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Publication numberUS20070081942 A1
Publication typeApplication
Application numberUS 11/601,669
Publication dateApr 12, 2007
Filing dateNov 20, 2006
Priority dateApr 4, 2000
Also published asCA2413211A1, CN1610743A, CN101643732A, DE60135315D1, EP1272636A2, EP1272636B1, EP1272636B8, US7268207, US7910551, US20020155447, US20090312263, WO2001074859A2, WO2001074859A3
Publication number11601669, 601669, US 2007/0081942 A1, US 2007/081942 A1, US 20070081942 A1, US 20070081942A1, US 2007081942 A1, US 2007081942A1, US-A1-20070081942, US-A1-2007081942, US2007/0081942A1, US2007/081942A1, US20070081942 A1, US20070081942A1, US2007081942 A1, US2007081942A1
InventorsMaurice Zauderer, Elizabeth Evans, Melinda Borrello
Original AssigneeUniversity Of Rochester
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Gene differentially expressed in breast and bladder cancer, and encoded polypeptides
US 20070081942 A1
Abstract
The present invention is directed to antibodies or antigen binding fragments thereof that specifically bind to the cancer specific antigen, C35. This invention also relates to a polynucleotide encoding the antibody or antigen binding fragment thereof as well as vectors and host cells comprising the polynucleotide. The present invention further relates to a composition comprising the antibody or antigen binding fragment thereof or further comprising a chemotherapeutic agent, specifically taxol. The invention is also directed to therapeutic and diagnostic methods of using antibodies against C35, preferably more than one anti-C35 antibody in combination with a chemotherapeutic agent, to treat C35 associated cancer.
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Claims(60)
1. An isolated antibody or antigen binding fragment thereof that specifically binds to an epitope of C35 (SEQ ID NO:2).
2. The antibody or fragment thereof of claim 1, wherein said epitope of C35 is selected from the group consisting of: amino acids S-9 to V-17, amino acids V-10 to V-17, amino acids E-16 to V-23, amino acids E-16 to R-24, amino acids E-16 to I-25, amino acids S-21 to F-35, amino acids C-30 to T-38, amino acids E-31 to Y-39, amino acids E-36 to A-43, amino acids A-37 to A-45, amino acids A-37 to V-46, amino acids Y-39 to V-46, amino acids S-44 to I-53, amino acids A-45 to I-53; amino acids G-52 to L-59, amino acids E-54 to T-62, amino acids S-57 to F-75, amino acids R-58 to I-67, amino acids G-61 to I-69, amino acids G-63 to F-83, amino acids E-66 to L-73, amino acids E-66 to V-74, amino acids F-83 to E-103, amino acids D-88 to A-96, amino acids L-89 to A-96, amino acids A-92 to T-101, amino acids R-95 to L-102, amino acids A-96 to K-104, amino acids K-104 to V-113, amino acids I-105 to V-113, amino acids I-105 to I-114, amino acids E-4 to P-12, amino acids S-21 to Y-29, amino acids G-22 to C-30, amino acids I-25 to C-33, amino acids T-38 to V-46, amino acids G-63 to G-71, amino acids F-65 to L-73, amino acids I-67 to F-75, amino acids K-77 to Y-85, amino acids Q-72 to E-86, amino acids G-81 to L-89, and amino acids K-104 to C-112, each of SEQ ID NO:2.
3. The antibody or fragment thereof of claim 2, wherein said epitope of C35 is amino acids A-96 to K-104 of SEQ ID NO: 2.
4. The antibody or fragment thereof of claim 2, wherein said epitope of C35 is amino acids K-104 to V-113 of SEQ ID NO: 2.
5. The antibody or fragment thereof of claim 2, wherein said epitope of C35 is amino acids I-105 to V-113 of SEQ ID NO: 2.
6. The antibody or fragment thereof of claim 2, wherein said epitope of C35 is amino acids I-105 to I-114 of SEQ ID NO: 2.
7. The antibody or fragment thereof of claim 2, wherein said epitope of C35 is K-104 to C-112 of SEQ ID NO: 2.
8. The antibody or fragment thereof of claim 1, wherein said antibody or fragment is selected from the group consisting of: a whole antibody, an Fab fragment, an F(ab′)2 fragment, an scFv fragment, and an Fv fragment.
9. The antibody or fragment thereof of claim 1, wherein said antibody is a fusion protein.
10. The antibody or fragment thereof of claim 1, wherein said antibody or fragment is humanized.
11. An isolated polynucleotide encoding the antibody or fragment thereof of claim 1.
12. An expression vector comprising the polynucleotide of claim 11.
13. A host cell comprising the expression vector of claim 12.
14. A method for producing an isolated antibody or antigen binding fragment thereof that specifically binds C35 comprising
(a) culturing the host cell of claim 13; and
(b) recovering said antibody or fragment thereof.
15. A composition comprising at least one isolated antibody or fragment thereof according to claim 1.
16. The composition of claim 15, wherein said composition comprises two antibodies or fragments thereof.
17. The composition of claim 15, further comprising an effective amount of a therapeutic agent.
18. The composition of claim 17, wherein said therapeutic agent is selected from the group consisting of: an anti-tumor drug, a cytotoxin, and a radioactive agent.
19. The composition of claim 18, wherein said therapeutic agent is a cytotoxin.
20. The composition of claim 19, wherein said cytotoxin is selected from the group consisting of: paclitaxel, ricin, doxorubicin, cytochalasin B, gramicidin D, ethidium bromide, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone, and glucocorticoid.
21. The composition of claim 20, wherein said cytotoxin is paclitaxel.
22. The composition of claim 17, wherein said therapeutic agent is conjugated to or complexed with said antibody or fragment thereof.
23. The composition of claim 15, wherein said composition further comprises a pharmaceutically acceptable carrier.
24. A method of treating a C35-associated cancer comprising administering the composition of claim 15 to a host in need of treatment thereof.
25. The method of claim 24, wherein said composition comprises two antibodies or fragments thereof.
26. The method of claim 24, wherein said composition further comprises a therapeutic agent.
27. The method of claim 26, wherein said therapeutic agent is selected from the group consisting of: an anti-tumor drug, a cytotoxin, and a radioactive agent.
28. The method of claim 27, wherein said therapeutic agent is a cytotoxin.
29. The method of claim 28, wherein said cytotoxin is selected from the group consisting of: paclitaxel, ricin, doxorubicin, cytochalasin B, gramicidin D, ethidium bromide, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone, and glucocorticoid.
30. The method of claim 29, wherein said cytotoxin is paclitaxel.
31. The method of claim 26, wherein said therapeutic agent is conjugated to or complexed with said antibody or fragment thereof.
32. The method of claim 24, wherein said cancer is selected from the group consisting of: breast cancer, ovarian cancer, bladder cancer, lung cancer, pancreatic cancer, and colon cancer.
33. The method of claim 32, wherein said cancer is breast cancer.
34. A method of detecting the presence of C35 in a sample, said method comprising:
(a) contacting said sample with the antibody or fragment thereof of claim 1; and
(b) assaying for binding of said antibody or fragment thereof to C35.
35. The method of claim 34, wherein said assaying step is performed by an assay selected from the group consisting of: ELISA, radioimmunoassay, immunofluorescence, and immunocytochemistry.
36. A diagnostic kit for detecting the presence of C35 in a sample, said kit comprising:
(a) the antibody or fragment thereof of claim 1;
(b) a conjugate comprising a specific binding partner for the C35 antibody and a label capable of producing a detectable signal.
37. An isolated antibody or antigen binding fragment thereof that competitively inhibits binding of an antibody or fragment thereof to an epitope of C35.
38. The antibody or fragment thereof of claim 37, wherein said epitope of C35 is selected from the group consisting of: amino acids S-9 to V-17, amino acids V-10 to V-17, amino acids E-16 to V-23, amino acids E-16 to R-24, amino acids E-16 to I-25, amino acids S-21 to F-35, amino acids C-30 to T-38, amino acids E-31 to Y-39, amino acids E-36 to A-43, amino acids A-37 to A-45, amino acids A-37 to V-46, amino acids Y-39 to V-46, amino acids S-44 to I-53, amino acids A-45 to I-53; amino acids G-52 to L-59, amino acids E-54 to T-62, amino acids S-57 to F-75, amino acids R-58 to I-67, amino acids G-61 to I-69, amino acids G-63 to F-83, amino acids E-66 to L-73, amino acids E-66 to V-74, amino acids F-83 to E-103, amino acids D-88 to A-96, amino acids L-89 to A-96, amino acids A-92 to T-101, amino acids R-95 to L-102, amino acids A-96 to K-104, amino acids K-104 to V-113, amino acids I-105 to V-113, amino acids I-105 to I-114, amino acids E-4 to P-12, amino acids S-21 to Y-29, amino acids G-22 to C-30, amino acids I-25 to C-33, amino acids T-38 to V-46, amino acids G-63 to G-71, amino acids F-65 to L-73, amino acids I-67 to F-75, amino acids K-77 to Y-85, amino acids Q-72 to E-86, amino acids G-81 to L-89, and amino acids K-104 to C-112, each of SEQ ID NO:2.
39. The antibody or fragment thereof of claim 38, wherein said epitope of C35 is amino acids A-96 to K-104 of SEQ ID NO: 2.
40. The antibody or fragment thereof of claim 38, wherein said epitope of C35 is amino acids K-104 to V-113 of SEQ ID NO: 2.
41. The antibody or fragment thereof of claim 38, wherein said epitope of C35 is amino acids I-105 to V-113 of SEQ ID NO: 2.
42. The antibody or fragment thereof of claim 38, wherein said epitope of C35 is amino acids I-105 to I-114 of SEQ ID NO: 2.
43. The antibody or fragment thereof of claim 38, wherein said epitope of C35 is amino acids K-104 to C-112 of SEQ ID NO: 2.
44. The antibody or fragment thereof of claim 38, wherein said antibody or fragment thereof is selected from the group consisting of: a whole antibody, an Fab fragment, an F(ab′)2 fragment, an scFv fragment, and an Fv fragment.
45. The antibody or fragment thereof of claim 38, wherein said antibody is a fusion protein.
46. The antibody or fragment thereof of claim 38, wherein said antibody or fragment is humanized.
47. A method of treating a C35-associated cancer, said method comprising administering to a subject in need thereof a therapeutic amount of an antibody or antigen binding fragment thereof that specifically binds to an epitope of C35.
48. The method of claim 47, wherein said epitope of C35 is selected from the group consisting of: amino acids S-9 to V-17, amino acids V-10 to V-17, amino acids E-16 to V-23, amino acids E-16 to R-24, amino acids E-16 to I-25, amino acids S-21 to F-35, amino acids C-30 to T-38, amino acids E-31 to Y-39, amino acids E-36 to A-43, amino acids A-37 to A-45, amino acids A-37 to V-46, amino acids Y-39 to V-46, amino acids S-44 to I-53, amino acids A-45 to I-53; amino acids G-52 to L-59, amino acids E-54 to T-62, amino acids S-57 to F-75, amino acids R-58 to I-67, amino acids G-61 to I-69, amino acids G-63 to F-83, amino acids E-66 to L-73, amino acids E-66 to V-74, amino acids F-83 to E-103, amino acids D-88 to A-96, amino acids L-89 to A-96, amino acids A-92 to T-101, amino acids R-95 to L-102, amino acids A-96 to K-104, amino acids K-104 to V-113, amino acids I-105 to V-113, amino acids I-105 to I-114, amino acids E-4 to P-12, amino acids S-21 to Y-29, amino acids G-22 to C-30, amino acids I-25 to C-33, amino acids T-38 to V-46, amino acids G-63 to G-71, amino acids F-65 to L-73, amino acids I-67 to F-75, amino acids K-77 to Y-85, amino acids Q-72 to E-86, amino acids G-81 to L-89, and amino acids K-104 to C-112, each of SEQ ID NO:2.
49. The method of claim 48, wherein said epitope of C35 is amino acids A-96 to K-104 of SEQ ID NO: 2.
50. The method of claim 48, wherein said epitope of C35 is amino acids K-104 to V-113 of SEQ ID NO: 2.
51. The method of claim 48, wherein said epitope of C35 is amino acids I-105 to V-113 of SEQ ID NO: 2.
52. The method of claim 48, wherein said epitope of C35 is amino acids I-105 to I-114 of SEQ ID NO: 2.
53. The method of claim 48, wherein said epitope of C35 is K-104 to C-112.
54. The method of claim 47, wherein said method comprises administering two antibodies or fragments thereof.
55. The method of claim 47, wherein said method further comprises administering an effective amount of a therapeutic agent.
56. The method of claim 55, wherein said therapeutic agent is selected from the group consisting of: an anti-tumor drug, a cytotoxin, and a radioactive agent.
57. The method of claim 56, wherein said therapeutic agent is a cytotoxin.
58. The method of claim 57, wherein said cytotoxin is selected from the group consisting of: paclitaxel, ricin, doxorubicin, cytochalasin B, gramicidin D, ethidium bromide, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone, and glucocorticoid.
59. The method of claim 58, wherein said cytotoxin is paclitaxel.
60. The method of claim 55, wherein said therapeutic agent is conjugated to or complexed with said antibody or fragment thereof.
Description
  • [0001]
    This application is a divisional application of U.S. application Ser. No. 09/824,787, filed Apr. 4, 2001, which claims the benefit of U.S. Provisional Application No. 60/194,463, filed Apr. 4, 2000, the entire contents of each of which is herein incorporated by reference.
  • BACKGROUND OF THE INVENTION
  • [0002]
    1. Field of the Invention
  • [0003]
    The present invention relates to a novel human gene that is differentially expressed in human breast and bladder carcinoma. More specifically, the present invention relates to a polynucleotide encoding a novel human polypeptide named C35. This invention also relates to C35 polypeptides, as well as vectors, host cells, antibodies directed to C35 polypeptides, and the recombinant methods for producing the same. The present invention further relates to diagnostic methods for detecting carcinomas, including human breast and bladder carcinomas. The present invention further relates to the formulation and use of the C35 gene and polypeptides in immunogenic compositions or vaccines, to induce antibody and cell-mediated immunity against target cells, such as tumor cells, that express the C35 gene. The invention further relates to screening methods for identifying agonists and antagonists of C35 activity.
  • [0004]
    2. Background Art
  • [0005]
    Cancer afflicts approximately 1.2 million people in the United States each year. About 50% of these cancers are curable with surgery, radiation therapy, and chemotherapy. Despite significant technical advances in these three types of treatments, each year more than 500,000 people will die of cancer in the United States alone. (Jaffee, E. M., Ann. N.Y. Acad. Sci. 886:67-72 (1999)). Because most recurrences are at distant sites such as the liver, brain, bone, and lung, there is an urgent need for improved systemic therapies.
  • [0006]
    The goal of cancer treatment is to develop modalities that specifically target tumor cells, thereby avoiding unnecessary side effects to normal tissue. Immunotherapy has the potential to provide an alternative systemic treatment for most types of cancer. The advantage of immunotherapy over radiation and chemotherapy is that it can act specifically against the tumor without causing normal tissue damage. One form of immunotherapy, vaccines, is particularly attractive because they can also provide for active immunization, which allows for amplification of the immune response. In addition, vaccines can generate a memory immune response.
  • [0007]
    The possibility that altered features of a tumor cell are recognized by the immune system as non-self and may induce protective immunity is the basis for attempts to develop cancer vaccines. Whether or not this is a viable strategy depends on how the features of a transformed cell are altered. Appreciation of the central role of mutation in tumor transformation gave rise to the hypothesis that tumor antigens arise as a result of random mutation in genetically unstable cells. Although random mutations might prove immunogenic, it would be predicted that these would induce specific immunity unique for each tumor. This would be unfavorable for development of broadly effective tumor vaccines. An alternate hypothesis, however, is that a tumor antigen may arise as a result of systematic and reproducible tissue specific gene deregulation that is associated with the transformation process. This could give rise to qualitatively or quantitatively different expression of shared antigens in certain types of tumors that might be suitable targets for immunotherapy. Early results, demonstrating that the immunogenicity of some experimental tumors could be traced to random mutations (De Plaen, et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988); Srivastava, & Old, Immunol. Today 9:78 (1989)), clearly supported the first hypothesis. There is, however, no a priori reason why random mutation and systematic gene deregulation could not both give rise to new immunogenic expression in tumors. Indeed, more recent studies in both experimental tumors (Sahasrabudhe et al., J. Immunol. 151:6202-6310 (1993); Torigoe et al., J. Immunol. 147:3251 (1991)) and human melanoma (van Der Bruggen et al., Science 254:1643-1647 (1991); Brichard et al., J. Exp. Med. 178:489-495 (1993); Kawakami et al., Proc. Natl. Acad. Sci. USA 91:3515-3519 (1994); Boel et al., Immunity 2:167-175 (1995); Van den Eynde et al., J. Exp. Med. 182: 689-698 (1995)) have clearly demonstrated expression of shared tumor antigens encoded by deregulated normal genes. The identification of MAGE-1 and other antigens common to different human melanoma holds great promise for the future development of multiple tumor vaccines.
  • [0008]
    In spite of the progress in melanoma, very few shared antigens recognized by cytotoxic T cells have been described for other human tumors. The major challenge is technological. The most widespread and to date most successful approach to identify immunogenic molecules uniquely expressed in tumor cells is to screen a cDNA library with tumor-specific CTLs (cytotoxic T lymphocytes). Application of this strategy has led to identification of several gene families expressed predominantly in human melanoma. Two major limitations of this approach, however, are that (1) screening requires labor intensive transfection of numerous small pools of recombinant DNA into separate target populations, which themselves often need to be modified to express one or more MHC molecules required for antigen presentation, in order to assay T cell stimulation by a minor component of some pool; and (2) with the possible exception of renal cell carcinoma, tumor-specific CTLs have been very difficult to isolate from either tumor infiltrating lymphocytes (TIL) or PBL of patients with other types of tumors, especially the epithelial cell carcinomas that comprise greater than 80% of human tumors. It appears that there may be tissue specific properties that result in tumor-specific CTLs being sequestered in melanoma.
  • [0009]
    Direct immunization with tumor-specific gene products may be essential to elicit an immune response against some shared tumor antigens. It has been argued that, if a tumor expressed strong antigens, it should have been eradicated prior to clinical manifestation. Perhaps then, tumors express only weak antigens. Immunologists have long been interested in the issue of what makes an antigen weak or strong. There have been two major hypotheses. Weak antigens may be poorly processed and fail to be presented effectively to T cells. Alternatively, the number of T cells in the organism with appropriate specificity might be inadequate for a vigorous response (a so-called “hole in the repertoire”). Elucidation of the complex cellular process whereby antigenic peptides associate with MHC molecules for transport to the cell surface and presentation to T cells has been one of the triumphs of modern immunology. These experiments have clearly established that failure of presentation due to processing defects or competition from other peptides could render a particular peptide less immunogenic. In contrast, it has, for technical reasons, been more difficult to establish that the frequency of clonal representation in the T cell repertoire is an important mechanism of low responsiveness. Recent studies demonstrating that the relationship between immunodominant and cryptic peptides of a protein antigen change in T cell receptor transgenic mice suggest, however, that the relative frequency of peptide-specific T cells can, indeed, be a determining factor in whether a particular peptide is cryptic or dominant in a T cell response. This has encouraging implications for development of vaccines. With present day methods, it would be a complex and difficult undertaking to modify the way in which antigenic peptides of a tumor are processed and presented to T cells. The relative frequency of a specific T cell population can, however, be directly and effectively increased by prior vaccination. This could, therefore, be the key manipulation required to render an otherwise cryptic response immunoprotective. These considerations of cryptic or sub-dominant antigens have special relevance in relation to possible immune evasion by tumors through tolerance induction. Evidence has been presented to suggest that tumor-specific T cells in the tumor-bearing host are anergic, possibly as a result of antigen presentation on non-professional APC (Morgan, D. J. et al., J. Immunol. 163:723-27 (1999); Sotomayor, E. M. et al., Proc. Natl. Acad. Sci. U.S.A. 96:11476-81 (1999); Lee, P. P. et al., Nature Medicine 5:677-85 (1999)). Prior tolerization of T cells specific for immunodominant antigens of a tumor may, therefore, account for the difficulty in developing successful strategies for immunotherapy of cancer. These observations suggest that T cells specific for immunodominant tumor antigens are less likely to be effective for immunotherapy of established tumors because they are most likely to have been tolerized. It may, therefore, be that T cells specific for sub-dominant antigens or T cells that are initially present at a lower frequency would prove more effective because they have escaped the tolerizing influence of a growing tumor.
  • [0010]
    Another major concern for the development of broadly effective human vaccines is the extreme polymorphism of HLA class I molecules. Class I MHC:cellular peptide complexes are the target antigens for specific CD8+ CTLs. The cellular peptides, derived by degradation of endogenously synthesized proteins, are translocated into a pre-Golgi compartment where they bind to class I MHC molecules for transport to the cell surface. The CD8 molecule contributes to the avidity of the interaction between T cell and target by binding to the α3 domain of the class I heavy chain. Since all endogenous proteins turn over, peptides derived from any cytoplasmic or nuclear protein may bind to an MHC molecule and be transported for presentation at the cell surface. This allows T cells to survey a much larger representation of cellular proteins than antibodies which are restricted to recognize conformational determinants of only those proteins that are either secreted or integrated at the cell membrane.
  • [0011]
    The T cell receptor antigen binding site interacts with determinants of both the peptide and the surrounding MHC. T cell specificity must, therefore, be defined in terms of an MHC:peptide complex. The specificity of peptide binding to MHC molecules is very broad and of relatively low affinity in comparison to the antigen binding sites of specific antibodies. Class I-bound peptides are generally 8-10 residues in length and accommodate amino acid side chains of restricted diversity at certain key positions that match pockets in the MHC peptide binding site. These key features of peptides that bind to a particular MHC molecule constitute a peptide binding motif.
  • [0012]
    Hence, there exists a need for methods to facilitate the induction and isolation of T cells specific for human tumors, cancers and infected cells and for methods to efficiently select the genes that encode the major target antigens recognized by these T cells in the proper MHC-context.
  • BRIEF SUMMARY OF THE INVENTION
  • [0013]
    The present invention relates to a novel polynucleotide, C35, that is differentially expressed in human breast and bladder carcinoma, and to the encoded polypeptide of C35. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant methods for producing C35 polypeptides and polynucleotides. The present invention further relates to the formulation and use of C35 polypeptides and polynucleotides in immunogenic compositions to induce antibodies and cell-mediated immunity against target cells, such as tumor cells, that express the C35 gene products. Also provided are diagnostic methods for detecting disorders relating to the C35 genes and polypeptides, including use as a prognostic marker for carcinomas, such as human breast carcinoma, and therapeutic methods for treating such disorders. The invention further relates to screening methods for identifying binding partners of C35.
  • BRIEF DESCRIPTION OF THE FIGURES
  • [0014]
    FIG. 1 (Panels A-B). Panel A shows the DNA coding sequence (SEQ ID NO:1) of C35. The sequence immediately upstream of the predicted ATG start codon is shown in lower case and conforms to the expected features described by Kozak, M., J. Biol. Chem. 266(30):19867-19870 (1991). Panel B shows the deduced amino acid sequence (SEQ ID NO:2) of C35.
  • [0015]
    FIG. 2. (Panel A to C). Panel A: C35 is overexpressed in Breast tumor cell lines. Upper Panel: 300 ng of poly-A RNA from 3 week old human thymus, normal breast epithelial cell line H16N2 from patient 21, and 4 breast tumor cell lines derived one year apart from primary or metastatic nodules of the same patient 21; 21NT, 21PT 21MT1, and 21MT2, was resolved on a 1% agarose/formaldehyde gel and transferred to a GeneScreen membrane. This blot was hybridized with a 32P labeled C35 probe. Hybridization was detected by exposing the blot to film for 15 hours. Lower Panel: To quantitate RNA loading, the same blot was stripped and re-hybridized with a 32P labeled probe for Glyceraldehyde-3 Phosphate Dehydrogenase (GAPDH). For each sample the C35 signal was normalized to the GAPDH signal. The numbers represent the fold expression of C35 in each sample relative to H16N2. Panel B: C35 is expressed at low levels in normal tissues. A Blot containing 1 microgram of poly-A RNA from each of the indicated adult normal tissues (Clontech) was hybridized with a 32P labeled C35 probe. Hybridization was detected by exposing the blot to film for 15 hours (upper panel), or 96 hours (lower panel). Panel C. C35 is overexpressed in primary Breast tumors. A blot containing 2 micrograms of poly-A RNA from 3 primary infiltrating ductal mammary carcinoma, T1, T2, T3 and 1 normal breast epithelium, N (Invitrogen) was hybridized with a 32P labeled C35 probe. To normalize loading a 32P labeled beta-Actin probe was included in the hybridization mix. Hybridization was detected by exposing the blot to film for 6 hours. The numbers represent the fold expression of C35 in each sample relative to normal breast epithelium.
  • [0016]
    FIG. 3. Expression of C35 in Breast Tumor Cell Lines. C35 is overexpressed in different breast tumor cell lines. Upper Panel: 300 ng of poly-A RNA from BT474 (ATCC HYB-20, mammary ductal carcinoma), SKBR3 (ATCC HTB-30, mammary adenocarcinoma), T47D (ATCC HTB-133, mammary ductal carcinoma), normal breast epithelial cell line H16N2 from patient 21, and 21-NT breast tumor cell line derived from primary tumor nodule of the same patient 21 was resolved on a 1% agarose/formaldehyde gel and transferred to a GeneScreen membrane. This blot was hybridized with a 32P labeled C35 probe. Hybridization was detected by exposing the blot to film for 15 hours. Lower Panel: To quantitate RNA loading, the same blot was stripped and re-hybridized with a 32P labeled probe for beta-actin. For each sample the C35 signal was normalized to the actin signal. The numbers represent the fold expression of C35 in each sample relative to H16N2.
  • [0017]
    FIG. 4 (Panels A-C): Surface Expression of C35 Protein Detected by Flow Cytometry. 1×105 breast tumor cells were stained with 3.5 microliters of antiserum raised in BALB/c mice against Line 1 mouse tumor cells transduced with retrovirus encoding human C35 or control, pre-bleed BALB/c serum. After a 30 minute incubation, cells were washed twice with staining buffer (PAB) and incubated with FITC-goat anti-mouse IgG (1 μg/sample) for 30 minutes. Samples were washed and analyzed on an EPICS Elite flow cytometer. Panel A: 21NT Panel B: SKBR3. Panel C: MDA-MB-231. These three breast tumor lines were selected to represent tumor cells that express high, intermediate and low levels of C35 RNA on Northern blots (see FIG. 3). Abbreviations: nms, ns; normal mouse serum; C35; C35 immune serum.
  • [0018]
    FIG. 5 (Panels A and B). CML Selected Recombinant Vaccinia cDNA Clones Stimulate Tumor Specific CTL. Panel A: CML Selected vaccinia clones were assayed for the ability, following infection of B/C.N, to stimulate tumor specific CTL to secrete interferon gamma. The amount of cytokine was measured by ELISA, and is represented as OD490 (14). An OD490 of 1.4 is approximately equal to 4 ng/ml of IFNg, and an OD490 of 0.65 is approximately equal to 1 ng/ml of IFNg. Panel B: CML selected clones sensitize host cells to lysis by tumor specific CTL. Monolayers of B/C.N in wells of a 6 well plate were infected with moi=1 of the indicated vaccinia virus clones. After 14 hours of infection the infected cells were harvested and along with the indicated control targets labeled with 51Cr. Target cells were incubated with the indicated ratios of tumor specific Cytotoxic T Lymphocytes for 4 hours at 37° C. and percentage specific lysis was determined (15). This experiment was repeated at least three times with similar results.
  • [0019]
    FIG. 6 (Panels A and B). The Tumor Antigen Is Encoded by a Ribosomal Protein L3 Gene. Sequence of H2.16 and rpL3 from amino acid position 45 to 56. Panel A: The amino acid (in single letter code) and nucleotide sequence of cDNA clone rpL3 (GenBank Accession no. Y00225). Panel B: A single nucleotide substitution at C170T of the H2.16 tumor cDNA is the only sequence change relative to the published L3 ribosomal allele. This substitution results in a T54I amino acid substitution in the protein.
  • [0020]
    FIG. 7 (Panels A and B). Identification of the Peptide Epitope Recognized by the Tumor Specific CTL. Panel A: CML assay to identify the peptide recognized by tumor specific CTL. Target cells were labeled with 51Cr (15). During the 51Cr incubation samples of B/C.N cells were incubated with 1 μM peptide L348-56(I54), 100 μM L348-56(T54) or 100 μM peptide L345-54(I54). Target cells were incubated with the indicated ratios of tumor specific Cytotoxic T Lymphocytes for 4 hours at 37° C. and percentage specific lysis was determined. This experiment was repeated at least three times with similar results. Panel B: Titration of peptide L348-56 (I54). Target cells were labeled with 51Cr. During the 51Cr incubation samples of B/C.N cells were incubated either with no peptide addition (D) or with the indicated concentrations (1 μM, 10 nM, 1 nM) of L348-56(I54) (▪), BCA 39 cells were included as a positive control (▴). Target cells were incubated with the indicated ratios of Tumor Specific Cytotoxic T Lymphocytes for 4 hours at 37° C. and percentage specific lysis was determined. The experiment was repeated twice with similar results.
  • [0021]
    FIG. 8 (Panels A-C). Analysis of L3 Expressed by Each Cell Line. Panel A: Sau3AI map of published rpL3 and H2.16. Shown above is the Sau3AI restriction map for the published ribosomal protein L3 gene (Top), and for H2.16 (Bottom). Digestion of cDNA for the published L3 sequence generates fragments of 200, 355, 348, 289, and 84 bp. The pattern for H2.16 is identical except for an extra Sau3AI site at position 168 caused by the C170T. This results in a 168 bp digestion product in place of the 200 bp fragment. Panel B: The BCA tumors express both L3 alleles. RT-PCR products generated from each cell line or from vH2.16 were generated using L3 specific primers and then digested with Sau3AI, and resolved on a 3% agarose gel for 2 hours at 80 volts. Panel C: The Immunogenic L3 allele is expressed at greatly reduced levels in B/C.N, BCB13, and Thymus. L3 specific RT-PCR products from each indicated sample were generated using a 32P end labeled 5 prime PCR primer. No PCR product was observed when RNA for each sample was used as template for PCR without cDNA synthesis, indicating that no sample was contaminated with genomic DNA. The PCR products were gel purified to ensure purity, digested with Sau3AI, and resolved on a 3% agarose gel for 15 hours at 60 volts. No PCR product was observed in a control PCR sample that had no template added to it. This result has been reproduced a total of 3 times.
  • [0022]
    FIG. 9 (Panels A-C). Immunization with iL3 is Immunoprotective. Panel A: Immunization with H2.16 induces tumor specific CTL. Balb/c mice (2/group) were immunized by subcutaneous injection with 5×106 pfu of vH2.16, or control vector v7.5/tk. Seven days later splenocytes were harvested and restimulated with peptide L348-56(I54) (26). Five days following the second restimulation the lymphocytes were tested in a chromium release assay as described in FIG. 11. The L348-56(I54) peptide was used at a 1 micromolar concentration, and the L348-56(T54) peptide was used at a 100 micromolar concentration. Similar results were obtained when the immunization experiment was repeated. Panels B and C: Female Balb/cByJ mice were immunized as indicated (27). The mice were challenged by SC injection with 200,000 viable BCA 34 tumor cells into the abdominal wall. Data is from day 35 post challenge. These data are representative of 4 independent experiments.
  • [0023]
    FIG. 10 (Panels A and B). Panel A: C35 coding sequence with translation; 5′ and 3′ untranslated regions are shown in lowercase letters. The predicted prenylation site, CVIL, at the 3′ terminus is boxed. Panel B: Genomic alignment of C35 gene on chromosome 17.
  • [0024]
    FIG. 11 (Panels A and B). C35 Expression in Breast Carcinoma. C35 was labeled with 32P in a random priming reaction and hybridized to Northern blots at 106 cpm/ml. Each blot was stripped and re-probed with GAPDH or Beta-actin to normalize MRNA loads. The numbers indicate densitometry ratios normalized against GAPDH/Beta-actin. A value of 1 has been assigned to normal cell line H16N2, and all values are relative to the level of expression in the normal cell line. Panel A: C35 expression in breast epithelial cell lines. Panel B: C35 expression in primary breast tissue/tumors. 300 ng mRNA was electrophoresed on 0.8% alkaline agarose gels, then blotted to Genescreen Plus, except leftmost panel of B loaded with 1 μg MRNA from 3 primary tumors and 1 normal tissue control (Real Tumor Blots, Invitrogen). Similar exposures are shown for all blots.
  • [0025]
    FIG. 12. C35 Expression in Bladder Carcinoma. C35 was labeled with 32P in a random priming reaction and hybridized to a Northern blot of tumor and normal RNA at 106 cpm/ml. The blot was stripped and re-probed with Beta-actin to normalize mRNA loads. The numbers indicate densitometry ratios normalized against Beta-actin. Values are relative to the level of expression in the normal bladder samples. 300 ng mRNA was electrophoresed on 0.8% alkaline agarose gels, then blotted to Genescreen Plus.
  • [0026]
    FIG. 13 (Panels A and B). FACS Analysis with Anti-C35 Antibodies. Panel A: Breast cell lines were stained with (top panel) sera from mice immunized with Line 1 cells infected with C35 recombinant retrovirus, and (bottom panel) 2C3 purified monoclonal antibody or isotype control. Panel B: Bladder cell lines stained with 2C3 purified monoclonal antibody or isotype control.
  • [0027]
    FIG. 14. Inhibition of Tumor Growth in Presence of 2C3 Antibody. 21NT breast tumor cells or H16N2 normal breast epithelial cells were incubated with the indicated concentrations of 2C3 anti-C35 monoclonal antibody or a non-specific isotype control antibody. Cell growth was measured by XTT assay following 72 hour incubation in the presence or absence of antibodies.
  • [0028]
    FIG. 15 (Panels A and B). CTL stimulated with C35 expressing dendritic cells specifically lyse C35+ Breast (21NT) and Bladder (ppT11A3) tumor cell lines, with minimal activity against normal breast (MEC), immortalized non-tumorigenic breast (H16N2) and bladder (SV-HUC) cell lines, or an NK sensitive cell line (K562). Panel A: T cell line 4 was generated from normal human PBL. Panel B: T cell clone 10G3 was selected from line 4 for C35-specific activity. Target cell lines MEC, ppT11A3 and SV-HUC are naturally HLA-A2 positive. Target cell lines 21NT and H16N2 were transected with HLA-A2 to provide a required MHC restriction element.
  • [0029]
    FIG. 16 (Panels A and B). Cytokine Release from T Cell Clone 10G3 upon Stimulation with Targets. Panel A: IFN-gamma secretion. Panel B: TNF-alpha secretion. Breast and bladder target cell lines were distinguished by the presence or absence of expression of HLA-A2 and C35 tumor antigen, an amino terminal 50 amino acid fragment of C35 (C35-50aa), or the irrelevant mouse L3 ribosomal protein. Each marker was either endogenously expressed or introduced by transfection of an HLA-A2.1 construct (pSV2.A2), or by infection with a vaccinia recombinant of C35 (vv.C35, vv.C35-50aa), L3 (vv.L3), or HLA-A2 (vv.A2)
  • [0030]
    FIG. 17 (Panels A (top) and B (Bottom)). Effect of anti-CD40 ligand antibody (anti-CD154) in blocking the reactivity of murine T cells to specific transplantation antigens. Significant cytotoxicity was induced against the control C3H alloantigens in both saline and anti-CD154 treated mice (Panel A (top), whereas a cytotoxic response to C57B1/6 was induced in the saline treated mice but not the anti-CD154 treated mice (Panel B (bottom)).
  • DETAILED DESCRIPTION OF THE INVENTION
  • [0000]
    Definitions
  • [0031]
    The following definitions are provided to facilitate understanding of certain terms used throughout this specification.
  • [0032]
    In the present invention, “isolated” refers to material removed from its native environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
  • [0033]
    In the present invention, a “membrane” C35 protein is one expressed on the cell surface through either direct or indirect association with the lipid bilayer, including, in particular, through prenylation of a carboxyl-terminal amino acid motif. Prenylation involves the covalent modification of a protein by the addition of either a famesyl or geranylgeranyl isoprenoid. Prenylation occurs on a cysteine residue located near the carboxyl-terminus of a protein. The C35 polypeptide contains the amino acids Cys-Val-Ile-Leu at positions 112-115, with the Leu being the C terminal residue of the polypeptide. The motif Cys-X-X-Leu, where “X” represents any aliphatic amino acid, results in the addition of a 20 carbon geranylgeranyl group onto the Cys residue. Generally, following addition of this lipid the three terminal amino acid residues are cleaved off the polypeptide, and the lipid group is methylated. Prenylation promotes the membrane localization of most proteins, with sequence motifs in the polypeptide being involved in directing the prenylated protein to the plasma, nuclear, or golgi membranes. Prenylation plays a role in protein-protein interactions, and many prenylated proteins are involved in signal transduction. Examples of prenylated proteins include Ras and the nuclear lamin B. (Zhang, F. L. and Casey, P. J., Ann. Rev. Biochem. 65:241-269 (1996)). The C35 protein has been detected on the surface of two breast tumor cell lines by fluorescence analysis employing as a primary reagent a mouse anti-human C35 antiserum (FIG. 4).
  • [0034]
    In the present invention, a “secreted” C35 protein refers to a protein capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as a C35 protein released into the extracellular space without necessarily containing a signal sequence. If the C35 secreted protein is released into the extracellular space, the C35 secreted protein can undergo extracellular processing to produce a “mature” C35 protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
  • [0035]
    As used herein, a C35 “polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:1. For example, the C35 polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a C35 “polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.
  • [0036]
    In specific embodiments, the polynucleotides of the invention are less than 300 nt, 200 nt, 100 nt, 50 nt, 15 nt, 10 nt, or 7 nt in length. In a further embodiment, polynucleotides of the invention comprise at least 15 contiguous nucleotides of C35 coding sequence, but do not comprise all or a portion of any C35 intron. In another embodiment, the nucleic acid comprising C35 coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the C35 gene in the genome).
  • [0037]
    In the present invention, the full length C35 coding sequence is identified as SEQ ID NO: 1.
  • [0038]
    A C35 “polynucleotide” also refers to isolated polynucleotides which encode the C35 polypeptides, and polynucleotides closely related thereto.
  • [0039]
    A C35 “polynucleotide” also refers to isolated polynucleotides which encode the amino acid sequence shown in SEQ ID NO: 2, or a biologically active fragment thereof.
  • [0040]
    A C35 “polynucleotide” also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO: 1, the complement thereof, or the cDNA within the deposited clone. “Stringent hybridization conditions” refers to an overnight incubation at 42Λ in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65Λ
  • [0041]
    Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3′ terminal polyA+ tract of a cDNA), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
  • [0042]
    The C35 polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, C35 polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the C35 polynucleotides can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. C35 polynucleotides may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
  • [0043]
    C35 polypeptides can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The C35 polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in the C35 polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given C35 polypeptide. Also, a given C35 polypeptide may contain many types of modifications. C35 polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic C35 polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, Proteins—Structure And Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
  • [0044]
    “SEQ ID NO: 1” refers to a C35 polynucleotide sequence while “SEQ ID NO: 2” refers to a C35 polypeptide sequence.
  • [0045]
    A C35 polypeptide “having biological activity” refers to polypeptides exhibiting activity similar to, but not necessarily identical to, an activity of a C35 polypeptide, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the C35 polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the C35 polypeptide (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the C35 polypeptide.)
  • [0000]
    C35 Polynucleotides and Polypeptides
  • [0046]
    A 348 base pair fragment of C35 was initially isolated by subtractive hybridization of poly-A RNA from tumor and normal mammary epithelial cell lines derived from the same patient with primary and infiltrating intraductal mammary carcinoma. Band, V. et al., Cancer Res. 50:7351-7357 (1990). Employing primers based on this sequence and that of an overlapping EST sequence (Accession No. W57569), a cDNA that includes the full-length C35 coding sequence was then amplified and cloned from the BT-20 breast tumor cell line (ATCC, HTB-19). This C35 cDNA contains the entire coding region identified as SEQ ID NO:1. The C35 clone includes, in addition to the 348 bp coding sequence, 167 bp of 3′ untranslated region. The open reading frame begins at an N-terminal methionine located at nucleotide position 1, and ends at a stop codon at nucleotide position 348 (FIG. 1). A representative clone containing all or most of the sequence for SEQ ID NO:1 was deposited with the American Type Culture Collection (“ATCC”) on Aug. 1, 2000, and was given the ATCC Deposit Number PTA-2310. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.
  • [0047]
    Therefore, SEQ ID NO: 1 and the translated SEQ ID NO: 2 are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO: 1 is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO: 1 or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:2 may be used to generate antibodies which bind specifically to C35, or to stimulate T cells which are specific for C35 derived peptides in association with MHC molecules on the cell surface.
  • [0048]
    Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
  • [0049]
    Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:1 and the predicted translated amino acid sequence identified as SEQ ID NO:2. The nucleotide sequence of the deposited C35 clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted C35 amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by the deposited clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human C35 cDNA, collecting the protein, and determining its sequence.
  • [0050]
    The present invention also relates to the C35 gene corresponding to SEQ ID NO:1, or the deposited clone. The C35 gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the C35 gene from appropriate sources of genomic material.
  • [0051]
    Also provided in the present invention are species homologs of C35. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homologue.
  • [0052]
    By “C35 polypeptide(s)” is meant all forms of C35 proteins and polypeptides described herein. The C35 polypeptides can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
  • [0053]
    The C35 polypeptides may be in the form of the membrane protein or a secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.
  • [0054]
    C35 polypeptides are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a C35 polypeptide, including the secreted polypeptide, can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). C35 polypeptides also can be purified from natural or recombinant sources using antibodies of the invention raised against the C35 protein in methods which are well known in the art.
  • [0000]
    Polynucleotide and Polypeptide Variants
  • [0055]
    “Variant” refers to a polynucleotide or polypeptide differing from the C35 polynucleotide or polypeptide, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the C35 polynucleotide or polypeptide.
  • [0056]
    By a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the C35 polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence shown of SEQ ID NO:1, the ORF (open reading frame), or any fragment specified as described herein.
  • [0057]
    As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci. 6:237-245 (1990). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.
  • [0058]
    If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
  • [0059]
    For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
  • [0060]
    By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • [0061]
    As a practical matter, whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in SEQ ID NO:2 or to the amino acid sequence encoded by deposited DNA clone, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci. 6:237-245 (1990). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.
  • [0062]
    If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.
  • [0063]
    For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
  • [0064]
    The C35 variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. C35 polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
  • [0065]
    Naturally occurring C35 variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) Also, allelic variants can occur as “tandem alleles” which are highly homologous sequences that occur at different loci on chromosomes of an organism. These allelic variants can vary at either the polynucleotide and/or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
  • [0066]
    Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the C35 polypeptides. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).
  • [0067]
    Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” (See, Abstract.) In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
  • [0068]
    Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
  • [0069]
    Thus, the invention further includes C35 polypeptide variants which show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as to have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
  • [0070]
    The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
  • [0071]
    The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.
  • [0072]
    As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
  • [0073]
    Besides conservative amino acid substitution, variants of C35 include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
  • [0074]
    For example, C35 polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)
  • [0000]
    Polynucleotide and Polypeptide Fragments
  • [0075]
    In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence contained in the deposited clone or shown in SEQ ID NO:1. The short nucleotide fragments are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in the deposited clone or the nucleotide sequence shown in SEQ ID NO:1. These nucleotide fragments are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., at least 50, 100, 150, 200, 250, 300 nucleotides) are preferred.
  • [0076]
    Moreover, representative examples of C35 polynucleotide fragments include, for example, fragments having a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, or 301 to the end of SEQ ID NO:1 or the cDNA contained in the deposited clone. In this context “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein.
  • [0077]
    In the present invention, a “polypeptide fragment” refers to a short amino acid sequence contained in SEQ ID NO:2 or encoded by the cDNA contained in the deposited clone. Protein fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, or 101 to the end of the coding region. Moreover, polypeptide fragments can comprise 9, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length. In this context “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
  • [0078]
    Preferred polypeptide fragments include the secreted C35 protein as well as the mature form. Further preferred polypeptide fragments include the secreted C35 protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both.
  • [0079]
    As mentioned above, even if deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of shortened C35 muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a C35 mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as 9 C35 amino acid residues may often evoke an immune response.
  • [0080]
    Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the C35 amino acid sequence shown in SEQ ID NO:2, up to the Threonine residue at position number 105 and polynucleotides encoding such polypeptides.
  • [0081]
    Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of the shortened C35 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a C35 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities.
  • [0082]
    Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the C35 polypeptide shown in SEQ ID NO:2, up to the valine residue at position number 10, and polynucleotides encoding such polypeptides
  • [0083]
    Moreover, the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini. In preferred embodiments, the invention is directed to polypeptides having residues: S-9 to V-17; V-10 to V-17; E-16 to V-23; E-16 to R-24; E-16 to I-25; S-21 to F-35; C-30 to T-38; E-31 to Y-39; E-36 to A-43; A-37 to A-45; A-37 to V-46; Y-39 to V-46; S-44 to I-53; A-45 to I-53; G-52 to L-59; E-54 to T-62; S-57 to F-75; R-58 to I-67; G-61 to I-69; G-63 to F-83; E-66 to L-73; E-66 to V-74; F-83 to E-103; D-88 to A-96; L-89 to A-96; A-92 to T-101; R-95 to L-102; A-96 to K-104; K-104 to V-113; I-105 to V-113; I-105 to I-114 of SEQ ID NO:2, and polynucleotides encoding such polypeptides.
  • [0084]
    Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases.
  • [0085]
    The human EST sequences referred to below were identified in a BLAST search of the EST database. These sequences are believed to be partial sequences of the cDNA inserts identified in the recited GenBank accession numbers. No homologous sequences were identified in a search of the annotated GenBank database. The Expect value (E) is a parameter that describes the number of hits one can “expect” to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences. In BLAST 2.0, the Expect value is also used instead of the P value (probability) to report the significance of matches. For example, an E value of 1 assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see 1 match with a similar score simply by chance.
  • [0086]
    For example, the following sequences are related to SEQ ID NO:1, GenBank Accession Nos.: AA971857 (SEQ ID NO:3); W57569 (SEQ ID NO:4); AI288765 (SEQ ID NO:5); W65390 (SEQ ID NO:6); W37432 (SEQ ID NO: 7); N42748 (SEQ ID NO:8); AA971638 (SEQ ID NO:9); R22331 (SEQ ID NO:10); AA308370 (SEQ ID NO:11); AA285089 (SEQ ID NO:12); R68901 (SEQ ID NO:13); AA037285 (SEQ ID NO:14); H94832 (SEQ ID NO:15); H96058 (SEQ ID NO:16); H56522 (SEQ ID NO:17); AA935328 (SEQ ID NO: 18); AW327450 (SEQ ID NO:19); AW406075 (SEQ ID NO:20); AW406223 (SEQ ID NO:21); AI909652 (SEQ ID NO:22); AA026773 (SEQ ID NO: 23); H96055 (SEQ ID NO:24); H12836 (SEQ ID NO:25); R22401 (SEQ ID NO:26); N34596 (SEQ ID NO:27); W32121 (SEQ ID NO:28); T84927 (SEQ ID NO:29); R63575 (SEQ ID NO:30); R23139 (SEQ ID NO:31); AA337071 (SEQ ID NO:32); AA813244 (SEQ ID NO:33); AA313422 (SEQ ID NO:34); N31910 (SEQ ID) NO:35); N42693 (SEQ ID NO:36); N32532 (SEQ ID NO:37); AA375119 (SEQ ID NO:38); R32153 (SEQ ID NO:39); R23369 (SEQ ID NO:40); AA393628 (SEQ ID NO:41); H12779 (SEQ ID NO:42); AI083674 (SEQ ID NO:43); AA284919 (SEQ ID NO:44); AA375286 (SEQ ID NO:45); AA830592 (SEQ ID NO:46); H95363 (SEQ ID NO:47); T92052 (SEQ ID NO:48); AI336555 (SEQ ID NO:49); AI285284 (SEQ ID NO:50); AA568537 (SEQ ID NO:51); AI041967 (SEQ ID NO:52); W44577 (SEQ ID NO:53); R22332 (SEQ ID NO:54); N27088 (SEQ ID NO:55); H96418 (SEQ ID NO:56); AI025384 (SEQ ID NO:57); AA707623 (SEQ ID NO:58); AI051009 (SEQ ID NO:59); AA026774 (SEQ ID NO:60); W51792 (SEQ ID NO:61); AI362693 (SEQ ID NO:62); AA911823 (SEQ ID NO:63); H96422 (SEQ ID NO:64); AI800991 (SEQ ID NO:65); AI525314 (SEQ ID NO:66); AI934846 (SEQ ID NO:67); AI937133 (SEQ ID NO:68); AW006797 (SEQ ID NO:69); AI914716 (SEQ ID NO:70); AI672936 (SEQ ID NO:71); W61294 (SEQ ID NO:72); AI199227 (SEQ ID NO:73); AI499727 (SEQ ID NO:74); R32154 (SEQ ID NO:75); AI439771 (SEQ ID NO:76); AA872671 (SEQ ID NO:77); AA502178 (SEQ ID NO:78); N26715 (SEQ ID NO:79); AA704668 (SEQ ID NO:80); R68799 (SEQ ID NO:81); H56704 (SEQ ID NO:82); A1360416 (SEQ ID NO:83).
  • [0087]
    Thus, in one embodiment the present invention is directed to polynucleotides comprising the polynucleotide fragments and full-length polynucleotide (e.g. the coding region) described herein exclusive of one or more of the above-recited ESTs.
  • [0088]
    Also preferred are C35 polypeptide and polynucleotide fragments characterized by structural or functional domains. Preferred embodiments of the invention include fragments that comprise MHC binding epitopes and prenylation sites.
  • [0089]
    Other preferred fragments are biologically active C35 fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the C35 polypeptide. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
  • [0000]
    Epitopes & Antibodies
  • [0090]
    Cellular peptides derived by degradation of endogenously synthesized proteins are translocated into a pre-Golgi compartment where they bind to Class I MHC molecules for transport to the cell surface. These class I MHC:peptide complexes are the target antigens for specific CD8+ cytotoxic T cells. Since all endogenous proteins “turn over,” peptides derived from any cytoplasmic or nuclear protein may bind to an MHC molecule and be transported for presentation at the cell surface. This allows T cells to survey a much larger representation of cellular proteins than antibodies which are restricted to recognize conformational determinants of only those proteins that are either secreted or integrated at the cell membrane. The T cell receptor antigen binding site interacts with determinants of both the peptide and the surrounding MHC. T cell specificity must, therefore, be defined in terms of an MHC:peptide complex. The specificity of peptide binding to MHC molecules is very broad and of relatively low affinity in comparison to the antigen binding site of specific antibodies. Class I-bound peptides are generally 8-10 residues in length that accommodate amino acid side chains of restricted diversity at certain key positions that match pockets in the MHC peptide binding site. These key features of peptides that bind to a particular MHC molecule constitute a peptide binding motif.
  • [0091]
    A number of computer algorithms have been described for identification of peptides in a larger protein that may satisfy the requirements of peptide binding motifs for specific MHC class I or MHC class II molecules. Because of the extensive polymorphism of MHC molecules, different peptides will often bind to different MHC molecules. Tables 1-3 list C35 peptides predicted to be MHC binding peptides using three different algorithms. Specifically, Tables 1 and 5 list C35 HLA Class I and II epitopes predicted using the rules found at the SYFPEITHI website and are based on the book “MHC Ligands and Peptide Motifs” by Rammensee, H. G., Bachmann, J. and Stevanovic, S. (Chapman & Hall, New York 1997). Table 2 lists predicted MHC binding peptides derived from the C35 sequence using the NIH BIMAS program available on the web. Finally, Tables 3 and 6 list predicted C35 peptides identified by the Tepitope program, a program for prediction of peptides that may bind to multiple different MHC class II molecules. Using Tepitope, four C35 peptides were identified as likely candidates for binding to a variety of HLA class II molecules. These peptides are, in general, longer than those binding to HLA class I and more degenerate in terms of binding to multiple HLA class II molecules. Unless expressly noted otherwise, all peptide sequences listed in Tables 1-6 refer to C35 peptide sequences appearing in SEQ ID NO:2 at the amino acid positions noted.
    TABLE 1
    C35 peptides predicted by SYFPEITHI website
    (score reflects ligation strength):
    Position 1 2 3 4 5 6 7 8 9 Score
    Class I MHC HLA-A*0201 nonamers
    9 S V A P P P E E V 23
    88 D L I E A I R R A 21
    37 A T Y L E L A S A 19
    97 S N G E T L E K I 18
    105 I T N S R P P C V 18
    2 S G E P G Q T S V 17
    45 A V K E Q Y P G I 17
    38 T Y L E L A S A V 16
    61 G T G A F E I E I 16
    85 Y E K D L I E A I 16
    65 F E I E I N G Q L 15
    107 N S R P P C V I L 15
    41 E L A S A V K E Q 14
    58 R L G G T G A F E 14
    59 L G G T G A F E I 14
    66 E I E I N G Q L V 14
    68 E I N G Q L V F S 14
    81 G G F P Y E K D L 14
    94 R R A S N G E T L 14
    HLA-A*0201 decamers
    58 R L G G T G A F E I 22
    96 A S N G E T L E K I 19
    104 K I T N S R P P C V 19
    37 A T Y L E L A S A V 18
    17 V E P G S G V R I V 17
    33 C G F E A T Y L E L 16
    44 S A V K E Q Y P G I 16
    92 A I R R A S N G E T 16
    39 Y L E L A S A V K E 15
    53 I E I E S R L G G T 15
    65 F E I E I N G Q L V 15
    105 I T N S R P P C V I 15
    1 M S G E P G Q T S V 14
    63 G A F E I E I N G Q 14
    68 E I N G Q L V F S K 14
    69 I N G Q L V F S K L 14
    83 F P Y E K D L I E A 14
    88 D L I E A I R R A S 14
    93 I R R A S N G E T L 14
    72 Q L V F S K L E N G 13
    89 L I E A I R R A S N 13
    8 T S V A P P P E E V 12
    16 E V E P G S G V R I 12
    50 Y P G I E I E S R L 12
    60 G G T G A F E I E I 12
    81 G G F P Y E K D L I 12
    106 T N S R P P C V I L 12
    HLA-A*0203 nonamers
    35 F E A T Y L E L A 12
    HLA-A*0203 decamers
    36 E A T Y L E L A S A 18
    HLA-A1 nonamers
    77 K L E N G G F P Y 29
    2 S G E P G Q T S V 18
    21 S G V R I V V E Y 18
    16 E V E P G S G V R 17
    29 Y C E P C G F E A 17
    42 L A S A V K E Q Y 17
    31 E P C G F E A T Y 16
    34 G F E A T Y L E L 16
    39 Y L E L A S A V K 14
    84 P Y E K D L I E A 14
    66 E I E I N G Q L V 13
    13 P P E E V E P G S 12
    46 V K E Q Y P G I E 12
    52 G I E I E S R L G 12
    96 A S N G E T L E K 12
    HLA-A1 decamers
    20 G S G V R I V V E Y 20
    29 Y C E P C G F E A T 19
    76 S K L E N G G F P Y 18
    2 S G E P G Q T S V A 17
    52 G I E I E S R L G G 17
    66 E I E I N G Q L V F 17
    41 E L A S A V K E Q Y 16
    46 V K E Q Y P G I E I 16
    16 E V E P G S G V R I 15
    30 C E P C G F E A T Y 15
    39 Y L E L A S A V K E 15
    77 K L E N G G F P Y E 14
    86 E K D L I E A I R R 14
    98 N G E T L E K I T N 14
    34 G F E A T Y L E L A 12
    64 A F E I E I N G Q L 12
    101 T L E K I T N S R P 12
    HLA-A26 nonamers
    68 E I N G Q L V F S 24
    100 E T L E K I T N S 24
    88 D L I E A I E R A 23
    54 E I E S R L G G T 22
    41 E L A S A V K E Q 21
    45 A V K E Q Y P G I 20
    31 E P C G F E A T Y 19
    34 G F E A T Y L E L 19
    73 L V F S K L E N G 19
    16 E V E P G S G V R 18
    77 K L E N G G F P Y 18
    66 E I E I N G Q L V 17
    21 S G V R I V V E Y 16
    37 A T Y L E L A S A 16
    24 R I V V E Y C E P 15
    9 S V A P P P E E V 14
    22 G V R I V V E Y C 14
    51 P G I E I E S R L 14
    70 N G Q L V F S K L 14
    57 S R L G G T G A F 13
    65 F E I E I N G Q L 13
    25 I V V E Y C E P C 12
    48 E Q Y P G I E I E 12
    67 I E I N G Q L V F 12
    75 F S K L E N G G F 12
    81 G G F P Y E K D L 12
    104 K I T N S R P P C 12
    105 I T N S R P P C V 12
    HLA-A26 decamers
    41 E L A S A V K E Q Y 27
    66 E I E I N G Q L V F 26
    68 E I N G Q L V F S K 23
    26 V V E Y C E P C G F 21
    16 E V E P G S G V R I 20
    88 D L I E A I R R A S 19
    100 E T L E K I T N S R 19
    74 V F S K L E N G G F 18
    33 C G F E A T Y L E L 17
    54 E I E S R L G G T G 17
    56 E S R L G G T G A F 17
    20 G S G V R I V V E Y 16
    31 E P C G F E A T Y L 16
    64 A F E I E I N G Q L 15
    69 I N G Q L V F S K L 15
    61 G T G A F E I E I N 14
    73 L V F S K L E N G G 14
    9 S V A P P P E E V E 13
    25 I V V E Y C E P C G 13
    45 A V K E Q Y P G I E 13
    72 Q L V F S K L E N G 13
    77 K L E N G G F P Y E 13
    79 E N G G F P Y E K D 13
    4 E P G Q T S V A P P 12
    7 Q T S V A P P P E E 12
    30 C E P C G F E A T Y 12
    36 E A T Y L E L A S A 12
    37 A T Y L E L A S A V 12
    76 S K L E N G G F P Y 12
    89 L I E A I R R A S N 12
    HLA-A3 nonamers
    39 Y L E L A S A V K 28
    77 K L E N G G F P Y 25
    16 E V E P G S G V R 24
    58 R L G G T G A F E 22
    67 I E I N G Q L V F 19
    96 A S N G E T L E K 18
    92 A I R R A S N G E 17
    9 S V A P P P E E V 16
    101 T L E K I T N S R 16
    22 G V R I V V E Y C 15
    31 E P C G F E A T Y 15
    45 A V K E Q Y P G I 15
    72 Q L V F S K L E N 15
    21 S G V R I V V E Y 14
    68 E I N G Q L V F S 14
    69 I N G Q L V F S K 14
    88 D L I E A I R R A 14
    91 E A I R R A S N G 14
    25 I V V E Y C E P C 13
    37 A T Y L E L A S A 13
    55 I E S R L G G T G 13
    57 S R L G G T G A F 13
    79 E N G G F P Y E K 13
    87 K D L I E A I R R 13
    104 K I T N S R P P C 13
    24 R I V V E Y C E P 12
    42 L A S A V K E Q Y 12
    66 E I E I N G Q L V 12
    89 L I E A I R R A S 12
    90 I E A I R R A S N 12
    94 R R A S N G E T L 12
    HLA-A3 decamers
    68 E I N G Q L V F S K 22
    16 E V E P G S G V R I 20
    38 T Y L E L A S A V K 20
    41 E L A S A V K E Q Y 20
    66 E I E I N G Q L V F 20
    9 S V A P P P E E V E 19
    58 R L G G T G A F E I 19
    39 Y L E L A S A V K E 18
    92 A I R R A S N G E T 18
    95 R A S N G E T L E K 18
    45 A V K E Q Y P G I E 17
    54 E I E S R L G G T G 16
    88 D L I E A I R R A S 16
    89 L I E A I R R A S N 16
    26 V V E Y C E P C G F 15
    37 A T Y L E L A S A V 15
    22 G V R I V V E Y C E 14
    77 K L E N G G F P Y E 14
    93 I R R A S N G E T L 14
    25 I V V E Y C E P C G 13
    30 C E P C G F E A T Y 13
    52 G I E I E S R L G G 13
    76 S K L E N G G F P Y 13
    78 L E N G G F P Y E K 13
    101 T L E K I T N S R P 13
    104 K I T N S R P P C V 13
    24 R I V V E Y C E P C 12
    72 Q L V F S K L E N G 12
    HLA-B*0702 nonamers
    18 E P G S G V R I V 19
    107 N S R P P C V I L 18
    4 E P G Q T S V A P 15
    11 A P P P E E V E P 15
    31 E P C G F E A T Y 14
    34 G F E A T Y L E L 13
    94 R R A S N G E T L 13
    12 P P P E E V E P G 12
    19 P G S G V R I V V 12
    32 P C G F E A T Y L 12
    83 F P Y E K D L I E 12
    106 T N S R P P C V I 12
    HLA-B*0702 decamers
    31 E P C G F E A T Y L 24
    50 Y P G I E I E S R L 21
    18 E P G S G V R I V V 20
    83 F P Y E K D L I E A 16
    4 E P G Q T S V A P P 15
    11 A P P P E E V E P G 15
    93 I R R A S N G E T L 14
    106 T N S R P P C V I L 14
    69 I N G Q L V F S K L 13
    33 C G F E A T Y L E L 12
    64 A F E I E I N G Q L 12
    HLA-B*08 octamers
    83 F P Y E K D L I 25
    66 E I E I N G Q L 16
    52 G I E I E S R L 15
    18 E P G S G V R I 14
    54 E I E S R L G G 14
    91 E A I R R A S N 14
    95 R A S N G E T L 14
    100 E T L E K I T N 14
    33 C G F E A T Y L 12
    45 A V K E Q Y P G 12
    58 R L G G T G A F 12
    68 E I N G Q L V F 12
    71 G Q L V F S K L 12
    75 F S K L E N G G 12
    82 G F P Y E K D L 12
    107 N S R P P C V I 12
    108 S R P P C V I L 12
    HLA-B*08 nonamers
    75 F S K L E N G G F 19
    83 F P Y E K D L I E 19
    45 A V K E Q Y P G I 18
    85 Y E K D L I E A I 18
    107 N S R P P C V I L 17
    100 E T L E K I T N S 15
    54 E I E S R L G G T 14
    65 F E I E I N G Q L 14
    91 E A I R R A S N G 14
    20 G S G V R I V V E 12
    34 G F E A T Y L E L 12
    51 P G I E I E S R L 12
    81 G G F P Y E K D L 12
    HLA-B*1510 nonamers
    107 N S R P P C V I L 15
    34 G F E A T Y L E L 13
    51 P G I E I E S R L 13
    81 G G F P Y E K D L 13
    94 R R A S N G E T L 13
    HLA-B*2705 nonamers
    57 S R L G G T G A F 26
    94 R R A S N G E T L 25
    67 I E I N G Q L V F 19
    87 K D L I E A I R R 19
    51 P G I E I E S R L 17
    81 G G F P Y E K D L 17
    65 F E I E I N G Q L 16
    69 I N G Q L V F S K 16
    96 A S N G E T L E K 16
    16 E V E P G S G V R 15
    34 G F E A T Y L E L 15
    50 Y P G I E I E S R 15
    70 N G Q L V F S K L 15
    101 T L E K I T N S R 15
    23 V R I V V E Y C E 14
    32 P C G F E A T Y L 14
    39 Y L E L A S A V K 14
    79 E N G G F P Y E K 14
    93 I R R A S N G E T 14
    21 S G V R I V V E Y 13
    27 V E Y C E P C G F 13
    75 F S K L E N G G F 13
    86 E K D L I E A I R 13
    107 N S R P P C V I L 13
    17 V E P G S G V R I 12
    31 E P C G F E A T Y 12
    77 K L E N G G F P Y 12
    HLA-B*2709 nonamers
    94 R R A S N G E T L 25
    57 S R L G G T G A F 20
    81 G G F P Y E K D L 16
    34 G F E A T Y L E L 14
    51 P G I E I E S R L 13
    65 F E I E I N G Q L 13
    23 V R I V V E Y C E 12
    107 N S R P P C V I L 12
    HLA-B*5101 nonamers
    18 E P G S G V R I V 21
    81 G G F P Y E K D L 21
    51 P G I E I E S R L 20
    70 N G Q L V F S K L 20
    19 P G S G V R I V V 19
    31 E P C G F E A T Y 19
    2 S G E P G Q T S V 18
    42 L A S A V K E Q Y 18
    59 L G G T G A F E I 18
    21 S G V R I V V E Y 14
    83 F P Y E K D L I E 14
    97 S N G E T L E K I 14
    13 P P E E V E P G S 13
    38 T Y L E L A S A V 13
    45 A V K E Q Y P G I 13
    63 G A F E I E I N G 13
    94 R R A S N G E T L 13
    12 P P P E E V E P G 12
    33 C G F E A T Y L E 12
    50 Y P G I E I E S R 12
    66 E I E I N G Q L V 12
    85 Y E K D L I E A I 12
    95 R A S N G E T L E 12
    105 I T N S R P P C V 12
    HLA-B*5101 octamers
    83 F P Y E K D L I 25
    95 R A S N G E T L 23
    10 V A P P P E E V 21
    18 E P G S G V R I 21
    33 C G F E A T Y L 21
    98 N G E T L E K I 19
    19 P G S G V R I V 18
    60 G G T G A F E I 18
    62 T G A F E I E I 18
    63 G A F E I E I N 14
    71 G Q L V F S K L 14
    48 E Q Y P G I E I 13
    67 I E I N G Q L V 13
    106 T N S R P P C V 12
    Class II MHC HLA-DRB1*0101 15-mers
    72 Q L V F S K L E N G G F P Y E 29
    37 A T Y L E L A S A V K E Q Y P 26
    26 V V E Y C E P C G F E A T Y L 25
    63 G A F E I E I N G Q L V F S K 25
    24 R I V V E Y C E P C G F E A T 24
    36 E A T Y L E L A S A V K E Q Y 24
    39 Y L E L A S A V K E Q Y P G I 24
    53 I E I E S R L G G T G A F E I 24
    56 E S R L G G T G A F E I E I N 24
    14 P E E V E P G S G V R I V V E 23
    43 A S A V K E Q Y P G I E I E S 23
    20 G S G V R I V V E Y C E P C G 20
    62 T G A F E I E I N G Q L V F S 20
    32 P C G F E A T Y L E L A S A V 19
    47 K E Q Y P G I E I E S R L G G 19
    64 A F E I E I N G Q L V F S K L 19
    82 G F P Y E K D L I E A I R R A 19
    34 G F E A T Y L E L A S A V K E 18
    54 E I E S R L G G T G A F E I E 18
    90 I E A I R R A S N G E T L E K 18
    99 G E T L E K I T N S R P P C V 18
    31 E P C G F E A T Y L E L A S A 17
    49 Q Y P G I E I E S R L G G T G 17
    58 R L G G T G A F E I E I N G Q 17
    66 E I E I N G Q L V F S K L E N 17
    67 I E I N G Q L V F S K L E N G 17
    68 E I N G Q L V F S K L E N G G 17
    84 P Y E K D L I E A I R R A S N 17
    86 E K D L I E A I R R A S N G E 17
    35 F E A T Y L E L A S A V K E Q 16
    74 V F S K L E N G G F P Y E K D 16
    87 K D L I E A I R R A S N G E T 16
    91 E A I R R A S N G E T L E K I 16
    1 M S G E P G Q T S V A P P P E 15
    4 E P G Q T S V A P P P E E V E 15
    11 A P P P E E V E P G S G V R I 15
    12 P P P E E V E P G S G V R I V 15
    29 Y C E P C G F E A T Y L E L A 15
    5 P G Q T S V A P P P E E V E P 14
    6 G Q T S V A P P P E E V E P G 14
    44 S A V K E Q Y P G I E I E S R 14
    52 G I E I E S R L G G T G A F E 14
    61 G T G A F E I E I N G Q L V F 13
    50 Y P G I E I E S R L G G T G A 12
    HLA-DRB1*0301 (DR17) 15-mers
    64 A F E I E I N G Q L V F S K L 26
    39 Y L E L A S A V K E Q Y P G I 25
    72 Q L V F S K L E N G G F P Y E 23
    62 T G A F E I E I N G Q L V F S 22
    24 R I V V E Y C E P C G F E A T 19
    71 G Q L V F S K L E N G G F P Y 19
    86 E K D L I E A I R R A S N G E 19
    7 Q T S V A P P P E E V E P G S 18
    23 V R I V V E Y C E P C G F E A 18
    50 Y P G I E I E S R L G G T G A 18
    90 I E A I R R A S N G E T L E K 18
    20 G S G V R I V V E Y C E P C G 17
    87 K D L I E A I R R A S N G E T 17
    99 G E T L E K I T N S R P P C V 16
    28 E Y C E P C G F E A T Y L E L 15
    37 A T Y L E L A S A V K E Q Y P 14
    48 E Q Y P G I E I E S R L G G T 14
    78 L E N G G F P Y E K D L I E A 14
    14 P E E V E P G S G V R I V V E 13
    70 N G Q L V F S K L E N G G F P 13
    43 A S A V K E Q Y P G I E I E S 12
    52 G I E I E S R L G G T G A F E 12
    54 E I E S R L G G T G A F E I E 12
    74 V F S K L E N G G F P Y E K D 12
    82 G F P Y E K D L T E A I R R A 12
    HLA-DRB1*0401 (DR4Dw4) 15-mers
    36 E A T Y L E L A S A V K E Q Y 28
    62 T G A F E I E I N G Q L V F S 28
    86 E K D L I E A I R R A S N G E 26
    87 K D L I E A I R R A S N G E T 26
    90 I E A I R R A S N G E T L E K 26
    72 Q L V F S K L E N G G F P Y E 22
    82 G F P Y E K D L I E A I R R A 22
    50 Y P G I E I E S R L G G T G A 20
    99 G E T L E K I T N S R P P C V 20
    26 V V E Y C E P C G F E A T Y L 16
    32 P C G F E A T Y L E L A S A V 16
    47 K E Q Y P G I E I E S R L G G 16
    80 N G G F P Y E K D L I E A I R 16
    14 P E E V E P G S G V R I V V E 14
    20 G S G V R I V V E Y C E P C G 14
    22 G V R I V V E Y C E P C G F E 14
    37 A T Y L E L A S A V K E Q Y P 14
    39 Y L E L A S A V K E Q Y P G I 14
    56 E S R L G G T G A F E I E I N 14
    64 A F E I E I N G Q L V F S K L 14
    66 E I E I N G Q L V F S K L E N 14
    10 V A P P P E E V E P G S G V R 12
    12 P P P E E V E P G S G V R I V 12
    16 E V E P G S G V R I V V E Y C 12
    29 Y C E P C G F E A T Y L E L A 12
    30 C E P C G F E A T Y L E L A S 12
    31 E P C G F E A T Y L E L A S A 12
    34 G F E A T Y L E L A S A V K E 12
    35 F E A T Y L E L A S A V K E Q 12
    42 L A S A V K E Q Y P G I E I E 12
    48 E Q Y P G I E I E S R L G G T 12
    49 Q Y P G I E I E S R L G G T G 12
    53 I E I E S R L G G T G A F E I 12
    58 R L G G T G A F E I E I N G Q 12
    59 L G G T G A F E I E I N G Q L 12
    61 G T G A F E I E I N G Q L V F 12
    63 G A F E I E I N G Q L V F S K 12
    67 I E I N G Q L V F S K L E N G 12
    68 E I N G Q L V F S K L E N G G 12
    69 I N G Q L V F S K L E N G G F 12
    85 Y E K D L I E A I R R A S N G 12
    93 I R R A S N G E T L E K I T N 12
    94 R R A S N G E T L E K I T N S 12
    96 A S N G E T L E K I T N S R P 12
    97 S N G E T L E K I T N S R P P 12
  • [0092]
    TABLE 2
    HLA peptide motif search results
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A1
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 77 KLENGGFPY 225.000
    2 16 EVEPGSGVR 90.000
    3 29 YCEPCGFEA 45.000
    4 39 YLELASAVK 36.000
    5 2 SGEPGQTSV 2.250
    6 26 VVEYCEPCG 1.800
    7 96 ASNGETLEK 1.500
    8 101 TLEKITNSR 0.900
    9 89 LIEAIRRAS 0.900
    10 54 EIESRLGGT 0.900
    11 66 EIEINGQLV 0.900
    12 52 GIEIESRLG 0.900
    13 86 EKDLIEAIR 0.500
    14 42 LASAVKEQY 0.500
    15 31 EPCGFEATY 0.250
    16 69 INGQLVFSK 0.250
    17 34 GFEATYLEL 0.225
    18 98 NGETLEKIT 0.225
    19 61 GTFAFEIEI 0.125
    20 79 ENGGFPYEK 0.100
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A1
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 66 IEIEnGQLVF 45.000
    2 16 EVEPgSGVRI 18.000
    3 29 YCEPcGFEAT 9.000
    4 26 VVEYcEPCGF 9.000
    5 52 GIEIeSRLGG 4.500
    6 2 SGEPgQTSVA 2.250
    7 89 LIEAiRRASN 1.800
    8 86 EKDLiEAIRR 1.250
    9 86 EKDLiEAIRR 1.250
    10 98 NGETlEKITN 1.125
    11 95 RASNgETLEK 1.000
    12 68 EINGqLVPSK 1.000
    13 54 EIESrLGGTG 0.900
    14 41 ELASaVKEQY 0.500
    15 100 ETLEkITNSR 0.250
    16 46 VKEQyPGIEI 0.225
    17 39 YLELaSAVKE 0.180
    18 77 KLENgGFPYE 0.180
    19 76 SKLEnGGFPY 0.125
    20 48 EQYPgIEIES 0.075
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A_0201
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 9 SVAPPPEEV 2.982
    2 104 KITNSRPPC 2.361
    3 105 ITNSRPPCV 1.642
    4 25 IVVEYCEPC 1.485
    5 65 FEIEINGQL 1.018
    6 47 KEQYPGIEI 0.710
    7 88 DLIEAIRRA 0.703
    8 59 LGGTGAFEI 0.671
    9 61 GTGAFEIEI 0.551
    10 81 GGFPYEKDL 0.516
    11 37 ATYLELASA 0.508
    12 35 FEATYLELA 0.501
    13 15 EEVEPGSGV 0.416
    14 17 VEPGSGVRI 0.345
    15 97 SNGETLEKI 0.315
    16 70 NGQLVFSKL 0.265
    17 22 GVRIVVEYC 0.205
    18 45 AVKEQYPGI 0.196
    19 85 YEKDLIEAI 0.151
    20 38 TYLELASAV 0.147
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A_0201
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 58 RLGGtGAFEI 60.510
    2 104 KETNsRPPCV 33.472
    3 83 FPYEkDLIEA 4.502
    4 83 FPYEkDLIEA 4.502
    5 33 CGFEaTYLEL 3.173
    6 1 MSGEpGQISV 3.165
    7 37 ATYLeLASAV 3.091
    8 50 YPGIeIESRL 0.641
    9 69 INGQlVFSKL 0.450
    10 17 VEPGsGVRIV 0.434
    11 24 RIVVeYCEPC 0.335
    12 53 IEIEsRLGGT 0.302
    13 60 GGTGaFGIEI 0.259
    14 8 TSVApPPEEV 0.222
    15 44 SAVKeQYPGI 0.217
    16 21 SGVRiVVEYC 0.201
    17 55 IESRlGGTGA 0.164
    18 80 NGGFpYEKDL 0.139
    19 81 GGFPyEDKLI 0.123
    20 105 ITNSrPPCVI 0.101
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A_0205
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 65 FEIEINGQL 8.820
    2 25 IVVEYCEPC 3.060
    3 9 SVAPPPEEV 2.000
    4 104 KITNSRPPC 1.500
    5 81 GGFPYEKDL 1.260
    6 45 AVKEQYPGI 1.200
    7 70 NGQLVESKL 0.700
    8 47 KEQYPGIEI 0.420
    9 105 ITNSRPPCV 0.340
    10 37 ATYLELASA 0.300
    11 35 FEATYLELA 0.252
    12 17 VEPGSGVRI 0.238
    13 61 GTGAFEIEI 0.200
    14 97 SNGETLEKI 0.150
    15 30 CEPCGFEAT 0.140
    16 85 YEKDLIEAI 0.126
    17 51 PGIEIESRL 0.105
    18 59 LGGTGAFEI 0.102
    19 22 GVEIVVEYC 0.100
    20 15 EEVEPGSGV 0.084
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A_0205
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 77 KLENGGFPY 225.000
    2 33 CGFEaTYLEL 6.300
    3 104 KITNsRPPCV 6.000
    4 53 IEIEsRLGGI 1.428
    5 83 FPYEkDLIEA 1.350
    6 58 RLGGtGAFEI 1.200
    7 69 INGQlVFSKL 1.190
    8 50 YPGIeIESRL 1.050
    9 37 ATYLeLASAV 0.600
    10 1 MSGEpGQTSV 0.510
    11 80 NGGFpYEKDL 0.420
    12 106 TRSRpPCVIL 0.350
    13 24 RIVVeYCEFC 0.300
    14 44 SAVKeQYPGI 0.200
    15 17 VEPGsGVRIV 0.190
    16 105 ITNSrPPCVI 0.170
    17 97 SNGEtLEKIT 0.150
    18 55 IESRlGGTGA 0.119
    19 60 GGTGaFEIEI 0.100
    20 92 AIRRaSNGET 0.100
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A24
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 34 GFEATYLEL 33.000
    2 49 QYPGIEIES 11.550
    3 70 NGQLVFSKL 11.088
    4 38 TYLELASAV 10.800
    5 82 GFPYEKDLI 7.500
    6 81 GGFPYEKDL 4.800
    7 107 NSRPPCVIL 4.800
    8 75 FSKLENGGF 2.000
    9 97 SNGETLEKI 1.320
    10 45 AVKEQYPGI 1.200
    11 61 GTGAFEIEI 1.100
    12 59 LGGTGAFEI 1.100
    13 65 FEIEINGQL 1.008
    14 51 PGIEIESRL 1.008
    15 106 TNSRPPCVI 1.000
    16 84 PYEKDLIEA 0.825
    17 94 RRASNGETL 0.800
    18 28 EYCEPCGPE 0.600
    19 32 PCGFEATYL 0.400
    20 47 KEQYPGIEI 0.300
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A24
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 64 AFEIeINGQL 42.000
    2 74 VFSKlENGGF 10.000
    3 84 PYEKdLIEAI 9.000
    4 69 INGQlVFSKL 7.392
    5 28 EICEpCGFEA 6.600
    6 50 YPGIeIESRL 5.600
    7 33 CGFEaTYLEL 5.280
    8 106 TNSRpPCVIL 4.000
    9 31 EPCGfEATYL 4.000
    10 80 NGGFpYEKDL 4.000
    11 26 VVEYcEPCGF 3.000
    12 66 EIEInGQLVF 3.000
    13 58 RLGGtGAFEI 2.200
    14 56 ESRLgGYGAF 2.000
    15 16 EVEPgSGVRI 1.800
    16 96 ASNGeTLEKI 1.650
    17 105 ITNSrPPCVI 1.500
    18 44 SAVKeQYPGI 1.500
    19 81 GGFPyEKDLI 1.200
    20 60 GGTGaFEIEI 1.100
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A3
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 77 KLENGGFPY 36.000
    2 39 YLELASAVK 20.000
    3 101 TLEKITNSR 6.000
    4 61 GTGAFEIEI 0.540
    5 69 INGQLVFSK 0.360
    6 96 ASNGETLEK 0.300
    7 22 GVRIVVEYC 0.270
    8 79 ENGGFPYER 0.162
    9 25 IVVEYCEPC 0.135
    10 45 AVKEQYPGI 0.090
    11 37 ATYLELASA 0.075
    12 42 LASAVKEQY 0.060
    13 104 KITNSRPPC 0.060
    14 50 YPGIEIESR 0.060
    15 72 QLVFSKLEN 0.060
    16 16 EVEPGSGVR 0.054
    17 31 EPCGFEATY 0.054
    18 9 SVAPPPEEV 0.045
    19 87 KDLIEAIRR 0.036
    20 27 VEYCEPCGF 0.030
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A3
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 68 EINGqLVFSK 8.100
    2 58 RLGGtGAFEI 2.700
    3 41 ELASaVKEQY 1.800
    4 78 IENGgFPYEK 0.810
    5 95 RASRgETLEK 0.400
    6 20 GSGVrIVVEY 0.270
    7 100 ETLEkITNSR 0.203
    8 26 VVEYcEPCGF 0.200
    9 77 KLENgGFPYE 0.180
    10 66 EIEInGQLVF 0.120
    11 24 RIVVeYCEPC 0.090
    12 104 KITNsRPPCV 0.060
    13 37 ATYLeLASAV 0.050
    14 38 TYLElASAVK 0.045
    15 83 FPYEkDLIEA 0.045
    16 105 ITNSrPPCVI 0.045
    17 72 QLVFsKLENG 0.045
    18 30 CEPCgFEATY 0.036
    19 22 GVRIvVEYCE 0.027
    20 16 EVEPgSGVRI 0.027
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A_1101
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 39 YLELASAVK 0.400
    2 69 INGQLVFSF 0.120
    3 16 EVEPGSGVR 0.120
    4 101 TLEKITNSR 0.080
    5 61 GTGAFEIEI 0.060
    6 50 YPGIEIESR 0.040
    7 96 ASNGETLEK 0.040
    8 87 KDLIEAIRR 0.036
    9 77 KLENGGFPY 0.036
    10 79 ENGGFPYEK 0.024
    11 9 SVAPPPEEV 0.020
    12 45 AVKEQYPGI 0.020
    13 37 ATYLELASA 0.020
    14 34 GFEATYLEL 0.012
    15 105 ITNSRPPCV 0.010
    16 22 GVRIVVEYC 0.006
    17 38 TYLELASAV 0.006
    18 82 GFPYEKDLI 0.006
    19 29 YCEPCGFEA 0.006
    20 73 LVFSKLENG 0.004
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A_3101
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 101 TLEKITNSR 2.000
    2 16 EVEPGSGVR 0.600
    3 50 YPGIEIESR 0.400
    4 87 KDLIEAIRR 0.240
    5 39 YLELASAVK 0.200
    6 77 KLENGGFPY 0.180
    7 37 ATYLELASA 0.060
    8 69 INGQLVFSK 0.024
    9 45 AVKEQYFGI 0.020
    10 61 GTGAFEIEI 0.020
    11 9 SVAPPPEEV 0.020
    12 24 RIVVEYCEP 0.012
    13 34 GFEATYLEL 0.012
    14 73 LVFSKLENG 0.012
    15 38 TYLELASAV 0.012
    16 105 ITNSRPPCV 0.010
    17 72 QLVFSKLEN 0.008
    18 82 GFPYEKDLI 0.006
    19 104 KITNSRPPC 0.006
    20 79 ENGGFPYEK 0.006
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A_3302
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 16 EVEPGSGVR 45.000
    2 101 TLEKITNSR 9.000
    3 50 YPGIEIESR 3.000
    4 66 EIEINGQLV 1.500
    5 56 ESRLGGTGA 1.500
    6 54 EIESRIGGT 1.500
    7 68 EINGQLVFS 1.500
    8 86 EKDLIEAIR 0.900
    9 41 ELASAVKEQ 0.900
    10 88 DLIEAIRRA 0.900
    11 96 ASNGETLEK 0.500
    12 22 GVRIVVEYC 0.500
    13 1 MSGEPGQTS 0.500
    14 89 LIEAIRRAS 0.500
    15 107 NSRPPCVIL 0.500
    16 9 SVAPPPEEV 0.500
    17 38 TYLELASAV 0.500
    18 25 IVVEYCEPC 0.500
    19 45 AVKEQYPGI 0.500
    20 49 QYPGIEIES 0.500
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A_3302
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 16 EVEPGSGVR 45.000
    2 101 TLEKITNSR 9.000
    3 50 YPGIEIESR 3.000
    4 66 EIEINGQLV 1.500
    5 56 ESRLGGTGA 1.500
    6 54 EIESRLGGT 1.500
    7 68 EINGQLVFS 1.500
    8 86 EKDLIEAIR 0.900
    9 41 ELASAVKEQ 0.900
    10 88 DLIEAIRRA 0.900
    11 96 ASNGETLEK 0.500
    12 22 GVRIVVEYC 0.500
    13 1 MSGEPGQTS 0.500
    14 89 LIEAIRRAS 0.500
    15 107 NSRPPCVIL 0.500
    16 9 SVAPPPEEV 0.500
    17 38 TYLELASAV 0.500
    18 25 IVVEYCEPC 0.500
    19 45 AVKEQYPGI 0.500
    20 49 QYPGIEIES 0.500
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A68.1
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 16 EVEPGSGVR 900.000
    2 9 SVAPPPEEV 12.000
    3 50 YPGIEIESR 10.000
    4 96 ASNGETLEK 9.000
    5 101 TLEKITNSR 5.000
    6 45 AVKEQYPGI 4.000
    7 79 ENGGFPYEK 3.600
    8 39 YLELASAVK 3.000
    9 61 GTGAFEIEI 3.000
    10 86 EKDLIEAIR 2.250
    11 69 INGQLVFSR 1.200
    12 87 NDLIEAIRR 1.000
    13 105 ITNSRPPCV 1.000
    14 37 ATYLELASA 1.000
    15 56 ESRLGGTGA 0.900
    16 25 IVVEYCEPC 0.800
    17 73 LVFSKLENG 0.800
    18 88 DLIEAIRRA 0.600
    19 18 EPGSGVRIV 0.600
    20 26 VVEYCEPCG 0.600
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A68.1
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 16 EVEPGSGVR 900.000
    2 9 SVAPPPEEV 12.000
    3 50 YPGIEIESR 10.000
    4 96 ASNGETLEK 9.000
    5 101 TLEKITNSR 5.000
    6 45 AVKEQYPGI 4.000
    7 79 ENGGFPYEK 3.600
    8 39 YLELASAVK 3.000
    9 61 GTGAFEIEI 3.000
    10 86 EKDLIEAIR 2.250
    11 69 INGQLVFSK 1.200
    12 87 KDLIEAIRR 1.000
    13 105 ITNSRPPCV 1.000
    14 37 ATYLELASA 1.000
    15 56 ESRLGGTGA 0.900
    16 25 IVVEYCEPC 0.800
    17 73 LVFSKLENG 0.800
    18 88 DLIEAIRRA 0.600
    19 18 EPGSGVRIV 0.600
    20 26 VVEYCEPCG 0.600
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected A68.1
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 100 ETLEkITNSR 300.000
    2 16 EVEPgSGVRI 18.000
    3 68 EINGqLVFSK 9.000
    4 15 EEVEpGSGVR 9.000
    5 95 RASNgETLEK 3.000
    6 85 YEKDlIEAIR 2.250
    7 9 SVAPpPEEVE 1.800
    8 86 EKDLiEAIRR 1.500
    9 73 LVFSkLENGG 1.200
    10 25 IVVEyCEPCG 1.200
    11 105 ITNSrPPCVI 1.000
    12 37 ATYLeLASAV 1.000
    13 78 LENGgFPYEK 0.900
    14 8 TSVApFPEEV 0.600
    15 22 GVRIvVEYCE 0.600
    16 18 EPGSgVRIVV 0.600
    17 1 MSGEpGQTSV 0.600
    18 38 TYLELASAVK 0.600
    19 49 QYPGiEISSR 0.500
    20 45 AVKEqYPGIE 0.400
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B14
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 94 RRASNGETL 20.000
    2 57 SRLGGTGAF 5.000
    3 100 ETLEKITNS 3.375
    4 105 ITNSRPPCV 2.000
    5 88 DLIEAIRRA 1.350
    6 18 EPGSGVRIV 1.200
    7 70 NGQLVFSKL 1.000
    8 81 GGFPYEKDL 1.000
    9 54 EIESRLGGT 0.900
    10 97 SNGETLEKI 0.600
    11 91 EAIRRASNG 0.450
    12 68 EINGQLVES 0.450
    13 65 FEIEINGQL 0.300
    14 23 VRIVVEYCE 0.300
    15 21 SGVRIVVEY 0.300
    16 51 PGIEIEGRL 0.300
    17 104 KITNSRPPC 0.250
    18 48 EQYPGIEIE 0.225
    19 93 IRRASNGET 0.200
    20 107 NSRPPCVIL 0.200
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B14
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 103 EKITnSRPPC 6.750
    2 33 CGFEaTYLEL 5.000
    3 93 IRRAsNGETL 4.000
    4 18 EPGSgVRIVV 3.000
    5 88 DLIEaIRRAS 2.250
    6 104 KITNsRPPCV 1.000
    7 106 TNSRpPCVIL 1.000
    8 50 YPGIeIESRL 1.000
    9 69 INGQlVFSKL 1.000
    10 37 ATYLeLASAV 1.000
    11 31 EPCGfEATYL 0.900
    12 48 EQYPgIEIES 0.750
    13 76 SKLEnGGFPY 0.750
    14 83 FPYEkDLIEA 0.750
    15 8 TSVApPPEEV 0.600
    16 96 ASNGeTLEKI 0.600
    17 44 SAVKeQYPGI 0.600
    18 57 SRLGgTGAFE 0.500
    19 53 IEIEsRLGGT 0.450
    20 21 SGVRiVVEYC 0.300
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_2705
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 94 RRASNGETL 6000.000
    2 57 SRLGGTGAF 1000.000
    3 93 IRRASNGET 200.000
    4 27 VEYCEPCGF 75.000
    5 77 KLENGGFPY 45.000
    6 39 YLELASAVK 30.000
    7 65 FEIEINGQL 30.000
    8 47 KEQYPGIEI 27.000
    9 69 INGQLVFSK 20.000
    10 23 VRIVVEYCE 20.000
    11 101 TLEKITNSR 15.000
    12 67 IEINGQLVF 15.000
    13 107 NSRPPCVIL 10.000
    14 96 ASNGETLEK 10.000
    15 85 YEKDLIEAI 9.000
    16 17 VEPGSGVRI 9.000
    17 81 GGFPYEKDL 7.500
    18 106 TNSRPPCVI 6.000
    19 97 SNGETLEKI 6.000
    20 75 PSKLENGGF 5.000
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_2705
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 93 IRRAsNGETL 2000.000
    2 94 PRASnGETLE 60.000
    3 78 LENGgFPYEK 30.000
    4 95 RASNgETLEK 30.000
    5 58 RLGGtGAFEI 27.000
    6 33 CGFFaTYLEL 25.000
    7 106 TNSRpPCVIL 20.000
    8 71 GQLVfSKLEN 20.000
    9 23 VRIVvEYCEP 20.000
    10 57 SRLGgTGAFE 20.000
    11 69 INGQlVFSKL 20.000
    12 30 CEPCgFEATY 15.000
    13 85 YEKDlIEAIR 15.000
    14 37 ATYLElASAV 15.000
    15 48 EQYPgIEIES 10.000
    16 50 YPGIeIESRL 10.000
    17 104 KITNsRPPCV 9.000
    18 65 FEIEiNGQLV 9.000
    19 81 GGFPyEKQLI 7.500
    20 83 FPYEkDLIEA 5.000
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_3501
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 31 EPCGFEATY 40.000
    2 75 FSKLENGGF 22.500
    3 107 NSRPPCVIL 15.000
    4 42 LASAVKEQY 6.000
    5 18 EPGSGVRIL 4.000
    6 45 AVKEQYPGI 2.400
    7 21 SGVRIVVEY 2.000
    8 56 ESRLGGTGA 1.500
    9 77 KLENGGFPY 1.200
    10 81 GGFPYEKDL 1.000
    11 1 MSGEPGQTS 1.000
    12 70 NGQLVFSKL 1.000
    13 97 SNGETLEKI 0.800
    14 83 FPYEKDLIE 0.400
    15 61 GTGAFEIEI 0.400
    16 59 LGGTGAFEI 0.400
    17 106 TNSRPPCVI 0.400
    18 50 YPGIEIESR 0.300
    19 22 GVIRVVEYC 0.300
    20 11 APPPEEVEP 0.300
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_3501
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 31 EPCFfEATYL 30.000
    2 50 YPGIeIESRL 20.000
    3 56 ESRLgGTFAF 15.000
    4 20 GSGVrIVVEY 10.000
    5 83 FPYEkDLIEA 6.000
    6 18 EPGSgVRIVV 4.000
    7 33 CGFEaTYLEL 2.000
    8 1 MSGEpGQTSV 2.000
    9 96 ASNGeTLEKI 2.000
    10 41 ELASaVKEQY 2.000
    11 44 SAVKeQYPGI 1.200
    12 69 INGQlVFSKL 1.000
    13 8 TSVApFPEEV 1.000
    14 80 NGGFpYEKDL 1.000
    15 106 TNSRpPCVIL 1.000
    16 58 RLGGtGAFEI 0.800
    17 81 GGFPyEKDLI 0.600
    18 26 VVEYeEPCGF 0.450
    19 36 EATYlELASA 0.450
    20 12 PPPEeVEPGS 0.400
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_3901
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 94 RRASNGETL 15.000
    2 34 GFEATYLEL 9.000
    3 38 TYLELASAV 4.000
    4 66 EIEINGQLV 3.000
    5 2 SGEPGQTSV 3.000
    6 97 SNGETLEKI 3.000
    7 70 NGQLVFSKL 3.000
    8 81 GGFPYEKDL 3.000
    9 18 EPGSGVRIV 1.500
    10 65 FEIEINGQL 1.200
    11 57 SRLGGTGAF 1.000
    12 106 TNSRPPCVI 1.000
    13 9 SVAPPPEEV 1.000
    14 59 LGGTGAFEI 1.000
    15 105 ITNSRPPCV 1.000
    16 107 NSRPPCVIL 0.900
    17 45 AVKEQYPGI 0.600
    18 51 PGIEIESRL 0.600
    19 88 DLIEAIRRA 0.600
    20 100 ETLEKITNS 0.600
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_3901
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 33 CGFEaTYLEL 12.000
    2 64 AFEIeINGQL 9.000
    3 93 IRRAsNGETL 4.500
    4 46 VKEQyPGIEI 3.000
    5 16 EVEPgSGVRI 3.000
    6 106 TNSRpPCVIL 3.000
    7 69 INGQlVFSKL 3.000
    8 31 EPCGfEATYL 3.000
    9 44 SAVKeQYPGI 2.000
    10 1 MSGEpGQTSV 2.000
    11 8 TSVApPPEEV 2.000
    12 37 ATYLeLASAV 2.000
    13 80 NGGFpYEKDL 1.500
    14 50 YPGIeIESRL 1.500
    15 96 ASNGeTLEKI 1.500
    16 58 RLGGtGAFEI 1.000
    17 105 ITNSrPPCVI 1.000
    18 81 GGFPyEKDLI 1.000
    19 104 KITNsRPPCV 1.000
    20 83 FPYEkDLIEA 0.600
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B40
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 65 FEIEINGQL 80.000
    2 3 GEPGQTSVA 40.000
    3 35 FEATYLELA 40.000
    4 15 EEVEPGSGV 24.000
    5 67 IEINGQLVF 16.000
    6 81 GGFPYEKDL 8.000
    7 27 VEYCEPCGF 8.000
    8 47 KEQYPGIEI 6.000
    9 17 VEPGSGVRI 4.000
    10 30 CEFCGFEAT 4.000
    11 99 GETLEKITN 2.400
    12 90 IEAIRRASN 2.400
    13 37 ATYLELASA 2.000
    14 85 YEKDLIEAI 2.000
    15 53 IEIESRLGG 1.600
    16 40 LELASAVKE 0.800
    17 107 NSRPPCVIL 0.750
    18 29 YCEPCGFEA 0.500
    19 70 NGQLVFSKL 0.500
    20 78 LENGGFPYE 0.400
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B40
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 55 IESRlGGTGA 20.000
    2 53 IEIEsRLGGT 16.000
    3 65 FEIEiNGQLV 16.000
    4 67 IEINgQLVFS 16.000
    5 99 GETLeKITNS 8.000
    6 35 FEATyLELAS 8.000
    7 87 DKLIeAIRRA 5.000
    8 17 VEPGsGVRIV 4.000
    9 30 CEPCgFEATY 4.000
    10 33 CGFEaTYLEL 2.000
    11 15 EEVEpGSGVR 1.600
    12 81 GGFPySKDLI 1.600
    13 27 VEYCePCGFE 1.200
    14 83 EPYEkDLIEA 1.000
    15 40 LELAsAVKEQ 0.800
    16 3 GEPGqTSVAP 0.800
    17 90 IEAIrRASGN 0.800
    18 106 TNSRpPCVIL 0.750
    19 8 TSVApPFEEV 0.600
    20 2 SGEPgQTSVA 0.500
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_5201
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 18 EPGSGVRIV 75.000
    2 67 IEINGQLVF 22.500
    3 59 LGGTGAFEI 11.250
    4 98 NGETLEKIT 11.000
    5 19 PGSGVRIVV 10.000
    6 106 TNSRPPCVI 10.000
    7 48 EQYPGIEIE 9.900
    8 2 SGEPGQTSV 9.000
    9 81 GGFPYEKDL 6.600
    10 38 TYLELASAV 4.800
    11 27 VEYCEPCGF 3.750
    12 83 FPYEKDLIE 3.000
    13 17 VEPGSGVRI 3.000
    14 70 NGQLVFSKL 2.400
    15 85 YEKDLIEAI 2.200
    16 3 GEPGQTSVA 2.200
    17 82 GFPYEKDLI 2.200
    18 97 SNGETLEKI 2.178
    19 61 GTGAFEIEI 1.800
    20 105 ITNSRPPCV 1.500
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_5201
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 18 EPGSgVRIVV 100.000
    2 17 VEPGsGVRIV 45.000
    3 81 GGFPyEKDLI 33.000
    4 105 ITNSrPPCVI 15.000
    5 37 ATYLeLASAV 12.000
    6 66 EIEInGQLVF 9.000
    7 33 CGFEaTYLEL 9.000
    8 60 GGTGaFEIEI 7.500
    9 2 SGEPgQTSVA 6.600
    10 83 EPYEkDLIEA 3.300
    11 1 MSGEpGQTSV 2.700
    12 97 SNGEtLEKIT 2.640
    13 65 FEIEiNGQLV 2.640
    14 50 YPGIeIESRL 2.400
    15 48 EQYPgIEIES 2.400
    16 106 TNSRpPCVIL 2.000
    17 96 ASNGeTLEKI 1.815
    18 58 RLGGtGAGEI 1.500
    19 8 TSVApPPEEV 1.320
    20 59 LGGTgAFEIE 1.238
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B60
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 65 FEIEINGQL 387.200
    2 17 VEPGSGVRI 17.600
    3 15 EEVEPGSGV 16.000
    4 47 KEQYPGIEI 16.000
    5 85 YEKDLIEAI 8.800
    6 107 NSRPPCVIL 8.000
    7 35 FEATYLELA 8.000
    8 70 NGQLVFSKL 4.840
    9 3 GEPGQTSVA 4.000
    10 81 GGFPYEKDL 4.000
    11 30 CEPCGFEAT 4.000
    12 67 IEINGQLVF 3.200
    13 90 IEAIRRASN 2.400
    14 99 GETLEKITN 2.400
    15 40 LELASAVKE 1.760
    16 53 IEIESRLGG 1.600
    17 51 PGIEIESRL 0.968
    18 55 IESRLGGTG 0.880
    19 34 GFEATYLEL 0.800
    20 94 RRASNGETL 0.800
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B60
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 65 FEIEiNGQLV 16.000
    2 106 TNSRpPCVIL 16.000
    3 53 IEIEsRLGGT 8.000
    4 33 CGFEaTYLEL 8.000
    5 17 VEPGsGVRIV 8.000
    6 55 IESRlGGTGA 8.000
    7 69 INGQlVFSKL 4.840
    8 50 YPGIeIESRL 4.840
    9 80 NGGFpYEKDL 4.000
    10 31 EPCGfEATYL 4.000
    11 35 FEATyLELAS 3.520
    12 67 IEINgQLVFS 3.200
    13 87 KDLIeAIRRA 1.100
    14 78 LENGgFPYEK 0.800
    15 15 SEVEpGSGVR 0.800
    16 99 GETLeKITNS 0.800
    17 30 CEPCgFEATY 0.800
    18 90 IEAIrRASNG 0.800
    19 3 GEPGqTSVAP 0.800
    20 40 LELAsAVKEQ 0.800
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B61
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 15 EEVEPGSGV 80.000
    2 35 GEATYLELA 40.000
    3 3 GEPGQTSVA 22.000
    4 65 FEIEINGQL 16.000
    5 85 YEKDLIEAI 16.000
    6 17 VEPGSGVRI 8.000
    7 47 KEQYPGIEI 8.000
    8 30 CEPCGFEAT 4.000
    9 99 GETLEKITN 2.640
    10 90 IEAIRRASN 2.400
    11 27 VEYCEPCGF 1.600
    12 67 IEINGQLVF 1.600
    13 2 SGEPGQTSV 1.000
    14 18 EPGSGVRIV 1.000
    15 105 ITNSRPPCV 1.000
    16 37 ATYLELASA 1.000
    17 53 IEIESRLGG 0.800
    18 40 LELASAVKE 0.800
    19 81 GGFPYEKDL 0.660
    20 29 YCEPCGFEA 0.500
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B61
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 65 FEIEiNGQLV 80.000
    2 17 VEPGsGVRIV 40.000
    3 55 IESRlGGTGA 20.000
    4 87 KDLIeAIRRA 10.000
    5 53 IEIEsRLGGT 8.000
    6 14 PEEVePGSGV 4.000
    7 99 GETLeKITNS 3.520
    8 37 ATYLeLASAV 2.000
    9 8 TSVApPPEEV 2.000
    10 67 IEINgQLVFS 1.600
    11 35 FEATyLELAS 1.600
    12 1 MSGEpGQTSV 1.000
    13 18 EPGSqVRIVV 1.000
    14 36 EATYlELASA 1.000
    15 83 FPYEkDLIEA 1.000
    16 15 EEVEpGSGVR 0.800
    17 27 VEYCePCGFE 0.800
    18 30 CEPCgFEATY 0.800
    19 90 IEAIrRASNG 0.800
    20 40 LELAsAVKEQ 0.800
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B62
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 77 KLENGGFPY 24.000
    2 21 SGVRIVVEY 4.800
    3 75 FSKLENGGF 3.000
    4 31 EPCGFEATY 2.640
    5 88 DLIEAIRRA 2.200
    6 42 LASAVKEQY 2.000
    7 48 EQYPGIEIE 0.960
    8 71 GQLVFSKLE 0.800
    9 6 GQTSVAPPP 0.800
    10 67 IEINGQLVF 0.686
    11 22 GVRIVVEYC 0.660
    12 58 RLGGTGAFE 0.480
    13 57 SRLGGTGAF 0.480
    14 18 EPGSGVRIV 0.400
    15 59 LGGTGAFEI 0.400
    16 56 ESRLGGTGA 0.360
    17 45 AVKEQYPGI 0.330
    18 104 KITNSRPPC 0.250
    19 72 QLVFSKLER 0.240
    20 61 GTGAFEIEI 0.240
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B62
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 41 ELASaVKEGI 40.000
    2 58 RLGGtGAFEI 9.600
    3 66 EIKInGQLVF 7.920
    4 56 ESRLgGTGAF 6.000
    5 20 GSGVrIVVEY 4.800
    6 92 AIRRaSNGET 1.500
    7 48 EQYPgIEIES 1.152
    8 26 VVEYcEPCGF 0.600
    9 24 RIVVeYCEPC 0.500
    10 104 KITNsRPPCV 0.500
    11 71 GQLVFSKLEN 0.480
    12 76 SKLEnGGFPY 0.440
    13 88 DLIEaIRRAS 0.440
    14 6 GQTSvAPPPE 0.400
    15 1 MSGEpGQTSV 0.264
    16 18 EPGSgVRIVV 0.264
    17 69 INGQlVFSKL 0.260
    18 21 SGVRiVVEYC 0.220
    19 30 CEPCgFEATY 0.220
    20 74 VFSKlENGGF 0.200
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B7
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 107 NSRPPCVIL 60.000
    2 45 AVKEQYPGI 6.000
    3 22 GVRIVVEYC 5.000
    4 70 NGQLVFSKL 4.000
    5 81 GGFPYEKDL 4.000
    6 18 EPGSGVRIV 4.000
    7 9 SVAPPPEEV 1.500
    8 56 ESRLGGTGA 1.000
    9 106 TNSRPPCVI 0.600
    10 11 APPPEEVEP 0.600
    11 25 IVVEYCEPC 0.500
    12 65 FEIEINGQL 0.400
    13 61 GTGAFEIEI 0.400
    14 31 EPCGFEATY 0.400
    15 94 RRASNGETL 0.400
    16 59 LGGTGAFEI 0.400
    17 51 PGIEIESRL 0.400
    18 32 PCGFEATYL 0.400
    19 97 SNGETLEKI 0.400
    20 92 AIRRASNGE 0.300
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B7
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 50 YPGIeIESRL 80.000
    2 31 EPCGfEATYL 80.000
    3 18 EPGSgVRIVV 6.000
    4 106 TNSRpPCVIL 6.000
    5 80 NGGFpYEKDL 4.000
    6 69 INGQlVFSKL 4.000
    7 93 IRRAsNGETL 4.000
    8 33 CGFEaTYLEL 4.000
    9 92 AIRRaSNGET 3.000
    10 83 FPYEkDLIEA 2.000
    11 44 SAVKeQYPGI 1.200
    12 96 ASNGeTLEKI 1.200
    13 11 APPPeEVEPG 0.600
    14 16 EVEPgSGVRI 0.600
    15 37 ATYLeLASAV 0.600
    16 105 ITNSrPPCVI 0.600
    17 22 GVRIvVEYCE 0.500
    18 60 GGTGeFEIEI 0.400
    19 81 GGFPyEKDLI 0.400
    20 58 KLGGtGAFEI 0.400
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B8
    length selected for subsequences  8
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 108
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 83 FPYEKDLI 0.000
    2 107 NSRPPCVI 1.000
    3 91 EAIRRASN 0.800
    4 20 GSGVRIVV 0.600
    5 18 EPGSGVRI 0.400
    6 95 RASNGETL 0.400
    7 100 ETLEKITN 0.300
    8 105 ITNSRPPC 0.200
    9 10 VAPPPEEV 0.120
    10 73 LVFSKLEN 0.100
    11 43 ASAVKEQY 0.100
    12 22 GVRIVVEY 0.100
    13 36 EATYLELA 0.080
    14 31 EPCGFEAT 0.080
    15 66 EIEINGQL 0.080
    16 4 EPGQTSVA 0.080
    17 33 CGFEATYL 0.060
    18 71 GQLVFSKL 0.060
    19 56 ESRLGGTG 0.040
    20 106 TNSRPPCV 0.030
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B8
    length selected for subsequences  8
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 108
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 83 FPYEKDLI 6.000
    2 107 NSRPPCVI 1.000
    3 91 EAIRRASN 0.800
    4 20 GSGVRIVV 0.600
    5 18 EPGSGVRI 0.400
    6 95 RASNGETL 0.400
    7 100 ETLEKITN 0.300
    8 105 ITNSRPPC 0.200
    9 10 VAPPPEEV 0.120
    10 73 LVFSKLEN 0.100
    11 43 ASAVKEQY 0.100
    12 22 GVRIVVEY 0.100
    13 36 EATYLELA 0.080
    14 31 EPCGFEAT 0.080
    15 66 EIEINGQL 0.080
    16 4 EPGQTSVA 0.080
    17 33 CGFEATYL 0.060
    18 71 GQLVFSKL 0.060
    19 56 ESRLGGTG 0.040
    20 106 TNSRPPCV 0.030
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected Cw_0702
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 20 GSGVrIVVEY 38.400
    2 30 CEPCgFEATY 16.000
    3 41 ELASaVKEQY 16.000
    4 50 YPGIeIESRL 7.920
    5 76 SKLEnGGFPY 4.000
    6 69 INGQlVFSKL 2.880
    7 18 EPGSgVRIVV 2.400
    8 33 CGFEaTYLEL 1.440
    9 80 NGGFpYEKDL 1.440
    10 56 ESRLgGTGAF 1.200
    11 93 IRRAgNGETL 1.200
    12 64 AFEIeINGQL 1.200
    13 66 EIEInGQLVF 1.000
    14 35 FEATyLELAS 0.960
    15 87 KDLIeAIRRA 0.800
    16 97 SNGEtLEKIT 0.800
    17 17 VEPGsGVRIV 0.800
    18 21 SGVRiVVEYC 0.800
    19 28 EYCEpCGFEA 0.720
    20 48 EQYPgIEIES 0.672
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B8
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 50 YPGIeIESRL 0.800
    2 93 IRRAsNGETL 0.400
    3 31 EPCGfEATYL 0.320
    4 104 KITNsRPPCV 0.300
    5 18 EPGSgVRIVV 0.240
    6 56 ESRLgGTGAP 0.200
    7 44 SAVKeQYPGI 0.200
    8 92 AIRRaSNGET 0.200
    9 69 INGQlVFSKL 0.200
    10 106 TNSRpPCVIL 0.200
    11 42 LASAvKEQYP 0.160
    12 33 CGFEaTYLEL 0.060
    13 105 ITNSrPPCVI 0.050
    14 58 RLGGtGAFEI 0.050
    15 96 ASNGeTLEKI 0.050
    16 1 MSGEpGQTSV 0.045
    17 75 FSKLeNGGFP 0.040
    18 80 NGGFpYEKDL 0.040
    19 72 QLVFsKLENG 0.040
    20 53 IEIEsRLGGT 0.030
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_2702
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 57 SRLGGTGAF 200.000
    2 94 RRASNGETL 180.000
    3 93 IRRASNGET 20.000
    4 27 VEYCEPCGF 15.000
    5 77 KLENGGFPY 9.000
    6 67 IEINGQLVF 3.000
    7 47 KEQYPGIEI 2.700
    8 23 VRIVVEYCE 2.000
    9 42 LASAVKEQY 1.000
    10 75 FSKLENGGF 1.000
    11 85 YEKDLIEAI 0.900
    12 17 VEPGSGVRI 0.900
    13 65 FEYEINGQL 0.900
    14 97 SNGETLEKI 0.600
    15 106 TNSRPPCVI 0.600
    16 37 ATYLELASA 0.500
    17 21 SGVRIVVEY 0.500
    18 107 NSRPPCVIL 0.300
    19 30 CEPCGFEAT 0.300
    20 48 EQYPGIETE 0.300
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_2702
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 93 IRRAsNGETL 60.000
    2 94 RRASnGETLE 6.000
    3 30 CEPCgFEATY 3.000
    4 58 RLGGtGAFEI 2.700
    5 23 VRIVvEYCEP 2.000
    6 57 SRLGgTGAFE 2.000
    7 48 EQYPgIEIES 1.500
    8 26 VVEYcEPCGF 1.000
    9 20 GSGVrIVVEY 1.000
    10 71 GQLVfSKLEN 1.000
    11 41 ELASaVKEQY 0.900
    12 33 CGFEaTYLEL 0.750
    13 81 GGFPyEKDLI 0.750
    14 106 TNSRpPCVIL 0.600
    15 69 INGQlVFSKL 0.600
    16 83 FPYEkDLIEA 0.500
    17 37 ATYLeLASAV 0.500
    18 55 IESRlGGTGA 0.300
    19 96 ASNGeTLEKI 0.300
    20 56 ESRLgGTGAF 0.300
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_3701
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 65 FEIEiNGQLV 10.000
    2 67 IEINgQLVES 5.000
    3 81 GGFPyEKDLI 5.000
    4 87 KDLIeAIRRA 4.000
    5 30 CEPCgFEATY 2.000
    6 17 VEPGsGVRIV 2.000
    7 50 YPGIeIESRL 1.500
    8 64 AFEIeINGQL 1.500
    9 69 INGQlVFSKL 1.500
    10 99 GETLeKITNS 1.000
    11 60 GGTGaFEIEI 1.000
    12 46 VKEQyPGIEI 1.000
    13 53 IEIEsRLGGT 1.000
    14 16 EVEPgSGVRI 1.000
    15 44 SAVKeQYPGI 1.000
    16 105 ITNSrPPCVI 1.000
    17 96 ASNGeTLEKI 1.000
    18 80 NGGFpYEKDL 1.000
    19 55 IESRlGGTGA 1.000
    20 31 EPCGfEATYL 1.000
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_3801
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 34 GFEATYLEL 6.000
    2 70 NGQLVFSKL 1.560
    3 38 TYLELASAV 1.040
    4 81 GGFPYEKDL 1.000
    5 97 SNGETLEKI 0.720
    6 66 EIEINGQLV 0.600
    7 2 SGEPGQTSV 0.600
    8 82 GFPYEKDLI 0.600
    9 49 QYPGIEIES 0.520
    10 18 EPGSGVRIV 0.400
    11 31 EPCGFEATY 0.400
    12 89 LIEAIRRAS 0.390
    13 98 NGETLEKIT 0.390
    14 77 KLENGGFPY 0.300
    15 61 GTGAFEIEI 0.300
    16 107 NSRPPCVIL 0.300
    17 75 FSKLENGGF 0.300
    18 106 TNSRPPCVI 0.300
    19 29 YCPECGFEA 0.300
    20 54 EIESRLGGP 0.300
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_3801
    length selected for subsequences  10
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 106
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 64 AFEIeINGQL 7.800
    2 31 RPCGfEATYL 4.800
    3 66 EIEInGQLVF 3.000
    4 26 VVEYcEPCGP 3.000
    5 50 YPGIeIESRL 2.600
    6 74 VFSKlENGGF 2.000
    7 33 CGFEaTYLEL 2.000
    8 69 INGQlVFSKL 1.560
    9 106 TNSRpPCVIL 1.000
    10 80 NGGFpYEKDL 1.000
    11 16 EVEPgSGVRI 0.900
    12 96 ASNGeTLEKI 0.720
    13 34 GFEAtYLELA 0.600
    14 60 GGTGaFEIEI 0.600
    15 58 RLGGtGAFEI 0.600
    16 18 EPGSgVRIVV 0.520
    17 83 FPYEkDLIEA 0.400
    18 28 EYCEpCGFEA 0.400
    19 1 MSGEpGQTSV 0.400
    20 2 SGEPgQTSVA 0.300
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_3902
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 70 NGQLVFSKL 2.400
    2 81 GGFPYEKDL 2.400
    3 94 RRASNGETL 2.000
    4 34 GFEATYLEL 2.000
    5 107 NSRPPCVIL 0.600
    6 57 SRLGGTGAF 0.500
    7 65 FEIEINGQL 0.480
    8 51 PGIEIESRL 0.240
    9 32 PCGFEATYL 0.200
    10 75 FSKLENGGF 0.150
    11 86 EKDLIEAIR 0.120
    12 6 GQTSVAPPP 0.120
    13 71 CQLVFSKLE 0.120
    14 46 VKEQYPGIE 0.120
    15 89 LIEAIRRAS 0.120
    16 21 SGVRIVVEY 0.120
    17 98 NGETLEKIT 0.120
    18 36 EATYLELAS 0.120
    19 38 TYLELASAV 0.120
    20 31 EPCGFEATY 0.120
    User Parameters and Scoring Information
    method selected to limit number of explicit number
    results
    number of results requested  20
    HLA molecule type selected B_3902
    length selected for subsequences  9
    to be scored
    echoing mode selected for input Y
    sequence
    echoing format numbered lines
    length of user's input peptide 115
    sequence
    number of subsequence scores 107
    calculated
    number of top-scoring subsequences  20
    reported back in scoring output
    table
    Scoring Results
    Score (Estimate of Half
    Time of Disassociation of
    Start Subsequence Residue a Molecule Containing This
    Rank Position Listing Subsequence)
    1 70 NGQLVFSKL 2.400
    2 81 GGFPYEKDL 2.400
    3 94 RRASNGETL 2.000
    4 34 GFEATYLEL 2.000
    5 107 NSRPPCVIL 0.600