from a panel of human cancer cell lines. Northern blots with 10 mg of total RNA/lane were probed with the 108P5H8 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Results show that 108P5H8 is expressed in all cell lines tested such as prostate, bladder, brain, lung, kidney, breast, testis and ovary cancer cell lines.
FIG. 14. Expression of 108P5H8 in patient cancer specimens and cancer cell lines. Expression of 108P5H8 was assayed in a panel of human cancers (T) and their respective matched normal tissues (N) on RNA dot blots. 108P5H8 expression was seen in prostate, kidney, uterus and stomach cancers. The expression detected in some normal adjacent tissues (isolated from diseased tissues), but not in normal tissues (isolated from healthy donors), may indicate that these tissues are not fully normal and that 108P5H8 may be expressed in early-stage tumors. 108P5H8 was also expressed in all 9 human cancer cell lines tested.
FIG. 15. Expression of 108P5H8 in prostate cancer patient specimens. RNA was extracted from prostate tumors (T) and matched normal adjacent tissue (NAT) isolated from prostate cancer patients. Northern blots with 10 mg of total RNA/lane were probed with 108P5H8 sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 108P5H8 in the two prostate tumors and in the normal matched tissues.
FIG. 16. 108P5H8 is not Androgen-Regulated. LNCaP cells were grown in charcoal-stripped medium and stimulated with the synthetic androgen mibolerone, for either 14 or 24 hours. Northern blots with 10 mg of total RNA/lane were probed with either the 108P5H8 sequence (A), or with the androgen-regulated gene PSA (B). A picture of the ethidium-bromide staining of the RNA gel is also presented (C). Results show expression of 108P5H8 is not regulated by androgen. The experimental samples were confirmed by testing for the expression of the androgen-regulated prostate cancer gene PSA (B). This experiment shows that, as expected, PSA levels go down in presence of charcoal-stripped serum, and expression is induced at 14 and 24 hours in presence of the synthetic androgen.
FIG. 17. Expression of 108P5H8 in cancer metastasis patient specimens. RNA was extracted from prostate cancer metastasis to lymph node obtained from two different patient, as well as from normal bladder (NB), normal kidney (NK), normal lung (NL), normal breast (NBr), normal ovary (NO), and normal pancreas (NPa). Northern blots with 10 mg of total RNA/lane were probed with 108P5H8 sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 108P5H8 in both cancer metastasis samples but not in normal tissues.
FIG. 18. Secondary structure and transmembrane prediction for 108P5H8 (SEQ ID. NO. : 2570). The secondary structure of 108P5H8 protein was predicted using the HNN—Hierarchical Neural Network method (Guermeur, 1997), accessed from the ExPasy molecular biology server. This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein in a given secondary structure is also given.
FIG. 19. Transmembrane prediction for 108P5H8. A. Schematic representation of the probability of existence of transmembrane regions and orientation of 108P5H8 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). B. Schematic representation of the probability of the existence of transmembrane regions and the extracellular and intracellular orientation of 108P5H8 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, CA: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed from the ExPasy molecular biology server. The results of the transmembrane prediction programs presented in A and B depict 108P5H8 as containing 6 transmembrane domains.
FIG. 20. Androgen-independent expression of 108P5H8 in prostate cancer cells. Western analysis of the indicated cell lysates were carried out with a 1:2000 dilution of an anti-108P5H8 polyclonal antibody derived from immunization of a rabbit with a GST-fusion protein encoding amino acids 1-112 of 108P5H8. 108P5H8 specific bands were developed by incubation with an anti-rabbit HRP-conjugated secondary antibody and visualized by enhanced chemiluminescence and exposure to autoradiography film. Indicated with an arrow is the full length 108P5H8 protein. 293T cells overexpressing Myc His-tagged 108P5H8 serves as a positive control
FIG. 21. Surface expression of 108P5H8 in prostate cancer cells. LNCaP and LAPC4 cells were subjected to flow cytometric and fluorescence microscopic analysis of 108P5H8 expression using an anti-108P5H8 polyclonal antibody or control rabbit IgG. Fluorescence was monitored following incubation with an FITC-conjugated anti-rabbit IgG secondary antibody
FIG. 22. Surface expression of 108P5H8 in 293T cells. 293T cells were transfected with either empty control vector or with pCDNA 3.1 encoding the 108P5H8 cDNA and subjected to flow cytometry and fluorescence microscopy using an anti-108P5H8 polyclonal antibody (1:100 dilution). Fluorescence was monitored following incubation with an FITC-conjugated anti-rabbit IgG secondary antibody. 293T-108P5H8 cells exhibited strong surface fluorescence.
FIG. 23. Expression of 108P5H8 in prostate and ovarian cancer patient specimens. Lysates from tumor (PCa) and normal adjacent tissue (NAT) from 2 prostate cancer patients and from a prostate cancer metastasis and tumor and normal adjacent tissue from an ovarian cancer patient were subjected to Western analysis using anti-108P5H8 polyclonal antibody as described in FIG. 20. Indicated with an arrow is a 48 kD band representing full length 1-08P5H8. 108P5H8 protein was present in the tumor tissue from the 2 prostate cancer patients and the metastasis sample and in the normal adjacent tissue of 1 of the patients. 108P5H8 was also expressed in the ovarian cancer sample but not in normal ovary. Low expression is seen in RNA positive 293T cells and strong expression in the overexpressed 293T-108P5H8 cells.
FIG. 24. Expression of 108P5H8 in engineered cell lines. PC3 human prostate cancer cells and NIH3T3 murine fibroblasts were engineered to stably express 108P5H8 through infection with retrovirus harboring the 108P5H8 cDNA. Stable lines were generated by G418 selection for neomycin resistance. 108P5H8 expression was verified by Western blot analysis with anti-108P5H8 polyclonal antibody as described in FIG. 20, using the respective cell lines expressing only the neomycin resistance gene as negative controls.
FIG. 25. 25A shows an alignment of 108P5H8 protein (SEQ ID.NO. : 2570) with the human zinc transporter 4, i.e. gi 11432533 (SEQ ID. NO. : 2579): 25B shows an alignment of 108P5H8 protein with the human zinc transporter ZNT4, i.e. gi 8134840 (SEQ ID. NO. : 2580): and 25C shows an alignment of 108P5H8 protein with the rat zinc transporter ZNT-4, i.e. gi 8134837 (SEQ ID. NO. : ?);.
FIG. 26. Detection of 108P5H8 protein by immunocytochemistry in LNCaP cells. Immunocytochemical staining of LNCaP cells (an androgen dependant prostate cancer cell line) showing expression of 108P5H8, which is not androgen regulated. LNCaP cell preparations were made from either cells grown in medium containing 10% fetal bovine serum (Panel A) or from cells grown for 72 hours in androgen free, serum depleted medium (by growing in charcoal dextran stripped medium) (Panel B) or from previously androgen starved cells which were subsequently stimulated with 10 mmol mibolerone, a synthetic androgen, for 48 hours (Panel C). LNCaP cells incubated with Rabbit IgG instead of rabbit antibody to 108P5H8 was included to show no non-specific binding of rabbit immunoglobulin to the cells (Panel D).
FIG. 27. Detection of 108P5H8 protein by immunohistochemistry in prostate cancer patient specimens. Immunohistochemical staining of frozen sections of a prostate carcinoma specimen (Gleason grade 6) showing expression of 108P5H8 in the neoplastic glands (Panel A) and no non-specific binding of rabbit immunoglobulin in the Rabbit IgG control (Panel B).
FIG. 28. FIGS. 28A & 28B show a vertical alignment comparison between nucleotide and amino acid sequences of the variants.



DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
I.) Definitions
II.) 108P5H8 Polynucleotides

    
    
        II.A.) Uses of 108P5H8 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 108P5H8-Encoding Nucleic Acid Molecules
            II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
        
        
    
    


III.) 108P5H8-related Proteins

    
    
        III.A.) Motif-bearing Protein Embodiments
        III.B.) Expression of 108P5H8-related Proteins
        III.C.) Modifications of 108P5H8-related Proteins
        III.D.) Uses of 108P5H8-related Proteins
    
    


IV.) 108P5H8 Antibodies
V.) 108P5H8 Cellular Immune Responses
VI.) 108P5H8 Transgenic Animals
VII.) Methods for the Detection of 108P5H8
VIII.) Methods for Monitoring the Status of 108P5H8-related Genes and Their Products
IX.) Identification of Molecules That Interact With 108P5H8
X.) Therapeutic Methods and Compositions

    
    
        X.A.) Anti-Cancer Vaccines
        X.B.) 108P5H8 as a Target for Antibody-Based Therapy
        X.C.) 108P5H8 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 108P5H8.
XII.) Inhibition of 108P5H8 Protein Function

    
    
        XII.A.) Inhibition of 108P5H8 With Intracellular Antibodies
        XII.B.) Inhibition of 108P5H8 with Recombinant Proteins
        XII.C.) Inhibition of 108P5H8 Transcription or Translation
        XII.D.) General Considerations for Therapeutic Strategies
    
    


XIII.) KITS
I.) Definitions:
Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.
The terms “advanced prostate cancer”, “locally advanced prostate cancer”, “advanced disease” and “locally advanced disease” mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.
“Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 108P5H8 (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 108P5H8. 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 108P5H8-related protein). For example an analog of a 108P5H8 protein can be specifically bound by an antibody or T cell that specifically binds to 108P5H8.
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-108P5H8 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.
An “antibody fragment” is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-108P5H8 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-108P5H8 antibody compositions with polyepitopic specificity.
The term “codon optimized sequences” refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an “expression enhanced sequences.”
The term “cytotoxic agent” refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to maytansinoids, yttrium, bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.
The term “homolog” refers to a molecule which exhibits, homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.
“Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, 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 108P5H8 genes or that encode polypeptides other than 108P5H8 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 108P5H8 polynucleotide. A protein is said to be “isolated,” for example, when physical, mechanical or chemical methods are employed to remove the 108P5H8 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 108P5H8 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 “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 an 108P5H8-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. In another embodiment, for example, the primary anchor residues of a peptide that will bind an HLA class II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length. The primary anchor positions for each motif and supermotif are set forth in Table IV. For example, analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif.
A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.
Non-limiting examples of small molecules include compounds that bind or interact with 108P5H8, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 108P5H8 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, 108P5H8 protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions
“Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
“Stringent conditions” or “high stringency conditions”, as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate),50 mM sodium phosphate (pH 6.8), 0.1% sodiumtpyrophosphate, 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.
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 108P5H8 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 “108P5H8-related proteins” of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different 108P5H8 proteins or fragments thereof, as well as fusion proteins of a 108P5H8 protein and a heterologous polypeptide are also included. Such 108P5H8 proteins are collectively referred to as the 108P5H8-related proteins, the proteins of the invention, or 108P5H8. The term “108P5H8-related protein” refers to a polypeptide fragment or an 108P5H8 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 or more than 100 amino acids.
II.) 108P5H8 Polynucleotides
One aspect of the invention provides polynucleotides corresponding or complementary to all or part of an 108P5H8 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding an 108P5H8-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to an 108P5H8 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to an 108P5H8 gene, mRNA, or to an 108P5H8 encoding polynucleotide, (collectively, “108P5H8 polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.
Embodiments of a 108P5H8 polynucleotide include: a 108P5H8 polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of 108P5H8 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 108P5H8 nucleotides comprise, without limitation:

    
    
        (I) a polynucleotide comprising, consisting essentially of, or consisting of a sequence as shown in FIG. 2 (SEQ ID Nos.: 2569, 2571, & 2573), wherein T can also be U;
        (II) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2A (SEQ ID NO.: 2569), from nucleotide residue number 253 through nucleotide residue number 1542, wherein T can also be U;
        (III) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2B (SEQ ID NO.: 2571), from nucleotide residue number 1 through nucleotide residue number 1290, wherein T can also be U;
        (IV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2C (SEQ ID NO.: 2573), from nucleotide residue number 1 through nucleotide residue number 1290, wherein T can also be U;
        (V) a polynucleotide that encodes an 108P5H8-related protein that is at least 90% homologous to an entire amino acid sequence shown in FIG. 2A-C (SEQ ID Nos.: 2570, 2572, & 2574);
        (VI) a polynucleotide that encodes an 108P5H8-related protein that is at least 90% identical to an entire amino acid sequence shown in FIG. 2A-C (SEQ ID Nos.: 2570, 2572, & 2574);
        (VII) a polynucleotide that encodes at least one peptide set forth in Tables V-XVIII, XXII, and XXIII;
        (VIII) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A or 3B in any whole number increment up to 429 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
        (IX) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A or 3B in any whole number increment up to 429 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
        (X) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A or 3B in any whole number increment up to 429 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
        (XI) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A or 3B in any whole number increment up to 429 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on FIG. 8;
        (XII) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A or 3B in any whole number increment up to 429 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
        (XIII) a polynucleotide that encodes a 108P5H8-related protein whose sequence is encoded by the cDNAs contained in the plasmid designated p108P5H8-C deposited with American Type Culture Collection as Accession No. PTA-2198;
        (XIV) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XIII);
        (XV) a polynucleotide that selectively hybridizes under stringent conditions to a polynucleotide of (I)-(XIV);
        (XVI) a peptide that is encoded by any of (I)-(XIII); and,
        (XLII) a polynucleotide of any of (I)-(XV) or peptide of (XVI) together with a pharmaceutical excipient and/or in a human unit dose form.
    
    


As used herein, a range is understood to specifically disclose all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 108P5H8 polynucleotides that encode specific portions of 108P5H8 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 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, 205, 210, 215, 220, 225, 250, 275, 300, 325, 350, 375, 400, 425, or 429 contiguous amino acids of variants 1, 2 or 3.
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 10of the 108P5H8 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 108P5H8 protein shown in FIG. 2, or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 108P5H8 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 108P5H8 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 108P5H8 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 108P5H8 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 108P5H8 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 108P5H8 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 108P5H8 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 108P5H8 protein shown in FIG. 2 or FIG. 3, in increments of about 10 amino acids, ending at the carboxyl terminal amino acid set forth in FIG. 2 or FIG. 3. Accordingly polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids 100 through the carboxyl terminal amino acid of the 108P5H8 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 108P5H8 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 108P5H8 protein 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 108P5H8 sequence as shown in FIG. 2.
Additional illustrative embodiments of the invention disclosed herein include 108P5H8 polynucleotide fragments encoding one or more of the biological motifs contained within a 108P5H8 protein sequence, including one or more of the motif-bearing subsequences of a 108P5H8 protein set forth in Tables V-XVIII, XXII, and XXIII. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 108P5H8 that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 108P5H8 N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.
II.A.) Uses of 108P5H8 Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 108P5H8 gene maps to the chromosomal location set forth in Example 3. For example, because the 108P5H8 gene maps to this chromosome, polynucleotides that encode different regions of the 108P5H8 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers. In certain genes, a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g. Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the 108P5H8 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 108P5H8 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 108P5H8 was shown to be highly expressed in bladder and other cancers, 108P5H8 polynucleotides are used in methods assessing the status of 108P5H8 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 108P5H8 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 108P5H8 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 108P5H8. 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 108P5H8 polynucleotides and polynucleotide sequences disclosed herein.
Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term “antisense” refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., 108P5H8. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 108P5H8 antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 108P5H8 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 108P5H8 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 108P5H8 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 108P5H8 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 108P5H8 antisense oligonucleotides of the present invention are 15 to 30 -mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 108P5H8 mRNA. Optionally, 108P5H8 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 108P5H8. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 108P5H8 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 this nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and 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 108P5H8 polynucleotide in a sample and as a means for detecting a cell expressing a 108P5H8 protein.
Examples of such probes include polypeptides comprising all or part of the human 108P5H8 cDNA sequence shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 108P5H8 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 108P5H8 mRNA.
The 108P5H8 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 108P5H8 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 108P5H8 polypeptides; as tools for modulating or inhibiting the expression of the 108P5H8 gene(s) and/or translation of the 108P5H8 transcript(s); and as therapeutic agents.
The present invention includes the use of any probe as described herein to identify and isolate a 108P5H8 or 108P5H8 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 108P5H8-Encoding Nucleic Acid Molecules
The 108P5H8 cDNA sequences described herein enable the isolation of other polynucleotides encoding 108P5H8 gene product(s), as well as the isolation of polynucleotides encoding 108P5H8 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 108P5H8 gene product as well as polynucleotides that encode analogs of 108P5H8-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding an 108P5H8 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 108P5H8 gene cDNAs can be identified by probing with a labeled 108P5H8 cDNA or a fragment thereof. For example, in one embodiment, a 108P5H8 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 108P5H8 gene. A 108P5H8 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 108P5H8 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 an 108P5H8 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 108P5H8 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 108P5H8 or a fragment, analog or homolog thereof can be used to generate 108P5H8 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 108P5H8 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, 108P5H8 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 108P5H8 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 108P5H8 and 108P5H8 mutations or analogs.
Recombinant human 108P5H8 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 108P5H8-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 108P5H8 or fragment, analog or homolog thereof, a 108P5H8-related protein is expressed in the 293T cells, and the recombinant 108P5H8 protein is isolated using standard purification methods (e.g., affinity purification using anti-108P5H8 antibodies). In another embodiment, a 108P5H8 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 108P5H8 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 108P5H8 coding sequence can be used for the generation of a secreted form of recombinant 108P5H8 protein.
As discussed herein, redundancy in the genetic code permits variation in 108P5H8 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.
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.) 108P5H8-Related Proteins
Another aspect of the present invention provides 108P5H8-related proteins. Specific embodiments of 108P5H8 proteins comprise a polypeptide having all or part of the amino acid sequence of human 108P5H8 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of 108P5H8 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 108P5H8 shown in FIG. 2 or FIG. 3.
In general, naturally occurring allelic variants of human 108P5H8 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a 108P5H8 protein contain conservative amino acid substitutions within the 108P5H8 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 108P5H8. One class of 108P5H8 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 108P5H8 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.
Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently-interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g. Table III herein; pages 13-15 “Biochemistry” 2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al. PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem May 19, 1995; 270(20):11882-6).
Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 108P5H8 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 108P5H8 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 108P5H8 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, 108P5H8 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a 108P5H8 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 an 108P5H8 variant also specifically binds to a 108P5H8 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 108P5H8 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 108P5H8-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 108P5H8 protein variants or analogs comprise one or more of the 108P5H8 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 108P5H8 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 108P5H8 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 108P5H8 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 108P5H8 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 108P5H8 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 108P5H8 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 108P5H8 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 108P5H8 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 108P5H8 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 108P5H8 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 108P5H8 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 108P5H8 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 108P5H8 protein shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a 108P5H8 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 108P5H8 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.
108P5H8-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 108P5H8-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 108P5H8 protein (or variants, homologs or analogs thereof).
III.A.) Motif-Bearing Protein Embodiments
Additional illustrative embodiments of the invention disclosed herein include 108P5H8 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 108P5H8 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., Epimatrix198  and EpimerTM, Brown University, and BIMAS).
Motif bearing subsequences of all 108P5H8 variant proteins are set forth and identified in Table XIX.
Table XX sets forth several frequently occurring motifs based on pfam searches. The columns of Table XX 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 108P5H8 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 108P5H8 motifs discussed above are associated with growth dysregulation and because 108P5H8 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 V-XVIII, XXII, and XXIII. CTL epitopes can be determined using specific algorithms to identify peptides within an 108P5H8 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; EpimatrixTM and EpimerTM, Brown University, and BIMAS.) 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, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue as defined in Table IV; substitute a less-preferred residue with a preferred residue as defined in Table IV; or substitute an originally-occurring preferred residue with another preferred residue as defined in Table IV. Substitutions can occur at primary anchor positions or at other positions in a peptide; see, 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 9733602 to Chesnut et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994); Kast et al., 1994 152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J. Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.
Related embodiments of the inventions include polypeptides comprising combinations of the different motifs set forth in Table XIX, and/or, one or more of the predicted CTL epitopes of Tables V-XVIII, Table XXII, Table XXIII, and/or, one or more of the T cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or the intervening sequences of the polypeptides. In addition, embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.
108P5H8-related proteins are embodied in many forms, preferably in isolated form. A purified 108P5H8 protein molecule will be substantially free of other proteins or molecules that impair the binding of 108P5H8 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 108P5H8-related proteins include purified 108P5H8-related proteins and functional, soluble 108P5H8-related proteins. In one embodiment, a functional, soluble 108P5H8 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
The invention also provides 108P5H8 proteins comprising biologically active fragments of a 108P5H8 amino acid sequence shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the starting 108P5H8 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 108P5H8 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.
108P5H8-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or on the basis of immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-108P5H8 antibodies, or T cells or in identifying cellular factors that bind to 108P5H8.
CTL epitopes can be determined using specific algorithms to identify peptides within an 108P5H8 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site at World Wide Web; the listings in Table IV(A)-(E); EpimatrixTM and EpimerTM, Brown University and BIMAS). Illustrating this, peptide epitopes from 108P5H8 that are presented in the context of human MHC class I molecules HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (Tables V-XVIII, XXII, and XXIII). Specifically, the complete amino acid sequence of the 108P5H8 protein and relevant portions of other variants, i.e., for HLA Class I predictions 9 flanking residues on either side of a point mutation, and for HLA Class II predictions 14 flanking residues on either side of a point mutation, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above; for HLA Class II the site SYFPEITHI was used.
The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules, in particular HLA-A2 (see, 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 108P5H8 predicted binding peptides are shown in Tables V-XVIII, XXII, and XXIII herein. In Tables V-XVIII, the top 50 ranking 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. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37° C. at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.
Actual binding of peptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigen-processing defective cell line T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa et al., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.
It is to be appreciated that every epitope predicted by the BIMAS site, EpimerTM and EpimatrixTM sites, or specified by the HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be “applied” to a 108P5H8 protein in accordance with the invention. As used in this context “applied” means that a 108P5H8 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 108P5H8 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 108P5H8-Related Proteins
In an embodiment described in the examples that follow, 108P5H8 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 108P5H8 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 108P5H8 protein in transfected cells. The secreted HIS-tagged 108P5H8 in the culture media can be purified, e.g., using a nickel column using standard techniques.
III.C.) Modifications of 108P5H8-Related Proteins
Modifications of 108P5H8-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 108P5H8 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of a 108P5H8 protein. Another type of covalent modification of a 108P5H8 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 108P5H8 comprises linking a 108P5H8 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 108P5H8-related proteins of the present invention can also be modified to form a chimeric molecule comprising 108P5H8 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 108P5H8 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 108P5H8. A chimeric molecule can comprise a fusion of a 108P5H8-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 108P5H8 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 108P5H8-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 Fe region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 108P5H8 polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
III.D.) Uses of 108P5H8-Related Proteins
The proteins of the invention have a number of different specific uses. As 108P5H8 is highly expressed in prostate and other cancers, 108P5H8-related proteins are used in methods that assess the status of 108P5H8 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 108P5H8 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 108P5H8-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 108P5H8 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, 108P5H8-related proteins that contain the amino acid residues of one or more of the biological motifs in a 108P5H8 protein are used to screen for factors that interact with that region of 108P5H8.
108P5H8 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of an 108P5H8 protein), for identifying agents or cellular factors that bind to 108P5H8 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 108P5H8 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 an 108P5H8 gene product. Antibodies raised against an 108P5H8 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 108P5H8 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 108P5H8-related nucleic acids or proteins are also used in generating HTL or CTL responses.
Various immunological assays useful for the detection of 108P5H8 proteins are used, including but not limited to various types of radioimmunoassay, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 108P5H8-expressing cells (e.g., in radioscintigraphic imaging methods). 108P5H8 proteins are also particularly useful in generating cancer vaccines, as further described herein.
IV.) 108P5H8 Antibodies
Another aspect of the invention provides antibodies that bind to 108P5H8-related proteins. Preferred antibodies specifically bind to a 108P5H8-related protein and do not bind (or bind weakly) to peptides or proteins that are not 108P5H8-related proteins. For example, antibodies that bind 108P5H8 can bind 108P5H8-related proteins such as the homologs or analogs thereof.
108P5H8 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 108P5H8 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 108P5H8 is involved, such as advanced or metastatic prostate cancers.
The invention also provides various immunological assays useful for the detection and quantification of 108P5H8 and mutant 108P5H8-related proteins. Such assays can comprise one or more 108P5H8 antibodies capable of recognizing and binding a 108P5H8-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.
Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.
In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 108P5H8 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 108P5H8 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 108P5H8 expressing cancers such as prostate cancer.
108P5H8 antibodies are also used in methods for purifying a 108P5H8-related protein and for isolating 108P5H8 homologues and related molecules. For example, a method of purifying a 108P5H8-related protein comprises incubating an 108P5H8 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 108P5H8-related protein under conditions that permit the 108P5H8 antibody to bind to the 108P5H8-related protein; washing the solid matrix to eliminate impurities; and eluting the 108P5H8-related protein from the coupled antibody. Other uses of 108P5H8 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 108P5H8 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 108P5H8-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 108P5H8 can also be used, such as a 108P5H8 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 108P5H8-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 108P5H8-related protein or 108P5H8 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 108P5H8 protein as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 108P5H8 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 108P5H8 amino acid sequence are used to identify hydrophilic regions in the 108P5H8 structure. Regions of a 108P5H8 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for the generation of 108P5H8 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 lining reagents such as those supplied by Pierce Chemical Co., Rockford, Ill., are effective. Administration of a 108P5H8 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.
108P5H8 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 108P5H8-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 108P5H8 protein can also be produced in the context of chimeric or complementarity determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 108P5H8 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 108P5H8 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 108P5H8 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued 19 Dec. 2000; U.S. Pat. No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No. 6,114,598 issued 5 Sep. 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.
Reactivity of 108P5H8 antibodies with an 108P5H8-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 108P5H8-related proteins, 108P5H8-expressing cells or extracts thereof. A 108P5H8 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 108P5H8 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.) 108P5H8 Cellular Immune Responses
The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the world-wide population. For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of immunology-related technology is provided.
A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV (see also, e.g., Southwood, et al., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web at URL syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999 November; 50(3-4):201-12, Review).
Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding cleft/groove of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e.g., Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991.)
Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s).
Thus, by a process of HLA motif identification, candidates for epitope-based vaccines have been identified; such candidates can be further evaluated by HLA-peptide binding assays to determine binding affinity and/or the time period of association of the epitope and its corresponding HLA molecule. Additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenicity.
Various strategies can be utilized to evaluate cellular immunogenicity, including:
1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158-1796, 1997; Kawashima, I. et al., Human Immunol. 59:1, 1998). This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a lymphokine- or 51Cr-release assay involving peptide sensitized target cells.
2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). For example, in such methods peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected using, e.g., a 51Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.
3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated and/or from chronically ill patients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response “naturally”, or from patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of “memory” T cells, as compared to “naive” T cells. At the end of the culture period, T cell activity is detected using assays including 51Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.
VI.) 108P5H8 Transgenic Animals
Nucleic acids that encode a 108P5H8-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 108P5H8 can be used to clone genomic DNA that encodes 108P5H8. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 108P5H8. 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 108P5H8 transgene incorporation with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding 108P5H8 can be used to examine the effect of increased expression of DNA that encodes 108P5H8. 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 108P5H8 can be used to construct a 108P5H8 “knock out” animal that has a defective or altered gene encoding 108P5H8 as a result of homologous recombination between the endogenous gene encoding 108P5H8 and altered genomic DNA encoding 108P5H8 introduced into an embryonic cell of the animal. For example, cDNA that encodes 108P5H8 can be used to clone genomic DNA encoding 108P5H8 in accordance with established techniques. A portion of the genomic DNA encoding 108P5H8 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 108P5H8 polypeptide.
VII.) Methods for the Detection of 108P5H8
Another aspect of the present invention relates to methods for detecting 108P5H8 polynucleotides and 108P5H8-related proteins, as well as methods for identifying a cell that expresses 108P5H8. The expression profile of 108P5H8 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 108P5H8 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 108P5H8 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 108P5H8 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 108P5H8 polynucleotides include, for example, a 108P5H8 gene or fragment thereof, 108P5H8 mRNA, alternative splice variant 108P5H8 mRNAs, and recombinant DNA or RNA molecules that contain a 108P5H8 polynucleotide. A number of methods for amplifying and/or detecting the presence of 108P5H8 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 an 108P5H8 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 an 108P5H8 polynucleotides as sense and antisense primers to amplify 108P5H8 cDNAs therein; and detecting the presence of the amplified 108P5H8 cDNA. Optionally, the sequence of the amplified 108P5H8 cDNA can be determined.
In another embodiment, a method of detecting a 108P5H8 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 108P5H8 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 108P5H8 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 192P1E1B nucleotide sequence (see, e.g., FIG. 2) and used for this purpose.
The invention also provides assays for detecting the presence of an 108P5H8 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 108P5H8-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 108P5H8-related protein in a biological sample comprises first contacting the sample with a 108P5H8 antibody, a 108P5H8-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a 108P5H8 antibody; and then detecting the binding of 108P5H8-related protein in the sample.
Methods for identifying a cell that expresses 108P5H8 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 108P5H8 gene comprises detecting the presence of 108P5H8 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 108P5H8 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 108P5H8, 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 108P5H8 gene comprises detecting the presence of 108P5H8-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 108P5H8-related proteins and cells that express 108P5H8-related proteins.
108P5H8 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 108P5H8 gene expression. For example, 108P5H8 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 108P5H8 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 108P5H8 expression by RT-PCR, nucleic acid hybridization or antibody binding.
VIII.) Methods for Monitoring the Status of 108P5H8-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 108P5H8 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 108P5H8 in a biological sample of interest can be compared, for example, to the status of 108P5H8 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 108P5H8 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. 9 Dec. 1996; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare 108P5H8 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 108P5H8 expressing cells) as well as the level, and biological activity of expressed gene products (such as 108P5H8 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 108P5H8 comprises a change in the location of 108P5H8 and/or 108P5H8 expressing cells and/or an increase in 108P5H8 mRNA and/or protein expression.
108P5H8 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 108P5H8 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 108P5H8 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 108P5H8 gene), Northern analysis and/or PCR analysis of 108P5H8 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 108P5H8 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 108P5H8 proteins and/or associations of 108P5H8 proteins with polypeptide binding partners). Detectable 108P5H8 polynucleotides include, for example, a 108P5H8 gene or fragment thereof, 108P5H8 mRNA, alternative splice variants, 108P5H8 mRNAs, and recombinant DNA or RNA molecules containing a 108P5H8 polynucleotide.
The expression profile of 108P5H8 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 108P5H8 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 108P5H8 status and diagnosing cancers that express 108P5H8, such as cancers of the tissues listed in Table I. For example, because 108P5H8 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 108P5H8 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 108P5H8 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.
The expression status of 108P5H8 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 108P5H8 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 108P5H8 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 108P5H8 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 108P5H8 expressing cells (e.g. those that express 108P5H8 mRNAs or proteins): This examination can provide evidence of dysregulated cellular growth, for example, when 108P5H8-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 108P5H8 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000);Su et al., Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995 August 154(2 Pt 1):474-8).
In one aspect, the invention provides methods for monitoring 108P5H8 gene products by determining the status of 108P5H8 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 108P5H8 gene products in a corresponding normal sample. The presence of aberrant 108P5H8 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 108P5H8 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 108P5H8 mRNA can, for example, be evaluated in tissue samples including but not limited to those listed in Table I. The presence of significant 108P5H8 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 108P5H8 mRNA or express it at lower levels.
In a related embodiment, 108P5H8 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 108P5H8 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 108P5H8 expressed in a corresponding normal sample. In one embodiment, the presence of 108P5H8 protein is evaluated, for example, using immunohistochemical methods. 108P5H8 antibodies or binding partners capable of detecting 108P5H8 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 108P5H8 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 108P5H8 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 108P5H8 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 108P5H8 gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S. Pat.No. 5,952,170 issued 17 Jan. 1995).
Additionally, one can examine the methylation status of a 108P5H8 gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5′ regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers 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 methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.
Gene amplification is an additional method for assessing the status of 108P5H8. 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 108P5H8 expression. The presence of RT-PCR amplifiable 108P5H8 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 108P5H8 mRNA or 108P5H8 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 108P5H8 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 108P5H8 in prostate or other tissue is examined, with the presence of 108P5H8 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 108P5H8 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 108P5H8 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 108P5H8 mRNA or 108P5H8 protein expressed by tumor cells, comparing the level so determined to the level of 108P5H8 mRNA or 108P5H8 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 108P5H8 mRNA or 108P5H8 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 108P5H8 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 108P5H8 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 108P5H8 mRNA or 108P5H8 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 108P5H8 mRNA or 108P5H8 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 108P5H8 mRNA or 108P5H8 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 108P5H8 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 108P5H8 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 108P5H8 gene and 108P5H8 gene products (or perturbations in 108P5H8 gene and 108P5H8 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 108P5H8 gene and 108P5H8 gene products (or perturbations in 108P5H8 gene and 108P5H8 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 108P5H8 gene and 108P5H8 gene products (or perturbations in 108P5H8 gene and 108P5H8 gene products) and another factor associated with malignancy entails detecting the overexpression of 108P5H8 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 108P5H8 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 108P5H8 and PSA mRNA in prostate tissue is examined, where the coincidence of 108P5H8 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 108P5H8 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 108P5H8 mRNA include in situ hybridization using labeled 108P5H8 riboprobes, Northern blot and related techniques using 108P5H8 polynucleotide probes, RT-PCR analysis using primers specific for 108P5H8, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify 108P5H8 mRNA expression. Any number of primers capable of amplifying 108P5H8 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 108P5H8 protein can be used in an immunohistochemical assay of biopsied tissue.
IX.) Identification of Molecules that Interact with 108P5H8
The 108P5H8 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 108P5H8, as well as pathways activated by 108P5H8 via any one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the “two-hybrid assay”). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523 issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S. Pat. No. 6,004,746 issued 21 Dec. 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e.g., Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).
Alternatively one can screen peptide libraries to identify molecules that interact with 108P5H8 protein sequences. In such methods, peptides that bind to 108P5H8 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 108P5H8 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 108P5H8 protein sequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3 Mar. 1998 and U.S.Pat.No. 5,733,731 issued 31 Mar. 1998.
Alternatively, cell lines that express 108P5H8 are used to identify protein-protein interactions mediated by 108P5H8. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 108P5H8 protein can be immunoprecipitated from 108P5H8-expressing cell lines using anti-108P5H8 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 108P5H8 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 108P5H8 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 108P5H8'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 108P5H8-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 108P5H8 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, Mass., 1992). Moreover, ligands that regulate 108P5H8 function can be identified based on their ability to bind 108P5H8 and activate a reporter construct. Typical methods are discussed for example in U.S. Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 108P5H8 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 108P5H8.
An embodiment of this invention comprises a method of screening for a molecule that interacts with an 108P5H8 amino acid sequence shown in FIG. 2 or FIG. 3, comprising the steps of contacting a population of molecules with a 108P5H8 amino acid sequence, allowing the population of molecules and the 108P5H8 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 108P5H8 amino acid sequence, and then separating molecules that do not interact with the 108P5H8 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 108P5H8 amino acid sequence. The identified molecule can be used to modulate a function performed by 108P5H8. In a preferred embodiment, the 108P5H8 amino acid sequence is contacted with a library of peptides.
X.) Therapeutic Methods and Compositions
The identification of 108P5H8 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in prostate and other cancers, opens a number of therapeutic approaches to the treatment of such cancers. As contemplated herein, 108P5H8 functions as a transcription factor involved in activating tumor-promoting genes or repressing genes that block tumorigenesis.
Accordingly, therapeutic approaches that inhibit the activity of a 108P5H8 protein are useful for patients suffering from a cancer that expresses 108P5H8. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 108P5H8 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 108P5H8 gene or translation of 108P5H8 mRNA.
X.A.) Anti-Cancer Vaccines
The invention provides cancer vaccines comprising a 108P5H8-related protein or 108P5H8-related nucleic acid. In view of the expression of 108P5H8, cancer vaccines prevent and/or treat 108P5H8-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 108P5H8-related protein, or an 108P5H8-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 108P5H8 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et al., Ann Med 1999 February 31(1):66-78; Maruyama et al., Cancer Immunol Immunother 2000 June 49(3):123-32) Briefly, such methods of generating an immune response (e.g. humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in a 108P5H8 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 108P5H8 immunogen contains a biological motif, see e.g., Tables V-XVIII, XXII, and XXIII, or a peptide of a size range from 108P5H8 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.
The entire 108P5H8 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M. -P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.
In patients with 108P5H8-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 108P5H8 protein that bind corresponding HLA alleles (see e.g., Table IV; EpimerTM and EpimatrixTM, Brown University; and, BIMAS,. In a preferred embodiment, a 108P5H8 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables V-XVIII, XXII, and XXIII 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 108P5H8 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 108P5H8 in a host, by contacting the host with a sufficient amount of at least one 108P5H8 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 108P5H8 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 108P5H8-related protein or a man-made multiepitopic peptide comprising: administering 108P5H8 immunogen (e.g. a 108P5H8 protein or a peptide fragment thereof, an 108P5H8 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 108P5H8 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes an 108P5H8 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 108P5H8, 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 108P5H8. Constructs comprising DNA encoding a 108P5H8-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 108P5H8 protein/immunogen. Alternatively, a vaccine comprises a 108P5H8-related protein. Expression of the 108P5H8-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 1 08P5H8 protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used. 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 108P5H8-related protein into the patient (e.g., intramuscularly or intradermally) to induce an anti-tumor response.
Vaccinia virus is used, for example, as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein immunogenic peptide, and thereby elicits a host immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.
Thus, gene delivery systems are used to deliver a 108P5H8-related nucleic acid molecule. In one embodiment, the full-length human 108P5H8 cDNA is employed. In another embodiment, 108P5H8 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 108P5H8 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 108P5H8 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 108P5H8 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 108P5H8 protein. Yet another embodiment involves engineering the overexpression of a 108P5H8 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 108P5H8 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.
X.B.) 108P5H8 as a Target for Antibody-Based Therapy
108P5H8 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 108P5H8 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 108P5H8-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 108P5H8 are useful to treat 108P5H8-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.
108P5H8 antibodies can be introduced into a patient such that the antibody binds to 108P5H8 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 108P5H8, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.
Those skilled in the art understand that antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of a 108P5H8 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. 108P5H8), 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-108P5H8 antibody) that binds to a marker (e.g. 108P5H8) 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 108P5H8, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 108P5H8 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-108P5H8 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, 108P5H8 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin (e.g., MylotargTM, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG4 kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).
Although 108P5H8 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 108P5H8 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 108P5H8 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 108P5H8 imaging, or other techniques that reliably indicate the presence and degree of 108P5H8 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-108P5H8 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-108P5H8 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-108P5H8 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 108P5H8. 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-108P5H8 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 108P5H8 antigen with high affinity but exhibit low or no antigenicity in the patient.
Therapeutic methods of the invention contemplate the administration of single anti-108P5H8 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-108P5H8 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-108P5H8 mAbs are administered in their “naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.
Anti-108P5H8 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-108P5H8 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-108P5H8 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 108P5H8 expression in the patient, the extent of circulating shed 108P5H8 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 108P5H8 in a given sample (e.g. the levels of circulating 108P5H8 antigen and/or 108P5H8 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-108P5H8 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 108P5H8-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-108P5H8 antibodies that mimic an epitope on a 108P5H8-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.) 108P5H8 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 108P5H8 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.
In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRETM (Epimmune, San Diego, Calif.) molecule (described e.g., in U.S. Pat. No. 5,736,142).
A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present-peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are transfected, e.g., with a minigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vaccine compositions, either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.
Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the following principles be balanced in order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.
1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For HLA Class I this includes 3-4 epitopes that come from at least one tumor associated antigen (TAA). For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one TAA (see, e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.
2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, often 200 nM or less; and for Class II an IC50 of 1000 nM or less.
3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.
4.) When selecting epitopes from cancer-related antigens it is often useful to select analogs because the patient may have developed tolerance to the native epitope.
5.) Of particular relevance are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A nested peptide sequence can comprise B cell, HLA class I and/or HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per sequence. Thus, an aspect is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.
6.) If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.
7.) Where the sequences of multiple variants of the same target protein are present, potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9 -mer core of a class II binding peptide be conserved in a designated percentage of the sequences-evaluated for a specific protein antigen.
X.C.1. Minigene Vaccines
A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention.
The use of multi-epitope minigenes is described below and in, Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding supermotif- and/or motif-beating epitopes derived 108P5H8, the PADRE® universal helper T cell epitope (or multiple HTL epitopes from 108P5H8) and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.
The immunogenicity of a multi-epitopic minigene can be confirmed in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.
For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.
The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.
Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.
Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.
In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.
In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRETM, Epimmune, San Diego, Calif.). Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.
Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g:, as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat No. 5,279,833; WO 91/06309; and Felgner, 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: 2582), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 2583), and Streptococcus 18 kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 2584). 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 to most preferably bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa (SEQ ID NO: 2585), 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 specifically prime 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 108P5H8. 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 108P5H8.
X.D. Adoptive Immunotherapy
Antigenic 108P5H8-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 108P5H8. 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 108P5H8. 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 108P5H8-associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses 108P5H8, a vaccine comprising 108P5H8-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 effectively stimulate a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.
The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50,000 μg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, has been eliminated or reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.
In certain embodiments, the peptides and compositions of the present invention are employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.
The vaccine compositions of the invention can also be used purely as prophylactic agents. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.
The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, intrathecal, or local (e.g. as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known-by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptide dose for initial immunization can be from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. For example, for nucleic acids an initial immunization may be performed using an expression vector in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5×109 pfu.
For antibodies, a treatment generally involves repeated administration of the anti-108P5H8 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated. Moreover, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-108P5H8 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 108P5H8 expression in the patient, the extent of circulating shed 108P5H8 antigen, the desired steady-state concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient. Non-limiting preferred human unit doses are, for example, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200 mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg, 800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, the dose is in a range of 2-5 mg/kg body weight, e.g., with follow on weekly doses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg body weight followed, e.g., in two, three or four weeks by weekly doses; 0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m2 of body area weekly; 1-600 mg m2 of body area weekly; 225-400 mg m2 of body area weekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks.
In one embodiment, human unit dose forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art a therapeutic effect depends on a number of factors, including the sequence of the polynucleotide, molecular weight of the polynucleotide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. Generally, for a polynucleotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit, greater than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral routes of administration may require higher doses of polynucleotide compared to more direct application to the nucleotide to diseased tissue, as do polynucleotides of increasing length.
In one embodiment, human unit dose forms of T-cells comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. A dose may be about 104 cells to about 106 cells, about 106 cells to about 108 cells, about 108 to about 1011 cells, or about 108 to about 5×1010 cells. A dose may also about 106 cells/m2 to about 1010 cells/m2, or about 106 cells/m2 to about 108 cells/m2.
Proteins(s) of the invention, and/or nucleic acids encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular tissue, such as lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.
For aerosol administration, immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01%-20% by weight, preferably about 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1%-20% by weight of the composition, preferably about 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
XI.) Diagnostic and Prognostic Embodiments of 108P5H8.
As disclosed herein, 108P5H8 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 Example 4).
108P5H8 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640(1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et al., Int J Mol Med July 4, 1999(1):99-102 and Minimoto et al., Cancer Detect Prev 2000;24(1):1-12). Therefore, this disclosure of 108P5H8 polynucleotides and polypeptides (as well as 108P5H8 polynucleotide probes and anti-108P5H8 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 108P5H8 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 108P5H8 polynucleotides described herein can be utilized in the same way to detect 108P5H8 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 108P5H8 polypeptides described herein can be utilized to generate anitbodies for use in detecting 108P5H8 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 108P5H8 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 108P5H8-expressing cells (lymph node) is found to contain 108P5H8-expressing cells such as the 108P5H8 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.
Alternatively 108P5H8 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 108P5H8 or express 108P5H8 at a different level are found to express 108P5H8 or have an increased expression of 108P5H8 (see, e.g., the 108P5H8 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 108P5H8) such as PSA, PSCA etc. (see, e.g.; Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).
Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 108P5H8 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 Example 4, where a 108P5H8 polynucleotide fragment is used as a probe to show the expression of 108P5H8 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. 1996 November -December 11(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g., a 108P5H8 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. 108P5H8 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 108P5H8 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 108P5H8 polypeptide shown in FIG. 3).
As shown herein, the 108P5H8 polynucleotides and polypeptides (as well as the 108P5H8 polynucleotide probes and anti-108P5H8 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 108P5H8 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 108P5H8 polynucleotides and polypeptides (as well as the 108P5H8 polynucleotide probes and anti-108P5H8 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 108P5H8 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 108P5H8 gene maps (see Example 3 below). Moreover, in addition to their use in diagnostic assays, the 108P5H8-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 28 Jun. 1996;80(1-2): 63-9).
Additionally, 108P5H8-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 108P5H8. 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 108P5H8 antigen. Antibodies or other molecules that react with 108P5H8 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.
XII.) Inhibition of 108P5H8 Protein Function
The invention includes various methods and compositions for inhibiting the binding of 108P5H8 to its binding partner or its association with other protein(s) as well as methods for inhibiting 108P5H8 function.
XII.A.) Inhibition of 108P5H8 with Intracellular Antibodies
In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 108P5H8 are introduced into 108P5H8 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-108P5H8 antibody is expressed intracellularly, binds to 108P5H8 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 precisely target the intrabody to the desired intracellular compartment. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif. Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.
In one embodiment, intrabodies are used to capture 108P5H8 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 108P5H8 intrabodies in order to achieve the desired targeting. Such 108P5H8 intrabodies are designed to bind specifically to a particular 108P5H8 domain. In another embodiment, cytosolic intrabodies that specifically bind to a 108P5H8 protein are used to prevent 108P5H8 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 108P5H8 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 108P5H8 with Recombinant Proteins
In another approach, recombinant molecules bind to 108P5H8 and thereby inhibit 108P5H8 function. For example, these recombinant molecules prevent or inhibit 108P5H8 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 108P5H8 specific antibody molecule. In a particular embodiment, the 108P5H8 binding domain of a 108P5H8 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 108P5H8 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 108P5H8, whereby the dimeric fusion protein specifically binds to 108P5H8 and blocks 108P5H8 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.
XII.C.) Inhibition of 108P5H8 Transcription or Translation
The present invention also comprises various methods and compositions for inhibiting the transcription of the 108P5H8 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 108P5H8 mRNA into protein.
In one approach, a method of inhibiting the transcription of the 108P5H8 gene comprises contacting the 108P5H8 gene with a 108P5H8 antisense polynucleotide. In another approach, a method of inhibiting 108P5H8 mRNA translation comprises contacting a 108P5H8 mRNA with an antisense polynucleotide. In another approach, a 108P5H8 specific ribozyme is used to cleave a 108P5H8 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 108P5H8 gene, such as 108P5H8 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 108P5H8 gene transcription factor are used to inhibit 108P5H8 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 108P5H8 by interfering with 108P5H8 transcriptional activation are also useful to treat cancers expressing 108P5H8. Similarly, factors that interfere with 108P5H8 processing are useful to treat cancers that express 108P5H8. 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 108P5H8 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 108P5H8 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 108P5H8 antisense polynucleotides, ribozymes, factors capable of interfering with 108P5H8 transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.
The above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens. The therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for all patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent well.
The anti-tumor activity of a particular composition (e.g., antisense, ribozyme, intrabody), or a combination of such compositions, can be evaluated using various in vitro and in vivo assay systems. In vitro assays that evaluate therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 108P5H8 to a binding partner, etc.
In vivo, the effect of a 108P5H8 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. 6,167,540 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.) Kits
For use in the diagnostic and therapeutic applications described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a 108P5H8-related protein or a 108P5H8 gene or message, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label. The kit can include all or part of the amino acid sequence of FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecules that encodes such amino acid sequences.
The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
A label can be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and can also indicate directions for either in vivo or in vitro use, such as those described above. Directions and or other information can also be included on an insert which is included with the kit.
EXAMPLES
Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which are intended to limit the scope of the invention.
Example 1
SSH-Generated Isolation of a cDNA Fragment of the 108P5H8 Gene
To isolate genes that are androgen regulated, the androgen-dependent prostate cancer cell line LNCaP was grown in media containing charcoal-stripped serum (steroid hormone depleted) for one week. The cells were subsequently stimulated with 10 nM mibolerone (synthetic androgen) for 9 h and were harvested as a source of mRNA. The 108P5H8 SSH cDNA sequence was derived from a subtraction consisting of LNCaP cells grown in presence of mibolerone minus LNCaP cells grown in absence of mibolerone.
The 108P5H8 SSH cDNA sequence of 448 bp (FIG. 1), showed homology only to ESTs in the dbEST database. The full length 108P5H8 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):
(SEQ ID NO: 2586)









5′TTTTGATCAAGCTT303′



 









Adaptor 1:
(SEQ ID NO: 2587)









5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′









(SEQ ID NO: 2588)









3′GGCCCGTCCTAG5′



 









Adaptor 2:
(SEQ ID NO: 2589)









5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′









(SEQ ID NO: 2590)









3′CGGCTCCTAG5′



 









PCR primer 1:
(SEQ ID NO: 2591)









5′CTAATACGACTCACTATAGGGC3′



 









Nested primer (NP) 1:
(SEQ ID NO: 2592)









5′TCGAGCGGCCGCCCGGGCAGGA3′



 









Nested primer (NP) 2:
(SEQ ID NO: 2593)









5′AGCGTGGTCGCGGCCGAGGA3′







Suppression Subtractive Hybridization:
Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that are androgen regulated. The SSH reaction utilized cDNA from LNCaP prostate cancer cells grown in presence of mibolerone minus LNCaP cells grown in absence of mibolerone.
The cDNA derived from LNCaP prostate cancer cells grown in absence of mibolerone was used as the source of the “driver” cDNA, while the LNCaP prostate cancer cells grown in presence of mibolerone 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 Adaptor 2 (10 μM), in separate ligation reactions, in a total volume of 10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.
The first hybridization was performed by adding 1.5 μl (600 ng) of driver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1- and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98° C. for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68° C. The two hybridizations were then mixed together with an additional 1 μl of fresh denatured driver cDNA and were allowed to hybridize overnight at 68° C. The second hybridization was then diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70° C. for 7 min. and stored at −20° C.
PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH:
To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 μl of the diluted final hybridization mix was added to 1 μl of PCR primer 1 (10 μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and 0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5 min., 94° C. for 25 sec., then 27 cycles of 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′-atatcgccgcgctcgtcgtcgacaa-3′ (SEQ ID NO: 2594) and 5′-agccacacgcagctcattgtagaagg-3′ (SEQ ID NO: 2595) 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 108P5H8 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 FIG. 10. RT-PCR expression analysis was performed on first strand cDNAs generated using pools of tissues from multiple samples. The cDNAs were shown to be normalized using beta-actin PCR. Strong expression of 108P5H8 was observed in prostate cancer xenograft pool, prostate cancer pool and in the 2 different prostate cancer metastasis samples. Lower expression was detected in bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis pool, pancreas cancer pool, VP1 and VP2.
Example 2
Full Length Cloning of 108P5H8
To isolate genes that are androgen regulated, the androgen-dependent prostate cancer cell line LNCaP was grown in media containing 2% charcoal-stripped serum (steroid hormone depleted) for one week. The cells were then stimulated with 10 nM Mibolerone (synthetic androgen) for 9 hours and were harvested for RNA.
The gene 108P5H8 was derived from an experiment where cDNA derived from LNCaP cells that was androgen-deprived (by growing in the presence of charcoal-stripped serum) was subtracted from cDNA derived from LNCaP cells that were stimulated with mibolerone for 9 hours. The SSH DNA sequence of 448 bp (FIG. 1) is novel and only exhibited homology to human EST sequences in the dbest database.
A full length 108P5H8 cDNA clone (108P5H8 v.1) of 2364 base pairs (bp) was cloned from a prostate library (Lambda ZAP Express, Stratagene) (FIG. 2). The cDNA encodes a putative open reading frame (ORF) of 429 amino acids.
108P5H8 variant 2 and variant 3 were identified. The nucleic acid and protein sequences of all 3 variants are presented in FIG. 3 and FIG. 4. The alignments of all 3 108P5H8 variants are presented in FIG. 4. The nucleic acid sequences of variants 1 and 2 encode identical protein. 108P5H8 v.3 has a base pair variation with a C at position 342 of v.1 converted into G in v.3. This nucleotide change converted amino acid position 30 from aspartic acid in the 108P5H8 v.1 and v.2 protein sequence, to glutamic acid in 108P5H8 v.3.
Analysis of 108P5H8 protein sequence using the PSORT program (http://psort.nibb.acjp:8800/form.html) reveals 6 predicted transmembrane domains. Sequence analysis of 108P5H8 reveals homology to the human zinc transporter protein ZnT4 (Huang and Gitschier, 1997, Nature Genetics 17:292). The 108P5H8 v.1 sequence includes novel 5′ UTR and 3′ UTR sequences, and the molecule contains 75% GC sequence, indicating possible translational regulatory sites.
To further confirm the parameters of a variant, a variety of techniques are available in the art, such as full-length cloning, proteomic validation, PCR-based validation, and 5′ RACE validation, etc. (see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta. 1999 Aug. 17;1433(1-2):321-6; Ferranti P, et al., Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(s1)-casein, Eur J Biochem. 1997 Oct. 1;249(1):1-7. For PCR-based Validation: Wellmann S, et al., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2004 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 108P5H8 maps is modulated in a particular cancer, the variants of 108P5H8 are modulated as well. Disclosed herein is that 108P5H8 has a particular expression profile. Variants of 108P5H8 that are structurally and/or functionally similar to 108P5H8 share this expression pattern, thus serving as tumor-associated markers/antigens.
Example 3
Chromosomal Localization
Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available, including fluorescent in situ hybridization (FISH), human/hamster Genebridge4 radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Ala.), human-rodent somatic cell hybrid panels such as is available from the Coriell Institute (Camden, N.J.), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Md.).
The chromosomal localization of 108P5H8 using the GeneBridge4 radiation hybrid panel was performed using the the following PCR primers:









(SEQ. ID. No.: 2596)













108P5H8.1
5′ TGCACACTGGACTTCGTAGAGTAA 3′




 









(SEQ. ID. No.: 2597)













108P5H8.2
5′ AAAGCTGTGAGAGTGGCTGAGAAA 3′







The resulting mapping vector for the 93 radiation hybrid panel DNAs was: 10010100110100010100000000000011010000000120110000101100010000101110001010001000002011 0110121
This mapping vector and the mapping program placed 108P5H8 to chromosome 15q 15 .2-q21.1.
Example 4
Expression Analysis of 108P5H8 in Normal Tissues and Patient Specimens
Expression of 108P5H8 was analyzed by RT-PCR (FIG. 10). First strand cDNA was prepared from vital pool 1 (VP1: liver, lung and kidney), vital pool 2 (VP2, pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI), prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, pancreas cancer pool, and from prostate cancer metastasis to lymph node from two different patients. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 108P5H8, was performed at 26 and 30 cycles of amplification. Strong expression of 108P5H8 was observed in prostate cancer xenograft pool, prostate cancer pool and in the 2 different prostate cancer metastasis samples. Lower expression was detected in bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis pool, pancreas cancer pool, VP1 and VP2.
Extensive Northern blot analysis of 108P5H8 in 16 human normal tissues confirmed the expression observed by RT-PCR (FIG. 11). An approximately 7 kb 108P5H8 transcript was strongly expressed in prostate. Significantly lower expression was detected in other tissues.
FIG. 11C shows expression of 108P5H8 in prostate cancer xenografts. RNA was extracted from prostate cancer xenografts, LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI. Northern blot with 10 μg of total RNA/lane was probed with 108P5H8 SSH sequence. Results showed expression of 108P5H8 in all four xenograft tissues. More detailed analysis of the xenografts shows that 108P5H8is highly expressed in the xenografts even when grown within the tibia of mice (FIG. 12). The expression is increased when the LAPC-4 xenograft is grown within a human bone implant (LAPC-4 AD2) It is possible that the human bone environment increases and/or induces the expression of 108P5H8. Northern blot analysis also showed that 108P5H8 is expressed in all human cancer cell lines tested such as prostate, bladder, brain, lung, kidney, breast, testis and ovary cancer cell lines (FIG. 13).
Expression of 108P5H8 was assayed in a panel of human cancers (T) and their respective matched normal tissues (N) on RNA dot blots (FIG. 14). 108P5H8 expression was detected in prostate, kidney, uterus and stomach cancers. The expression detected in some normal adjacent tissues (isolated from diseased tissues), but not in normal tissues (isolated from healthy donors), may indicate that these tissues are not fully normal and that 108P5H8 may be expressed in early stage tumors. 108P5H8 was also expressed in all 9 human cancer cell lines tested.
To test expression of 108P5H8 in patient cancer specimens, RNA was extracted from prostate cancer tumors (T) and their matched normal adjacent tissue (NAT). Northern blots with 10 μg of total RNA/lane were probed with 108P5H8 SSH sequence (FIG. 15). Results showed expression of 108P5H8 in all prostate patient specimens tested.
108P5H8 was isolated from an experiment where cDNA derived from LNCaP cells that was androgen-deprived (by growing in the presence of charcoal-stripped serum) was subtracted from cDNA derived from LNCaP cells that were stimulated with mibolerone. To assess whether 108P5H8 is androgen-regulated, LNCaP cells were grown in charcoal-stripped medium and stimulated with the synthetic androgen mibolerone, for either 14 or 24 hours (FIG. 16). Northern blots with 10 μg of total RNA/lane were probed with either the 108P5H8 sequence (FIG. 16A). Results show expression of 108P5H8 is not regulated by androgen. The experimental samples were confirmed by testing for the expression of the androgen-regulated prostate cancer gene PSA (FIG. 16B). This experiment shows that, as expected, PSA levels go down in presence of charcoal-stripped serum, and expression is induced at 14 and 24 hours in presence of the synthetic androgen. A picture of the ethidium-bromide staining of the RNA gel is also presented (FIG. 16C).
FIG. 17 shows expression of 108P5H8 in cancer metastasis patient specimens. RNA was extracted from prostate cancer metastasis to lymph node isolated from 2 different patients, as well as from normal bladder (NB), normal kidney (NK), normal lung (NL), normal breast (NBr), normal ovary (NO), and normal pancreas (NPa). Northern blots with 10 μg of total RNA/lane was probed with 108P5H8 sequence. The results show expression of 108P5H8 in both cancer metastasis samples but not in normal tissues.
108P5H8 expression showed prostate restricted expression. Its strong expression detected in normal prostate and prostate cancer tissues and the low expression detected in other normal tissues indicate that 108P5H8 is therapeutic and prophylactic target and a diagnostic and prognostic marker for human cancers.
Example 5
Production of Recombinant 108P5H8 in Prokaryotic Systems
To express recombinant 108P5H8 in prokaryotic cells, the full or partial length 108P5H8 cDNA sequences can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 108P5H8 are expressed in these constructs, amino acids 1 to 429 of variant 1 or variant 2; or amino acids 1 to 388 of variant 4, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 108P5H8, variants, or analogs thereof. In certain embodiments a region of 108P5H8 is expressed that encodes an amino acid not shared amongst at least one of the variants, such as a construct encoding the D to E mutation at amino acid 30.
A. In Vitro Transcription and Translation Constructs:
pCRII: To generate 108P5H8 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) are generated encoding either all or fragments of a 108P5H8 cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 108P5H8 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 108P5H8 at the RNA level. Transcribed 108P5H8 RNA representing the cDNA amino acid coding region of the 108P5H8 gene is used in in vitro translation systems such as the TnTTM Coupled Reticulolysate System (Promega, Corp., Madison, Wis.) to synthesize 108P5H8 protein.
B. Bacterial Constructs:
pGEX Constructs: To generate recombinant 108P5H8 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of a 108P5H8 cDNA protein coding sequence are fused to the GST gene by cloning into pGEX-6P-1 or any other GST-fusion vector of the pGEX family (Amersham Pharmacia Biotech, Piscataway, N.J.). These constructs allow controlled expression of recombinant 108P5H8 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 108P5H8-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in E. coli. 
In one embodiment, a GST-fusion protein was constructed and expressed that encoded amino acids 1-112. This protein was used as an immunogen for generation of a 108P5H8 specific polyclonal antibody as described in example 8.
pMAL Constructs: To generate, in bacteria, recombinant 108P5H8 proteins that are fused to maltose-binding protein (MBP), all or parts of a 108P5H8 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2× and pMAL-p2× vectors (New England Biolabs, Beverly, Mass.). These constructs allow controlled expression of recombinant 108P5H8 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 108P5H8. The pMAL-c2× and pMAL-p2× 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 108P5H8 in bacterial cells, all or parts of a 108P5H8 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wis.). These vectors allow tightly controlled expression of recombinant 108P5H8 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 a 108P5H8 protein are expressed as amino-terminal fusions to NusA.
C. Yeast Constructs:
pESC Constructs: To express 108P5H8 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of a 108P5H8 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 108P5H8. 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 108P5H8 in the yeast species Saccharomyces pombe, all or parts of a 108P5H8 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 108P5H8 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 6
Production of Recombinant 108P5H8 in Eukaryotic Systems
A. Mammalian Constructs:
To express recombinant 108P5H8 in eukaryotic cells, the full or partial length 108P5H8 cDNA sequences can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 108P5H8 are expressed in these constructs, amino acids 1 to 429 of variant, variant 2 or variant 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 108P5H8, variants, or analogs thereof. In certain embodiments a region of 108P5H8 is expressed that encodes an amino acid not shared amongst at least two variants.
The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates are probed with an anti-His epitope tag antibody or with anti-108P5H8 polyclonal antibodies to verify protein expression.
pcDNA4/HisMax Constructs: To express 108P5H8 in mammalian cells, a 108P5H8 ORF, or portions thereof, of 108P5H8 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 108P5H8 in mammalian cells, a 108P5H8 ORF, or portions thereof, of 108P5H8 with a consensus Kozak translation initiation site were cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6× His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. The pcDNA3.1/MycHis construct encoding 108P5H8 was transfected into 293T cells. Expression of 108P5H8 was assayed by flow cytometry and using anti-His antibody as well as polyclonal anti-108P5H8 antibody (FIG. 22). Results show that 108P5H8 protein was expressed and was localized to the cell surface.
pcDNA3.1/CT-GFP-TOPO Construct: To express 108P5H8 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 108P5H8 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 pcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. Additional constructs with an amino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a 108P5H8 protein.
PAPtag: A 108P5H8 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 108P5H8 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 108P5H8 protein. The resulting recombinant 108P5H8 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 108P5H8 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 108P5H8 ORF, or portions thereof, is cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 108P5H8 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 108P5H8 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 108P5H8 proteins. Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli. 
PsecFc: A 108P5H8 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 108P5H8 proteins, while fusing the IgGK signal sequence to N-terminus. 108P5H8 fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 108P5H8 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 108P5H8 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 108P5H8 constitutively, 108P5H8 ORF, or portions thereof, of 108P5H8 were cloned into pSRα constructs. Amphotropic and ecotropic retroviruses are 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, 108P5H8, 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. Results of expression of 108P5H8 protein driven from the pSRα in PC3 and NIH3T3 cells are shown in FIG. 24.
Additional pSRα constructs are made that fuse an epitope tag such as the FLAGTM tag to the carboxyl-terminus of 108P5H8 sequences to allow detection using anti-Flag antibodies. For example, the FLAGTM sequence 5′ gat tac aag gat gac gac gat aag 3′ (SEQ. ID. No.: 2598) 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 108P5H8 proteins.
Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 108P5H8. High virus titer leading to high level expression of 108P5H8 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 108P5H8 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, 108P5H8 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 108P5H8 in mammalian cells, coding sequences of 108P5H8, 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 108P5H8. These vectors are thereafter used to control expression of 108P5H8 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
B. Baculovirus Expression Systems
To generate recombinant 108P5H8 proteins in a baculovirus expression system, 108P5H8 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-108P5H8 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 108P5H8 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 108P5H8 protein can be detected using anti-108P5H8 or anti-His-tag antibody. 108P5H8 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 108P5H8.
Example 7
Antigenicity Profiles and Secondary Structure
FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 depict graphically five amino acid profiles of the 108P5H8 amino acid sequence (variant 1), each assessment is available by accessing the ProtScale website on the ExPasy molecular biology server.
These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7, Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG. 8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of 108P5H8 protein. Each of the above amino acid profiles of 108P5H8 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 profiles, and values less than 0.5 on the Hydropathicity profile). Such regions are likely to be exposed to the aqueous environment, be present on the surface of the protein, and thus available for immune recognition, such as by antibodies.
Average Flexibility (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 full length 108P5H8 protein (variant 1) 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-108P5H8 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 108P5H8 protein. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 429 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 429 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 429 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 429 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on FIG. 8; and, a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 429 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 108P5H8, namely the predicted presence and location of alpha helices, extended strands, and random coils, is predicted from the primary amino acid sequence of 108P5H8 variant 1 using the HNN—Hierarchical Neural Network method (Guermeur, 1997 ), accessed from the ExPasy molecular biology server. The analysis indicates that 108P5H8 is composed of 49.88% alpha helix, 11.66% extended strand, and 38.46% random coil (FIG. 18).
Analysis for the potential presence of transmembrane domains in 108P5H8 was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server. The programs predict the presence of 6 transmembrane domains in 108P5H8. Shown graphically in FIGS. 19A and 19B are the results of analysis using the TMpred and TMHMM prediction programs, respectively, depicting the location of the 6 transmembrane domains. The results of each program, namely the amino acids encoding the transmembrane domains are summarized in Table XXI.
Example 8
Generation of 108P5H8 Polyclonal Antibodies
Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full length 108P5H8 protein, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled “Antigenicity Profiles and Secondary Structure”). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9 for amino acid profiles that indicate such regions of 108P5H8).
For example, 108P5H8 recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 108P5H8 are used as antigens to generate polyclonal antibodies in New Zealand White rabbits. Such regions often reside in extracellular and intracellular loops between transmembrane domains. For example, such regions include, but are not limited to, amino acids 1-112 (intracellular amino terminus), amino acids 139-152 (1st extracellular loop), amino acids 201-214 (second extracellular loop), amino acids 294-307 (third extracellular loop), or amino acids 336-429 (carboxyl terminus). 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 294-307 of 108P5H8 is conjugated to KLH and used to immunize the rabbit. Alternatively the immunizing agent may include all or portions of a 108P5H8 protein, analogs or fusion proteins thereof. For example, a 108P5H8 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.
In one embodiment, a GST-fusion protein encoding amino acids 1-112 of 108P5H8 was produced and purified and used to immunize a rabbit. This polyclonal antibody specifically recognized both recombinant and endogenous 108P5H8 protein in cells and tissues. FIG. 20 shows non-androgen-regulated expression of 108P5H8 in the prostate cancer cell lines LNCaP and LAPC4. The expression of 108P5H8 is cell surface as detected by the polyclonal antibody in LNCaP and LAPC4 cells (FIG. 21) and when overexpressed in 293T cells (FIG. 22). FIG. 23 shows expression in prostate patient cancer samples, including metastatic disease, indicating the protein is a therapeutic target in both androgen-dependent and independent prostate cancer. 108P5H8 is also expressed in ovarian cancer, but not in normal ovary (FIG. 23), indicating the protein is a therapeutic and diagnostic target in this disease as well.
In addition to GST-fusions, other recombinant bacterial fusion proteins that can be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled “Production of 108P5H8 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 108P5H8 in Eukaryotic Systems”), and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, the first extracellular loop of 108P5H8 (amino acids 139-152) is cloned into the Tag5 mammalian secretion-vector. The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tag5 108P5H8 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 Tag5 108P5H8 protein or KLH-coupled peptide encoding amino acids 294-307, the full-length 108P5H8 cDNA is cloned into pcDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled “Production of Recombinant 108P5H8 in Eukaryotic Systems”). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-108P5H8 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determine specific reactivity to denatured 108P5H8 protein using the Western blot technique (FIG. 20). Immunoprecipitation and flow cytometric analyses of 293T and other recombinant 108P5H8-expressing cells determine recognition of native protein by the antiserum (FIG. 22, FIG. 24). In addition, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 108P5H8 are carried out to test specificity (FIG. 20, FIG. 21).
The anti-serum from the Tag5 108P5H8 immunized rabbit is affinity purified by passage over a column composed of the Tag5 antigen covalently coupled to Affigel matrix (BioRad, Hercules, Calif.). The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Serum from rabbits immunized with the GST-fusion protein was purified by depletion of antibodies reactive to the fusion partner sequence (GST) by passage over an affinity column containing the GST alone and then by passage back over a GST-108P5H8 column to isolate gene specific antibodies. 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 9
Generation of 108P5H8 Monoclonal Antibodies (mAbs)
In one embodiment, therapeutic mAbs to 108P5H8 comprise those that react with epitopes of the protein that would disrupt or modulate the biological function of 108P5H8, for example those that would disrupt its interaction with ligands, proteins, or substrates that mediate its biological activity. Immunogens for generation of such mAbs include those designed to encode or contain an entire 108P5H8 protein or its variants or regions of 108P5H8 protein predicted to be exposed on the cell surface and/or antigenic from computer analysis of the amino acid sequence (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9, and the Example entitled “Antigenicity Profiles and Secondary Structure”). Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, cells expressing high levels of 108P5H8, such as 293T-108P5H8 or 300.19-108P5H8 murine Pre-B cells, are used to immunize mice.
To generate mAbs to 108P5H8, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 107 108P5H8-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 μg of protein immunogen or 107 cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In addition to the above protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding 108P5H8 sequence is used to immunize mice by direct injection of the plasmid DNA. For example, the predicted third extracellular loop, amino acids 294-307 of 108P5H8, is cloned into the Tag5 mammalian secretion vector and the recombinant vector is used as immunogen. In another example the amino acids are cloned into an Fc-fusion secretion vector in which a 108P5H8 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 108P5H8.
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, monoclonal antibodies are derived from immunization of mice with 300.19 cells engineered to express high levels of 108P5H8 (>30,000 molecules per cell). Balb C mice are initially immunized intraperitoneally with 107 cells mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 107 cells mixed in incomplete Freund's adjuvant for a total of three immunizations. Reactivity and specificity of serum to the full length 108P5H8 protein is monitored by Western blotting, immunoprecipitation and flow cytometry using various cells engineered to overexpress 108P5H8 protein (FIG. 24). Mice showing the strongest reactivity are rested and given a final injection of cells in PBS and then sacrificed four days later. The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (see, e.g., Harlow and Lane, 1988). Supernatants from HAT selected growth wells are, screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 108P5H8 specific antibody-producing clones.
The binding affinity of a 108P5H8 monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 108P5H8 monoclonal antibodies preferred, e.g., 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 10
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.
Example 11
Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes
HLA vaccine compositions of the invention can include multiple epitopes. The multiple epitopes can comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification and confirmation of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.
Computer Searches and Algorithms for Identification of Supermotif and/or Motif-Bearing Epitopes
The searches performed to identify the motif-bearing peptide sequences in the Example entitled “Antigenicity Profiles” and Tables V-XVIII, XXII, and XXIII employ the protein sequence data from the gene product of 108P5H8 set forth in FIGS. 2 and 3.
Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated 108P5H8 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 108P5H8 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 108P5H8 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 108P5H8 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-Baring Epitopes
To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine compositions. An analysis of the 108P5H8 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 12
Confirmation of Immunogenicity
Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology:
Target Cell Lines for Cellular Screening:
The 0.221 A2.1 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 Dyna1 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-1 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 200 μCi of 51Cr sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37° C. Labeled target cells are resuspended at 106 per ml and diluted 1:10 with K562 cells at a-concentration of 3.3×106/ml (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 μl) and effectors (100 μl) are plated in 96 well round-bottom plates and incubated for 5 hours at 37° C. At that time, 100 μl of supernatant are collected from each well and percent lysis is determined according to the formula:

[(cpm of the test sample−cpm of the spontaneous 51Cr release sample)/(cpm of the maximal 51Cr release sample−cpm of the spontaneous 51Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeled targets with 1% Triton X-100 and media alone, respectively. A positive culture is defined as one in which the specific lysis (sample-background) is 10% or higher in the case of individual wells and is 15% or more at the two highest E:T ratios when expanded cultures are assayed.
In situ Measurement of Human IFNγ Production as an Indicator of Peptide-Specific and Endogenous Recognition
Immulon 2 plates are coated with mouse anti-human IFNγ monoclonal antibody (4 μg/ml 0.1M NaHCO3, pH8.2) overnight at 4° C. The plates are washed with Ca2+, Mg2+-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 μl/well) and targets (100 μl/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used at a concentration of 1×106 cells/ml. The plates are incubated for 48 hours at 37° C. with 5% CO2.
Recombinant human IFN-gamma is added to the standard wells starting at 400 pg or 1200 pg/100 microliter/well and the plate incubated for two hours at 37° C. The plates are washed and 100 μl of biotinylated mouse anti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3% FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1:4000) are added and the plates incubated for one hour at room temperature. The plates are then washed 6× with wash buffer, 100 microliter/well developing solution (TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 microliter/well 1M H3PO4 and read at OD450. A culture is considered positive if it measured at least 50 pg of IFN-gamma/well above background and is twice the background level of expression.
CTL Expansion.
Those cultures that demonstrate specific lytic activity against peptide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, 5×104 CD8+ cells are added to a T25 flask containing the following: 1×106 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2×105 irradiated (8,000 rad) EBV-transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640 containing 10% (v/v) human AB serum, non-essential amino acids, sodium pyruvate, 25 μM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 hours later at a final concentration of 200 IU/ml and every three days thereafter with fresh media at 50 IU/ml. The cells are split if the cell concentration exceeds 1×106/ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the 51Cr release assay or at 1×106/ml in the in situ IFNγ assay using the same targets as before the expansion.
Cultures are expanded in the absence of anti-CD3+ as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5×104 CD8+ cells are added to a T25 flask containing the following: 1×106 autologous PBMC per ml which have been peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. and irradiated (4,200 rad); 2×105 irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640 containing 10% (v/v) human AB serum, non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.
Immunogenicity of A2 Supermotif-Bearing Peptides
A2-supermotif cross-reactive binding peptides are tested in the cellular assay for the ability to induce peptide-specific CTL in normal individuals. In this analysis, a peptide is typically considered to be an epitope if it induces peptide-specific CTLs in at least individuals, and preferably, also recognizes the endogenously expressed peptide.
Immunogenicity can also be confirmed using PBMCs isolated from patients bearing a tumor that expresses 108P5H8. 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 13
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 108P5H8-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 14
Identification and Confirmation of 108P5H8-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 108P5H8-derived, HLA class II HTL epitopes, a 108P5H8 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 108P5H8-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. 108P5H8-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 108P5H8 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 15
Immunogenicity of 108P5H8-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 108P5H8-expressing tumors.
Example 16
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, Al is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%. 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 17
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 108P5H8 expression vectors.
The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 108P5H8 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 18
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 108P5H8-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 108P5H8-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 19
Selection of CTL and HTL Epitopes for Inclusion in an 108P5H8-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 108P5H8 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 108P5H8. For example, if it has been observed that patients who spontaneously clear 108P5H8 generate an immune response to at least three (3) from 108P5H8 antigen, then three or four (3-4) 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 BIAS 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 108P5H8, 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 108P5H8.
Example 20
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 108P5H8, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 108P5H8 to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.
Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the Ii protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.
This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.
The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.
For example, a minigene is prepared as follows. For a first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended: In an example using eight oligonucleotides, i.e., four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM (NH4)2SO4, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.
Example 21
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 22
Peptide Composition for Prophylactic Uses
Vaccine compositions of the present invention can be used to prevent 108P5H8 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 108P5H8-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 108P5H8-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 23
Polyepitopic Vaccine Compositions Derived from Native 108P5H8 Sequences
A native 108P5H8 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 is selected; it 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 108P5H8 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 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 108P5H8, 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 24
Polyepitopic Vaccine Compositions from Multiple Antigens
The 108P5H8 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 108P5H8 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 108P5H8 as well as tumor-associated antigens that are often expressed with a target cancer associated with 108P5H8 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 25
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 108P5H8. 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, 108P5H8 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising an 168P5H8 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 36 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 108P5H8 epitope, and thus the status of exposure to 108P5H8, or exposure to a vaccine that elicits a protective or therapeutic response.
Example 26
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 108P5H8-associated disease or who have been vaccinated with an 108P5H8 vaccine.
For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 108P5H8 vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.
PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.
In the microculture format, 4×105 PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rIL-2 and 105 irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific 51Cr release, based on comparison with non-diseased control subjects as previously described (Rehermarm, 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 108P5H8 or an 108P5H8 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 108P5H8 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 27
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 28
Phase II Trials in Patients Expressing 108P5H8
Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 108P5H8. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 108P5H8, 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 108P5H8.
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 108P5H8-associated disease.
Example 29
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 108P5H8 is generated.
Example 30
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 108P5H8 protein from which the epitopes in the vaccine are derived.
For example, a cocktail of epitope-comprising peptides is administered ex vivo to PBMC, or isolated DC therefrom. A pharmaceutical to facilitate harvesting of DC can be used, such as ProgenipoietinTM (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides, and prior to reinfusion into patients, the DC are washed to remove unbound peptides.
As appreciated clinically, and readily determined by one of skill based on clinical outcomes, the number of DC reinfused into the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 and Prostate 32:272, 1997). Although 2-50×106 DC per patient are typically administered, larger number of DC, such as 107 or 108 can also be provided. Such cell populations typically contain between 50-90% DC.
In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For example, PBMC generated after treatment with an agent such as ProgenipoietinTM are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 108 to 1010. Generally, the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if ProgenipoietinTM mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5×106 DC, then the patient will be injected with a total of 2.5×108 peptide-loaded PBMC. The percent DC mobilized by an agent such as ProgenipoietinTM is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.
Ex Vivo Activation of CTL/HTL Responses
Alternatively, ex vivo CTL or HTL responses to 108P5H8 antigens can be induced by incubating, in tissue culture, the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., tumor cells.
Example 31
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. 108P5H8. 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 108P5H8 to isolate peptides corresponding to 108P5H8 that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.
As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.
Example 32
Complementary Polynucleotides
Sequences complementary to the 108P5H8-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 108P5H8. 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 108P5H8. 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 108P5H8-encoding transcript.
Example 33
Purification of Naturally-Occurring or Recombinant 108P5H8 Using 108P5H8 Specific Antibodies
Naturally occurring or recombinant 108P5H8 is substantially purified by immunoaffinity chromatography using antibodies specific for 108P5H8. An immunoaffinity column is constructed by covalently coupling anti-108P5H8 antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing 108P5H8 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 108P5H8 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/108P5H8 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 34
Identification of Molecules which Interact with 108P5H8
108P5H8, 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 108P5H8, washed, and any wells with labeled 108P5H8 complex are assayed. Data obtained using different concentrations of 108P5H8 are used to calculate values for the number, affinity, and association of 108P5H8 with the candidate molecules.
Example 35
In Vivo Assay for 108P5H8 Tumor Growth Promotion
The effect of a 108P5H8 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, including prostate, kidney, colon and bladder. For example, SCID mice can be injected SQ on each flank with 1×106 prostate, kidney, colon or bladder cancer cells (such as PC3, DU145, SCaBER, UM-UC-3, HT1376, SK-CO, Caco, RT4, T24, Caki, A-498 and SW839 cells) containing tkNeo empty vector or 108P5H8.
At least two strategies can be used:

    
    
        (1) Constitutive 108P5H8 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 108P5H8-expressing cells grow at a faster rate and/or that tumors produced by 108P5H8-expressing cells 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 108P5H8 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 PC3-108P5H8 can be compared in the prostate of SCID mice. Such experiments reveal the effect of 108P5H8 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 for example, 108P5H8 antibodies or intrabodies, and 108P5H8 antisense molecules or ribozymes, or 108P5H8 directed small molecules, on cells that express a 108P5H8 protein.
Example 36
108P5H8 Monoclonal Antibody-Mediated Inhibition of Human Xenograft Tumors in Vivo
The significant expression of 108P5H8, in cancer tissues, together with its restricted expression in normal tissues along with its cell surface expression makes 108P5H8 an excellent target for antibody therapy. Similarly, 108P5H8 is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-108P5H8 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), prostate cancer cell lines transfected with 108P5H8 (such as PC3-108P5H8, DU145-108P5H8), in human kidney cancer xenografts (AGS-K3, AGS-K6),.kidney cancer metastases to lymph node (AGS-K6 met) xenografts, and kidney cancer cell lines transfected with 108P5H8 (769P-108P5H8, A498-108P5H8).
Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in mouse subcutaneous or 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-108P5H8 mAbs inhibit formation of both the androgen-dependent LAPC-9 and androgen-independent PC3-108P5H8 tumor xenografts. Anti-108P5H8 mAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-108P5H8 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 ). These results indicate the use of anti-108P5H8 mAbs in the treatment of prostate cancer.
Administration of the anti-108P5H8 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 108P5H8 is an attractive target for immunotherapy and demonstrate the therapeutic use of anti-108P5H8 mAbs for the treatment of local and metastatic cancer. This example demonstrates that unconjugated 108P5H8 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 108P5H8 mAbs
Materials and Methods
108P5H8 Monoclonal Antibodies:
Monoclonal antibodies are obtained against 108P5H8, such as described in the Example entitled “Generation of 108P5H8 Monoclonal Antibodies (mAbs)” or may be obtained commercially. The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 108P5H8. Epitope mapping data for the anti-108P5H8 mAbs, as determined by ELISA and Western analysis, recognize epitopes on a 108P5H8 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 s.c. trocar implant (Craft, N., et al., 1999, Cancer Res. 59:5030-5036). Single-cell suspensions of tumor cells are prepared as described in Craft, et al. The prostate carcinoma cell lines PC3 and DU145 (American Type Culture Collection) are 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.
PC3-108P5H8, DU145-108P5H8 and A498-108P5H8 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-108P5H8 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, PC3, PC3-108P5H8, DU145 or DU145-108P5H8 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-108P5H8 mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, Tex.). (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078 ).
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. The mice are segregated into groups for appropriate treatments, with anti-108P5H8 or control mAbs being injected i.p.
Anti-108P5H8 mAbs Inhibit Growth of 108P5H8-Expressing Xenograft-Cancer Tumors
The effect of anti-108P5H8 mAbs on tumor formation is tested by using orthotopic models, e.g., LAPC-9 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-108P5H8 Ab, b) PBS or c) control non-specific monoclonal antibody 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).
Mice bearing established orthotopic LAPC-9 tumors are administered one to three injections per week of 500-1000 μg of either anti-108P5H8 mAb, control antibody or PBS two-to three times per week over a 4-8 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-108P5H8 antibodies on initiation and/or progression of prostate and kidney cancer in xenograft mouse models. Anti-108P5H8 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-108P5H8 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-108P5H8 mAbs are efficacious-on major clinically relevant end points (tumor growth), prolongation of survival, and health.
Example 37
Therapeutic and Diagnostic Use of Anti-108P5H8 Antibodies in Humans
Anti-108P5H8 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-108P5H8 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 108P5H8 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-108P5H8 antibodies are therefore used in diagnostic applications such as immunohistochemistry of biopsy specimens to detect cancer from suspect patients.
As determined by immunofluorescence, anti-108P5H8 mAb specifically binds to carcinoma cells. Thus, anti-108P5H8 antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of 108P5H8. Shedding or release of an extracellular domain of 108P5H8 into the extracellular milieu, such as that seen for alkaline phosphodiesterase BIO (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 108P5H8 by anti-108P5H8 antibodies in serum and/or urine samples from suspect patients.
Anti-108P5H8 antibodies that specifically bind 108P5H8 are used in therapeutic applications for the treatment of cancers that express 108P5H8. Anti-108P5H8 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-108P5H8 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., LAPC9 (see, e.g., the Example entitled “Monoclonal Antibody-mediated Inhibition of Prostate and Kidney Tumors In vivo.”) Conjugated and unconjugated anti-108P5H8 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 38
Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas Through Use of Human Anti-108P5H8 Antibodies in Vivo
Antibodies are used in accordance with the present invention which recognize an epitope on 108P5H8, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 108P5H8 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-108P5H8 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-108P5H8 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-108P5H8 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-108P5H8 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-108P5H8 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 108P5H8. In connection with the use of the anti-108P5H8 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)-108P5H8 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 108P5H8 (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-108P5H8 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-108P5H8 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-108P5H8 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-108P5H8 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-108P5H8 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-108P5H8 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-108P5H8 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 108P5H8 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 108P5H8. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-108P5H8 antibodies are found to be safe upon human administration.
Example 39
Human Clinical Trial Adjunctive Therapy with Human Anti-108P5H8 Antibody and Chemotherapeutic Agent
A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-108P5H8 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-108P5H8 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent, such as cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-108P5H8 antibody with dosage of antibody escalating from approximately about 25 mg/m2to about 275 mg/m2 over the course of the treatment in accordance with the following schedule:


















Day0
Day7
Day14
Day21
Day28
Day35






















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 108P5H8. 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-108P5H8 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.
Example 40
Human Clinical Trial: Monotherapy with Human Anti-108P5H8 Antibody
Anti-108P5H8 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-108P5H8 antibodies.
Example 41
Human Clinical Trial: Diagnostic Imaging with Anti-108P5H8 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-108P5H8 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 42
Homology Comparison of 108P5H8 to Known Sequences
The 108P5H8 protein is a six-transmembrane type 3 cell surface protein, consisting of 429 amino acids (table XXI). The 108P5H8 protein has 2 variant forms (FIG. 3), with 108P5H8v.3 differing from 108P5H8 v.1 by one amino acid at position 30 (D to E). This alteration in amino acid at position 30 corresponds to a point mutation at nucleic acid position 90, making variant 3 a true SNP. Both 108P5H8 variants, 108P5H8 v.1 and 108P5H8 v.3 have a calculated molecular weight of 47.5 kDa, and pI of 6.11, and contain an ion efflux motif between amino acid 114-417. Proteins carrying the ion efflux motif are found to increase tolerance to divalent metal ions such as cadmium, zinc, and cobalt. These proteins are thought to be efflux pumps that remove these ions from cells (Kunito T et al, Biosci Biotechnol Biochem 1996, 60:699).
The 108P5H8 protein variants show homology to human zinc transporter 4 (gi 11432533); with 108P5H8v.1 sharing 100% identity and 100% homology with that protein over the entire length of the protein (FIG. 25). 108P5H8v.2 share 100% identity and 100% homology with human zinc transporter ZNT4-gi 8134840 over the entire protein. As with the two 108P5H8 variants, ZNT4 (gi 8134840) and ZNT4-gi 11432533 differ by one amino acid at position 30, showing the same D to E change observed in 108P5H8v.3 and 108P5H8v.1. Based on sequence homology, 108P5H8 is conserved in various, species, showing high homology to Rat ZNT4 (90% identify shown in FIG. 25) and mouse ZNT4 (91% identity with gi 8134841).
Zinc has been shown to play an important role in the physiology and pathology of prostate epithelial cells. Zinc ions regulate the activity of chromatin and plasmalemma structures in seminal plasma, and participate in spermadhesin function (Holody D and Strzezek J. Acta Biochini Pol 1999, 46:935). In relation to prostate cancer, zinc was found to inhibit the activity of aminopeptidase N in prostate cancer cells (Ishii K et al, Int J Cancer 2001, 92:49). Efflux of Zn++ from the prostate by 108P5H8 or ZNT4 enhances the endogenous activity of aminopeptidase N, thereby increasing matrix degradation and tissue invasion by prostate cancer cells. In addition to its role in invasion, ZNT4 regulates apoptosis and proliferation of prostate cells. Accumulation of Zn++ within the prostate induces apoptosis of normal epithelial cells (Feng P et al, Mol Urol 2000, 4:31). Enhanced expression of ZNT4 in prostate cancer cells and the resulting efflux of Zn++, allow reduced apoptosis, survival and proliferation of prostate cancer cells. Finally, intracellular Zn++ concentrations play a direct role in regulating gene transcription by zinc finger proteins.
This information indicates that 108P5H8 plays a role in the growth of cancer cells, supports cell survival, and regulates gene transcription by regulating events in the nucleus. Accordingly, when 108P5H8 functions as a regulator of cell transformation, tumor formation, or as a modulator of transcription involved in activating genes associated with inflammation, tumorigenesis or proliferation, 108P5H8 is used for therapeutic, diagnostic, prognostic and/or preventative purposes.
Example 43
Identification and Confirmation of Signal Transduction Pathways
Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways. (see, e.g., J. Neurochem. 2001; 76:217-223). Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 108P5H8 and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by 108P5H8, including phospholipid pathways such as P13K, AKT, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as well as mitogenic/survival cascades such as ERK, p38, etc. (Cell Growth Differ. 2000, 11:279; J. Biol. Chem. 1999, 274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913).
To confirm that 108P5H8 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.

    
    
        1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress
        2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation
        3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress
        4. ARE-luc, androgen receptor; steroids/MAPK; growth/differentiation/apoptosis
        5. p53-luc, p53; SAPK; growth/differentiation/apoptosis
        6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
    
    


Gene-mediated effects can be assayed in cells showing mRNA expression. Luciferase reporter plasmids can be introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer.
Signaling pathways activated by 108P5H8 are mapped and used for the identification and validation of therapeutic targets. When 108P5H8 is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 44
Involvement in Tumor Progression
The 108P5H8 gene contributes to the growth of cancer cells. The role of 108P5H8 in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate cell lines, as well as NIH 3T3 cells engineered to stably express 108P5H8. Parental cells lacking 108P5H8 and cells expressing 108P5H8 are evaluated for cell growth using a well-documented proliferation assay (Fraser, S. P., et al., Prostate 2000; 44:61, Johnson, D. E., et al., Anticancer Drugs 1996, 7:288).
To confirm the role of 108P5H8 in the transformation process, its effect in colony forming assays is investigated. Parental NIH-3T3 cells lacking 108P5H8 are compared to NIH-3T3 cells expressing 108P5H8, 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 108P5H8 in invasion and metastasis of cancer cells, a well-established assay is used, e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010). Control cells, including prostate, colon, bladder and kidney cell lines lacking 108P5H8 are compared to corresponding cells expressing 108P5H8. Cells are loaded with the fluorescent dye, calcein, and plated in the top well of the Transwell insert coated with a basement membrane analog. Invasion is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population.
108P5H8 can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 108P5H8 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, 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 108P5H8, including normal and tumor prostate, colon and lung cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, flutamide, etc., and protein synthesis inhibitors, such as cycloheximide. Cells are stained with annexin V-FITC and cell death is measured by FACS analysis. Modulation of cell death by 108P5H8 plays a critical role in regulating tumor progression and tumor load.
When 108P5H8 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). 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 108P5H8 in angiogenesis, enhancement or inhibition, is confirmed.
For example, endothelial cells engineered to express 108P5H8 are evaluated using tube formation and proliferation-assays. The effect of 108P5H8 is also confirmed in animal models in vivo. For example, cells either expressing or lacking 108P5H8 are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques. 108P5H8 affects angiogenesis, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes
Example 46
Regulation of Transcription
The cellular localization of 108P5H8 (Table XXI) and its ability to regulate intracellular zinc ion concentrations, 108P5H8 is effectively used as a modulator of the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, e.g., by studying gene expression in cells expressing or lacking 108P5H8. For this purpose, two types of experiments are performed.
In the first set of experiments, RNA from parental and 108P5H8-expressing cells are extracted and hybridized to commercially available gene arrays (Clontech) (Smid-Koopman, E., et al., Br. J. Cancer, 2000, 83:246). Resting cells as well as cells treated with FBS or androgen are compared. Differentially expressed genes are identified in accordance with procedures known in the art. The differentially expressed genes are then mapped to biological pathways (Chen, K., et al. Thyroid 2001, 11:41.).
In the second set of experiments, specific transcriptional pathway activation is evaluated using commercially available (Stratagene) luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc, ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters contain consensus binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways, and represent a good tool to ascertain pathway activation and screen for positive and negative modulators of pathway activation.
Accordingly, it is found that 108P5H8 plays a role in gene regulation, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 47
Involvement in Cell Adhesion
Cell adhesion plays a critical role in tissue colonization and metastasis. 108P5H8 participates in cellular organization, and as a consequence cell adhesion and motility. To confirm the role of 108P5H8 in the regulation of cell adhesion, control cells lacking 108P5H8 are compared to cells expressing 108P5H8, 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 in 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 108P5H8 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 108P5H8 is involved in these processes. Thus, 108P5H8 serves as a diagnostic, prognostic, preventative and/or therapeutic modality.
Example 48
Protein-Protein Association
Several ion transporters have been shown to interact with other proteins, thereby regulating gene transcription, gene sequence, as well as cell growth. Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate with 108P5H8. Immunoprecipitates from cells expressing 108P5H8 and cells lacking 108P5H8 are compared for specific protein-protein associations.
Studies are performed to confirm the extent of association of 108P5H8 with effector molecules, such as nuclear proteins, transcription factors, kinases, phsophates, etc. Studies comparing 108P5H8 positive and 108P5H8 negative cells as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, growth factors, androgen and anti-integrin Ab reveal unique interactions.
In addition, protein-protein interactions are confirmed using two yeast hybrid methodology (Curr. Opin. Chem. Biol. 1999, 3:64). A vector carrying a library of proteins fused to the activation domain of a transcription factor is introduced into yeast expressing a 108P5H8-DNA-binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by colorimetric reporter activity. Specific association with effector molecules and transcription factors directs one of skill to the mode of action of 108P5H8, and thus identifies therapeutic, prognostic, preventative and/or diagnostic targets for cancer. This and similar assays are also used to identify and screen for small molecules that interact with 108P5H8.
Thus it is found that 108P5H8 associates with proteins and small molecules. Accordingly, 108P5H8 and these proteins and small molecules are used for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 49
Ion Flux Activity
To confirm that 108P5H8 functions as an ion channel, FACS analysis and electrophysiology techniques are used (Gergely L, Cook L, Agnello V. Clin Diagn Lab Immunol. 1997;4:70; Skryma R, et al. J Physiol. 2000, 527: 71). Using FACS analysis and commercially available indicators (Molecular Probes), parental cells and cells expressing genes under consideration are compared for their ability to transport calcium, and zinc. Prostate, colon, bladder and kidney normal and tumor cell lines are used in these studies. For example cells loaded with calcium responsive indicators such as Fluo4 and Fura red are incubated in the presence or absence of ions and analyzed by flow cytometry. Information derived from these experiments provides a mechanism by which cancer cells are regulated. This is particularly true in the case of calcium, as calcium channel inhibitors have been reported to induce the death of certain cancer cells, including prostate cancer cell lines (Batra S, Popper L D, Hartley-Asp B. Prostate. 1991, 19: 299). It is possible to determine efflux and influx of zinc using fluoZin 1, a fluorescent Zn++ indicator detected by FACS in a manner similar to Fluo4 above, or using 65Zn. Prostate, kidney, bladder or colon cells, engineered to express or lack 108P5H8, will be incubated in the presence of 65Zn. Cells will be evaluated over time for uptake and efflux of 65Zn (Kim A H et al, Brain Res. 2000, 886:99; Grass G et al, J Bacteriol. 2001, 183:4664).
Using electrophysiology, uninjected oocytes and oocytes injected with gene-specific cRNA are compared for ion channel activity. Patch/voltage clamp assays are performed on oocytes in the presence or absence of selected ions, including calcium, zinc, etc. Ion channel activators (such as cAMP/GMP, forskolin, TPA, etc) and inhibitors (such as calcicludine, conotoxin, TEA, tetrodotoxin, etc) are used to evaluate the function of 108P5H8 as ion channels (Schweitz H. et al. Proc. Natl. Acad. Sci. 1994. 91:878; Skryma R. et al. Prostate. 1997. 33:112).
Using any of the assays listed above, we can evaluate the effect of antibodies directed against 108P5H8 on ion transport. Similarly, these assays can be used to identify and evaluate small molecule that modulate ion and protein transport.
When 108P5H8 functions as an ion channel, it is used as a target for diagnostic, preventative and therapeutic purposes.
Example 50
Detection of 108P5H8 Protein in LNCaP Cells, a Prostate Cancer Cell Line by Immunocytochemistry
To assess the expression of 108P5H8 protein in a prostate cancer cell line, preparations of cytocentrifuged LNCaP cells were stained using a rabbit polyclonal antibody to 108P5H8. Preparations of LNCaP cells were made from three differently treated cell populations to assess whether 108P5H8 is androgen regulated. The LNCaP cell preparations were made from cells grown in medium containing 10% fetal bovine serum; from cells grown for 72 hours in androgen free, serum depleted medium (by growing in charcoal dextran stripped medium); or from previously androgen starved cells which were subsequently stimulated with 10 mmol mibolerone (a synthetic androgen) for 48 hours. The cells were spun down, washed twice (in buffer), resuspended (in buffer) and then centrifuged onto slides (1000 rpm for 2 minutes), allowed to dry and then fixed in acetone for 10 minutes. The cells were then incubated with rabbit polyclonal 108P5H8 for 3 hours (FIG. 26A-C), or rabbit IgG (FIG. 26D). The slides were washed three times in buffer then incubated in DAKO EnVision+TM peroxidase conjugated goat anti-rabbit secondary antibody (DAKO Corporation, Carpenteria, Calif.) for 1 hour. The cells were then washed in buffer, developed using the DAB kit (SIGMA Chemicals), counterstained using hematoxylin, and analyzed by bright field microscopy. The results showed strong expression of 108P5H8 in all three LNCaP cell preparations demonstrating that expression of 108P5H8 can be detected in this prostate cancer cell line and is not androgen related. This indicates that antibodies to 108P5H8 are useful in detecting non-androgen related cancer of the prostate; the protein is a useful marker.
Example 51
Detection of 108P5H8 Protein in Prostate Cancer Patient Specimens by Immunohistochemisttry
To assess the expression of 108P5H8 protein, prostate cancer specimens were obtained from prostate cancer patients and stained using a rabbit polyclonal antibody to 108P5H8. Frozen tissues were then cut into 4 micron sections and fixed in acetone for 10 minutes. The sections were then incubated with rabbit polyclonal 108P5H8 for 3 hours (FIG. 27A), or rabbit IgG (FIG. 27B). The slides were washed three times in buffer then incubated in DAKO EnVision+TM peroxidase conjugated goat anti-rabbit secondary antibody (DAKO Corporation, Carpenteria, Calif.) for 1 hour. The sections were then washed in buffer, developed using the DAB kit (SIGMA Chemicals), counterstained using hematoxylin, and analyzed by bright field microscopy. The results showed strong expression in the neoplastic glands of the prostate (FIG. 27A). These results further confirm the utility of 108P5H8 as a prostate tumor marker.
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
Table I: Tissues that Express 108P5H8 When Malignant

    
    
        Prostate
        Bladder
        Kidney
        Colon
        Lung
        Ovary
        Breast
        Pancreas
        Uterus
        Stomach
    
    









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.



























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 (A)







POSITION
POSITION



POSITION
3 (Primary
C Terminus



2 (Primary Anchor)
Anchor)
(Primary 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)







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


Super-


motif





Italicized residues indicate less preferred or “tolerated” residues.













TABLE V (A)







HLA PEPTIDE SCORING RESULTS - 108P5H8 - A1, 9-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE
ID#














1
23
LNDTSAFDF
6.250
1.


2
148
LTDLSAIIL
6.250
2.


3
141
MTDALHMLT
6.250
3.


4
303
IADPICTYV
5.000
4.


5
94
SCDNCSKQR
5.000
5.


6
356
SVEDLNIWS
4.500
6.


7
192
YILMGFLLY
2.500
7.


8
377
QLIPGSSSK
2.000
8.


9
178
RLEVLSAMI
1.800
9.


10
54
GSEAPERPV
1.350
10.


11
184
AMISVLLVY
1.250
11.


12
35
AGDEGLSRF
1.250
12.


13
285
SVGVLIAAY
1.000
13.


14
113
RLTIAAVLY
1.000
14.


15
76
LLDQDLPLT
1.000
15.


16
101
QREILKQRK
0.900
16.


17
331
ILEGVPSHL
0.900
17.


18
166
KSPTKRFTF
0.750
18.


19
31
FSDEAGDEG
0.750
19.


20
214
NGDIMLITA
0.625
20.


21
91
KVDSCDNCS
0.500
21.


22
34
EAGDEGLSR
0.500
22.


23
116
IAAVLYLLF
0.500
23.


24
288
VLIAAYIIR
0.500
24.


25
405
RCTIQLQSY
0.500
25.


26
340
NVDYIKEAL
0.500
26.


27
226
VAVNVIMGF
0.500
27.


28
289
LIAAYIIRF
0.500
28.


29
396
LLLNTFGMY
0.500
29.


30
125
MIGELVGGY
0.500
30.


31
397
LLNTFGMYR
0.500
31.


32
302
KIADPICTY
0.500
32.


33
83
LTNSQLSLK
0.500
33.


34
189
LLVYILMGF
0.500
34.


35
162
WLSSKSPTK
0.400
35.


36
267
DSLAVRAAF
0.300
36.


37
3
GSGAWKRLK
0.300
37.


38
70
QADDDSLLD
0.250
38.


39
406
CTIQLQSYR
0.250
39.


40
335
VPSHLNVDY
0.250
40.


41
126
IGELVGGYI
0.225
41.


42
290
IAAYIIRFK
0.200
42.


43
310
YVFSLLVAF
0.200
43.


44
248
SLPSNSPTR
0.200
44.


45
197
FLLYEAVQR
0.200
45.


46
158
LLALWLSSK
0.200
46.


47
98
CSKQREILK
0.150
47.


48
265
GQDSLAVRA
0.150
48.


49
247
HSLPSNSPT
0.150
49.


50
414
RQEVDRTCA
0.135
50.
















TABLE VI (A)







HLA PEPTIDE SCORING RESULTS - 108P5H8 - A1, 10-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE
ID#














1
303
IADPICTYVF
100.000
51.


2
91
KVDSCDNCSK
10.000
52.


3
148
LTDLSAIILT
6.250
53.


4
36
GDEGLSRFNK
4.500
54.


5
178
RLEVLSAMIS
4.500
55.


6
247
HSLPSNSPTR
3.000
56.


7
54
GSEAPERPVN
2.700
57.


8
76
LLDQDLPLTN
2.500
58.


9
183
SAMISVLLVY
2.500
59.


10
70
QADDDSLLDQ
2.500
60.


11
284
QSVGVLIAAY
1.500
61.


12
340
NVDYIKEALM
1.000
62.


13
334
GVPSHLNVDY
1.000
63.


14
356
SVEDLNIWSL
0.900
64.


15
331
ILEGVPSHLN
0.900
65.


16
31
FSDEAGDEGL
0.750
66.


17
141
MTDALHMLTD
0.625
67.


18
396
LLLNTFGMYR
0.500
68.


19
225
GVAVNVIMGF
0.500
69.


20
22
FLNDTSAFDF
0.500
70.


21
287
GVLIAAYIIR
0.500
71.


22
346
EALMKIEDVY
0.500
72.


23
51
ADDGSEAPER
0.500
73.


24
188
VLLVYILMGF
0.500
74.


25
395
HLLLNTFGMY
0.500
75.


26
288
VLIAAYIIRF
0.500
76.


27
115
TIAAVLYLLF
0.500
77.


28
126
IGELVGGYIA
0.450
78.


29
165
SKSPTKRFTF
0.250
79.


30
202
AVQRTIHMNY
0.250
80.


31
124
FMIGELVGGY
0.250
81.


32
279
LGDLVQSVGV
0.250
82.


33
298
KPEYKIADPI
0.225
83.


34
210
NYEINGDIML
0.225
84.


35
157
TLLALWLSSK
0.200
85.


36
289
LIAAYIIRFK
0.200
86.


37
350
KIEDVYSVED
0.180
87.


38
93
DSCDNCSKQR
0.150
88.


39
312
FSLLVAFTTF
0.150
89.


40
414
RQEVDRTCAN
0.135
90.


41
15
RKDDAPLFLN
0.125
91.


42
156
LTLLALWLSS
0.125
92.


43
83
LTNSQLSLKV
0.125
93.


44
172
FTFGFHRLEV
0.125
94.


45
214
NGDIMLITAA
0.125
95.


46
308
CTYVFSLLVA
0.125
96.


47
23
LNDTSAFDFS
0.125
97.


48
35
AGDEGLSRFN
0.125
98.


49
191
VYILMGFLLY
0.125
99.


50
97
NCSKQREILK
0.100
100.
















TABLE VII (A)







HLA PEPTIDE SCORING RESULTS - 108P5H8 - A2, 9-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE
ID#














1
278
ALGDLVQSV
655.875
101.


2
268
SLAVRAAFV
382.536
102.


3
198
LLYEAVQRT
382.282
103.


4
75
SLLDQDLPL
324.068
104.


5
140
IMTDALHML
247.333
105.


6
122
LLFMIGELV
214.366
106.


7
155
ILTLLALWL
199.738
107.


8
218
MLITAAVGV
118.238
108.


9
121
YLLFMIGEL
108.713
109.


10
323
IIWDTVVII
78.258
110.


11
409
QLQSYRQEV
69.552
111.


12
402
GMYRCTIQL
49.371
112.


13
118
AVLYLLFMI
45.057
113.


14
181
VLSAMISVL
34.246
114.


15
185
MISVLLVYI
30.849
115.


16
147
MLTDLSAII
29.814
116.


17
190
LVYILMGFL
16.722
117.


18
69
LQADDDSLL
15.096
118.


19
194
LMGFLLYEA
14.029
119.


20
275
FVHALGDLV
13.717
120.


21
308
CTYVFSLLV
11.747
121.


22
76
LLDQDLPLT
11.655
122.


23
137
SLAIMTDAL
10.468
123.


24
68
TLQADDDSL
10.468
124.


25
315
LVAFTTFRI
9.001
125.


26
183
SAMISVLLV
8.221
126.


27
327
TVVIILEGV
6.859
127.


28
115
TIAAVLYLL
6.756
128.


29
153
AIILTLLAL
6.756
129.


30
319
TTFRIIWDT
6.606
130.


31
114
LTIAAVLYL
6.381
131.


32
87
QLSLKVDSC
5.599
132.


33
364
SLTSGKSTA
4.968
133.


34
357
VEDLNIWSL
4.872
134.


35
312
FSLLVAFTT
4.802
135.


36
193
ILMGFLLYE
4.506
136.


37
180
EVLSAMISV
3.884
137.


38
150
DLSAIILTL
3.685
138.


39
389
VQSKANHLL
3.682
139.


40
395
HLLLNTFGM
3.625
140.


41
303
IADPICTYV
3.613
141.


42
227
AVNVIMGFL
3.074
142.


43
49
VVADDGSEA
3.030
143.


44
133
YIANSLAIM
2.963
144.


45
13
MLRKDDAPL
2.760
145.


46
216
DIMLITAAV
2.654
146.


47
139
AIMTDALHM
2.527
147.


48
187
SVLLVYILM
2.413
148.


49
331
ILEGVPSHL
2.324
149.


50
146
HMLTDLSAI
2.180
150.
















TABLE VIII (A)







HLA PEPTIDE SCORING RESULTS - 108P5H8 - A2, 10-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE
ID#














1
302
KIADPICTYV
754.791
151.


2
121
YLLFMIGELV
580.050
152.


3
217
IMLITAAVGV
315.959
153.


4
197
FLLYEAVQRT
291.716
154.


5
113
RLTIAAVLYL
270.234
155.


6
75
SLLDQDLPLT
260.008
156.


7
323
IIWDTVVIIL
160.242
157.


8
310
YVFSLLVAFT
140.388
158.


9
314
LLVAFTTFRI
102.867
159.


10
154
IILTLLALWL
101.617
160.


11
184
AMISVLLVYI
95.315
161.


12
330
IILEGVPSHL
75.751
162.


13
193
ILMGFLLYEA
71.872
163.


14
39
GLSRFNKLRV
69.552
164.


15
194
LMGFLLYEAV
62.765
165.


16
160
ALWLSSKSPT
61.852
166.


17
147
MLTDLSAIIL
61.047
167.


18
189
LLVYILMGFL
59.722
168.


19
190
LVYILMGFLL
58.977
169.


20
12
SMLRKDDAPL
57.085
170.


21
198
LLYEAVQRTI
46.539
171.


22
408
IQLQSYRQEV
44.356
172.


23
140
IMTDALHMLT
37.513
173.


24
181
VLSAMISVLL
36.316
174.


25
139
AIMTDALHML
24.997
175.


26
68
TLQADDDSLL
21.362
176.


27
397
LLNTFGMYRC
19.425
177.


28
172
FTFGFHRLEV
16.441
178.


29
219
LITAAVGVAV
16.258
179.


30
227
AVNVIMGFLL
10.841
180.


31
364
SLTSGKSTAI
10.433
181.


32
356
SVEDLNIWSL
8.461
182.


33
318
FTTFRIIWDT
8.213
183.


34
22
FLNDTSAFDF
8.152
184.


35
125
MIGELVGGYI
7.149
185.


36
348
LMKIEDVYSV
6.874
186.


37
345
KEALMKIEDV
5.335
187.


38
179
LEVLSAMISV
5.288
188.


39
285
SVGVLIAAYI
5.021
189.


40
144
ALHMLTDLSA
4.968
190.


41
133
YIANSLAIMT
4.713
191.


42
185
MISVLLVYIL
4.709
192.


43
150
DLSAIILTLL
3.685
193.


44
389
VQSKANHLLL
3.682
194.


45
129
LVGGYIANSL
3.178
195.


46
319
TTFRIIWDTV
2.977
196.


47
307
ICTYVFSLLV
2.933
197.


48
5
GAWKRLKSML
2.463
198.


49
401
FGMYRCTIQL
2.373
199.


50
332
LEGVPSHLNV
2.299
200.
















TABLE IX (A)







HLA PEPTIDE SCORING RESULTS - 108P5H8 - A3, 9-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE
ID#














1
158
LLALWLSSK
90.000
201.


2
377
QLIPGSSSK
45.000
202.


3
184
AMISVLLVY
27.000
203.


4
397
LLNTFGMYR
24.000
204.


5
162
WLSSKSPTK
20.000
205.


6
39
GLSRFNKLR
18.000
206.


7
402
GMYRCTIQL
18.000
207.


8
314
LLVAFTTFR
18.000
208.


9
189
LLVYILMGF
13.500
209.


10
288
VLIAAYIIR
12.000
210.


11
361
NIWSLTSGK
10.000
211.


12
347
ALMKIEDVY
9.000
212.


13
313
SLLVAFTTF
9.000
213.


14
197
FLLYEAVQR
6.000
214.


15
396
LLLNTFGMY
5.400
215.


16
113
RLTIAAVLY
4.000
216.


17
248
SLPSNSPTR
4.000
217.


18
192
YILMGFLLY
3.600
218.


19
302
KIADPICTY
2.700
219.


20
198
LLYEAVQRT
2.250
220.


21
294
IIRFKPEYK
2.000
221.


22
289
LIAAYIIRF
1.800
222.


23
293
YIIRFKPEY
1.800
223.


24
234
FLLNQSGHR
1.800
224.


25
75
SLLDQDLPL
1.800
225.


26
122
LLFMIGELV
1.500
226.


27
310
YVFSLLVAF
1.500
227.


28
83
LTNSQLSLK
1.500
228.


29
146
HMLTDLSAI
1.350
229.


30
331
ILEGVPSHL
1.350
230.


31
155
ILTLLALWL
1.200
231.


32
181
VLSAMISVL
0.900
232.


33
395
HLLLNTFGM
0.900
233.


34
194
LMGFLLYEA
0.900
234.


35
323
IIWDTVVII
0.900
235.


36
140
IMTDALHML
0.900
236.


37
150
DLSAIILTL
0.810
237.


38
287
GVLIAAYII
0.810
238.


39
100
KQREILKQR
0.608
239.


40
278
ALGDLVQSV
0.600
240.


41
409
QLQSYRQEV
0.600
241.


42
137
SLAIMTDAL
0.600
242.


43
68
TLQADDDSL
0.600
243.


44
285
SVGVLIAAY
0.600
244.


45
178
RLEVLSAMI
0.600
245.


46
119
VLYLLFMIG
0.600
246.


47
147
MLTDLSAII
0.600
247.


48
13
MLRKDDAPL
0.600
248.


49
87
QLSLKVDSC
0.600
249.


50
170
KRFTFGFHR
0.540
250.
















TABLE X (A)







HLA PEPTIDE SCORING RESULTS - 108P5H8 - A3, 10-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE
ID#














1
157
TLLALWLSSK
135.000
251.


2
396
LLLNTFGMYR
36.000
252.


3
288
VLIAAYIIRF
27.000
253.


4
313
SLLVAFTTFR
18.000
254.


5
188
VLLVYILMGF
13.500
255.


6
124
FMIGELVGGY
8.100
256.


7
22
FLNDTSAFDF
6.000
257.


8
91
KVDSCDNCSK
6.000
258.


9
104
ILKQRKVKAR
6.000
259.


10
395
HLLLNTFGMY
5.400
260.


11
162
WLSSKSPTKR
4.000
261.


12
287
GVLIAAYIIR
3.600
262.


13
113
RLTIAAVLYL
3.600
263.


14
293
YIIRFKPEYK
3.000
264.


15
82
PLTNSQLSLK
3.000
265.


16
100
KQREILKQRK
2.700
266.


17
323
IIWDTVVIIL
2.700
267.


18
314
LLVAFTTFRI
2.700
268.


19
225
GVAVNVIMGF
2.700
269.


20
193
ILMGFLLYEA
2.025
270.


21
13
MLRKDDAPLF
2.000
271.


22
348
LMKIEDVYSV
1.800
272.


23
184
AMISVLLVYI
1.350
273.


24
334
GVPSHLNVDY
1.200
274.


25
147
MLTDLSAIIL
1.200
275.


26
39
GLSRFNKLRV
1.200
276.


27
202
AVQRTIHMNY
1.200
277.


28
20
PLFLNDTSAF
1.000
278.


29
376
IQLIPGSSSK
0.900
279.


30
190
LVYILMGFLL
0.900
280.


31
181
VLSAMISVLL
0.900
281.


32
12
SMLRKDDAPL
0.900
282.


33
119
VLYLLFMIGE
0.900
283.


34
397
LLNTFGMYRC
0.900
284.


35
146
HMLTDLSAII
0.900
285.


36
198
LLYEAVQRTI
0.675
286.


37
399
NTFGMYRCTI
0.675
287.


38
68
TLQADDDSLL
0.600
288.


39
364
SLTSGKSTAI
0.600
289.


40
268
SLAVRAAFVH
0.600
290.


41
270
AVRAAFVHAL
0.540
291.


42
185
MISVLLVYIL
0.540
292.


43
160
ALWLSSKSPT
0.500
293.


44
402
GMYRCTIQLQ
0.450
294.


45
289
LIAAYIIRFK
0.450
295.


46
338
HLNVDYIKEA
0.450
296.


47
121
YLLFMIGELV
0.450
297.


48
197
FLLYEAVQRT
0.450
298.


49
377
QLIPGSSSKW
0.450
299.


50
150
DLSAIILTLL
0.405
300.
















TABLE XI (A)







HLA PEPTIDE SCORING RESULTS - 108P5H8 - A11, 9-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE
ID#














1
83
LTNSQLSLK
1.000
301.


2
361
NIWSLTSGK
0.800
302.


3
377
QLIPGSSSK
0.600
303.


4
294
IIRFKPEYK
0.400
304.


5
162
WLSSKSPTK
0.400
305.


6
158
LLALWLSSK
0.400
306.


7
342
DYIKEALMK
0.360
307.


8
406
CTIQLQSYR
0.300
308.


9
288
VLIAAYIIR
0.240
309.


10
8
KRLKSMLRK
0.180
310.


11
287
GVLIAAYII
0.180
311.


12
100
KQREILKQR
0.180
312.


13
397
LLNTFGMYR
0.160
313.


14
39
GLSRFNKLR
0.120
314.


15
197
FLLYEAVQR
0.120
315.


16
234
FLLNQSGHR
0.120
316.


17
314
LLVAFTTFR
0.120
317.


18
205
RTIHMNYEI
0.090
318.


19
118
AVLYLLFMI
0.090
319.


20
103
EILKQRKVK
0.090
320.


21
248
SLPSNSPTR
0.080
321.


22
1
MAGSGAWKR
0.080
322.


23
170
KRFTFGFHR
0.072
323.


24
337
SHLNVDYIK
0.060
324.


25
315
LVAFTTFRI
0.060
325.


26
109
KVKARLTIA
0.060
326.


27
187
SVLLVYILM
0.060
327.


28
37
DEGLSRFNK
0.054
328.


29
402
GMYRCTIQL
0.048
329.


30
190
LVYILMGFL
0.040
330.


31
310
YVFSLLVAF
0.040
331.


32
308
CTYVFSLLV
0.040
332.


33
384
SKWEEVQSK
0.040
333.


34
98
CSKQREILK
0.040
334.


35
132
GYIANSLAI
0.036
335.


36
327
TVVIILEGV
0.030
336.


37
114
LTIAAVLYL
0.030
337.


38
34
EAGDEGLSR
0.024
338.


39
275
FVHALGDLV
0.020
339.


40
94
SCDNCSKQR
0.020
340.


41
49
VVADDGSEA
0.020
341.


42
282
LVQSVGVLI
0.020
342.


43
290
IAAYIIRFK
0.020
343.


44
223
AVGVAVNVI
0.020
344.


45
92
VDSCDNCSK
0.020
345.


46
285
SVGVLIAAY
0.020
346.


47
340
NVDYIKEAL
0.020
347.


48
227
AVNVIMGFL
0.020
348.


49
270
AVRAAFVHA
0.020
349.


50
101
QREILKQRK
0.020
350.
















TABLE XII (A)







HLA PEPTIDE SCORING RESULTS -


108P5H8 - A11, 10-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID#














1
91
KVDSCDNCSK
6.000
351.


2
287
GVLIAAYIIR
3.600
352.


3
100
KQREILKQRK
1.800
353.


4
376
IQLIPGSSSK
0.900
354.


5
157
TLLALWLSSK
0.600
355.


6
293
YIIRFKPEYK
0.600
356.


7
97
NCSKQREILK
0.400
357.


8
396
LLLNTFGMYR
0.240
358.


9
36
GDEGLSRFNK
0.180
359.


10
196
GFLLYEAVQR
0.180
360.


11
233
GFLLNQSGHR
0.180
361.


12
102
REILKQRKVK
0.135
362.


13
225
GVAVNVIMGF
0.120
363.


14
190
LVYILMGFLL
0.120
364.


15
109
KVKARLTIAA
0.120
365.


16
410
LQSYRQEVDR
0.120
366.


17
405
RCTIQLQSYR
0.120
367.


18
313
SLLVAFTTFR
0.120
368.


19
162
WLSSKSPTKR
0.080
369.


20
341
VDYIKEALMK
0.080
370.


21
360
LNIWSLTSGK
0.060
371.


22
334
GVPSHLNVDY
0.060
372.


23
227
AVNVIMGFLL
0.060
373.


24
253
SPTRGSGCER
0.040
374.


25
7
WKRLKSMLRK
0.040
375.


26
104
ILKQRKVKAR
0.040
376.


27
308
CTYVFSLLVA
0.040
377.


28
282
LVQSVGVLIA
0.040
378.


29
289
LIAAYIIRFK
0.040
379.


30
172
FTFGFHRLEV
0.040
380.


31
356
SVEDLNIWSL
0.040
381.


32
82
PLTNSQLSLK
0.040
382.


33
202
AVQRTIHMNY
0.040
383.


34
161
LWLSSKSPTK
0.030
384.


35
48
VVVADDGSEA
0.030
385.


36
114
LTIAAVLYLL
0.030
386.


37
353
DVYSVEDLNI
0.024
387.


38
39
GLSRFNKLRV
0.024
388.


39
113
RLTIAAVLYL
0.024
389.


40
319
TTFRIIWDTV
0.020
390.


41
223
AVGVAVNVIM
0.020
391.


42
383
SSKWEEVQSK
0.020
392.


43
270
AVRAAFVHAL
0.020
393.


44
399
NTFGMYRCTI
0.020
394.


45
83
LTNSQLSLKV
0.020
395.


46
285
SVGVLIAAYI
0.020
396.


47
129
LVGGYIANSL
0.020
397.


48
340
NVDYIKEALM
0.020
398.


49
314
LLVAFTTFRI
0.018
399.


50
322
RIIWDTVVII
0.018
400.
















TABLE XIII (A)







HLA PEPTIDE SCORING RESULTS - 108P5H8 - A24, 9-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID#














1
191
VYILMGFLL
300.000
401.


2
199
LYEAVQRTI
105.000
402.


3
132
GYIANSLAI
75.000
403.


4
354
VYSVEDLNI
50.000
404.


5
171
RFTFGFHRL
48.000
405.


6
210
NYEINGDIM
37.500
406.


7
274
AFVHALGDL
30.000
407.


8
21
LFLNDTSAF
15.000
408.


9
106
KQRKVKARL
11.200
409.


10
38
EGLSRFNKL
9.504
410.


11
331
ILEGVPSHL
8.400
411.


12
227
AVNVIMGFL
8.400
412.


13
309
TYVFSLLVA
7.500
413.


14
75
SLLDQDLPL
7.200
414.


15
186
ISVLLVYIL
7.200
415.


16
388
EVQSKANHL
7.200
416.


17
130
VGGYIANSL
6.720
417.


18
307
ICTYVFSLL
6.720
418.


19
121
YLLFMIGEL
6.600
419.


20
153
AIILTLLAL
6.000
420.


21
166
KSPTKRFTF
6.000
421.


22
281
DLVQSVGVL
6.000
422.


23
68
TLQADDDSL
6.000
423.


24
143
DALHMLTDL
6.000
424.


25
228
VNVIMGFLL
6.000
425.


26
80
DLPLTNSQL
6.000
426.


27
114
LTIAAVLYL
6.000
427.


28
150
DLSAIILTL
5.600
428.


29
340
NVDYIKEAL
5.600
429.


30
324
IWDTVVIIL
5.600
430.


31
115
TIAAVLYLL
5.600
431.


32
370
STAIVHIQL
5.600
432.


33
151
LSAIILTLL
5.600
433.


34
182
LSAMISVLL
5.600
434.


35
300
EYKIADPIC
5.000
435.


36
400
TFGMYRCTI
5.000
436.


37
412
SYRQEVDRT
5.000
437.


38
6
AWKRLKSML
4.800
438.


39
190
LVYILMGFL
4.800
439.


40
69
LQADDDSLL
4.800
440.


41
155
ILTLLALWL
4.800
441.


42
140
IMTDALHML
4.800
442.


43
189
LLVYILMGF
4.200
443.


44
226
VAVNVIMGF
4.200
444.


45
267
DSLAVRAAF
4.200
445.


46
13
MLRKDDAPL
4.000
446.


47
402
GMYRCTIQL
4.000
447.


48
2
AGSGAWKRL
4.000
448.


49
97
NCSKQREIL
4.000
449.


50
390
QSKANHLLL
4.000
450.
















TABLE XIV (A)







HLA PEPTIDE SCORING RESULTS -


108P5H8 - A24, 10-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID#














1
120
LYLLFMIGEL
330.000
451.


2
210
NYEINGDIML
300.000
452.


3
309
TYVFSLLVAF
180.000
453.


4
342
DYIKEALMKI
82.500
454.


5
132
GYIANSLAIM
37.500
455.


6
173
TFGFHRLEVL
20.000
456.


7
60
RPVNGAHPTL
12.000
457.


8
369
KSTAIVHIQL
11.200
458.


9
111
KARLTIAAVL
11.200
459.


10
339
LNVDYIKEAL
10.080
460.


11
330
IILEGVPSHL
10.080
461.


12
191
VYILMGFLLY
9.000
462.


13
392
KANHLLLNTF
8.640
463.


14
226
VAVNVIMGFL
8.400
464.


15
114
LTIAAVLYLL
8.400
465.


16
292
AYIIRFKPEY
8.250
466.


17
113
RLTIAAVLYL
8.000
467.


18
189
LLVYILMGFL
7.200
468.


19
356
SVEDLNIWSL
7.200
469.


20
154
IILTLLALWL
7.200
470.


21
403
MYRCTIQLQS
7.000
471.


22
412
SYRQEVDRTC
7.000
472.


23
129
LVGGYIANSL
6.720
473.


24
323
IIWDTVVIIL
6.720
474.


25
305
DPICTYVFSL
6.000
475.


26
152
SAIILTLLAL
6.000
476.


27
401
FGMYRCTIQL
6.000
477.


28
12
SMLRKDDAPL
6.000
478.


29
68
TLQADDDSLL
6.000
479.


30
354
VYSVEDLNIW
6.000
480.


31
81
LPLTNSQLSL
6.000
481.


32
227
AVNVIMGFLL
6.000
482.


33
139
AIMTDALHML
6.000
483.


34
136
NSLAIMTDAL
6.000
484.


35
388
EVQSKANHLL
6.000
485.


36
180
EVLSAMISVL
6.000
486.


37
74
DSLLDQDLPL
6.000
487.


38
150
DLSAIILTLL
5.600
488.


39
181
VLSAMISVLL
5.600
489.


40
300
EYKIADPICT
5.000
490.


41
185
MISVLLVYIL
4.800
491.


42
5
GAWKRLKSML
4.800
492.


43
147
MLTDLSAIIL
4.800
493.


44
31
FSDEAGDEGL
4.800
494.


45
298
KPEYKIADPI
4.200
495.


46
188
VLLVYILMGF
4.200
496.


47
273
AAFVHALGDL
4.000
497.


48
270
AVRAAFVHAL
4.000
498.


49
1
MAGSGAWKRL
4.000
499.


50
96
DNCSKQREIL
4.000
500.
















TABLE XV (A)







HLA PEPTIDE SCORING RESULTS - 108P5H8 - B7, 9-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID#














1
227
AVNVIMGFL
60.000
501.


2
106
KQRKVKARL
40.000
502.


3
13
MLRKDDAPL
40.000
503.


4
190
LVYILMGFL
20.000
504.


5
388
EVQSKANHL
20.000
505.


6
270
AVRAAFVHA
15.000
506.


7
2
AGSGAWKRL
12.000
507.


8
143
DALHMLTDL
12.000
508.


9
153
AIILTLLAL
12.000
509.


10
139
AIMTDALHM
9.000
510.


11
117
AAVLYLLFM
9.000
511.


12
340
NVDYIKEAL
6.000
512.


13
97
NCSKQREIL
6.000
513.


14
111
KARLTIAAV
6.000
514.


15
118
AVLYLLFMI
6.000
515.


16
223
AVGVAVNVI
6.000
516.


17
187
SVLLVYILM
5.000
517.


18
150
DLSAIILTL
4.000
518.


19
307
ICTYVFSLL
4.000
519.


20
186
ISVLLVYIL
4.000
520.


21
130
VGGYIANSL
4.000
521.


22
151
LSAIILTLL
4.000
522.


23
182
LSAMISVLL
4.000
523.


24
390
QSKANHLLL
4.000
524.


25
155
ILTLLALWL
4.000
525.


26
402
GMYRCTIQL
4.000
526.


27
75
SLLDQDLPL
4.000
527.


28
69
LQADDDSLL
4.000
528.


29
181
VLSAMISVL
4.000
529.


30
370
STAIVHIQL
4.000
530.


31
114
LTIAAVLYL
4.000
531.


32
281
DLVQSVGVL
4.000
532.


33
80
DLPLTNSQL
4.000
533.


34
389
VQSKANHLL
4.000
534.


35
38
EGLSRFNKL
4.000
535.


36
115
TIAAVLYLL
4.000
536.


37
140
IMTDALHML
4.000
537.


38
68
TLQADDDSL
4.000
538.


39
174
FGFHRLEVL
4.000
539.


40
228
VNVIMGFLL
4.000
540.


41
121
YLLFMIGEL
4.000
541.


42
137
SLAIMTDAL
4.000
542.


43
5
GAWKRLKSM
3.000
543.


44
201
EAVQRTIHM
3.000
544.


45
57
APERPVNGA
2.700
545.


46
61
PVNGAHPTL
2.000
546.


47
282
LVQSVGVLI
2.000
547.


48
287
GVLIAAYII
2.000
548.


49
315
LVAFTTFRI
2.000
549.


50
40
LSRFNKLRV
2.000
550.
















TABLE XVI (A)







HLA PEPTIDE SCORING RESULTS - 108P5H8 - B7, 10-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID#














1
270
AVRAAFVHAL
600.000
551.


2
111
KARLTIAAVL
120.000
552.


3
60
RPVNGAHPTL
80.000
553.


4
81
LPLTNSQLSL
80.000
554.


5
305
DPICTYVFSL
80.000
555.


6
227
AVNVIMGFLL
60.000
556.


7
273
AAFVHALGDL
36.000
557.


8
139
AIMTDALHML
36.000
558.


9
388
EVQSKANHLL
20.000
559.


10
180
EVLSAMISVL
20.000
560.


11
190
LVYILMGFLL
20.000
561.


12
129
LVGGYIANSL
20.000
562.


13
223
AVGVAVNVIM
15.000
563.


14
5
GAWKRLKSML
12.000
564.


15
1
MAGSGAWKRL
12.000
565.


16
152
SAIILTLLAL
12.000
566.


17
401
FGMYRCTIQL
12.000
567.


18
226
VAVNVIMGFL
12.000
568.


19
335
VPSHLNVDYI
8.000
569.


20
356
SVEDLNIWSL
6.000
570.


21
19
APLFLNDTSA
6.000
571.


22
96
DNCSKQREIL
6.000
572.


23
294
IIRFKPEYKI
6.000
573.


24
113
RLTIAAVLYL
4.000
574.


25
150
DLSAIILTLL
4.000
575.


26
339
LNVDYIKEAL
4.000
576.


27
240
GHRHSHSHSL
4.000
577.


28
147
MLTDLSAIIL
4.000
578.


29
330
IILEGVPSHL
4.000
579.


30
114
LTIAAVLYLL
4.000
580.


31
189
LLVYILMGFL
4.000
581.


32
12
SMLRKDDAPL
4.000
582.


33
74
DSLLDQDLPL
4.000
583.


34
185
MISVLLVYIL
4.000
584.


35
260
CERNHGQDSL
4.000
585.


36
181
VLSAMISVLL
4.000
586.


37
323
IIWDTVVIIL
4.000
587.


38
68
TLQADDDSLL
4.000
588.


39
136
NSLAIMTDAL
4.000
589.


40
154
IILTLLALWL
4.000
590.


41
369
KSTAIVHIQL
4.000
591.


42
389
VQSKANHLLL
4.000
592.


43
117
AAVLYLLFMI
3.600
593.


44
222
AAVGVAVNVI
3.600
594.


45
138
LAIMTDALHM
3.000
595.


46
116
IAAVLYLLFM
3.000
596.


47
298
KPEYKIADPI
2.400
597.


48
315
LVAFTTFRII
2.000
598.


49
285
SVGVLIAAYI
2.000
599.


50
40
LSRFNKLRVV
2.000
600.
















TABLE XVII (A)







HLA PEPTIDE SCORING RESULTS - 108P5H8 - B35, 9-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID#














1
335
VPSHLNVDY
40.000
601.


2
390
QSKANHLLL
15.000
602.


3
164
SSKSPTKRF
15.000
603.


4
166
KSPTKRFTF
10.000
604.


5
302
KIADPICTY
8.000
605.


6
355
YSVEDLNIW
7.500
606.


7
201
EAVQRTIHM
6.000
607.


8
5
GAWKRLKSM
6.000
608.


9
203
VQRTIHMNY
6.000
609.


10
117
AAVLYLLFM
6.000
610.


11
106
KQRKVKARL
6.000
611.


12
267
DSLAVRAAF
5.000
612.


13
151
LSAIILTLL
5.000
613.


14
182
LSAMISVLL
5.000
614.


15
186
ISVLLVYIL
5.000
615.


16
13
MLRKDDAPL
4.500
616.


17
60
RPVNGAHPT
4.000
617.


18
125
MIGELVGGY
4.000
618.


19
113
RLTIAAVLY
4.000
619.


20
405
RCTIQLQSY
4.000
620.


21
111
KARLTIAAV
3.600
621.


22
143
DALHMLTDL
3.000
622.


23
69
LQADDDSLL
3.000
623.


24
116
IAAVLYLLF
3.000
624.


25
40
LSRFNKLRV
3.000
625.


26
226
VAVNVIMGF
3.000
626.


27
139
AIMTDALHM
3.000
627.


28
343
YIKEALMKI
2.400
628.


29
383
SSKWEEVQS
2.250
629.


30
19
APLFLNDTS
2.000
630.


31
192
YILMGFLLY
2.000
631.


32
293
YIIRFKPEY
2.000
632.


33
184
AMISVLLVY
2.000
633.


34
75
SLLDQDLPL
2.000
634.


35
187
SVLLVYILM
2.000
635.


36
224
VGVAVNVIM
2.000
636.


37
81
LPLTNSQLS
2.000
637.


38
133
YIANSLAIM
2.000
638.


39
347
ALMKIEDVY
2.000
639.


40
305
DPICTYVFS
2.000
640.


41
285
SVGVLIAAY
2.000
641.


42
140
IMTDALHML
2.000
642.


43
395
HLLLNTFGM
2.000
643.


44
396
LLLNTFGMY
2.000
644.


45
68
TLQADDDSL
1.500
645.


46
371
TAIVHIQLI
1.200
646.


47
316
VAFTTFRII
1.200
647.


48
322
RIIWDTVVI
1.200
648.


49
97
NCSKQREIL
1.000
649.


50
181
VLSAMISVL
1.000
650.
















TABLE XVIII (A)







HLA PEPTIDE SCORING RESULTS -


108P5H8 - B35, 10-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID#














1
60
RPVNGAHPTL
40.000
651.


2
305
DPICTYVFSL
20.000
652.


3
81
LPLTNSQLSL
20.000
653.


4
167
SPTKRFTFGF
20.000
654.


5
111
KARLTIAAVL
18.000
655.


6
369
KSTAIVHIQL
10.000
656.


7
284
QSVGVLIAAY
10.000
657.


8
186
ISVLLVYILM
10.000
658.


9
138
LAIMTDALHM
9.000
659.


10
335
VPSHLNVDYI
8.000
660.


11
392
KANHLLLNTF
6.000
661.


12
34
EAGDEGLSRF
6.000
662.


13
346
EALMKIEDVY
6.000
663.


14
116
IAAVLYLLFM
6.000
664.


15
183
SAMISVLLVY
6.000
665.


16
136
NSLAIMTDAL
5.000
666.


17
74
DSLLDQDLPL
5.000
667.


18
312
FSLLVAFTTF
5.000
668.


19
163
LSSKSPTKRF
5.000
669.


20
298
KPEYKIADPI
4.800
670.


21
13
MLRKDDAPLF
4.500
671.


22
209
MNYEINGDIM
4.000
672.


23
40
LSRFNKLRVV
3.000
673.


24
270
AVRAAFVHAL
3.000
674.


25
273
AAFVHALGDL
3.000
675.


26
226
VAVNVIMGFL
3.000
676.


27
5
GAWKRLKSML
3.000
677.


28
1
MAGSGAWKRL
3.000
678.


29
152
SAIILTLLAL
3.000
679.


30
31
FSDEAGDEGL
3.000
680.


31
19
APLFLNDTSA
2.000
681.


32
4
SGAWKRLKSM
2.000
682.


33
113
RLTIAAVLYL
2.000
683.


34
395
HLLLNTFGMY
2.000
684.


35
147
MLTDLSAIIL
2.000
685.


36
334
GVPSHLNVDY
2.000
686.


37
66
HPTLQADDDS
2.000
687.


38
339
LNVDYIKEAL
2.000
688.


39
249
LPSNSPTRGS
2.000
689.


40
323
IIWDTVVIIL
2.000
690.


41
330
IILEGVPSHL
2.000
691.


42
202
AVQRTIHMNY
2.000
692.


43
124
FMIGELVGGY
2.000
693.


44
22
FLNDTSAFDF
2.000
694.


45
223
AVGVAVNVIM
2.000
695.


46
164
SSKSPTKRFT
1.500
696.


47
390
QSKANHLLLN
1.500
697.


48
12
SMLRKDDAPL
1.500
698.


49
68
TLQADDDSLL
1.500
699.


50
316
VAFTTFRIIW
1.500
700.
















TABLE V (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - A1, 9-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID#





1
23
LNDTSAFEF
6.250
701.


2
28
AFEFSDEAG
0.045
702.


3
25
DTSAFEFSD
0.013
703.


4
28
SAFEFSDEA
0.010
704.


5
22
FLNDTSAFE
0.002
705.


6
26
TSAFEFSDE
0.002
706.


7
24
NDTSAFEFS
0.001
707.


8
29
FEFSDEAGD
0.000
708.


9
30
EFSDEAGDE
0.000
709.
















TABLE VI (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - A1, 10-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID#














1
22
FLNDTSAFEF
0.500
710.


2
23
LNDTSAFEFS
0.125
711.


3
26
TSAFEFSDEA
0.015
712.


4
27
SAFEFSDEAG
0.010
713.


5
28
AFEFSDEAGD
0.009
714.


6
25
DTSAFEFSDE
0.003
715.


7
30
EFSDEAGDEG
0.001
716.


8
24
NDTSAFEFSD
0.000
717.


9
21
LFLNDTSAFE
0.000
718.


10
29
FEFSDEAGDE
0.000
719.
















TABLE VII (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - A2, 9-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID#





1
27
SAFEFSDEA
1.949
720.


2
22
FLNDTSAFE
1.546
721.


3
29
FEFSDEAGD
0.005
722.


4
23
LNDTSAFEF
0.002
723.


5
24
NDTSAFEFS
0.001
724.


6
25
DTSAFEFSD
0.000
725.


7
26
TSAFEFSDE
0.000
726.


8
28
AFEFSDEAG
0.000
727.


9
30
EFSDEAGDE
0.000
728.
















TABLE VIII (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - A2, 10-MERS














SCORE (ESTIMATE






OF HALF TIME OF





DISASSOCIATION




SUBSEQUENCE
OF A MOLECULE



START
RESIDUE
CONTAINING THIS
SEQ.


RANK
POSITION
LISTING
SEQUENCE)
ID #














1
22
FLNDTSAFEF
8.152
729.


2
26
TSAFEFSDEA
0.060
730.


3
27
SAFEFSDEAG
0.008
731.


4
23
LNDTSAFEFS
0.002
732.


5
29
FEFSDEAGDE
0.001
733.


6
24
NDTSAFEFSD
0.000
734.


7
21
LFLNDTSAFE
0.000
735.


8
25
DTSAFEFSDE
0.000
736.


9
30
EFSDEAGDEG
0.000
737.


10
28
AFEFSDEAGD
0.000
738.
















TABLE IX (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - A3, 9-MERS














SCORE (ESTIMATE 






OF HALF TIME OF





DISASSOCIATION




SUBSEQUENCE
OF A MOLECULE



START
RESIDUE
CONTAINING THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID #














1
27
SAFEFSDEA
0.045
739.


2
22
FLNDTSAFE
0.020
740.


3
23
LNDTSAFEF
0.012
741.


4
25
DTSAFEFSD
0.003
742.


5
29
FEFSDEAGD
0.000
743.


6
26
TSAFEFSDE
0.000
744.


7
24
NDTSAFEFS
0.000
745.


8
28
AFEFSDEAG
0.000
746.


9
30
EFSDEAGDE
0.000
747.
















TABLE X (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - A3, 10-MERS














SCORE (ESTIMATE






OF HALF TIME OF





DISASSOCIATION




SUBSEQUENCE
OF A MOLECULE



START
RESIDUE
CONTAINING THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID #














1
22
FLNDTSAFEF
6.000
748.


2
26
TSAFEFSDEA
0.003
749.


3
27
SAFEFSDEAG
0.002
750.


4
25
DTSAFEFSDE
0.001
751.


5
23
LNDTSAFEFS
0.000
752.


6
24
NDTSAFEFSD
0.000
753.


7
29
FEFSDEAGDE
0.000
754.


8
21
LFLNDTSAFE
0.000
755.


9
28
AFEFSDEAGD
0.000
756.


10
30
EFSDEAGDEG
0.000
757.
















TABLE XI (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - All, 9-MERS














SCORE (ESTIMATE






OF HALF TIME OF





DISASSOCIATION




SUBSEQUENCE
OF A MOLECULE



START
RESIDUE
CONTAINING THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID #














1
27
SAFEFSDEA
0.004
758.


2
23
LNDTSAFEF
0.001
759.


3
25
DTSAFEFSD
0.001
760.


4
22
FLNDTSAFE
0.000
761.


5
28
AFEFSDEAG
0.000
762.


6
29
FEFSDEAGD
0.000
763.


7
30
EFSDEAGDE
0.000
764.


8
24
NDTSAFEFS
0.000
765.


9
26
TSAFEFSDE
0.000
766.
















TABLE XII (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - All, 10-MERS














SCORE (ESTIMATE






OF HALF TIME OF





DISASSOCIATION




SUBSEQUENCE
OF A MOLECULE



START
RESIDUE
CONTAINING THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID #














1
22
FLNDTSAFEF
0.012
767.


2
27
SAFEFSDEAG
0.000
768.


3
21
LFLNDTSAFE
0.000
769.


4
25
DTSAFEFSDE
0.000
770.


5
28
AFEFSDEAGD
0.000
771.


6
26
TSAFEFSDEA
0.000
772.


7
29
FEFSDEAGDE
0.000
773.


8
24
NDTSAFEFSD
0.000
774.


9
30
EFSDEAGDEG
0.000
775.


10
23
LNDTSAFEFS
0.000
776.
















TABLE XIII (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - A24, 9-MERS














SCORE (ESTIMATE






OF HALF TIME OF





DISASSOCIATION




SUBSEQUENCE
OF A MOLECULE



START
RESIDUE
CONTAINING THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID #














1
23
LNDTSAFEF
2.200
777.


2
27
SAFEFSDEA
0.132
778.


3
28
AFEFSDEAG
0.075
779.


4
30
EFSDEAGDE
0.060
780.


5
22
FLNDTSAFE
0.018
781.


6
24
NDTSAFEFS
0.012
782.


7
26
TSAFEFSDE
0.012
783.


8
25
DTSAFEFSD
0.010
784.


9
29
FEFSDEAGD
0.001
785.
















TABLE XIV (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - A24, 10-MERS














SCORE (ESTIMATE






OF HALF TIME OF





DISASSOCIATION




SUBSEQUENCE
OF A MOLECULE



START
RESIDUE
CONTAINING THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID #














1
22
FLNDTSAFEF
3.960
786.


2
23
LNDTSAFEFS
0.120
787.


3
26
TSAFEFSDEA
0.110
788.


4
28
AFEFSDEAGD
0.075
789.


5
21
LFLNDTSAFE
0.075
790.


6
30
EFSDEAGDEG
0.066
791.


7
27
SAFEFSDEAG
0.012
792.


8
25
DTSAFEFSDE
0.012
793.


9
24
NDTSAFEFSD
0.001
794.


10
29
FEFSDEAGDE
0.001
795.
















TABLE XV (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - B7, 9-MERS














SCORE (ESTIMATE






OF HALF TIME OF





DISASSOCIATION




SUBSEQUENCE
OF A MOLECULE



START
RESIDUE
CONTAINING THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID #














1
27
SAFEFSDEA
0.300
796.


2
22
FLNDTSAFE
0.010
797.


3
26
TSAFEFSDE
0.010
798.


4
25
DTSAFEFSD
0.010
799.


5
23
LNDTSAFEF
0.006
800.


6
24
NDTSAFEFS
0.002
801.


7
30
EFSDEAGDE
0.001
802.


8
29
FEFSDEAGD
0.001
803.


9
28
AFEFSDEAG
0.001
804.
















TABLE XVI (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - B7, 10-MERS














SCORE (ESTIMATE






OF HALF TIME OF





DISASSOCIATION




SUBSEQUENCE
OF A MOLECULE



START
RESIDUE
CONTAINING THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID #














1
26
TSAFEFSDEA
0.100
805.


2
27
SAFEFSDEAG
0.030
806.


3
22
FLNDTSAFEF
0.020
807.


4
25
DTSAFEFSDE
0.010
808.


5
23
LNDTSAFEFS
0.006
809.


6
30
EFSDEAGDEG
0.001
810


7
24
NDTSAFEFSD
0.001
811.


8
29
FEFSDEAGDE
0.001
812.


9
21
LFLNDTSAFE
0.001
813.


10
28
AFEFSDEAGD
0.001
814.
















TABLE XVII (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - B35, 9-MERS














SCORE (ESTIMATE






OF HALF TIME OF





DISASSOCIATION




SUBSEQUENCE
OF A MOLECULE



START
RESIDUE
CONTAINING THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE)
ID #














1
276
SAFEFSDEA
0.600
815.


2
23
LNDTSAFEF
0.300
816.


3
26
TSAFEFSDE
0.075
817.


4
22
FLNDTSAFE
0.020
818.


5
24
NDTSAFEFS
0.010
819.


6
25
DTSAFEFSD
0.010
820.


7
30
EFSDEAGDE
0.003
821.


8
29
FEFSDEAGD
0.002
822.


9
28
AFEFSDEAG
0.000
823.
















TABLE XVIII (B)







unique to variant 3 relative to variants 1 and 2


HLA PEPTIDE SCORING RESULTS - 108P5H8 - B35, 10-MERS














SCORE






(ESTIMATE





OF HALF





TIME OF





DISASSOCIATION





OF A





MOLECULE




SUBSEQUENCE
CONTAINING



START
RESIDUE
THIS
SEQ.


RANK
POSITION
LISTING
SUBSEQUENCE
ID#














1
22
FLNDTSAFEF
2.000
824.


2
26
TSAFEFSDEA
0.500
825.


3
27
SAFEFSDEAG
0.060
826.


4
23
LNDTSAFEFS
0.030
827.


5
25
DTSAFEFSDE
0.015
828.


6
30
EFSDEAGDEG
0.002
829.


7
29
FEFSDEAGDE
0.002
830.


8
24
NDTSAFEFSD
0.001
831.


9
21
LFLNDTSAFE
0.001
832.


10
28
AFEFSDEAGD
0.000
833.
















TABLE XIX





Motifs and Post-translational Modifications of 108P5H8







N-glycosylation site


Number of matches: 3










1
24-27
NDTS
(SEQ. ID. No. 834)


2
 97-100
NCSK
(SEQ. ID. No. 835)


3
237-240
NQSG
(SEQ. ID. No. 836)







cAMP- and cGMP-dependent protein kinase phosphorylation site











170-173
KRFT
(SEQ. ID. No. 837)







Protein kinase C phosphorylation site


Number of matches: 7









1
89-91
SLK


2
164-166
SSK


3
383-385
SSK


4
169-171
TKR


5
320-322
TFR


6
367-369
SGK


7
164-166
SSK







Casein kinase II phosphorylation site


Number of matches: 5










1
27-30
SAFD
(SEQ. ID. No. 838)


2
75-78
SLLD
(SEQ. ID. No. 839)


3
258-261
SGCE
(SEQ. ID. No. 840)


4
356-359
SVED
(SEQ. ID. No. 841)


5
384-387
SKWE
(SEQ. ID. No. 842)







N-myristoylation site


Number of matches: 6










1
64-69
GAHPTL
(SEQ. ID. No. 843)


2
131-136
GGYIAN
(SEQ. ID. No. 844)


3
225-230
GVAVNV
(SEQ. ID. No. 845)


4
259-264
GCERNH
(SEQ. ID. No. 846)


5
287-292
GVLIAA
(SEQ. ID. No. 847)


6
402-407
GMYRCT
(SEQ. ID. No. 848)







Leucine zipper pattern











69-90
LQADDDSLLDQDLPLTNSQLSL
(SEQ. ID. No. 849)
















TABLE XX







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 repat
Cytoplasmic protein, associates integral





membrane proteins to the cytoskeleton


oxidored_q1
32%
NADH-
membrane associated. Involved in proton




Ubiquinone/plastoquinone
translocation across the membrane




complex I), various chains


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





residue loop flanked on both sides by a 12





residue alpha-helical domain


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





catalytic aspartyl residue


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




(20 copies)
formation of connective tissue. The





sequence consists of the G-X-Y and the





polypeptide chains forms a triple helix.


fn3
20%
Fibronectin type III domain
Located in the extracellular ligand-binding





region of receptors and is about 200 amino





acid residues long with two pairs of





cysteines involved in disulfide bonds


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




(rhodopsin family)
with the N-terminus located extracellularly





while the C-terminus is cytoplasmic.





Signal through G proteins
















TABLE XXI







Properties of 108P5H8


Motifs and localization apply to 108P5H8 variants 1 and 2.










Bioinformatic




Program
Outcome













ORF
ORF Finder
1290




(includes stop)


Protein Length
n/a
429 amino acids


Transmembrane
TM Pred
6 TM, at amino acids


region

114-130, 147-163,




181-200, 217-236,




273-295, 306-324



HMMTop
6 TM, at amino acid




113-130, 135-164,




179-202, 215-236,




271-296, 306-331



Sosui
6 TM, at amino acid




113-135, 141-163,




180-202, 215-237,




272-294, 308-330



TMHMM
6 TM, at amino acids




114-136, 146-165,




178-200, 215-237,




273-295, 310-332


Signal Peptide
Signal P
indicates no signal


pI
pI/MW tool
pI 6.11


Molecular weight
pI/MW tool
47.5 kDa


Localization
PSORT
Plasma membrane




60%



PSORT II
Plasma membrane




43%



iPSORT
No signal motif


Motifs
Pfam
Ribosomal protein




L34; Cation efflux




family



Prints
Rhodopsin



Blocks
No motif



Prosite
No motif
















TABLE XXII







MHC Class 1 nonamer and decamer analysis of 108P5H8 for selected


alleles. Listed are scores that fall within


the top 50% (rounded up) of all scores for the selected allele.





























SEQ. ID.


Pos
1
2
3
4
5
6
7
8
9
score
No.













HLA-A*0201 nonamers



















278
A
L
G
D
L
V
Q
S
V
30
1968


121
Y
L
L
F
M
I
G
E
L
29
1969


153
A
I
I
L
T
L
L
A
L
28
1970


137
S
L
A
I
M
T
D
A
L
27
1971


 75
S
L
L
D
Q
D
L
P
L
26
1972


150
D
L
S
A
I
I
L
T
L
26
1973


268
S
L
A
V
R
A
A
F
V
26
1974


323
I
I
W
D
T
V
V
I
I
26
1975


115
T
I
A
A
V
L
Y
L
L
25
1976


140
I
M
T
D
A
L
H
M
L
25
1977


181
V
L
S
A
M
I
S
V
L
25
1978


218
M
L
I
T
A
A
V
G
V
25
1979


343
Y
I
K
E
A
L
M
K
I
25
1980


114
L
T
I
A
A
V
L
Y
L
23
1981


122
L
L
F
M
I
G
E
L
V
23
1982


146
H
M
L
T
D
L
S
A
I
23
1983


155
I
L
T
L
L
A
L
W
L
23
1984


185
M
I
S
V
L
L
V
Y
I
23
1985


198
L
L
Y
E
A
V
Q
R
T
23
1986


216
D
I
M
L
I
T
A
A
V
23
1987


281
D
L
V
Q
S
V
G
V
L
23
1988


330
I
I
L
E
G
V
P
S
H
23
1989


331
I
L
E
G
V
P
S
H
L
23
1990


409
Q
L
Q
S
Y
R
Q
E
V
23
1991


 13
M
L
R
K
D
D
A
P
L
22
1992


 68
T
L
Q
A
D
D
D
S
L
22
1993


111
K
A
R
L
T
I
A
A
V
22
1994


133
Y
I
A
N
S
L
A
I
M
22
1995


183
S
A
M
I
S
V
L
L
V
22
1996


306
P
I
C
T
Y
V
F
S
L
22
1997


322
R
I
I
W
D
T
V
V
I
22
1998


402
G
M
Y
R
C
T
I
Q
L
22
1999


 76
L
L
D
Q
D
L
P
L
T
21
2000


147
M
L
T
D
L
S
A
I
I
21
2001


193
I
L
M
G
F
L
L
Y
E
21
2002


194
L
M
G
F
L
L
Y
E
A
21
2003


220
I
T
A
A
V
G
V
A
V
21
2004


349
M
K
I
E
D
V
Y
S
V
21
2005


118
A
V
L
Y
L
L
F
M
I
20
2006


124
F
M
I
G
E
L
V
G
G
20
2007


158
L
L
A
L
W
L
S
S
K
20
2008


178
R
L
E
V
L
S
A
M
I
20
2009


212
E
I
N
G
D
I
M
L
I
20
2010


222
A
A
V
G
V
A
V
N
V
20
2011


271
V
R
A
A
F
V
H
A
L
20
2012


 80
D
L
P
L
T
N
S
Q
L
19
2013


 82
P
L
T
N
S
Q
L
S
L
19
2014


104
I
L
K
Q
R
K
V
K
A
19
2015


143
D
A
L
H
M
L
T
D
L
19
2016


154
I
I
L
T
L
L
A
L
W
19
2017


188
V
L
L
V
Y
I
L
M
G
19
2018


223
A
V
G
V
A
V
N
V
I
19
2019


302
K
I
A
D
P
I
C
T
Y
19
2020


327
T
V
V
I
I
L
E
G
V
19
2021


364
S
L
T
S
G
K
S
T
A
19
2022


395
H
L
L
L
N
T
F
G
M
19
2023


 41
S
R
F
N
K
L
R
V
V
18
2024


151
L
S
A
I
I
L
T
L
L
18
2025


186
I
S
V
L
L
V
Y
I
L
18
2026


190
L
V
Y
I
L
M
G
F
L
18
2027


230
V
I
M
G
F
L
L
N
Q
18
2028


303
I
A
D
P
I
C
T
Y
V
18
2029


346
E
A
L
M
K
I
E
D
V
18
2030


365
L
T
S
G
K
S
T
A
I
18
2031


370
S
T
A
I
V
H
I
Q
L
18
2032


371
T
A
I
V
H
I
Q
L
I
18
2033


377
Q
L
I
P
G
S
S
S
K
18
2034


 45
K
L
R
V
V
V
A
D
D
17
2035


 61
P
V
N
G
A
H
P
T
L
17
2036


 87
Q
L
S
L
K
V
D
S
C
17
2037


184
A
M
I
S
V
L
L
V
Y
17
2038


205
R
T
I
H
M
N
Y
E
I
17
2039


219
L
I
T
A
A
V
G
V
A
17
2040


227
A
V
N
V
I
M
G
F
L
17
2041


289
L
I
A
A
Y
I
I
R
F
17
2042


308
C
T
Y
V
F
S
L
L
V
17
2043


315
L
V
A
F
T
T
F
R
I
17
2044


357
V
E
D
L
N
I
W
S
L
17
2045


112
A
R
L
T
I
A
A
V
L
16
2046


117
A
A
V
L
Y
L
L
F
M
16
2047


125
M
I
G
E
L
V
G
G
Y
16
2048


139
A
I
M
T
D
A
L
H
M
16
2049


148
L
T
D
L
S
A
I
I
L
16
2050


152
S
A
I
I
L
T
L
L
A
16
2051


157
T
L
L
A
L
W
L
S
S
16
2052


174
F
G
F
H
R
L
E
V
L
16
2053


182
L
S
A
M
I
S
V
L
L
16
2054


189
L
L
V
Y
I
L
M
G
F
16
2055


211
Y
E
I
N
G
D
I
M
L
16
2056


274
A
F
V
H
A
L
G
D
L
16
2057


275
F
V
H
A
L
G
D
L
V
16
2058


280
G
D
L
V
Q
S
V
G
V
16
2059


282
L
V
Q
S
V
G
V
L
I
16
2060


287
G
V
L
I
A
A
Y
I
I
16
2061


293
Y
I
I
R
F
K
P
E
Y
16
2062


313
S
L
L
V
A
F
T
T
F
16
2063


324
I
W
D
T
V
V
I
I
L
16
2064


392
K
A
N
H
L
L
L
N
T
16
2065


 5
G
A
W
K
R
L
K
S
M
15
2066


 9
R
L
K
S
M
L
R
K
D
15
2067


 22
F
L
N
D
T
S
A
F
D
15
2068


 27
S
A
F
D
F
S
D
E
A
15
2069


 69
L
Q
A
D
D
D
S
L
L
15
2070


102
R
E
I
L
K
Q
R
K
V
15
2071


129
L
V
G
G
Y
I
A
N
S
15
2072


144
A
L
H
M
L
T
D
L
S
15
2073


180
E
V
L
S
A
M
I
S
V
15
2074


192
Y
I
L
M
G
F
L
L
Y
15
2075


231
I
M
G
F
L
L
N
Q
S
15
2076


248
S
L
P
S
N
S
P
T
R
15
2077


295
I
R
F
K
P
E
Y
K
I
15
2078


310
Y
V
F
S
L
L
V
A
F
15
2079


314
L
L
V
A
F
T
T
F
R
15
2080


368
G
K
S
T
A
I
V
H
I
15
2081










HLA-A*0203 nonamers



















 49
V
V
A
D
D
G
S
E
A
12
850


 63
N
G
A
H
P
T
L
Q
A
12
851


110
V
K
A
R
L
T
I
A
A
12
852


 27
S
A
F
D
F
S
D
E
A
11
853


152
S
A
I
I
L
T
L
L
A
11
854


 57
A
P
E
R
P
V
N
G
A
10
855


270
A
V
R
A
A
F
V
H
A
10
856


 11
K
S
M
L
R
K
D
D
A
9
857


 20
P
L
F
L
N
D
T
S
A
9
858


 43
F
N
K
L
R
V
V
V
A
9
859


104
I
L
K
Q
R
K
V
K
A
9
860


109
K
V
K
A
R
L
T
I
A
9
861


127
G
E
L
V
G
G
Y
I
A
9
862


131
G
G
Y
I
A
N
S
L
A
9
863


136
N
S
L
A
I
M
T
D
A
9
864


145
L
H
M
L
T
D
L
S
A
9
865


176
F
H
R
L
E
V
L
S
A
9
866


194
L
M
G
F
L
L
Y
E
A
9
867


214
N
G
D
I
M
L
I
T
A
9
868


215
G
D
I
M
L
I
T
A
A
9
869


219
L
I
T
A
A
V
G
V
A
9
870


262
R
N
H
G
Q
D
S
L
A
9
871


265
G
Q
D
S
L
A
V
R
A
9
872


266
Q
D
S
L
A
V
R
A
A
9
873


283
V
Q
S
V
G
V
L
I
A
9
874


284
Q
S
V
G
V
L
I
A
A
9
875


296
R
F
K
P
E
Y
K
I
A
9
876


309
T
Y
V
F
S
L
L
V
A
9
877


339
L
N
V
D
Y
I
K
E
A
9
878


364
S
L
T
S
G
K
S
T
A
9
879


385
K
W
E
E
V
Q
S
K
A
9
880


414
R
Q
E
V
D
R
T
C
A
9
881










HLA-A1 nonamers



















192
Y
I
L
M
G
F
L
L
Y
30
882


184
A
M
I
S
V
L
L
V
Y
25
883


148
L
T
D
L
S
A
I
I
L
20
884


396
L
L
L
N
T
F
G
M
Y
20
885


 70
Q
A
D
D
D
S
L
L
D
18
886


113
R
L
T
I
A
A
V
L
Y
18
887


141
M
T
D
A
L
H
M
L
T
18
888


347
A
L
M
K
I
E
D
V
Y
18
889


 71
A
D
D
D
S
L
L
D
Q
17
890


285
S
V
G
V
L
I
A
A
Y
17
891


293
Y
I
I
R
F
K
P
E
Y
17
892


324
I
W
D
T
V
V
I
I
L
17
893


335
V
P
S
H
L
N
V
D
Y
17
894


356
S
V
E
D
L
N
I
W
S
17
895


405
R
C
T
I
Q
L
Q
S
Y
17
896


 15
R
K
D
D
A
P
L
F
L
16
897


 31
F
S
D
E
A
G
D
E
G
16
898


125
M
I
G
E
L
V
G
G
Y
16
899


203
V
Q
R
T
I
H
M
N
Y
16
900


302
K
I
A
D
P
I
C
T
Y
16
901


 54
G
S
E
A
P
E
R
P
V
15
902










HLA-A26 nonamers



















310
Y
V
F
S
L
L
V
A
F
30
903


125
M
I
G
E
L
V
G
G
Y
28
904


281
D
L
V
Q
S
V
G
V
L
28
905


285
S
V
G
V
L
I
A
A
Y
27
906


388
E
V
Q
S
K
A
N
H
L
27
907


150
D
L
S
A
I
I
L
T
L
26
908


289
L
I
A
A
Y
I
I
R
F
26
909


302
K
I
A
D
P
I
C
T
Y
26
910


 80
D
L
P
L
T
N
S
Q
L
25
911


133
Y
I
A
N
S
L
A
I
M
25
912


153
A
I
I
L
T
L
L
A
L
25
913


114
L
T
I
A
A
V
L
Y
L
24
914


189
L
L
V
Y
I
L
M
G
F
24
915


192
Y
I
L
M
G
F
L
L
Y
24
916


293
Y
I
I
R
F
K
P
E
Y
24
917


115
T
I
A
A
V
L
Y
L
L
23
918


190
L
V
Y
I
L
M
G
F
L
23
919


212
E
I
N
G
D
I
M
L
I
23
920


306
P
I
C
T
Y
V
F
S
L
23
921


396
L
L
L
N
T
F
G
M
Y
23
922


168
P
T
K
R
F
T
F
G
F
22
923


180
E
V
L
S
A
M
I
S
V
22
924


187
S
V
L
L
V
Y
I
L
M
22
925


227
A
V
N
V
I
M
G
F
L
22
926


313
S
L
L
V
A
F
T
T
F
22
927


 61
P
V
N
G
A
H
P
T
L
21
928


121
Y
L
L
F
M
I
G
E
L
21
929


128
E
L
V
G
G
Y
I
A
N
21
930


181
V
L
S
A
M
I
S
V
L
21
931


326
D
T
V
V
I
I
L
E
G
21
932


 38
E
G
L
S
R
F
N
K
L
20
933


148
L
T
D
L
S
A
I
I
L
20
934


331
I
L
E
G
V
P
S
H
L
20
935


340
N
V
D
Y
I
K
E
A
L
20
936


370
S
T
A
I
V
H
I
Q
L
20
937


 35
A
G
D
E
G
L
S
R
F
19
938


113
R
L
T
I
A
A
V
L
Y
19
939


139
A
I
M
T
D
A
L
H
M
19
940


171
R
F
T
F
G
F
H
R
L
19
941


343
Y
I
K
E
A
L
M
K
I
19
942


347
A
L
M
K
I
E
D
V
Y
19
943


352
E
D
V
Y
S
V
E
D
L
19
944


416
E
V
D
R
T
C
A
N
C
19
945


 13
M
L
R
K
D
D
A
P
L
18
946


 21
L
F
L
N
D
T
S
A
F
18
947


 75
S
L
L
D
Q
D
L
P
L
18
948


 82
P
L
T
N
S
Q
L
S
L
18
949


103
E
I
L
K
Q
R
K
V
K
18
950


129
L
V
G
G
Y
I
A
N
S
18
951


137
S
L
A
I
M
T
D
A
L
18
952


143
D
A
L
H
M
L
T
D
L
18
953


216
D
I
M
L
I
T
A
A
V
18
954


267
D
S
L
A
V
R
A
A
F
18
955


274
A
F
V
H
A
L
G
D
L
18
956


330
I
I
L
E
G
V
P
S
H
18
957


399
N
T
F
G
M
Y
R
C
T
18
958


 25
D
T
S
A
F
D
F
S
D
17
959


 68
T
L
Q
A
D
D
D
S
L
17
960


 83
L
T
N
S
Q
L
S
L
K
17
961


155
I
L
T
L
L
A
L
W
L
17
962


184
A
M
I
S
V
L
L
V
Y
17
963


230
V
I
M
G
F
L
L
N
Q
17
964


353
D
V
Y
S
V
E
D
L
N
17
965


395
H
L
L
L
N
T
F
G
M
17
966


405
R
C
T
I
Q
L
Q
S
Y
17
967


 73
D
D
S
L
L
D
Q
D
L
16
968


116
I
A
A
V
L
Y
L
L
F
16
969


118
A
V
L
Y
L
L
F
M
I
16
970


154
I
I
L
T
L
L
A
L
W
16
971


177
H
R
L
E
V
L
S
A
M
16
972


185
M
I
S
V
L
L
V
Y
I
16
973


198
L
L
Y
E
A
V
Q
R
T
16
974


201
E
A
V
Q
R
T
I
H
M
16
975


225
G
V
A
V
N
V
I
M
G
16
976


226
V
A
V
N
V
I
M
G
F
16
977


229
N
V
I
M
G
F
L
L
N
16
978


270
A
V
R
A
A
F
V
H
A
16
979


278
A
L
G
D
L
V
Q
S
V
16
980


319
T
T
F
R
I
I
W
D
T
16
981


323
I
I
W
D
T
V
V
I
I
16
982


334
G
V
P
S
H
L
N
V
D
16
983


350
K
I
E
D
V
Y
S
V
E
16
984


378
L
I
P
G
S
S
S
K
W
16
985


 9
R
L
K
S
M
L
R
K
D
15
986


 17
D
D
A
P
L
F
L
N
D
15
987


 76
L
L
D
Q
D
L
P
L
T
15
988


109
K
V
K
A
R
L
T
I
A
15
989


158
L
L
A
L
W
L
S
S
K
15
990


202
A
V
Q
R
T
I
H
M
N
15
991


261
E
R
N
H
G
Q
D
S
L
15
992


327
T
V
V
I
I
L
E
G
V
15
993


373
I
V
H
I
Q
L
I
P
G
15
994


393
A
N
H
L
L
L
N
T
F
15
995


406
C
T
I
Q
L
Q
S
Y
R
15
996










HLA-A3 nonamers



















377
Q
L
I
P
G
S
S
S
K
35
997


113
R
L
T
I
A
A
V
L
Y
28
998


158
L
L
A
L
W
L
S
S
K
27
999


197
F
L
L
Y
E
A
V
Q
R
26
1000


103
E
I
L
K
Q
R
K
V
K
25
1001


162
W
L
S
S
K
S
P
T
K
25
1002


294
I
I
R
F
K
P
E
Y
K
25
1003


302
K
I
A
D
P
I
C
T
Y
23
1004


322
R
I
I
W
D
T
V
V
I
23
1005


347
A
L
M
K
I
E
D
V
Y
23
1006


270
A
V
R
A
A
F
V
H
A
22
1007


313
S
L
L
V
A
F
T
T
F
22
1008


192
Y
I
L
M
G
F
L
L
Y
21
1009


285
S
V
G
V
L
I
A
A
Y
21
1010


330
I
I
L
E
G
V
P
S
H
21
1011


361
N
I
W
S
L
T
S
G
K
21
1012


396
L
L
L
N
T
F
G
M
Y
21
1013


 45
K
L
R
V
V
V
A
D
D
20
1014


155
I
L
T
L
L
A
L
W
L
20
1015


184
A
M
I
S
V
L
L
V
Y
20
1016


218
M
L
I
T
A
A
V
G
V
20
1017


268
S
L
A
V
R
A
A
F
V
20
1018


293
Y
I
I
R
F
K
P
E
Y
20
1019


342
D
Y
I
K
E
A
L
M
K
20
1020


350
K
I
E
D
V
Y
S
V
E
20
1021


364
S
L
T
S
G
K
S
T
A
20
1022


104
I
L
K
Q
R
K
V
K
A
19
1023


109
K
V
K
A
R
L
T
I
A
19
1024


150
D
L
S
A
I
I
L
T
L
19
1025


153
A
I
I
L
T
L
L
A
L
19
1026


157
T
L
L
A
L
W
L
S
S
19
1027


181
V
L
S
A
M
I
S
V
L
19
1028


248
S
L
P
S
N
S
P
T
R
19
1029


288
V
L
I
A
A
Y
I
I
R
19
1030


310
Y
V
F
S
L
L
V
A
F
19
1031


328
V
V
I
I
L
E
G
V
P
19
1032


329
V
I
I
L
E
G
V
P
S
19
1033


331
I
L
E
G
V
P
S
H
L
19
1034


 8
K
R
L
K
S
M
L
R
K
18
1035


 49
V
V
A
D
D
G
S
E
A
18
1036


178
R
L
E
V
L
S
A
M
I
18
1037


229
N
V
I
M
G
F
L
L
N
18
1038


234
F
L
L
N
Q
S
G
H
R
18
1039


282
L
V
Q
S
V
G
V
L
I
18
1040


359
D
L
N
I
W
S
L
T
S
18
1041


397
L
L
N
T
F
G
M
Y
R
18
1042


 39
G
L
S
R
F
N
K
L
R
17
1043


 75
S
L
L
D
Q
D
L
P
L
17
1044


 80
D
L
P
L
T
N
S
Q
L
17
1045


139
A
I
M
T
D
A
L
H
M
17
1046


193
I
L
M
G
F
L
L
Y
E
17
1047


202
A
V
Q
R
T
I
H
M
N
17
1048


223
A
V
G
V
A
V
N
V
I
17
1049


235
L
L
N
Q
S
G
H
R
H
17
1050


314
L
L
V
A
F
T
T
F
R
17
1051


411
Q
S
Y
R
Q
E
V
D
R
17
1052










HLA-B*0702 nonamers



















57
A
P
E
R
P
V
N
G
A
22
1053


 60
R
P
V
N
G
A
H
P
T
18
1054


 2
A
G
S
G
A
W
K
R
L
16
1055


 13
M
L
R
K
D
D
A
P
L
15
1056


 15
R
K
D
D
A
P
L
F
L
15
1057


106
K
Q
R
K
V
K
A
R
L
15
1058


150
D
L
S
A
I
I
L
T
L
15
1059


153
A
I
I
L
T
L
L
A
L
15
1060


 97
N
C
S
K
Q
R
E
I
L
14
1061


112
A
R
L
T
I
A
A
V
L
14
1062


114
L
T
I
A
A
V
L
Y
L
14
1063


167
S
P
T
K
R
F
T
F
G
14
1064


181
V
L
S
A
M
I
S
V
L
14
1065


270
A
V
R
A
A
F
V
H
A
14
1066


271
V
R
A
A
F
V
H
A
L
14
1067


335
V
P
S
H
L
N
V
D
Y
14
1068


 61
P
V
N
G
A
H
P
T
L
13
1069


 63
N
G
A
H
P
T
L
Q
A
13
1070


 75
S
L
L
D
Q
D
L
P
L
13
1071


137
S
L
A
I
M
T
D
A
L
13
1072


155
I
L
T
L
L
A
L
W
L
13
1073


182
L
S
A
M
I
S
V
L
L
13
1074


220
I
T
A
A
V
G
V
A
V
13
1075


227
A
V
N
V
I
M
G
F
L
13
1076


249
L
P
S
N
S
P
T
R
G
13
1077


305
D
P
I
C
T
Y
V
F
S
13
1078


324
I
W
D
T
V
V
I
I
L
13
1079


331
I
L
E
G
V
P
S
H
L
13
1080


389
V
Q
S
K
A
N
H
L
L
13
1081


390
Q
S
K
A
N
H
L
L
L
13
1082


 6
A
W
K
R
L
K
S
M
L
12
1083


 19
A
P
L
F
L
N
D
T
S
12
1084


 38
E
G
L
S
R
F
N
K
L
12
1085


 42
R
F
N
K
L
R
V
V
V
12
1086


 73
D
D
S
L
L
D
Q
D
L
12
1087


 82
P
L
T
N
S
Q
L
S
L
12
1088


115
T
I
A
A
V
L
Y
L
L
12
1089


151
L
S
A
I
I
L
T
L
L
12
1090


171
R
F
T
F
G
F
H
R
L
12
1091


186
I
S
V
L
L
V
Y
I
L
12
1092


222
A
A
V
G
V
A
V
N
V
12
1093


274
A
F
V
H
A
L
G
D
L
12
1094


281
D
L
V
Q
S
V
G
V
L
12
1095


298
K
P
E
Y
K
I
A
D
P
12
1096


307
I
C
T
Y
V
F
S
L
L
12
1097


340
N
V
D
Y
I
K
E
A
L
12
1098


352
E
D
V
Y
S
V
E
D
L
12
1099


365
L
T
S
G
K
S
T
A
I
12
1100


379
I
P
G
S
S
S
K
W
E
12
1101


 32
S
D
E
A
G
D
E
G
L
11
1102


 54
G
S
E
A
P
E
R
P
V
11
1103


 68
T
L
Q
A
D
D
D
S
L
11
1104


 69
L
Q
A
D
D
D
S
L
L
11
1105


108
R
K
V
K
A
R
L
T
I
11
1106


111
K
A
R
L
T
I
A
A
V
11
1107


117
A
A
V
L
Y
L
L
F
M
11
1108


130
V
G
G
Y
I
A
N
S
L
11
1109


139
A
I
M
T
D
A
L
H
M
11
1110


140
I
M
T
D
A
L
H
M
L
11
1111


143
D
A
L
H
M
L
T
D
L
11
1112


148
L
T
D
L
S
A
I
I
L
11
1113


174
F
G
F
H
R
L
E
V
L
11
1114


176
F
H
R
L
E
V
L
S
A
11
1115


190
L
V
Y
I
L
M
G
F
L
11
1116


191
V
Y
I
L
M
G
F
L
L
11
1117


223
A
V
G
V
A
V
N
V
I
11
1118


228
V
N
V
I
M
G
F
L
L
11
1119


261
E
R
N
H
G
Q
D
S
L
11
1120


283
V
Q
S
V
G
V
L
I
A
11
1121


295
I
R
F
K
P
E
Y
K
I
11
1122


306
P
I
C
T
Y
V
F
S
L
11
1123


311
V
F
S
L
L
V
A
F
T
11
1124


322
R
I
I
W
D
T
V
V
I
11
1125


357
V
E
D
L
N
I
W
S
L
11
1126


370
S
T
A
I
V
H
I
Q
L
11
1127


388
E
V
Q
S
K
A
N
H
L
11
1128


402
G
M
Y
R
C
T
I
Q
L
11
1129










HLA-B*08 nonamers



















104
I
L
K
Q
R
K
V
K
A
26
1130


 13
M
L
R
K
D
D
A
P
L
22
1131


 43
F
N
K
L
R
V
V
V
A
22
1132


343
Y
I
K
E
A
L
M
K
I
22
1133


388
E
V
Q
S
K
A
N
H
L
22
1134


107
Q
R
K
V
K
A
R
L
T
21
1135


294
I
I
R
F
K
P
E
Y
K
21
1136


 6
A
W
K
R
L
K
S
M
L
20
1137


 38
E
G
L
S
R
F
N
K
L
20
1138


390
Q
S
K
A
N
H
L
L
L
20
1139


 89
S
L
K
V
D
S
C
D
N
19
1140


137
S
L
A
I
M
T
D
A
L
19
1141


164
S
S
K
S
P
T
K
R
F
19
1142


174
F
G
F
H
R
L
E
V
L
19
1143


 75
S
L
L
D
Q
D
L
P
L
18
1144


 80
D
L
P
L
T
N
S
Q
L
18
1145


 87
Q
L
S
L
K
V
D
S
C
18
1146


 98
C
S
K
Q
R
E
I
L
K
18
1147


109
K
V
K
A
R
L
T
I
A
18
1148


121
Y
L
L
F
M
I
G
E
L
18
1149


155
I
L
T
L
L
A
L
W
L
18
1150


181
V
L
S
A
M
I
S
V
L
18
1151


298
K
P
E
Y
K
I
A
D
P
18
1152


 7
W
K
R
L
K
S
M
L
R
17
1153


 56
E
A
P
E
R
P
V
N
G
17
1154


 96
D
N
C
S
K
Q
R
E
I
17
1155


150
D
L
S
A
I
I
L
T
L
17
1156


268
S
L
A
V
R
A
A
F
V
17
1157


281
D
L
V
Q
S
V
G
V
L
17
1158


331
I
L
E
G
V
P
S
H
L
17
1159


346
E
A
L
M
K
I
E
D
V
17
1160


365
L
T
S
G
K
S
T
A
I
17
1161


 9
R
L
K
S
M
L
R
K
D
16
1162


 14
L
R
K
D
D
A
P
L
F
16
1163


 68
T
L
Q
A
D
D
D
S
L
16
1164


 82
P
L
T
N
S
Q
L
S
L
16
1165


106
K
Q
R
K
V
K
A
R
L
16
1166


153
A
I
I
L
T
L
L
A
L
16
1167


162
W
L
S
S
K
S
P
T
K
16
1168


166
K
S
P
T
K
R
F
T
F
16
1169


167
S
P
T
K
R
F
T
F
G
16
1170


168
P
T
K
R
F
T
F
G
F
16
1171


306
P
I
C
T
Y
V
F
S
L
15
1172


313
S
L
L
V
A
F
T
T
F
15
1173


 5
G
A
W
K
R
L
K
S
M
14
1174


 45
K
L
R
V
V
V
A
D
D
14
1175


115
T
I
A
A
V
L
Y
L
L
14
1176


143
D
A
L
H
M
L
T
D
L
14
1177


201
E
A
V
Q
R
T
I
H
M
14
1178


352
E
D
V
Y
S
V
E
D
L
14
1179


 12
S
M
L
R
K
D
D
A
P
13
1180


105
L
K
Q
R
K
V
K
A
R
13
1181


111
K
A
R
L
T
I
A
A
V
13
1182


178
R
L
E
V
L
S
A
M
I
13
1183


186
I
S
V
L
L
V
Y
I
L
13
1184


189
L
L
V
Y
I
L
M
G
F
13
1185


212
E
I
N
G
D
I
M
L
I
13
1186


258
S
G
C
E
R
N
H
G
Q
13
1187


271
V
R
A
A
F
V
H
A
L
13
1188


323
I
I
W
D
T
V
V
I
I
13
1189


357
V
E
D
L
N
I
W
S
L
13
1190


383
S
S
K
W
E
E
V
Q
S
13
1191










HLA-B*1510 nonamers



















331
I
L
E
G
V
P
S
H
L
15
1192


 2
A
G
S
G
A
W
K
R
L
14
1193


106
K
Q
R
K
V
K
A
R
L
14
1194


181
V
L
S
A
M
I
S
V
L
14
1195


276
V
H
A
L
G
D
L
V
Q
14
1196


281
D
L
V
Q
S
V
G
V
L
14
1197


 61
P
V
N
G
A
H
P
T
L
13
1198


 97
N
C
S
K
Q
R
E
I
L
13
1199


121
Y
L
L
F
M
I
G
E
L
13
1200


140
I
M
T
D
A
L
H
M
L
13
1201


150
D
L
S
A
I
I
L
T
L
13
1202


171
R
F
T
F
G
F
H
R
L
13
1203


182
L
S
A
M
I
S
V
L
L
13
1204


186
I
S
V
L
L
V
Y
I
L
13
1205


271
V
R
A
A
F
V
H
A
L
13
1206


324
I
W
D
T
V
V
I
I
L
13
1207


374
V
H
I
Q
L
I
P
G
S
13
1208


 15
R
K
D
D
A
P
L
F
L
12
1209


 32
S
D
E
A
G
D
E
G
L
12
1210


 69
L
Q
A
D
D
D
S
L
L
12
1211


112
A
R
L
T
I
A
A
V
L
12
1212


115
T
I
A
A
V
L
Y
L
L
12
1213


137
S
L
A
I
M
T
D
A
L
12
1214


155
I
L
T
L
L
A
L
W
L
12
1215


174
F
G
F
H
R
L
E
V
L
12
1216


207
I
H
M
N
Y
E
I
N
G
12
1217


211
Y
E
I
N
G
D
I
M
L
12
1218


227
A
V
N
V
I
M
G
F
L
12
1219


244
S
H
S
H
S
L
P
S
N
12
1220


246
S
H
S
L
P
S
N
S
P
12
1221


261
E
R
N
H
G
Q
D
S
L
12
1222


263
N
H
G
Q
D
S
L
A
V
12
1223


307
I
C
T
Y
V
F
S
L
L
12
1224


340
N
V
D
Y
I
K
E
A
L
12
1225


352
E
D
V
Y
S
V
E
D
L
12
1226


357
V
E
D
L
N
I
W
S
L
12
1227


389
V
Q
S
K
A
N
H
L
L
12
1228


 6
A
W
K
R
L
K
S
M
L
11
1229


 13
M
L
R
K
D
D
A
P
L
11
1230


 38
E
G
L
S
R
F
N
K
L
11
1231


 68
T
L
Q
A
D
D
D
S
L
11
1232


 73
D
D
S
L
L
D
Q
D
L
11
1233


 75
S
L
L
D
Q
D
L
P
L
11
1234


114
L
T
I
A
A
V
L
Y
L
11
1235


151
L
S
A
I
I
L
T
L
L
11
1236


153
A
I
I
L
T
L
L
A
L
11
1237


176
F
H
R
L
E
V
L
S
A
11
1238


190
L
V
Y
I
L
M
G
F
L
11
1239


240
G
H
R
H
S
H
S
H
S
11
1240


242
R
H
S
H
S
H
S
L
P
11
1241


306
P
I
C
T
Y
V
F
S
L
11
1242


370
S
T
A
I
V
H
I
Q
L
11
1243


388
E
V
Q
S
K
A
N
H
L
11
1244


390
Q
S
K
A
N
H
L
L
L
11
1245


402
G
M
Y
R
C
T
I
Q
L
11
1246


 35
A
G
D
E
G
L
S
R
F
10
1247


 65
A
H
P
T
L
Q
A
D
D
10
1248


 80
D
L
P
L
T
N
S
Q
L
10
1249


 82
P
L
T
N
S
Q
L
S
L
10
1250


116
I
A
A
V
L
Y
L
L
F
10
1251


130
V
G
G
Y
I
A
N
S
L
10
1252


143
D
A
L
H
M
L
T
D
L
10
1253


145
L
H
M
L
T
D
L
S
A
10
1254


148
L
T
D
L
S
A
I
I
L
10
1255


191
V
Y
I
L
M
G
F
L
L
10
1256


228
V
N
V
I
M
G
F
L
L
10
1257


241
H
R
H
S
H
S
H
S
L
10
1258


274
A
F
V
H
A
L
G
D
L
10
1259


289
L
I
A
A
Y
I
I
R
F
10
1260


310
Y
V
F
S
L
L
V
A
F
10
1261


337
S
H
L
N
V
D
Y
I
K
10
1262


394
N
H
L
L
L
N
T
F
G
10
1263


164
S
S
K
S
P
T
K
R
F
9
1264


 5
G
A
W
K
R
L
K
S
M
8
1265


 14
L
R
K
D
D
A
P
L
F
8
1266


133
Y
I
A
N
S
L
A
I
M
8
1267


166
K
S
P
T
K
R
F
T
F
8
1268


177
H
R
L
E
V
L
S
A
M
8
1269


201
E
A
V
Q
R
T
I
H
M
8
1270


210
N
Y
E
I
N
G
D
I
M
8
1271


224
V
G
V
A
V
N
V
I
M
8
1272


267
D
S
L
A
V
R
A
A
F
8
1273


304
A
D
P
I
C
T
Y
V
F
8
1274


313
S
L
L
V
A
F
T
T
F
8
1275


 21
L
F
L
N
D
T
S
A
F
7
1276


 55
S
E
A
P
E
R
P
V
N
7
1277


221
T
A
A
V
G
V
A
V
N
7
1278


226
V
A
V
N
V
I
M
G
F
7
1279


393
A
N
H
L
L
L
N
T
F
7
1280


395
H
L
L
L
N
T
F
G
M
7
1281










HLA-B*2705 nonamers



















 8
K
R
L
K
S
M
L
R
K
30
1282


112
A
R
L
T
I
A
A
V
L
27
1283


295
I
R
F
K
P
E
Y
K
I
26
1284


170
K
R
F
T
F
G
F
H
R
25
1285


261
E
R
N
H
G
Q
D
S
L
25
1286


101
Q
R
E
I
L
K
Q
R
K
24
1287


177
H
R
L
E
V
L
S
A
M
24
1288


 14
L
R
K
D
D
A
P
L
F
22
1289


241
H
R
H
S
H
S
H
S
L
22
1290


271
V
R
A
A
F
V
H
A
L
21
1291


106
K
Q
R
K
V
K
A
R
L
19
1292


 35
A
G
D
E
G
L
S
R
F
18
1293


171
R
F
T
F
G
F
H
R
L
18
1294


377
Q
L
I
P
G
S
S
S
K
18
1295


406
C
T
I
Q
L
Q
S
Y
R
18
1296


163
L
S
S
K
S
P
T
K
R
17
1297


233
G
F
L
L
N
Q
S
G
H
17
1298


330
I
I
L
E
G
V
P
S
H
17
1299


342
D
Y
I
K
E
A
L
M
K
17
1300


402
G
M
Y
R
C
T
I
Q
L
17
1301


 1
M
A
G
S
G
A
W
K
R
16
1302


 41
S
R
F
N
K
L
R
V
V
16
1303


100
K
Q
R
E
I
L
K
Q
R
16
1304


121
Y
L
L
F
M
I
G
E
L
16
1305


150
D
L
S
A
I
I
L
T
L
16
1306


153
A
I
I
L
T
L
L
A
L
16
1307


186
I
S
V
L
L
V
Y
I
L
16
1308


197
F
L
L
Y
E
A
V
Q
R
16
1309


211
Y
E
I
N
G
D
I
M
L
16
1310


226
V
A
V
N
V
I
M
G
F
16
1311


256
R
G
S
G
C
E
R
N
H
16
1312


281
D
L
V
Q
S
V
G
V
L
16
1313


289
L
I
A
A
Y
I
I
R
F
16
1314


310
Y
V
F
S
L
L
V
A
F
16
1315


321
F
R
I
I
W
D
T
V
V
16
1316


357
V
E
D
L
N
I
W
S
L
16
1317


387
E
E
V
Q
S
K
A
N
H
16
1318


393
A
N
H
L
L
L
N
T
F
16
1319


 2
A
G
S
G
A
W
K
R
L
15
1320


 5
G
A
W
K
R
L
K
S
M
15
1321


 15
R
K
D
D
A
P
L
F
L
15
1322


 38
E
G
L
S
R
F
N
K
L
15
1323


 82
P
L
T
N
S
Q
L
S
L
15
1324


103
E
I
L
K
Q
R
K
V
K
15
1325


108
R
K
V
K
A
R
L
T
I
15
1326


143
D
A
L
H
M
L
T
D
L
15
1327


155
I
L
T
L
L
A
L
W
L
15
1328


174
F
G
F
H
R
L
E
V
L
15
1329


181
V
L
S
A
M
I
S
V
L
15
1330


184
A
M
I
S
V
L
L
V
Y
15
1331


205
R
T
I
H
M
N
Y
E
I
15
1332


290
I
A
A
Y
I
I
R
F
K
15
1333


302
K
I
A
D
P
I
C
T
Y
15
1334


313
S
L
L
V
A
F
T
T
F
15
1335


331
I
L
E
G
V
P
S
H
L
15
1336


337
S
H
L
N
V
D
Y
I
K
15
1337


404
Y
R
C
T
I
Q
L
Q
S
15
1338










HLA-B*2709 nonamers



















112
A
R
L
T
I
A
A
V
L
25
1339


295
I
R
F
K
P
E
Y
K
I
22
1340


 14
L
R
K
D
D
A
P
L
F
21
1341


241
H
R
H
S
H
S
H
S
L
21
1342


271
V
R
A
A
F
V
H
A
L
21
1343


 41
S
R
F
N
K
L
R
V
V
20
1344


177
H
R
L
E
V
L
S
A
M
20
1345


261
E
R
N
H
G
Q
D
S
L
20
1346


321
F
R
I
I
W
D
T
V
V
19
1347


 8
K
R
L
K
S
M
L
R
K
16
1348


171
R
F
T
F
G
F
H
R
L
16
1349


402
G
M
Y
R
C
T
I
Q
L
16
1350


 15
R
K
D
D
A
P
L
F
L
15
1351


287
G
V
L
I
A
A
Y
I
I
15
1352


322
R
I
I
W
D
T
V
V
I
15
1353


108
R
K
V
K
A
R
L
T
I
14
1354


155
I
L
T
L
L
A
L
W
L
14
1355


170
K
R
F
T
F
G
F
H
R
14
1356


186
I
S
V
L
L
V
Y
I
L
14
1357


205
R
T
I
H
M
N
Y
E
I
14
1358


280
G
D
L
V
Q
S
V
G
V
14
1359


 38
E
G
L
S
R
F
N
K
L
13
1360


 42
R
F
N
K
L
R
V
V
V
13
1361


 75
S
L
L
D
Q
D
L
P
L
13
1362


102
R
E
I
L
K
Q
R
K
V
13
1363


106
K
Q
R
K
V
K
A
R
L
13
1364


114
L
T
I
A
A
V
L
Y
L
13
1365


132
G
Y
I
A
N
S
L
A
I
13
1366


153
A
I
I
L
T
L
L
A
L
13
1367


222
A
A
V
G
V
A
V
N
V
13
1368


404
Y
R
C
T
I
Q
L
Q
S
13
1369


 2
A
G
S
G
A
W
K
R
L
12
1370


 46
L
R
V
V
V
A
D
D
G
12
1371


 82
P
L
T
N
S
Q
L
S
L
12
1372


121
Y
L
L
F
M
I
G
E
L
12
1373


130
V
G
G
Y
I
A
N
S
L
12
1374


139
A
I
M
T
D
A
L
H
M
12
1375


140
I
M
T
D
A
L
H
M
L
12
1376


143
D
A
L
H
M
L
T
D
L
12
1377


150
D
L
S
A
I
I
L
T
L
12
1378


174
F
G
F
H
R
L
E
V
L
12
1379


178
R
L
E
V
L
S
A
M
I
12
1380


182
L
S
A
M
I
S
V
L
L
12
1381


190
L
V
Y
I
L
M
G
F
L
12
1382


227
A
V
N
V
I
M
G
F
L
12
1383


255
T
R
G
S
G
C
E
R
N
12
1384


274
A
F
V
H
A
L
G
D
L
12
1385


281
D
L
V
Q
S
V
G
V
L
12
1386


299
P
E
Y
K
I
A
D
P
I
12
1387


307
I
C
T
Y
V
F
S
L
L
12
1388


310
Y
V
F
S
L
L
V
A
F
12
1389


324
I
W
D
T
V
V
I
I
L
12
1390


333
E
G
V
P
S
H
L
N
V
12
1391


349
M
K
I
E
D
V
Y
S
V
12
1392


352
E
D
V
Y
S
V
E
D
L
12
1393


368
G
K
S
T
A
I
V
H
I
12
1394


381
G
S
S
S
K
W
E
E
V
12
1395


388
E
V
Q
S
K
A
N
H
L
12
1396


418
D
R
T
C
A
N
C
Q
S
12
1397










HLA-B*5101 nonamers



















316
V
A
F
T
T
F
R
I
I
26
1398


143
D
A
L
H
M
L
T
D
L
25
1399


222
A
A
V
G
V
A
V
N
V
23
1400


183
S
A
M
I
S
V
L
L
V
22
1401


346
E
A
L
M
K
I
E
D
V
22
1402


371
T
A
I
V
H
I
Q
L
I
22
1403


303
I
A
D
P
I
C
T
Y
V
21
1404


111
K
A
R
L
T
I
A
A
V
20
1405


126
I
G
E
L
V
G
G
Y
I
20
1406


 38
E
G
L
S
R
F
N
K
L
18
1407


174
F
G
F
H
R
L
E
V
L
18
1408


199
L
Y
E
A
V
Q
R
T
I
18
1409


209
M
N
Y
E
I
N
G
D
I
18
1410


286
V
G
V
L
I
A
A
Y
I
18
1411


323
I
I
W
D
T
V
V
I
I
18
1412


 18
D
A
P
L
F
L
N
D
T
17
1413


343
Y
I
K
E
A
L
M
K
I
17
1414


 96
D
N
C
S
K
Q
R
E
I
16
1415


159
L
A
L
W
L
S
S
K
S
16
1416


195
M
G
F
L
L
Y
E
A
V
16
1417


221
T
A
A
V
G
V
A
V
N
16
1418


223
A
V
G
V
A
V
N
V
I
16
1419


249
L
P
S
N
S
P
T
R
G
16
1420


269
L
A
V
R
A
A
F
V
H
16
1421


281
D
L
V
Q
S
V
G
V
L
16
1422


291
A
A
Y
I
I
R
F
K
P
16
1423


295
I
R
F
K
P
E
Y
K
I
16
1424


299
P
E
Y
K
I
A
D
P
I
16
1425


305
D
P
I
C
T
Y
V
F
S
16
1426


333
E
G
V
P
S
H
L
N
V
16
1427


 2
A
G
S
G
A
W
K
R
L
15
1428


 19
A
P
L
F
L
N
D
T
S
15
1429


 41
S
R
F
N
K
L
R
V
V
15
1430


 56
E
A
P
E
R
P
V
N
G
15
1431


 81
L
P
L
T
N
S
Q
L
S
15
1432


130
V
G
G
Y
I
A
N
S
L
15
1433


150
D
L
S
A
I
I
L
T
L
15
1434


277
H
A
L
G
D
L
V
Q
S
15
1435


282
L
V
Q
S
V
G
V
L
I
15
1436


365
L
T
S
G
K
S
T
A
I
15
1437


 1
M
A
G
S
G
A
W
K
R
14
1438


 5
G
A
W
K
R
L
K
S
M
14
1439


 53
D
G
S
E
A
P
E
R
P
14
1440


108
R
K
V
K
A
R
L
T
I
14
1441


134
I
A
N
S
L
A
I
M
T
14
1442


146
H
M
L
T
D
L
S
A
I
14
1443


220
I
T
A
A
V
G
V
A
V
14
1444


226
V
A
V
N
V
I
M
G
F
14
1445


290
I
A
A
Y
I
I
R
F
K
14
1446


315
L
V
A
F
T
T
F
R
I
14
1447


322
R
I
I
W
D
T
V
V
I
14
1448


324
I
W
D
T
V
V
I
I
L
14
1449


368
G
K
S
T
A
I
V
H
I
14
1450


379
I
P
G
S
S
S
K
W
E
14
1451


400
T
F
G
M
Y
R
C
T
I
14
1452


 42
R
F
N
K
L
R
V
V
V
13
1453


 57
A
P
E
R
P
V
N
G
A
13
1454


 80
D
L
P
L
T
N
S
Q
L
13
1455


112
A
R
L
T
I
A
A
V
L
13
1456


116
I
A
A
V
L
Y
L
L
F
13
1457


117
A
A
V
L
Y
L
L
F
M
13
1458


118
A
V
L
Y
L
L
F
M
I
13
1459


138
L
A
I
M
T
D
A
L
H
13
1460


167
S
P
T
K
R
F
T
F
G
13
1461


181
V
L
S
A
M
I
S
V
L
13
1462


185
M
I
S
V
L
L
V
Y
I
13
1463


273
A
A
F
V
H
A
L
G
D
13
1464


279
L
G
D
L
V
Q
S
V
G
13
1465


287
G
V
L
I
A
A
Y
I
I
13
1466


308
C
T
Y
V
F
S
L
L
V
13
1467


321
F
R
I
I
W
D
T
V
V
13
1468


331
I
L
E
G
V
P
S
H
L
13
1469


335
V
P
S
H
L
N
V
D
Y
13
1470


349
M
K
I
E
D
V
Y
S
V
13
1471


































SEQ. ID.


Pos
1
2
3
4
5
6
7
8
9
0
score
No.













HLA-A*0201 decamers




















139
A
I
M
T
D
A
L
H
M
L
26
1472


184
A
M
I
S
V
L
L
V
Y
I
26
1473


193
I
L
M
G
F
L
L
Y
E
A
26
1474


323
I
I
W
D
T
V
V
I
I
L
26
1475


348
L
M
K
I
E
D
V
Y
S
V
26
1476


198
L
L
Y
E
A
V
Q
R
T
I
25
1477


217
I
M
L
I
T
A
A
V
G
V
25
1478


 12
S
M
L
R
K
D
D
A
P
L
24
1479


 75
S
L
L
D
Q
D
L
P
L
T
24
1480


113
R
L
T
I
A
A
V
L
Y
L
24
1481


152
S
A
I
I
L
T
L
L
A
L
24
1482


277
H
A
L
G
D
L
V
Q
S
V
24
1483


322
R
I
I
W
D
T
V
V
I
I
24
1484


330
I
I
L
E
G
V
P
S
H
L
24
1485


114
L
T
I
A
A
V
L
Y
L
L
23
1486


121
Y
L
L
F
M
I
G
E
L
V
23
1487


150
D
L
S
A
I
I
L
T
L
L
23
1488


154
I
I
L
T
L
L
A
L
W
L
23
1489


181
V
L
S
A
M
I
S
V
L
L
23
1490


185
M
I
S
V
L
L
V
Y
I
L
23
1491


 39
G
L
S
R
F
N
K
L
R
V
22
1492


219
L
I
T
A
A
V
G
V
A
V
22
1493


270
A
V
R
A
A
F
V
H
A
L
22
1494


302
K
I
A
D
P
I
C
T
Y
V
22
1495


356
S
V
E
D
L
N
I
W
S
L
22
1496


129
L
V
G
G
Y
I
A
N
S
L
21
1497


172
F
T
F
G
F
H
R
L
E
V
21
1498


189
L
L
V
Y
I
L
M
G
F
L
21
1499


222
A
A
V
G
V
A
V
N
V
I
21
1500


273
A
A
F
V
H
A
L
G
D
L
21
1501


 68
T
L
Q
A
D
D
D
S
L
L
20
1502


 83
L
T
N
S
Q
L
S
L
K
V
20
1503


110
V
K
A
R
L
T
I
A
A
V
20
1504


120
L
Y
L
L
F
M
I
G
E
L
20
1505


146
H
M
L
T
D
L
S
A
I
I
20
1506


147
M
L
T
D
L
S
A
I
I
L
20
1507


194
L
M
G
F
L
L
Y
E
A
V
20
1508


197
F
L
L
Y
E
A
V
Q
R
T
20
1509


218
M
L
I
T
A
A
V
G
V
A
20
1510


294
I
I
R
F
K
P
E
Y
K
I
20
1511


314
L
L
V
A
F
T
T
F
R
I
20
1512


338
H
L
N
V
D
Y
I
K
E
A
20
1513


364
S
L
T
S
G
K
S
T
A
I
20
1514


365
L
T
S
G
K
S
T
A
I
V
20
1515


370
S
T
A
I
V
H
I
Q
L
I
20
1516


144
A
L
H
M
L
T
D
L
S
A
19
1517


149
T
D
L
S
A
I
I
L
T
L
19
1518


182
L
S
A
M
I
S
V
L
L
V
19
1519


281
D
L
V
Q
S
V
G
V
L
I
19
1520


117
A
A
V
L
Y
L
L
F
M
I
18
1521


124
F
M
I
G
E
L
V
G
G
Y
18
1522


125
M
I
G
E
L
V
G
G
Y
I
18
1523


153
A
I
I
L
T
L
L
A
L
W
18
1524


157
T
L
L
A
L
W
L
S
S
K
18
1525


188
V
L
L
V
Y
I
L
M
G
F
18
1526


221
T
A
A
V
G
V
A
V
N
V
18
1527


326
D
T
V
V
I
I
L
E
G
V
18
1528


329
V
I
I
L
E
G
V
P
S
H
18
1529


 81
L
P
L
T
N
S
Q
L
S
L
17
1530


111
K
A
R
L
T
I
A
A
V
L
17
1531


116
I
A
A
V
L
Y
L
L
F
M
17
1532


160
A
L
W
L
S
S
K
S
P
T
17
1533


180
E
V
L
S
A
M
I
S
V
L
17
1534


190
L
V
Y
I
L
M
G
F
L
L
17
1535


192
Y
I
L
M
G
F
L
L
Y
E
17
1536


227
A
V
N
V
I
M
G
F
L
L
17
1537


230
V
I
M
G
F
L
L
N
Q
S
17
1538


280
G
D
L
V
Q
S
V
G
V
L
17
1539


288
V
L
I
A
A
Y
I
I
R
F
17
1540


305
D
P
I
C
T
Y
V
F
S
L
17
1541


310
Y
V
F
S
L
L
V
A
F
T
17
1542


319
T
T
F
R
I
I
W
D
T
V
17
1543


345
K
E
A
L
M
K
I
E
D
V
17
1544


399
N
T
F
G
M
Y
R
C
T
I
17
1545


 5
G
A
W
K
R
L
K
S
M
L
16
1546


133
Y
I
A
N
S
L
A
I
M
T
16
1547


137
S
L
A
I
M
T
D
A
L
H
16
1548


142
T
D
A
L
H
M
L
T
D
L
16
1549


145
L
H
M
L
T
D
L
S
A
I
16
1550


158
L
L
A
L
W
L
S
S
K
S
16
1551


208
H
M
N
Y
E
I
N
G
D
I
16
1552


211
Y
E
I
N
G
D
I
M
L
I
16
1553


215
G
D
I
M
L
I
T
A
A
V
16
1554


285
S
V
G
V
L
I
A
A
Y
I
16
1555


289
L
I
A
A
Y
I
I
R
F
K
16
1556


351
I
E
D
V
Y
S
V
E
D
L
16
1557


359
D
L
N
I
W
S
L
T
S
G
16
1558


377
Q
L
I
P
G
S
S
S
K
W
16
1559


408
I
Q
L
Q
S
Y
R
Q
E
V
16
1560


 40
L
S
R
F
N
K
L
R
V
V
15
1561


 41
S
R
F
N
K
L
R
V
V
V
15
1562


 67
P
T
L
Q
A
D
D
D
S
L
15
1563


 76
L
L
D
Q
D
L
P
L
T
N
15
1564


103
E
I
L
K
Q
R
K
V
K
A
15
1565


104
I
L
K
Q
R
K
V
K
A
R
15
1566


128
E
L
V
G
G
Y
I
A
N
S
15
1567


155
I
L
T
L
L
A
L
W
L
S
15
1568


226
V
A
V
N
V
I
M
G
F
L
15
1569


279
L
G
D
L
V
Q
S
V
G
V
15
1570


282
L
V
Q
S
V
G
V
L
I
A
15
1571


306
P
I
C
T
Y
V
F
S
L
L
15
1572


315
L
V
A
F
T
T
F
R
I
I
15
1573


342
D
Y
I
K
E
A
L
M
K
I
15
1574


347
A
L
M
K
I
E
D
V
Y
S
15
1575


367
S
G
K
S
T
A
I
V
H
I
15
1576


 31
F
S
D
E
A
G
D
E
G
L
14
1577


 79
Q
D
L
P
L
T
N
S
Q
L
14
1578


105
L
K
Q
R
K
V
K
A
R
L
14
1579


122
L
L
F
M
I
G
E
L
V
G
14
1580


140
I
M
T
D
A
L
H
M
L
T
14
1581


148
L
T
D
L
S
A
I
I
L
T
14
1582


177
H
R
L
E
V
L
S
A
M
I
14
1583


179
L
E
V
L
S
A
M
I
S
V
14
1584


220
I
T
A
A
V
G
V
A
V
N
14
1585


234
F
L
L
N
Q
S
G
H
R
H
14
1586


240
G
H
R
H
S
H
S
H
S
L
14
1587


262
R
N
H
G
Q
D
S
L
A
V
14
1588


268
S
L
A
V
R
A
A
F
V
H
14
1589


308
C
T
Y
V
F
S
L
L
V
A
14
1590


313
S
L
L
V
A
F
T
T
F
R
14
1591


331
I
L
E
G
V
P
S
H
L
N
14
1592


339
L
N
V
D
Y
I
K
E
A
L
14
1593


372
A
I
V
H
I
Q
L
I
P
G
14
1594


396
L
L
L
N
T
F
G
M
Y
R
14
1595


397
L
L
N
T
F
G
M
Y
R
C
14
1596


402
G
M
Y
R
C
T
I
Q
L
Q
14
1597


 1
M
A
G
S
G
A
W
K
R
L
13
1598


 4
S
G
A
W
K
R
L
K
S
M
13
1599


 14
L
R
K
D
D
A
P
L
F
L
13
1600


 22
F
L
N
D
T
S
A
F
D
F
13
1601


 56
E
A
P
E
R
P
V
N
G
A
13
1602


 60
R
P
V
N
G
A
H
P
T
L
13
1603


119
V
L
Y
L
L
F
M
I
G
E
13
1604


132
G
Y
I
A
N
S
L
A
I
M
13
1605


134
I
A
N
S
L
A
I
M
T
D
13
1606


136
N
S
L
A
I
M
T
D
A
L
13
1607


138
L
A
I
M
T
D
A
L
H
M
13
1608


170
K
R
F
T
F
G
F
H
R
L
13
1609


173
T
F
G
F
H
R
L
E
V
L
13
1610


187
S
V
L
L
V
Y
I
L
M
G
13
1611


212
E
I
N
G
D
I
M
L
I
T
13
1612


213
I
N
G
D
I
M
L
I
T
A
13
1613


235
L
L
N
Q
S
G
H
R
H
S
13
1614


267
D
S
L
A
V
R
A
A
F
V
13
1615


278
A
L
G
D
L
V
Q
S
V
G
13
1616


321
F
R
I
I
W
D
T
V
V
I
13
1617


332
L
E
G
V
P
S
H
L
N
V
13
1618


335
V
P
S
H
L
N
V
D
Y
I
13
1619


350
K
I
E
D
V
Y
S
V
E
D
13
1620


353
D
V
Y
S
V
E
D
L
N
I
13
1621


391
S
K
A
N
H
L
L
L
N
T
13
1622


392
K
A
N
H
L
L
L
N
T
F
13
1623










HLA-A*0203 decamers




















109
K
V
K
A
R
L
T
I
A
A
19
1624


214
N
G
D
I
M
L
I
T
A
A
19
1625


265
G
Q
D
S
L
A
V
R
A
A
19
1626


283
V
Q
S
V
G
V
L
I
A
A
19
1627


110
V
K
A
R
L
T
I
A
A
V
17
1628


215
G
D
I
M
L
I
T
A
A
V
17
1629


266
Q
D
S
L
A
V
R
A
A
F
17
1630


284
Q
S
V
G
V
L
I
A
A
Y
17
1631


 10
L
K
S
M
L
R
K
D
D
A
10
1632


 19
A
P
L
F
L
N
D
T
S
A
10
1633


 26
T
S
A
F
D
F
S
D
E
A
10
1634


 42
R
F
N
K
L
R
V
V
V
A
10
1635


 48
V
V
V
A
D
D
G
S
E
A
10
1636


 56
E
A
P
E
R
P
V
N
G
A
10
1637


 62
V
N
G
A
H
P
T
L
Q
A
10
1638


103
E
I
L
K
Q
R
K
V
K
A
10
1639


108
R
K
V
K
A
R
L
T
I
A
10
1640


126
I
G
E
L
V
G
G
Y
I
A
10
1641


130
V
G
G
Y
I
A
N
S
L
A
10
1642


135
A
N
S
L
A
I
M
T
D
A
10
1643


144
A
L
H
M
L
T
D
L
S
A
10
1644


151
L
S
A
I
I
L
T
L
L
A
10
1645


175
G
F
H
R
L
E
V
L
S
A
10
1646


193
I
L
M
G
F
L
L
Y
E
A
10
1647


213
I
N
G
D
I
M
L
I
T
A
10
1648


218
M
L
I
T
A
A
V
G
V
A
10
1649


261
E
R
N
H
G
Q
D
S
L
A
10
1650


264
H
G
Q
D
S
L
A
V
R
A
10
1651


269
L
A
V
R
A
A
F
V
H
A
10
1652


282
L
V
Q
S
V
G
V
L
I
A
10
1653


295
I
R
F
K
P
E
Y
K
I
A
10
1654


308
C
T
Y
V
F
S
L
L
V
A
10
1655


338
H
L
N
V
D
Y
I
K
E
A
10
1656


363
W
S
L
T
S
G
K
S
T
A
10
1657


384
S
K
W
E
E
V
Q
S
K
A
10
1658


413
Y
R
Q
E
V
D
R
T
C
A
10
1659


 11
K
S
M
L
R
K
D
D
A
P
9
1660


 20
P
L
F
L
N
D
T
S
A
F
9
1661


 27
S
A
F
D
F
S
D
E
A
G
9
1662


 43
F
N
K
L
R
V
V
V
A
D
9
1663


 49
V
V
A
D
D
G
S
E
A
P
9
1664


 57
A
P
E
R
P
V
N
G
A
H
9
1665


 63
N
G
A
H
P
T
L
Q
A
D
9
1666


104
I
L
K
Q
R
K
V
K
A
R
9
1667


127
G
E
L
V
G
G
Y
I
A
N
9
1668


131
G
G
Y
I
A
N
S
L
A
I
9
1669


136
N
S
L
A
I
M
T
D
A
L
9
1670


145
L
H
M
L
T
D
L
S
A
I
9
1671


152
S
A
I
I
L
T
L
L
A
L
9
1672


176
F
H
R
L
E
V
L
S
A
M
9
1673


194
L
M
G
F
L
L
Y
E
A
V
9
1674


219
L
I
T
A
A
V
G
V
A
V
9
1675


262
R
N
H
G
Q
D
S
L
A
V
9
1676


270
A
V
R
A
A
F
V
H
A
L
9
1677


296
R
F
K
P
E
Y
K
I
A
D
9
1678


309
T
Y
V
F
S
L
L
V
A
F
9
1679


339
L
N
V
D
Y
I
K
E
A
L
9
1680


364
S
L
T
S
G
K
S
T
A
I
9
1681


385
K
W
E
E
V
Q
S
K
A
N
9
1682


414
R
Q
E
V
D
R
T
C
A
N
9
1683










HLA-A1 decamers




















191
V
Y
I
L
M
G
F
L
L
Y
27
1684


183
S
A
M
I
S
V
L
L
V
Y
24
1685


141
M
T
D
A
L
H
M
L
T
D
22
1686


148
L
T
D
L
S
A
I
I
L
T
22
1687


284
Q
S
V
G
V
L
I
A
A
Y
20
1688


395
H
L
L
L
N
T
F
G
M
Y
20
1689


 16
K
D
D
A
P
L
F
L
N
D
19
1690


112
A
R
L
T
I
A
A
V
L
Y
18
1691


124
F
M
I
G
E
L
V
G
G
Y
18
1692


 76
L
L
D
Q
D
L
P
L
T
N
17
1693


301
Y
K
I
A
D
P
I
C
T
Y
17
1694


404
Y
R
C
T
I
Q
L
Q
S
Y
17
1695


 54
G
S
E
A
P
E
R
P
V
N
16
1696


 70
Q
A
D
D
D
S
L
L
D
Q
16
1697


202
A
V
Q
R
T
I
H
M
N
Y
16
1698


292
A
Y
I
I
R
F
K
P
E
Y
16
1699


324
I
W
D
T
V
V
I
I
L
E
16
1700


334
G
V
P
S
H
L
N
V
D
Y
16
1701


346
E
A
L
M
K
I
E
D
V
Y
16
1702


 31
F
S
D
E
A
G
D
E
G
L
15
1703


172
F
T
F
G
F
H
R
L
E
V
15
1704


210
N
Y
E
I
N
G
D
I
M
L
15
1705


303
I
A
D
P
I
C
T
Y
V
F
15
1706


 32
S
D
E
A
G
D
E
G
L
S
14
1707


 83
L
T
N
S
Q
L
S
L
K
V
14
1708


331
I
L
E
G
V
P
S
H
L
N
14
1709


344
I
K
E
A
L
M
K
I
E
D
14
1710


 3
G
S
G
A
W
K
R
L
K
S
13
1711


 23
L
N
D
T
S
A
F
D
F
S
13
1712


182
L
S
A
M
I
S
V
L
L
V
13
1713


308
C
T
Y
V
F
S
L
L
V
A
13
1714


357
V
E
D
L
N
I
W
S
L
T
13
1715










HLA-A26 decamers




















180
E
V
L
S
A
M
I
S
V
L
30
1716


225
G
V
A
V
N
V
I
M
G
F
27
1717


150
D
L
S
A
I
I
L
T
L
L
26
1718


115
T
I
A
A
V
L
Y
L
L
F
25
1719


288
V
L
I
A
A
Y
I
I
R
F
25
1720


388
E
V
Q
S
K
A
N
H
L
L
25
1721


114
L
T
I
A
A
V
L
Y
L
L
24
1722


188
V
L
L
V
Y
I
L
M
G
F
24
1723


356
S
V
E
D
L
N
I
W
S
L
24
1724


 34
E
A
G
D
E
G
L
S
R
F
23
1725


139
A
I
M
T
D
A
L
H
M
L
23
1726


270
A
V
R
A
A
F
V
H
A
L
23
1727


306
P
I
C
T
Y
V
F
S
L
L
23
1728


323
I
I
W
D
T
V
V
I
I
L
23
1729


334
G
V
P
S
H
L
N
V
D
Y
23
1730


395
H
L
L
L
N
T
F
G
M
Y
23
1731


 20
P
L
F
L
N
D
T
S
A
F
22
1732


128
E
L
V
G
G
Y
I
A
N
S
22
1733


185
M
I
S
V
L
L
V
Y
I
L
22
1734


202
A
V
Q
R
T
I
H
M
N
Y
22
1735


212
E
I
N
G
D
I
M
L
I
T
22
1736


330
I
I
L
E
G
V
P
S
H
L
22
1737


 25
D
T
S
A
F
D
F
S
D
E
21
1738


129
L
V
G
G
Y
I
A
N
S
L
21
1739


305
D
P
I
C
T
Y
V
F
S
L
21
1740


326
D
T
V
V
I
I
L
E
G
V
21
1741


 13
M
L
R
K
D
D
A
P
L
F
20
1742


 67
P
T
L
Q
A
D
D
D
S
L
20
1743


113
R
L
T
I
A
A
V
L
Y
L
20
1744


189
L
L
V
Y
I
L
M
G
F
L
20
1745


340
N
V
D
Y
I
K
E
A
L
M
20
1746


 22
F
L
N
D
T
S
A
F
D
F
19
1747


 37
D
E
G
L
S
R
F
N
K
L
19
1748


103
E
I
L
K
Q
R
K
V
K
A
19
1749


124
F
M
I
G
E
L
V
G
G
Y
19
1750


223
A
V
G
V
A
V
N
V
I
M
19
1751


359
D
L
N
I
W
S
L
T
S
G
19
1752


154
I
I
L
T
L
L
A
L
W
L
18
1753


173
T
F
G
F
H
R
L
E
V
L
18
1754


190
L
V
Y
I
L
M
G
F
L
L
18
1755


227
A
V
N
V
I
M
G
F
L
L
18
1756


284
Q
S
V
G
V
L
I
A
A
Y
18
1757


310
Y
V
F
S
L
L
V
A
F
T
18
1758


329
V
I
I
L
E
G
V
P
S
H
18
1759


353
D
V
Y
S
V
E
D
L
N
I
18
1760


 68
T
L
Q
A
D
D
D
S
L
L
17
1761


 72
D
D
D
S
L
L
D
Q
D
L
17
1762


147
M
L
T
D
L
S
A
I
I
L
17
1763


153
A
I
I
L
T
L
L
A
L
W
17
1764


181
V
L
S
A
M
I
S
V
L
L
17
1765


216
D
I
M
L
I
T
A
A
V
G
17
1766


229
N
V
I
M
G
F
L
L
N
Q
17
1767


230
V
I
M
G
F
L
L
N
Q
S
17
1768


322
R
I
I
W
D
T
V
V
I
I
17
1769


343
Y
I
K
E
A
L
M
K
I
E
17
1770


416
E
V
D
R
T
C
A
N
C
Q
17
1771


 80
D
L
P
L
T
N
S
Q
L
S
16
1772


191
V
Y
I
L
M
G
F
L
L
Y
16
1773


205
R
T
I
H
M
N
Y
E
I
N
16
1774


220
I
T
A
A
V
G
V
A
V
N
16
1775


281
D
L
V
Q
S
V
G
V
L
I
16
1776


301
Y
K
I
A
D
P
I
C
T
Y
16
1777


309
T
Y
V
F
S
L
L
V
A
F
16
1778


346
E
A
L
M
K
I
E
D
V
Y
16
1779


370
S
T
A
I
V
H
I
Q
L
I
16
1780


373
I
V
H
I
Q
L
I
P
G
S
16
1781


387
E
E
V
Q
S
K
A
N
H
L
16
1782


404
Y
R
C
T
I
Q
L
Q
S
Y
16
1783


 96
D
N
C
S
K
Q
R
E
I
L
15
1784


104
I
L
K
Q
R
K
V
K
A
R
15
1785


118
A
V
L
Y
L
L
F
M
I
G
15
1786


132
G
Y
I
A
N
S
L
A
I
M
15
1787


141
M
T
D
A
L
H
M
L
T
D
15
1788


148
L
T
D
L
S
A
I
I
L
T
15
1789


152
S
A
I
I
L
T
L
L
A
L
15
1790


168
P
T
K
R
F
T
F
G
F
H
15
1791


170
K
R
F
T
F
G
F
H
R
L
15
1792


172
F
T
F
G
F
H
R
L
E
V
15
1793


187
S
V
L
L
V
Y
I
L
M
G
15
1794


193
I
L
M
G
F
L
L
Y
E
A
15
1795


282
L
V
Q
S
V
G
V
L
I
A
15
1796


289
L
I
A
A
Y
I
I
R
F
K
15
1797


315
L
V
A
F
T
T
F
R
I
I
15
1798


342
D
Y
I
K
E
A
L
M
K
I
15
1799


407
T
I
Q
L
Q
S
Y
R
Q
E
15
1800










HLA-A3 decamers




















157
T
L
L
A
L
W
L
S
S
K
29
1801


 91
K
V
D
S
C
D
N
C
S
K
25
1802


293
Y
I
I
R
F
K
P
E
Y
K
25
1803


 82
P
L
T
N
S
Q
L
S
L
K
24
1804


102
R
E
I
L
K
Q
R
K
V
K
23
1805


202
A
V
Q
R
T
I
H
M
N
Y
23
1806


268
S
L
A
V
R
A
A
F
V
H
23
1807


341
V
D
Y
I
K
E
A
L
M
K
22
1808


278
A
L
G
D
L
V
Q
S
V
G
21
1809


289
L
I
A
A
Y
I
I
R
F
K
21
1810


328
V
V
I
I
L
E
G
V
P
S
21
1811


377
Q
L
I
P
G
S
S
S
K
W
21
1812


395
H
L
L
L
N
T
F
G
M
Y
21
1813


396
L
L
L
N
T
F
G
M
Y
R
21
1814


 13
M
L
R
K
D
D
A
P
L
F
20
1815


137
S
L
A
I
M
T
D
A
L
H
20
1816


234
F
L
L
N
Q
S
G
H
R
H
20
1817


334
G
V
P
S
H
L
N
V
D
Y
20
1818


376
I
Q
L
I
P
G
S
S
S
K
20
1819


 20
P
L
F
L
N
D
T
S
A
F
19
1820


 45
K
L
R
V
V
V
A
D
D
G
19
1821


154
I
I
L
T
L
L
A
L
W
L
19
1822


180
E
V
L
S
A
M
I
S
V
L
19
1823


198
L
L
Y
E
A
V
Q
R
T
I
19
1824


216
D
I
M
L
I
T
A
A
V
G
19
1825


270
A
V
R
A
A
F
V
H
A
L
19
1826


275
F
V
H
A
L
G
D
L
V
Q
19
1827


288
V
L
I
A
A
Y
I
I
R
F
19
1828


322
R
I
I
W
D
T
V
V
I
I
19
1829


 22
F
L
N
D
T
S
A
F
D
F
18
1830


 48
V
V
V
A
D
D
G
S
E
A
18
1831


 76
L
L
D
Q
D
L
P
L
T
N
18
1832


104
I
L
K
Q
R
K
V
K
A
R
18
1833


112
A
R
L
T
I
A
A
V
L
Y
18
1834


113
R
L
T
I
A
A
V
L
Y
L
18
1835


115
T
I
A
A
V
L
Y
L
L
F
18
1836


144
A
L
H
M
L
T
D
L
S
A
18
1837


153
A
I
I
L
T
L
L
A
L
W
18
1838


178
R
L
E
V
L
S
A
M
I
S
18
1839


187
S
V
L
L
V
Y
I
L
M
G
18
1840


190
L
V
Y
I
L
M
G
F
L
L
18
1841


218
M
L
I
T
A
A
V
G
V
A
18
1842


219
L
I
T
A
A
V
G
V
A
V
18
1843


313
S
L
L
V
A
F
T
T
F
R
18
1844


329
V
I
I
L
E
G
V
P
S
H
18
1845


 7
W
K
R
L
K
S
M
L
R
K
17
1846


 47
R
V
V
V
A
D
D
G
S
E
17
1847


100
K
Q
R
E
I
L
K
Q
R
K
17
1848


109
K
V
K
A
R
L
T
I
A
A
17
1849


111
K
A
R
L
T
I
A
A
V
L
17
1850


122
L
L
F
M
I
G
E
L
V
G
17
1851


129
L
V
G
G
Y
I
A
N
S
L
17
1852


160
A
L
W
L
S
S
K
S
P
T
17
1853


188
V
L
L
V
Y
I
L
M
G
F
17
1854


196
G
F
L
L
Y
E
A
V
Q
R
17
1855


223
A
V
G
V
A
V
N
V
I
M
17
1856


281
D
L
V
Q
S
V
G
V
L
I
17
1857


285
S
V
G
V
L
I
A
A
Y
I
17
1858


287
G
V
L
I
A
A
Y
I
I
R
17
1859


330
I
I
L
E
G
V
P
S
H
L
17
1860


347
A
L
M
K
I
E
D
V
Y
S
17
1861


353
D
V
Y
S
V
E
D
L
N
I
17
1862


375
H
I
Q
L
I
P
G
S
S
S
17
1863


 9
R
L
K
S
M
L
R
K
D
D
16
1864


 39
G
L
S
R
F
N
K
L
R
V
16
1865


 87
Q
L
S
L
K
V
D
S
C
D
16
1866


118
A
V
L
Y
L
L
F
M
I
G
16
1867


161
L
W
L
S
S
K
S
P
T
K
16
1868


162
W
L
S
S
K
S
P
T
K
R
16
1869


181
V
L
S
A
M
I
S
V
L
L
16
1870


225
G
V
A
V
N
V
I
M
G
F
16
1871


227
A
V
N
V
I
M
G
F
L
L
16
1872


229
N
V
I
M
G
F
L
L
N
Q
16
1873


327
T
V
V
I
I
L
E
G
V
P
16
1874


350
K
I
E
D
V
Y
S
V
E
D
16
1875


360
L
N
I
W
S
L
T
S
G
K
16
1876


409
Q
L
Q
S
Y
R
Q
E
V
D
16
1877


416
E
V
D
R
T
C
A
N
C
Q
16
1878


 2
A
G
S
G
A
W
K
R
L
K
15
1879


 33
D
E
A
G
D
E
G
L
S
R
15
1880


 68
T
L
Q
A
D
D
D
S
L
L
15
1881


119
V
L
Y
L
L
F
M
I
G
E
15
1882


121
Y
L
L
F
M
I
G
E
L
V
15
1883


128
E
L
V
G
G
Y
I
A
N
S
15
1884


158
L
L
A
L
W
L
S
S
K
S
15
1885


183
S
A
M
I
S
V
L
L
V
Y
15
1886


301
Y
K
I
A
D
P
I
C
T
Y
15
1887


 61
P
V
N
G
A
H
P
T
L
Q
14
1888


 75
S
L
L
D
Q
D
L
P
L
T
14
1889


103
E
I
L
K
Q
R
K
V
K
A
14
1890


133
Y
I
A
N
S
L
A
I
M
T
14
1891


147
M
L
T
D
L
S
A
I
I
L
14
1892


193
I
L
M
G
F
L
L
Y
E
A
14
1893


284
Q
S
V
G
V
L
I
A
A
Y
14
1894


302
K
I
A
D
P
I
C
T
Y
V
14
1895


331
I
L
E
G
V
P
S
H
L
N
14
1896


356
S
V
E
D
L
N
I
W
S
L
14
1897


359
D
L
N
I
W
S
L
T
S
G
14
1898


383
S
S
K
W
E
E
V
Q
S
K
14
1899










HLA-B*0702 decamers




















 60
R
P
V
N
G
A
H
P
T
L
22
1900


 81
L
P
L
T
N
S
Q
L
S
L
22
1901


305
D
P
I
C
T
Y
V
F
S
L
21
1902


 19
A
P
L
F
L
N
D
T
S
A
19
1903


335
V
P
S
H
L
N
V
D
Y
I
19
1904


167
S
P
T
K
R
F
T
F
G
F
18
1905


298
K
P
E
Y
K
I
A
D
P
I
18
1906


270
A
V
R
A
A
F
V
H
A
L
17
1907


111
K
A
R
L
T
I
A
A
V
L
15
1908


181
V
L
S
A
M
I
S
V
L
L
15
1909


389
V
Q
S
K
A
N
H
L
L
L
15
1910


113
R
L
T
I
A
A
V
L
Y
L
14
1911


150
D
L
S
A
I
I
L
T
L
L
14
1912


 14
L
R
K
D
D
A
P
L
F
L
13
1913


 57
A
P
E
R
P
V
N
G
A
H
13
1914


 74
D
S
L
L
D
Q
D
L
P
L
13
1915


129
L
V
G
G
Y
I
A
N
S
L
13
1916


136
N
S
L
A
I
M
T
D
A
L
13
1917


139
A
I
M
T
D
A
L
H
M
L
13
1918


152
S
A
I
I
L
T
L
L
A
L
13
1919


154
I
I
L
T
L
L
A
L
W
L
13
1920


185
M
I
S
V
L
L
V
Y
I
L
13
1921


249
L
P
S
N
S
P
T
R
G
S
13
1922


351
I
E
D
V
Y
S
V
E
D
L
13
1923


 12
S
M
L
R
K
D
D
A
P
L
12
1924


 37
D
E
G
L
S
R
F
N
K
L
12
1925


 42
R
F
N
K
L
R
V
V
V
A
12
1926


142
T
D
A
L
H
M
L
T
D
L
12
1927


149
T
D
L
S
A
I
I
L
T
L
12
1928


170
K
R
F
T
F
G
F
H
R
L
12
1929


173
T
F
G
F
H
R
L
E
V
L
12
1930


180
E
V
L
S
A
M
I
S
V
L
12
1931


222
A
A
V
G
V
A
V
N
V
I
12
1932


227
A
V
N
V
I
M
G
F
L
L
12
1933


240
G
H
R
H
S
H
S
H
S
L
12
1934


260
C
E
R
N
H
G
Q
D
S
L
12
1935


262
R
N
H
G
Q
D
S
L
A
V
12
1936


273
A
A
F
V
H
A
L
G
D
L
12
1937


280
G
D
L
V
Q
S
V
G
V
L
12
1938


323
I
I
W
D
T
V
V
I
I
L
12
1939


365
L
T
S
G
K
S
T
A
I
V
12
1940


369
K
S
T
A
I
V
H
I
Q
L
12
1941


379
I
P
G
S
S
S
K
W
E
E
12
1942


401
F
G
M
Y
R
C
T
I
Q
L
12
1943


 1
M
A
G
S
G
A
W
K
R
L
11
1944


 5
G
A
W
K
R
L
K
S
M
L
11
1945


 31
F
S
D
E
A
G
D
E
G
L
11
1946


 62
V
N
G
A
H
P
T
L
Q
A
11
1947


 68
T
L
Q
A
D
D
D
S
L
L
11
1948


 72
D
D
D
S
L
L
D
Q
D
L
11
1949


 79
Q
D
L
P
L
T
N
S
Q
L
11
1950


 96
D
N
C
S
K
Q
R
E
I
L
11
1951


105
L
K
Q
R
K
V
K
A
R
L
11
1952


114
L
T
I
A
A
V
L
Y
L
L
11
1953


116
I
A
A
V
L
Y
L
L
F
M
11
1954


135
A
N
S
L
A
I
M
T
D
A
11
1955


172
F
T
F
G
F
H
R
L
E
V
11
1956


189
L
L
V
Y
I
L
M
G
F
L
11
1957


212
E
I
N
G
D
I
M
L
I
T
11
1958


223
A
V
G
V
A
V
N
V
I
M
11
1959


226
V
A
V
N
V
I
M
G
F
L
11
1960


266
Q
D
S
L
A
V
R
A
A
F
11
1961


303
I
A
D
P
I
C
T
Y
V
F
11
1962


306
P
I
C
T
Y
V
F
S
L
L
11
1963


330
I
I
L
E
G
V
P
S
H
L
11
1964


339
L
N
V
D
Y
I
K
E
A
L
11
1965


387
E
E
V
Q
S
K
A
N
H
L
11
1966


388
E
V
Q
S
K
A
N
H
L
L
11
1967










MHC Class 1 nonamer and decamer


analysis of 108P5H8 flanking the D


to E mutation at amino acid 30.


Listed are scores that fall within


the top 50% (rounded up) of all


scores for a selected allele of


the 108P5H8 variant 1 sequence


that does not contain the mutation.





























SEQ. ID.


Pos
1
2
3
4
5
6
7
8
9
score
No.













HLA-A*0201 nonamers



















 27
S
A
F
E
F
S
D
E
A
16
2082


 22
F
L
N
D
T
S
A
F
E
15
2083










HLA-A*0203 nonamers



















 27
S
A
F
E
F
S
D
E
A
11
2084










HLA-A26 nonamers



















 25
D
T
S
A
F
E
F
S
D
17
2085










HLA-B*1510 nonamers



















 23
L
N
D
T
S
A
F
E
F
7
2086










HLA-B*5101 decamers



















 27
S
A
F
E
F
S
D
E
A
13
2087


































SEQ. ID.


Pos
1
2
3
4
5
6
7
8
9
0
score
No.













HLA-A*0201 decamers




















 22
F
L
N
D
T
S
A
F
E
F
15
2088










HLA-A1 decamers




















 23
L
N
D
T
S
A
F
E
F
S
13
2089










HLA-A26 decamers




















 25
D
T
S
A
F
E
F
S
D
E
21
2090


 22
F
L
N
D
T
S
A
F
E
F
20
2091










HLA-A3 decamers




















 22
F
L
N
D
T
S
A
F
E
F
18
2092
















TABLE XXIII







MHC Class II 15-mer analysis of 108P5H8 for selected alleles. Listed are


scores that fall within the top 50% (rounded up) of all scores for the selected allele.









































SEQ. ID


Pos
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
score
No.













HLA-DRB1*0101 15-mers

























326
D
T
V
V
I
I
L
E
G
V
P
S
H
L
N
36
2093


188
V
L
L
V
Y
I
L
M
G
F
L
L
Y
E
A
35
2094


145
L
H
M
L
T
D
L
S
A
I
I
L
T
L
L
33
2095


123
L
F
M
I
G
E
L
V
G
G
Y
I
A
N
S
32
2096


152
S
A
I
I
L
T
L
L
A
L
W
L
S
S
K
32
2097


176
F
H
R
L
E
V
L
S
A
M
I
S
V
L
L
32
2098


283
V
Q
S
V
G
V
L
I
A
A
Y
I
I
R
F
32
2099


119
V
L
Y
L
L
F
M
I
G
E
L
V
G
G
Y
31
2100


225
G
V
A
V
N
V
I
M
G
F
L
L
N
Q
S
31
2101


359
D
L
N
I
W
S
L
T
S
G
K
S
T
A
I
31
2102


373
I
V
H
I
Q
L
I
P
G
S
S
S
K
W
E
31
2103


318
F
T
T
F
R
I
I
W
D
T
V
V
I
I
L
30
2104


 40
L
S
R
F
N
K
L
R
V
V
V
A
D
D
G
29
2105


215
G
D
I
M
L
I
T
A
A
V
G
V
A
V
N
29
2106


 11
K
S
M
L
R
K
D
D
A
P
L
F
L
N
D
28
2107


179
L
E
V
L
S
A
M
I
S
V
L
L
V
Y
I
28
2108


173
T
F
G
F
H
R
L
E
V
L
S
A
M
I
S
27
2109


272
R
A
A
F
V
H
A
L
G
D
L
V
Q
S
V
27
2110


309
T
Y
V
F
S
L
L
V
A
F
T
T
F
R
I
27
2111


371
T
A
I
V
H
I
Q
L
I
P
G
S
S
S
K
27
2112


153
A
I
I
L
T
L
L
A
L
W
L
S
S
K
S
26
2113


325
W
D
T
V
V
I
I
L
E
G
V
P
S
H
L
26
2114


 4
S
G
A
W
K
R
L
K
S
M
L
R
K
D
D
25
2115


 20
P
L
F
L
N
D
T
S
A
F
D
F
S
D
E
25
2116


 46
L
R
V
V
V
A
D
D
G
S
E
A
P
E
R
25
2117


101
Q
R
E
I
L
K
Q
R
K
V
K
A
R
L
T
25
2118


142
T
D
A
L
H
M
L
T
D
L
S
A
I
I
L
25
2119


192
Y
I
L
M
G
F
L
L
Y
E
A
V
Q
R
T
25
2120


217
I
M
L
I
T
A
A
V
G
V
A
V
N
V
I
25
2121


279
L
G
D
L
V
Q
S
V
G
V
L
I
A
A
Y
25
2122


345
K
E
A
L
M
K
I
E
D
V
Y
S
V
E
D
25
2123


392
K
A
N
H
L
L
L
N
T
F
G
M
Y
R
C
25
2124


 43
F
N
K
L
R
V
V
V
A
D
D
G
S
E
A
24
2125


 56
E
A
P
E
R
P
V
N
G
A
H
P
T
L
Q
24
2126


124
F
M
I
G
E
L
V
G
G
Y
I
A
N
S
L
24
2127


127
G
E
L
V
G
G
Y
I
A
N
S
L
A
I
M
24
2128


156
L
T
L
L
A
L
W
L
S
S
K
S
P
T
K
24
2129


183
S
A
M
I
S
V
L
L
V
Y
I
L
M
G
F
24
2130


284
Q
S
V
G
V
L
I
A
A
Y
I
I
R
F
K
24
2131


354
V
Y
S
V
E
D
L
N
I
W
S
L
T
S
G
24
2132


362
I
W
S
L
T
S
G
K
S
T
A
I
V
H
I
24
2133


 18
D
A
P
L
F
L
N
D
T
S
A
F
D
F
S
23
2134


 47
R
V
V
V
A
D
D
G
S
E
A
P
E
R
P
23
2135


 78
D
Q
D
L
P
L
T
N
S
Q
L
S
L
K
V
23
2136


107
Q
R
K
V
K
A
R
L
T
I
A
A
V
L
Y
23
2137


148
L
T
D
L
S
A
I
I
L
T
L
L
A
L
W
23
2138


149
T
D
L
S
A
I
I
L
T
L
L
A
L
W
L
23
2139


157
T
L
L
A
L
W
L
S
S
K
S
P
T
K
R
23
2140


187
S
V
L
L
V
Y
I
L
M
G
F
L
L
Y
E
23
2141


213
I
N
G
D
I
M
L
I
T
A
A
V
G
V
A
23
2142


214
N
G
D
I
M
L
I
T
A
A
V
G
V
A
V
23
2143


277
H
A
L
G
D
L
V
Q
S
V
G
V
L
I
A
23
2144


280
G
D
L
V
Q
S
V
G
V
L
I
A
A
Y
I
23
2145


312
F
S
L
L
V
A
F
T
T
F
R
I
I
W
D
23
2146


351
I
E
D
V
Y
S
V
E
D
L
N
I
W
S
L
23
2147


383
S
S
K
W
E
E
V
Q
S
K
A
N
H
L
L
23
2148


 81
L
P
L
T
N
S
Q
L
S
L
K
V
D
S
C
22
2149


113
R
L
T
I
A
A
V
L
Y
L
L
F
M
I
G
22
2150


144
A
L
H
M
L
T
D
L
S
A
I
I
L
T
L
22
2151


182
L
S
A
M
I
S
V
L
L
V
Y
I
L
M
G
22
2152


207
I
H
M
N
Y
E
I
N
G
D
I
M
L
I
T
22
2153


208
H
M
N
Y
E
I
N
G
D
I
M
L
I
T
A
22
2154


232
M
G
F
L
L
N
Q
S
G
H
R
H
S
H
S
22
2155


243
H
S
H
S
H
S
L
P
S
N
S
P
T
R
G
22
2156


265
G
Q
D
S
L
A
V
R
A
A
F
V
H
A
L
22
2157


328
V
V
I
I
L
E
G
V
P
S
H
L
N
V
D
22
2158


338
H
L
N
V
D
Y
I
K
E
A
L
M
K
I
E
22
2159


372
A
I
V
H
I
Q
L
I
P
G
S
S
S
K
W
22
2160


 99
S
K
Q
R
E
I
L
K
Q
R
K
V
K
A
R
21
2161


305
D
P
I
C
T
Y
V
F
S
L
L
V
A
F
T
21
2162


340
N
V
D
Y
I
K
E
A
L
M
K
I
E
D
V
21
2163


111
K
A
R
L
T
I
A
A
V
L
Y
L
L
F
M
20
2164


134
I
A
N
S
L
A
I
M
T
D
A
L
H
M
L
20
2165


141
M
T
D
A
L
H
M
L
T
D
L
S
A
I
I
20
2166


175
G
F
H
R
L
E
V
L
S
A
M
I
S
V
L
20
2167


194
L
M
G
F
L
L
Y
E
A
V
Q
R
T
I
H
20
2168


198
L
L
Y
E
A
V
Q
R
T
I
H
M
N
Y
E
20
2169


260
C
E
R
N
H
G
Q
D
S
L
A
V
R
A
A
20
2170


292
A
Y
I
I
R
F
K
P
E
Y
K
I
A
D
P
20
2171


298
K
P
E
Y
K
I
A
D
P
I
C
T
Y
V
F
20
2172


301
Y
K
I
A
D
P
I
C
T
Y
V
F
S
L
L
20
2173


307
I
C
T
Y
V
F
S
L
L
V
A
F
T
T
F
20
2174


329
V
I
I
L
E
G
V
P
S
H
L
N
V
D
Y
20
2175


346
E
A
L
M
K
I
E
D
V
Y
S
V
E
D
L
20
2176


348
L
M
K
I
E
D
V
Y
S
V
E
D
L
N
I
20
2177


356
S
V
E
D
L
N
I
W
S
L
T
S
G
K
S
20
2178


360
L
N
I
W
S
L
T
S
G
K
S
T
A
I
V
20
2179


401
F
G
M
Y
R
C
T
I
Q
L
Q
S
Y
R
Q
20
2180


104
I
L
K
Q
R
K
V
K
A
R
L
T
I
A
A
19
2181


109
K
V
K
A
R
L
T
I
A
A
V
L
Y
L
L
19
2182


117
A
A
V
L
Y
L
L
F
M
I
G
E
L
V
G
19
2183


118
A
V
L
Y
L
L
F
M
I
G
E
L
V
G
G
19
2184


150
D
L
S
A
I
I
L
T
L
L
A
L
W
L
S
19
2185


165
S
K
S
P
T
K
R
F
T
F
G
F
H
R
L
19
2186


171
R
F
T
F
G
F
H
R
L
E
V
L
S
A
M
19
2187


233
G
F
L
L
N
Q
S
G
H
R
H
S
H
S
H
19
2188


 17
D
D
A
P
L
F
L
N
D
T
S
A
F
D
F
18
2189


 28
A
F
D
F
S
D
E
A
G
D
E
G
L
S
R
18
2190


130
V
G
G
Y
I
A
N
S
L
A
I
M
T
D
A
18
2191


184
A
M
I
S
V
L
L
V
Y
I
L
M
G
F
L
18
2192


186
I
S
V
L
L
V
Y
I
L
M
G
F
L
L
Y
18
2193


189
L
L
V
Y
I
L
M
G
F
L
L
Y
E
A
V
18
2194


212
E
I
N
G
D
I
M
L
I
T
A
A
V
G
V
18
2195


223
A
V
G
V
A
V
N
V
I
M
G
F
L
L
N
18
2196


268
S
L
A
V
R
A
A
F
V
H
A
L
G
D
L
18
2197


313
S
L
L
V
A
F
T
T
F
R
I
I
W
D
T
18
2198


336
P
S
H
L
N
V
D
Y
I
K
E
A
L
M
K
18
2199










HLA-DRB1*0301 (DR17) 15-mers

























 11
K
S
M
L
R
K
D
D
A
P
L
F
L
N
D
30
2200


 66
H
P
T
L
Q
A
D
D
D
S
L
L
D
Q
D
29
2201


137
S
L
A
I
M
T
D
A
L
H
M
L
T
D
L
28
2202


332
L
E
G
V
P
S
H
L
N
V
D
Y
I
K
E
27
2203


386
W
E
E
V
Q
S
K
A
N
H
L
L
L
N
T
26
2204


144
A
L
H
M
L
T
D
L
S
A
I
I
L
T
L
24
2205


 87
Q
L
S
L
K
V
D
S
C
D
N
C
S
K
Q
23
2206


 47
R
V
V
V
A
D
D
G
S
E
A
P
E
R
P
22
2207


 72
D
D
D
S
L
L
D
Q
D
L
P
L
T
N
S
22
2208


 74
D
S
L
L
D
Q
D
L
P
L
T
N
S
Q
L
22
2209


152
S
A
I
I
L
T
L
L
A
L
W
L
S
S
K
22
2210


320
T
F
R
I
I
W
D
T
V
V
I
I
L
E
G
22
2211


 10
L
K
S
M
L
R
K
D
D
A
P
L
F
L
N
21
2212


 19
A
P
L
F
L
N
D
T
S
A
F
D
F
S
D
21
2213


 46
L
R
V
V
V
A
D
D
G
S
E
A
P
E
R
21
2214


113
R
L
T
I
A
A
V
L
Y
L
L
F
M
I
G
21
2215


178
R
L
E
V
L
S
A
M
I
S
V
L
L
V
Y
21
2216


210
N
Y
E
I
N
G
D
I
M
L
I
T
A
A
V
21
2217


328
V
V
I
I
L
E
G
V
P
S
H
L
N
V
D
21
2218


393
A
N
H
L
L
L
N
T
F
G
M
Y
R
C
T
21
2219


 12
S
M
L
R
K
D
D
A
P
L
F
L
N
D
T
20
2220


 18
D
A
P
L
F
L
N
D
T
S
A
F
D
F
S
20
2221


101
Q
R
E
I
L
K
Q
R
K
V
K
A
R
L
T
20
2222


111
K
A
R
L
T
I
A
A
V
L
Y
L
L
F
M
20
2223


122
L
L
F
M
I
G
E
L
V
G
G
Y
I
A
N
20
2224


145
L
H
M
L
T
D
L
S
A
I
I
L
T
L
L
20
2225


179
L
E
V
L
S
A
M
I
S
V
L
L
V
Y
I
20
2226


186
I
S
V
L
L
V
Y
I
L
M
G
F
L
L
Y
20
2227


187
S
V
L
L
V
Y
I
L
M
G
F
L
L
Y
E
20
2228


268
S
L
A
V
R
A
A
F
V
H
A
L
G
D
L
20
2229


299
P
E
Y
K
I
A
D
P
I
C
T
Y
V
F
S
20
2230


336
P
S
H
L
N
V
D
Y
I
K
E
A
L
M
K
20
2231


 7
W
K
R
L
K
S
M
L
R
K
D
D
A
P
L
19
2232


 20
P
L
F
L
N
D
T
S
A
F
D
F
S
D
E
19
2233


120
L
Y
L
L
F
M
I
G
E
L
V
G
G
Y
I
19
2234


127
G
E
L
V
G
G
Y
I
A
N
S
L
A
I
M
19
2235


148
L
T
D
L
S
A
I
I
L
T
L
L
A
L
W
19
2236


183
S
A
M
I
S
V
L
L
V
Y
I
L
M
G
F
19
2237


188
V
L
L
V
Y
I
L
M
G
F
L
L
Y
E
A
19
2238


200
Y
E
A
V
Q
R
T
I
H
M
N
Y
E
I
N
19
2239


225
G
V
A
V
N
V
I
M
G
F
L
L
N
Q
S
19
2240


286
V
G
V
L
I
A
A
Y
I
I
R
F
K
P
E
19
2241


292
A
Y
I
I
R
F
K
P
E
Y
K
I
A
D
P
19
2242


304
A
D
P
I
C
T
Y
V
F
S
L
L
V
A
F
19
2243


338
H
L
N
V
D
Y
I
K
E
A
L
M
K
I
E
19
2244


353
D
V
Y
S
V
E
D
L
N
I
W
S
L
T
S
19
2245


 79
Q
D
L
P
L
T
N
S
Q
L
S
L
K
V
D
18
2246


158
L
L
A
L
W
L
S
S
K
S
P
T
K
R
F
18
2247


204
Q
R
T
I
H
M
N
Y
E
I
N
G
D
I
M
18
2248


223
A
V
G
V
A
V
N
V
I
M
G
F
L
L
N
18
2249


229
N
V
I
M
G
F
L
L
N
Q
S
G
H
R
H
18
2250


321
F
R
I
I
W
D
T
V
V
I
I
L
E
G
V
18
2251


325
W
D
T
V
V
I
I
L
E
G
V
P
S
H
L
18
2252


354
V
Y
S
V
E
D
L
N
I
W
S
L
T
S
G
18
2253


 37
D
E
G
L
S
R
F
N
K
L
R
V
V
V
A
17
2254


 77
L
D
Q
D
L
P
L
T
N
S
Q
L
S
L
K
17
2255


 89
S
L
K
V
D
S
C
D
N
C
S
K
Q
R
E
17
2256


196
G
F
L
L
Y
E
A
V
Q
R
T
I
H
M
N
17
2257


221
T
A
A
V
G
V
A
V
N
V
I
M
G
F
L
17
2258


264
H
G
Q
D
S
L
A
V
R
A
A
F
V
H
A
17
2259


294
I
I
R
F
K
P
E
Y
K
I
A
D
P
I
C
17
2260


344
I
K
E
A
L
M
K
I
E
D
V
Y
S
V
E
17
2261


407
T
I
Q
L
Q
S
Y
R
Q
E
V
D
R
T
C
17
2262


163
L
S
S
K
S
P
T
K
R
F
T
F
G
F
H
16
2263


171
R
F
T
F
G
F
H
R
L
E
V
L
S
A
M
16
2264


206
T
I
H
M
N
Y
E
I
N
G
D
I
M
L
I
16
2265


258
S
G
C
E
R
N
H
G
Q
D
S
L
A
V
R
16
2266


276
V
H
A
L
G
D
L
V
Q
S
V
G
V
L
I
16
2267


337
S
H
L
N
V
D
Y
I
K
E
A
L
M
K
I
16
2268


 3
G
S
G
A
W
K
R
L
K
S
M
L
R
K
D
15
2269


 26
T
S
A
F
D
F
S
D
E
A
G
D
E
G
L
15
2270


 94
S
C
D
N
C
S
K
Q
R
E
I
L
K
Q
R
15
2271


103
E
I
L
K
Q
R
K
V
K
A
R
L
T
I
A
15
2272


151
L
S
A
I
I
L
T
L
L
A
L
W
L
S
S
15
2273


290
I
A
A
Y
I
I
R
F
K
P
E
Y
K
I
A
15
2274










HLA-DRB1*0401 (DR4Dw4) 15-mers

























383
S
S
K
W
E
E
V
Q
S
K
A
N
H
L
L
28
2275


138
L
A
I
M
T
D
A
L
H
M
L
T
D
L
S
26
2276


144
A
L
H
M
L
T
D
L
S
A
I
I
L
T
L
26
2277


196
G
F
L
L
Y
E
A
V
Q
R
T
I
H
M
N
26
2278


206
T
I
H
M
N
Y
E
I
N
G
D
I
M
L
I
26
2279


221
T
A
A
V
G
V
A
V
N
V
I
M
G
F
L
26
2280


312
F
S
L
L
V
A
F
T
T
F
R
I
I
W
D
26
2281


329
V
I
I
L
E
G
V
P
S
H
L
N
V
D
Y
26
2282


359
D
L
N
I
W
S
L
T
S
G
K
S
T
A
I
26
2283


 4
S
G
A
W
K
R
L
K
S
M
L
R
K
D
D
22
2284


 19
A
P
L
F
L
N
D
T
S
A
F
D
F
S
D
22
2285


 40
L
S
R
F
N
K
L
R
V
V
V
A
D
D
G
22
2286


118
A
V
L
Y
L
L
F
M
I
G
E
L
V
G
G
22
2287


159
L
A
L
W
L
S
S
K
S
P
T
K
R
F
T
22
2288


173
T
F
G
F
H
R
L
E
V
L
S
A
M
I
S
22
2289


197
F
L
L
Y
E
A
V
Q
R
T
I
H
M
N
Y
22
2290


272
R
A
A
F
V
H
A
L
G
D
L
V
Q
S
V
22
2291


298
K
P
E
Y
K
I
A
D
P
I
C
T
Y
V
F
22
2292


309
T
Y
V
F
S
L
L
V
A
F
T
T
F
R
I
22
2293


318
F
T
T
F
R
I
I
W
D
T
V
V
I
I
L
22
2294


340
N
V
D
Y
I
K
E
A
L
M
K
I
E
D
V
22
2295


401
F
G
M
Y
R
C
T
I
Q
L
Q
S
Y
R
Q
22
2296


 37
D
E
G
L
S
R
F
N
K
L
R
V
V
V
A
20
2297


 46
L
R
V
V
V
A
D
D
G
S
E
A
P
E
R
20
2298


 47
R
V
V
V
A
D
D
G
S
E
A
P
E
R
P
20
2299


 59
E
R
P
V
N
G
A
H
P
T
L
Q
A
D
D
20
2300


 66
H
P
T
L
Q
A
D
D
D
S
L
L
D
Q
D
20
2301


 74
D
S
L
L
D
Q
D
L
P
L
T
N
S
Q
L
20
2302


 78
D
Q
D
L
P
L
T
N
S
Q
L
S
L
K
V
20
2303


 89
S
L
K
V
D
S
C
D
N
C
S
K
Q
R
E
20
2304


111
K
A
R
L
T
I
A
A
V
L
Y
L
L
F
M
20
2305


123
L
F
M
I
G
E
L
V
G
G
Y
I
A
N
S
20
2306


137
S
L
A
I
M
T
D
A
L
H
M
L
T
D
L
20
2307


142
T
D
A
L
H
M
L
T
D
L
S
A
I
I
L
20
2308


145
L
H
M
L
T
D
L
S
A
I
I
L
T
L
L
20
2309


148
L
T
D
L
S
A
I
I
L
T
L
L
A
L
W
20
2310


152
S
A
I
I
L
T
L
L
A
L
W
L
S
S
K
20
2311


153
A
I
I
L
T
L
L
A
L
W
L
S
S
K
S
20
2312


155
I
L
T
L
L
A
L
W
L
S
S
K
S
P
T
20
2313


156
L
T
L
L
A
L
W
L
S
S
K
S
P
T
K
20
2314


176
F
H
R
L
E
V
L
S
A
M
I
S
V
L
L
20
2315


178
R
L
E
V
L
S
A
M
I
S
V
L
L
V
Y
20
2316


179
L
E
V
L
S
A
M
I
S
V
L
L
V
Y
I
20
2317


182
L
S
A
M
I
S
V
L
L
V
Y
I
L
M
G
20
2318


183
S
A
M
I
S
V
L
L
V
Y
I
L
M
G
F
20
2319


187
S
V
L
L
V
Y
I
L
M
G
F
L
L
Y
E
20
2320


188
V
L
L
V
Y
I
L
M
G
F
L
L
Y
E
A
20
2321


191
V
Y
I
L
M
G
F
L
L
Y
E
A
V
Q
R
20
2322


192
Y
I
L
M
G
F
L
L
Y
E
A
V
Q
R
T
20
2323


195
M
G
F
L
L
Y
E
A
V
Q
R
T
I
H
M
20
2324


210
N
Y
E
I
N
G
D
I
M
L
I
T
A
A
V
20
2325


216
D
I
M
L
I
T
A
A
V
G
V
A
V
N
V
20
2326


217
I
M
L
I
T
A
A
V
G
V
A
V
N
V
I
20
2327


225
G
V
A
V
N
V
I
M
G
F
L
L
N
Q
S
20
2328


228
V
N
V
I
M
G
F
L
L
N
Q
S
G
H
R
20
2329


229
N
V
I
M
G
F
L
L
N
Q
S
G
H
R
H
20
2330


233
G
F
L
L
N
Q
S
G
H
R
H
S
H
S
H
20
2331


268
S
L
A
V
R
A
A
F
V
H
A
L
G
D
L
20
2332


273
A
A
F
V
H
A
L
G
D
L
V
Q
S
V
G
20
2333


276
V
H
A
L
G
D
L
V
Q
S
V
G
V
L
I
20
2334


280
G
D
L
V
Q
S
V
G
V
L
I
A
A
Y
I
20
2335


283
V
Q
S
V
G
V
L
I
A
A
Y
I
I
R
F
20
2336


291
A
A
Y
I
I
R
F
K
P
E
Y
K
I
A
D
20
2337


308
C
T
Y
V
F
S
L
L
V
A
F
T
T
F
R
20
2338


311
V
F
S
L
L
V
A
F
T
T
F
R
I
I
W
20
2339


320
T
F
R
I
I
W
D
T
V
V
I
I
L
E
G
20
2340


326
D
T
V
V
I
I
L
E
G
V
P
S
H
L
N
20
2341


336
P
S
H
L
N
V
D
Y
I
K
E
A
L
M
K
20
2342


338
H
L
N
V
D
Y
I
K
E
A
L
M
K
I
E
20
2343


345
K
E
A
L
M
K
I
E
D
V
Y
S
V
E
D
20
2344


346
E
A
L
M
K
I
E
D
V
Y
S
V
E
D
L
20
2345


348
L
M
K
I
E
D
V
Y
S
V
E
D
L
N
I
20
2346


351
I
E
D
V
Y
S
V
E
D
L
N
I
W
S
L
20
2347


354
V
Y
S
V
E
D
L
N
I
W
S
L
T
S
G
20
2348


362
I
W
S
L
T
S
G
K
S
T
A
I
V
H
I
20
2349


373
I
V
H
I
Q
L
I
P
G
S
S
S
K
W
E
20
2350


 16
K
D
D
A
P
L
F
L
N
D
T
S
A
F
D
18
2351


 24
N
D
T
S
A
F
D
F
S
D
E
A
G
D
E
18
2352


 34
E
A
G
D
E
G
L
S
R
F
N
K
L
R
V
18
2353


 77
L
D
Q
D
L
P
L
T
N
S
Q
L
S
L
K
18
2354


 86
S
Q
L
S
L
K
V
D
S
C
D
N
C
S
K
18
2355


 99
S
K
Q
R
E
I
L
K
Q
R
K
V
K
A
R
18
2356


128
E
L
V
G
G
Y
I
A
N
S
L
A
I
M
T
18
2357


129
L
V
G
G
Y
I
A
N
S
L
A
I
M
T
D
18
2358


134
I
A
N
S
L
A
I
M
T
D
A
L
H
M
L
18
2359


149
T
D
L
S
A
I
I
L
T
L
L
A
L
W
L
18
2360


170
K
R
F
T
F
G
F
H
R
L
E
V
L
S
A
18
2361


175
G
F
H
R
L
E
V
L
S
A
M
I
S
V
L
18
2362


213
I
N
G
D
I
M
L
I
T
A
A
V
G
V
A
18
2363


255
T
R
G
S
G
C
E
R
N
H
G
Q
D
S
L
18
2364


277
H
A
L
G
D
L
V
Q
S
V
G
V
L
I
A
18
2365


305
D
P
I
C
T
Y
V
F
S
L
L
V
A
F
T
18
2366


353
D
V
Y
S
V
E
D
L
N
I
W
S
L
T
S
18
2367


356
S
V
E
D
L
N
I
W
S
L
T
S
G
K
S
18
2368


367
S
G
K
S
T
A
I
V
H
I
Q
L
I
P
G
18
2369


382
S
S
S
K
W
E
E
V
Q
S
K
A
N
H
L
18
2370


387
E
E
V
Q
S
K
A
N
H
L
L
L
N
T
F
18
2371


392
K
A
N
H
L
L
L
N
T
F
G
M
Y
R
C
18
2372


404
Y
R
C
T
I
Q
L
Q
S
Y
R
Q
E
V
D
18
2373


411
Q
S
Y
R
Q
E
V
D
R
T
C
A
N
C
Q
18
2374


412
S
Y
R
Q
E
V
D
R
T
C
A
N
C
Q
S
18
2375


 28
A
F
D
F
S
D
E
A
G
D
E
G
L
S
R
16
2376


130
V
G
G
Y
I
A
N
S
L
A
I
M
T
D
A
16
2377


169
T
K
R
F
T
F
G
F
H
R
L
E
V
L
S
16
2378


171
R
F
T
F
G
F
H
R
L
E
V
L
S
A
M
16
2379


208
H
M
N
Y
E
I
N
G
D
I
M
L
I
T
A
16
2380


231
I
M
G
F
L
L
N
Q
S
G
H
R
H
S
H
16
2381


294
I
I
R
F
K
P
E
Y
K
I
A
D
P
I
C
16
2382


315
L
V
A
F
T
T
F
R
I
I
W
D
T
V
V
16
2383


322
R
I
I
W
D
T
V
V
I
I
L
E
G
V
P
16
2384


352
E
D
V
Y
S
V
E
D
L
N
I
W
S
L
T
16
2385


360
L
N
I
W
S
L
T
S
G
K
S
T
A
I
V
16
2386


398
L
N
T
F
G
M
Y
R
C
T
I
Q
L
Q
S
16
2387


410
L
Q
S
Y
R
Q
E
V
D
R
T
C
A
N
C
16
2388


107
Q
R
K
V
K
A
R
L
T
I
A
A
V
L
Y
15
2389


386
W
E
E
V
Q
S
K
A
N
H
L
L
L
N
T
15
2390


 7
W
K
R
L
K
S
M
L
R
K
D
D
A
P
L
14
2391


 11
K
S
M
L
R
K
D
D
A
P
L
F
L
N
D
14
2392


 18
D
A
P
L
F
L
N
D
T
S
A
F
D
F
S
14
2393


 43
F
N
K
L
R
V
V
V
A
D
D
G
S
E
A
14
2394


 45
K
L
R
V
V
V
A
D
D
G
S
E
A
P
E
14
2395


 73
D
D
S
L
L
D
Q
D
L
P
L
T
N
S
Q
14
2396


 80
D
L
P
L
T
N
S
Q
L
S
L
K
V
D
S
14
2397


 87
Q
L
S
L
K
V
D
S
C
D
N
C
S
K
Q
14
2398


101
Q
R
E
I
L
K
Q
R
K
V
K
A
R
L
T
14
2399


113
R
L
T
I
A
A
V
L
Y
L
L
F
M
I
G
14
2400


116
I
A
A
V
L
Y
L
L
F
M
I
G
E
L
V
14
2401


117
A
A
V
L
Y
L
L
F
M
I
G
E
L
V
G
14
2402


119
V
L
Y
L
L
F
M
I
G
E
L
V
G
G
Y
14
2403


120
L
Y
L
L
F
M
I
G
E
L
V
G
G
Y
I
14
2404


122
L
L
F
M
I
G
E
L
V
G
G
Y
I
A
N
14
2405


126
I
G
E
L
V
G
G
Y
I
A
N
S
L
A
I
14
2406


127
G
E
L
V
G
G
Y
I
A
N
S
L
A
I
M
14
2407


131
G
G
Y
I
A
N
S
L
A
I
M
T
D
A
L
14
2408


158
L
L
A
L
W
L
S
S
K
S
P
T
K
R
F
14
2409


185
M
I
S
V
L
L
V
Y
I
L
M
G
F
L
L
14
2410


200
Y
E
A
V
Q
R
T
I
H
M
N
Y
E
I
N
14
2411


214
N
G
D
I
M
L
I
T
A
A
V
G
V
A
V
14
2412


215
G
D
I
M
L
I
T
A
A
V
G
V
A
V
N
14
2413


227
A
V
N
V
I
M
G
F
L
L
N
Q
S
G
H
14
2414


279
L
G
D
L
V
Q
S
V
G
V
L
I
A
A
Y
14
2415


285
S
V
G
V
L
I
A
A
Y
I
I
R
F
K
P
14
2416


286
V
G
V
L
I
A
A
Y
I
I
R
F
K
P
E
14
2417


287
G
V
L
I
A
A
Y
I
I
R
F
K
P
E
Y
14
2418


300
E
Y
K
I
A
D
P
I
C
T
Y
V
F
S
L
14
2419


304
A
D
P
I
C
T
Y
V
F
S
L
L
V
A
F
14
2420


313
S
L
L
V
A
F
T
T
F
R
I
I
W
D
T
14
2421


321
F
R
I
I
W
D
T
V
V
I
I
L
E
G
V
14
2422


325
W
D
T
V
V
I
I
L
E
G
V
P
S
H
L
14
2423


327
T
V
V
I
I
L
E
G
V
P
S
H
L
N
V
14
2424


328
V
V
I
I
L
E
G
V
P
S
H
L
N
V
D
14
2425


332
L
E
G
V
P
S
H
L
N
V
D
Y
I
K
E
14
2426


341
V
D
Y
I
K
E
A
L
M
K
I
E
D
V
Y
14
2427


370
S
T
A
I
V
H
I
Q
L
I
P
G
S
S
S
14
2428


371
T
A
I
V
H
I
Q
L
I
P
G
S
S
S
K
14
2429


375
H
I
Q
L
I
P
G
S
S
S
K
W
E
E
V
14
2430


376
I
Q
L
I
P
G
S
S
S
K
W
E
E
V
Q
14
2431


395
H
L
L
L
N
T
F
G
M
Y
R
C
T
I
Q
14
2432


400
T
F
G
M
Y
R
C
T
I
Q
L
Q
S
Y
R
14
2433


407
T
I
Q
L
Q
S
Y
R
Q
E
V
D
R
T
C
14
2434


414
R
Q
E
V
D
R
T
C
A
N
C
Q
S
S
S
14
2435










HLA-DRB1*1101 15-mers

























197
F
L
L
Y
E
A
V
Q
R
T
I
H
M
N
Y
25
2436


 40
L
S
R
F
N
K
L
R
V
V
V
A
D
D
G
23
2437


169
T
K
R
F
T
F
G
F
H
R
L
E
V
L
S
22
2438


173
T
F
G
F
H
R
L
E
V
L
S
A
M
I
S
22
2439


 43
F
N
K
L
R
V
V
V
A
D
D
G
S
E
A
21
2440


101
Q
R
E
I
L
K
Q
R
K
V
K
A
R
L
T
21
2441


153
A
I
I
L
T
L
L
A
L
W
L
S
S
K
S
21
2442


233
G
F
L
L
N
Q
S
G
H
R
H
S
H
S
H
21
2443


276
V
H
A
L
G
D
L
V
Q
S
V
G
V
L
I
21
2444


288
V
L
I
A
A
Y
I
I
R
F
K
P
E
Y
K
21
2445


 7
W
K
R
L
K
S
M
L
R
K
D
D
A
P
L
20
2446


 8
K
R
L
K
S
M
L
R
K
D
D
A
P
L
F
20
2447


120
L
Y
L
L
F
M
I
G
E
L
V
G
G
Y
I
20
2448


176
F
H
R
L
E
V
L
S
A
M
I
S
V
L
L
20
2449


411
Q
S
Y
R
Q
E
V
D
R
T
C
A
N
C
Q
20
2450


116
I
A
A
V
L
Y
L
L
F
M
I
G
E
L
V
19
2451


214
N
G
D
I
M
L
I
T
A
A
V
G
V
A
V
19
2452


280
G
D
L
V
Q
S
V
G
V
L
I
A
A
Y
I
19
2453


322
R
I
I
W
D
T
V
V
I
I
L
E
G
V
P
19
2454


325
W
D
T
V
V
I
I
L
E
G
V
P
S
H
L
19
2455


359
D
L
N
I
W
S
L
T
S
G
K
S
T
A
I
19
2456


142
T
D
A
L
H
M
L
T
D
L
S
A
I
I
L
18
2457


185
M
I
S
V
L
L
V
Y
I
L
M
G
F
L
L
18
2458


229
N
V
I
M
G
F
L
L
N
Q
S
G
H
R
H
18
2459


290
I
A
A
Y
I
I
R
F
K
P
E
Y
K
I
A
18
2460


294
I
I
R
F
K
P
E
Y
K
I
A
D
P
I
C
18
2461


309
T
Y
V
F
S
L
L
V
A
F
T
T
F
R
I
18
2462


326
D
T
V
V
I
I
L
E
G
V
P
S
H
L
N
18
2463


345
K
E
A
L
M
K
I
E
D
V
Y
S
V
E
D
18
2464


367
S
G
K
S
T
A
I
V
H
I
Q
L
I
P
G
18
2465


370
S
T
A
I
V
H
I
Q
L
I
P
G
S
S
S
18
2466


373
I
V
H
I
Q
L
I
P
G
S
S
S
K
W
E
18
2467


 4
S
G
A
W
K
R
L
K
S
M
L
R
K
D
D
17
2468


138
L
A
I
M
T
D
A
L
H
M
L
T
D
L
S
17
2469


189
L
L
V
Y
I
L
M
G
F
L
L
Y
E
A
V
17
2470


269
L
A
V
R
A
A
F
V
H
A
L
G
D
L
V
17
2471


318
F
T
T
F
R
I
I
W
D
T
V
V
I
I
L
17
2472


 28
A
F
D
F
S
D
E
A
G
D
E
G
L
S
R
16
2473


 37
D
E
G
L
S
R
F
N
K
L
R
V
V
V
A
16
2474


 98
C
S
K
Q
R
E
I
L
K
Q
R
K
V
K
A
16
2475


121
Y
L
L
F
M
I
G
E
L
V
G
G
Y
I
A
16
2476


383
S
S
K
W
E
E
V
Q
S
K
A
N
H
L
L
16
2477


401
F
G
M
Y
R
C
T
I
Q
L
Q
S
Y
R
Q
16
2478


 1
M
A
G
S
G
A
W
K
R
L
K
S
M
L
R
15
2479


145
L
H
M
L
T
D
L
S
A
I
I
L
T
L
L
15
2480


239
S
G
H
R
H
S
H
S
H
S
L
P
S
N
S
15
2481


323
I
I
W
D
T
V
V
I
I
L
E
G
V
P
S
15
2482


397
L
L
N
T
F
G
M
Y
R
C
T
I
Q
L
Q
15
2483


 34
E
A
G
D
E
G
L
S
R
F
N
K
L
R
V
14
2484


 83
L
T
N
S
Q
L
S
L
K
V
D
S
C
D
N
14
2485


123
L
F
M
I
G
E
L
V
G
G
Y
I
A
N
S
14
2486


158
L
L
A
L
W
L
S
S
K
S
P
T
K
R
F
14
2487


183
S
A
M
I
S
V
L
L
V
Y
I
L
M
G
F
14
2488


200
Y
E
A
V
Q
R
T
I
H
M
N
Y
E
I
N
14
2489


225
G
V
A
V
N
V
I
M
G
F
L
L
N
Q
S
14
2490


235
L
L
N
Q
S
G
H
R
H
S
H
S
H
S
L
14
2491


237
N
Q
S
G
H
R
H
S
H
S
H
S
L
P
S
14
2492


246
S
H
S
L
P
S
N
S
P
T
R
G
S
G
C
14
2493


248
S
L
P
S
N
S
P
T
R
G
S
G
C
E
R
14
2494


254
P
T
R
G
S
G
C
E
R
N
H
G
Q
D
S
14
2495


342
D
Y
I
K
E
A
L
M
K
I
E
D
V
Y
S
14
2496


361
N
I
W
S
L
T
S
G
K
S
T
A
I
V
H
14
2497


 44
N
K
L
R
V
V
V
A
D
D
G
S
E
A
P
13
2498


 71
A
D
D
D
S
L
L
D
Q
D
L
P
L
T
N
13
2499


 78
D
Q
D
L
P
L
T
N
S
Q
L
S
L
K
V
13
2500


113
R
L
T
I
A
A
V
L
Y
L
L
F
M
I
G
13
2501


119
V
L
Y
L
L
F
M
I
G
E
L
V
G
G
Y
13
2502


124
F
M
I
G
E
L
V
G
G
Y
I
A
N
S
L
13
2503


128
E
L
V
G
G
Y
I
A
N
S
L
A
I
M
T
13
2504


135
A
N
S
L
A
I
M
T
D
A
L
H
M
L
T
13
2505


141
M
T
D
A
L
H
M
L
T
D
L
S
A
I
I
13
2506


148
L
T
D
L
S
A
I
I
L
T
L
L
A
L
W
13
2507


149
T
D
L
S
A
I
I
L
T
L
L
A
L
W
L
13
2508


156
L
T
L
L
A
L
W
L
S
S
K
S
P
T
K
13
2509


179
L
E
V
L
S
A
M
I
S
V
L
L
V
Y
I
13
2510


188
V
L
L
V
Y
I
L
M
G
F
L
L
Y
E
A
13
2511


216
D
I
M
L
I
T
A
A
V
G
V
A
V
N
V
13
2512


263
N
H
G
Q
D
S
L
A
V
R
A
A
F
V
H
13
2513


273
A
A
F
V
H
A
L
G
D
L
V
Q
S
V
G
13
2514


283
V
Q
S
V
G
V
L
I
A
A
Y
I
I
R
F
13
2515


308
C
T
Y
V
F
S
L
L
V
A
F
T
T
F
R
13
2516


329
V
I
I
L
E
G
V
P
S
H
L
N
V
D
Y
13
2517


336
P
S
H
L
N
V
D
Y
I
K
E
A
L
M
K
13
2518


338
H
L
N
V
D
Y
I
K
E
A
L
M
K
I
E
13
2519


410
L
Q
S
Y
R
Q
E
V
D
R
T
C
A
N
C
13
2520


 17
D
D
A
P
L
F
L
N
D
T
S
A
F
D
F
12
2521


 46
L
R
V
V
V
A
D
D
G
S
E
A
P
E
R
12
2522


 47
R
V
V
V
A
D
D
G
S
E
A
P
E
R
P
12
2523


 56
E
A
P
E
R
P
V
N
G
A
H
P
T
L
Q
12
2524


 75
S
L
L
D
Q
D
L
P
L
T
N
S
Q
L
S
12
2525


103
E
I
L
K
Q
R
K
V
K
A
R
L
T
I
A
12
2526


107
Q
R
K
V
K
A
R
L
T
I
A
A
V
L
Y
12
2527


117
A
A
V
L
Y
L
L
F
M
I
G
E
L
V
G
12
2528


126
I
G
E
L
V
G
G
Y
I
A
N
S
L
A
I
12
2529


152
S
A
I
I
L
T
L
L
A
L
W
L
S
S
K
12
2530


155
I
L
T
L
L
A
L
W
L
S
S
K
S
P
T
12
2531


157
T
L
L
A
L
W
L
S
S
K
S
P
T
K
R
12
2532


182
L
S
A
M
I
S
V
L
L
V
Y
I
L
M
G
12
2533


187
S
V
L
L
V
Y
I
L
M
G
F
L
L
Y
E
12
2534


191
V
Y
I
L
M
G
F
L
L
Y
E
A
V
Q
R
12
2535


192
Y
I
L
M
G
F
L
L
Y
E
A
V
Q
R
T
12
2536


204
Q
R
T
I
H
M
N
Y
E
I
N
G
D
I
M
12
2537


211
Y
E
I
N
G
D
I
M
L
I
T
A
A
V
G
12
2538


212
E
I
N
G
D
I
M
L
I
T
A
A
V
G
V
12
2539


222
A
A
V
G
V
A
V
N
V
I
M
G
F
L
L
12
2540


228
V
N
V
I
M
G
F
L
L
N
Q
S
G
H
R
12
2541


243
H
S
H
S
H
S
L
P
S
N
S
P
T
R
G
12
2542


315
L
V
A
F
T
T
F
R
I
I
W
D
T
V
V
12
2543


348
L
M
K
I
E
D
V
Y
S
V
E
D
L
N
I
12
2544


351
I
E
D
V
Y
S
V
E
D
L
N
I
W
S
L
12
2545


352
E
D
V
Y
S
V
E
D
L
N
I
W
S
L
T
12
2546


354
V
Y
S
V
E
D
L
N
I
W
S
L
T
S
G
12
2547


356
S
V
E
D
L
N
I
W
S
L
T
S
G
K
S
12
2548


357
V
E
D
L
N
I
W
S
L
T
S
G
K
S
T
12
2549


371
T
A
I
V
H
I
Q
L
I
P
G
S
S
S
K
12
2550


372
A
I
V
H
I
Q
L
I
P
G
S
S
S
K
W
12
2551


391
S
K
A
N
H
L
L
L
N
T
F
G
M
Y
R
12
2552









MHC class II analysis of 108P5H8 flanking the D to E mutation at amino acid 30. Listed are scores that fall within the top 50% (rounded up) of all scores for a selected allele of the 108P5H8 variant 1 sequence that does not contain the mutation.













































SEQ. ID


Pos
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
score
No.


















HLA-DRB1*0101 15-mers

























20
P
L
F
L
N
D
T
S
A
F
E
F
S
D
E
25
2553


18
D
A
P
L
F
L
N
D
T
S
A
F
E
F
S
23
2554


17
D
D
A
P
L
F
L
N
D
T
S
A
F
E
F
18
2555


28
A
F
E
F
S
D
E
A
G
D
E
G
L
S
R
18
2556










HLA-DRB1*0301 (DR17) 15-mers

























19
A
P
L
F
L
N
D
T
S
A
F
E
F
S
D
21
2557


18
D
A
P
L
F
L
N
D
T
S
A
F
E
F
S
20
2558


20
P
L
F
L
N
D
T
S
A
F
E
F
S
D
E
19
2559


26
T
S
A
F
E
F
S
D
E
A
G
D
E
G
L
15
2560










HLA-DRB1*0401 (DR4Dw4) 15-mers

























19
A
P
L
F
L
N
D
T
S
A
F
E
F
S
D
22
2561


16
K
D
D
A
P
L
F
L
N
D
T
S
A
F
E
18
2562


24
N
D
T
S
A
F
E
F
S
D
E
A
G
D
E
18
2563


28
A
F
E
F
S
D
E
A
G
D
E
G
L
S
R
16
2564


18
D
A
P
L
F
L
N
D
T
S
A
F
E
F
S
14
2565










HLA-DRB1*1101 15-mers

























28
A
F
E
F
S
D
E
A
G
D
E
G
L
S
R
16
2566


17
D
D
A
P
L
F
L
N
D
T
S
A
F
E
F
12
2567

## Claims

1. An isolated monoclonal antibody or antibody fragment that specifically binds to a protein having an amino acid sequence of SEQ ID NO: 2570.

2. The antibody or antibody fragment of claim 1, wherein the monoclonal antibody is recombinantly produced.

3. The antibody or antibody fragment of claim 1, wherein the antibody or fragment is labeled with a detectable marker.

4. The antibody or antibody fragment of claim 1, wherein the fragment is selected from the group consisting of Fab, F(ab′)2, Fv and sFv.

5. The antibody or antibody fragment of claim 1, wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody.

6. A hybridoma that produces monoclonal antibody that specifically binds to a protein having an amino acid sequence of SEQ ID NO: 2570.

7. The antibody or antibody fragment of claim 2, wherein the monoclonal antibody is a single chain monoclonal antibody.

8. The antibody or antibody fragment of claim 1, wherein the antibody or fragment is labeled with an agent.

9. The antibody or antibody fragment of claim 8, wherein the cytotoxic agent is selected from the group consisting of radioactive isotopes, chemotherapeutic agents and toxins.

10. The antibody or antibody fragment of claim 9, wherein the radioactive isotope is selected from the group consisting of 211At, 131I, 125I, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P and radioactive isotopes of Lu.

11. The antibody or antibody fragment of claim 9, wherein the chemotherapeutic agent is selected from the group consisting of taxol, actinomycin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, gelonin, and calicheamicin.

12. The antibody or antibody fragment of claim 9, wherein the toxin is selected from the group consisting of diphtheria toxin, enomycin, phenomycin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, mitogellin, modeccin A chain, and alpha-sarcin.