Patent application title: CONSTRUCTION AND METHODS OF USE OF A THERAPEUTIC CANCER VACCINE LIBRARY COMPRISING FUSION-SPECIFIC VACCINES
Inventors:
IPC8 Class: AA61K3900FI
USPC Class:
1 1
Class name:
Publication date: 2019-01-31
Patent application number: 20190030147
Abstract:
Methods described herein relate to constructing therapeutic
fusion-specific vaccine libraries, selecting a therapeutic
fusion-specific vaccine for a cancer patient, and/or constructing a de
novo therapeutic fusion-specific vaccine for patients having a gene
fusion that is absent from a fusion-specific vaccine library.Claims:
1. A method for constructing a therapeutic fusion-specific vaccine
library for a cancer patient, the method comprising: a) identifying one
or more tumor-specific gene fusions from patient data; b) identifying one
or more fusion-derived neoantigens, wherein the fusion-derived neoantigen
comprises amino acid sequences corresponding to one or more
tumor-specific gene fusions; c) obtaining information relating to
interaction between fusion-derived neoantigens with one or more HLA
alleles; and d) selecting at least one fusion-derived neoantigen for the
therapeutic fusion-specific vaccine library.
2. A method of selecting a therapeutic fusion-specific vaccine for a cancer patient, the method comprising: a) determining the presence of one or more tumor-specific gene fusions in a biological sample from the cancer patient; b) obtaining information relating to one or more HLA alleles of the cancer patient; c) analyzing a library of fusion-specific vaccines, wherein each fusion-specific vaccine in the library comprises one or more fusion-derived neoantigens having a nucleic acid sequence and/or an amino acid sequence corresponding to one or more tumor-specific gene fusions; and d) selecting at least one fusion-specific vaccine in the library as the therapeutic fusion-specific vaccine for the cancer patient by selecting the fusion-specific vaccine comprising one or more fusion-derived neoantigens that i) correspond to one or more tumor-specific gene fusions of the cancer patient, and ii) bind one or more HLA alleles of the cancer patient.
3. A method of constructing a de novo therapeutic fusion-specific vaccine for a cancer patient, the method comprising: a) determining the presence of one or more tumor-specific gene fusions in a biological sample from the cancer patient; b) obtaining information relating to one or more HLA alleles of the cancer patient; c) constructing at least one fusion-specific vaccine as the de novo therapeutic fusion-specific vaccine for the cancer patient by constructing the fusion-specific vaccine comprising one or more fusion-derived neoantigens that i) correspond to one or more tumor-specific gene fusions of the cancer patient, and ii) bind one or more HLA alleles of the cancer patient.
4. The method of any one of the foregoing, further comprising administering the therapeutic fusion-specific vaccine to the cancer patient.
5. The method of claim 2 or 3, wherein the biological sample is a tissue sample or fluid sample taken from the cancer patient.
6. The method of claim 2 or 3, wherein the biological sample is analyzed by DNA sequencing, RNA sequencing, and/or protein sequencing.
7. The method of claim 2 or 3, wherein one or more tumor-specific gene fusions are identified in the biological sample.
8. The method of any one of the foregoing claims, wherein information relating to one or more HLA alleles of the cancer patient are obtained by analysis of the biological sample.
9. The method of any one of the foregoing claims, wherein the fusion-specific vaccine comprises one or more nucleic acid sequences corresponding to one or more tumor-specific gene fusions.
10. The method of any one of the foregoing claims, wherein the fusion-specific vaccine comprises one or more peptides having amino acid sequences corresponding to one or more tumor-specific gene fusions.
11. The method of any one of the foregoing claims, wherein the fusion-specific vaccine comprises one or more peptides selected from Table A.
12. The method of any one of the foregoing claims, wherein the fusion-specific vaccine comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 peptides corresponding to one or more tumor-specific gene fusions.
13. The method of any one of the foregoing claims, wherein the fusion-specific vaccine comprises 20 or more peptides corresponding to one or more tumor-specific gene fusions.
14. The method of any one of the foregoing claims, wherein the fusion-specific vaccine comprises one or more peptides that bind to one or more HLA alleles of the cancer patient with an IC50 less than 500 nM.
15. The method of any one of the foregoing claims, wherein the fusion-specific vaccine comprises one or more peptides that bind to one or more HLA alleles of the cancer patient with an IC50 less than 250 nM.
16. The method of any one of the foregoing claims, wherein the fusion-specific vaccine comprises one or more peptides that bind to one or more HLA alleles of the cancer patient with an IC50 less than 100 nM.
17. The method of any one of the foregoing claims, wherein the fusion-specific vaccine comprises one or more peptides that bind to one or more HLA alleles of the cancer patient with an IC50 less than 50 nM.
18. The method of any of the foregoing claims, wherein the library of fusion-specific vaccines comprises at least 2-10 fusion-specific vaccines corresponding to distinct fusion-derived neoantigens.
19. The method of any of the foregoing claims, wherein the library of fusion-specific vaccines comprises at least 5-10 fusion-specific vaccines corresponding to distinct fusion-derived neoantigens.
20. The method of any of the foregoing claims, wherein the library of fusion-specific vaccines comprises at least 10-50 fusion-specific vaccines corresponding to distinct fusion-derived neoantigens.
21. The method of any of the foregoing claims, wherein the library of fusion-specific vaccines comprises at least 50-100 fusion-specific vaccines corresponding to distinct fusion-derived neoantigens.
22. The method of any of the foregoing claims, wherein the library of fusion-specific vaccines comprises 100 or more fusion-specific vaccines corresponding to distinct fusion-derived neoantigens.
23. The method of any of the foregoing claims, wherein the fusion-specific vaccine comprises one or more peptides having 8-10 amino acids.
24. The method of any of the foregoing claims, wherein the fusion-specific vaccine comprises one or more peptides having 10-25 amino acids.
25. The method of any of the foregoing claims, wherein the fusion-specific vaccine comprises one or more peptides having 25-50 amino acids.
26. The method of any of the foregoing claims, wherein the fusion-specific vaccine comprises one or more peptides having 50-100 amino acids.
27. The method of any of the foregoing claims, wherein the fusion-specific vaccine comprises one or more peptides having 100 or more amino acids.
28. The method of any of the foregoing claims, wherein the fusion-specific vaccine comprises one or more peptides having an amino acid sequence corresponding to a one or more tumor-specific gene fusions and one or more addition amino acid sequences.
29. The method of any of the foregoing claims, wherein the fusion-specific vaccine comprises two or more peptides having an amino acid sequence corresponding to two or more tumor-specific gene fusions and one or more addition amino acid sequences.
30. The method of any of the foregoing claims, wherein the fusion-specific vaccine comprises one or more peptides having a modification.
31. The method of any of the foregoing claims, wherein the fusion-specific vaccine comprises one or more adjuvants.
32. The method of any of the foregoing claims, wherein the fusion-specific vaccine comprises the peptide having a concentration of 100 ng-10 mg.
33. The method of any of the foregoing claims, wherein the fusion-specific vaccine is administered once to the cancer patient.
34. The method of any of the foregoing claims, wherein the cancer patient is treated with two or more doses of the fusion-specific vaccine.
35. The method of any one of the foregoing claims, wherein the library of fusion-specific vaccines comprises fusion-derived neoantigens corresponding to gene fusions that are present in a type of cancer.
36. The method of any one of the foregoing claims, wherein the type of cancer is melanoma, skin cancer, head and neck cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, testicular cancer, brain tumor, glioma, glioblastoma, solitary fibrous tumor, bladder cancer, colorectal cancer, renal cell carcinoma, sarcoma, myeloma, leukemia, and/or lymphoma.
37. The method of any one of the foregoing claims, wherein the type of cancer is a metastatic tumor.
38. The method of any one of the foregoing claims, wherein the type of cancer is any tumor type.
39. The method of any one of the foregoing claims, wherein the cancer patient is administered dendritic cells or other antigen presenting cells that have been pulsed with at least one fusion-derived neoantigens from the fusion-specific vaccine library.
40. The method of any one of the foregoing claims, wherein autologous T-cells can be collected from the cancer patient, stimulated ex vivo with fusion-derived neoantigens, and stimulated autologous T-cells are transfused into the cancer patient.
41. The method of any one of the foregoing claims, wherein one or more fusion-specific vaccines are DNA or RNA molecules that encode fusion-derived neoantigens.
42. The method of any one of the foregoing claims, wherein the cancer patient is administered dendritic cells or other antigen presenting cells comprising at least one fusion-derived neoantigens from the fusion-specific vaccine library.
Description:
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn. 119(e) of the filing date of U.S. provisional patent application Ser. No. 62/319,774, entitled "CONSTRUCTION AND METHODS OF USE OF A THERAPEUTIC CANCER VACCINE LIBRARY COMPRISING FUSION-SPECIFIC VACCINES," filed Apr. 7, 2016, the entire contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The methods described herein are directed toward the field of immunotherapy for cancer patients.
BACKGROUND
[0003] Recent advances in personalized genomic sequencing and cancer genomic sequencing technologies have made it possible to obtain patient-specific information. This information can be used to prepare patient-specific medicines including patient-specific cancer vaccines that are developed based on the genome of the patient and the mutation information gathered from the patient's biological sample.
SUMMARY
[0004] Provided herein, inter alia, are methods for constructing therapeutic fusion-specific vaccine libraries, selecting a therapeutic fusion-specific vaccine for a cancer patient, and/or constructing a de novo therapeutic fusion-specific vaccine for patients having a gene fusion that is absent from a fusion-specific vaccine library.
[0005] In some embodiments, the method provides compositions for providing therapeutic fusion-specific vaccine libraries that can be used as sources of vaccines for many cancer patients.
[0006] In some aspects, the specification provides a method for constructing a therapeutic fusion-specific vaccine library for a cancer patient, the method comprising (a) identifying one or more tumor-specific gene fusions from publicly available data, (b) identifying one or more fusion junction sequences, wherein the fusion junction sequences comprise nucleic acid sequences and/or amino acid sequences corresponding to one or more tumor-specific gene fusions, (c) obtaining information relating to interaction between fusion junction sequences with one or more HLA alleles, and (d) selecting at least one fusion junction sequence for the therapeutic fusion-specific vaccine library.
[0007] In some aspects, the specification provides a method for selecting a therapeutic fusion-specific vaccine for a cancer patient, the method comprising (a) determining the presence of one or more tumor-specific gene fusions in a biological sample from the cancer patient, (b) obtaining information relating to one or more HLA alleles of the cancer patient, (c) analyzing a library of fusion-specific vaccines, wherein each fusion-specific vaccine in the library comprises one or more fusion junction sequences having a nucleic acid sequences and/or an amino acid sequence corresponding to one or more tumor-specific gene fusions, and (d) selecting at least one fusion-specific vaccine in the library as the therapeutic fusion-specific vaccine for the cancer patient by selecting the fusion-specific vaccine comprising (i) one or more fusion junction sequences corresponding to one or more tumor-specific gene fusions of the cancer patient, and (ii) one or more fusion junction sequences that bind one or more HLA alleles of the cancer patient.
[0008] In some aspects, the specification provides a method for constructing a de novo therapeutic fusion-specific vaccine for a cancer patient, the method comprising (a) determining the presence of one or more tumor-specific gene fusions in a biological sample from the cancer patient, (b) obtaining information relating to one or more HLA alleles of the cancer patient, (c) determining that the cancer patient's fusion junction sequence is not present in the library of fusion-specific vaccines, and (d) constructing at least one fusion-specific vaccine as the de novo therapeutic fusion-specific vaccine for the cancer patient by constructing the fusion-specific vaccine comprising (i) one or more fusion junction sequences corresponding to one or more tumor-specific gene fusions of the cancer patient, and (ii) one or more fusion junction sequences that bind one or more HLA alleles of the cancer patient.
[0009] In some embodiments, the method further comprises administering the therapeutic fusion-specific vaccine to the cancer patient.
[0010] In some embodiments, the biological sample is a tissue sample or fluid sample taken from the cancer patient. In some embodiments, the biological sample is analyzed by DNA sequencing, RNA sequencing, and/or protein sequencing. In some embodiments, one or more tumor-specific gene fusions are identified in the biological sample. In some embodiments, information relating to one or more HLA alleles of the cancer patient are obtained by analysis of the biological sample.
[0011] In some embodiments, the fusion-specific vaccine comprises one or more nucleic acid sequences corresponding to one or more tumor-specific gene fusions. In some embodiments, the fusion-specific vaccine comprises one or more peptides having amino acid sequences corresponding to one or more tumor-specific gene fusions. In some embodiments, the fusion-specific vaccine comprises one or more peptides selected from Table A.
[0012] In some embodiments, the fusion-specific vaccine comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 peptides corresponding to one or more tumor-specific gene fusions. In some embodiments, the fusion-specific vaccine comprises 20 or more peptides corresponding to one or more tumor-specific gene fusions.
[0013] In some embodiments, the fusion-specific vaccine comprises one or more peptides that bind to one or more HLA alleles of the cancer patient with an IC.sub.50 less than 500 nM. In some embodiments, the fusion-specific vaccine comprises one or more peptides that bind to one or more HLA alleles of the cancer patient with an IC.sub.50 less than 250 nM. In some embodiments, the fusion-specific vaccine comprises one or more peptides that bind to one or more HLA alleles of the cancer patient with an IC.sub.50 less than 100 nM. In some embodiments, the fusion-specific vaccine comprises one or more peptides that bind to one or more HLA alleles of the cancer patient with an IC.sub.50 less than 50 nM.
[0014] In some embodiments, the library of fusion-specific vaccines comprises at least 2-10 fusion-specific vaccines having distinct fusion junction sequences. In some embodiments, the library of fusion-specific vaccines comprises at least 5-10 fusion-specific vaccines corresponding to distinct fusion junction sequences. In some embodiments, the library of fusion-specific vaccines comprises at least 10-50 fusion-specific vaccines corresponding to distinct fusion junction sequences. In some embodiments, the library of fusion-specific vaccines comprises at least 50-100 fusion-specific vaccines corresponding to distinct fusion junction sequences. In some embodiments, the library of fusion-specific vaccines comprises 100 or more fusion-specific vaccines corresponding to distinct fusion junction sequences.
[0015] In some embodiments, the fusion-specific vaccine comprises one or more peptides having 8-10 amino acids. In some embodiments, the fusion-specific vaccine comprises one or more peptides having 10-25 amino acids. In some embodiments, the fusion-specific vaccine comprises one or more peptides having 25-50 amino acids. In some embodiments, the fusion-specific vaccine comprises one or more peptides having 50-100 amino acids. In some embodiments, the fusion-specific vaccine comprises one or more peptides having 100 or more amino acids. In some embodiments, the fusion-specific vaccine comprises a combination of two or more peptides having numbers of amino acids described herein.
[0016] In some embodiments, the fusion-specific vaccine comprises one or more peptides having an amino acid sequence corresponding to a one or more tumor-specific gene fusions and one or more addition amino acid sequences. In some embodiments, the fusion-specific vaccine comprises two or more peptides having an amino acid sequence corresponding to two or more tumor-specific gene fusions and one or more addition amino acid sequences.
[0017] In some embodiments, the fusion-specific vaccine comprises one or more peptides having a modification. In some embodiments, the fusion-specific vaccine comprises one or more adjuvants. In some embodiments, the fusion-specific vaccine comprises the peptide having a concentration of 100 ng-10 mg.
[0018] In some embodiments, the fusion-specific vaccine is administered once to the cancer patient. In some embodiments, the cancer patient is treated with two or more doses of the fusion-specific vaccine.
[0019] In some embodiments, the library of fusion-specific vaccines comprises peptides having an amino acid sequence corresponding to gene fusions that are present in a type of cancer. In some embodiments, the type of cancer is melanoma, skin cancer, head and neck cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, testicular cancer, brain tumor, glioma, glioblastoma, solitary fibrous tumor, bladder cancer, colorectal cancer, renal cell carcinoma, sarcoma, myeloma, leukemia, and/or lymphoma. In some embodiments, the type of cancer is a metastatic tumor. In some embodiments, the type of cancer is any tumor type.
[0020] In some embodiments, the cancer patient is administered dendritic cells or other antigen presenting cells that have been pulsed with at least one fusion junction peptide having a sequence from the fusion-specific vaccine library. In some embodiments, the cancer patient is administered dendritic cells or other antigen presenting cells comprising at least one fusion junction peptide having a sequence from the fusion-specific vaccine library. In some embodiments, autologous T-cells can be collected from the cancer patient, stimulated ex vivo with fusion junction sequences, and stimulated autologous T-cells are transfused into the cancer patient.
BRIEF DESCRIPTION OF DRAWINGS
[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0022] FIG. 1 illustrates a non-limiting example of a typical strategy for generating personalized neoantigenic vaccines including 3 steps: (1) detection of mutations from sequencing data and mutation calling; (2) identification of personal neoantigens; and (3) vaccine composition.
[0023] FIG. 2 illustrates a non-limiting example of a strategy for constructing a vaccine library comprising (1) screening of publicly available databases containing cancer sample sequences, information relating to genomic aberrations in cancer, and/or information relating to gene fusions in cancer; (2) extracting gene fusion incidence data; (3) analyzing fusion breakpoint peptides and MHC interaction; (4) selecting fusion breakpoint peptides based on affinity for MHC proteins; (5) calculating population coverage of selected peptides; and (6) incorporation of selected peptides into vaccine library.
[0024] FIG. 3 illustrates a non-limiting scheme for selecting patients for treatment with a vaccine library comprising (1) examining patient's cancer genome for gene fusions; (2) determining patient's HLA genotype; (3) matching vaccine library to patient's gene fusion and HLA type, and (4) administering a vaccine from the vaccine library to the patient or de novo vaccine construction from the patient's tumor specific mutations or selection of another treatment.
[0025] FIG. 4 shows a non-limiting example of a therapeutic cancer vaccine comprising BCR/ABL1 fusion peptides. The method described in the specification is applied to identification of BCR/ABL1 fusion peptides for the treatment of CML. Three breakpoints in BCR and ABL1 genes are marked with arrows (1). Fusion at these breakpoints yields two BCR/ABL1 gene fusions that are present in 90% of CML patients which comprises 45,000 patients in the U.S. and 950,00 patients worldwide (2). The method discloses two BCR/ABL1 fusion peptides selected based on allele coverage and patient coverage that are present in 68% of CML patients which means that 30,000 patients in the U.S. and 650,000 patients worldwide are eligible for treatment with a cancer vaccine comprising these two peptides (3). The sequences correspond to SEQ ID NOs: 610 to 611 from top to bottom, respectively.
[0026] FIG. 5 shows a non-limiting example of an environment in which embodiments of the present methods may operate.
[0027] FIG. 6 shows a non-limiting example of a data structure for storing data associated with one or more patients, in accordance with some embodiments of the present methods.
[0028] FIG. 7 shows flow chart of an illustrative process for matching patient data and library data, in accordance with some embodiments of the present methods.
[0029] FIG. 8 shows a block diagram of an illustrative computer system that may be used in implementing aspects of the present methods.
DETAILED DESCRIPTION
[0030] Applicant has recognized that patient-specific medicines, such as patient-specific vaccines, although effective, can be very expensive. As illustrated in FIG. 1, generating a patient specific vaccine typically involves a series of steps that are tailored for each individual patient, including detecting mutations from patient sequence data, identifying patient-specific neoantigens, and designing and synthesizing a patient-specific vaccine composition. In contrast, Applicant has developed methods and compositions for providing simple vaccine libraries that can be used as sources of vaccines for many cancer patients. Applicant has recognized that many cancers involve gene fusions, regardless of whether the cancers also include point mutations or other genetic rearrangements. Applicant has developed a method involving a vaccine library comprising a plurality of fusion junction sequences corresponding to the protein products of gene fusions associated with cancers in many patients. Aspects of the application are based, at least in part, on the recognition that gene fusions are present in many cancers and that a library of fusion-specific vaccines can be generated to represent a significant percentage of cancers without requiring de novo construction of personalized cancer vaccines for patients having cancers associated with these gene fusions.
[0031] Accordingly, aspects of the application relate to methods and compositions for providing fusion-specific cancer vaccines to patients without resorting to personalized vaccine development for each patient. In some embodiments, a library of fusion-specific vaccines is provided and a patient is evaluated to determine whether the patient will be responsive to at least one of the vaccines in the library. In some embodiments, the presence of one or more cancer-associated gene fusions is identified in the patient. In some embodiments, the HLA genotype of the patient is evaluated to determine whether the patient will be responsive to one or more fusion-specific vaccines corresponding to a cancer-associated gene fusion identified in the patient. In some embodiments, if the patient is determined to be responsive to at least one fusion-specific vaccine in the library, then one or more of the vaccines is administered to the patient. In some embodiments, if the patient is determined not to be responsive to at least one fusion-specific vaccine in the library, then a personalized vaccine can be prepared de novo for the patient.
A Fusion-Specific Vaccine Library
[0032] In some aspects, the specification provides a method for constructing a therapeutic fusion-specific vaccine library for a cancer patient, including (a) identifying one or more tumor-specific gene fusions from publicly available data, (b) identifying one or more fusion junction sequences, wherein the fusion junction sequences comprise nucleic acid sequences and/or amino acid sequences corresponding to one or more tumor-specific gene fusions, (c) obtaining information relating to interaction between fusion junction sequences with one or more HLA alleles, and (d) selecting at least one fusion junction sequence for the therapeutic fusion-specific vaccine library. For example, FIG. 2 illustrates a non-limiting embodiment for constructing a vaccine library that includes screening publicly available databases containing cancer sample sequences, information relating to genomic aberrations in cancer, and/or information relating to gene fusions in cancer; extracting gene fusion incidence data; analyzing fusion breakpoint peptides and MHC interaction; selecting fusion breakpoint peptides based on affinity for MHC proteins; calculating population coverage of selected peptides; and incorporation of selected peptides into vaccine library. It should be appreciated that one or more of these steps may be omitted or modified depending on the vaccine library being developed. For example, sequence and related information can be obtained from any suitable source including public, private, customized, or other libraries containing cancer-related sequence information. In some embodiments, gene fusion incidence data does not need to be used. In some embodiments, population coverage does not need to be calculated. Accordingly, in some embodiments, a library can be designed by i) selecting fusion peptides of interest (e.g., based on one or more criteria, for example the types of cancer to be covered, the target patient population, the geographic region, the size and range of peptides to be included in the library, etc., or any combination thereof), and ii) obtaining or calculating MHC interaction information for those peptides. In some embodiments, the library can by synthesized or assembled using any suitable synthetic techniques (e.g., any suitable peptide chemistry, recombinant expression techniques, or any combination thereof).
[0033] In some aspects, the specification provides a method for selecting a therapeutic fusion-specific vaccine for a cancer patient, the method comprising (a) determining the presence of one or more tumor-specific gene fusions in a biological sample from the cancer patient, (b) obtaining information relating to one or more HLA alleles of the cancer patient, (c) analyzing a library of fusion-specific vaccines, wherein each fusion-specific vaccine in the library comprises one or more fusion junction sequences having a nucleic acid sequences and/or an amino acid sequence corresponding to one or more tumor-specific gene fusions, and (d) selecting at least one fusion-specific vaccine in the library as the therapeutic fusion-specific vaccine for the cancer patient by selecting the fusion-specific vaccine comprising (i) one or more fusion junction sequences corresponding to one or more tumor-specific gene fusions of the cancer patient, and (ii) one or more fusion junction sequences that bind one or more HLA alleles of the cancer patient. FIG. 3 illustrates a non-limiting example of a method of selecting a vaccine for a patient from a vaccine library described in this application. In some embodiments, i) a patient's cancer genome (e.g., the genome of a cancer in the patient, for example obtained from a biopsy or other sample obtained from cancer tissue in the patient) is analyzed to detect the presence of one or more gene fusions, ii) the patient's HLA genotype is determined (e.g., by sequencing one or more HLA loci of the patient, or obtained from whole genome sequence data for the patient, and iii) the library and the patient's HLA genotype are evaluated to determine with the library contains one or more peptides that match the patient's HLA genotype (e.g., based on one or more matching criteria that can be selected by the user, for example by a physician or a healthcare organization). If the library contains one or more peptides that are suitable for the patient (e.g., that correspond to one or more tumor specific mutations/neoantigens and that are HLA matched, for example based on specified HLA match criteria), then a vaccine containing one or more of those peptides can be selected from the library and administered to the patient. In the event that the library does not contain any peptides that are suitable for the patient (e.g., based on the tumor specific mutations in the patient that are not represented in the library and/or based on a failure to meet the specified HLA match criteria) then a de novo vaccine can be designed and synthesized for the patient (e.g., based on the patient's tumor specific mutations/neoantigens and/or HLA type) and/or a different therapy can be selected for the patient.
[0034] In some aspects, the specification provides a method for constructing a de novo therapeutic fusion-specific vaccine for a cancer patient, the method comprising (a) determining the presence of one or more tumor-specific gene fusions in a biological sample from the cancer patient, (b) obtaining information relating to one or more HLA alleles of the cancer patient, (c) determining that the cancer patient's fusion junction sequence is not present in the library of fusion-specific vaccines, and (d) constructing at least one fusion-specific vaccine as the de novo therapeutic fusion-specific vaccine for the cancer patient by constructing the fusion-specific vaccine comprising (i) one or more fusion junction sequences corresponding to one or more tumor-specific gene fusions of the cancer patient, and (ii) one or more fusion junction sequences that bind one or more HLA alleles of the cancer patient.
[0035] In some embodiments, the fusion-specific vaccine comprises one or more nucleic acid sequences corresponding to one or more tumor-specific gene fusions. In some embodiments, the fusion-specific vaccine comprises one or more peptides having amino acid sequences corresponding to one or more tumor-specific gene fusions. In some embodiments, the fusion-specific vaccine comprises one or more peptides selected from Table A. In some embodiments, one or more of the peptides listed in Table A is included in a peptide vaccine library. In some embodiments, one or more peptides in a peptide vaccine library consists of or includes a fragment of a peptide listed in Table A. In some embodiments, a fragment includes at least one amino acid (e.g., at least 2, 3, 4, 5 or more amino acids) from the N-terminal portion of a fusion peptide and at least one amino acid (e.g., at least 2, 3, 4, 5 or more amino acids) from the C-terminal portion of a fusion peptide. A vaccine library can include a plurality separate receptacles each containing a different peptide or a different combination of two or more peptides. Accordingly, a library can be a collection of receptacles, for example, wells, vials, tubes, or other containers capable of storing one or more different peptides, for example in the form of dry preparations (e.g., powder or crystalline form), liquid preparations (e.g., aqueous, organic, or other solutions), suspensions, or a combination thereof in different containers. In some embodiments, the receptacles are separate. In some embodiments, the receptacles are connected (e.g., in the form of an array, for example a 2D array). Receptacles can be made of any suitable material (e.g., glass, plastic, polymer, or other suitable material). In some embodiments, different peptides may be provided in different solutions (e.g., different salt concentrations, different pHs, etc.) depending on their solubility properties. Peptides in receptacles in a library can be provided at any suitable concentration (for example, nanomolar (e.g., from 1-1,000 nM), micromolar (e.g., from 1-1,000 .mu.M), millimolar (e.g., 1-1,000 mM), or molar (e.g., 1M or higher)). Different peptides may be provided at different concentrations. In some embodiments, a peptide in a library is in a solution without an adjuvant or other agent, and an adjuvant or other agent may be added when the vaccine is prepared. However, in some embodiments, a peptide preparation in a library may also contain an adjuvant and/or other agent (e.g., a solubilizing or stabilizing agent, a therapeutic agent, or other agent).
[0036] Once a suitable combination of peptides has been determined or selected for a patient as described in this application, a vaccine can be prepared for the patient by retrieving (e.g., using a pipette or other suitable device for transferring the peptide(s)) from the appropriate library receptacles an appropriate amount of the one or more peptides that were identified to be suitable for that patient. The resulting combination of peptide(s) can be transferred to one or more suitable containers (e.g., syringes, infusion pumps, etc.) for administration to the patient. During this process, a peptide solution from the library may be diluted (e.g., around 5.times., 10.times., 100.times., or other dilution) in a physiologically acceptable buffer for administration to a patient. An adjuvant also may be added at this stage. This procedure can be followed for a plurality of patients, for example at a hospital or other medical center.
[0037] Accordingly, in some embodiments a library can include a plurality (e.g., all, or a subset of) the peptides in Table A and a vaccine can be selected from that library based on the criteria described in this application. However, other libraries can be used (e.g., containing one or more of the peptides listed in Table A, and/or one or more other neoantigenic peptides) as described in this application.
TABLE-US-00001 TABLE A Peptides for vaccine library. SEQ ID NO. Long peptide for vaccine Exons Fusion name 1 AIKSKNVDVNVKDEETEVKSEEGPGWTILR E5:E5 ACBD6/RRP15 2 EAPLNPKANREKMTQDPLLGMLDGREDLER E3:E7 ACTB/GLI1 3 EAPLNPKANREKMTQPISGIPSPDESPKRA E3:E5 4 EAPLNPKANREKMTQLKSELDMLVGKCREE E3:E6 5 PRAVFPSIVGRPRHQLKSELDMLVGKCREE E2:E6 6 QTKVLSASQAFAAQRDSITQHKVCAPENYL E5:E14 AGPAT5/MCPH1 7 NDKLQKELNVLKSEQDLIRDQGFRGDGGST E8:E9 AKAP9/BRAF 8 QERERDPQQEQERERIDDVIDEIISLESSY E7:E5 ASPSCR1/TFE3 9 QERERDPQQEQERERLPVSGNLLDVYSSQG E7:E6 10 SSQKAHGILARRPSYRTHG E3:E10 ATF1/EWSR1 11 MSVPTPIYQTSSGQYSSFRQDHPSSMGVYG E4:E8 12 RNVQDFKRASEEITKTTQDGLDWLIST E10:E11 ATG4C/FBX038 13 TPAQLYTLQPKLPITVYRRKHQELQAMQME E7:E20 ATIC/ALK 14 TQLPHIPVAVRVGCLQYYRTDSDNQAILSE E9:E21 BBS9/PKD1L1 15 KTRVYRDTAEPNWNEKPFSGQ E15:E2 BCR/ABL1 16 RKQTGVFGVKIAVVTKSPSAASSI E18:E2 17 EITPRRQSMTVKKGESYTFLISSDYERAEW E8:E12 18 KLQTVHSIPLTINKEAGSIEALQRPVASDF E13:E2 19 KTRVYRDTAEPNWNELDPQALQDRDWQRTV E15:E17 20 QWSHQQRVGDLFQKLNLRARSNKDAKDPTT E4:E6 21 SRDALVSGALESTKATKAKPFSGQ E2:E2 22 SNKDAKDPTTKNSLEKALQRPVASDFEPQG E6:E2 23 ASEETYLSHLEALLLKPFSGQ E3:E2 24 EITPRRQSMTVKKGEKPFSGQ E8:E2 25 KLQTVHSIPLTINKEGEKLRVLGYNHNGEW E13:E3 26 GVATDIQALKAAFDVRPSPCEDRHWPGLAL E19:E1 27 GVATDIQALKAAFDVKALQRPVASDFEPQG E19:E2 28 QIWPNDGEGAFHGDAGEKLRVLGYNHNGEW E1:E3 29 LNVIVHSATGFKQSSSEKLRVLGYNHNGEW E14:E3 30 LNVIVHSATGFKQSSKALQRPVASDFEPQG E14:E2 31 KLQTVHSIPLTINKEEALQRPVASDFEPQG E13:E2 32 QIWPNDGEGAFHGDAEALQRPVASDFEPQG E1:E2 33 QIWPNDGEGAFHGDADYELLTENDMLPNMR E1:E19 BCR/JAK2 34 QIWPNDGEGAFHGDAVLQERIPWVPPECIE E1:E17 35 APNVHINTIEPVNIDNDLRLQMEAQRICLS E8:E9 BRAF/AKAP9 36 RYVKSCLQKKQRKPFSSALPGPDMSMKPSA E10:E2 BRD3/NUTM1 37 SSSESSSSDSEDSETASALPGPDMSMKPSA E11:E2 38 SEFREGVRKIAREQKVYRRKHQELQAMQME E17:E20 CARS/ALK 39 LQEENRDLRKASVTIEDPKWEFPRKNLVLG E1:E12 CCDC6/RET 40 EEEFLTNELSRKLMQEDPKWEFPRKNLVLG E2:E12 41 SRHSLEQKPTDAPPKVVVHRRLKNQKSAKEG E6:E35 CD74/ROS1 42 SRHSLEQKPTDAPPKAGVPNKPGIPKLLEG E6:E32 43 SRHSLEQKPTDAPPKDDFWIPETSFILTII E6:E34 44 VHPDALNRFGKTALQLHVHACLLTRKQEDC E2:E3 CDKN2D/WDFY2 45 VHPDALNRFGKTALQLHVHACLLTRKQEDC E2:E3 46 VHPDALNRFGKTALQLHVHACLLTRKQEDC E2:E3 47 LAENITQERDSLMCLDLIRDQGFRGDGGST E16:E9 CEP89/BRAF 48 TQEEEDEILPRKDYEKTLGRRDSSDDWEIP E2:E11 CLCN6/BRAF 49 EERSVLNNQLLEMKKSTLPTQEEIENLPAF E20:E36 CLIP1/ROS1 50 RKEEEQATETQPIVYVYRRKHQELQAMQME E31:E20 CLTC/ALK 51 AMPYFIQVMKEYLTKGVYRRKHQELQAMQM E30:E20 52 AMPYFIQVMKEYLTKCTAGSTRSCKPCRWS E30:E20 53 CVAYERGQCDLELINLPVSGNLLDVYSSQG E17:E6 CLTC/TFE3 54 GKAGERGVPGPPGAVGDPIPEELYEMLSDH E26:E2 COL1A1/PDGFB 55 GPSGPRGLPGPPGAPGDPIPEELYEMLSDH E7:E2 56 GAPGAKGARGSAGPPGDPIPEELYEMLSDH E37:E2 57 GPSGPAGPTGARGAPGDPIPEELYEMLSDH E34:E2 58 GPPGPAGPAGPPGPIGDPIPEELYEMLSDH E36:E2 59 GPPGPRGRTGDAGPVGDPIPEELYEMLSDH E47:E2 60 GPAGPPGFPGAVGAKGDPIPEELYEMLSDH E16:E2 61 GPRGANGAPGNDGAKGDPIPEELYEMLSDH E31:E2 62 GKPGRPGERGPPGPQGDPIPEELYEMLSDH E10:E2 63 YDEKSTGGISVPGPMGDPIPEELYEMLSDH E6:E2 64 GPQGPGGPPGPKGNSGDPIPEELYEMLSDH E19:E2 65 GLPGTAGLPGMKGHRGDPIPEELYEMLSDH E11:E2 66 GNPGADGQPGAKGANGDPIPEELYEMLSDH E18:E2 67 KRHVWFGESMTDGFQGDPIPEELYEMLSDH E49:E2 68 RTCRDLKMCHSDWKSGGPHSRGAL E48:E2 69 GFQGPPGEPGEPGASGDPIPEELYEMLSDH E8:E2 70 GEPGSPGENGAPGQMGDPIPEELYEMLSDH E13:E2 71 GHRGFSGLQGPPGPPGDPIPEELYEMLSDH E45:E2 72 GPIGPPGPAGAPGDKGDPIPEELYEMLSDH E33:E2 73 GQAGVMGFPGPKGAAGDPIPEELYEMLSDH E25:E2 74 PPGPPGPPGPPGLGGGDPIPEELYEMLSDH E5:E2 75 GKDGEAGAQGPPGPAGDPIPEELYEMLSDH E27:E2 76 GQRGERGFPGLPGPSGDPIPEELYEMLSDH E40:E2 77 GPPGLAGPPGESGREGDPIPEELYEMLSDH E41:E2 78 GPAGPVGPVGARGPAGDPIPEELYEMLSDH E44:E2 79 GVPGDLGAPGPSGARGDPIPEELYEMLSDH E30:E2 80 GLPGERGRPGAPGPAGDPIPEELYEMLSDH E14:E2 81 GPAGEKGSPGADGPAGDPIPEELYEMLSDH E39:E2 82 GARGNDGATGAAGPPGDPIPEELYEMLSDH E15:E2 83 GEPGPPGPAGAAGPAGDPIPEELYEMLSDH E17:E2 84 GAPGSKGDTGAKGEPGDPIPEELYEMLSDH E20:E2 85 GAEGSPGRDGSPGAKGDPIPEELYEMLSDH E42:E2 86 GEAGRPGEAGLPGAKGDPIPEELYEMLSDH E23:E2 87 GFPGAAGRVGPPGPSGDPIPEELYEMLSDH E38:E2 88 GPAGPPGEAGKPGEQGDPIPEELYEMLSDH E29:E2 89 GEQGPSGASGPAGPRGDPIPEELYEMLSDH E46:E2 90 GPVGPAGKSGDRGETGDPIPEELYEMLSDH E43:E2 91 GERGAAGLPGPKGDRGDPIPEELYEMLSDH E32:E2 92 THGQEEGQVEGQDEDRWTVV E1:E2 COL1A1/USP6 93 THGQEEGQVEGQDEDTKNTIK E1:E1 94 TLLLLAVTLCLATCQYVVPKWEGLDSTLFHE E1:E3 COL1A2/PLAG1 95 TLLLLAVTLCLATCQCCLLVLPWPYLAPRM E1:E2 96 APSALSSSPLLTAPHRK E5:E7 CREB3L2/FUS 97 APSALSSSPLLTAPHVTMAKINPP E5:E6 98 ELSEPGDGEALMYHTALGTKDHVMTPNRII E1:E8 99 ELSEPGDGEALMYHTALGTKDHVMTP E1:E8 100 TRAFEQLMTDLTLSRLQGSLKRKQVVNLSP E1:E2 CRTC3/MAML2 101 SPPWDQDRRMMFPPPGILTNVI E20:E10 CTAGE5/SIP1 102 PPPSGIATLVSGIAGVYRRKHQELQAMQME E26:E20 DCTN1/ALK 103 DCVLVLLLMPRLICKCTAGSTRSCKPCRWS E16:E20 104 RHLVFPLLEFLSVKELVMYQIPFARVVCLV E1:E3 EIF3E/RSPO2 105 RHLVFPLLEFLSVKEVRGGEMLIALN E1:E2 106 NKYIMSNSGDYEILYLYRRKHQELQAMQME E20:E20 EML4/ALK 107 ILRGTFNDGFQIEVQCTAGSTRSCKPCRWS E15:E20 108 HTDGNEQLSVMRYSIVYRRKHQELQAMQME E18:E20 109 VTKTADKHKDVIINQVSPTPEPHLPLSLIL E6:E19 110 SEDHVASVKKSVSSKVYRRKHQELQAMQME E2:E20 111 IILWDHDLNPEREIECTAGSTRSCKPCRWS E14:E20 112 WSKTTVEPTPGKGPKVYRRKHQELQAMQME E13:E20 113 VTKTADKHKDVIINQVYRRKHQELQAMQME E6:E20 114 STSTQSKSSSGSAHFGPPRMQWRSPPG E10:E2 EPC1/PHF1 115 ECEQAERLGAVDESLRKYIFKPRTV E5:E5 ERO1L/FERMT2 116 ALRMEEDSIRLPAHLPKNRGIAIPVDLDSQ E2:E35 ETV6/ITPR2 117 IHTQPEVILHQNHEEGRKHPYSWECMCQKY E4:E16 ETV6/JAK2 118 IHTQPEVILHQNHEEVLQERIPWVPPECIE E4:E17 119 VSPPEEHAMPIGRIADYELLTENDMLPNMR E5:E19 120 VSPPEEHAMPIGRIADKSNLLVFRTNGVSD E5:E12 121 VSPPEEHAMPIGRIADVQHIKRRDIVLKRE E5:E15 ETV6/NTRK3 122 IHTQPEVILHQNHEEGPVAVISGEEDSASP E4:E14 123 VSPPEEHAMPIGRIAGPVAVISGEEDSASP E5:E14 124 IHTQPEVILHQNHEEDVQHIKRRDIVLKRE E4:E15 125 APSQYSQQSSSYGQQIAIAPNGALQLASPG E7:E5 EWSR1/ATF1 126 APSQYSQQSSSYGQQKKLLENVAERRKNM E7:E7 127 GMGSAGERGGFNKPGEKF E9:E4 128 GGMSRGGRGGGRGGMGKILKDLSSEDTRGR E8:E4 129 APSQYSQQSSSYGQQIAITQGGAIQLANNG E7:E7 EWSR1/CREB1 130 NKPGGPMDEGPDLDLVFKKEVYLHTSPHLK E10:E2 EWSR1/DDIT3 131 LPPREGRGMPPPLRGVFKKEVYLHTSPHLK E13:E2 132 APSQYSQQSSSYGQQMFKKEVYLHTSPHLK E7:E2 133 APSQYSQQSSSYGQQTAQPSPSTVPKTEDQ E7:E9 EWSR1/ERG 134 APSQYSQQSSSYGQQNPYQILGPTSSRLAN E7:E10 135 NKPGGPMDEGPDLDLDLPYEPPRRSAWTGH E10:E8 136 APSQYSQQSSSYGQQSSGQIQLWQFLLELL E7:E11 137 APSQYSQQSSSYGQQNLPYEPPRRSAWTGH E7:E8 138 APSQYSQQSSSYGQQRDIKQEPGMYREGPT E7:E12 EWSR1/ETV1 139 NKPGGPMDEGPDLDLDPVGDGLFKDGKNPS E10:E2 EWSR1/FEV 140 APSQYSQQSSSYGQQNPVGDGLFKDGKNPS E7:E2 141 GGMSRGGRGGGRGGMGIPVTASLPPLEWPL E8:E3 EWSR1/NFATC2 142 YEDPPTAKAAVEWFDDMPCVQAQYSPSPPG E12:E3 EWSR1/NR4A3 143 APSQYSQQSSSYGQQRPMDEGPDLDLGPPV E7:E10 144 GMGSAGERGGFNKPGGPPVDPDEDSDNSAI E9:E11 145 LPPREGRGMPPPLRGDMPCVQAQYSPSPPG E13:E3 146 NKPGGPMDEGPDLDLDMPCVQAQYSPSPPG E10:E3 147 YEDPPTAKAAVEWFDEPTAEEGSPASPGPE E12:E2 148 APSQYSQQSSSYGQQKPTAEEGSPASPGPE E7:E2 149 APSQYSQQSSSYGQQSGRDGISTSKRQKS E7:E5 EWSR1/PBX1 150 GGMSRGGRGGGRGGMGRKRRNFNKQATEIL E8:E5 151 APSQYSQQSSSYGQQNVKWGKLRDYQVRGL E7:E5 EWSR1/SMARCA5 152 GGMSRGGRGGGRGGMGGTNLGKKKQHICHI E8:E6 EWSR1/SP3 153 YGQTAYATSYGQPPTEGTSTGYTTPTAPQA E4:E5 EWSR1/WT1 154 AYPAYGQQPAATAPTSSAGERGGFNKPG E5:E9 155 AYPAYGQQPAATAPTSYSSTQPTSYDQSSY E5:E7 156 APSQYSQQSSSYGQQNEKKDIDHETVVEEQ E7:E2 EWSR1/YY1 157 GGMSRGGRGGGRGGMGRHRPWG E8:E5 EWSR1/ZNF444 158 LRLQDYEEKTKKAERDDFWIPETSFILTII E10:E34 EZR/ROS1 159 TSSLHGSSLHRPSTEDLIRDQGFRGDGGST E2:E9 FAM131B/BRAF 160 MGCIGSRTVGSTTGLSATPPASLPG E1:E10 161 STEQTRTDFSWDGINDLIRDQGFRGDGGST E3:E9 162 MASSGERTPSNALDPRWRSLW E1:E17 FBXL18/RNF216 163 RLSEARLSQRDLSPTDLIRDQGFRGDGGST E13:E9 FCHSD1/BRAF 164 ATLCTARPSPTLPEQDWPKWEGLDSTLFHE E2:E3 FGFR1/PLAG1 165 ATLCTARPSPTLPEQGCLLVLPWPYLAPRM E2:E2 166 LVEDLDRIVALTSNQGLLESSAEKAPVSVS E17:E7 FGFR1/TACC1 167 ARDIHHIDYYKKTTNLDAKKSPLALLAQTC E14:E2 FGFR1/ZNF703 168 LVEDLDRVLTVTSTDNVMEQFNPGLRNLIN E17:E2 FGFR3/BAIAP2L1 169 LVEDLDRVLTVTSTDFKESALRKQSLYLKF E17:E8 FGFR3/TACC3 170 LVEDLDRVLTVTSTDVPGPPPGVPAPGGPP E17:E10 171 LVEDLDRVLTVTSTDVKATQEENRELRSRC E17:E11 172 RKAGDFHRNDSIYEEPQQVVQKKPAQEETE E8:E4 FHIT/HMGA2 173 YFKDKGDSNSSAGWKVMGLLTNHGGVPHQP E1:E8 FOXO1/PAX3 174 QYNSSSGGGGGGGGGVAIAPNGALQLASPG E5:E5 FUS/ATF1 175 EPRGRGGGRGGRGGMGPSGPPAFAAHPSEQ E6:E5 FUS/CREB3L2 176 EPRGRGGGRGGRGGMGNCRDQALWS E6:E6 177 EPRGRGGGRGGRGGMGPQWTTCICRPPLRA E6:E5 178 EPRGRGGGRGGRGGMGCLPSGPPAFAAHPS E6:E5 179 QYNSSSGGGGGGGGGAPVDHLHLPPTPPSS E5:E5 180 QYNSSSGGGGGGGGGETAGIRPSGPDRGGE E5:E6 181 GGMGGSDRGGFNKFGAPVDHLHLPPTPPSS E7:E5
182 QYNSSSGGGGGGGGGVFKKEVYLHTSPHLK E5:E2 FUS/DDIT3 183 EPRGRGGGRGGRGGMGVQEGSVSSYITTPE E6:E2 184 GGMGGSDRGGFNKFGVFKKEVYLHTSPHLK E7:E2 185 GYGQSSYSSYGQSQNMFKKEVYLHTSPHLK E3:E2 186 FNRGGGNGRGGRGRGVFKKEVYLHTSPHLK E11:E2 187 IESVADYFKQIGIIKTDPTAEMAAESLPFS E9:E3 188 NKFGGPRDQGSRHDSVFKKEVYLHTSPHLK E8:E2 189 EPRGRGGGRGGRGGMGGSDRGGFNKFG E6:E7 FUS/ERG 190 GGMGGSDRGGFNKFGDLPYEPPRRSAWTGH E7:E8 191 NKFGGPRDQGSRHDSAAQPSPSTVPKTEDQ E8:E9 192 QYNSSSGGGGGGGGGDLPYEPPRRSAWTGH E5:E8 193 GGMGGSDRGGFNKFGDPYQILGPTSSRLAN E7:E10 194 GGMGGSDRGGFNKFGGSGQIQLWQFLLELL E7:E11 195 EPRGRGGGRGGRGGMGQWPDPALAVPPGAP E6:E11 196 FDDPPSAKAAIDWFDDPVGDGLFKDGKNPS E10:E2 FUS/FEV 197 RAENACVPPFTIEVKKTLGRRDSSDDWEIP E2:E11 GATM/BRAF 198 PLEGDMSSPNSTGIQIMFETFNTPAMYVAI E6:E4 GLI1/ACTB 199 MIPHPQSRGPFPTCQIMFETFNTPAMYVAI E5:E4 200 GKPRNVALITGITGQPLHICQP E1:E22 GMDS/PDE8B 201 EDGEKAAREVKLLLLGSTTGLSATPPASLP E1:E10 GNAI1/BRAF 202 QKLMGQIHQLRSELQEDPKWEFPRKNLVLG E7:E12 GOLGA5/RET 203 GARLAAKYLDKELAGSTLPTQEEIENLPAF E4:E36 GOPC/ROS1 204 GASCKDTSGEIKVLQVWHRRLKNQKSAKEG E8:E35 205 LINIMIEPQATRKAQDAIRSHSESASPSAL E16:E8 HACLI/RAFI 206 EVLRNLSSPGWENISSLLFVSKFFEHHPIP E4:E7 HERPUDI/BRAF 207 MTVDHLKMLHTAGGKAFNNPRPGQLGRLLP E4:E13 HEY1/NCOA2 208 VVASTISGKSQIEETVYRRKHQELQAMQME E28:E20 HIP1/ALK 209 KKHYELAGVAEGWEEVYRRKHQELQAMQME E30:E20 210 HGATTCLRAPPEPADLYRRKHQELQAMQME E21:E20 211 SDSAQGSDVSLTACKDDFWIPETSFILTII E9:E34 HLA-A/ROS1 212 EATGEKRPRGRPRKWVTVKVPQKNS E3:E13 HMGA2/ALDH2 213 PKGSKNKSPSKAAQKVHQERLYQEYNFSKA E2:E6 HMGA2/CCNB1IP1 214 EATGEKRPRGRPRKWNFFRLNYLEVCH E3:E4 HMGA2/COX6C 215 EATGEKRPRGRPRKWVVVSTTVNVDGHVLA E3:E8 HMGA2/EBF1 216 EATGEKRPRGRPRKWLQKHDKEDFPASWRS E3:E9 HMGA2/FHIT 217 EATGEKRPRGRPRKWGSVKSAGPTTAREQV E3:E4 HMGA2/LHFP 218 PRKWPQQVVQKKPAQSWYLG E4:E12 HMGA2/NFIB 219 EATGEKRPRGRPRKWSWYLG E3:E12 220 EATGEKRPRGRPRKWVILTNQITTHLSGAL E3:E8 HMGA2/RAD51B 221 EATGEKRPRGRPRKWLSASLAVVHMEPAMN E3:E9 HMGA2/WIF1 222 EATGEKRPRGRPRKWGTKPASYMP E3:E10 223 EATGEKRPRGRPRKWAEYFYEFLSLRSLDK E3:E3 224 EATGEKRPRGRPRKWLFKLVSHVLENRMGW E3:E4 225 PRKWPQQVVQKKPAQGTKPASYMP E4:E10 226 MESQRGRNCNEKPTNVR E1:E4 HNRNPA2B1/ETV1 227 KANAARSQLETYKRQEDPKWEFPRKNLVLG E11:E12 HOOK3/RET 228 SRSPPAENEVSTPMQKKKGGRGLMTENTMR E7:E3 IL6R/ATP8B2 229 SVEGVVRILLEHYYKAWKKRWFILRSGRMS E2:E2 INTS4/GAB2 230 SFRAIIRDLNSLFTPDCRLLWDYVYQLLSD E18:E6 JAK2/ETV6 231 NIIFQFTKCCPPKPKETGSDFSMFEALRDT E11:E25 JAK2/PCM1 232 SPKDFNKYFLTFAVEEAESGNISQKSDEED E10:E37 233 PPITPSSSFRSSTPTGPPRMQWRSPPG E3:E2 JAZF1/PHF1 234 PPITPSSSFRSSTPTEPTQIYRFLRTRNLI E3:E2 JAZF1/SUZ12 235 DLAEIGIAVGNNDVKEDPKWEFPRKNLVLG E15:E12 KIF5B/RET 236 VRSKNMARRGHSAQIDPLCDELCRTVIAAA E24:E11 237 LRKLFVQDLATRVKKEDPKWEFPRKNLVLG E22:E12 238 FLENNLEQLTKVHKQEDPKWEFPRKNLVLG E23:E12 239 VRSKNMARRGHSAQIDVAEEAGCPLSCAVS E24:E8 240 ENEKELAACQLRISQEDPKWEFPRKNLVLG E16:E12 241 EILTRAHEREFGSVDVYRRKHQELQAMQME E9:E20 KLC1/ALK 242 TLEGELHDLRGQVAKTLTAHLETRWRRRTK E2:E10 LMNA/NTRK1 243 TSSGAGFFLPQHDSSVWHRRLKNQKSAKEG E16:E35 LRIG3/ROS1 244 RGGRGGREFADFEYRDLIRDQGFRGDGGST E9:E9 LSM14A/BRAF 245 RKDEELTSSQRDLAVRDLKLDNILLDAEGH E6:E12 MBOAT2/PRKCE 246 QKKKAKSQQYKGHKKRTGWAPPTFLLYQFA E16:E1 MBTD1/CXorf67 247 SHHSSHKKRKNKNRHRPPQDAMAQPPRLSR E5:E2 MEAF6/PHF1 248 VLHPMDAAQRSQHIKKTLGRRDSSDDWEIP E4:E11 MKRN1/BRAF 249 KNLLEFAETLQFIDSFLNTSSNHENSDLEM E9:E11 MYB/NFIB 250 AFTVPKNRSLASPLQSWYLG E15:E12 251 AFTVPKNRSLASPLQPNGSGQVVGKVPGHF E15:E9 252 AFTVPKNRSLASPLQSSQLELHLHLHRCHF E15:E8 253 LMSTENELKGQQVLPLRICDWTMNQNGRHL E8:E11 254 AFTVPKNRSLASPLQPTQPQAHLKPIDMWD E15:E10 255 AFTVPKNRSLASPLQLRICDWTMNQNGRHL E15:E11 256 LMSTENELKGQQVLPPNGSGQVVGKVPGHF E8:E9 257 FQENGPPLLKKIKQESWYLG E13:E12 258 FQENGPPLLKKIKQELRICDWTMNQNGRHL E13:E11 259 LMSTENELKGQQVLPSWYLG E8:E12 260 EALNHRIVQQAKEMTVWHRRLKNQKSAKEG E23:E35 MYO5A/ROS1 261 FDSKRREGKQLSLHEATSKSQIMSLWGLVS E2:E2 NAB2/STAT6 262 DSASLSGESLDGHLQAEQMGKDGRGYVPAT E5:E17 263 QVARESTYLSSLKGSRQPPSPRSCLCGVWS E3:E2 264 QVARESTYLSSLKGSRPQVYPPHSHSIPPY E3:E19 265 DSASLSGESLDGHLQGTNHFLPQSSRCLPW E5:E18 266 PEELGGPPLKKLKQEGPTTSYPRAPDAYHG E4:E18 267 FDSKRREGKQLSLHECLCTA E2:E6 268 QVARESTYLSSLKGSRDQPLPTPELQMPTM E3:E18 269 PEELGGPPLKKLKQEGVPGRLRRLLLQLG E4:E3 270 QVARESTYLSSLKGSRPHLQMPPSLGQMSL E3:E20 271 DSASLSGESLDGHLQGSPQIENIQPFSAKD E5:E16 272 QVARESTYLSSLKGSS E3:E17 273 PEELGGPPLKKLKQEATSKSQIMSLWGLVS E4:E2 274 PEELGGPPLKKLKQESIYQRDPLKLVATFR E4:E4 275 FDSKRREGKQLSLHEFRHLPMPFHWKQEEL E2:E5 276 VGRLSPCVPAKPPLAAEQMGKDGRGYVPAT E6:E17 277 VGRLSPCVPAKPPLAGNLQVPDHVSVGSGL E6:E2 278 VGRLSPCVPAKPPLAGSPQIENIQPFSAKD E6:E16 279 PEELGGPPLKKLKQEFLFSVSSELQGGTWV E4:E1 280 VGRLSPCVPAKPPLAGTNHFLPQSSRCLPW E6:E18 281 PSRKPLDSRVLNAVKYYGTAANDIGDTTNR E4:E13 NACC2/NTRK2 282 KGETITGLLQEFDVQEALSVVSEDQSLFEC E3:E4 NDRG1/ERG 283 DVDLAEVKPLVEKGEEALSVVSEDQSLFEC E2:E4 284 PIAMALANVVPCSQWVFTKYGKCYMFNSGE E27:E2 NF1/ACCN1 285 PPTWPKPLVPAIPICSSAGERGGFNKPGGP E2:E9 NFATC2/EWSR1 286 MRRQQEGFKGTFPDALPVSGNLLDVYSSQG E9:E6 NONO/TFE3 287 GEVDADCMDVNVRGPAGPSRTGYLHPPSPG E30:E14 NOTCH1/GABBR2 288 KCGSGPVHISGQHLVVYRRKHQELQAMQME E5:E20 NPM1/ALK 289 VAPPTTAASSVEEPEGHRESNSRLPGPPEG E5:E11 NTN1/ACLY 290 FMDNPFEFNPEDPIPDKLKCTKEEHLCTQR E9:E8 NTRK1/TPM3 291 PQYFRQGHNCHKPDTYCRLLWDYVYQLLSD E14:E6 NTRK3/ETV6 292 MDRDLSYNLLEDLPSFSV E1:E12 NUP107/LGR5 293 SNPMNPTIGNGLSPQNSIRHNLSLHSKFIR E7:E2 PAX3/FOXO1 294 SNPMNPTIGNGLSPQFTADLDQFDQLLPTL E7:E14 PAX3/NCOA1 295 YQLSETSYQPTSIPQGLPELELEAIDNQFG E6:E15 296 SNPMNPTIGNGLSPQPGSELDNLEEILDDL E7:E12 PAX3/NCOA2 297 PSNHMNPVSNGLSPQNSIRHNLSLHSKFIR E7:E2 PAX7/FOXO1 298 LGRNLSTHQTYPVVAGREMVGPTLPGYPPH E8:E10 PAX8/PPARG 299 AGSPDTESPVLVNDYRENVIEYKHCLITKN E36:E11 PCM1/JAK2 300 AGSPDTESPVLVNDYEIELSSLREALSFVS E36:E9 301 LEKIIKCNRSTEISSVLQERIPWVPPECIE E24:E17 302 KKRNSTQLKSRVKNINKSNLLVFRTNGVSD E23:E12 303 NSELTPSESLATTDDEIELSSLREALSFVS E26:E9 304 RQIKAIMKEVIPFLKEDPKWEFPRKNLVLG E29:E12 PCM1/RET 305 GVACEPLPDRYTVSEESNGWK E13:E4 PLXND1/TMCC1 306 LQPMAGTCPAPEIHAIERLEVSSLAQTSSA E12:E4 307 DSERLQYEKKLKSTKCTAGSTRSCKPCRWS E8:E20 PPFIBP1/ALK 308 YKKMQDTVVLAQGKKVYRRKHQELQAMQME E12:E20 PPFIBP1/ALK 309 LVCKMKGEGVEIVDRVWHRRLKNQKSAKEG E9:E35 PPFIBP1/ROS1 310 KEPVKIAAPELHKGDVQTHLENPTRYHLQQ E1:E4 PRCC/TFE3 311 PQEIAPDASFIDDEAVQTHLENPTRYHLQQ E4:E4 312 DSEEDEPTKKKTILQLPVSGNLLDVYSSQG E2:E6 313 KEPVKIAAPELHKGDIDDVIDEIISLESSY E1:E5 314 KLWGIDRDSYRRILMEDPKWEFPRKNLVLG E7:E12 PRKAR1A/RET 315 SPWPLLGSAQGQFSAVHPNVSQGCQGGCAT E1:E2 PTPRK/RSPO3 316 TGLPGPPLITRTKCAVHPNVSQGCQGGCAT E7:E2 317 FGEKLFSGVLMDLSKSTLPTQEEIENLPAF E1:E36 PWWP2A/ROS1 318 ATSILEYPIEPSGVLGPASVISNDDDSASP E6:E16 QKI/NTRK2 319 EASSLKYLAEEFSIPEPTGEPSPKRPRGRP E7:E2 RAD51B/HMGA2 320 EASSLKYLAEEFSIPKAEATGEKRPRGRPR E7:E3 RAD51B/HMGA2 321 HMVSTTLPVDSRMIESTANPETPNSTISRE E7:E2 RAF1/DAZL 322 DGYQGSQTFHGAPLTVYRRKHQELQAMQME E18:E20 RANBP2/ALK 323 ASYDDPYKKAVAMSKR E2:E5 RBM14/PACS1 324 DSMENQVSVDAFKILDMEAQQVNEAESARE E11:E8 RET/GOLGA5 325 DSMENQVSVDAFKILKCERLLLYLYCHELS E11:E17 RET/TRIM33 326 EHRKELGPYVFREAQVNKLELELESAKQKF E22:E24 RGS22/SYCP1 327 FIQKIRYTNARDRNQDLIRDQGFRGDGGST E3:E9 RNF130/BRAF 328 TVQGSNIFERTEVLADDFWIPETSFILTII E4:E34 SDC4/ROS1 329 TVQGSNIFERTEVLAAGVPNKPGIPKLLEG E4:E32 330 GQESDDFELSGSGDLDDFWIPETSFILTII E2:E34 331 GQESDDFELSGSGDLVWHRRLKNQKSAKEG E2:E35 332 GQESDDFELSGSGDLAGVPNKPGIPKLLEG E2:E32 333 PGHMHTQVPPYPQPQLYRRKHQELQAMQME E19:E20 SEC31A/ALK 334 GTLPAASELPASQRTVYRRKHQELQAMQME E20:E20 335 GTLPAASELPASQRTVLQERIPWVPPECIE E20:E17 SEC31A/JAK2 336 MAATDLERFSNSYNNSQAPSPGLGS E1:E19 SEPT8/AFF4 337 VPPATMSGSMMGSDMLPVSGNLLDVYSSQG E9:E6 SFPQ/TFE3 338 VPPATMSGSMMGSDMIDDVIDEIISLESSY E9:E5 339 RQREESYSRMGYMDPIDDVIDEIISLESSY E7:E5 340 LEDMRSNNVEDCKMMVIHPKTDEQRCRLQE E17:E4 SLC26A6/PRKAR2A 341 VCSLDILSSAFQLVGDDFWIPETSFILTII E4:E34 SLC34A2/ROS1 342 VCSLDILSSAFQLVGAGVPNKPGIPKLLEG E4:E32 343 ILPYTLASLYHREKQLIAMDSAITLWQFLL E4: E2 SLC45A3/ELK4 344 LSSIRPPRLEGENTQDLIRDQGFRGDGGST E10:E9 SND1/BRAF 345 LSSIRPPRLEGENTQKTLGRRDSSDDWEIP E10:E11 346 PTANLDQKDKQFVAKDLIRDQGFRGDGGST E9:E9
347 SKKEVPIHRVADISGKTLGRRDSSDDWEIP E14:E11 348 SKKEVPIHRVADISGDLIRDQGFRGDGGST E14:E9 349 YSHKLFNGSMEAFIKRPRGQRDSSYYWEIE E12:E10 SRGAP3/RAF1 350 KQTLGEGERAECGTTRMQFEVTANQPHLQP E11:E8 351 GPPQPPQQRPYGYDQEWVSKSPSHLSCVIN E10:E4 SS18/SSX1 352 GPPQPPQQRPYGYDQLNILR E10:E5 353 QYPGQQGYPGQQQGYVEHPQMTFGRLHRII E9:E5 354 QYPGQQGYPGQQQGYGFKVTLPPFMCNKQA E9:E4 355 GPPQPPQQRPYGYDQIMPKKPAEDENDSKG E10:E6 356 TAPSAQQQRPYGYEQIMPKKPAEDENDSKG E10:E6 SS18L1/SSX1 357 GDGMPVGPVPPGFFQRENVIEYKHCLITKN E5:E11 SSBP2/JAK2 358 ETCEHSSEAKAFHDYRENVIEYKHCLITKN E4:E11 359 NITLGEPPGFLHSWWCEKMSLNINTV E3:E11 360 HPQMTFGRLHRIIPKGQYGNYQQ E5:E11 SSX1/SS18 361 YPKKPKDEAFRSHYKQFEEGLLDRCPAPGP E16:E7 STAT6/NAB2 362 TELNQGDMKPPSYDSVYRRKHQELQAMQME E3:E20 STRN/ALK 363 CTEKGTWRESTLTCTVLLQIL E6:E2 SUSD1/ROD1 364 RFAKKMDKMVQKKNAIGQNGKDWIPLSSTK E1:E3 TCEA1/PLAG1 365 HEGLSPTPFMNSNLMDMPCVQAQYSPSPPG E5:E3 TCF12/NR4A3 366 GYKILIPKGSYGRVKVS E7:E8 TECTA/TBCEL 367 SYLPGGTTGLQLPSTPVDREPVDREPVVCH E5:E8 TFE3/ASPSCR1 368 SPMALLTIGSSSEKEPVDREPVDREPVVCH E4:E8 369 QPPYTGAQTQAGQIEVYRRKHQELQAMQME E6:E20 TFG/ALK 370 KNVMSAFGLTDDQVSVYRRKHQELQAMQME E5:E20 371 EPPGEPGPSTNIPENVYRRKHQELQAMQME E4:E20 372 QAPPQQPQQYGIQYSDMPCVQAQYSPSPPG E7:E3 TFG/NR4A3 373 KNVMSAFGLTDDQVSDTNSTSGDPVEKKDE E5:E10 TFG/NTRK1 374 MALNSEALSVVSEDQSLFEC E2:E4 TMPRSS2/ERG 375 MALNSPSGSEQLVDGLAY E2:E3 376 FLVGAALAAGLLWKFRTLLMNAVWPKAGRW E4:E5 277 FLVGAALAAGLLWKFRSLISCE E4:E4 378 DGVSHCPGGEDENRCGSLISCE E5:E4 379 MALNSVIPGSLETRGKPC E2:E2 380 VCTQPKSPSGTVCTSRSLISCE E3:E4 381 VCTQPKSPSGTVCTSSYSRIFGDPRKAVLT E3:E2 382 MALNSLRYLTMMSSLYQTIR E2:E6 TMPRSS2/ETV1 383 VCTQPKSPSGTVCTSRGSLFPQKLLNAETS E3:E2 384 KGEPHHELPPGSTKRVPASVQLHTAVEMHH E8:E9 TP53/NTRK1 385 DAQAGKEPGGSRAHSSPGQCAAAHGGGDAP E10:E9 386 QPKKKPLDGEYFTLQSRPVCSCTRRWRCTT E9:E9 387 ERSVAKLEKTIDDLEVYRRKHQELQAMQME E7:E20 TPM3/ALK 388 ERSVAKLEKTIDDLEDTNSTSGDPVEKKDE E7:E10 TPM3/NTRK1 389 ERSVAKLEKTIDDLEVWHRRLKNQKSAKEG E8:E35 TPM3/ROS1 390 LQKLEEAEKAADESESTLPTQEEIENLPAF E2:E36 391 PRMQGPIQQPSISHQEDPKWEFPRKNLVLG E9:E12 TRIM24/RET 392 QLEEKQQQPTRELLQEDPKWEFPRKNLVLG E3:E12 393 KKGKTAQGLSPVDQREDPKWEFPRKNLVLG E16:E12 394 VISAENWKPATKTDQGLLKMTEYKLVVVGA E3:E2 UBE2L3/KRAS 395 RNELLGDDGNSSENQSNKVPVVQHPHHVHP E4:E5 VTI1A/TCF7L2 396 MRSYKQEMGKLETDFSNKVPVVQHPHHVHP E3:E5 397 MRSYKQEMGKLETDFYLQMKWPLLDVQAGS E3:E4 398 MVANVEKQLEEAKELSNKVPVVQHPHHVHP E2:E5 399 LRDNLTLWTSDMQGDAYPALGPGVTANPGT E5:E2 YWHAE/FAM22A 400 LRDNLTLWTSDMQGDAYPVLGPGVTANPGT E5:E2 YWHAE/NUTM2B 401 IKTLEGEFSVTMWSSGPMDEGPDLDLGPPV E1:E10 YY1/EWSR1 402 FGSTRGSLDKPDSFMGEYSVGNKHRDPFEA E10:E7 ZC3H7B/BCOR 403 QCKTETQESQAFQERGSTTGLSATPPASLP E3:E10 ZSCAN30/BRAF
[0038] In some embodiments, the fusion-specific vaccine library comprises 2 or more (e.g., 2-5, 5-10, 10-25, 25-50, 50-100, 100-500, 500-1,000 or more) different fusion junction sequences that can be used as therapeutic vaccines for treating cancer.
[0039] In some embodiments, a method was developed for constructing a fusion-specific vaccine library from publicly available data such as but not limited to whole genome sequencing (WGS) data and/or RNA-sequencing data. In some embodiments, sources of publicly available data are databases such as but not limited to COSMIC (cancer.sanger.ac.uk/cosmic/), TCGA Fusion Gene Data Portal (54.84.12.177/PanCanFusV2/), and/or FusionCancer Database (donglab.ecnu.edu.cn/databases/FusionCancer/).
[0040] In some embodiments, neoantigenic peptides having amino acid sequences corresponding to the gene fusion junction can be generated. In some embodiments, a library of recurrent fusion-derived neoantigenic peptide vaccines can be constructed containing all cancer-associated neoantigenic peptides or subsets thereof. In some embodiments, a library can contain 2 or more different fusion peptides (e.g., 2-5, 5-10, 10-25, 25-50, 50-100, 100-500, 500-1,000 or more different fusion peptides).
[0041] In some embodiments, methods are disclosed for construction of a fusion-derived neoantigenic vaccine library, selection of patients and/or peptides for vaccination, and subsequent treating of patients with neoantigenic vaccines.
[0042] In some embodiments, methods for identifying recurrent fusion derived neoepitopes and constructing a vaccine library include several steps (FIG. 2). In some embodiments, methods comprise screening publicly available databases containing information regarding tumor whole genome sequencing (WGS) and/or RNA-sequencing data. In some embodiments, fusion genes and fusion transcripts can be de novo discovered using WGS and/or RNA-sequencing data. In some embodiments, methods integrate publicly available fusion-detection algorithms followed by heuristic filtering.
[0043] In some embodiments, methods comprise screening one or more publicly available databases containing information relating to annotated fusion genes, fusion transcripts, breakpoint coordinates of tumor-specific fusion genes, fusion proteins, and/or any resources containing information about recurrent fusions and their presence in tumor(s). In some embodiments, methods comprise screening peer-review articles. In some embodiments, methods comprise screening proprietary databases and/or patient sequence information.
[0044] In some embodiments, methods comprise further validation of fusion genes detected by screening publicly available databases containing WGS and/or RNA-sequencing data.
[0045] In some embodiments, methods comprise extracting fusion incidence data in cancer(s) from public data sources (e.g., cancer.org and/or cancer.net), proprietary information resources (e.g., uptodate.com) and/or peer-reviewed literature.
[0046] In some embodiments, methods comprise translating recurrent fusions to chimeric proteins, identifying long peptides at fusion breakpoints, and predicting peptide binding affinity to MHC class I proteins and/or MHC class II proteins using bioinformatics algorithms. In some embodiments, methods comprise determining peptide binding affinity to MHC proteins using biochemical techniques in vitro.
[0047] In some embodiments, methods comprise generating a set of T-cell epitopes by selection of fusion-derived neoepitopes having an IC.sub.50 less than 500 nM. In some embodiments, the epitopes can bind HLA proteins and be recognized by T-cells. In some embodiments, IC.sub.50 thresholds for MHC class I-peptide interaction are used for selecting peptides. In some embodiments, MHC class II-peptide interaction are used for selecting peptides.
[0048] In some embodiments, methods comprise determining a binding affinity of peptide-HLA class I major genes comprising HLA-A, HLA-B, and/or HLA-C. In some embodiments, methods comprise determining a binding affinity of peptide-HLA class I minor genes comprising HLA-E, HLA-F, and/or HLA-G. In some embodiments, methods comprise determining a binding affinity of peptide-HLA class II genes comprising HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, and/or HLA-DR. In some embodiments, methods comprise determining a binding affinity of the peptide to any antigen presenting protein.
[0049] In some embodiments, population coverage for each long peptide is calculated based on HLA allele frequency, wherein the HLA protein can bind to the fusion-derived neoepitopes with IC.sub.50<500 nM. In some embodiments, patient coverage is calculated based on HLA allele frequency and gene fusion incidence rates in tumors. In some embodiments, the patient coverage is the percentage of patients having a particular tumor or tumors comprising a fusion-derived neoantigen included in the library. In some embodiments, patients having a particular tumor or tumors comprising a fusion-derived neoantigen included in the library are vaccinated with one or more fusion-specific vaccines from the library.
[0050] In some embodiments, fusion-specific vaccines from the library comprise fusion-derived long peptides having 25-30 amino acids. In some embodiments, fusion-specific vaccines from the library comprise fusion-derived long peptides having 30-50 amino acids. In some embodiments, fusion-specific vaccines from the library comprise fusion-derived long peptides having 50-100 amino acids. In some embodiments, fusion-specific vaccines from the library comprise fusion-derived long peptides encompassing the entire fusion protein.
[0051] In some embodiments, fusion-derived neoantigens can be modified by anchor residues, with additional amino acid sequences like T-helper epitopes that can be included in polypeptide for enhancing of in vivo processing, binding to MHC, and/or immunogenicity.
[0052] In some embodiments, fusion-derived long peptides in the library are characterized by their association with particular fusion genes from which peptides are derived. In some embodiments, fusion-derived long peptides in the library are characterized by listing the HLA proteins that can effectively present neoepitopes derived from the fusion-derived long peptide. In some embodiments, fusion-derived long peptides in the library are characterized by the population coverage of the fusion-derived long peptide.
[0053] In some embodiments, a vaccine library contains fusion-derived long peptides or polypeptides constructed of fusion-derived neoepitopes at the fusion breakpoint. In some embodiments, fusion-derived long peptides or polypeptides in the library are described by cancer types. In some embodiments, fusion-derived long peptides or polypeptides in the library are described by short peptides derived from long peptides, wherein the short peptides interact with MHC class I and/or MHC class II proteins. In some embodiments, fusion-derived long peptides or polypeptides in the library are described by the population coverage.
[0054] In some embodiments, the interactions between short peptides and MHC proteins are characterized by an IC.sub.50, wherein the short peptides bind to MHC proteins with an IC.sub.50<500 nM. In some embodiments, fusion-derived peptides are formulated with an appropriate carrier or adjuvant. In some embodiments, fusion-derived neoantigens are DNA and/or RNA. In some embodiments, a single vaccine can contain one or more long peptides. In some embodiments, a fusion-derived long peptide can produce several shorter peptides that bind to MHC proteins. In some embodiments, a vaccine targets one or more neoantigens. In some embodiments, a vaccine library targets more neoantigens than the number of vaccines in the library.
Peptide Length
[0055] In some embodiments, a fusion-derived peptide in a vaccine has a length of 7-11 amino acids. In some embodiments, the peptide has a length of 7 amino acids. In some embodiments, the peptide has a length of 8 amino acids. In some embodiments, the peptide has a length of 9 amino acids. In some embodiments, the peptide has a length of 10 amino acids. In some embodiments, the peptide has a length of 11 amino acids. In some embodiments the peptide length is 25 or more amino acids, 50 or more amino acids, 75 or more amino acids, or 100 or more amino acids. In some embodiments, the peptides are processed to a shorter length intracellularly. In some embodiments the neoantigenic fusion-derived peptides in the library can include, but are not limited to, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more amino acids. In some embodiments, long peptides in the library correspond to shorter peptides that bind to MHC proteins. In some embodiments, a peptide in a vaccine can be of any suitable length that is sufficient to be immunogenic in a patient.
Peptide Synthesis and Modification
[0056] In some embodiments, fusion-derived peptides are in neutral (uncharged) forms or in forms that are salts. Methods for synthesizing peptides are well known in the art. In one embodiment, the peptides provided herein are synthesized using an appropriate solid-state synthetic procedure (see for example, Steward and Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco, Calif. (1968); Merrifield (1967) Recent Progress in Hormone Res 23: 451). However, any other suitable synthetic technique may be used. In some embodiments, one or more peptides provided herein are synthesized by expressing the peptides through standard molecular biological techniques. In some embodiments, one or more peptides provided herein are isolated from natural sources.
[0057] In some embodiments, fusion-derived peptides are modified. Modifications should not reduce biological activity of peptides and their ability to induce tumor-specific immune responses. Modifications include but are not limited to phosphorylation, glycosylation, and oxidation. In some embodiments, peptides described herein are linked to one or more additional moieties (e.g., that help promote or stimulate an immune response, stabilize or solubilize the peptide, etc., or any combination thereof).
[0058] In some embodiments, fusion-derived peptides are modified for enhancing their in vivo processing, binding to MHC, and/or immunogenicity. In some embodiments, fusion-derived peptide modifications include modification to anchor residues. In some embodiments, anchor residues are position 2 and position 8 in peptides comprising 9-10 amino acid residues. In some embodiments, peptide modifications comprise adding amino acid sequences like T-helper epitopes upstream or downstream.
[0059] In some embodiments, fusion-derived neoantigens comprise polypeptides consisting of repeating fusion-derived neoantigenic short peptides.
Patient Selection
[0060] In some embodiments, a patient is eligible for treatment with one or more fusion-specific vaccines from the library when the patient's tumor biopsy comprises one or more gene fusions corresponding to fusion-derived peptides or fusion-derived neoantigens present in the library.
[0061] In some embodiments, a patient is eligible for treatment with fusion-specific vaccines from the library when the long peptide in the library can bind to the HLA alleles of the patient and be presented to the patient's immune system. In some embodiments, long peptides in the library are characterized by the HLA alleles which can bind the long peptide derivatives and present them to the immune system.
[0062] In some embodiments, the long peptide or polypeptide at the point of junction must be able to produce neoantigens for a particular patient. In some embodiments, the patient is eligible for vaccination if at least one HLA allele of the patient matches the HLA allele of the particular fusion-derived neoantigen. In some embodiments, up to six HLA alleles of the patient match the HLA alleles of the particular fusion-derived neoantigen.
[0063] In some embodiments, a method of selecting a patient for vaccination with a therapeutic fusion-specific vaccine from a fusion-specific vaccine library is based on one or more factors (e.g., as illustrated in FIG. 3). In some embodiments, selecting a patient for vaccination with a therapeutic peptide vaccine from a fusion-specific vaccine library is based on genetic analysis of a patient's tumor sample (e.g., biopsy) and normal tissue sample. In some embodiments, genetic analysis is performed using one or more techniques that detects fusion genes, fusion transcripts, and/or fusion proteins in a patient's tumor. In some embodiments, genetic analysis is performed with whole genome sequencing, RNA sequencing, proteome sequencing, fluorescence in situ hybridization, pyrosequencing, qPCR, Sanger sequencing, or any known analysis method that allows identification of fusion genes or/and transcripts presented in a tumor sample and absent in a normal tissue sample.
[0064] In some embodiments, one or more gene fusions found with WGS and/or RNA sequencing are validated with in vitro techniques. In some embodiments, fusions identified by screening public and proprietary databases for information about the presence of a particular fusion in a particular cancer type are validated with in vitro techniques.
[0065] In some embodiments, selecting a patient for vaccination with a therapeutic peptide vaccine from a fusion-specific vaccine library is based on measuring the expression level of the fusion gene or transcript. In some embodiments, the expression level of the fusion gene or transcript is measured by RNA sequencing and/or any technique that measures the expression level of the fusion transcript(s) and/or the corresponding chimeric protein(s). In some embodiments, the fusion is expressed.
[0066] In some embodiments, selecting a patient for vaccination with a therapeutic fusion-specific vaccine from a fusion-specific vaccine library is based on the patient's HLA genotype. In some embodiments, the patient is HLA genotyped using one or more serological tests, PCR, WGS, WES, RNA sequencing, and/or any other technique that determines the patient's HLA genes and/or HLA proteins. In some embodiments, the patient's HLA genotyping can be performed in silico with patient's tumor sequencing data comprising transcriptome, exome, and/or genome sequencing data. In some embodiments, HLA genotyping should have at least a 4-digit resolution for matching a patient's HLA alleles to vaccines in the library. However, other thresholds can be used for HLA matching.
[0067] In some embodiments, selecting a patient for vaccination with a therapeutic fusion-specific vaccine from fusion-specific vaccine library is based on matching of patient's HLA alleles with HLA alleles that bind to fusion-derived long peptides in the vaccine library. In some embodiments, HLA proteins can bind to peptides derived from the fusion gene detected in a patient's tumor and bind to short peptides derived from long peptides presented in the library. In some embodiments, the patient is eligible for vaccination if at least one HLA allele of the patient matches the HLA allele of the particular fusion-derived peptide. In some embodiments, up to six HLA alleles of the patient matches the HLA alleles of the particular fusion-derived peptide.
[0068] In some embodiments, a patient is selected for vaccination with a therapeutic fusion-specific vaccine from the fusion-specific vaccine library if one or more of the fusion-derived long peptide(s) in library are found in the patient's tumor biopsy as part of the expressed fusion protein (e.g., a fusion gene or fusion transcript) and not found in normal tissue(s). In some embodiments, a patient is selected for vaccination with a therapeutic fusion-specific vaccine from the fusion-specific vaccine library if the patient has one or more HLA proteins that can effectively bind and present to the immune system one or more fusion-derived neoantigens that are present in the library. In some embodiments, at least one HLA allele of the patient must match one of the HLA alleles that bind to the fusion-derived neoantigens in the vaccine library.
[0069] In some embodiments, long peptides in a library might not bind to a patient's HLA proteins, whereas shorter peptides may bind to one or more of the patient's HLA proteins, and the patient is eligible for vaccination. For example, 30-mer peptides (peptides that are 30 amino acids long) cannot bind to MHC class I proteins, but derivatives of the 30-mer peptides, such as 8-mer, 9-mer, 10-mer, and/or 11-mer peptides will bind to the patient's HLA proteins and can be used to vaccinate the patient.
[0070] In some embodiments, a patient may have multiple HLA proteins that match multiple tumor-specific recurrent fusion-derived long peptides present in the library.
[0071] In some embodiments, protein mass spectrometry can be used for identifying fusion-derived peptides bound to a patient's HLA proteins. In some embodiments, peptides are eluted from HLA molecules on cancer cells and then analyzed with mass spectrometry and matched with peptides in the vaccine library.
[0072] In some embodiments, no appropriate vaccine in the library matches a particular patient and a "de novo" method for identifying tumor-specific chimeric protein-derived neoepitopes in a patient can be used and a patient-specific personalized vaccine composition can be generated.
[0073] In some embodiments, patient tumor-specific chimeric protein-derived neoepitopes (e.g., neoantigens) can be determined by sequencing RNA and/or DNA isolated from the patient's tumor biopsy sample(s) and/or normal tissue sample(s).
[0074] In some embodiments, fusion transcripts which are present in a tumor sample and absent from normal tissue are identified using established bioinformatical algorithms. In some embodiments, false positive fusion transcripts are filtered out using WGS data, homology of fused genes at RNA level, or other available approaches. In some embodiments, false positive fusion transcripts identified with in silico fusion detection are filtered out using publicly available fusion genes and fusion transcripts and chimeric proteins databases.
[0075] In some embodiments, in vitro validation of in silico detected fusion transcripts is performed using qPCR and/or Sanger sequencing with DNA and/or RNA isolated from patient's tumor sample(s) and normal tissue sample(s).
[0076] In some embodiments, a set of patient tumor-specific fusion-derived peptides corresponding to a chimeric protein's breakpoint is generated. In some embodiments, a peptide represents one or more novel tumor-specific sequences that are not presented in normal tissues and contains at least one amino acid of one fusion partner, for example from the N-terminal side or from the C-terminal side of the breakpoint, and several amino acids of the other fusion partner (e.g., from the C-terminal side or from the N-terminal side of the breakpoint).
[0077] In some embodiments, the patient is HLA genotyped using one or more in vitro techniques such as serological tests or in silico techniques based on WGS, WES or RNA sequencing data.
[0078] In some embodiments, binding affinity (or any other parameter characterizing peptide--MHC interaction process) is predicted by means of highly validated bioinformatical algorithms that can be found elsewhere including iedb.org, or cbs.dtu.dk/services/. In some embodiments, the expression level of all identified fusion transcripts corresponding to the patient's tumor-specific fusion-derived peptides are measured using RNA sequencing data or any available technique for measuring expression level of fusion transcript and/or corresponding chimeric protein. In some embodiments, the patient's tumor-specific fusion-derived neoepitopes are expression products of the fusion gene transcript and have IC.sub.50 to patient's HLA proteins less than 500, less than 250, less than 150,or less than 50 nM.
[0079] In some embodiments, a patient's tumor-specific vaccine composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any appropriate number of patient tumor-specific fusion-derived peptides, wherein the composition optionally comprises one or more DNA or RNA constructs encoding such peptides.
[0080] In some embodiments, a variety of bioinformatical algorithms and pipelines have been established for identifying tumor-specific fusions and generating corresponding neoepitopes.
Peptide Selection
[0081] In some embodiments, gene fusion peptides are selected based on IC.sub.50 for HLA proteins. In some embodiments, gene fusion peptides having an IC.sub.50 of more than 500 nM are considered non-binding. In some embodiments, peptides with an IC.sub.50 of less than 500 nM are considered binding. In some embodiments, peptide vaccine compositions comprise gene fusion peptides having IC.sub.50 less than 500 nM, less than 450 nM, less than 400 nM, less than 350 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM, less than 10 nM, or less than 1 nM for one or more HLA proteins of a patient are selected for a vaccine for that patient (e.g., one or more of these concentration thresholds can be used for determining peptide matches for a patient). However, other IC.sub.50 thresholds (e.g., higher, lower, or intermediate concentrations) can be used to determine whether a peptide is a match for a patient.
[0082] In some embodiments, a gene fusion peptide IC.sub.50 is predicted using established computational methods. In some embodiments, computational methods include NetMHC (e.g., cbs.dtu.dk/services/NetMHC/) and NetMHCpan (e.g., cbs.dtu.dk/services/NetMHCpan/). In some embodiments, a gene fusion peptide IC.sub.50 is determined in vitro using one or more biochemical assays. Methods described herein are not limited by the techniques used to identify the gene fusion peptide IC.sub.50.
[0083] In some embodiments, binding of gene fusion peptides to HLA proteins is determined from biological samples by eluting peptides from HLA proteins and analyzing them using mass spectrometry.
[0084] In some embodiments, gene fusion peptides are selected based on patient coverage. In some embodiments, gene fusion peptides having patient coverage greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% are selected.
[0085] In some embodiments, the vaccine library can be expanded with the same methods described herein to incorporate new recurrent fusion data as it emerges.
Biological Samples
[0086] Methods provided herein detect gene fusions in biological samples. Biological samples, as used herein, refer to samples taken or derived from a patient. These samples may be tissue samples or they may be fluid samples (e.g., a bodily fluid). Examples of biological fluid samples are whole blood, plasma, serum, urine, sputum, phlegm, saliva, tears, and other bodily fluids. In some embodiments, the biological sample is a whole blood sample, or a sample of white blood cells from a subject. In some embodiments, the biological sample is a tumor, a fragment of a tumor, a tumor biopsy, or a tumor cell(s).
Treatment
[0087] Other aspects of the specification relate to methods of treatment for a patient with cancer. Cancer includes, but is not limited to: melanoma, skin cancer, head and neck cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, testicular cancer, brain tumor, glioma, glioblastoma, solitary fibrous tumor, bladder cancer, colorectal cancer, renal cell carcinoma, sarcoma, myeloma, leukemia, and/or lymphoma.
[0088] In some embodiments, the treatment is regimented to modulate the patient's immune system to induce a response against a tumor comprising a gene fusion. In some embodiments, treatment comprises administering a peptide vaccine one or more times to a patient. In some embodiments, an initial vaccine can be administered followed by one or more booster administrations. In some embodiments, a vaccine can be administered once or more daily, weekly, monthly, or at other time intervals or other frequencies. The frequency of administration and the duration of a treatment (e.g., once, one week, 1-4 weeks, 1-12 months or longer) can depend on the severity of the disease, the effectiveness of the vaccine, and/or the health of the patient. In some embodiments, treatment comprises administering a peptide vaccine at day 1, 4, 8, 15, and 21 and at week 12, 20, 30, and 40.
[0089] In some embodiments, vaccines can be administered intravenously (i.v.), intradermally (i.d.), parenterally, intraperitoneally (i.p.), subcutaneously (s.c.), intramuscularly (i.m.), orally, sublingually, intranasally, rectally, or intravaginally. The treatment is not limited to the method of vaccine administration.
[0090] In some embodiments, treatment comprises therapeutic or prophylactic vaccination of a patient with a tumor or at risk of developing cancer, and inducing antitumor immune responses in the patient. In some embodiments, treatment comprises preventive vaccination of the patient against a tumor by one or more peptides from the library or vaccine composition identified with methods described herein.
[0091] In some embodiments, treatment comprises treating a patient with a fusion-derived peptide or vaccine composition in combination with one or more additional therapies. In some embodiments, an additional therapy is an FDA approved therapy. In some embodiments, an additional therapy is an experimental therapy that is not yet approved. In some embodiments, vaccination of a patient is combined with one or more targeted therapies, chemotherapies, radiation, immunotherapy, or with hematopoietic stem cell transplantation. In some embodiments, any other suitable therapy for a particular tumor and patient may be combined with vaccination. In some embodiments, suitable therapies that can be combined with vaccination include but are not limited to immunotherapies, for example checkpoint blockade therapies, for example, but not limited to, anti-PD1 (e.g., nivolumab (Opdivo.RTM.), pembrolizumab (Keytruda.RTM.), pidilizumab etc), anti-CTLA4 (e.g., ipilimumab (Yervoy.RTM.), tremelimumab), or other checkpoint inhibitors such as BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, IMP321, MGA271.
[0092] One aspect of the specification provides methods for treating a patient having a tumor and eliciting an antitumor immune response in the patient by administering dendritic cells (or other antigen presenting cells) that have been pulsed with at least one fusion-derived peptide (or coding mRNA or DNA) from the vaccine library. The dendritic cells (or other antigen presenting cells) pulsed in this manner comprise at least one fusion-derived peptide (or coding mRNA or DNA) from the vaccine library.
[0093] In some embodiments, the vaccine composition comprises at least two distinct peptides wherein each peptide corresponds to the amino acid sequence at the point of junction and each peptide is equal to or less than 50 amino acids in length.
[0094] In some embodiments, peptides or vaccine compositions are administered at concentrations sufficient to induce an antitumor immune response in the patient. Concentrations of peptides and vaccine components and dosing regimens can be easily determined by anyone skilled in the art.
[0095] In some embodiments, treatment comprises administering a vaccine having a peptide concentration of 1-10 mg per injection. In some embodiments, peptide doses per patient for one vaccination are 100 ng or more (e.g., 100-500 ng, 500-1,000 ng, or more), 1 .mu.g or more (e.g., 1-10 .mu.g, 10-100 .mu.g, 100-500 .mu.g, 500-1,000 .mu.g, or more), 1 mg or more (e.g., 1-10 mg, 10-50 mg, 50-100 mg, or more), or 100 mg or more. In some embodiments, peptide doses per patient are adjusted depending on patient condition, patient weight, cancer stage and severity. In some embodiments, peptide doses and treatment regimen can vary depending on a patient's response to therapy. In some embodiments, peptide doses and treatment regimen can vary depending on a patient's condition.
[0096] The concentration of peptides provided in the pharmaceutical formulations can vary widely, e.g., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
[0097] In some embodiments, the peptide concentration in the vaccine (e.g., for each peptide, or the combined total peptide concentration) is around 1 nM, around 250 nM, around 500 nM, around 750 nM, around 1 around 250 around 500 around 750 around 1 mM, around 250 mM, around 500 mM, around 750 mM, or around 1M, or more or an intermediate concentration. In some embodiments, the peptide dose per patient is around 1 ng, around 250 ng, around 500 ng, around 750 ng, around 1 .mu.g, around 250 .mu.g, around 500 .mu.g, around 750 .mu.g, around 1 mg, around 250 mg, around 500 mg, around 750 mg, around 1,000 mg, or more or an intermediate amount.
[0098] In some embodiments, an initial dose is (e.g., for therapeutic or prophylactic administration) from about 1.0m to about 50,000m of peptide for a 70 kg patient, followed by subsequent doses (e.g., boosting doses) of from about 1.0m to about 10,000m of peptide pursuant to a regimen (e.g., a boosting regimen) over weeks to months depending upon the patient's response and condition. However, in some embodiments, subsequent doses can be the same as the initial dose. It should be appreciated that equivalent dosages for patients in different weight ranges can be calculated. In some embodiments, a typical dose (e.g., daily dose) is at least 0.1 .mu.g/kg, at least 1 .mu.g/kg, at least 10 .mu.g/kg, at least 25 .mu.g/kg, at least 50 .mu.g/kg, at least 100 .mu.g/kg, at least 250 .mu.g/kg, at least 500 .mu.g/kg, at least 750 .mu.g/kg, at least 1 mg/kg, at least 10 mg/kg, or more or an intermediate range between any of these values.
[0099] In some embodiments, DNA or mRNA encoding a fusion-derived peptide or polypeptide is administered to a patient. In some embodiments, DNA or mRNA encoding a fusion-derived peptide or polypeptide is incorporated into a vector. In some embodiments, DNA or mRNA comprises additional sequences including but not limited to a T-helper sequence. In some embodiments, DNA or mRNA comprises standard regulatory sequences that ensure expression in a particular cell type. In some embodiments, DNA or mRNA comprises additional sequences encoding proteins such as IL-2 or GM-CSF that can increase a patient's immune response. In some embodiments, DNA or mRNA is transfected into mammalian cells with techniques including but not limited to electroporation.
[0100] In some embodiments, autologous T-cells can be collected from a patient and stimulated ex vivo with fusion-derived peptides and/or DNA or RNA encoding the fusion-derived peptides. In some embodiments, autologous T-cells can be collected from a patient and stimulated ex vivo with fusion-derived peptides and/or DNA or RNA encoding the fusion-derived peptides and the stimulated T-cells are infused into the patient.
[0101] In some embodiments, DNA or RNA encoding the fusion-derived peptides comprises attenuated viral vectors. In some embodiments, viral vectors comprise BCG, vaccinia, and/or fowlpox.
[0102] In some embodiments, the library can be used for treating subjects diagnosed with cancer, wherein the subject is human. In some embodiments, the subject can be a mouse, cat, dog, horse, primate, or any animal.
Use of a Therapeutic Fusion-Specific Vaccine for Treating Cancer
[0103] To practice the method disclosed herein, an effective amount of the pharmaceutical composition (e.g., vaccine) described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route (e.g., intravenous administration, subcutaneous, intradermal, or other route).
[0104] The subject to be treated by the methods described herein can be a human patient having cancer. Examples of cancer includes, but is not limited to, melanoma, skin cancer, head and neck cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer testicular cancer, brain tumor, glioma, glioblastoma, solitary fibrous tumor, bladder cancer, colorectal cancer, renal cell carcinoma, sarcoma, myeloma, leukemia, and/or lymphoma.
[0105] The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject who needs the treatment may be a human patient having a cancer. A subject having a cancer can be identified by routine medical examination, e.g., laboratory tests, PET scan, biopsy, PET scans, CT scans, or ultrasounds. A subject having a cancer might show one or more symptoms of the disorder, e.g., unexplained weight loss, fever, fatigue, pain, skin changes, unusual bleeding or discharge, and/or thickening or lumps in parts of the body. A subject at risk for a cancer can be a subject having one or more of the risk factors for that disorder. For example, risk factors associated with cancer include (a) viral infection (e.g., herpes virus infection), (b) age, (c) family history, (d) heavy alcohol consumption, (e) obesity, and (f) tobacco use.
[0106] "An effective amount" as used herein refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
[0107] Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the peptide and to prevent the peptide being attacked by the host's immune system. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and/or suppression and/or amelioration and/or delay of a cancer. Alternatively, sustained continuous release formulations of a fusion-specific vaccine may be appropriate. Various formulations and devices for achieving sustained release are known in the art.
[0108] In one example, dosages for a fusion-specific vaccine as described herein may be determined empirically in individuals who have been given one or more administration(s) of a fusion-specific vaccine. Individuals are given incremental dosages of the fusion-specific vaccine. To assess efficacy of the fusion-specific vaccine, an indicator of a cancer (such as tumor formation or tumor growth) can be followed.
[0109] In some embodiments, dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the fusion-specific vaccine used) can vary over time. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate a cancer, or a symptom thereof.
[0110] For the purpose of the present disclosure, the appropriate dosage of a fusion-specific vaccine will depend on the specific fusion-specific vaccine(s) (or compositions thereof) employed, the type and severity of cancer, whether the fusion-specific vaccine is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antagonist, and the discretion of the attending physician. Typically the clinician will administer a fusion-specific vaccine, until a dosage is reached that achieves the desired result. Administration of a fusion-specific vaccine can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of a fusion-specific vaccine may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing cancer.
[0111] As used herein, the term "treating" refers to the application or administration of a composition including one or more active agents to a subject, who has a cancer, a symptom of a cancer, or a predisposition toward a cancer, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward a cancer.
[0112] Alleviating a cancer includes delaying the development or progression of the disease, or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, "delaying" the development of a disease (such as lung cancer) means to defer, hinder, slow, retard, stabilize, and/or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individuals being treated. A method that "delays" or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.
[0113] "Development" or "progression" of a disease means initial manifestations and/or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. "Development" includes occurrence, recurrence, and onset. As used herein "onset" or "occurrence" of a cancer includes initial onset and/or recurrence.
[0114] In some embodiments, the fusion-specific vaccine described herein is administered to a subject in need of the treatment at an amount sufficient to induce a immunological response against a tumor by at least 5% (e.g., 15%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater). In other embodiments, the fusion-specific vaccine is administered in an amount effective in reducing tumor metastasis. Alternatively, the fusion-specific vaccine is administered in an amount effective in reducing tumor formation or tumor growth.
[0115] Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical composition to the subject, depending upon the type of disease to be treated or the site of the disease. This composition can also be administered via other conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.
[0116] Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipients is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients. Intramuscular preparations, e.g., a sterile formulation of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution.
[0117] In one embodiment, a fusion-specific vaccine is administered via site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable depot sources of the fusion-specific vaccine or local delivery catheters, such as infusion catheters, an indwelling catheter, or a needle catheter, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct application. See, e.g., PCT Publication No. WO 00/53211 and U.S. Pat. No. 5,981,568.
[0118] Targeted delivery of therapeutic compositions containing a polynucleotide, expression vector, or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeutic compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. In some embodiments, concentration ranges of about 500 ng to about 50 mg, about 1 .mu.g to about 2 mg, about 5 .mu.g to about 500 .mu.g, and about 20 .mu.g to about 100 .mu.g of DNA or more can also be used during a gene therapy protocol.
[0119] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and/or enhancers. Expression of the coding sequence can be either constitutive or regulated.
[0120] Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EP Patent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.
[0121] Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968. Additional approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.
[0122] It is also apparent that an expression vector can be used to direct expression of any of the fusion-specific vaccines described herein. The particular dosage regimen, i.e., dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject's medical history.
[0123] In some embodiments, more than one fusion-specific vaccine may be administered to a subject in need of the treatment. The fusion-specific vaccine can correspond to the same tumor junction sequence or different tumor junction sequences. At least one, at least two, at least three, at least four, at least five different fusion-specific vaccines can be co-administered. Generally, those fusion-specific vaccines have complementary activities that do not adversely affect each other. Fusion-specific vaccines can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.
[0124] Treatment efficacy can be assessed by methods well-known in the art, e.g., monitoring tumor growth or formation in a patient subjected to the treatment.
Combination Therapy
[0125] Also provided herein are combined therapies using any of the fusion-specific vaccines described herein and another anti-cancer therapeutic agent, such as those described herein. The term combination therapy, as used herein, embraces administration of these agents (e.g., a fusion-specific vaccine and an anti-cancer therapeutic agent) in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the agents, in a substantially simultaneous manner. In some embodiments, combination therapies include two or more therapeutic agents (e.g., vaccines or other agents) combined in the same composition. However, in many embodiments, different therapeutic agents are provided in separate compositions. Sequential or substantially simultaneous administration of each agent can be affected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular, subcutaneous routes, and direct absorption through mucous membrane tissues. The agents can be administered by the same route or by different routes. For example, a first agent (e.g., a fusion-specific vaccine) can be administered orally, and a second agent (e.g., an anti-cancer agent) can be administered intravenously.
[0126] As used herein, the term "sequential" means, unless otherwise specified, characterized by a regular sequence or order, e.g., if a dosage regimen includes the administration of a fusion-specific vaccine and an anti-cancer agent, a sequential dosage regimen could include administration of the fusion-specific vaccine before, simultaneously, substantially simultaneously, or after administration of the anti-cancer agent, but both agents will be administered in a regular sequence or order. The term "separate" means, unless otherwise specified, to keep apart one from the other. The term "simultaneously" means, unless otherwise specified, happening or done at the same time, i.e., the agents of the invention are administered at the same time. The term "substantially simultaneously" means that the agents are administered within minutes of each other (e.g., within 10 minutes of each other) and intends to embrace joint administration as well as consecutive administration, but if the administration is consecutive it is separated in time for only a short period (e.g., the time it would take a medical practitioner to administer two agents separately). As used herein, concurrent administration and substantially simultaneous administration are used interchangeably. Sequential administration refers to temporally separated administration of the agents described herein.
[0127] Combination therapy can also embrace the administration of the agents described herein (e.g., a fusion-specific vaccine and an anti-cancer agent) in further combination with other biologically active ingredients (e.g., a different anti-cancer agent) and non-drug therapies (e.g., surgery).
[0128] It should be appreciated that any combination of a fusion-specific vaccine and another anti-cancer agent (e.g., a chemotherapeutic agent) may be used in any sequence for treating a cancer. The combinations described herein may be selected on the basis of a number of factors, which include but are not limited to the effectiveness of inducing an immune response, reducing tumor formation or tumor growth, and/or alleviating at least one symptom associated with the cancer, or the effectiveness for mitigating the side effects of another agent of the combination. For example, a combined therapy described herein may reduce any of the side effects associated with each individual members of the combination, for example, a side effect associated with the anti-cancer agent.
[0129] In some embodiments, another anti-cancer therapeutic agent is a chemotherapy, a radiation therapy, a surgical therapy and/or an immunotherapy. Examples of the chemotherapeutic agents include, but are not limited to, Carboplatin or Cisplatin, Docetaxel, Gemcitabine, Nab-Paclitaxel, Paclitaxel, Pemetrexed, and Vinorelbine. Examples of radiation therapy include, but are not limited to, ionizing radiation, gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes and radiosensitizers. Examples of a surgical therapy include, but are not limited to, a curative surgery (e.g., tumor removal surgery), a preventive surgery, a laparoscopic surgery, and a laser surgery. Examples of an immunotherapy include, but are not limited to, a PD-1 inhibitor or a PD-L1 inhibitor, adoptive cell transfer, and therapeutic cancer vaccines.
[0130] Additional examples of chemotherapy include, but are not limited to, Platinating agents, such as Carboplatin, Oxaliplatin, Cisplatin, Nedaplatin, Satraplatin, Lobaplatin, Triplatin, Tetranitrate, Picoplatin, Prolindac, Aroplatin and other derivatives; Topoisomerase I inhibitors, such as Camptothecin, Topotecan, irinotecan/SN38, rubitecan, Belotecan, and other derivatives; Topoisomerase II inhibitors, such as Etoposide (VP-16), Daunorubicin, a doxorubicin agent (e.g., doxorubicin, doxorubicin HCl, doxorubicin analogs, or doxorubicin and salts or analogs thereof in liposomes), Mitoxantrone, Aclarubicin, Epirubicin, Idarubicin, Amrubicin, Amsacrine, Pirarubicin, Valrubicin, Zorubicin, Teniposide and other derivatives; Antimetabolites, such as Folic family (Methotrexate, Pemetrexed, Raltitrexed, Aminopterin, and relatives); Purine antagonists (Thioguanine, Fludarabine, Cladribine, 6-Mercaptopurine, Pentostatin, clofarabine and relatives) and Pyrimidine antagonists (Cytarabine, Floxuridine, Azacitidine, Tegafur, Carmofur, Capacitabine, Gemcitabine, hydroxyurea, 5-Fluorouracil (5FU), and relatives); Alkylating agents, such as Nitrogen mustards (e.g., Cyclophosphamide, Melphalan, Chlorambucil, mechlorethamine, Ifosfamide, mechlorethamine, Trofosfamide, Prednimustine, Bendamustine, Uramustine, Estramustine, and relatives); nitrosoureas (e.g., Carmustine, Lomustine, Semustine, Fotemustine, Nimustine, Ranimustine, Streptozocin, and relatives); Triazenes (e.g., Dacarbazine, Altretamine, Temozolomide, and relatives); Alkyl sulphonates (e.g., Busulfan, Mannosulfan, Treosulfan, and relatives); Procarbazine; Mitobronitol, and Aziridines (e.g., Carboquone, Triaziquone, ThioTEPA, triethylenemalamine, and relatives); Antibiotics, such as Hydroxyurea, Anthracyclines (e.g., doxorubicin agent, daunorubicin, epirubicin and other derivatives); Anthracenediones (e.g., Mitoxantrone and relatives); Streptomyces family (e.g., Bleomycin, Mitomycin C, Actinomycin, Plicamycin); and Ultraviolet light.
Pharmaceutical Compositions
[0131] In some embodiments, methods are provided for constructing a fusion-derived vaccine library which can be used for treating a patient having cancer and eliciting an immune response in the patient. In some embodiments, the vaccine comprises fusion-derived long peptides or polypeptides comprising neoantigens peptides matched to patients HLA and which can induce an antitumor immune response in the patient. In some embodiments, the immune response can be detected by analyzing IFN-.gamma. production or tumor killing activity of T-cells in vitro.
[0132] In some embodiments, the vaccine comprises one fusion-derived long peptide at a breakpoint or several shorter derivatives of the long peptide. In some embodiments, the vaccine comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 neoantigenic peptides that can be bound by the patient's HLA class I and/or class II proteins. It should be appreciated that the vaccine comprising one or more neoantigenic peptides may further comprise one or more additional therapeutic agents. Alternatively, or in addition, on or more additional therapeutic agents may be administered separately
[0133] In some embodiments, a vaccine comprises two or more different peptides that are selected for a patient, wherein the peptide are mixed together in a single vaccine composition for administration to the patient. However, in some embodiments, two or more different peptides can be administered separately to a patient. It should be appreciated that a vaccine can contain any suitable concentration of each of the two or more peptides. In some embodiments, the concentration of each peptide in the vaccine is identical or similar. However, in some embodiments, different peptides can be provided at different concentrations.
[0134] In some embodiments, the vaccine comprises fusion-derived long peptides or polypeptides that are processed into shorter neoantigenic peptides which can be bound to at least one HLA class I protein of the patient. In some embodiments, the vaccine comprises fusion-derived long peptides or polypeptides that are processed into shorter neoantigenic peptides which can be bound to one or more, two or more, three or more, four or more, five or more, or six HLA class I protein of the patient. In some embodiments, peptides can bind to HLA class II proteins and induce helper T-cell response.
[0135] In some embodiments, a vaccine comprises several different fusion-derived long peptides or polypeptides corresponding to several different fusion genes present in the patient's tumor. In some embodiments, compositions comprises one or more, or two or more neoantigenic fusion-derived peptides.
[0136] In some embodiments, vaccine compositions comprises any adjuvant that can be used to activate an immune response in a patient. In some embodiments, an adjuvant can be any substance or any compound incorporated into a vaccine composition that increases the immune response of the patient to the fusion-derived peptides. Examples of adjuvants include, but are not limited to, poly-ICLC, Poly (I:C), GM-CSF, BCG, monophosphoryl lipid A, MF59, Freund's adjuvants, Montanide IMS, Montanide ISA 206, Montanide ISA-51, Montanide ISA 50V, Aluminium salts, Alhydrogel, Rehydrogel HPA, PLGA, MPL1, AS04, AS01B, AS02A, P3CSK4, CpG-ODN, Imiquimod ISS, ONTAK, LipoVac, Amplivax, CpG7909, dSLIM, IC30, IC31, SRL172, virus particles, YF-17D, AS15, OK-432, IMP321, OM-174, cytokines, and other adjuvants that can be found in the field using any source (e.g., peer-reviewed literature or medical guidelines).
[0137] In some embodiments, fusion-derived peptides are administered separately from one or more adjuvant(s). In some embodiments, fusion-derived peptides are administered along with one or more adjuvant(s). In some embodiments, vaccine compositions comprise one or more adjuvants and one or more fusion-derived peptides.
[0138] In some embodiments, vaccine compositions comprise different carriers such as polysaccharides, polypeptides, proteins, liposomes, or antigen presenting cells. In some embodiments, peptides might be combined with carriers that increase their immunogenicity, biological activity, and/or stability. In some embodiments, carriers include, but are not limited to, proteins such as bovine serum albumin (BSA), human serum albumin, immunoglobulins, transferrin, insulin; and/or carbohydrates such as lactose, cellulose, starch. In some embodiments, carriers are proteins such as diphtheria toxoid, tetanus toxoid, influenza virus proteins, and/or herpes virus proteins. In some embodiments, vaccine compositions comprise any carriers that are safe and acceptable for patients.
[0139] In some embodiments, vaccine compositions comprise at least one or more autologous antigen presenting cells (APC) pulsed with respective fusion-derived peptides from library. Pulsing APC cells may be performed in any manner known in the field. See, for example, Boczkowski et al, J Exp. Med. 1996 Aug. 1; 184(2): 465-72; see also: O'Neill and Bhardwaj, Adoptive Immunotherapy: Methods and Protocols, 2005, Volume 109 pp 97-112, which are incorporated by reference herein for this purpose. In some embodiments, vaccine compositions comprise at least one or more autologous antigen presenting cells (APC) comprising fusion-derived peptides from library. In some embodiments, APCs can be dendritic cells, macrophages, B-cells, or any professional antigen presenting cell. In some embodiments, APCs can be loaded with DNA or mRNA encoding respective fusion-derived peptide(s) of a vaccine library. In some embodiments, vaccine compositions comprise autologous peripheral blood mononuclear cells (PBMC).
[0140] In another aspect, vaccine compositions comprise components directed to the personal needs of a particular patient. In some embodiments, the personal needs of a particular patient include but are not limited to allergies, previous treatments, and/or comorbidities.
[0141] In some embodiments, fusion-derived peptides from a library can be combined with any other tumor-specific neoantigens for a particular patient that are not included in the vaccine library. In some embodiments, fusion-derived peptides from a library can be combined with tumor-specific neoantigens for a particular patient that have been identified de novo from WGS, WES, or RNA sequencing data.
[0142] In some embodiments, vaccine compositions can be prepared in sterilized water. In some embodiments, vaccine compositions can be prepared in buffered solutions such as but not limited to 0.9% saline.
[0143] One or more of the fusion-specific vaccines provided herein can be mixed with a pharmaceutically acceptable carrier (excipient), including buffer, to form a pharmaceutical composition for use in treating cancer. "Acceptable" means that the carrier must be compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Pharmaceutically acceptable excipients (carriers) including buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.
[0144] For example, a pharmaceutical composition described herein contains one or more fusion-specific vaccines that correspond to one or more tumor-specific gene fusions. In one example, a pharmaceutical composition described herein contains more than one fusion-specific vaccines that recognize a tumor-specific gene fusion. In another example, the pharmaceutical composition comprises at least two fusion-specific vaccines that correspond to different tumor-specific gene fusions. Alternatively or in addition, the pharmaceutical composition comprises one or more peptide vaccines or other anticancer agents. It should be appreciated that any of the pharmaceutical compositions provided herein may be administered alone, separately or in a combination.
[0145] The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20.sup.th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN.TM. (polysorbate), PLURONICS.TM. (poloxamers) or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.
[0146] In some examples, the pharmaceutical composition described herein comprises liposomes containing the fusion-specific vaccine, which can be prepared by methods known in the art, such as described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
[0147] The active ingredients (e.g., a fusion-specific vaccine) may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are known in the art, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).
[0148] In other examples, the pharmaceutical composition described herein can be formulated in sustained-release format. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT.TM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
[0149] The pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. However, other sterilization techniques, including but not limited to heat, chemical, and/or radiation based sterilization techniques may be used. Therapeutic vaccine compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
[0150] The pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.
[0151] For preparing solid compositions such as tablets, the principal active ingredient can be mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described herein containing from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
[0152] Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g., Tween.TM. 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span.TM. 20, 40, 60, 80 or 85). Compositions with a surface-active agent will conveniently comprise between 0.05 and 5% surface-active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.
[0153] Suitable emulsions may be prepared using commercially available fat emulsions, such as Intralipid.TM., Liposyn.TM., Infonutrol.TM., Lipofundin.TM. and Lipiphysan.TM.. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%. The fat emulsion can comprise fat droplets between 0.1 and 1.0 .im, particularly 0.1 and 0.5 .im, and have a pH in the range of 5.5 to 8.0.
[0154] The emulsion compositions can be those prepared by mixing a fusion-specific vaccine with Intralipid.TM. (a lipid emulsion) or the components thereof (soybean oil, egg phospholipids, glycerol and water).
[0155] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
[0156] Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulised by use of gases. Nebulised solutions may be breathed directly from the nebulising device or the nebulising device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
Kits for Use in Treating Cancer
[0157] The present disclosure also provides kits for use in alleviating cancer. Such kits can include one or more containers comprising a fusion-specific vaccine. In some embodiments, the kit can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of administration of the fusion-specific vaccine to treat, delay the onset, or alleviate a cancer according to any of the methods described herein. The kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has cancer. In still other embodiments, the instructions comprise a description of administering a fusion-specific vaccine to an individual having, suspected of having, or at risk for a cancer.
[0158] The instructions relating to the use of a fusion-specific vaccine generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
[0159] The label or package insert indicates that the composition is used for treating, delaying the onset and/or alleviating a cancer. Instructions may be provided for practicing any of the methods described herein.
[0160] The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a fusion-specific vaccine.
[0161] Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture comprising contents of the kits described herein.
[0162] Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.
Computational Methods
[0163] It should be appreciated that the various aspects and concepts of the present methods described herein may be implemented in any of numerous ways, and are not limited to any particular implementation technique. Examples of specific implementations are described below for illustrative purposes only, but the aspects of the methods described herein are not limited to these illustrative implementations.
[0164] FIG. 5 shows a non-limiting illustrative environment 500 in which embodiments of the present methods may operate. In the illustrative environment, healthcare provider 502 may use any device 504 to obtain information from system 510. Healthcare provider 502 may obtain information from system 510 one time or multiple times. Information between healthcare provider 502 and system 510 may be initiated in any suitable way.
[0165] In some embodiments, device 504 may be any suitable device that healthcare provider 502 may use to obtain information from system 510. For instance, device 504 may be a dedicated mobile phone (e.g., a cellular phone), a mobile smart phone, a personal digital assistant (PDA), a laptop computer, a tablet computer, or any other mobile device capable of supporting an exchange of information through a communications medium (e.g., a wired or wireless communications medium). In some embodiments, healthcare provider 502 is not limited to obtaining information by using a mobile device and may use any device capable of obtaining information. For instance, healthcare provider 502 may obtain information from 510 by using a telephone connected to landline, a desktop computer connected to a network such as the Internet, or using any other device capable of communicating with system 510. It should also be recognized that if healthcare provider 502 obtains information from system 510, healthcare provider 502 is not limited to using the same device. Thus, healthcare provider 502 may obtain information using one or a plurality of different devices, each of which may be any suitable device capable of obtaining information.
[0166] Information may be any suitable information relating to patient data and/or library data. Information is not limited by the type of communications infrastructure or communications medium used to obtain the information. For example, information may be patient information such as but not limited to a patient's identity, cancer type, gene fusion, and/or HLA allele information. As another example, information may be library information such as but not limited to peptide sequence information, peptide-HLA affinities, sequences of DNA and/or RNA encoding peptide sequence information, and/or gene fusion information.
[0167] In the illustrated embodiment, device 504 communicates with system 510 through a communication medium 508. Communication medium 508 may be any suitable network or other type of communication medium. For example, communication medium 508 may be a cellular network, a local area network, a circuit-switched telephone network, a public-switched telephone network, the Internet, some combination of any of the foregoing, or any other communication medium capable of supporting telecommunications.
[0168] In the illustrated embodiment, device 504 is communicatively coupled to communication medium 508 via wireless connection 506 and system 510 is communicatively coupled to communication medium 508 using a wired connection 516. This is merely for illustration, as device 504 and system 510 each may be communicatively coupled to communication medium 508 in any suitable way including wired and/or wireless connections.
[0169] System 510 comprises one or more computers (e.g., servers) each configured to perform processing related to supporting matching patient data and library data. In the illustrated embodiment, system 510 comprises a single server 512, though in other embodiments any suitable number of servers may be used and distributed in any manner. For instance, in some embodiments, system 510 may comprise at least ten, at least one hundred, at least one thousand, or at least ten thousand servers. For embodiments in which system 510 comprises multiple servers, the servers need not be located in the same physical location and may be distributed across multiple physical locations.
[0170] Each server, such as server 512, may be configured (alone or in combination with one or more other servers) to support one or more applications and may be configured to execute any other programs. For example, one or more of the servers may be configured to execute programs for supporting functionality related to patient data, library data, patient and library matching, and/or any other functions used in embodiments of the present methods.
[0171] As previously discussed, a healthcare provider, such as healthcare provider 502, may conduct one or multiple analyses with system 510. Multiple analyses may take place over any suitable time period of any suitable length. In some embodiments, system 510 calculates HLA-peptide affinities. In some embodiments, system 510 calculates HLA-peptide matching and a healthcare provider obtains this information. In some embodiments, a healthcare provider receives matched HLA-peptide information from system 510.
[0172] As discussed herein, system 510 may obtain any suitable data associated with one or more healthcare providers (e.g., information identifying healthcare provider 502, patient information, library information, patient and library matching information, etc.). System 510 may obtain any of the above-described data, associated with one or more healthcare providers, in any suitable way. For example, system 510 may extract this data from information collected from healthcare provider 502. In this case, system 510 may store at least a portion of the obtained data on one or more storage devices 516 so that it may be retrieved from the one or more storage devices for subsequent use as discussed herein.
[0173] Storage device(s) 516 may be any suitable storage device(s) or article(s) of manufacture capable of storing information. For example, storage device(s) 516 may be any non-transitory computer readable storage medium or media such as a computer memory (RAM and/or ROM), one or more hard disk drives, one or more optical disks (CDs and/or DVDs), one or more magnetic tapes, one or more flash memories, one or more circuit configurations in Field Programmable Gate Arrays and/or any other suitable device(s).
[0174] In the illustrated embodiment, storage device(s) 516 are shown as being part of system 510 and as being connected to server 512 by wired connection 514. It should be appreciated that all embodiments are not limited in this respect. For example, in other embodiments, storage device(s) 516 may be connected to server 512 using any suitable type of connection or communication medium, and may be internal or external to system 510. When storage device(s) 516 are external to system 510, the system may be communicatively coupled to storage device(s) 516 to obtain information from them in any suitable manner.
[0175] While FIG. 5 shows only a single healthcare provider using a single device 504, it should be recognized that embodiments of the present method are not limited in this respect and that system 510 may be configured to service any number of healthcare providers (e.g., hundreds, thousands, or greater) who may be using any number of devices (e.g., hundreds, thousands, or greater). Data associated with one or more patients made by each healthcare provider may be stored on at least one storage device (e.g., storage device 516 and/or on devices that the healthcare provider(s) use).
[0176] Regardless of where data associated with one or more patients involving one or more healthcare providers may be stored, such data may be stored in any suitable form. FIG. 6 shows a non-limiting example of a data structure 600 that may be used to store data associated with one or more patients in accordance with one embodiment. While in the illustrated embodiment data structure 600 is shown in the form of a table, this is not a limitation of the present method, as any suitable data structure may be used to store the data.
[0177] Data structure 600 may store any suitable data associated with one or more patients between any of one or more healthcare providers and system 510. In one non-limiting example described herein, data structure 600 may store patient identification data 602, patient data 604, library data 606, and/or data matching 608. In some embodiments, patient identification data 602 comprises information relating to patient identification such as name and date of birth. In some embodiments, patient data 604 comprises information relating to a patient's cancer type, gene fusion information, and/or HLA allele information. In some embodiments, library data 606 comprises information relating to peptide sequence, peptide-HLA affinity, sequences of DNA and/or RNA encoding peptides, gene fusion sequences, gene fusion cancer types, and IC.sub.50. In some embodiments, data matching 608 comprises information relating to peptide population coverage, allele coverage, and information matching peptides to HLA alleles, peptides to gene fusions, and/or peptides to cancer types. However, it should be recognized that these examples are not limiting, and that data structure 600 may store any other data associated with one or more patients.
[0178] For each patient, data structure 600 may store one or more data records. A data record may store any suitable data associated with a patient and may store such data in any suitable format (e.g., in one or more fields). Each field may store any suitable amount of data and any suitable type of data and, for example, may store alphanumeric data representing the data directly, or a pointer to another location where data may be stored. In some instances, data structure 600 may store the same type of data record for each patient, but this is not a limitation on the data structure or other aspects of the methods described herein, as in other embodiments, data structure 600 may store one type of data record for one patient and another type of data record for another patient. For example, a data record for one patient may store a different amount of data than another data record for another patient.
[0179] Data structure 600 may store any suitable number of data records. In FIG. 6, data structure 600 is shown as storing four data records, but it should be appreciated that the data structure may store many more (e.g., hundreds, thousands, millions, etc.) data records.
[0180] Each data record in data structure 600 is shown as having four fields; however, this is for purposes of illustration only and is not a limitation of the present methods as any suitable number of fields may be used. For instance, any suitable number of fields may be used to store information identifying the patient, and any suitable number of fields may be used to store data associated with the patient.
[0181] FIG. 7 shows a flow chart of an illustrative process for matching patient data and library data, in accordance with some embodiments of the present method.
[0182] Process 700 begins in act 702, where patient data may be received from a healthcare provider. The patient data may be received by a system, such as system 510, and may be received in any suitable way. Next, process 700 proceeds to act 704, where library information is obtained.
[0183] After the library information is obtained in act 704, process 700 proceeds to decision block 706, where it is determined whether patient information obtained in act 702 matches library information obtained in act 704. For example, it may be determined, in decision block 706, whether the patient is a candidate for treatment with one or more peptide vaccines from the library. As disclosed herein, such data may include patient allele data, peptide sequence information, and/or any of the other types of data described herein.
[0184] If it is determined, in decision block 706, that library data does not match data associated with one or more patients, process 700 proceeds to act 712, where a patient may be identified as a candidate for de novo vaccine generation and treatment or a candidate for alternative treatment.
[0185] If it is determined in decision block 706 that library data matches patient data, process 700 proceeds to act 708, where at least a portion of the library data may be retrieved. The data to retrieve may be any suitable data and may be determined in any suitable way. After peptide library data associated with one or more patients is retrieved in act 708, process 700 proceeds to act 710, where peptide information is sent to the healthcare provider.
[0186] An illustrative implementation of a computer system 810 that may be used in connection with any of the embodiments of the method described herein is shown in FIG. 8. The computer system 800 may include one or more processors 810 and one or more articles of manufacture that comprise non-transitory computer-readable storage media (e.g., memory 820 and one or more non-volatile storage media 830). Processor 810 may control writing data to and reading data from memory 820 and non-volatile storage device 830 in any suitable manner, as the aspects of the method described herein are not limited in this respect.
[0187] To perform any of the functionality described herein, processor 810 may execute one or more processor-executable instructions stored in one or more non-transitory computer-readable storage media (e.g., memory 820), which may serve as non-transitory computer-readable storage media storing processor-executable instructions for execution by processor 810.
[0188] The terms "program" or "software" are used herein in a generic sense to refer to any type of computer code or set of processor-executable instructions that can be employed to program a computer or other processor to implement various aspects of embodiments as discussed herein. Additionally, it should be appreciated that according to one aspect, one or more computer programs that when executed perform methods described herein need not reside on a single computer or processor, but may be distributed in a modular fashion among different computers or processors to implement various aspects of the present method.
Programs for Fusion Detection in RNA Sequence and/or WGS Data
[0189] In some embodiments, any one of the more than 30 publicly available programs for fusion detection can be used (programs available at omicstools (omictools.com/gene-fusion-detection-category). In some embodiments, more than one program for fusion detection is used. In some embodiments, the results from different fusion detection programs may significantly diverge.
[0190] In some embodiments, fusion detection programs are alignment-based programs that map reads to a reference and infer breakpoint positions from discordantly aligned reads. The majority of existing programs are alignment-based programs. However, alignment-based programs may differ in references (genome, transcriptome or both) or aligners, e.g., STAR (ncbi.nlm.nih.gov/pmc/articles/PMC3530905/), TopHat2 (ncbi.nlm.nih.gov/pmc/articles/PMC4053844/) or bowtie2 (ncbi.nlm.nih.gov/pmc/articles/PMC3322381/) and therefore give very diverse results.
[0191] In some embodiments, de novo assembly-based programs perform transcriptome assembly and align obtained sequences to a reference transcriptome and thus find chimeric transcripts. In some embodiments, the de novo assembly-based program JAFFA (genomemedicine.biomedcentral.com/articles/10.1186/s13073-015-0167-x) can be used in `assembly` mode.
[0192] In some embodiments, fusion detection programs are a hybrid of alignment-based and de-novo assembly-based approaches. In some embodiments, the hybrid approach first aligns reads to known transcriptome similar to the alignment-based approach. In some embodiments, the hybrid approach re-assembles unaligned reads and fragments spanning a breakpoint to new sequences. In some embodiments, the hybrid approach re-assembles unaligned reads and fragments spanning a breakpoint to new sequences using TRUP (ncbi.nlm.nih.gov/pmc/articles/PMC4300615/).
[0193] In some embodiments, fusion callers may be used for de novo assembly. In some embodiments, fusion callers consume a lot of resources and time. In some embodiments, fusion callers and alignment-based tools gave the same results.
[0194] In some embodiments, a program has filters that influence the results produced by the program. In some embodiments, a program has its own set of filters with specific default values. In some embodiments, a program has a filter for low fusion coverage that filters out low frequency junction or spanning reads. In some embodiments, a program has a filter for homology that filters out fusion partners with long homological regions. In some embodiments, a program has a filter for minimal distance between fusion partners that filters out potential read through transcripts. In some embodiments, a program has a promiscuity filter that filters out fusions containing partner transcripts which are presented in many other fusions. In some embodiments, a program generates a program-specific list of frequent false-positives.
[0195] In some embodiments, multiple fusion detection programs are used to determine a gene fusion recurrence rate. In some embodiments, four fusion detection programs are used to identify gene fusions. In some embodiments, a gene fusion is detected by two out of four fusion detection programs. In some embodiments, the fusion detection programs used are ChimeraScan (ncbi.nlm.nih.gov/pmc/articles/PMC3187648/), SOAP-fuse (ncbi.nlm.nih.gov/pmc/articles/PMC4054009/), and Integrate (genome.cshlp.org/content/early/2015/11/10/gr.186114.114.abstract).
Algorithm for Calculating Fusion-Derived Peptide Lengths
[0196] In some embodiments, fusion-derived peptide lengths are calculated from gene fusions. In some embodiments, computational algorithm (1) is used for calculating fusion-derived peptide lengths,
S ( X , L ) = Right ( SA 1 , 3 ( L - 1 ) + RF ( Right ( SA 1 , 1 ) ) Left ( SB 2 , BT ) , BT = RF ( Right ( SA 1 , 1 ) ) = RF ( Left ( SB 2 , 1 ) ) .fwdarw. 3 ( L - 1 ) + 3 - RF ( Left ( SB 2 , 1 ) ) RF ( Right ( SA 1 , 1 ) ) .noteq. RF ( Left ( SB 2 , 1 ) ) .fwdarw. 3 - RF ( Right ( SA 1 , 1 ) ) , + LRF ( Shift ( SB 2 , 3 - RF ( Right ( SA 1 , 1 ) ) ) ) ( 1 ) ##EQU00001##
wherein X is a fusion of genes A and B, SA.sub.1 is a transcript sequence of A to the left of the fusion breakpoint, SA.sub.2 is a transcript sequence of A to the right of the fusion breakpoint, SB.sub.1 is a transcript sequences of B to the left of the fusion breakpoint, SB.sub.2 is a transcript sequence of B to the right of the fusion breakpoint, SA.sub.1.andgate.SB.sub.2 is a fusion transcript sequence, SA.sub.1.andgate.SA.sub.2 is a transcript sequence of gene A, SB.sub.1.andgate.SB.sub.2 is a transcript sequence of gene B, L is the peptide length, Right and Left return a given number of bases to rightmost or leftmost, Shift skips a given number of bases on the left, RF is a position of a base in open reading frame, and LRF is the length of ORF. Fusion X yields a joint set of mutant peptides {S(X,L.sub.i)} where peptide length L.sub.i varies in some predefined range L.sub.min . . . L.sub.max.
Algorithm for HLA Genotyping
[0197] In some embodiments, HLA genotyping is calculated in silico from genome, exome, transcriptome, and/or HLA sequencing data. In some embodiments, a set of predicted HLA alleles that a patient may have is generated using computation algorithm (2),
CA k .di-elect cons. CA , i CA k .di-elect cons. A ij .gtoreq. H 2 ( 2 ) ##EQU00002##
[0198] wherein H.sub.i is a HLA genotyping algorithm, A.sub.ij is a set of HLA alleles predicted by algorithm H.sub.i, and CA is a set of HLA alleles. In some embodiments, A.sub.ij should have 4-digit precision (e.g., HLA-A0201). In some embodiments, the set of HLA alleles CA is used for matching patient and vaccine.
Algorithms for Predicting MHC-Peptide Binding
[0199] Algorithms are available for predicting MHC-peptide binding affinity and (algorithms available at cancerimmunity.org/resources/webtools/or cbs.dtu.dk/services/). In some embodiments, MHC-peptide binding was predicted with NetMHC and NetMHCpan programs (NetMHC (ncbi.nlm.nih.gov/pubmed/26258412, NetMHCpan ncbi.nlm.nih.gov/pubmed/25588790).
[0200] In some embodiments, MHC molecules of class I consist of a heavy chain and a light chain and bind to peptides comprising 8 to 11 amino acids. In some embodiments, MHC molecule of class I bind to peptides comprising 9 or 10 amino acids. In some embodiments, long peptides or entire proteins are degraded in the cell, bound to MHC proteins, and presented to cytotoxic T-cells.
[0201] In some embodiments, HLA proteins bind peptides comprising 8 to 14 amino acids. In some embodiments, peptides with predicted IC.sub.50<500 nM are considered to bind to HLA proteins. In some embodiments, peptides with predicted IC.sub.50<50 nM are considered as strong binders to HLA proteins. In some embodiments, peptides with predicted IC.sub.50>500 nM are considered as non-binders to HLA proteins. In some embodiments, non-binding peptides are filtered out from a list of peptides. In some embodiments, a population coverage for each long peptide is calculated using only those HLA proteins that can bind its short derivatives. In some embodiments, a long peptide comprising 30 amino acids can be shortened into peptides comprising 9 amino acids for predicting binding to HLA proteins. In some embodiments, mutant peptides are compared to wild type counterparts for binding affinity.
Algorithm for Calculating Population Coverage of Fusion-Derived Peptides
[0202] In some embodiments, a population coverage of fusion-derived peptides is calculated. In some embodiments, a population coverage of fusion-derived peptides is an estimate of how many patients will be eligible candidates for vaccination with the fusion-derived peptide. In some embodiments, a population coverage of fusion-derived peptides is calculated from the incidence of the corresponding gene fusion. In some embodiments, a population coverage of fusion-derived peptides is calculated from the allele frequency of HLA proteins that bind to the fusion-derived peptide. In some embodiments, a population coverage of fusion-derived peptides is calculated using computation algorithm (3),
P=.SIGMA..sub.iQ(F.sub.i)(1-.SIGMA..sub.j'(1-Q(A.sub.ij')).sup.2 (3)
wherein A.sub.ij'=.orgate..sub.k (A.sub.jjk), A.sub.ij' is a set of alleles matching peptides yielded by fusion F.sub.i, F.sub.i is a set of fusions yielding a set of peptides D.sub.ij comprising the given peptide vaccine, A.sub.ijk is the HLA alleles that can bind peptide D.sub.ij, Q(A.sub.ijk) is the allele frequency of allele A.sub.ijk, Q(F.sub.i) is the incidence rate of fusion F.sub.i, and P is the probability that a patient will be a candidate for treatment with the peptide vaccine.
Definitions
[0203] "Fusion transcript" refers to a product of fusion gene. Fusion transcript and chimeric transcript are used interchangeably.
[0204] "Fusion protein" or "chimeric protein" refers to a product of a fusion transcript and may emerge from genomic rearrangement, read through transcript, or trans-splicing. Fusion protein and chimeric protein are used interchangeably.
[0205] "Fusion variant" refers to a variant at particular breakpoints in fusion partner genes. "Fusion exonic variant" refers to a variant at breakpoints leading to fusion of the same partner gene exons.
[0206] "Point of junction" and "breakpoint" refer to the point at which two genes, transcripts, or proteins are fused together.
[0207] "Administering" or "administration" means providing a material to a subject in a manner that is pharmacologically useful.
[0208] "At risk" patients are patients that a health practitioner believes has a chance of having a disease, disorder or condition as provided herein.
[0209] "HLA proteins" refers to human leukocyte antigen (HLA) proteins that bind peptides resulting from proteolytic cleavage of protein antigens and representing potential T-cell epitopes, transporting them to the cell surface and presenting them there to specific cells, in particular cytotoxic T-lymphocytes or T-helper cells. HLA proteins are products of HLA genes that are classified into two gene groups coding for different proteins, namely proteins of HLA class I and proteins of HLA class II. HLA class I proteins and HLA class II proteins are specialized for different antigen sources. HLA class I proteins present endogenously synthesized antigens, for example viral proteins and tumor antigens. HLA class II proteins present protein antigens originating from exogenous sources, for example bacterial products. "HLA protein" and "MHC protein" are used interchangeably.
[0210] "Vaccine" refers to a composition for generating immunity for the prophylaxis and/or treatment of diseases. Vaccines are compositions comprising antigens and are intended to be used in humans or animals for generating specific defense and protective substance by vaccination.
[0211] "Peptide" refers to a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the .alpha.--amino and carboxyl groups of adjacent amino acids. Peptides can be a variety of lengths, either in neutral (uncharged) forms or in forms which are salts, and either free of modifications or containing modifications, subject to the condition that the modification not destroy the biological activity of the peptide as described herein. It should be appreciated that a "peptide sequence" or "sequence" may be used to refer to a "peptide" having a particular sequence in the present disclosure in some contexts. In some embodiments, a "long peptide" refers to a peptide comprising about 11-30 amino acid residues corresponding to the fusion breakpoint. In some embodiments, the long peptide is processed into a neoantigenic peptide comprising about 8-11 amino acid residues.
[0212] "Neoantigen" refers to a tumor antigen which is produced from tumor-specific mutations in expressed genes. "Fusion-derived", "fusion-specific", and "neoantigen" are used interchangeably. "Neoantigenic peptides" refers to peptides that comprise tumor-specific amino acid sequences (e.g., that contain a fusion junction that is only found in cancer cells due to the genetic rearrangement that is only present in the cancer cells). "Fusion-derived peptide", "fusion-specific peptide", and "neoantigenic peptide" are used interchangeably.
[0213] "IC.sub.50" refers to the concentration of gene fusion peptide necessary for inhibiting binding of a standard peptide to the HLA protein by fifty percent.
[0214] "Patient coverage" refers to the percentage of patients (e.g., in a population, for example in a population that is being treated or evaluated) that have a gene fusion corresponding to a fusion-specific neoantigen present in the vaccine library and that have HLA proteins capable of binding the fusion-specific neoantigen.
[0215] "Pulsing" refers to the process of antigen loading onto a cell, wherein the antigen may be a nucleic acid or a peptide.
EXAMPLES
Example 1: Therapeutic Cancer Vaccines Comprising Fusion-Specific Peptides
[0216] A therapeutic cancer vaccine should guide the immune response and be readily combine with other therapies. Therapeutic cancer vaccines can be composed of processed tumor cells, peptides, whole proteins, DNA, or mRNA. Vaccine antigens include tumor-associated antigens (TAA), differentiation antigens, or cancer-testis antigens. However, many antigens that are present in cancer cells are also present in peripheral organs, thus specifically targeting cancer cells is difficult. The first and only FDA approved therapeutic cancer vaccine called "Provenge" showed modest efficacy in prostate cancer (ncbi.nlm.nih.gov/pubmed/16809734).
[0217] Advances in sequencing technologies and bioinformatics algorithms have made it possible to identify mutations in whole cancer genomes. However, "de novo" identification of mutations from whole genome (WGS), whole exome (WES) or RNA sequencing data is expensive and time consuming. Many patients are in need of immediate treatment, especially patients with aggressive tumors like lung cancer, advanced melanoma, or acute leukemia.
[0218] Fusion genes have a higher reoccurrence rate than the reoccurrence rates of missense mutations or frameshift mutations. Highly mutated genes including TP53, PIK3CA, EGFR, KRAS, BRAF, and NRAS can be found across different tumor types, yet the reoccurrence rate of mutations in these genes is low. Moreover, recurrent fusion genes and recurrent driver mutations are often mutually exclusive. Thus, recurrent fusions are attractive as a source of neoantigens for the construction of "off-the-shelf" vaccine libraries for at least two reasons: high recurrence rate and the ability to drive cancer (ncbi.nlm.nih.gov/pubmed/25500544). The following examples provide a background for the construction and methods of use of a therapeutic cancer vaccine library comprising fusion-specific vaccines.
Murine Neoantigen Peptide Vaccines
[0219] In one study (ncbi.nlm.nih.gov/pubmed/25428507), neoantigenic peptides were identified and used for vaccination of mice. The study showed that two 3-methylcholanthrene-induced sarcoma cell lines d42m1-T3 and F244 were effectively rejected with anti-PD-1 and/or anti-CTLA-4 therapies. The immunological nature of the rejection was confirmed with multiple experimental approaches including depleting CD4+ and CD8+ T-cells with mouse antibodies, performing experiments in mice lacking T, B, natural killer T-cells or dendritic cells, and generating immunological memory in the mice.
[0220] The study identified neoantigens responsible for the checkpoint blockade therapy-mediated tumor rejection using a genomic approach. The approach was initially established for identifying TSA responsible for spontaneous rejection of d42m1 sarcomas (ncbi.nlm.nih.gov/pubmed/22318521). The study included features including mutation calling from exome sequencing data, translating mutations to corresponding protein sequences, predicting peptide-MHC class I binding affinity for each epitope, prioritizing epitopes, and filtering out epitopes with lower expression level and lower binding affinity in comparison to the wild-type peptides. Two mutant epitopes for murine MHC H-2 Kb were identified in this study: ITYTWTRL (SEQ ID NO: 612) (A506T mutation in alpha-1,3-glucosyltransferase) and VGFNFRTL (SEQ ID NO: 613) (G1254V mutation in laminin alpha subunit 4). Both epitopes were shown to be targets of CD8+ T-cells generated as a consequence of anti-PD1 treatment by experiments including MHC tetramer-binding assays, mass spectrometry detection of epitopes, and ELISPOT assays. Mice were vaccinated with two peptide vaccines based on the identified mutant epitopes and 9/10 mice rejected their tumors and survived. As a negative control, mice were vaccinated with human papillomavirus 28-mer peptides and only 1/10 mice survived. In multiple experiments of mice vaccination, the survival rate for mice vaccinated with therapeutic neoantigenic vaccines was 85% while the survival rate of mice vaccinated with human papillomavirus peptides was only 10%.
[0221] In another study (ncbi.nlm.nih.gov/pubmed/25901682), a bioinformatics approach was used to detect tumor-specific mutations from cancer exome sequencing data for B 16F10 tumor (melanoma) and colon carcinoma model CT26 in three independent mouse models with different MHC backgrounds. Mutant epitopes were prioritized using both MHC class I and class II prediction algorithms. A considerable fraction of detected mutations produced immunogenic neoantigens which can be recognized by both CD8+ and CD4+ T-cells. Vaccination with the identified immunogenic neoantigens produced anti-tumor activity and complete tumor rejection. About two-thirds of the mice vaccinated with the neoantigenic peptides were alive at day 100 while all untreated mice died at day 65. Interestingly, depletion experiments showed that CD4+ instead of CD8+ T-cells played a major role in anti-cancer response efficacy. Also, immunization with neoantigens encoded by mRNA elicited higher anti-tumor activity than immunization with peptides only. Taken together, this study showed that both MHC class I and class II restricted epitopes can be selected based on binding affinity and can display high anti-tumor potency.
[0222] In another study (ncbi.nlm.nih.gov/pubmed/25428506), neoepitopes were identified using whole-exome and transcriptome sequencing analysis with mass spectrometry. This approach identified neoepitopes in MC-38 and TRAMP-C1 mouse tumor cell lines using mutation calling from sequencing data, identification of expressed neoantigens using RNA-sequencing data, prediction of MHC class I binding interactions, and filtering of immunogenic epitopes. Six epitopes were predicted to bind with an IC.sub.50 less than 500 nM. Immunization of C57BL/6 mice with peptides encoding all six mutated epitopes elicited CD8+ T-cell responses in three out of six mice. Vaccination with long peptides encoding mutant epitopes had therapeutic and preventive effects. Subsequent challenging of vaccinated mice with MC-38 tumor cells didn't result in tumor spreading or growth compared to mice immunized with adjuvant only which experienced tumor spreading and growth.
[0223] As described herein, bioinformatics pipelines for detecting and prioritizing neoantigens in whole cancer exomes and/or transcriptomes have made it possible to select targets for vaccination using solely in silico simulation steps. Computational pipelines validated in mice models have improved mouse survival rates and inspired confidence in the potential success of vaccination with personalized neoantigenic vaccines in humans.
Average Bioinformatics Pipelines for Neoantigen Prediction
[0224] The following two examples illustrate a non-limiting schematic pipeline used for neoantigen generation from sequencing data (FIG. 1).
[0225] In one study (ncbi.nlm.nih.gov/pubmed/24894089), epitope-MHC class I binding prediction algorithms were applied to a large set of neoantigens which had been known to induce strong anti-tumor activity or long-term cancer control. The study analyzed 40 epitopes previously identified as cytotoxic T-cell targets in the literature including 35 epitopes resulting from missense mutations and 5 epitopes resulting from frame-shift mutations. All epitopes were restricted by HLA class I alleles and were present in non-small cell lung cancer, melanoma, renal cell carcinoma, bladder cancer, B-cell acute lymphoblastic leukemia, multiple myeloma, and chronic lymphocytic leukemia. All epitopes were neoantigens that had been identified from cloning, mass spectrometry experiments, genomic mutations, and epitope-binding predictions. Using artificial neural network based prediction algorithms, 27 of 31 of the epitopes recognized by T-cells were predicted to have binding affinity of less than 500 nM to MHC class I proteins. Two-thirds of the patients experienced spontaneous complete tumor regression or tumor regression following therapy. Thus, in silico peptide-MHC class I binding predictions provided a reliable tool to identify neoantigens from exome and/or transcriptome data, and inclusion of such neoantigens in vaccine formulations provided tumor regression in patients.
[0226] In another study (ncbi.nlm.nih.gov/pubmed/24891321), neoantigens in human cancer genomes were identified using bioinformatics. A bioinformatics pipeline was applied to 91 whole exomes obtained from chronic lymphocytic leukemias (CLL) biopsies from a group of patients who developed durable remission after allo-HSCT. On average 20 neoantigens derived from 16 missense mutations were predicted to bind HLA class I proteins and more than 50% of all neoantigen were confirmed to bind HLA proteins with biochemical studies. The general steps of the computational pipeline were DNA sequencing, mutation calling, peptide-MHC class I predictions, and experimental validation of binding peptides. From a total of 1838 detected mutations, 3 general classes of mutations were identified including missense, frameshift and splice site mutations. The most abundant class of mutations were missense mutations which represented more than 90% of all detected mutations in cancer genomes (or exomes). A computational pipeline was applied to 2488 whole exome sequences from 13 tumor types and to a large set of HLA alleles. The analysis revealed that one point mutation produced on average 1 to 5 neoantigens and 1 frameshift mutation produced 4 neoantigens with an IC.sub.50<50 nM. Neoantigens derived from frameshift mutations and gene fusions are expected to be more specific and more immunogenic because there is no corresponding wild type peptide. The study demonstrated that neoantigens derived from frameshift mutations and from gene fusions can be predicted using computational methods.
Vaccines Comprising Modified Fusion-Derived Peptides
[0227] The Wilms tumor gene 1 (WT1) can be used as a biomarker in different cancer types including acute myeloid leukemia (AML). In the clinical trial NCT00398138, WT1-positive patients with AML were treated with synthetic analogs of WT1 protein fragments in combination with adjuvants. Synthetic peptide analogs of WT1 protein fragments were designed to bind HLA molecules better than wild-type counterparts and were shown to more effectively induce a WT1-specific T-cell response compared to wild-type counterparts.
[0228] Anchor-modified peptides derived from PASD1 were shown to induce a cytotoxic T-cell response against tumor cells in AML patients. Interestingly, their non-modified wild-type analogs demonstrated no detectable binding to HLA proteins. These two examples demonstrate that neoantigenic peptide modifications can lead to an increase in peptide immunogenicity.
Vaccines Based on Fusion-Derived Neoantigens
[0229] The Philadelphia chromosome is the result of a translocation between chromosomes 22 and 9. This translocation is present in several cancers types and most notably the translocation is present in .about.95% of chronic myelogenous leukemia (CML) cases. The translocation produces a fusion between BCR and ABL1 genes. The ABL1 kinase is constitutively active in the BCR/ABL1 fusion protein and this upregulation of activity leads to dysregulation of cell division and development of cancer.
[0230] The sequence spanning the BCR/ABL1 breakpoint is absent in normal tissues and is present only in cancer cells, thus it may be recognized and attacked by the immune system. In a series of recent studies, peptides spanning the BCR/ABL breakpoint were shown to stimulate the immune response.
[0231] Cancer specific gene fusions have been used as neoantigens in several anti-cancer vaccines (Table 1). The EWS/FLI1 gene fusion is present in more than 85% of Ewing's sarcoma tumors (ncbi.nlm.nih.gov/pubmed/17250957) and the PAX-3/FKHR gene fusion is present in 55% of patients with alveolar rhabdomyosarcoma (ARMS) (ncbi.nlm.nih.gov/pubmed/12039929).
TABLE-US-00002 TABLE 1 List of fusions used as neoantigens in anti-cancer vaccines. Gene Fusion Patent BCR/ABL-1 [1-5] google.com.ar/patents/U.S. Pat. No. 5,997,869 EWS/FLI-1 (ncbi.nlm.nih.gov/pubmed/12039929) google.com.ar/patents/U.S. Pat. No. 5,997,869 PAX-3/FKHR (ncbi.nlm.nih.gov/pubmed/12039929) google.com.ar/patents/U.S. Pat. No. 5,997,869 TMPRSS2/ERG (ncbi.nlm.nih.gov/pubmed/24149465)
Example 2: Follicular Lymphoma (FL) Gene Fusions are Present in Multiple Cancer Types
[0232] Detection of recurrent chimeric transcripts was performed on 34 follicular lymphoma (FL) transcriptome samples from SRP048820 (ncbi.nlm.nih.gov/pubmed/25607463) and SRP056293 (ncbi.nlm.nih.gov/pmc/articles/PMC4676270/). Transcriptome samples included read through transcripts, products of trans-splicing, and chimeric transcripts formed as a result of genomic rearrangements. Chimeric transcripts were identified as recurrent if present in one or more of the FL transcriptome samples. Chimeric transcripts were identified as present in the sample if detected using two or more software programs, wherein the software programs were ChimeraScan, SOAPFuse, and Integrate.
[0233] An average of 28-29 fusion candidates were detected per sample. A total of 308 fusion candidates were detected with 149 of the fusion candidates found in more than one sample. A number of gene fusions present in the FL samples are also found in other cancer types including gastric, colon, breast, and prostate cancers (Table 2).
[0234] Neoantigenic peptides for each recurrent fusion protein having a predicted IC50 less than 500 nM to HLA class I proteins were selected. The prediction analysis can be performed using various length peptides, for example a peptide with 9 amino acids corresponding to 9 amino acids of the fusion junction. HLA class I alleles that bind to peptides with an IC.sub.50 less than 500 nM are identified as binding (Table 3). All fusion-derived peptides were novel and could be placed into a vaccine library. Patients are eligible for vaccination if they have a gene fusion corresponding to a fusion-derived peptide in the library and if the patient has at least one MHC class I alleles capable of binding to the fusion-derived peptide in the library. Methods of selecting a patient for vaccination and vaccine compositions are described herein.
TABLE-US-00003 TABLE 2 Recurrent Fusions Detected in Follicular Lymphoma (FL) Samples Breakpoint Number of Samples Non-FL Cancer Type Gene Fusion Coordinates Fusion/Total (References) DUS4L/BCAP29 chr7: 107217037 4/34 Gastric Cancer chr7: 107221204 (ncbi.nlm.nih.gov/pubmed/24240688) RRM2/C2orf48 chr2: 10269281 2/34 BT474 and SKBR3 cell lines chr2: 10281981 (ncbi.nlm.nih.gov/pubmed/23956304) PRKAA1/TTC33 chr5: 40764616 13/34 Colon, Kidney, Stomach, Breast, chr5: 40747121 Ovary, Uterus Cancers (ncbi.nlm.nih.gov/pubmed/23226102) MTG1/LOC619207 chr10: 135216277 8/34 Breast Cancer chr10: 135269741 (ncbi.nlm.nih.gov/pubmed/22496456) CIRBP/C19orf24 chr19: 1274439 14/34 Prostate Cancer chr19: 1277182 (google.com/patents/WO2012149522A1?cl=en)
TABLE-US-00004 TABLE 3 Predicted HLA Alleles of Recurrent Fusion-Derived Peptides. Gene Number of HLA class I alleles Fusion Fusion Sequence peptides (9 AA) (IC50 < 500 nM) DUS4L/ VWRITGTDGVKKKMTLQW 13 HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:06, BCAP29 AAV HLA-A*02:11, HLA-A*02:12, HLA-A*02:16, HLA-A*02:19, (SEQ ID NO.: 404) HLA-A*02:50, HLA-A*11:01, HLA-B*15:03, HLA-B*27:20, HLA-B*39:01 RRM2/ RLMLELGFSKVLGDREVQS 37 HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, C2orf48 RWSPGPRGDSTPVREMETN HLA-A*02:06, HLA-A*02:11, HLA-A*02:12, HPPSVRG HLA-A*02:16, HLA-A*02:19, HLA-A*02:50, (SEQ ID NO.: 405) HLA-A*30:01, HLA-A*31:01, HLA-A*32:07, HLA-A*33:01, HLA-A*68:01, HLA-B*07:02, HLA-B*15:03, HLA-B*18:01, HLA-B*27:20, HLA-B*40:01, HLA-B*40:02, HLA-B*40:13, HLA-B*42:01, HLA-B*44:02, HLA-B*45:01, HLA-B*73:01, HLA-C*03:03, HLA-C*07:01, HLA-C*12:03 PRKAA1/ TYLLDFRSIDEWLPLGGRG 34 HLA-A*02:03, HLA-A*02:06, HLA-A*02:11, TTC33 KLVRRSQRSLPSSLKLKLLM HLA-A*02:16, HLA-A*02:50, HLA-A*03:01, RRM HLA-A*11:01, HLA-A*30:01, HLA-A*31:01, (SEQ ID NO.: 406) HLA-A*32:07, HLA-A*32:15, HLA-A*33:01, HLA-A*68:23, HLA-B*07:02, HLA-B*08:01, HLA-B*15:01, HLA-B*15:03, HLA-B*15:17, HLA-B*27:05, HLA-B*27:20, HLA-B*35:01, HLA-B*39:01, HLA-B*40:13, HLA-B*42:01, HLA-B*53:01, HLA-B*57:01, HLA-B*58:01 HLA-C*03:03, HLA-C*12:03,HLA-C*15:02 MTG1/ LYTLNKHQRFGWTRCSEAG 208 HLA-A*31:01, HLA-B*27:20, HLA-C*06:02 LOC619207 VQTQHVRRSGVGPTPRGM GIRVQPGVDAGRGLCRVQA AGLRPCRGRPQVCPAAWR DGPALASQRVLPGQRVLPL GVQPWLMVPEPVPPRMGG GRAVLQRHFPGAPAGEGPQ SLRGTPRDQKREWGGPPLC PACGGGHGVLPGAGVRPCA PGPPPGRGRRQEVPGLQGY RAHHPQLQTGQQLPQRLRP AAGRRGGLLR (SEQ ID NO.: 407) CIRBP/ non-coding 0 -- C19orf24
Example 3: Fusion-Derived Peptides Having High Percentages of Patient Population Coverage
[0235] Two fusion transcripts can be found in solitary fibrous tumors. The transcripts are formed from fusion of exon 4 of the Nab2 gene (5' partner gene) with exon 2 of the Stat6 gene (3' partner gene) and exon 6 of the Nab2 gene (5' partner gene) with exon 16 of the Stat6 gene (3' partner gene). The amino acid sequence of the Nab2/Stat6 fusion protein is shown below. A novel amino acid sequence is generated when exon 4 of Nab2 is fused with exon 2 of Stat6 because of the presence of an untranslated region at the beginning of exon 2 of Stat6. Exon 6 of the Nab2 gene is fused with exon 16 of the Stat6 gene in frame.
TABLE-US-00005 Nab2/Stat6 (Nab2 exon 4 to Stat6 exon 2) fusion protein (COSF1547) (SEQ ID NO.: 408) MHRAPSPTAEQPPGGGDSARRTLQPRLKPSARAMALPRTLGELQLYRVLQ RANLLSYYETFIQQGGDDVQQLCEAGEEEFLEIMALVGMATKPLHVRRLQ KALREWATNPGLFSQPVPAVPVSSIPLFKISETAGTRKGSMSNGHGSPGE KAGSARSFSPKSPLELGEKLSPLPGGPGAGDPRIWPGRSTPESDVGAGGE EEAGSPPFSPPAGGGVPEGTGAGGLAAGGTGGGPDRLEPEMVRMVVESVE RIFRSFPRGDAGEVTSLLKLNKKLARSVGHIFEMDDNDSQKEEEIRKYSI IYGRFDSKRREGKQLSLHELTINEAAAQFCMRDNTLLLRRVELFSLSRQV ARESTYLSSLKGSRLHPEELGGPPLKKLKQEATSKSQIMSLWGLVSKMPP EKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCCNLASALLSDTVQ HLQASVGEQGEGSTILQHISTLESIYQRDPLKLVATFRQILQGEKKAVME QFRHLPMPFHWKQEELKFKTGLRRLQHRVGEIHLLREALQKGAEAGQVSL HSLIETPANGTGPSEALAMLLQETTGELEAAKALVLKRIQIWKRQQQLAG NGAPFEESLAPLQERCESLVDIYSQLQQEVGAAGGELEPKTRASLTGRLD EVLRTLVTSCFLVEKQPPQVLKTQTKFQAGVRFLLGLRFLGAPAKPPLVR ADMVTEKQARELSVPQGPGAGAESTGEIINNTVPLENSIPGNCCSALFKN LLLKKIKRCERKGTESVTEEKCAVLFSASFTLGPGKLPIQLQALSLPLVV IVHGNQDNNAKATILWDNAFSEMDRVPFVVAERVPWEKMCETLNLKFMAE VGTNRGLLPEHFLFLAQKIFNDNSLSMEAFQHRSVSWSQFNKEILLGRGF TFWQWFDGVLDLTKRCLRSYWSDRLIIGFISKQYVTSLLLNEPDGTFLLR FSDSEIGGITIAHVIRGQDGSPQIENIQPFSAKDLSIRSLGDRIRDLAQL KNLYPKKPKDEAFRSHYKPEQMGKDGRGYVPATIKMTVERDQPLPTPELQ MPTMVPSYDLGMAPDSSMSMQLGPDMVPQVYPPHSHSIPPYQGLSPEESV NVLSAFQEPHLQMPPSLGQMSLPFDQPHPQGLLPCQPQEHAVSSPDPLLC SDVTMVEDSCLSQPVTAFPQGTWIGEDIFPPLLPPTEQDLTKLLLEGQGE SGGGSLGAQPLLQPSHYGQSGISMSHMDLRANPSW Nab2/Stat6 (Nab2 exon 6 to Stat6 exon 16) fusion protein (COSF1561) (SEQ ID NO.: 409) MHRAPSPTAEQPPGGGDSARRTLQPRLKPSARAMALPRTLGELQLYRVLQ RANLLSYYETFIQQGGDDVQQLCEAGEEEFLEIMALVGMATKPLHVRRLQ KALREWATNPGLFSQPVPAVPVSSIPLFKISETAGTRKGSMSNGHGSPGE KAGSARSFSPKSPLELGEKLSPLPGGPGAGDPRIWPGRSTPESDVGAGGE EEAGSPPFSPPAGGGVPEGTGAGGLAAGGTGGGPDRLEPEMVRMVVESVE RIFRSFPRGDAGEVTSLLKLNKKLARSVGHIFEMDDNDSQKEEEIRKYSI IYGRFDSKRREGKQLSLHELTINEAAAQFCMRDNTLLLRRVELFSLSRQV ARESTYLSSLKGSRLHPEELGGPPLKKLKQEVGEQSHPEIQQPPPGPESY VPPYRPSLEEDSASLSGESLDGHLQAVGSCPRLTPPPADLPLALPAHGLW SRHILQQTLMDEGLRLARLVSHDRVGRLSPCVPAKPPLAGSPQIENIQPF SAKDLSIRSLGDRIRDLAQLKNLYPKKPKDEAFRSHYKPEQMGKDGRGYV PATIKMTVERDQPLPTPELQMPTMVPSYDLGMAPDSSMSMQLGPDMVPQV YPPHSHSIPPYQGLSPEESVNVLSAFQEPHLQMPPSLGQMSLPFDQPHPQ GLLPCQPQEHAVSSPDPLLCSDVTMVEDSCLSQPVTAFPQGTWIGEDIFP PLLPPTEQDLTKLLLEGQGESGGGSLGAQPLLQPSHYGQSGISMSHMDLR ANPSW
[0236] In the case of COSF1547, a long peptide having a sequence GPPLKKLKQEATSKSQIMSLWGLVSKM (SEQ ID NO.: 410) was used for generating neoantigenic peptides (8-11 amino acids) for predicting peptide binding to MHC class I proteins. The example below contains predicted neoantigenic peptides having 9 amino acids.
TABLE-US-00006 ##STR00001##
[0237] In the case of COSF1561, a long peptide having a sequence PCVPAKPPLAGSPQIENIQP (SEQ ID NO: 614) was used for generating neoantigenic peptides having 8-11 amino acids for predicting peptide binding to MHC class I proteins.
[0238] A set of neoantigenic peptides comprising 8-11 amino acids was generated for all available MHC class I alleles using prediction algorithms described herein and filtering out of all epitopes with IC.sub.50>500 nM. Those MHC class I proteins that are able to effectively bind Nab/Stat6 fusion-derived neoepitopes are listed in the Table 4.
[0239] For Nab2/Stat6 COSF1547, the allele coverage was calculated to be 94% meaning that 94% of the population can effectively present neoantigenic peptides derived from the long peptide spanning the Nab2/Stat6 fusion breakpoint. The Nab2/Stat6 COSF1547 fusion incidence rate is around 34% in solitary fibrous tumors. The patient coverage of the fusion-derived long peptide from Nab2/Stat6 COSF1547 is around 32%. For COSF1561, patient coverage of the fusion-derived long peptide is about 20%.
[0240] Both recurrent Nab2/Stat6 fusion-derived long peptides spanning fusion breakpoints are original and placed into the vaccine library. Results for another less frequent Nab2/Stat6 fusion (COSF1561, exon 6 of Nab2 to exon 17 of Stat6) are also shown in Table 4.
TABLE-US-00007 TABLE 4 Nab/Stat6 fusion-derived peptides predicted to bind HLA alleles. Long peptide spanning HLA class I alleles fusion breakpoint and 8-11-mer neoantigenic that bind neoantigens Fusion yielding neoantigens peptides (SEQ ID NO.) (IC50 < 500 nM) Nab2/Stat6 GPPLKKLKQEATSKSQIMS ATSKSQIM (429) HLA-A*02:01 COSF1547 LWGLVSKM EATSKSQI (430) HLA-A*02:02 (SEQ ID NO.: 426) IMSLWGLV (431) HLA-A*02:03 KSQIMSLW (432) HLA-A*02:06 QIMSLWGL (433) HLA-A*02:11 SKSQIMSL (434) HLA-A*02:17 EATSKSQIM (435) HLA-A*03:01 KLKQEATSK (436) HLA-A*11:01 QEATSKSQI (437) HLA-A*30:01 QIMSLWGLV (438) HLA-A*32:01 SQIMSLWGL (439) HLA-A*68:01 TSKSQIMSL (440) HLA-A*68:02 ATSKSQIMSL (441) HLA-A*69:01 IMSLWGLVSK (442) HLA-B*15:01 KKLKQEATSK (443) HLA-B*15:03 KQEATSKSQI (444) HLA-B*15:17 KSQIMSLWGL (445) HLA-B*35:01 QEATSKSQIM (446) HLA-B*39:01 SQIMSLWGLV (447) HLA-B*40:01 TSKSQIMSLW (448) HLA-B*40:02 ATSKSQIMSLW (449) HLA-B*44:02 EATSKSQIMSL (450) HLA-B*45:01 IMSLWGLVSKM (451) HLA-B*48:01 KQEATSKSQIM (452) HLA-B*57:01 KSQIMSLWGLV (453) HLA-B*58:01 LKQEATSKSQI (454) HLA-C*03:03 QEATSKSQIMS (455) HLA-C*12:03 QIMSLWGLVSK (456) HLA-C*15:02 SQIMSLWGLVS (457) Nab2/Stat6 PCVPAKPPLAGSPQIENIQP PLAGSPQI (458) HLA-A*02:11 COSF1561 (SEQ ID NO.: 427) PPLAGSPQI (459) HLA-B*07:02 KPPLAGSPQI (460) HLA-B*15:17 LAGSPQIENI (461) HLA-B*42:01 GSPQIENIQPF (462) HLA-C*03:03 PLAGSPQIENI (463) HLA-C*12:03 Nab2/Stat6 PCVPAKPPLAAEQMGKDG AEQMGKDG (464) HLA-A*03:01 COSF1561 RGY LAAEQMGK (465) HLA-A*68:01 (SEQ ID NO.: 428) PPLAAEQM (466) HLA-B*07:02 KPPLAAEQM (467) HLA-B*35:01 PLAAEQMGK (468) HLA-B*42:01 VPAKPPLAA (469) HLA-B*44:02 AEQMGKDGRGY (470) HLA-B*44:03 LAAEQMGKDGR (471) HLA-B*45:01 PAKPPLAAEQM (472) HLA-C*03:03 HLA-C*12:03
Example 4: Prostate Cancer Vaccine Compositions Comprising TMPRSS2/ERG Fusion Peptides
[0241] In total, 8 exonic variants of the TMPRSS2/ERG fusion from the COSMIC database were analyzed. The majority of the discovered antigenic peptides were not previously published. The TMPRSS2/ERG fusion is specific to prostate cancer and has quite a high incidence rate. The major protein coding exonic variant is estimated to be found in over 10% of cases. Further analysis showed that a vaccine based on TMPRSS2/ERG fusion-derived neoantigens can be used for about 20,000 prostate cancer patients a year in the USA.
TABLE-US-00008 TABLE 5 Population coverage of TMPRSS2/ERG fusion peptides. Long peptide spanning fusion breakpoint and Population yielding neoantigens HLA class (alleles that bind covered by (number of neoantigens) neoantigens (IC50 < 500 nM) the alleles DGVSHCPGGEDENRCGSLISCE (3) B*40:01, B*40:02, C*12:03 18% (SEQ ID NO.: 473) FLVGAALAAGLLWKFRSLISCE (13) A*02:01, A*02:02, A*02:03, A*02:06, A*02:11, 73% (SEQ ID NO.: 474) A*02:17, A*30:01, A*31:01,A*33:01, A*68:01, B*08:01, B*15:03, C*03:03, C*12:03 FLVGAALAAGLLWKFRTLLMNAVW A*02:01, A*02:02, A*02:03, A*02:06, A*02:11, 92% PKAGRW (22) A*02:17, A*03:01, A*11:01, A*24:03, A*30:01, (SEQ ID NO.: 475) A*31:01, A*32:01, A*33:01, A*68:01, A*69:01, B*08:01, B*15:03, B*15:17, B*27:05, B*39:01, B*57:01, B*58:01, C*03:03, C*12:03, C*14:02, C*15:02 MALNSEALSVVSEDQSLFEC (9) A*02:01, A*02:02, A*02:03, A*02:06, A*02:11, 67% (SEQ ID NO.: 476) A*02:17, A*68:02, A*69:01, B*15:03, B*15:17, B*35:01, B*40:02, B*45:01, B*53:01, C*03:03, C*12:03, C*14:02, C*15:02 MALNSPSGSEQLVDGLAY (11) A*01:01, A*02:02, A*02:11, A*02:17, A*26:01, 88% (SEQ ID NO.: 477) A*29:02, A*68:02, B*07:02, B*15:01, B*15:03, B*15:17, B*18:01, B*35:01, B*40:01, B*40:02, B*42:01, B*44:02, B*44:03, B*45:01, C*03:03, C*15:02 MALNSVIPGSLETRGKPC (10) A*02:01, A*02:02, A*02:03, A*02:06, A*02:11, 78% (SEQ ID NO.: 478) A*11:01, A*30:01, A*31:01, A*68:01, A*69:01, B*15:01, B*15:03, B*15:17, B*35:01, C*03:03, C*12:03, C*14:02 VCTQPKSPSGTVCTSRSLISCE (5) A*31:01, A*68:02, B*15:17, C*03:03 17% (SEQ ID NO.: 479) VCTQPKSPSGTVCTSSYSRIFGDPRK A*02:01, A*02:02, A*02:03, A*02:11, A*03:01, 98% AVLT (60) A*11:01, A*29:02, A*30:01, A*30:02, A*31:01, (SEQ ID NO.: 480) A*32:01, A*33:01, A*68:01, A*68:02, B*07:02, B*15:01, B*15:03, B*15:17, B*35:01, B*42:01, B*44:03, B*53:01, B*57:01, B*58:01, C*03:03, C*05:01, C*07:02, C*08:02, C*12:03, C*14:02, C*15:02
Example 5: Universal Cancer Vaccine Compositions Comprising ETV6/NTRK3 Fusion Peptides
[0242] Four exonic variants of the ETV6/NTRK3 fusion from the COSMIC database were analyzed to predict neoantigen peptides. All of the predicted neoantigen peptides were not previously published. According to the COSMIC database, this fusion was found in congenital (infantile) fibrosarcoma, mesoblastic nephroma, breast ductal secretory carcinoma, mammary analogue secretory carcinoma of salivary glands, thyroid carcinoma, and intestine carcinoma. This demonstrated that neoantigen vaccines based on a single fusion can target a wide spectrum of tumor types.
TABLE-US-00009 TABLE 6 ETV6/NTRK3 fusion-derived peptides predicted to bind HLA alleles. Long peptide spanning fusion breakpoint and Population yielding neoantigens HLA class I alleles that bind covered by (number of neoantigens) neoantigens (IC50 < 500 nM) the alleles IHTQPEVILHQNHEEDVQH A*02:02, A*02:03, A*02:11, A*33:01, A*68:01, B*15:03, 68% IKRRDIVLKRE (8) B*40:01, B*44:02, C*06:02, C*07:01, C*12:03 (SEQ ID NO.: 481) IHTQPEVILHQNHEEGPVA A*02:02, A*02:03, A*02:11, A*02:17, B*15:03, B*15:09, 48% VISGEEDSASP (10) B*39:01, B*40:01, B*40:02, B*45:01, C*07:01, C*12:03, (SEQ ID NO.: 482) C*14:02 VSPPEEHAMPIGRIADVQH A*02:03, A*02:06, A*02:11, A*03:01, A*11:01, A*30:01, 92% IKRRDIVLKRE (14) A*31:01, A*33:01, A*68:01, A*68:02, B*07:02, B*08:01, (SEQ ID NO.: 483) B*27:05, B*35:01, B*42:01, B*58:01, C*03:03, C*06:02, C*07:01, C*12:03, C*15:02 VSPPEEHAMPIGRIADVQH A*02:02, A*02:03, A*02:06, A*02:11, A*02:17, A*30:01, 66% IKRRDIVLKRE (11) A*32:01, A*68:02, A*69:01, B*07:02, B*15:01, B*15:17, (SEQ ID NO.: 484) B*27:05, B*35:01, B*39:01, B*42:01, B*58:01, C*03:03, C*12:03, C*14:02, C*15:02
Example 6: Identification of Novel Peptides in Previously Studied Gene Fusion (FUS/ERG)
[0243] FUS/ERG fusion can be found, for example, in acute myeloid leukaemia and occurs due to fusion of exon 7 of the FUS gene with exon 11 of the ERG gene. The amino acid sequence of the FUS/ERG fusion protein is shown below.
TABLE-US-00010 FUS/ERG (Fus exon 7 to exon 11 of Erg) fusion protein (COSF315) (SEQ ID NO.: 485) MASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGY GQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGY GQQPAPSSTSGSYGSSSQSSSYGQPQSGSYSQQPSYGGQQQSYGQQQSYN PPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMSSGGGSGGGYGNQDQS GGGGSGGYGQQDRGGRGRGGSGGGGGGGGGGYNRSSGGYEPRGRGGGRGG RGGMGGSDRGGFNKFGGSGQIQLWQFLLELLSDSSNSSCITWEGTNGEFK MTDPDEVARRWGERKSKPNMNYDKLSRALRYYYDKNIMTKVHGKRYAYKF DFHGIAQALQPHPPESSLYKYPSDLPYMGSYHAHPQKMNFVAPHPPALPV TSSSFFAAPNPYWNSPTGGIYPNTRLPTSHMPSHLGTYY
[0244] The fusion-derived long peptide having the sequence SDRGGFNKFGGSGQIQLWQF (SEQ ID NO: 615) was used for generating neoantigenic peptides (8-11 amino acids) for predicting peptide binding to MHC class I proteins. Results are shown in the Table 7. In the case of acute myeloid leukaemia, patient coverage of the fusion-derived long peptide is about 10%. Recurrent FUS/ERG fusion-derived long peptides are original and placed into the vaccine library.
TABLE-US-00011 TABLE 7 FUS/ERG fusion-derived peptides predicted to bind HLA alleles. HLA class I Long peptide spanning neoantigenic peptides alleles that bind fusion breakpoint and (8-11 amino acids) neoantigens Fusion yielding neoantigens (SEQ ID NO.) (IC50 <500 nM) FUS/ERG GPPLKKLKQEATSKSQIMSLW GSGQIQLW (487) HLA-A*24:02 COSF315 GLVSKM KFGGSGQI (488) HLA-A*24:03 (SEQ ID NO.: 486) FGGSGQIQL (489) HLA-B*15:17 GGSGQIQLW (490) HLA-B*39:01 KFGGSGQIQL (491) HLA-B*57:01 KFGGSGQIQLW (492) HLA-B*58:01 NKFGGSGQIQL (493) HLA-C*03:03 HLA-C*14:02
Example 7: Peptides Corresponding to Multiple Gene Fusion Junctions Increase Patient Coverage
[0245] FUS/DDIT3 fusions can be found, for example, in myxoid-round cell liposarcoma. The most frequent subtypes of this fusion are formed due to junction of exon 5 of Fus with exon 2 of DDIT3, exon 7 of Fus with exon 2 of DDIT3, and exon 8 of Fus with exon 2 of DDIT3. The amino acid sequences of the FUS/DDIT3 fusion proteins are shown below. In each fusion protein, a stop codon occurs soon after the breakpoint due to a frameshift. Neoantigens predictions were calculated using long peptides having 10 amino acids to the left and all amino acids to the right from the fusion junction.
TABLE-US-00012 FUS/DDIT3 (Fus exon 5 to exon 2 of DDIT3) fusion protein (COSF300) (SEQ ID NO.: 494) MASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGY GQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGY GQQPAPSSTSGSYGSSSQSSSYGQPQSGSYSQQPSYGGQQQSYGQQQSYN PPQGYGQQNQYNSSSGGGGGGGGGVFKKEVYLHTSPHLKAD FUS/DDIT3 (Fus exon 7 to exon 2 of DDIT3) fusion protein (COSF302) (SEQ ID NO.: 495) MASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGY GQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGY GQQPAPSSTSGSYGSSSQSSSYGQPQSGSYSQQPSYGGQQQSYGQQQSYN PPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMSSGGGSGGGYGNQDQS GGGGSGGYGQQDRGGRGRGGSGGGGGGGGGGYNRSSGGYEPRGRGGGRGG RGGMGGSDRGGFNKFGVFKKEVYLHTSPHLKAD FUS/DDIT3 (Fus exon 8 to exon 2 of DDIT3) fusion protein (COSF301) (SEQ ID NO.: 496) MASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGY GQSSYSSYGQSQNTGYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGY GQQPAPSSTSGSYGSSSQSSSYGQPQSGSYSQQPSYGGQQQSYGQQQSYN PPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMSSGGGSGGGYGNQDQS GGGGSGGYGQQDRGGRGRGGSGGGGGGGGGGYNRSSGGYEPRGRGGGRGG RGGMGGSDRGGFNKFGGPRDQGSRHDSVFKKEVYLHTSPHLKAD
[0246] Fusion-derived long peptides that were used for generating neoantigenic peptides having 8-11 amino acids for predicting peptide binding to MHC class I are shown in Table 8. Patient coverage with three fusion-derived long peptides is around 50% for myxoid-round cell liposarcoma. Recurrent FUS/DDIT3 fusion-derived peptides spanning fusion breakpoints are original and placed into vaccine library.
TABLE-US-00013 TABLE 8 FUS/DDIT3 fusion-derived peptides predicted to bind HLA alleles. Long peptide spanning fusion breakpoint and neoantigens peptides HLA class I alleles that bind Fusion yielding neoantigens (8-11 amino acids) neoantigens (IC50 < 500 nM) FUS/DDIT3 SGGGGGGGGGVFK HTSPHLKA (498) HLA-A*02:01, HLA-A*02:02, COSF300 KEVYLHTSPHLKAD KEVYLHTS (499) HLA-A*02:03, HLA-A*02:06, (SEQ ID NO.: 497) VFKKEVYL (500) HLA-A*02:11, HLA-A*02:17, VYLHTSPH (501) HLA-A*03:01, HLA-A*11:01, YLHTSPHL (502) HLA-A*23:01, HLA-A*24:02, EVYLHTSPH (503) HLA-A*24:03, HLA-A*26:01, GVFKKEVYL (504) HLA-A*29:02, HLA-A*30:01, KEVYLHTSP (505) HLA-A*33:01, HLA-A*66:01, VYLHTSPHL (506) HLA-A*68:01, HLA-A*68:02, YLHTSPHLK (507) HLA-A*69:01, HLA-B*15:01, EVYLHTSPHL (508) HLA-B*15:03, HLA-B*15:03, YLHTSPHLKA (509) HLA-B*40:01, HLA-B*40:02, EVYLHTSPHLK (510) HLA-B*44:03, HLA-B*45:01, KEVYLHTSPHL (511) HLA-C*03:03, HLA-C*12:03, GVFKKEVY (512) HLA-C*14:02 FUS/DDIT3 SDRGGENKFGVEKK FNKFGVFK (514) HLA-A*02:01, HLA-A*02:02, COSF302 EVYLHTSPHLKAD GFNKFGVF (515) HLA-A*02:03, HLA-A*02:06, (SEQ ID NO.: 513) GVFKKEVY (516) HLA-A*02:11, HLA-A*02:17, HTSPHLKA (517) HLA-A*03:01, HLA-A*11:01, KEVYLHTS (518) HLA-A*23:01, HLA-A*24:02, NKFGVFKK (519) HLA-A*24:03, HLA-A*26:01, VFKKEVYL (520) HLA-A*29:02, HLA-A*30:01, VYLHTSPH (521) HLA-A*31:01, HLA-A*33:01, YLHTSPHL (522) HLA-A*66:01, HLA-A*68:01, EVYLHTSPH (523) HLA-A*68:02, HLA-A*69:01, FNKFGVFKK (524) HLA-B*15:01, HLA-B*15:03, GFNKFGVFK (525) HLA-B*40:01, HLA-B*40:02, GGFNKFGVF (526) HLA-B*44:03, HLA-B*45:01, GVFKKEVYL (527) HLA-C*03:03, HLA-C*12:03, KEVYLHTSP (528) HLA-C*14:02 VYLHTSPHL (529) YLHTSPHLK (530) EVYLHTSPHL (531) FGVFKKEVYL (532) GFNKFGVFKK (533) YLHTSPHLKA (534) EVYLHTSPHLK (535) FNKFGVFKKEV (536) GGFNKFGVFKK (537) KEVYLHTSPHL (538) KFGVFKKEVYL (539) NKFGVFKKEVY (540) RGGFNKFGVFK (541) FUS/DDIT3 PRDQGSRHDSVFKK DSVFKKEV (543) HLA-A*02:01, HLA-A*02:02, COSF301 EVYLHTSPHLKAD GSRHDSVF (544) HLA-A*02:03, HLA-A*02:06, (SEQ ID NO.: 542) HTSPHLKA (545) HLA-A*02:11, HLA-A*02:17, KEVYLHTS (546) HLA-A*03:01, HLA-A*11:01, SRHDSVFK (547) HLA-A*23:01, HLA-A*24:02, SVFKKEVY (548) HLA-A*24:03, HLA-A*26:01, VFKKEVYL (549) HLA-A*29:02, HLA-A*30:01, VYLHTSPH (550) HLA-A*33:01, HLA-A*66:01, YLHTSPHL (551) HLA-A*68:01, HLA-A*68:02, EVYLHTSPH (552) HLA-A*69:01, HLA-B*15:01, GSRHDSVFK (553) HLA-B*15:03, HLA-B*15:17, KEVYLHTSP (554) HLA-B*27:05, HLA-B*40:01, QGSRHDSVF (555) HLA-B*40:02, HLA-B*44:03, SRHDSVFKK (556) HLA-B*45:01, HLA-C*03:03, SVFKKEVYL (557) HLA-C*06:02, HLA-C*07:01, VYLHTSPHL (558) HLA-C*12:03, HLA-C*14:02, YLHTSPHLK (559) HLA-C*15:02 DQGSRHDSVF (560) EVYLHTSPHL (561) GSRHDSVFKK (562) RHDSVFKKEV (563) YLHTSPHLKA (564) EVYLHTSPHLK (565) KEVYLHTSPHL (566) RDQGSRHDSVF (567) SRHDSVFKKEV (568)
Example 8: Identification of Novel Peptides in Previously Studied Gene Fusion (BCR/ABL1)
[0247] While the BCR/ABL1 fusion and its immunogenic properties have been described, the most comprehensive neoantigens were discovered by the disclosed method, namely two fusion peptides that are present in 68% of patients (FIG. 4). In this non-limiting example, the method described in the specification is applied to identification of BCR/ABL1 fusion peptides for the treatment of CML. Three breakpoints in BCR and ABL1 genes are marked with arrows in FIG. 4. Fusion at these breakpoints yields two BCR/ABL1 gene fusions that are present in 90% of CML patients which comprises 45,000 patients in the U.S. and 950,00 patients worldwide (2). The method discloses two BCR/ABL1 fusion peptides selected based on allele coverage and patient coverage that are present in 68% of CML patients which means that 30,000 patients in the U.S. and 650,000 patients worldwide are eligible for treatment with a cancer vaccine comprising these two peptides shown in FIG. 4. The sequences correspond to SEQ ID NOs: 610 to 611 from top to bottom, respectively.
[0248] The COSMIC database contains over 18 exonic variants of the BCR/ABL1 fusion that were analyzed and yielded neoantigen peptides. Half of the fusion exonic variants yielded peptides that were not previously published. According to COSMIC, the fusion variants are found in CML and ALL (including T-ALL and B-ALL).
TABLE-US-00014 TABLE 9 BCR/ABL1 fusion-derived peptides predicted to bind HLA alleles. Long peptide spanning fusion Population breakpoint and yielding neoantigens HLA class I alleles that bind covered by (number of neoantigens) neoantigens (IC50 <500 nM) the alleles ASEETYLSHLEALLLKPFSGQ (6) A*03:01, A*11:01, A*23:01, A*24:03, 79% (SEQ ID NO.: 569) A*30:01, A*68:01, B*15:01, B*15:03, B*18:01, B*35:01, B*40:01, B*40:02, B*44:02, B*44:03, C*14:02 EITPRRQSMTVKKGEKPFSGQ (6) A*11:01, A*68:01, B*15:01, B*15:03, 37% (SEQ ID NO.: 570) B*15:17, C*03:03, C*14:02 EITPRRQSMTVKKGESYTFLISSDY A*23:01, A*25:01, A*26:01, A*29:02, 77% ERAEW (17) A*30:01, A*30:02, A*32:01, A*68:01, (SEQ ID NO.: 571) B*15:01, B*15:03, B*15:17, B*35:01, B*40:01, B*40:02, B*44:02, B*44:03, B*45:01, B*57:01, B*58:01, C*03:03 GVATDIQALKAAFDVKALQRPVAS A*02:02, A*02:03, A*02:11, A*03:01, 64% DFEPQG (14) A*11:01, A*30:01, A*31:01, A*68:01, (SEQ ID NO.: 572) A*68:02, B*15:01, B*15:03, B*15:17, B*39:01, B*58:01, C*03:03, C*12:03 GVATDIQALKAAFDVRPSPCEDRH A*31:01, A*68:01, B*15:01, B*15:03, 88% WPGLAL (15) B*15:09, B*15:17, B*35:01, B*38:01, (SEQ ID NO.: 573) B*39:01, B*40:01, B*40:02, B*42:01, B*48:01, B*53:01, B*58:01, C*03:03, C*06:02, C*07:01, C*07:02, C*12:03, C*14:02 KLQTVHSIPLTINKEAGSIEALQRPV A*02:11, A*02:17, A*11:01, A*68:01, 53% ASDF (14) A*68:02, B*15:03, B*15:17, B*35:01, (SEQ ID NO.: 574) B*39:01, B*40:01, B*40:02, B*42:01, B*45:01, C*03:03, C*12:03, C*15:02 KLQTVHSIPLTINKEEALQRPVASD A*31:01, A*68:01, B*07:02, B*15:17, 64% FEPQG (11) B*35:01, B*40:02, B*42:01, B*44:02, (SEQ ID NO.: 575) B*45:01, C*03:03, C*14:02 KLQTVHSIPLTINKEGEKLRVLGYN A*11:01, A*31:01, A*68:01, B*15:17, 54% HNGEW (10) B*40:02, B*44:02, B*44:03, C*03:03, (SEQ ID NO.: 576) C*15:02 KTRVYRDTAEPNWNEKPFSGQ (5) A*68:01, B*15:03, B*35:01, B*42:01, 32% (SEQ ID NO.: 577) B*53:01, C*03:03, C*12:03 KTRVYRDTAEPNWNELDPQALQD A*68:02, A*69:01, B*39:01, B*40:01, 45% RDWQRTV (5) B*40:02, B*44:02, C*03:03, C*12:03 (SEQ ID NO.: 578) LNVIVHSATGFKQSSKALQRPVAS A*02:11, A*03:01, A*11:01, A*30:01, 63% DFEPQG (17) A*31:01, A*68:01, A*68:02, B*15:03, (SEQ ID NO.: 579) B*15:09, B*15:17, B*39:01, B*58:01, C*03:03, C*12:03, C*14:02, C*15:02 LNVIVHSATGFKQSSSEKLRVLGY A*01:01, A*11:01, A*30:01, A*31:01, 88% NHNGEW (18) A*68:01, B*15:01, B*15:03, B*15:09, (SEQ ID NO.: 580) B*15:17, B*18:01, B*39:01, B*44:02, B*44:03, C*03:03, C*06:02, C*07:01, C*15:02 QIWPNDGEGAFHGDAEALQRPVAS A*24:03, A*68:02, B*15:09, B*35:01, 88% DFEPQG (11) B*38:01, B*39:01, B*40:01, B*40:02, (SEQ ID NO.: 581) B*44:02, B*45:01, C*03:03, C*06:02, C*07:01, C*07:02, C*12:03, C*14:02 QIWPNDGEGAFHGDAGEKLRVLG A*24:03, B*15:09, B*15:17, B*38:01, 74% YNHNGEW (7) B*39:01, B*44:02, B*44:03, C*03:03, (SEQ ID NO.: 582) C*06:02, C*07:01, C*12:03, C*14:02 QWSHQQRVGDLFQKLNLRARSNK A*02:06, A*02:11, A*03:01, A*11:01, 50% DAKDPTT (8) A*30:01, A*31:01, A*33:01, A*68:01, (SEQ ID NO.: 583) B*15:03 RKQTGVFGVKIAVVTKSPSAASSI A*02:03, A*02:06, A*11:01, A*30:01, 41% (10) A*31:01, B*15:03, B*15:17, B*39:01, (SEQ ID NO.: 584) C*03:03, C*12:03, C*14:02, C*15:02 SNKDAKDPTTKNSLEKALQRPVAS A*02:02, A*02:03, A*02:11, A*03:01, 68% DFEPQG (10) A*11:01, A*30:01, A*68:01, A*69:01, (SEQ ID NO.: 585) B*15:03, B*15:17, B*39:01, B*40:02, B*44:02, B*58:01, C*03:03, C*12:03, C*14:02, C*15:02 SRDALVSGALESTKATKAKPFSGQ A*03:01, A*11:01, A*30:01, A*31:01, 63% (SEQ ID NO.: 586) A*68:01, B*15:01, B*15:03, B*15:17, B*57:01, B*58:01, C*03:03, C*14:02
Example 9: Method Validation Using EWSR1/FLI1 Gene Fusion
[0249] The EWSR1/FLI1 fusion can be found, for example, in Ewing's sarcoma. Two most frequent types of EWSR1/FLI1 fusions occur due to junction of exon 7 of the EWSR1 gene with exon 5 or exon 6 of the FLI1 gene. The amino acid sequences of the fusion proteins are shown below.
[0250] Fusion-derived peptides having 8-10 amino acids corresponding to the fusion junction were used for predicting peptide binding to MHC class I proteins. Results are shown in Table 10. Some of the immunogenic EWSR1/FLI1 derived peptides were previously disclosed by patent application U.S. Pat. No. 5,997,869A and were also successfully discovered using methods described herein.
TABLE-US-00015 EWSR1/FLI1 (EWSR1 exon 7 to exon 6 of FLI1) fusion protein (COSF166) (SEQ ID NO.: 587) MASTDYSTYSQAAAQQGYSAYTAQPTQGYAQTTQAYGQQSYGTYGQPTDV SYTQAQTTATYGQTAYATSYGQPPTGYTTPTAPQAYSQPVQGYGTGAYDT TTATVTTTQASYAAQSAYGTQPAYPAYGQQPAATAPTRPQDGNKPTETSQ PQSSTGGYNQPSLGYGQSNYSYPQVPGSYPMQPVTAPPSYPPTSYSSTQP TSYDQSSYSQQNTYGQPSSYGQQSSYGQQSSYGQQPPTSYPPQTGSYSQA PSQYSQQSSSYGQQNPSYDSVRRGAWGNNMNSGLNKSPPLGGAQTISKNT EQRPQPDPYQILGPTSSRLANPGSGQIQLWQFLLELLSDSANASCITWEG TNGEFKMTDPDEVARRWGERKSKPNMNYDKLSRALRYYYDKNIMTKVHGK RYAYKFDFHGIAQALQPHPTESSMYKYPSDISYMPSYHAHQQKVNFVPPH PSSMPVTSSSFFGAASQYWTSPTGGIYPNPNVPRHPNTHVPSHLGSYY EWSR1/FLI1 (EWSR1 exon 7 to exon 5 of FLI1) fusion protein (COSF168) (SEQ ID NO.: 588) MASTDYSTYSQAAAQQGYSAYTAQPTQGYAQTTQAYGQQSYGTYGQPTDV SYTQAQTTATYGQTAYATSYGQPPTGYTTPTAPQAYSQPVQGYGTGAYDT TTATVTTTQASYAAQSAYGTQPAYPAYGQQPAATAPTRPQDGNKPTETSQ PQSSTGGYNQPSLGYGQSNYSYPQVPGSYPMQPVTAPPSYPPTSYSSTQP TSYDQSSYSQQNTYGQPSSYGQQSSYGQQSSYGQQPPTSYPPQTGSYSQA PSQYSQQSSSYGQQSSLLAYNTTSHTDQSSRLSVKEDPSYDSVRRGAWGN NMNSGLNKSPPLGGAQTISKNTEQRPQPDPYQILGPTSSRLANPGSGQIQ LWQFLLELLSDSANASCITWEGTNGEFKMTDPDEVARRWGERKSKPNMNY DKLSRALRYYYDKNIMTKVHGKRYAYKFDFHGIAQALQPHPTESSMYKYP SDISYMPSYHAHQQKVNFVPPHPSSMPVTSSSFFGAASQYWTSPTGGIYP NPNVPRHPNTHVPSHLGSYY
TABLE-US-00016 TABLE 10 EWSR1/FLI1 fusion-derived peptides predicted to bind HLA alleles. Long peptide spanning HLA class I alleles fusion breakpoint and 8-11-mer neo- that bind neoantigens Fusion yielding neoantigens antigenic peptides (IC50 < 500 nM) EWSR1/FLI1 SQQSSSYGQQNPSYDSVRRG SSYGQQNPSY (591) HLA-A*11:01 COSF166 (SEQ ID NO.: 589) SYGQQNPSY (592) HLA-A*29:02 SSYGQQNPSY (593) HLA-A*31:01 YGQQNPSY (594) HLA-A*68:01 NPSYDSVR (595) HLA-B*15:01 QQNPSYDSV (596) HLA-B*35:01 NPSYDSVRR (597) HLA-C*12:03 YGQQNPSY (598) QQNPSYDSVR (599) EWSR1/FLI1 SQQSSSYGQQSSLLAYNTTS GQQSSLLAY (600) HLA-A*24:02 COSF168 (SEQ ID NO.: 590) QQSSLLAY (601) HLA-A*29:02 SSSYGQQSSL (602) HLA-B*15:01 SSYGQQSSL (603) HLA-B*35:01 SSYGQQSSL (604) HLA-C*03:03 SSYGQQSSLL (605) HLA-C*06:02 SYGQQSSL (606) HLA-C*07:01 SYGQQSSLL (607) HLA-C*07:02 YGQQSSLL (608) YGQQSSLLAY (609)
Other Embodiments
[0251] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
[0252] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
EQUIVALENTS
[0253] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
[0254] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
[0255] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
[0256] The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."
[0257] The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
[0258] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.
[0259] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
[0260] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
[0261] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., "comprising") are also contemplated, in alternative embodiments, as "consisting of" and "consisting essentially of" the feature described by the open-ended transitional phrase. For example, if the disclosure describes "a composition comprising A and B", the disclosure also contemplates the alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B".
Sequence CWU
1
1
615130PRTArtificial SequenceSynthetic Polypeptide 1Ala Ile Lys Ser Lys Asn
Val Asp Val Asn Val Lys Asp Glu Glu Thr 1 5
10 15 Glu Val Lys Ser Glu Glu Gly Pro Gly Trp Thr
Ile Leu Arg 20 25 30
230PRTArtificial SequenceSynthetic Polypeptide 2Glu Ala Pro Leu Asn Pro
Lys Ala Asn Arg Glu Lys Met Thr Gln Asp 1 5
10 15 Pro Leu Leu Gly Met Leu Asp Gly Arg Glu Asp
Leu Glu Arg 20 25 30
330PRTArtificial SequenceSynthetic Polypeptide 3Glu Ala Pro Leu Asn Pro
Lys Ala Asn Arg Glu Lys Met Thr Gln Pro 1 5
10 15 Ile Ser Gly Ile Pro Ser Pro Asp Glu Ser Pro
Lys Arg Ala 20 25 30
430PRTArtificial SequenceSynthetic Polypeptide 4Glu Ala Pro Leu Asn Pro
Lys Ala Asn Arg Glu Lys Met Thr Gln Leu 1 5
10 15 Lys Ser Glu Leu Asp Met Leu Val Gly Lys Cys
Arg Glu Glu 20 25 30
530PRTArtificial SequenceSynthetic Polypeptide 5Pro Arg Ala Val Phe Pro
Ser Ile Val Gly Arg Pro Arg His Gln Leu 1 5
10 15 Lys Ser Glu Leu Asp Met Leu Val Gly Lys Cys
Arg Glu Glu 20 25 30
630PRTArtificial SequenceSynthetic Polypeptide 6Gln Thr Lys Val Leu Ser
Ala Ser Gln Ala Phe Ala Ala Gln Arg Asp 1 5
10 15 Ser Ile Thr Gln His Lys Val Cys Ala Pro Glu
Asn Tyr Leu 20 25 30
730PRTArtificial SequenceSynthetic Polypeptide 7Asn Asp Lys Leu Gln Lys
Glu Leu Asn Val Leu Lys Ser Glu Gln Asp 1 5
10 15 Leu Ile Arg Asp Gln Gly Phe Arg Gly Asp Gly
Gly Ser Thr 20 25 30
830PRTArtificial SequenceSynthetic Polypeptide 8Gln Glu Arg Glu Arg Asp
Pro Gln Gln Glu Gln Glu Arg Glu Arg Ile 1 5
10 15 Asp Asp Val Ile Asp Glu Ile Ile Ser Leu Glu
Ser Ser Tyr 20 25 30
930PRTArtificial SequenceSynthetic Polypeptide 9Gln Glu Arg Glu Arg Asp
Pro Gln Gln Glu Gln Glu Arg Glu Arg Leu 1 5
10 15 Pro Val Ser Gly Asn Leu Leu Asp Val Tyr Ser
Ser Gln Gly 20 25 30
1019PRTArtificial SequenceSynthetic Polypeptide 10Ser Ser Gln Lys Ala His
Gly Ile Leu Ala Arg Arg Pro Ser Tyr Arg 1 5
10 15 Thr His Gly 1130PRTArtificial
SequenceSynthetic Polypeptide 11Met Ser Val Pro Thr Pro Ile Tyr Gln Thr
Ser Ser Gly Gln Tyr Ser 1 5 10
15 Ser Phe Arg Gln Asp His Pro Ser Ser Met Gly Val Tyr Gly
20 25 30 1227PRTArtificial
SequenceSynthetic Polypeptide 12Arg Asn Val Gln Asp Phe Lys Arg Ala Ser
Glu Glu Ile Thr Lys Thr 1 5 10
15 Thr Gln Asp Gly Leu Asp Trp Leu Ile Ser Thr 20
25 1330PRTArtificial SequenceSynthetic
Polypeptide 13Thr Pro Ala Gln Leu Tyr Thr Leu Gln Pro Lys Leu Pro Ile Thr
Val 1 5 10 15 Tyr
Arg Arg Lys His Gln Glu Leu Gln Ala Met Gln Met Glu 20
25 30 1430PRTArtificial SequenceSynthetic
Polypeptide 14Thr Gln Leu Pro His Ile Pro Val Ala Val Arg Val Gly Cys Leu
Gln 1 5 10 15 Tyr
Tyr Arg Thr Asp Ser Asp Asn Gln Ala Ile Leu Ser Glu 20
25 30 1521PRTArtificial SequenceSynthetic
Polypeptide 15Lys Thr Arg Val Tyr Arg Asp Thr Ala Glu Pro Asn Trp Asn Glu
Lys 1 5 10 15 Pro
Phe Ser Gly Gln 20 1624PRTArtificial SequenceSynthetic
Polypeptide 16Arg Lys Gln Thr Gly Val Phe Gly Val Lys Ile Ala Val Val Thr
Lys 1 5 10 15 Ser
Pro Ser Ala Ala Ser Ser Ile 20
1730PRTArtificial SequenceSynthetic Polypeptide 17Glu Ile Thr Pro Arg Arg
Gln Ser Met Thr Val Lys Lys Gly Glu Ser 1 5
10 15 Tyr Thr Phe Leu Ile Ser Ser Asp Tyr Glu Arg
Ala Glu Trp 20 25 30
1830PRTArtificial SequenceSynthetic Polypeptide 18Lys Leu Gln Thr Val His
Ser Ile Pro Leu Thr Ile Asn Lys Glu Ala 1 5
10 15 Gly Ser Ile Glu Ala Leu Gln Arg Pro Val Ala
Ser Asp Phe 20 25 30
1930PRTArtificial SequenceSynthetic Polypeptide 19Lys Thr Arg Val Tyr Arg
Asp Thr Ala Glu Pro Asn Trp Asn Glu Leu 1 5
10 15 Asp Pro Gln Ala Leu Gln Asp Arg Asp Trp Gln
Arg Thr Val 20 25 30
2030PRTArtificial SequenceSynthetic Polypeptide 20Gln Trp Ser His Gln Gln
Arg Val Gly Asp Leu Phe Gln Lys Leu Asn 1 5
10 15 Leu Arg Ala Arg Ser Asn Lys Asp Ala Lys Asp
Pro Thr Thr 20 25 30
2124PRTArtificial SequenceSynthetic Polypeptide 21Ser Arg Asp Ala Leu Val
Ser Gly Ala Leu Glu Ser Thr Lys Ala Thr 1 5
10 15 Lys Ala Lys Pro Phe Ser Gly Gln
20 2230PRTArtificial SequenceSynthetic Polypeptide 22Ser
Asn Lys Asp Ala Lys Asp Pro Thr Thr Lys Asn Ser Leu Glu Lys 1
5 10 15 Ala Leu Gln Arg Pro Val
Ala Ser Asp Phe Glu Pro Gln Gly 20 25
30 2321PRTArtificial SequenceSynthetic Polypeptide 23Ala Ser
Glu Glu Thr Tyr Leu Ser His Leu Glu Ala Leu Leu Leu Lys 1 5
10 15 Pro Phe Ser Gly Gln
20 2421PRTArtificial SequenceSynthetic Polypeptide 24Glu Ile Thr
Pro Arg Arg Gln Ser Met Thr Val Lys Lys Gly Glu Lys 1 5
10 15 Pro Phe Ser Gly Gln
20 2530PRTArtificial SequenceSynthetic Polypeptide 25Lys Leu Gln Thr
Val His Ser Ile Pro Leu Thr Ile Asn Lys Glu Gly 1 5
10 15 Glu Lys Leu Arg Val Leu Gly Tyr Asn
His Asn Gly Glu Trp 20 25
30 2630PRTArtificial SequenceSynthetic Polypeptide 26Gly Val Ala Thr Asp
Ile Gln Ala Leu Lys Ala Ala Phe Asp Val Arg 1 5
10 15 Pro Ser Pro Cys Glu Asp Arg His Trp Pro
Gly Leu Ala Leu 20 25 30
2730PRTArtificial SequenceSynthetic Polypeptide 27Gly Val Ala Thr Asp Ile
Gln Ala Leu Lys Ala Ala Phe Asp Val Lys 1 5
10 15 Ala Leu Gln Arg Pro Val Ala Ser Asp Phe Glu
Pro Gln Gly 20 25 30
2830PRTArtificial SequenceSynthetic Polypeptide 28Gln Ile Trp Pro Asn Asp
Gly Glu Gly Ala Phe His Gly Asp Ala Gly 1 5
10 15 Glu Lys Leu Arg Val Leu Gly Tyr Asn His Asn
Gly Glu Trp 20 25 30
2930PRTArtificial SequenceSynthetic Polypeptide 29Leu Asn Val Ile Val His
Ser Ala Thr Gly Phe Lys Gln Ser Ser Ser 1 5
10 15 Glu Lys Leu Arg Val Leu Gly Tyr Asn His Asn
Gly Glu Trp 20 25 30
3030PRTArtificial SequenceSynthetic Polypeptide 30Leu Asn Val Ile Val His
Ser Ala Thr Gly Phe Lys Gln Ser Ser Lys 1 5
10 15 Ala Leu Gln Arg Pro Val Ala Ser Asp Phe Glu
Pro Gln Gly 20 25 30
3130PRTArtificial SequenceSynthetic Polypeptide 31Lys Leu Gln Thr Val His
Ser Ile Pro Leu Thr Ile Asn Lys Glu Glu 1 5
10 15 Ala Leu Gln Arg Pro Val Ala Ser Asp Phe Glu
Pro Gln Gly 20 25 30
3230PRTArtificial SequenceSynthetic Polypeptide 32Gln Ile Trp Pro Asn Asp
Gly Glu Gly Ala Phe His Gly Asp Ala Glu 1 5
10 15 Ala Leu Gln Arg Pro Val Ala Ser Asp Phe Glu
Pro Gln Gly 20 25 30
3330PRTArtificial SequenceSynthetic Polypeptide 33Gln Ile Trp Pro Asn Asp
Gly Glu Gly Ala Phe His Gly Asp Ala Asp 1 5
10 15 Tyr Glu Leu Leu Thr Glu Asn Asp Met Leu Pro
Asn Met Arg 20 25 30
3430PRTArtificial SequenceSynthetic Polypeptide 34Gln Ile Trp Pro Asn Asp
Gly Glu Gly Ala Phe His Gly Asp Ala Val 1 5
10 15 Leu Gln Glu Arg Ile Pro Trp Val Pro Pro Glu
Cys Ile Glu 20 25 30
3530PRTArtificial SequenceSynthetic Polypeptide 35Ala Pro Asn Val His Ile
Asn Thr Ile Glu Pro Val Asn Ile Asp Asn 1 5
10 15 Asp Leu Arg Leu Gln Met Glu Ala Gln Arg Ile
Cys Leu Ser 20 25 30
3630PRTArtificial SequenceSynthetic Polypeptide 36Arg Tyr Val Lys Ser Cys
Leu Gln Lys Lys Gln Arg Lys Pro Phe Ser 1 5
10 15 Ser Ala Leu Pro Gly Pro Asp Met Ser Met Lys
Pro Ser Ala 20 25 30
3730PRTArtificial SequenceSynthetic Polypeptide 37Ser Ser Ser Glu Ser Ser
Ser Ser Asp Ser Glu Asp Ser Glu Thr Ala 1 5
10 15 Ser Ala Leu Pro Gly Pro Asp Met Ser Met Lys
Pro Ser Ala 20 25 30
3830PRTArtificial SequenceSynthetic Polypeptide 38Ser Glu Phe Arg Glu Gly
Val Arg Lys Ile Ala Arg Glu Gln Lys Val 1 5
10 15 Tyr Arg Arg Lys His Gln Glu Leu Gln Ala Met
Gln Met Glu 20 25 30
3930PRTArtificial SequenceSynthetic Polypeptide 39Leu Gln Glu Glu Asn Arg
Asp Leu Arg Lys Ala Ser Val Thr Ile Glu 1 5
10 15 Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn Leu
Val Leu Gly 20 25 30
4030PRTArtificial SequenceSynthetic Polypeptide 40Glu Glu Glu Phe Leu Thr
Asn Glu Leu Ser Arg Lys Leu Met Gln Glu 1 5
10 15 Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn Leu
Val Leu Gly 20 25 30
4130PRTArtificial SequenceSynthetic Polypeptide 41Ser Arg His Ser Leu Glu
Gln Lys Pro Thr Asp Ala Pro Pro Lys Val 1 5
10 15 Trp His Arg Arg Leu Lys Asn Gln Lys Ser Ala
Lys Glu Gly 20 25 30
4230PRTArtificial SequenceSynthetic Polypeptide 42Ser Arg His Ser Leu Glu
Gln Lys Pro Thr Asp Ala Pro Pro Lys Ala 1 5
10 15 Gly Val Pro Asn Lys Pro Gly Ile Pro Lys Leu
Leu Glu Gly 20 25 30
4330PRTArtificial SequenceSynthetic Polypeptide 43Ser Arg His Ser Leu Glu
Gln Lys Pro Thr Asp Ala Pro Pro Lys Asp 1 5
10 15 Asp Phe Trp Ile Pro Glu Thr Ser Phe Ile Leu
Thr Ile Ile 20 25 30
4430PRTArtificial SequenceSynthetic Polypeptide 44Val His Pro Asp Ala Leu
Asn Arg Phe Gly Lys Thr Ala Leu Gln Leu 1 5
10 15 His Val His Ala Cys Leu Leu Thr Arg Lys Gln
Glu Asp Cys 20 25 30
4530PRTArtificial SequenceSynthetic Polypeptide 45Val His Pro Asp Ala Leu
Asn Arg Phe Gly Lys Thr Ala Leu Gln Leu 1 5
10 15 His Val His Ala Cys Leu Leu Thr Arg Lys Gln
Glu Asp Cys 20 25 30
4630PRTArtificial SequenceSynthetic Polypeptide 46Val His Pro Asp Ala Leu
Asn Arg Phe Gly Lys Thr Ala Leu Gln Leu 1 5
10 15 His Val His Ala Cys Leu Leu Thr Arg Lys Gln
Glu Asp Cys 20 25 30
4730PRTArtificial SequenceSynthetic Polypeptide 47Leu Ala Glu Asn Ile Thr
Gln Glu Arg Asp Ser Leu Met Cys Leu Asp 1 5
10 15 Leu Ile Arg Asp Gln Gly Phe Arg Gly Asp Gly
Gly Ser Thr 20 25 30
4830PRTArtificial SequenceSynthetic Polypeptide 48Thr Gln Glu Glu Glu Asp
Glu Ile Leu Pro Arg Lys Asp Tyr Glu Lys 1 5
10 15 Thr Leu Gly Arg Arg Asp Ser Ser Asp Asp Trp
Glu Ile Pro 20 25 30
4930PRTArtificial SequenceSynthetic Polypeptide 49Glu Glu Arg Ser Val Leu
Asn Asn Gln Leu Leu Glu Met Lys Lys Ser 1 5
10 15 Thr Leu Pro Thr Gln Glu Glu Ile Glu Asn Leu
Pro Ala Phe 20 25 30
5030PRTArtificial SequenceSynthetic Polypeptide 50Arg Lys Glu Glu Glu Gln
Ala Thr Glu Thr Gln Pro Ile Val Tyr Val 1 5
10 15 Tyr Arg Arg Lys His Gln Glu Leu Gln Ala Met
Gln Met Glu 20 25 30
5130PRTArtificial SequenceSynthetic Polypeptide 51Ala Met Pro Tyr Phe Ile
Gln Val Met Lys Glu Tyr Leu Thr Lys Gly 1 5
10 15 Val Tyr Arg Arg Lys His Gln Glu Leu Gln Ala
Met Gln Met 20 25 30
5230PRTArtificial SequenceSynthetic Polypeptide 52Ala Met Pro Tyr Phe Ile
Gln Val Met Lys Glu Tyr Leu Thr Lys Cys 1 5
10 15 Thr Ala Gly Ser Thr Arg Ser Cys Lys Pro Cys
Arg Trp Ser 20 25 30
5330PRTArtificial SequenceSynthetic Polypeptide 53Cys Val Ala Tyr Glu Arg
Gly Gln Cys Asp Leu Glu Leu Ile Asn Leu 1 5
10 15 Pro Val Ser Gly Asn Leu Leu Asp Val Tyr Ser
Ser Gln Gly 20 25 30
5430PRTArtificial SequenceSynthetic Polypeptide 54Gly Lys Ala Gly Glu Arg
Gly Val Pro Gly Pro Pro Gly Ala Val Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
5530PRTArtificial SequenceSynthetic Polypeptide 55Gly Pro Ser Gly Pro Arg
Gly Leu Pro Gly Pro Pro Gly Ala Pro Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
5630PRTArtificial SequenceSynthetic Polypeptide 56Gly Ala Pro Gly Ala Lys
Gly Ala Arg Gly Ser Ala Gly Pro Pro Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
5730PRTArtificial SequenceSynthetic Polypeptide 57Gly Pro Ser Gly Pro Ala
Gly Pro Thr Gly Ala Arg Gly Ala Pro Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
5830PRTArtificial SequenceSynthetic Polypeptide 58Gly Pro Pro Gly Pro Ala
Gly Pro Ala Gly Pro Pro Gly Pro Ile Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
5930PRTArtificial SequenceSynthetic Polypeptide 59Gly Pro Pro Gly Pro Arg
Gly Arg Thr Gly Asp Ala Gly Pro Val Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
6030PRTArtificial SequenceSynthetic Polypeptide 60Gly Pro Ala Gly Pro Pro
Gly Phe Pro Gly Ala Val Gly Ala Lys Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
6130PRTArtificial SequenceSynthetic Polypeptide 61Gly Pro Arg Gly Ala Asn
Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
6230PRTArtificial SequenceSynthetic Polypeptide 62Gly Lys Pro Gly Arg Pro
Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
6330PRTArtificial SequenceSynthetic Polypeptide 63Tyr Asp Glu Lys Ser Thr
Gly Gly Ile Ser Val Pro Gly Pro Met Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
6430PRTArtificial SequenceSynthetic Polypeptide 64Gly Pro Gln Gly Pro Gly
Gly Pro Pro Gly Pro Lys Gly Asn Ser Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
6530PRTArtificial SequenceSynthetic Polypeptide 65Gly Leu Pro Gly Thr Ala
Gly Leu Pro Gly Met Lys Gly His Arg Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
6630PRTArtificial SequenceSynthetic Polypeptide 66Gly Asn Pro Gly Ala Asp
Gly Gln Pro Gly Ala Lys Gly Ala Asn Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
6730PRTArtificial SequenceSynthetic Polypeptide 67Lys Arg His Val Trp Phe
Gly Glu Ser Met Thr Asp Gly Phe Gln Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
6824PRTArtificial SequenceSynthetic Polypeptide 68Arg Thr Cys Arg Asp Leu
Lys Met Cys His Ser Asp Trp Lys Ser Gly 1 5
10 15 Gly Pro His Ser Arg Gly Ala Leu
20 6930PRTArtificial SequenceSynthetic Polypeptide 69Gly
Phe Gln Gly Pro Pro Gly Glu Pro Gly Glu Pro Gly Ala Ser Gly 1
5 10 15 Asp Pro Ile Pro Glu Glu
Leu Tyr Glu Met Leu Ser Asp His 20 25
30 7030PRTArtificial SequenceSynthetic Polypeptide 70Gly Glu
Pro Gly Ser Pro Gly Glu Asn Gly Ala Pro Gly Gln Met Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu
Tyr Glu Met Leu Ser Asp His 20 25
30 7130PRTArtificial SequenceSynthetic Polypeptide 71Gly His Arg
Gly Phe Ser Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr
Glu Met Leu Ser Asp His 20 25
30 7230PRTArtificial SequenceSynthetic Polypeptide 72Gly Pro Ile Gly
Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu
Met Leu Ser Asp His 20 25
30 7330PRTArtificial SequenceSynthetic Polypeptide 73Gly Gln Ala Gly Val
Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met
Leu Ser Asp His 20 25 30
7430PRTArtificial SequenceSynthetic Polypeptide 74Pro Pro Gly Pro Pro Gly
Pro Pro Gly Pro Pro Gly Leu Gly Gly Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
7530PRTArtificial SequenceSynthetic Polypeptide 75Gly Lys Asp Gly Glu Ala
Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
7630PRTArtificial SequenceSynthetic Polypeptide 76Gly Gln Arg Gly Glu Arg
Gly Phe Pro Gly Leu Pro Gly Pro Ser Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
7730PRTArtificial SequenceSynthetic Polypeptide 77Gly Pro Pro Gly Leu Ala
Gly Pro Pro Gly Glu Ser Gly Arg Glu Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
7830PRTArtificial SequenceSynthetic Polypeptide 78Gly Pro Ala Gly Pro Val
Gly Pro Val Gly Ala Arg Gly Pro Ala Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
7930PRTArtificial SequenceSynthetic Polypeptide 79Gly Val Pro Gly Asp Leu
Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
8030PRTArtificial SequenceSynthetic Polypeptide 80Gly Leu Pro Gly Glu Arg
Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
8130PRTArtificial SequenceSynthetic Polypeptide 81Gly Pro Ala Gly Glu Lys
Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
8230PRTArtificial SequenceSynthetic Polypeptide 82Gly Ala Arg Gly Asn Asp
Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
8330PRTArtificial SequenceSynthetic Polypeptide 83Gly Glu Pro Gly Pro Pro
Gly Pro Ala Gly Ala Ala Gly Pro Ala Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
8430PRTArtificial SequenceSynthetic Polypeptide 84Gly Ala Pro Gly Ser Lys
Gly Asp Thr Gly Ala Lys Gly Glu Pro Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
8530PRTArtificial SequenceSynthetic Polypeptide 85Gly Ala Glu Gly Ser Pro
Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
8630PRTArtificial SequenceSynthetic Polypeptide 86Gly Glu Ala Gly Arg Pro
Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
8730PRTArtificial SequenceSynthetic Polypeptide 87Gly Phe Pro Gly Ala Ala
Gly Arg Val Gly Pro Pro Gly Pro Ser Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
8830PRTArtificial SequenceSynthetic Polypeptide 88Gly Pro Ala Gly Pro Pro
Gly Glu Ala Gly Lys Pro Gly Glu Gln Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
8930PRTArtificial SequenceSynthetic Polypeptide 89Gly Glu Gln Gly Pro Ser
Gly Ala Ser Gly Pro Ala Gly Pro Arg Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
9030PRTArtificial SequenceSynthetic Polypeptide 90Gly Pro Val Gly Pro Ala
Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
9130PRTArtificial SequenceSynthetic Polypeptide 91Gly Glu Arg Gly Ala Ala
Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly 1 5
10 15 Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met Leu
Ser Asp His 20 25 30
9219PRTArtificial SequenceSynthetic Polypeptide 92Thr His Gly Gln Glu Glu
Gly Gln Val Glu Gly Gln Asp Glu Asp Arg 1 5
10 15 Trp Thr Trp 9321PRTArtificial
SequenceSynthetic Polypeptide 93Thr His Gly Gln Glu Glu Gly Gln Val Glu
Gly Gln Asp Glu Asp Thr 1 5 10
15 Lys Asn Thr Ile Lys 20 9430PRTArtificial
SequenceSynthetic Polypeptide 94Thr Leu Leu Leu Leu Ala Val Thr Leu Cys
Leu Ala Thr Cys Gln Tyr 1 5 10
15 Trp Pro Lys Trp Glu Gly Leu Asp Ser Thr Leu Phe His Glu
20 25 30 9530PRTArtificial
SequenceSynthetic Polypeptide 95Thr Leu Leu Leu Leu Ala Val Thr Leu Cys
Leu Ala Thr Cys Gln Cys 1 5 10
15 Cys Leu Leu Val Leu Pro Trp Pro Tyr Leu Ala Pro Arg Met
20 25 30 9617PRTArtificial
SequenceSynthetic Polypeptide 96Ala Pro Ser Ala Leu Ser Ser Ser Pro Leu
Leu Thr Ala Pro His Arg 1 5 10
15 Lys 9724PRTArtificial SequenceSynthetic Polypeptide 97Ala
Pro Ser Ala Leu Ser Ser Ser Pro Leu Leu Thr Ala Pro His Val 1
5 10 15 Thr Met Ala Lys Ile Asn
Pro Pro 20 9830PRTArtificial
SequenceSynthetic Polypeptide 98Glu Leu Ser Glu Pro Gly Asp Gly Glu Ala
Leu Met Tyr His Thr Ala 1 5 10
15 Leu Gly Thr Lys Asp His Val Met Thr Pro Asn Arg Ile Ile
20 25 30 9926PRTArtificial
SequenceSynthetic Polypeptide 99Glu Leu Ser Glu Pro Gly Asp Gly Glu Ala
Leu Met Tyr His Thr Ala 1 5 10
15 Leu Gly Thr Lys Asp His Val Met Thr Pro 20
25 10030PRTArtificial SequenceSynthetic Polypeptide
100Thr Arg Ala Phe Glu Gln Leu Met Thr Asp Leu Thr Leu Ser Arg Leu 1
5 10 15 Gln Gly Ser Leu
Lys Arg Lys Gln Val Val Asn Leu Ser Pro 20
25 30 10122PRTArtificial SequenceSynthetic Polypeptide
101Ser Pro Pro Trp Asp Gln Asp Arg Arg Met Met Phe Pro Pro Pro Gly 1
5 10 15 Ile Leu Thr Asn
Val Ile 20 10230PRTArtificial SequenceSynthetic
Polypeptide 102Pro Pro Pro Ser Gly Ile Ala Thr Leu Val Ser Gly Ile Ala
Gly Val 1 5 10 15
Tyr Arg Arg Lys His Gln Glu Leu Gln Ala Met Gln Met Glu 20
25 30 10330PRTArtificial
SequenceSynthetic Polypeptide 103Asp Cys Val Leu Val Leu Leu Leu Met Pro
Arg Leu Ile Cys Lys Cys 1 5 10
15 Thr Ala Gly Ser Thr Arg Ser Cys Lys Pro Cys Arg Trp Ser
20 25 30 10430PRTArtificial
SequenceSynthetic Polypeptide 104Arg His Leu Val Phe Pro Leu Leu Glu Phe
Leu Ser Val Lys Glu Leu 1 5 10
15 Val Met Tyr Gln Ile Pro Phe Ala Arg Val Val Cys Leu Val
20 25 30 10526PRTArtificial
SequenceSynthetic Polypeptide 105Arg His Leu Val Phe Pro Leu Leu Glu Phe
Leu Ser Val Lys Glu Val 1 5 10
15 Arg Gly Gly Glu Met Leu Ile Ala Leu Asn 20
25 10630PRTArtificial SequenceSynthetic Polypeptide
106Asn Lys Tyr Ile Met Ser Asn Ser Gly Asp Tyr Glu Ile Leu Tyr Leu 1
5 10 15 Tyr Arg Arg Lys
His Gln Glu Leu Gln Ala Met Gln Met Glu 20
25 30 10730PRTArtificial SequenceSynthetic Polypeptide
107Ile Leu Arg Gly Thr Phe Asn Asp Gly Phe Gln Ile Glu Val Gln Cys 1
5 10 15 Thr Ala Gly Ser
Thr Arg Ser Cys Lys Pro Cys Arg Trp Ser 20
25 30 10830PRTArtificial SequenceSynthetic Polypeptide
108His Thr Asp Gly Asn Glu Gln Leu Ser Val Met Arg Tyr Ser Ile Val 1
5 10 15 Tyr Arg Arg Lys
His Gln Glu Leu Gln Ala Met Gln Met Glu 20
25 30 10930PRTArtificial SequenceSynthetic Polypeptide
109Val Thr Lys Thr Ala Asp Lys His Lys Asp Val Ile Ile Asn Gln Val 1
5 10 15 Ser Pro Thr Pro
Glu Pro His Leu Pro Leu Ser Leu Ile Leu 20
25 30 11030PRTArtificial SequenceSynthetic Polypeptide
110Ser Glu Asp His Val Ala Ser Val Lys Lys Ser Val Ser Ser Lys Val 1
5 10 15 Tyr Arg Arg Lys
His Gln Glu Leu Gln Ala Met Gln Met Glu 20
25 30 11130PRTArtificial SequenceSynthetic Polypeptide
111Ile Ile Leu Trp Asp His Asp Leu Asn Pro Glu Arg Glu Ile Glu Cys 1
5 10 15 Thr Ala Gly Ser
Thr Arg Ser Cys Lys Pro Cys Arg Trp Ser 20
25 30 11230PRTArtificial SequenceSynthetic Polypeptide
112Trp Ser Lys Thr Thr Val Glu Pro Thr Pro Gly Lys Gly Pro Lys Val 1
5 10 15 Tyr Arg Arg Lys
His Gln Glu Leu Gln Ala Met Gln Met Glu 20
25 30 11330PRTArtificial SequenceSynthetic Polypeptide
113Val Thr Lys Thr Ala Asp Lys His Lys Asp Val Ile Ile Asn Gln Val 1
5 10 15 Tyr Arg Arg Lys
His Gln Glu Leu Gln Ala Met Gln Met Glu 20
25 30 11427PRTArtificial SequenceSynthetic Polypeptide
114Ser Thr Ser Thr Gln Ser Lys Ser Ser Ser Gly Ser Ala His Phe Gly 1
5 10 15 Pro Pro Arg Met
Gln Trp Arg Ser Pro Pro Gly 20 25
11525PRTArtificial SequenceSynthetic Polypeptide 115Glu Cys Glu Gln Ala
Glu Arg Leu Gly Ala Val Asp Glu Ser Leu Arg 1 5
10 15 Lys Tyr Ile Phe Lys Pro Arg Thr Val
20 25 11630PRTArtificial SequenceSynthetic
Polypeptide 116Ala Leu Arg Met Glu Glu Asp Ser Ile Arg Leu Pro Ala His
Leu Pro 1 5 10 15
Lys Asn Arg Gly Ile Ala Ile Pro Val Asp Leu Asp Ser Gln 20
25 30 11730PRTArtificial
SequenceSynthetic Polypeptide 117Ile His Thr Gln Pro Glu Val Ile Leu His
Gln Asn His Glu Glu Gly 1 5 10
15 Arg Lys His Pro Tyr Ser Trp Glu Cys Met Cys Gln Lys Tyr
20 25 30 11830PRTArtificial
SequenceSynthetic Polypeptide 118Ile His Thr Gln Pro Glu Val Ile Leu His
Gln Asn His Glu Glu Val 1 5 10
15 Leu Gln Glu Arg Ile Pro Trp Val Pro Pro Glu Cys Ile Glu
20 25 30 11930PRTArtificial
SequenceSynthetic Polypeptide 119Val Ser Pro Pro Glu Glu His Ala Met Pro
Ile Gly Arg Ile Ala Asp 1 5 10
15 Tyr Glu Leu Leu Thr Glu Asn Asp Met Leu Pro Asn Met Arg
20 25 30 12030PRTArtificial
SequenceSynthetic Polypeptide 120Val Ser Pro Pro Glu Glu His Ala Met Pro
Ile Gly Arg Ile Ala Asp 1 5 10
15 Lys Ser Asn Leu Leu Val Phe Arg Thr Asn Gly Val Ser Asp
20 25 30 12130PRTArtificial
SequenceSynthetic Polypeptide 121Val Ser Pro Pro Glu Glu His Ala Met Pro
Ile Gly Arg Ile Ala Asp 1 5 10
15 Val Gln His Ile Lys Arg Arg Asp Ile Val Leu Lys Arg Glu
20 25 30 12230PRTArtificial
SequenceSynthetic Polypeptide 122Ile His Thr Gln Pro Glu Val Ile Leu His
Gln Asn His Glu Glu Gly 1 5 10
15 Pro Val Ala Val Ile Ser Gly Glu Glu Asp Ser Ala Ser Pro
20 25 30 12330PRTArtificial
SequenceSynthetic Polypeptide 123Val Ser Pro Pro Glu Glu His Ala Met Pro
Ile Gly Arg Ile Ala Gly 1 5 10
15 Pro Val Ala Val Ile Ser Gly Glu Glu Asp Ser Ala Ser Pro
20 25 30 12430PRTArtificial
SequenceSynthetic Polypeptide 124Ile His Thr Gln Pro Glu Val Ile Leu His
Gln Asn His Glu Glu Asp 1 5 10
15 Val Gln His Ile Lys Arg Arg Asp Ile Val Leu Lys Arg Glu
20 25 30 12530PRTArtificial
SequenceSynthetic Polypeptide 125Ala Pro Ser Gln Tyr Ser Gln Gln Ser Ser
Ser Tyr Gly Gln Gln Ile 1 5 10
15 Ala Ile Ala Pro Asn Gly Ala Leu Gln Leu Ala Ser Pro Gly
20 25 30 12629PRTArtificial
SequenceSynthetic Polypeptide 126Ala Pro Ser Gln Tyr Ser Gln Gln Ser Ser
Ser Tyr Gly Gln Gln Lys 1 5 10
15 Lys Leu Leu Glu Asn Val Ala Glu Arg Arg Lys Asn Met
20 25 12718PRTArtificial
SequenceSynthetic Polypeptide 127Gly Met Gly Ser Ala Gly Glu Arg Gly Gly
Phe Asn Lys Pro Gly Glu 1 5 10
15 Lys Phe 12830PRTArtificial SequenceSynthetic Polypeptide
128Gly Gly Met Ser Arg Gly Gly Arg Gly Gly Gly Arg Gly Gly Met Gly 1
5 10 15 Lys Ile Leu Lys
Asp Leu Ser Ser Glu Asp Thr Arg Gly Arg 20
25 30 12930PRTArtificial SequenceSynthetic Polypeptide
129Ala Pro Ser Gln Tyr Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Ile 1
5 10 15 Ala Ile Thr Gln
Gly Gly Ala Ile Gln Leu Ala Asn Asn Gly 20
25 30 13030PRTArtificial SequenceSynthetic Polypeptide
130Asn Lys Pro Gly Gly Pro Met Asp Glu Gly Pro Asp Leu Asp Leu Val 1
5 10 15 Phe Lys Lys Glu
Val Tyr Leu His Thr Ser Pro His Leu Lys 20
25 30 13130PRTArtificial SequenceSynthetic Polypeptide
131Leu Pro Pro Arg Glu Gly Arg Gly Met Pro Pro Pro Leu Arg Gly Val 1
5 10 15 Phe Lys Lys Glu
Val Tyr Leu His Thr Ser Pro His Leu Lys 20
25 30 13230PRTArtificial SequenceSynthetic Polypeptide
132Ala Pro Ser Gln Tyr Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Met 1
5 10 15 Phe Lys Lys Glu
Val Tyr Leu His Thr Ser Pro His Leu Lys 20
25 30 13330PRTArtificial SequenceSynthetic Polypeptide
133Ala Pro Ser Gln Tyr Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Thr 1
5 10 15 Ala Gln Pro Ser
Pro Ser Thr Val Pro Lys Thr Glu Asp Gln 20
25 30 13430PRTArtificial SequenceSynthetic Polypeptide
134Ala Pro Ser Gln Tyr Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Asn 1
5 10 15 Pro Tyr Gln Ile
Leu Gly Pro Thr Ser Ser Arg Leu Ala Asn 20
25 30 13530PRTArtificial SequenceSynthetic Polypeptide
135Asn Lys Pro Gly Gly Pro Met Asp Glu Gly Pro Asp Leu Asp Leu Asp 1
5 10 15 Leu Pro Tyr Glu
Pro Pro Arg Arg Ser Ala Trp Thr Gly His 20
25 30 13630PRTArtificial SequenceSynthetic Polypeptide
136Ala Pro Ser Gln Tyr Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Ser 1
5 10 15 Ser Gly Gln Ile
Gln Leu Trp Gln Phe Leu Leu Glu Leu Leu 20
25 30 13730PRTArtificial SequenceSynthetic Polypeptide
137Ala Pro Ser Gln Tyr Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Asn 1
5 10 15 Leu Pro Tyr Glu
Pro Pro Arg Arg Ser Ala Trp Thr Gly His 20
25 30 13830PRTArtificial SequenceSynthetic Polypeptide
138Ala Pro Ser Gln Tyr Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Arg 1
5 10 15 Asp Ile Lys Gln
Glu Pro Gly Met Tyr Arg Glu Gly Pro Thr 20
25 30 13930PRTArtificial SequenceSynthetic Polypeptide
139Asn Lys Pro Gly Gly Pro Met Asp Glu Gly Pro Asp Leu Asp Leu Asp 1
5 10 15 Pro Val Gly Asp
Gly Leu Phe Lys Asp Gly Lys Asn Pro Ser 20
25 30 14030PRTArtificial SequenceSynthetic Polypeptide
140Ala Pro Ser Gln Tyr Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Asn 1
5 10 15 Pro Val Gly Asp
Gly Leu Phe Lys Asp Gly Lys Asn Pro Ser 20
25 30 14130PRTArtificial SequenceSynthetic Polypeptide
141Gly Gly Met Ser Arg Gly Gly Arg Gly Gly Gly Arg Gly Gly Met Gly 1
5 10 15 Ile Pro Val Thr
Ala Ser Leu Pro Pro Leu Glu Trp Pro Leu 20
25 30 14230PRTArtificial SequenceSynthetic Polypeptide
142Tyr Glu Asp Pro Pro Thr Ala Lys Ala Ala Val Glu Trp Phe Asp Asp 1
5 10 15 Met Pro Cys Val
Gln Ala Gln Tyr Ser Pro Ser Pro Pro Gly 20
25 30 14330PRTArtificial SequenceSynthetic Polypeptide
143Ala Pro Ser Gln Tyr Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Arg 1
5 10 15 Pro Met Asp Glu
Gly Pro Asp Leu Asp Leu Gly Pro Pro Val 20
25 30 14430PRTArtificial SequenceSynthetic Polypeptide
144Gly Met Gly Ser Ala Gly Glu Arg Gly Gly Phe Asn Lys Pro Gly Gly 1
5 10 15 Pro Pro Val Asp
Pro Asp Glu Asp Ser Asp Asn Ser Ala Ile 20
25 30 14530PRTArtificial SequenceSynthetic Polypeptide
145Leu Pro Pro Arg Glu Gly Arg Gly Met Pro Pro Pro Leu Arg Gly Asp 1
5 10 15 Met Pro Cys Val
Gln Ala Gln Tyr Ser Pro Ser Pro Pro Gly 20
25 30 14630PRTArtificial SequenceSynthetic Polypeptide
146Asn Lys Pro Gly Gly Pro Met Asp Glu Gly Pro Asp Leu Asp Leu Asp 1
5 10 15 Met Pro Cys Val
Gln Ala Gln Tyr Ser Pro Ser Pro Pro Gly 20
25 30 14730PRTArtificial SequenceSynthetic Polypeptide
147Tyr Glu Asp Pro Pro Thr Ala Lys Ala Ala Val Glu Trp Phe Asp Glu 1
5 10 15 Pro Thr Ala Glu
Glu Gly Ser Pro Ala Ser Pro Gly Pro Glu 20
25 30 14830PRTArtificial SequenceSynthetic Polypeptide
148Ala Pro Ser Gln Tyr Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Lys 1
5 10 15 Pro Thr Ala Glu
Glu Gly Ser Pro Ala Ser Pro Gly Pro Glu 20
25 30 14929PRTArtificial SequenceSynthetic Polypeptide
149Ala Pro Ser Gln Tyr Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Ser 1
5 10 15 Gly Arg Asp Gly
Ile Ser Thr Ser Lys Arg Gln Lys Ser 20 25
15030PRTArtificial SequenceSynthetic Polypeptide 150Gly Gly
Met Ser Arg Gly Gly Arg Gly Gly Gly Arg Gly Gly Met Gly 1 5
10 15 Arg Lys Arg Arg Asn Phe Asn
Lys Gln Ala Thr Glu Ile Leu 20 25
30 15130PRTArtificial SequenceSynthetic Polypeptide 151Ala Pro Ser
Gln Tyr Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Asn 1 5
10 15 Val Lys Trp Gly Lys Leu Arg Asp
Tyr Gln Val Arg Gly Leu 20 25
30 15230PRTArtificial SequenceSynthetic Polypeptide 152Gly Gly Met Ser
Arg Gly Gly Arg Gly Gly Gly Arg Gly Gly Met Gly 1 5
10 15 Gly Thr Asn Leu Gly Lys Lys Lys Gln
His Ile Cys His Ile 20 25
30 15330PRTArtificial SequenceSynthetic Polypeptide 153Tyr Gly Gln Thr
Ala Tyr Ala Thr Ser Tyr Gly Gln Pro Pro Thr Glu 1 5
10 15 Gly Thr Ser Thr Gly Tyr Thr Thr Pro
Thr Ala Pro Gln Ala 20 25
30 15428PRTArtificial SequenceSynthetic Polypeptide 154Ala Tyr Pro Ala
Tyr Gly Gln Gln Pro Ala Ala Thr Ala Pro Thr Ser 1 5
10 15 Ser Ala Gly Glu Arg Gly Gly Phe Asn
Lys Pro Gly 20 25
15530PRTArtificial SequenceSynthetic Polypeptide 155Ala Tyr Pro Ala Tyr
Gly Gln Gln Pro Ala Ala Thr Ala Pro Thr Ser 1 5
10 15 Tyr Ser Ser Thr Gln Pro Thr Ser Tyr Asp
Gln Ser Ser Tyr 20 25 30
15630PRTArtificial SequenceSynthetic Polypeptide 156Ala Pro Ser Gln Tyr
Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Asn 1 5
10 15 Glu Lys Lys Asp Ile Asp His Glu Thr Val
Val Glu Glu Gln 20 25 30
15722PRTArtificial SequenceSynthetic Polypeptide 157Gly Gly Met Ser Arg
Gly Gly Arg Gly Gly Gly Arg Gly Gly Met Gly 1 5
10 15 Arg His Arg Pro Trp Gly 20
15830PRTArtificial SequenceSynthetic Polypeptide 158Leu Arg Leu
Gln Asp Tyr Glu Glu Lys Thr Lys Lys Ala Glu Arg Asp 1 5
10 15 Asp Phe Trp Ile Pro Glu Thr Ser
Phe Ile Leu Thr Ile Ile 20 25
30 15930PRTArtificial SequenceSynthetic Polypeptide 159Thr Ser Ser Leu
His Gly Ser Ser Leu His Arg Pro Ser Thr Glu Asp 1 5
10 15 Leu Ile Arg Asp Gln Gly Phe Arg Gly
Asp Gly Gly Ser Thr 20 25
30 16025PRTArtificial SequenceSynthetic Polypeptide 160Met Gly Cys Ile
Gly Ser Arg Thr Val Gly Ser Thr Thr Gly Leu Ser 1 5
10 15 Ala Thr Pro Pro Ala Ser Leu Pro Gly
20 25 16130PRTArtificial SequenceSynthetic
Polypeptide 161Ser Thr Glu Gln Thr Arg Thr Asp Phe Ser Trp Asp Gly Ile
Asn Asp 1 5 10 15
Leu Ile Arg Asp Gln Gly Phe Arg Gly Asp Gly Gly Ser Thr 20
25 30 16221PRTArtificial
SequenceSynthetic Polypeptide 162Met Ala Ser Ser Gly Glu Arg Thr Pro Ser
Asn Ala Leu Asp Pro Arg 1 5 10
15 Trp Arg Ser Leu Trp 20 16330PRTArtificial
SequenceSynthetic Polypeptide 163Arg Leu Ser Glu Ala Arg Leu Ser Gln Arg
Asp Leu Ser Pro Thr Asp 1 5 10
15 Leu Ile Arg Asp Gln Gly Phe Arg Gly Asp Gly Gly Ser Thr
20 25 30 16430PRTArtificial
SequenceSynthetic Polypeptide 164Ala Thr Leu Cys Thr Ala Arg Pro Ser Pro
Thr Leu Pro Glu Gln Asp 1 5 10
15 Trp Pro Lys Trp Glu Gly Leu Asp Ser Thr Leu Phe His Glu
20 25 30 16530PRTArtificial
SequenceSynthetic Polypeptide 165Ala Thr Leu Cys Thr Ala Arg Pro Ser Pro
Thr Leu Pro Glu Gln Gly 1 5 10
15 Cys Leu Leu Val Leu Pro Trp Pro Tyr Leu Ala Pro Arg Met
20 25 30 16630PRTArtificial
SequenceSynthetic Polypeptide 166Leu Val Glu Asp Leu Asp Arg Ile Val Ala
Leu Thr Ser Asn Gln Gly 1 5 10
15 Leu Leu Glu Ser Ser Ala Glu Lys Ala Pro Val Ser Val Ser
20 25 30 16730PRTArtificial
SequenceSynthetic Polypeptide 167Ala Arg Asp Ile His His Ile Asp Tyr Tyr
Lys Lys Thr Thr Asn Leu 1 5 10
15 Asp Ala Lys Lys Ser Pro Leu Ala Leu Leu Ala Gln Thr Cys
20 25 30 16830PRTArtificial
SequenceSynthetic Polypeptide 168Leu Val Glu Asp Leu Asp Arg Val Leu Thr
Val Thr Ser Thr Asp Asn 1 5 10
15 Val Met Glu Gln Phe Asn Pro Gly Leu Arg Asn Leu Ile Asn
20 25 30 16930PRTArtificial
SequenceSynthetic Polypeptide 169Leu Val Glu Asp Leu Asp Arg Val Leu Thr
Val Thr Ser Thr Asp Phe 1 5 10
15 Lys Glu Ser Ala Leu Arg Lys Gln Ser Leu Tyr Leu Lys Phe
20 25 30 17030PRTArtificial
SequenceSynthetic Polypeptide 170Leu Val Glu Asp Leu Asp Arg Val Leu Thr
Val Thr Ser Thr Asp Val 1 5 10
15 Pro Gly Pro Pro Pro Gly Val Pro Ala Pro Gly Gly Pro Pro
20 25 30 17130PRTArtificial
SequenceSynthetic Polypeptide 171Leu Val Glu Asp Leu Asp Arg Val Leu Thr
Val Thr Ser Thr Asp Val 1 5 10
15 Lys Ala Thr Gln Glu Glu Asn Arg Glu Leu Arg Ser Arg Cys
20 25 30 17230PRTArtificial
SequenceSynthetic Polypeptide 172Arg Lys Ala Gly Asp Phe His Arg Asn Asp
Ser Ile Tyr Glu Glu Pro 1 5 10
15 Gln Gln Val Val Gln Lys Lys Pro Ala Gln Glu Glu Thr Glu
20 25 30 17330PRTArtificial
SequenceSynthetic Polypeptide 173Tyr Phe Lys Asp Lys Gly Asp Ser Asn Ser
Ser Ala Gly Trp Lys Val 1 5 10
15 Met Gly Leu Leu Thr Asn His Gly Gly Val Pro His Gln Pro
20 25 30 17430PRTArtificial
SequenceSynthetic Polypeptide 174Gln Tyr Asn Ser Ser Ser Gly Gly Gly Gly
Gly Gly Gly Gly Gly Val 1 5 10
15 Ala Ile Ala Pro Asn Gly Ala Leu Gln Leu Ala Ser Pro Gly
20 25 30 17530PRTArtificial
SequenceSynthetic Polypeptide 175Glu Pro Arg Gly Arg Gly Gly Gly Arg Gly
Gly Arg Gly Gly Met Gly 1 5 10
15 Pro Ser Gly Pro Pro Ala Phe Ala Ala His Pro Ser Glu Gln
20 25 30 17625PRTArtificial
SequenceSynthetic Polypeptide 176Glu Pro Arg Gly Arg Gly Gly Gly Arg Gly
Gly Arg Gly Gly Met Gly 1 5 10
15 Asn Cys Arg Asp Gln Ala Leu Trp Ser 20
25 17730PRTArtificial SequenceSynthetic Polypeptide 177Glu Pro
Arg Gly Arg Gly Gly Gly Arg Gly Gly Arg Gly Gly Met Gly 1 5
10 15 Pro Gln Trp Thr Thr Cys Ile
Cys Arg Pro Pro Leu Arg Ala 20 25
30 17830PRTArtificial SequenceSynthetic Polypeptide 178Glu Pro Arg
Gly Arg Gly Gly Gly Arg Gly Gly Arg Gly Gly Met Gly 1 5
10 15 Cys Leu Pro Ser Gly Pro Pro Ala
Phe Ala Ala His Pro Ser 20 25
30 17930PRTArtificial SequenceSynthetic Polypeptide 179Gln Tyr Asn Ser
Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala 1 5
10 15 Pro Val Asp His Leu His Leu Pro Pro
Thr Pro Pro Ser Ser 20 25
30 18030PRTArtificial SequenceSynthetic Polypeptide 180Gln Tyr Asn Ser
Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu 1 5
10 15 Thr Ala Gly Ile Arg Pro Ser Gly Pro
Asp Arg Gly Gly Glu 20 25
30 18130PRTArtificial SequenceSynthetic Polypeptide 181Gly Gly Met Gly
Gly Ser Asp Arg Gly Gly Phe Asn Lys Phe Gly Ala 1 5
10 15 Pro Val Asp His Leu His Leu Pro Pro
Thr Pro Pro Ser Ser 20 25
30 18230PRTArtificial SequenceSynthetic Polypeptide 182Gln Tyr Asn Ser
Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Val 1 5
10 15 Phe Lys Lys Glu Val Tyr Leu His Thr
Ser Pro His Leu Lys 20 25
30 18330PRTArtificial SequenceSynthetic Polypeptide 183Glu Pro Arg Gly
Arg Gly Gly Gly Arg Gly Gly Arg Gly Gly Met Gly 1 5
10 15 Val Gln Glu Gly Ser Val Ser Ser Tyr
Ile Thr Thr Pro Glu 20 25
30 18430PRTArtificial SequenceSynthetic Polypeptide 184Gly Gly Met Gly
Gly Ser Asp Arg Gly Gly Phe Asn Lys Phe Gly Val 1 5
10 15 Phe Lys Lys Glu Val Tyr Leu His Thr
Ser Pro His Leu Lys 20 25
30 18530PRTArtificial SequenceSynthetic Polypeptide 185Gly Tyr Gly Gln
Ser Ser Tyr Ser Ser Tyr Gly Gln Ser Gln Asn Met 1 5
10 15 Phe Lys Lys Glu Val Tyr Leu His Thr
Ser Pro His Leu Lys 20 25
30 18630PRTArtificial SequenceSynthetic Polypeptide 186Phe Asn Arg Gly
Gly Gly Asn Gly Arg Gly Gly Arg Gly Arg Gly Val 1 5
10 15 Phe Lys Lys Glu Val Tyr Leu His Thr
Ser Pro His Leu Lys 20 25
30 18730PRTArtificial SequenceSynthetic Polypeptide 187Ile Glu Ser Val
Ala Asp Tyr Phe Lys Gln Ile Gly Ile Ile Lys Thr 1 5
10 15 Asp Pro Thr Ala Glu Met Ala Ala Glu
Ser Leu Pro Phe Ser 20 25
30 18830PRTArtificial SequenceSynthetic Polypeptide 188Asn Lys Phe Gly
Gly Pro Arg Asp Gln Gly Ser Arg His Asp Ser Val 1 5
10 15 Phe Lys Lys Glu Val Tyr Leu His Thr
Ser Pro His Leu Lys 20 25
30 18927PRTArtificial SequenceSynthetic Polypeptide 189Glu Pro Arg Gly
Arg Gly Gly Gly Arg Gly Gly Arg Gly Gly Met Gly 1 5
10 15 Gly Ser Asp Arg Gly Gly Phe Asn Lys
Phe Gly 20 25 19030PRTArtificial
SequenceSynthetic Polypeptide 190Gly Gly Met Gly Gly Ser Asp Arg Gly Gly
Phe Asn Lys Phe Gly Asp 1 5 10
15 Leu Pro Tyr Glu Pro Pro Arg Arg Ser Ala Trp Thr Gly His
20 25 30 19130PRTArtificial
SequenceSynthetic Polypeptide 191Asn Lys Phe Gly Gly Pro Arg Asp Gln Gly
Ser Arg His Asp Ser Ala 1 5 10
15 Ala Gln Pro Ser Pro Ser Thr Val Pro Lys Thr Glu Asp Gln
20 25 30 19230PRTArtificial
SequenceSynthetic Polypeptide 192Gln Tyr Asn Ser Ser Ser Gly Gly Gly Gly
Gly Gly Gly Gly Gly Asp 1 5 10
15 Leu Pro Tyr Glu Pro Pro Arg Arg Ser Ala Trp Thr Gly His
20 25 30 19330PRTArtificial
SequenceSynthetic Polypeptide 193Gly Gly Met Gly Gly Ser Asp Arg Gly Gly
Phe Asn Lys Phe Gly Asp 1 5 10
15 Pro Tyr Gln Ile Leu Gly Pro Thr Ser Ser Arg Leu Ala Asn
20 25 30 19430PRTArtificial
SequenceSynthetic Polypeptide 194Gly Gly Met Gly Gly Ser Asp Arg Gly Gly
Phe Asn Lys Phe Gly Gly 1 5 10
15 Ser Gly Gln Ile Gln Leu Trp Gln Phe Leu Leu Glu Leu Leu
20 25 30 19530PRTArtificial
SequenceSynthetic Polypeptide 195Glu Pro Arg Gly Arg Gly Gly Gly Arg Gly
Gly Arg Gly Gly Met Gly 1 5 10
15 Gln Trp Pro Asp Pro Ala Leu Ala Val Pro Pro Gly Ala Pro
20 25 30 19630PRTArtificial
SequenceSynthetic Polypeptide 196Phe Asp Asp Pro Pro Ser Ala Lys Ala Ala
Ile Asp Trp Phe Asp Asp 1 5 10
15 Pro Val Gly Asp Gly Leu Phe Lys Asp Gly Lys Asn Pro Ser
20 25 30 19730PRTArtificial
SequenceSynthetic Polypeptide 197Arg Ala Glu Asn Ala Cys Val Pro Pro Phe
Thr Ile Glu Val Lys Lys 1 5 10
15 Thr Leu Gly Arg Arg Asp Ser Ser Asp Asp Trp Glu Ile Pro
20 25 30 19830PRTArtificial
SequenceSynthetic Polypeptide 198Pro Leu Glu Gly Asp Met Ser Ser Pro Asn
Ser Thr Gly Ile Gln Ile 1 5 10
15 Met Phe Glu Thr Phe Asn Thr Pro Ala Met Tyr Val Ala Ile
20 25 30 19930PRTArtificial
SequenceSynthetic Polypeptide 199Met Ile Pro His Pro Gln Ser Arg Gly Pro
Phe Pro Thr Cys Gln Ile 1 5 10
15 Met Phe Glu Thr Phe Asn Thr Pro Ala Met Tyr Val Ala Ile
20 25 30 20022PRTArtificial
SequenceSynthetic Polypeptide 200Gly Lys Pro Arg Asn Val Ala Leu Ile Thr
Gly Ile Thr Gly Gln Pro 1 5 10
15 Leu His Ile Cys Gln Pro 20
20130PRTArtificial SequenceSynthetic Polypeptide 201Glu Asp Gly Glu Lys
Ala Ala Arg Glu Val Lys Leu Leu Leu Leu Gly 1 5
10 15 Ser Thr Thr Gly Leu Ser Ala Thr Pro Pro
Ala Ser Leu Pro 20 25 30
20230PRTArtificial SequenceSynthetic Polypeptide 202Gln Lys Leu Met Gly
Gln Ile His Gln Leu Arg Ser Glu Leu Gln Glu 1 5
10 15 Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn
Leu Val Leu Gly 20 25 30
20330PRTArtificial SequenceSynthetic Polypeptide 203Gly Ala Arg Leu Ala
Ala Lys Tyr Leu Asp Lys Glu Leu Ala Gly Ser 1 5
10 15 Thr Leu Pro Thr Gln Glu Glu Ile Glu Asn
Leu Pro Ala Phe 20 25 30
20430PRTArtificial SequenceSynthetic Polypeptide 204Gly Ala Ser Cys Lys
Asp Thr Ser Gly Glu Ile Lys Val Leu Gln Val 1 5
10 15 Trp His Arg Arg Leu Lys Asn Gln Lys Ser
Ala Lys Glu Gly 20 25 30
20530PRTArtificial SequenceSynthetic Polypeptide 205Leu Ile Asn Ile Met
Ile Glu Pro Gln Ala Thr Arg Lys Ala Gln Asp 1 5
10 15 Ala Ile Arg Ser His Ser Glu Ser Ala Ser
Pro Ser Ala Leu 20 25 30
20630PRTArtificial SequenceSynthetic Polypeptide 206Glu Val Leu Arg Asn
Leu Ser Ser Pro Gly Trp Glu Asn Ile Ser Ser 1 5
10 15 Leu Leu Phe Val Ser Lys Phe Phe Glu His
His Pro Ile Pro 20 25 30
20730PRTArtificial SequenceSynthetic Polypeptide 207Met Thr Val Asp His
Leu Lys Met Leu His Thr Ala Gly Gly Lys Ala 1 5
10 15 Phe Asn Asn Pro Arg Pro Gly Gln Leu Gly
Arg Leu Leu Pro 20 25 30
20830PRTArtificial SequenceSynthetic Polypeptide 208Val Val Ala Ser Thr
Ile Ser Gly Lys Ser Gln Ile Glu Glu Thr Val 1 5
10 15 Tyr Arg Arg Lys His Gln Glu Leu Gln Ala
Met Gln Met Glu 20 25 30
20930PRTArtificial SequenceSynthetic Polypeptide 209Lys Lys His Tyr Glu
Leu Ala Gly Val Ala Glu Gly Trp Glu Glu Val 1 5
10 15 Tyr Arg Arg Lys His Gln Glu Leu Gln Ala
Met Gln Met Glu 20 25 30
21030PRTArtificial SequenceSynthetic Polypeptide 210His Gly Ala Thr Thr
Cys Leu Arg Ala Pro Pro Glu Pro Ala Asp Leu 1 5
10 15 Tyr Arg Arg Lys His Gln Glu Leu Gln Ala
Met Gln Met Glu 20 25 30
21130PRTArtificial SequenceSynthetic Polypeptide 211Ser Asp Ser Ala Gln
Gly Ser Asp Val Ser Leu Thr Ala Cys Lys Asp 1 5
10 15 Asp Phe Trp Ile Pro Glu Thr Ser Phe Ile
Leu Thr Ile Ile 20 25 30
21225PRTArtificial SequenceSynthetic Polypeptide 212Glu Ala Thr Gly Glu
Lys Arg Pro Arg Gly Arg Pro Arg Lys Trp Val 1 5
10 15 Thr Val Lys Val Pro Gln Lys Asn Ser
20 25 21330PRTArtificial SequenceSynthetic
Polypeptide 213Pro Lys Gly Ser Lys Asn Lys Ser Pro Ser Lys Ala Ala Gln
Lys Val 1 5 10 15
His Gln Glu Arg Leu Tyr Gln Glu Tyr Asn Phe Ser Lys Ala 20
25 30 21427PRTArtificial
SequenceSynthetic Polypeptide 214Glu Ala Thr Gly Glu Lys Arg Pro Arg Gly
Arg Pro Arg Lys Trp Asn 1 5 10
15 Phe Phe Arg Leu Asn Tyr Leu Glu Val Cys His 20
25 21530PRTArtificial SequenceSynthetic
Polypeptide 215Glu Ala Thr Gly Glu Lys Arg Pro Arg Gly Arg Pro Arg Lys
Trp Val 1 5 10 15
Val Val Ser Thr Thr Val Asn Val Asp Gly His Val Leu Ala 20
25 30 21630PRTArtificial
SequenceSynthetic Polypeptide 216Glu Ala Thr Gly Glu Lys Arg Pro Arg Gly
Arg Pro Arg Lys Trp Leu 1 5 10
15 Gln Lys His Asp Lys Glu Asp Phe Pro Ala Ser Trp Arg Ser
20 25 30 21730PRTArtificial
SequenceSynthetic Polypeptide 217Glu Ala Thr Gly Glu Lys Arg Pro Arg Gly
Arg Pro Arg Lys Trp Gly 1 5 10
15 Ser Val Lys Ser Ala Gly Pro Thr Thr Ala Arg Glu Gln Val
20 25 30 21820PRTArtificial
SequenceSynthetic Polypeptide 218Pro Arg Lys Trp Pro Gln Gln Val Val Gln
Lys Lys Pro Ala Gln Ser 1 5 10
15 Trp Tyr Leu Gly 20 21920PRTArtificial
SequenceSynthetic Polypeptide 219Glu Ala Thr Gly Glu Lys Arg Pro Arg Gly
Arg Pro Arg Lys Trp Ser 1 5 10
15 Trp Tyr Leu Gly 20 22030PRTArtificial
SequenceSynthetic Polypeptide 220Glu Ala Thr Gly Glu Lys Arg Pro Arg Gly
Arg Pro Arg Lys Trp Val 1 5 10
15 Ile Leu Thr Asn Gln Ile Thr Thr His Leu Ser Gly Ala Leu
20 25 30 22130PRTArtificial
SequenceSynthetic Polypeptide 221Glu Ala Thr Gly Glu Lys Arg Pro Arg Gly
Arg Pro Arg Lys Trp Leu 1 5 10
15 Ser Ala Ser Leu Ala Val Val His Met Glu Pro Ala Met Asn
20 25 30 22224PRTArtificial
SequenceSynthetic Polypeptide 222Glu Ala Thr Gly Glu Lys Arg Pro Arg Gly
Arg Pro Arg Lys Trp Gly 1 5 10
15 Thr Lys Pro Ala Ser Tyr Met Pro 20
22330PRTArtificial SequenceSynthetic Polypeptide 223Glu Ala Thr Gly
Glu Lys Arg Pro Arg Gly Arg Pro Arg Lys Trp Ala 1 5
10 15 Glu Tyr Phe Tyr Glu Phe Leu Ser Leu
Arg Ser Leu Asp Lys 20 25
30 22430PRTArtificial SequenceSynthetic Polypeptide 224Glu Ala Thr Gly
Glu Lys Arg Pro Arg Gly Arg Pro Arg Lys Trp Leu 1 5
10 15 Phe Lys Leu Val Ser His Val Leu Glu
Asn Arg Met Gly Trp 20 25
30 22524PRTArtificial SequenceSynthetic Polypeptide 225Pro Arg Lys Trp
Pro Gln Gln Val Val Gln Lys Lys Pro Ala Gln Gly 1 5
10 15 Thr Lys Pro Ala Ser Tyr Met Pro
20 22617PRTArtificial SequenceSynthetic
Polypeptide 226Met Glu Ser Gln Arg Gly Arg Asn Cys Asn Glu Lys Pro Thr
Asn Val 1 5 10 15
Arg 22730PRTArtificial SequenceSynthetic Polypeptide 227Lys Ala Asn Ala
Ala Arg Ser Gln Leu Glu Thr Tyr Lys Arg Gln Glu 1 5
10 15 Asp Pro Lys Trp Glu Phe Pro Arg Lys
Asn Leu Val Leu Gly 20 25
30 22830PRTArtificial SequenceSynthetic Polypeptide 228Ser Arg Ser Pro
Pro Ala Glu Asn Glu Val Ser Thr Pro Met Gln Lys 1 5
10 15 Lys Lys Gly Gly Arg Gly Leu Met Thr
Glu Asn Thr Met Arg 20 25
30 22930PRTArtificial SequenceSynthetic Polypeptide 229Ser Val Glu Gly
Val Val Arg Ile Leu Leu Glu His Tyr Tyr Lys Ala 1 5
10 15 Trp Lys Lys Arg Trp Phe Ile Leu Arg
Ser Gly Arg Met Ser 20 25
30 23030PRTArtificial SequenceSynthetic Polypeptide 230Ser Phe Arg Ala
Ile Ile Arg Asp Leu Asn Ser Leu Phe Thr Pro Asp 1 5
10 15 Cys Arg Leu Leu Trp Asp Tyr Val Tyr
Gln Leu Leu Ser Asp 20 25
30 23130PRTArtificial SequenceSynthetic Polypeptide 231Asn Ile Ile Phe
Gln Phe Thr Lys Cys Cys Pro Pro Lys Pro Lys Glu 1 5
10 15 Thr Gly Ser Asp Phe Ser Met Phe Glu
Ala Leu Arg Asp Thr 20 25
30 23230PRTArtificial SequenceSynthetic Polypeptide 232Ser Pro Lys Asp
Phe Asn Lys Tyr Phe Leu Thr Phe Ala Val Glu Glu 1 5
10 15 Ala Glu Ser Gly Asn Ile Ser Gln Lys
Ser Asp Glu Glu Asp 20 25
30 23327PRTArtificial SequenceSynthetic Polypeptide 233Pro Pro Ile Thr
Pro Ser Ser Ser Phe Arg Ser Ser Thr Pro Thr Gly 1 5
10 15 Pro Pro Arg Met Gln Trp Arg Ser Pro
Pro Gly 20 25 23430PRTArtificial
SequenceSynthetic Polypeptide 234Pro Pro Ile Thr Pro Ser Ser Ser Phe Arg
Ser Ser Thr Pro Thr Glu 1 5 10
15 Pro Thr Gln Ile Tyr Arg Phe Leu Arg Thr Arg Asn Leu Ile
20 25 30 23530PRTArtificial
SequenceSynthetic Polypeptide 235Asp Leu Ala Glu Ile Gly Ile Ala Val Gly
Asn Asn Asp Val Lys Glu 1 5 10
15 Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn Leu Val Leu Gly
20 25 30 23630PRTArtificial
SequenceSynthetic Polypeptide 236Val Arg Ser Lys Asn Met Ala Arg Arg Gly
His Ser Ala Gln Ile Asp 1 5 10
15 Pro Leu Cys Asp Glu Leu Cys Arg Thr Val Ile Ala Ala Ala
20 25 30 23730PRTArtificial
SequenceSynthetic Polypeptide 237Leu Arg Lys Leu Phe Val Gln Asp Leu Ala
Thr Arg Val Lys Lys Glu 1 5 10
15 Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn Leu Val Leu Gly
20 25 30 23830PRTArtificial
SequenceSynthetic Polypeptide 238Phe Leu Glu Asn Asn Leu Glu Gln Leu Thr
Lys Val His Lys Gln Glu 1 5 10
15 Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn Leu Val Leu Gly
20 25 30 23930PRTArtificial
SequenceSynthetic Polypeptide 239Val Arg Ser Lys Asn Met Ala Arg Arg Gly
His Ser Ala Gln Ile Asp 1 5 10
15 Val Ala Glu Glu Ala Gly Cys Pro Leu Ser Cys Ala Val Ser
20 25 30 24030PRTArtificial
SequenceSynthetic Polypeptide 240Glu Asn Glu Lys Glu Leu Ala Ala Cys Gln
Leu Arg Ile Ser Gln Glu 1 5 10
15 Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn Leu Val Leu Gly
20 25 30 24130PRTArtificial
SequenceSynthetic Polypeptide 241Glu Ile Leu Thr Arg Ala His Glu Arg Glu
Phe Gly Ser Val Asp Val 1 5 10
15 Tyr Arg Arg Lys His Gln Glu Leu Gln Ala Met Gln Met Glu
20 25 30 24230PRTArtificial
SequenceSynthetic Polypeptide 242Thr Leu Glu Gly Glu Leu His Asp Leu Arg
Gly Gln Val Ala Lys Thr 1 5 10
15 Leu Thr Ala His Leu Glu Thr Arg Trp Arg Arg Arg Thr Lys
20 25 30 24330PRTArtificial
SequenceSynthetic Polypeptide 243Thr Ser Ser Gly Ala Gly Phe Phe Leu Pro
Gln His Asp Ser Ser Val 1 5 10
15 Trp His Arg Arg Leu Lys Asn Gln Lys Ser Ala Lys Glu Gly
20 25 30 24430PRTArtificial
SequenceSynthetic Polypeptide 244Arg Gly Gly Arg Gly Gly Arg Glu Phe Ala
Asp Phe Glu Tyr Arg Asp 1 5 10
15 Leu Ile Arg Asp Gln Gly Phe Arg Gly Asp Gly Gly Ser Thr
20 25 30 24530PRTArtificial
SequenceSynthetic Polypeptide 245Arg Lys Asp Glu Glu Leu Thr Ser Ser Gln
Arg Asp Leu Ala Val Arg 1 5 10
15 Asp Leu Lys Leu Asp Asn Ile Leu Leu Asp Ala Glu Gly His
20 25 30 24630PRTArtificial
SequenceSynthetic Polypeptide 246Gln Lys Lys Lys Ala Lys Ser Gln Gln Tyr
Lys Gly His Lys Lys Arg 1 5 10
15 Thr Gly Trp Ala Pro Pro Thr Phe Leu Leu Tyr Gln Phe Ala
20 25 30 24730PRTArtificial
SequenceSynthetic Polypeptide 247Ser His His Ser Ser His Lys Lys Arg Lys
Asn Lys Asn Arg His Arg 1 5 10
15 Pro Pro Gln Asp Ala Met Ala Gln Pro Pro Arg Leu Ser Arg
20 25 30 24830PRTArtificial
SequenceSynthetic Polypeptide 248Val Leu His Pro Met Asp Ala Ala Gln Arg
Ser Gln His Ile Lys Lys 1 5 10
15 Thr Leu Gly Arg Arg Asp Ser Ser Asp Asp Trp Glu Ile Pro
20 25 30 24930PRTArtificial
SequenceSynthetic Polypeptide 249Lys Asn Leu Leu Glu Phe Ala Glu Thr Leu
Gln Phe Ile Asp Ser Phe 1 5 10
15 Leu Asn Thr Ser Ser Asn His Glu Asn Ser Asp Leu Glu Met
20 25 30 25020PRTArtificial
SequenceSynthetic Polypeptide 250Ala Phe Thr Val Pro Lys Asn Arg Ser Leu
Ala Ser Pro Leu Gln Ser 1 5 10
15 Trp Tyr Leu Gly 20 25130PRTArtificial
SequenceSynthetic Polypeptide 251Ala Phe Thr Val Pro Lys Asn Arg Ser Leu
Ala Ser Pro Leu Gln Pro 1 5 10
15 Asn Gly Ser Gly Gln Val Val Gly Lys Val Pro Gly His Phe
20 25 30 25230PRTArtificial
SequenceSynthetic Polypeptide 252Ala Phe Thr Val Pro Lys Asn Arg Ser Leu
Ala Ser Pro Leu Gln Ser 1 5 10
15 Ser Gln Leu Glu Leu His Leu His Leu His Arg Cys His Phe
20 25 30 25330PRTArtificial
SequenceSynthetic Polypeptide 253Leu Met Ser Thr Glu Asn Glu Leu Lys Gly
Gln Gln Val Leu Pro Leu 1 5 10
15 Arg Ile Cys Asp Trp Thr Met Asn Gln Asn Gly Arg His Leu
20 25 30 25430PRTArtificial
SequenceSynthetic Polypeptide 254Ala Phe Thr Val Pro Lys Asn Arg Ser Leu
Ala Ser Pro Leu Gln Pro 1 5 10
15 Thr Gln Pro Gln Ala His Leu Lys Pro Ile Asp Met Trp Asp
20 25 30 25530PRTArtificial
SequenceSynthetic Polypeptide 255Ala Phe Thr Val Pro Lys Asn Arg Ser Leu
Ala Ser Pro Leu Gln Leu 1 5 10
15 Arg Ile Cys Asp Trp Thr Met Asn Gln Asn Gly Arg His Leu
20 25 30 25630PRTArtificial
SequenceSynthetic Polypeptide 256Leu Met Ser Thr Glu Asn Glu Leu Lys Gly
Gln Gln Val Leu Pro Pro 1 5 10
15 Asn Gly Ser Gly Gln Val Val Gly Lys Val Pro Gly His Phe
20 25 30 25720PRTArtificial
SequenceSynthetic Polypeptide 257Phe Gln Glu Asn Gly Pro Pro Leu Leu Lys
Lys Ile Lys Gln Glu Ser 1 5 10
15 Trp Tyr Leu Gly 20 25830PRTArtificial
SequenceSynthetic Polypeptide 258Phe Gln Glu Asn Gly Pro Pro Leu Leu Lys
Lys Ile Lys Gln Glu Leu 1 5 10
15 Arg Ile Cys Asp Trp Thr Met Asn Gln Asn Gly Arg His Leu
20 25 30 25920PRTArtificial
SequenceSynthetic Polypeptide 259Leu Met Ser Thr Glu Asn Glu Leu Lys Gly
Gln Gln Val Leu Pro Ser 1 5 10
15 Trp Tyr Leu Gly 20 26030PRTArtificial
SequenceSynthetic Polypeptide 260Glu Ala Leu Asn His Arg Ile Val Gln Gln
Ala Lys Glu Met Thr Val 1 5 10
15 Trp His Arg Arg Leu Lys Asn Gln Lys Ser Ala Lys Glu Gly
20 25 30 26130PRTArtificial
SequenceSynthetic Polypeptide 261Phe Asp Ser Lys Arg Arg Glu Gly Lys Gln
Leu Ser Leu His Glu Ala 1 5 10
15 Thr Ser Lys Ser Gln Ile Met Ser Leu Trp Gly Leu Val Ser
20 25 30 26230PRTArtificial
SequenceSynthetic Polypeptide 262Asp Ser Ala Ser Leu Ser Gly Glu Ser Leu
Asp Gly His Leu Gln Ala 1 5 10
15 Glu Gln Met Gly Lys Asp Gly Arg Gly Tyr Val Pro Ala Thr
20 25 30 26330PRTArtificial
SequenceSynthetic Polypeptide 263Gln Val Ala Arg Glu Ser Thr Tyr Leu Ser
Ser Leu Lys Gly Ser Arg 1 5 10
15 Gln Pro Pro Ser Pro Arg Ser Cys Leu Cys Gly Val Trp Ser
20 25 30 26430PRTArtificial
SequenceSynthetic Polypeptide 264Gln Val Ala Arg Glu Ser Thr Tyr Leu Ser
Ser Leu Lys Gly Ser Arg 1 5 10
15 Pro Gln Val Tyr Pro Pro His Ser His Ser Ile Pro Pro Tyr
20 25 30 26530PRTArtificial
SequenceSynthetic Polypeptide 265Asp Ser Ala Ser Leu Ser Gly Glu Ser Leu
Asp Gly His Leu Gln Gly 1 5 10
15 Thr Asn His Phe Leu Pro Gln Ser Ser Arg Cys Leu Pro Trp
20 25 30 26630PRTArtificial
SequenceSynthetic Polypeptide 266Pro Glu Glu Leu Gly Gly Pro Pro Leu Lys
Lys Leu Lys Gln Glu Gly 1 5 10
15 Pro Thr Thr Ser Tyr Pro Arg Ala Pro Asp Ala Tyr His Gly
20 25 30 26720PRTArtificial
SequenceSynthetic Polypeptide 267Phe Asp Ser Lys Arg Arg Glu Gly Lys Gln
Leu Ser Leu His Glu Cys 1 5 10
15 Leu Cys Thr Ala 20 26830PRTArtificial
SequenceSynthetic Polypeptide 268Gln Val Ala Arg Glu Ser Thr Tyr Leu Ser
Ser Leu Lys Gly Ser Arg 1 5 10
15 Asp Gln Pro Leu Pro Thr Pro Glu Leu Gln Met Pro Thr Met
20 25 30 26929PRTArtificial
SequenceSynthetic Polypeptide 269Pro Glu Glu Leu Gly Gly Pro Pro Leu Lys
Lys Leu Lys Gln Glu Gly 1 5 10
15 Val Pro Gly Arg Leu Arg Arg Leu Leu Leu Gln Leu Gly
20 25 27030PRTArtificial
SequenceSynthetic Polypeptide 270Gln Val Ala Arg Glu Ser Thr Tyr Leu Ser
Ser Leu Lys Gly Ser Arg 1 5 10
15 Pro His Leu Gln Met Pro Pro Ser Leu Gly Gln Met Ser Leu
20 25 30 27130PRTArtificial
SequenceSynthetic Polypeptide 271Asp Ser Ala Ser Leu Ser Gly Glu Ser Leu
Asp Gly His Leu Gln Gly 1 5 10
15 Ser Pro Gln Ile Glu Asn Ile Gln Pro Phe Ser Ala Lys Asp
20 25 30 27216PRTArtificial
SequenceSynthetic Polypeptide 272Gln Val Ala Arg Glu Ser Thr Tyr Leu Ser
Ser Leu Lys Gly Ser Ser 1 5 10
15 27330PRTArtificial SequenceSynthetic Polypeptide 273Pro Glu
Glu Leu Gly Gly Pro Pro Leu Lys Lys Leu Lys Gln Glu Ala 1 5
10 15 Thr Ser Lys Ser Gln Ile Met
Ser Leu Trp Gly Leu Val Ser 20 25
30 27430PRTArtificial SequenceSynthetic Polypeptide 274Pro Glu Glu
Leu Gly Gly Pro Pro Leu Lys Lys Leu Lys Gln Glu Ser 1 5
10 15 Ile Tyr Gln Arg Asp Pro Leu Lys
Leu Val Ala Thr Phe Arg 20 25
30 27530PRTArtificial SequenceSynthetic Polypeptide 275Phe Asp Ser Lys
Arg Arg Glu Gly Lys Gln Leu Ser Leu His Glu Phe 1 5
10 15 Arg His Leu Pro Met Pro Phe His Trp
Lys Gln Glu Glu Leu 20 25
30 27630PRTArtificial SequenceSynthetic Polypeptide 276Val Gly Arg Leu
Ser Pro Cys Val Pro Ala Lys Pro Pro Leu Ala Ala 1 5
10 15 Glu Gln Met Gly Lys Asp Gly Arg Gly
Tyr Val Pro Ala Thr 20 25
30 27730PRTArtificial SequenceSynthetic Polypeptide 277Val Gly Arg Leu
Ser Pro Cys Val Pro Ala Lys Pro Pro Leu Ala Gly 1 5
10 15 Asn Leu Gln Val Pro Asp His Val Ser
Val Gly Ser Gly Leu 20 25
30 27830PRTArtificial SequenceSynthetic Polypeptide 278Val Gly Arg Leu
Ser Pro Cys Val Pro Ala Lys Pro Pro Leu Ala Gly 1 5
10 15 Ser Pro Gln Ile Glu Asn Ile Gln Pro
Phe Ser Ala Lys Asp 20 25
30 27930PRTArtificial SequenceSynthetic Polypeptide 279Pro Glu Glu Leu
Gly Gly Pro Pro Leu Lys Lys Leu Lys Gln Glu Phe 1 5
10 15 Leu Phe Ser Val Ser Ser Glu Leu Gln
Gly Gly Thr Trp Val 20 25
30 28030PRTArtificial SequenceSynthetic Polypeptide 280Val Gly Arg Leu
Ser Pro Cys Val Pro Ala Lys Pro Pro Leu Ala Gly 1 5
10 15 Thr Asn His Phe Leu Pro Gln Ser Ser
Arg Cys Leu Pro Trp 20 25
30 28130PRTArtificial SequenceSynthetic Polypeptide 281Pro Ser Arg Lys
Pro Leu Asp Ser Arg Val Leu Asn Ala Val Lys Tyr 1 5
10 15 Tyr Gly Thr Ala Ala Asn Asp Ile Gly
Asp Thr Thr Asn Arg 20 25
30 28230PRTArtificial SequenceSynthetic Polypeptide 282Lys Gly Glu Thr
Ile Thr Gly Leu Leu Gln Glu Phe Asp Val Gln Glu 1 5
10 15 Ala Leu Ser Val Val Ser Glu Asp Gln
Ser Leu Phe Glu Cys 20 25
30 28330PRTArtificial SequenceSynthetic Polypeptide 283Asp Val Asp Leu
Ala Glu Val Lys Pro Leu Val Glu Lys Gly Glu Glu 1 5
10 15 Ala Leu Ser Val Val Ser Glu Asp Gln
Ser Leu Phe Glu Cys 20 25
30 28430PRTArtificial SequenceSynthetic Polypeptide 284Pro Ile Ala Met
Ala Leu Ala Asn Val Val Pro Cys Ser Gln Trp Val 1 5
10 15 Phe Thr Lys Tyr Gly Lys Cys Tyr Met
Phe Asn Ser Gly Glu 20 25
30 28530PRTArtificial SequenceSynthetic Polypeptide 285Pro Pro Thr Trp
Pro Lys Pro Leu Val Pro Ala Ile Pro Ile Cys Ser 1 5
10 15 Ser Ala Gly Glu Arg Gly Gly Phe Asn
Lys Pro Gly Gly Pro 20 25
30 28630PRTArtificial SequenceSynthetic Polypeptide 286Met Arg Arg Gln
Gln Glu Gly Phe Lys Gly Thr Phe Pro Asp Ala Leu 1 5
10 15 Pro Val Ser Gly Asn Leu Leu Asp Val
Tyr Ser Ser Gln Gly 20 25
30 28730PRTArtificial SequenceSynthetic Polypeptide 287Gly Glu Val Asp
Ala Asp Cys Met Asp Val Asn Val Arg Gly Pro Ala 1 5
10 15 Gly Pro Ser Arg Thr Gly Tyr Leu His
Pro Pro Ser Pro Gly 20 25
30 28830PRTArtificial SequenceSynthetic Polypeptide 288Lys Cys Gly Ser
Gly Pro Val His Ile Ser Gly Gln His Leu Val Val 1 5
10 15 Tyr Arg Arg Lys His Gln Glu Leu Gln
Ala Met Gln Met Glu 20 25
30 28930PRTArtificial SequenceSynthetic Polypeptide 289Val Ala Pro Pro
Thr Thr Ala Ala Ser Ser Val Glu Glu Pro Glu Gly 1 5
10 15 His Arg Glu Ser Asn Ser Arg Leu Pro
Gly Pro Pro Glu Gly 20 25
30 29030PRTArtificial SequenceSynthetic Polypeptide 290Phe Met Asp Asn
Pro Phe Glu Phe Asn Pro Glu Asp Pro Ile Pro Asp 1 5
10 15 Lys Leu Lys Cys Thr Lys Glu Glu His
Leu Cys Thr Gln Arg 20 25
30 29130PRTArtificial SequenceSynthetic Polypeptide 291Pro Gln Tyr Phe
Arg Gln Gly His Asn Cys His Lys Pro Asp Thr Tyr 1 5
10 15 Cys Arg Leu Leu Trp Asp Tyr Val Tyr
Gln Leu Leu Ser Asp 20 25
30 29218PRTArtificial SequenceSynthetic Polypeptide 292Met Asp Arg Asp
Leu Ser Tyr Asn Leu Leu Glu Asp Leu Pro Ser Phe 1 5
10 15 Ser Val 29330PRTArtificial
SequenceSynthetic Polypeptide 293Ser Asn Pro Met Asn Pro Thr Ile Gly Asn
Gly Leu Ser Pro Gln Asn 1 5 10
15 Ser Ile Arg His Asn Leu Ser Leu His Ser Lys Phe Ile Arg
20 25 30 29430PRTArtificial
SequenceSynthetic Polypeptide 294Ser Asn Pro Met Asn Pro Thr Ile Gly Asn
Gly Leu Ser Pro Gln Phe 1 5 10
15 Thr Ala Asp Leu Asp Gln Phe Asp Gln Leu Leu Pro Thr Leu
20 25 30 29530PRTArtificial
SequenceSynthetic Polypeptide 295Tyr Gln Leu Ser Glu Thr Ser Tyr Gln Pro
Thr Ser Ile Pro Gln Gly 1 5 10
15 Leu Pro Glu Leu Glu Leu Glu Ala Ile Asp Asn Gln Phe Gly
20 25 30 29630PRTArtificial
SequenceSynthetic Polypeptide 296Ser Asn Pro Met Asn Pro Thr Ile Gly Asn
Gly Leu Ser Pro Gln Pro 1 5 10
15 Gly Ser Glu Leu Asp Asn Leu Glu Glu Ile Leu Asp Asp Leu
20 25 30 29730PRTArtificial
SequenceSynthetic Polypeptide 297Pro Ser Asn His Met Asn Pro Val Ser Asn
Gly Leu Ser Pro Gln Asn 1 5 10
15 Ser Ile Arg His Asn Leu Ser Leu His Ser Lys Phe Ile Arg
20 25 30 29830PRTArtificial
SequenceSynthetic Polypeptide 298Leu Gly Arg Asn Leu Ser Thr His Gln Thr
Tyr Pro Val Val Ala Gly 1 5 10
15 Arg Glu Met Val Gly Pro Thr Leu Pro Gly Tyr Pro Pro His
20 25 30 29930PRTArtificial
SequenceSynthetic Polypeptide 299Ala Gly Ser Pro Asp Thr Glu Ser Pro Val
Leu Val Asn Asp Tyr Arg 1 5 10
15 Glu Asn Val Ile Glu Tyr Lys His Cys Leu Ile Thr Lys Asn
20 25 30 30030PRTArtificial
SequenceSynthetic Polypeptide 300Ala Gly Ser Pro Asp Thr Glu Ser Pro Val
Leu Val Asn Asp Tyr Glu 1 5 10
15 Ile Glu Leu Ser Ser Leu Arg Glu Ala Leu Ser Phe Val Ser
20 25 30 30130PRTArtificial
SequenceSynthetic Polypeptide 301Leu Glu Lys Ile Ile Lys Cys Asn Arg Ser
Thr Glu Ile Ser Ser Val 1 5 10
15 Leu Gln Glu Arg Ile Pro Trp Val Pro Pro Glu Cys Ile Glu
20 25 30 30230PRTArtificial
SequenceSynthetic Polypeptide 302Lys Lys Arg Asn Ser Thr Gln Leu Lys Ser
Arg Val Lys Asn Ile Asn 1 5 10
15 Lys Ser Asn Leu Leu Val Phe Arg Thr Asn Gly Val Ser Asp
20 25 30 30330PRTArtificial
SequenceSynthetic Polypeptide 303Asn Ser Glu Leu Thr Pro Ser Glu Ser Leu
Ala Thr Thr Asp Asp Glu 1 5 10
15 Ile Glu Leu Ser Ser Leu Arg Glu Ala Leu Ser Phe Val Ser
20 25 30 30430PRTArtificial
SequenceSynthetic Polypeptide 304Arg Gln Ile Lys Ala Ile Met Lys Glu Val
Ile Pro Phe Leu Lys Glu 1 5 10
15 Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn Leu Val Leu Gly
20 25 30 30521PRTArtificial
SequenceSynthetic Polypeptide 305Gly Val Ala Cys Glu Pro Leu Pro Asp Arg
Tyr Thr Val Ser Glu Glu 1 5 10
15 Ser Asn Gly Trp Lys 20 30630PRTArtificial
SequenceSynthetic Polypeptide 306Leu Gln Pro Met Ala Gly Thr Cys Pro Ala
Pro Glu Ile His Ala Ile 1 5 10
15 Glu Arg Leu Glu Val Ser Ser Leu Ala Gln Thr Ser Ser Ala
20 25 30 30730PRTArtificial
SequenceSynthetic Polypeptide 307Asp Ser Glu Arg Leu Gln Tyr Glu Lys Lys
Leu Lys Ser Thr Lys Cys 1 5 10
15 Thr Ala Gly Ser Thr Arg Ser Cys Lys Pro Cys Arg Trp Ser
20 25 30 30830PRTArtificial
SequenceSynthetic Polypeptide 308Tyr Lys Lys Met Gln Asp Thr Val Val Leu
Ala Gln Gly Lys Lys Val 1 5 10
15 Tyr Arg Arg Lys His Gln Glu Leu Gln Ala Met Gln Met Glu
20 25 30 30930PRTArtificial
SequenceSynthetic Polypeptide 309Leu Val Cys Lys Met Lys Gly Glu Gly Val
Glu Ile Val Asp Arg Val 1 5 10
15 Trp His Arg Arg Leu Lys Asn Gln Lys Ser Ala Lys Glu Gly
20 25 30 31030PRTArtificial
SequenceSynthetic Polypeptide 310Lys Glu Pro Val Lys Ile Ala Ala Pro Glu
Leu His Lys Gly Asp Val 1 5 10
15 Gln Thr His Leu Glu Asn Pro Thr Arg Tyr His Leu Gln Gln
20 25 30 31130PRTArtificial
SequenceSynthetic Polypeptide 311Pro Gln Glu Ile Ala Pro Asp Ala Ser Phe
Ile Asp Asp Glu Ala Val 1 5 10
15 Gln Thr His Leu Glu Asn Pro Thr Arg Tyr His Leu Gln Gln
20 25 30 31230PRTArtificial
SequenceSynthetic Polypeptide 312Asp Ser Glu Glu Asp Glu Pro Thr Lys Lys
Lys Thr Ile Leu Gln Leu 1 5 10
15 Pro Val Ser Gly Asn Leu Leu Asp Val Tyr Ser Ser Gln Gly
20 25 30 31330PRTArtificial
SequenceSynthetic Polypeptide 313Lys Glu Pro Val Lys Ile Ala Ala Pro Glu
Leu His Lys Gly Asp Ile 1 5 10
15 Asp Asp Val Ile Asp Glu Ile Ile Ser Leu Glu Ser Ser Tyr
20 25 30 31430PRTArtificial
SequenceSynthetic Polypeptide 314Lys Leu Trp Gly Ile Asp Arg Asp Ser Tyr
Arg Arg Ile Leu Met Glu 1 5 10
15 Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn Leu Val Leu Gly
20 25 30 31530PRTArtificial
SequenceSynthetic Polypeptide 315Ser Pro Trp Pro Leu Leu Gly Ser Ala Gln
Gly Gln Phe Ser Ala Val 1 5 10
15 His Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys Ala Thr
20 25 30 31630PRTArtificial
SequenceSynthetic Polypeptide 316Thr Gly Leu Pro Gly Pro Pro Leu Ile Thr
Arg Thr Lys Cys Ala Val 1 5 10
15 His Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys Ala Thr
20 25 30 31730PRTArtificial
SequenceSynthetic Polypeptide 317Phe Gly Glu Lys Leu Phe Ser Gly Val Leu
Met Asp Leu Ser Lys Ser 1 5 10
15 Thr Leu Pro Thr Gln Glu Glu Ile Glu Asn Leu Pro Ala Phe
20 25 30 31830PRTArtificial
SequenceSynthetic Polypeptide 318Ala Thr Ser Ile Leu Glu Tyr Pro Ile Glu
Pro Ser Gly Val Leu Gly 1 5 10
15 Pro Ala Ser Val Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro
20 25 30 31930PRTArtificial
SequenceSynthetic Polypeptide 319Glu Ala Ser Ser Leu Lys Tyr Leu Ala Glu
Glu Phe Ser Ile Pro Glu 1 5 10
15 Pro Thr Gly Glu Pro Ser Pro Lys Arg Pro Arg Gly Arg Pro
20 25 30 32030PRTArtificial
SequenceSynthetic Polypeptide 320Glu Ala Ser Ser Leu Lys Tyr Leu Ala Glu
Glu Phe Ser Ile Pro Lys 1 5 10
15 Ala Glu Ala Thr Gly Glu Lys Arg Pro Arg Gly Arg Pro Arg
20 25 30 32130PRTArtificial
SequenceSynthetic Polypeptide 321His Met Val Ser Thr Thr Leu Pro Val Asp
Ser Arg Met Ile Glu Ser 1 5 10
15 Thr Ala Asn Pro Glu Thr Pro Asn Ser Thr Ile Ser Arg Glu
20 25 30 32230PRTArtificial
SequenceSynthetic Polypeptide 322Asp Gly Tyr Gln Gly Ser Gln Thr Phe His
Gly Ala Pro Leu Thr Val 1 5 10
15 Tyr Arg Arg Lys His Gln Glu Leu Gln Ala Met Gln Met Glu
20 25 30 32316PRTArtificial
SequenceSynthetic Polypeptide 323Ala Ser Tyr Asp Asp Pro Tyr Lys Lys Ala
Val Ala Met Ser Lys Arg 1 5 10
15 32430PRTArtificial SequenceSynthetic Polypeptide 324Asp Ser
Met Glu Asn Gln Val Ser Val Asp Ala Phe Lys Ile Leu Asp 1 5
10 15 Met Glu Ala Gln Gln Val Asn
Glu Ala Glu Ser Ala Arg Glu 20 25
30 32530PRTArtificial SequenceSynthetic Polypeptide 325Asp Ser Met
Glu Asn Gln Val Ser Val Asp Ala Phe Lys Ile Leu Lys 1 5
10 15 Cys Glu Arg Leu Leu Leu Tyr Leu
Tyr Cys His Glu Leu Ser 20 25
30 32630PRTArtificial SequenceSynthetic Polypeptide 326Glu His Arg Lys
Glu Leu Gly Pro Tyr Val Phe Arg Glu Ala Gln Val 1 5
10 15 Asn Lys Leu Glu Leu Glu Leu Glu Ser
Ala Lys Gln Lys Phe 20 25
30 32730PRTArtificial SequenceSynthetic Polypeptide 327Phe Ile Gln Lys
Ile Arg Tyr Thr Asn Ala Arg Asp Arg Asn Gln Asp 1 5
10 15 Leu Ile Arg Asp Gln Gly Phe Arg Gly
Asp Gly Gly Ser Thr 20 25
30 32830PRTArtificial SequenceSynthetic Polypeptide 328Thr Val Gln Gly
Ser Asn Ile Phe Glu Arg Thr Glu Val Leu Ala Asp 1 5
10 15 Asp Phe Trp Ile Pro Glu Thr Ser Phe
Ile Leu Thr Ile Ile 20 25
30 32930PRTArtificial SequenceSynthetic Polypeptide 329Thr Val Gln Gly
Ser Asn Ile Phe Glu Arg Thr Glu Val Leu Ala Ala 1 5
10 15 Gly Val Pro Asn Lys Pro Gly Ile Pro
Lys Leu Leu Glu Gly 20 25
30 33030PRTArtificial SequenceSynthetic Polypeptide 330Gly Gln Glu Ser
Asp Asp Phe Glu Leu Ser Gly Ser Gly Asp Leu Asp 1 5
10 15 Asp Phe Trp Ile Pro Glu Thr Ser Phe
Ile Leu Thr Ile Ile 20 25
30 33130PRTArtificial SequenceSynthetic Polypeptide 331Gly Gln Glu Ser
Asp Asp Phe Glu Leu Ser Gly Ser Gly Asp Leu Val 1 5
10 15 Trp His Arg Arg Leu Lys Asn Gln Lys
Ser Ala Lys Glu Gly 20 25
30 33230PRTArtificial SequenceSynthetic Polypeptide 332Gly Gln Glu Ser
Asp Asp Phe Glu Leu Ser Gly Ser Gly Asp Leu Ala 1 5
10 15 Gly Val Pro Asn Lys Pro Gly Ile Pro
Lys Leu Leu Glu Gly 20 25
30 33330PRTArtificial SequenceSynthetic Polypeptide 333Pro Gly His Met
His Thr Gln Val Pro Pro Tyr Pro Gln Pro Gln Leu 1 5
10 15 Tyr Arg Arg Lys His Gln Glu Leu Gln
Ala Met Gln Met Glu 20 25
30 33430PRTArtificial SequenceSynthetic Polypeptide 334Gly Thr Leu Pro
Ala Ala Ser Glu Leu Pro Ala Ser Gln Arg Thr Val 1 5
10 15 Tyr Arg Arg Lys His Gln Glu Leu Gln
Ala Met Gln Met Glu 20 25
30 33530PRTArtificial SequenceSynthetic Polypeptide 335Gly Thr Leu Pro
Ala Ala Ser Glu Leu Pro Ala Ser Gln Arg Thr Val 1 5
10 15 Leu Gln Glu Arg Ile Pro Trp Val Pro
Pro Glu Cys Ile Glu 20 25
30 33625PRTArtificial SequenceSynthetic Polypeptide 336Met Ala Ala Thr
Asp Leu Glu Arg Phe Ser Asn Ser Tyr Asn Asn Ser 1 5
10 15 Gln Ala Pro Ser Pro Gly Leu Gly Ser
20 25 33730PRTArtificial SequenceSynthetic
Polypeptide 337Val Pro Pro Ala Thr Met Ser Gly Ser Met Met Gly Ser Asp
Met Leu 1 5 10 15
Pro Val Ser Gly Asn Leu Leu Asp Val Tyr Ser Ser Gln Gly 20
25 30 33830PRTArtificial
SequenceSynthetic Polypeptide 338Val Pro Pro Ala Thr Met Ser Gly Ser Met
Met Gly Ser Asp Met Ile 1 5 10
15 Asp Asp Val Ile Asp Glu Ile Ile Ser Leu Glu Ser Ser Tyr
20 25 30 33930PRTArtificial
SequenceSynthetic Polypeptide 339Arg Gln Arg Glu Glu Ser Tyr Ser Arg Met
Gly Tyr Met Asp Pro Ile 1 5 10
15 Asp Asp Val Ile Asp Glu Ile Ile Ser Leu Glu Ser Ser Tyr
20 25 30 34030PRTArtificial
SequenceSynthetic Polypeptide 340Leu Glu Asp Met Arg Ser Asn Asn Val Glu
Asp Cys Lys Met Met Val 1 5 10
15 Ile His Pro Lys Thr Asp Glu Gln Arg Cys Arg Leu Gln Glu
20 25 30 34130PRTArtificial
SequenceSynthetic Polypeptide 341Val Cys Ser Leu Asp Ile Leu Ser Ser Ala
Phe Gln Leu Val Gly Asp 1 5 10
15 Asp Phe Trp Ile Pro Glu Thr Ser Phe Ile Leu Thr Ile Ile
20 25 30 34230PRTArtificial
SequenceSynthetic Polypeptide 342Val Cys Ser Leu Asp Ile Leu Ser Ser Ala
Phe Gln Leu Val Gly Ala 1 5 10
15 Gly Val Pro Asn Lys Pro Gly Ile Pro Lys Leu Leu Glu Gly
20 25 30 34330PRTArtificial
SequenceSynthetic Polypeptide 343Ile Leu Pro Tyr Thr Leu Ala Ser Leu Tyr
His Arg Glu Lys Gln Leu 1 5 10
15 Ile Ala Met Asp Ser Ala Ile Thr Leu Trp Gln Phe Leu Leu
20 25 30 34430PRTArtificial
SequenceSynthetic Polypeptide 344Leu Ser Ser Ile Arg Pro Pro Arg Leu Glu
Gly Glu Asn Thr Gln Asp 1 5 10
15 Leu Ile Arg Asp Gln Gly Phe Arg Gly Asp Gly Gly Ser Thr
20 25 30 34530PRTArtificial
SequenceSynthetic Polypeptide 345Leu Ser Ser Ile Arg Pro Pro Arg Leu Glu
Gly Glu Asn Thr Gln Lys 1 5 10
15 Thr Leu Gly Arg Arg Asp Ser Ser Asp Asp Trp Glu Ile Pro
20 25 30 34630PRTArtificial
SequenceSynthetic Polypeptide 346Pro Thr Ala Asn Leu Asp Gln Lys Asp Lys
Gln Phe Val Ala Lys Asp 1 5 10
15 Leu Ile Arg Asp Gln Gly Phe Arg Gly Asp Gly Gly Ser Thr
20 25 30 34730PRTArtificial
SequenceSynthetic Polypeptide 347Ser Lys Lys Glu Val Pro Ile His Arg Val
Ala Asp Ile Ser Gly Lys 1 5 10
15 Thr Leu Gly Arg Arg Asp Ser Ser Asp Asp Trp Glu Ile Pro
20 25 30 34830PRTArtificial
SequenceSynthetic Polypeptide 348Ser Lys Lys Glu Val Pro Ile His Arg Val
Ala Asp Ile Ser Gly Asp 1 5 10
15 Leu Ile Arg Asp Gln Gly Phe Arg Gly Asp Gly Gly Ser Thr
20 25 30 34930PRTArtificial
SequenceSynthetic Polypeptide 349Tyr Ser His Lys Leu Phe Asn Gly Ser Met
Glu Ala Phe Ile Lys Arg 1 5 10
15 Pro Arg Gly Gln Arg Asp Ser Ser Tyr Tyr Trp Glu Ile Glu
20 25 30 35030PRTArtificial
SequenceSynthetic Polypeptide 350Lys Gln Thr Leu Gly Glu Gly Glu Arg Ala
Glu Cys Gly Thr Thr Arg 1 5 10
15 Met Gln Phe Glu Val Thr Ala Asn Gln Pro His Leu Gln Pro
20 25 30 35130PRTArtificial
SequenceSynthetic Polypeptide 351Gly Pro Pro Gln Pro Pro Gln Gln Arg Pro
Tyr Gly Tyr Asp Gln Glu 1 5 10
15 Trp Val Ser Lys Ser Pro Ser His Leu Ser Cys Val Ile Asn
20 25 30 35220PRTArtificial
SequenceSynthetic Polypeptide 352Gly Pro Pro Gln Pro Pro Gln Gln Arg Pro
Tyr Gly Tyr Asp Gln Leu 1 5 10
15 Asn Ile Leu Arg 20 35330PRTArtificial
SequenceSynthetic Polypeptide 353Gln Tyr Pro Gly Gln Gln Gly Tyr Pro Gly
Gln Gln Gln Gly Tyr Val 1 5 10
15 Glu His Pro Gln Met Thr Phe Gly Arg Leu His Arg Ile Ile
20 25 30 35430PRTArtificial
SequenceSynthetic Polypeptide 354Gln Tyr Pro Gly Gln Gln Gly Tyr Pro Gly
Gln Gln Gln Gly Tyr Gly 1 5 10
15 Phe Lys Val Thr Leu Pro Pro Phe Met Cys Asn Lys Gln Ala
20 25 30 35530PRTArtificial
SequenceSynthetic Polypeptide 355Gly Pro Pro Gln Pro Pro Gln Gln Arg Pro
Tyr Gly Tyr Asp Gln Ile 1 5 10
15 Met Pro Lys Lys Pro Ala Glu Asp Glu Asn Asp Ser Lys Gly
20 25 30 35630PRTArtificial
SequenceSynthetic Polypeptide 356Thr Ala Pro Ser Ala Gln Gln Gln Arg Pro
Tyr Gly Tyr Glu Gln Ile 1 5 10
15 Met Pro Lys Lys Pro Ala Glu Asp Glu Asn Asp Ser Lys Gly
20 25 30 35730PRTArtificial
SequenceSynthetic Polypeptide 357Gly Asp Gly Met Pro Val Gly Pro Val Pro
Pro Gly Phe Phe Gln Arg 1 5 10
15 Glu Asn Val Ile Glu Tyr Lys His Cys Leu Ile Thr Lys Asn
20 25 30 35830PRTArtificial
SequenceSynthetic Polypeptide 358Glu Thr Cys Glu His Ser Ser Glu Ala Lys
Ala Phe His Asp Tyr Arg 1 5 10
15 Glu Asn Val Ile Glu Tyr Lys His Cys Leu Ile Thr Lys Asn
20 25 30 35926PRTArtificial
SequenceSynthetic Polypeptide 359Asn Ile Thr Leu Gly Glu Pro Pro Gly Phe
Leu His Ser Trp Trp Cys 1 5 10
15 Glu Lys Met Ser Leu Asn Ile Asn Thr Val 20
25 36023PRTArtificial SequenceSynthetic Polypeptide
360His Pro Gln Met Thr Phe Gly Arg Leu His Arg Ile Ile Pro Lys Gly 1
5 10 15 Gln Tyr Gly Asn
Tyr Gln Gln 20 36130PRTArtificial
SequenceSynthetic Polypeptide 361Tyr Pro Lys Lys Pro Lys Asp Glu Ala Phe
Arg Ser His Tyr Lys Gln 1 5 10
15 Phe Glu Glu Gly Leu Leu Asp Arg Cys Pro Ala Pro Gly Pro
20 25 30 36230PRTArtificial
SequenceSynthetic Polypeptide 362Thr Glu Leu Asn Gln Gly Asp Met Lys Pro
Pro Ser Tyr Asp Ser Val 1 5 10
15 Tyr Arg Arg Lys His Gln Glu Leu Gln Ala Met Gln Met Glu
20 25 30 36321PRTArtificial
SequenceSynthetic Polypeptide 363Cys Thr Glu Lys Gly Thr Trp Arg Glu Ser
Thr Leu Thr Cys Thr Val 1 5 10
15 Leu Leu Gln Ile Leu 20 36430PRTArtificial
SequenceSynthetic Polypeptide 364Arg Phe Ala Lys Lys Met Asp Lys Met Val
Gln Lys Lys Asn Ala Ile 1 5 10
15 Gly Gln Asn Gly Lys Asp Trp Ile Pro Leu Ser Ser Thr Lys
20 25 30 36530PRTArtificial
SequenceSynthetic Polypeptide 365His Glu Gly Leu Ser Pro Thr Pro Phe Met
Asn Ser Asn Leu Met Asp 1 5 10
15 Met Pro Cys Val Gln Ala Gln Tyr Ser Pro Ser Pro Pro Gly
20 25 30 36617PRTArtificial
SequenceSynthetic Polypeptide 366Gly Tyr Lys Ile Leu Ile Pro Lys Gly Ser
Tyr Gly Arg Val Lys Val 1 5 10
15 Ser 36730PRTArtificial SequenceSynthetic Polypeptide 367Ser
Tyr Leu Pro Gly Gly Thr Thr Gly Leu Gln Leu Pro Ser Thr Pro 1
5 10 15 Val Asp Arg Glu Pro Val
Asp Arg Glu Pro Val Val Cys His 20 25
30 36830PRTArtificial SequenceSynthetic Polypeptide 368Ser Pro
Met Ala Leu Leu Thr Ile Gly Ser Ser Ser Glu Lys Glu Pro 1 5
10 15 Val Asp Arg Glu Pro Val Asp
Arg Glu Pro Val Val Cys His 20 25
30 36930PRTArtificial SequenceSynthetic Polypeptide 369Gln Pro Pro
Tyr Thr Gly Ala Gln Thr Gln Ala Gly Gln Ile Glu Val 1 5
10 15 Tyr Arg Arg Lys His Gln Glu Leu
Gln Ala Met Gln Met Glu 20 25
30 37030PRTArtificial SequenceSynthetic Polypeptide 370Lys Asn Val Met
Ser Ala Phe Gly Leu Thr Asp Asp Gln Val Ser Val 1 5
10 15 Tyr Arg Arg Lys His Gln Glu Leu Gln
Ala Met Gln Met Glu 20 25
30 37130PRTArtificial SequenceSynthetic Polypeptide 371Glu Pro Pro Gly
Glu Pro Gly Pro Ser Thr Asn Ile Pro Glu Asn Val 1 5
10 15 Tyr Arg Arg Lys His Gln Glu Leu Gln
Ala Met Gln Met Glu 20 25
30 37230PRTArtificial SequenceSynthetic Polypeptide 372Gln Ala Pro Pro
Gln Gln Pro Gln Gln Tyr Gly Ile Gln Tyr Ser Asp 1 5
10 15 Met Pro Cys Val Gln Ala Gln Tyr Ser
Pro Ser Pro Pro Gly 20 25
30 37330PRTArtificial SequenceSynthetic Polypeptide 373Lys Asn Val Met
Ser Ala Phe Gly Leu Thr Asp Asp Gln Val Ser Asp 1 5
10 15 Thr Asn Ser Thr Ser Gly Asp Pro Val
Glu Lys Lys Asp Glu 20 25
30 37420PRTArtificial SequenceSynthetic Polypeptide 374Met Ala Leu Asn
Ser Glu Ala Leu Ser Val Val Ser Glu Asp Gln Ser 1 5
10 15 Leu Phe Glu Cys 20
37518PRTArtificial SequenceSynthetic Polypeptide 375Met Ala Leu Asn Ser
Pro Ser Gly Ser Glu Gln Leu Val Asp Gly Leu 1 5
10 15 Ala Tyr 37630PRTArtificial
SequenceSynthetic Polypeptide 376Phe Leu Val Gly Ala Ala Leu Ala Ala Gly
Leu Leu Trp Lys Phe Arg 1 5 10
15 Thr Leu Leu Met Asn Ala Val Trp Pro Lys Ala Gly Arg Trp
20 25 30 37722PRTArtificial
SequenceSynthetic Polypeptide 377Phe Leu Val Gly Ala Ala Leu Ala Ala Gly
Leu Leu Trp Lys Phe Arg 1 5 10
15 Ser Leu Ile Ser Cys Glu 20
37822PRTArtificial SequenceSynthetic Polypeptide 378Asp Gly Val Ser His
Cys Pro Gly Gly Glu Asp Glu Asn Arg Cys Gly 1 5
10 15 Ser Leu Ile Ser Cys Glu 20
37918PRTArtificial SequenceSynthetic Polypeptide 379Met Ala Leu
Asn Ser Val Ile Pro Gly Ser Leu Glu Thr Arg Gly Lys 1 5
10 15 Pro Cys 38022PRTArtificial
SequenceSynthetic Polypeptide 380Val Cys Thr Gln Pro Lys Ser Pro Ser Gly
Thr Val Cys Thr Ser Arg 1 5 10
15 Ser Leu Ile Ser Cys Glu 20
38130PRTArtificial SequenceSynthetic Polypeptide 381Val Cys Thr Gln Pro
Lys Ser Pro Ser Gly Thr Val Cys Thr Ser Ser 1 5
10 15 Tyr Ser Arg Ile Phe Gly Asp Pro Arg Lys
Ala Val Leu Thr 20 25 30
38220PRTArtificial SequenceSynthetic Polypeptide 382Met Ala Leu Asn Ser
Leu Arg Tyr Leu Thr Met Met Ser Ser Leu Tyr 1 5
10 15 Gln Thr Ile Arg 20
38330PRTArtificial SequenceSynthetic Polypeptide 383Val Cys Thr Gln Pro
Lys Ser Pro Ser Gly Thr Val Cys Thr Ser Arg 1 5
10 15 Gly Ser Leu Phe Pro Gln Lys Leu Leu Asn
Ala Glu Thr Ser 20 25 30
38430PRTArtificial SequenceSynthetic Polypeptide 384Lys Gly Glu Pro His
His Glu Leu Pro Pro Gly Ser Thr Lys Arg Val 1 5
10 15 Pro Ala Ser Val Gln Leu His Thr Ala Val
Glu Met His His 20 25 30
38530PRTArtificial SequenceSynthetic Polypeptide 385Asp Ala Gln Ala Gly
Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser 1 5
10 15 Pro Gly Gln Cys Ala Ala Ala His Gly Gly
Gly Asp Ala Pro 20 25 30
38630PRTArtificial SequenceSynthetic Polypeptide 386Gln Pro Lys Lys Lys
Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ser 1 5
10 15 Arg Pro Val Cys Ser Cys Thr Arg Arg Trp
Arg Cys Thr Thr 20 25 30
38730PRTArtificial SequenceSynthetic Polypeptide 387Glu Arg Ser Val Ala
Lys Leu Glu Lys Thr Ile Asp Asp Leu Glu Val 1 5
10 15 Tyr Arg Arg Lys His Gln Glu Leu Gln Ala
Met Gln Met Glu 20 25 30
38830PRTArtificial SequenceSynthetic Polypeptide 388Glu Arg Ser Val Ala
Lys Leu Glu Lys Thr Ile Asp Asp Leu Glu Asp 1 5
10 15 Thr Asn Ser Thr Ser Gly Asp Pro Val Glu
Lys Lys Asp Glu 20 25 30
38930PRTArtificial SequenceSynthetic Polypeptide 389Glu Arg Ser Val Ala
Lys Leu Glu Lys Thr Ile Asp Asp Leu Glu Val 1 5
10 15 Trp His Arg Arg Leu Lys Asn Gln Lys Ser
Ala Lys Glu Gly 20 25 30
39030PRTArtificial SequenceSynthetic Polypeptide 390Leu Gln Lys Leu Glu
Glu Ala Glu Lys Ala Ala Asp Glu Ser Glu Ser 1 5
10 15 Thr Leu Pro Thr Gln Glu Glu Ile Glu Asn
Leu Pro Ala Phe 20 25 30
39130PRTArtificial SequenceSynthetic Polypeptide 391Pro Arg Met Gln Gly
Pro Ile Gln Gln Pro Ser Ile Ser His Gln Glu 1 5
10 15 Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn
Leu Val Leu Gly 20 25 30
39230PRTArtificial SequenceSynthetic Polypeptide 392Gln Leu Glu Glu Lys
Gln Gln Gln Pro Thr Arg Glu Leu Leu Gln Glu 1 5
10 15 Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn
Leu Val Leu Gly 20 25 30
39330PRTArtificial SequenceSynthetic Polypeptide 393Lys Lys Gly Lys Thr
Ala Gln Gly Leu Ser Pro Val Asp Gln Arg Glu 1 5
10 15 Asp Pro Lys Trp Glu Phe Pro Arg Lys Asn
Leu Val Leu Gly 20 25 30
39430PRTArtificial SequenceSynthetic Polypeptide 394Val Ile Ser Ala Glu
Asn Trp Lys Pro Ala Thr Lys Thr Asp Gln Gly 1 5
10 15 Leu Leu Lys Met Thr Glu Tyr Lys Leu Val
Val Val Gly Ala 20 25 30
39530PRTArtificial SequenceSynthetic Polypeptide 395Arg Asn Glu Leu Leu
Gly Asp Asp Gly Asn Ser Ser Glu Asn Gln Ser 1 5
10 15 Asn Lys Val Pro Val Val Gln His Pro His
His Val His Pro 20 25 30
39630PRTArtificial SequenceSynthetic Polypeptide 396Met Arg Ser Tyr Lys
Gln Glu Met Gly Lys Leu Glu Thr Asp Phe Ser 1 5
10 15 Asn Lys Val Pro Val Val Gln His Pro His
His Val His Pro 20 25 30
39730PRTArtificial SequenceSynthetic Polypeptide 397Met Arg Ser Tyr Lys
Gln Glu Met Gly Lys Leu Glu Thr Asp Phe Tyr 1 5
10 15 Leu Gln Met Lys Trp Pro Leu Leu Asp Val
Gln Ala Gly Ser 20 25 30
39830PRTArtificial SequenceSynthetic Polypeptide 398Met Val Ala Asn Val
Glu Lys Gln Leu Glu Glu Ala Lys Glu Leu Ser 1 5
10 15 Asn Lys Val Pro Val Val Gln His Pro His
His Val His Pro 20 25 30
39930PRTArtificial SequenceSynthetic Polypeptide 399Leu Arg Asp Asn Leu
Thr Leu Trp Thr Ser Asp Met Gln Gly Asp Ala 1 5
10 15 Tyr Pro Ala Leu Gly Pro Gly Val Thr Ala
Asn Pro Gly Thr 20 25 30
40030PRTArtificial SequenceSynthetic Polypeptide 400Leu Arg Asp Asn Leu
Thr Leu Trp Thr Ser Asp Met Gln Gly Asp Ala 1 5
10 15 Tyr Pro Val Leu Gly Pro Gly Val Thr Ala
Asn Pro Gly Thr 20 25 30
40130PRTArtificial SequenceSynthetic Polypeptide 401Ile Lys Thr Leu Glu
Gly Glu Phe Ser Val Thr Met Trp Ser Ser Gly 1 5
10 15 Pro Met Asp Glu Gly Pro Asp Leu Asp Leu
Gly Pro Pro Val 20 25 30
40230PRTArtificial SequenceSynthetic Polypeptide 402Phe Gly Ser Thr Arg
Gly Ser Leu Asp Lys Pro Asp Ser Phe Met Gly 1 5
10 15 Glu Tyr Ser Val Gly Asn Lys His Arg Asp
Pro Phe Glu Ala 20 25 30
40330PRTArtificial SequenceSynthetic Polypeptide 403Gln Cys Lys Thr Glu
Thr Gln Glu Ser Gln Ala Phe Gln Glu Arg Gly 1 5
10 15 Ser Thr Thr Gly Leu Ser Ala Thr Pro Pro
Ala Ser Leu Pro 20 25 30
40421PRTArtificial SequenceSynthetic Polypeptide 404Val Trp Arg Ile Thr
Gly Thr Asp Gly Val Lys Lys Lys Met Thr Leu 1 5
10 15 Gln Trp Ala Ala Val 20
40545PRTArtificial SequenceSynthetic Polypeptide 405Arg Leu Met Leu Glu
Leu Gly Phe Ser Lys Val Leu Gly Asp Arg Glu 1 5
10 15 Val Gln Ser Arg Trp Ser Pro Gly Pro Arg
Gly Asp Ser Thr Pro Val 20 25
30 Arg Glu Met Glu Thr Asn His Pro Pro Ser Val Arg Gly
35 40 45 40642PRTArtificial
SequenceSynthetic Polypeptide 406Thr Tyr Leu Leu Asp Phe Arg Ser Ile Asp
Glu Trp Leu Pro Leu Gly 1 5 10
15 Gly Arg Gly Lys Leu Val Arg Arg Ser Gln Arg Ser Leu Pro Ser
Ser 20 25 30 Leu
Lys Leu Lys Leu Leu Met Arg Arg Met 35 40
407216PRTArtificial SequenceSynthetic Polypeptide 407Leu Tyr Thr Leu
Asn Lys His Gln Arg Phe Gly Trp Thr Arg Cys Ser 1 5
10 15 Glu Ala Gly Val Gln Thr Gln His Val
Arg Arg Ser Gly Val Gly Pro 20 25
30 Thr Pro Arg Gly Met Gly Ile Arg Val Gln Pro Gly Val Asp
Ala Gly 35 40 45
Arg Gly Leu Cys Arg Val Gln Ala Ala Gly Leu Arg Pro Cys Arg Gly 50
55 60 Arg Pro Gln Val Cys
Pro Ala Ala Trp Arg Asp Gly Pro Ala Leu Ala 65 70
75 80 Ser Gln Arg Val Leu Pro Gly Gln Arg Val
Leu Pro Leu Gly Val Gln 85 90
95 Pro Trp Leu Met Val Pro Glu Pro Val Pro Pro Arg Met Gly Gly
Gly 100 105 110 Arg
Ala Val Leu Gln Arg His Phe Pro Gly Ala Pro Ala Gly Glu Gly 115
120 125 Pro Gln Ser Leu Arg Gly
Thr Pro Arg Asp Gln Lys Arg Glu Trp Gly 130 135
140 Gly Pro Pro Leu Cys Pro Ala Cys Gly Gly Gly
His Gly Val Leu Pro 145 150 155
160 Gly Ala Gly Val Arg Pro Cys Ala Pro Gly Pro Pro Pro Gly Arg Gly
165 170 175 Arg Arg
Gln Glu Val Pro Gly Leu Gln Gly Tyr Arg Ala His His Pro 180
185 190 Gln Leu Gln Thr Gly Gln Gln
Leu Pro Gln Arg Leu Arg Pro Ala Ala 195 200
205 Gly Arg Arg Gly Gly Leu Leu Arg 210
215 4081235PRTArtificial SequenceSynthetic Polypeptide 408Met
His Arg Ala Pro Ser Pro Thr Ala Glu Gln Pro Pro Gly Gly Gly 1
5 10 15 Asp Ser Ala Arg Arg Thr
Leu Gln Pro Arg Leu Lys Pro Ser Ala Arg 20
25 30 Ala Met Ala Leu Pro Arg Thr Leu Gly Glu
Leu Gln Leu Tyr Arg Val 35 40
45 Leu Gln Arg Ala Asn Leu Leu Ser Tyr Tyr Glu Thr Phe Ile
Gln Gln 50 55 60
Gly Gly Asp Asp Val Gln Gln Leu Cys Glu Ala Gly Glu Glu Glu Phe 65
70 75 80 Leu Glu Ile Met Ala
Leu Val Gly Met Ala Thr Lys Pro Leu His Val 85
90 95 Arg Arg Leu Gln Lys Ala Leu Arg Glu Trp
Ala Thr Asn Pro Gly Leu 100 105
110 Phe Ser Gln Pro Val Pro Ala Val Pro Val Ser Ser Ile Pro Leu
Phe 115 120 125 Lys
Ile Ser Glu Thr Ala Gly Thr Arg Lys Gly Ser Met Ser Asn Gly 130
135 140 His Gly Ser Pro Gly Glu
Lys Ala Gly Ser Ala Arg Ser Phe Ser Pro 145 150
155 160 Lys Ser Pro Leu Glu Leu Gly Glu Lys Leu Ser
Pro Leu Pro Gly Gly 165 170
175 Pro Gly Ala Gly Asp Pro Arg Ile Trp Pro Gly Arg Ser Thr Pro Glu
180 185 190 Ser Asp
Val Gly Ala Gly Gly Glu Glu Glu Ala Gly Ser Pro Pro Phe 195
200 205 Ser Pro Pro Ala Gly Gly Gly
Val Pro Glu Gly Thr Gly Ala Gly Gly 210 215
220 Leu Ala Ala Gly Gly Thr Gly Gly Gly Pro Asp Arg
Leu Glu Pro Glu 225 230 235
240 Met Val Arg Met Val Val Glu Ser Val Glu Arg Ile Phe Arg Ser Phe
245 250 255 Pro Arg Gly
Asp Ala Gly Glu Val Thr Ser Leu Leu Lys Leu Asn Lys 260
265 270 Lys Leu Ala Arg Ser Val Gly His
Ile Phe Glu Met Asp Asp Asn Asp 275 280
285 Ser Gln Lys Glu Glu Glu Ile Arg Lys Tyr Ser Ile Ile
Tyr Gly Arg 290 295 300
Phe Asp Ser Lys Arg Arg Glu Gly Lys Gln Leu Ser Leu His Glu Leu 305
310 315 320 Thr Ile Asn Glu
Ala Ala Ala Gln Phe Cys Met Arg Asp Asn Thr Leu 325
330 335 Leu Leu Arg Arg Val Glu Leu Phe Ser
Leu Ser Arg Gln Val Ala Arg 340 345
350 Glu Ser Thr Tyr Leu Ser Ser Leu Lys Gly Ser Arg Leu His
Pro Glu 355 360 365
Glu Leu Gly Gly Pro Pro Leu Lys Lys Leu Lys Gln Glu Ala Thr Ser 370
375 380 Lys Ser Gln Ile Met
Ser Leu Trp Gly Leu Val Ser Lys Met Pro Pro 385 390
395 400 Glu Lys Val Gln Arg Leu Tyr Val Asp Phe
Pro Gln His Leu Arg His 405 410
415 Leu Leu Gly Asp Trp Leu Glu Ser Gln Pro Trp Glu Phe Leu Val
Gly 420 425 430 Ser
Asp Ala Phe Cys Cys Asn Leu Ala Ser Ala Leu Leu Ser Asp Thr 435
440 445 Val Gln His Leu Gln Ala
Ser Val Gly Glu Gln Gly Glu Gly Ser Thr 450 455
460 Ile Leu Gln His Ile Ser Thr Leu Glu Ser Ile
Tyr Gln Arg Asp Pro 465 470 475
480 Leu Lys Leu Val Ala Thr Phe Arg Gln Ile Leu Gln Gly Glu Lys Lys
485 490 495 Ala Val
Met Glu Gln Phe Arg His Leu Pro Met Pro Phe His Trp Lys 500
505 510 Gln Glu Glu Leu Lys Phe Lys
Thr Gly Leu Arg Arg Leu Gln His Arg 515 520
525 Val Gly Glu Ile His Leu Leu Arg Glu Ala Leu Gln
Lys Gly Ala Glu 530 535 540
Ala Gly Gln Val Ser Leu His Ser Leu Ile Glu Thr Pro Ala Asn Gly 545
550 555 560 Thr Gly Pro
Ser Glu Ala Leu Ala Met Leu Leu Gln Glu Thr Thr Gly 565
570 575 Glu Leu Glu Ala Ala Lys Ala Leu
Val Leu Lys Arg Ile Gln Ile Trp 580 585
590 Lys Arg Gln Gln Gln Leu Ala Gly Asn Gly Ala Pro Phe
Glu Glu Ser 595 600 605
Leu Ala Pro Leu Gln Glu Arg Cys Glu Ser Leu Val Asp Ile Tyr Ser 610
615 620 Gln Leu Gln Gln
Glu Val Gly Ala Ala Gly Gly Glu Leu Glu Pro Lys 625 630
635 640 Thr Arg Ala Ser Leu Thr Gly Arg Leu
Asp Glu Val Leu Arg Thr Leu 645 650
655 Val Thr Ser Cys Phe Leu Val Glu Lys Gln Pro Pro Gln Val
Leu Lys 660 665 670
Thr Gln Thr Lys Phe Gln Ala Gly Val Arg Phe Leu Leu Gly Leu Arg
675 680 685 Phe Leu Gly Ala
Pro Ala Lys Pro Pro Leu Val Arg Ala Asp Met Val 690
695 700 Thr Glu Lys Gln Ala Arg Glu Leu
Ser Val Pro Gln Gly Pro Gly Ala 705 710
715 720 Gly Ala Glu Ser Thr Gly Glu Ile Ile Asn Asn Thr
Val Pro Leu Glu 725 730
735 Asn Ser Ile Pro Gly Asn Cys Cys Ser Ala Leu Phe Lys Asn Leu Leu
740 745 750 Leu Lys Lys
Ile Lys Arg Cys Glu Arg Lys Gly Thr Glu Ser Val Thr 755
760 765 Glu Glu Lys Cys Ala Val Leu Phe
Ser Ala Ser Phe Thr Leu Gly Pro 770 775
780 Gly Lys Leu Pro Ile Gln Leu Gln Ala Leu Ser Leu Pro
Leu Val Val 785 790 795
800 Ile Val His Gly Asn Gln Asp Asn Asn Ala Lys Ala Thr Ile Leu Trp
805 810 815 Asp Asn Ala Phe
Ser Glu Met Asp Arg Val Pro Phe Val Val Ala Glu 820
825 830 Arg Val Pro Trp Glu Lys Met Cys Glu
Thr Leu Asn Leu Lys Phe Met 835 840
845 Ala Glu Val Gly Thr Asn Arg Gly Leu Leu Pro Glu His Phe
Leu Phe 850 855 860
Leu Ala Gln Lys Ile Phe Asn Asp Asn Ser Leu Ser Met Glu Ala Phe 865
870 875 880 Gln His Arg Ser Val
Ser Trp Ser Gln Phe Asn Lys Glu Ile Leu Leu 885
890 895 Gly Arg Gly Phe Thr Phe Trp Gln Trp Phe
Asp Gly Val Leu Asp Leu 900 905
910 Thr Lys Arg Cys Leu Arg Ser Tyr Trp Ser Asp Arg Leu Ile Ile
Gly 915 920 925 Phe
Ile Ser Lys Gln Tyr Val Thr Ser Leu Leu Leu Asn Glu Pro Asp 930
935 940 Gly Thr Phe Leu Leu Arg
Phe Ser Asp Ser Glu Ile Gly Gly Ile Thr 945 950
955 960 Ile Ala His Val Ile Arg Gly Gln Asp Gly Ser
Pro Gln Ile Glu Asn 965 970
975 Ile Gln Pro Phe Ser Ala Lys Asp Leu Ser Ile Arg Ser Leu Gly Asp
980 985 990 Arg Ile
Arg Asp Leu Ala Gln Leu Lys Asn Leu Tyr Pro Lys Lys Pro 995
1000 1005 Lys Asp Glu Ala Phe
Arg Ser His Tyr Lys Pro Glu Gln Met Gly 1010 1015
1020 Lys Asp Gly Arg Gly Tyr Val Pro Ala Thr
Ile Lys Met Thr Val 1025 1030 1035
Glu Arg Asp Gln Pro Leu Pro Thr Pro Glu Leu Gln Met Pro Thr
1040 1045 1050 Met Val
Pro Ser Tyr Asp Leu Gly Met Ala Pro Asp Ser Ser Met 1055
1060 1065 Ser Met Gln Leu Gly Pro Asp
Met Val Pro Gln Val Tyr Pro Pro 1070 1075
1080 His Ser His Ser Ile Pro Pro Tyr Gln Gly Leu Ser
Pro Glu Glu 1085 1090 1095
Ser Val Asn Val Leu Ser Ala Phe Gln Glu Pro His Leu Gln Met 1100
1105 1110 Pro Pro Ser Leu Gly
Gln Met Ser Leu Pro Phe Asp Gln Pro His 1115 1120
1125 Pro Gln Gly Leu Leu Pro Cys Gln Pro Gln
Glu His Ala Val Ser 1130 1135 1140
Ser Pro Asp Pro Leu Leu Cys Ser Asp Val Thr Met Val Glu Asp
1145 1150 1155 Ser Cys
Leu Ser Gln Pro Val Thr Ala Phe Pro Gln Gly Thr Trp 1160
1165 1170 Ile Gly Glu Asp Ile Phe Pro
Pro Leu Leu Pro Pro Thr Glu Gln 1175 1180
1185 Asp Leu Thr Lys Leu Leu Leu Glu Gly Gln Gly Glu
Ser Gly Gly 1190 1195 1200
Gly Ser Leu Gly Ala Gln Pro Leu Leu Gln Pro Ser His Tyr Gly 1205
1210 1215 Gln Ser Gly Ile Ser
Met Ser His Met Asp Leu Arg Ala Asn Pro 1220 1225
1230 Ser Trp 1235 409755PRTArtificial
SequenceSynthetic Polypeptide 409Met His Arg Ala Pro Ser Pro Thr Ala Glu
Gln Pro Pro Gly Gly Gly 1 5 10
15 Asp Ser Ala Arg Arg Thr Leu Gln Pro Arg Leu Lys Pro Ser Ala
Arg 20 25 30 Ala
Met Ala Leu Pro Arg Thr Leu Gly Glu Leu Gln Leu Tyr Arg Val 35
40 45 Leu Gln Arg Ala Asn Leu
Leu Ser Tyr Tyr Glu Thr Phe Ile Gln Gln 50 55
60 Gly Gly Asp Asp Val Gln Gln Leu Cys Glu Ala
Gly Glu Glu Glu Phe 65 70 75
80 Leu Glu Ile Met Ala Leu Val Gly Met Ala Thr Lys Pro Leu His Val
85 90 95 Arg Arg
Leu Gln Lys Ala Leu Arg Glu Trp Ala Thr Asn Pro Gly Leu 100
105 110 Phe Ser Gln Pro Val Pro Ala
Val Pro Val Ser Ser Ile Pro Leu Phe 115 120
125 Lys Ile Ser Glu Thr Ala Gly Thr Arg Lys Gly Ser
Met Ser Asn Gly 130 135 140
His Gly Ser Pro Gly Glu Lys Ala Gly Ser Ala Arg Ser Phe Ser Pro 145
150 155 160 Lys Ser Pro
Leu Glu Leu Gly Glu Lys Leu Ser Pro Leu Pro Gly Gly 165
170 175 Pro Gly Ala Gly Asp Pro Arg Ile
Trp Pro Gly Arg Ser Thr Pro Glu 180 185
190 Ser Asp Val Gly Ala Gly Gly Glu Glu Glu Ala Gly Ser
Pro Pro Phe 195 200 205
Ser Pro Pro Ala Gly Gly Gly Val Pro Glu Gly Thr Gly Ala Gly Gly 210
215 220 Leu Ala Ala Gly
Gly Thr Gly Gly Gly Pro Asp Arg Leu Glu Pro Glu 225 230
235 240 Met Val Arg Met Val Val Glu Ser Val
Glu Arg Ile Phe Arg Ser Phe 245 250
255 Pro Arg Gly Asp Ala Gly Glu Val Thr Ser Leu Leu Lys Leu
Asn Lys 260 265 270
Lys Leu Ala Arg Ser Val Gly His Ile Phe Glu Met Asp Asp Asn Asp
275 280 285 Ser Gln Lys Glu
Glu Glu Ile Arg Lys Tyr Ser Ile Ile Tyr Gly Arg 290
295 300 Phe Asp Ser Lys Arg Arg Glu Gly
Lys Gln Leu Ser Leu His Glu Leu 305 310
315 320 Thr Ile Asn Glu Ala Ala Ala Gln Phe Cys Met Arg
Asp Asn Thr Leu 325 330
335 Leu Leu Arg Arg Val Glu Leu Phe Ser Leu Ser Arg Gln Val Ala Arg
340 345 350 Glu Ser Thr
Tyr Leu Ser Ser Leu Lys Gly Ser Arg Leu His Pro Glu 355
360 365 Glu Leu Gly Gly Pro Pro Leu Lys
Lys Leu Lys Gln Glu Val Gly Glu 370 375
380 Gln Ser His Pro Glu Ile Gln Gln Pro Pro Pro Gly Pro
Glu Ser Tyr 385 390 395
400 Val Pro Pro Tyr Arg Pro Ser Leu Glu Glu Asp Ser Ala Ser Leu Ser
405 410 415 Gly Glu Ser Leu
Asp Gly His Leu Gln Ala Val Gly Ser Cys Pro Arg 420
425 430 Leu Thr Pro Pro Pro Ala Asp Leu Pro
Leu Ala Leu Pro Ala His Gly 435 440
445 Leu Trp Ser Arg His Ile Leu Gln Gln Thr Leu Met Asp Glu
Gly Leu 450 455 460
Arg Leu Ala Arg Leu Val Ser His Asp Arg Val Gly Arg Leu Ser Pro 465
470 475 480 Cys Val Pro Ala Lys
Pro Pro Leu Ala Gly Ser Pro Gln Ile Glu Asn 485
490 495 Ile Gln Pro Phe Ser Ala Lys Asp Leu Ser
Ile Arg Ser Leu Gly Asp 500 505
510 Arg Ile Arg Asp Leu Ala Gln Leu Lys Asn Leu Tyr Pro Lys Lys
Pro 515 520 525 Lys
Asp Glu Ala Phe Arg Ser His Tyr Lys Pro Glu Gln Met Gly Lys 530
535 540 Asp Gly Arg Gly Tyr Val
Pro Ala Thr Ile Lys Met Thr Val Glu Arg 545 550
555 560 Asp Gln Pro Leu Pro Thr Pro Glu Leu Gln Met
Pro Thr Met Val Pro 565 570
575 Ser Tyr Asp Leu Gly Met Ala Pro Asp Ser Ser Met Ser Met Gln Leu
580 585 590 Gly Pro
Asp Met Val Pro Gln Val Tyr Pro Pro His Ser His Ser Ile 595
600 605 Pro Pro Tyr Gln Gly Leu Ser
Pro Glu Glu Ser Val Asn Val Leu Ser 610 615
620 Ala Phe Gln Glu Pro His Leu Gln Met Pro Pro Ser
Leu Gly Gln Met 625 630 635
640 Ser Leu Pro Phe Asp Gln Pro His Pro Gln Gly Leu Leu Pro Cys Gln
645 650 655 Pro Gln Glu
His Ala Val Ser Ser Pro Asp Pro Leu Leu Cys Ser Asp 660
665 670 Val Thr Met Val Glu Asp Ser Cys
Leu Ser Gln Pro Val Thr Ala Phe 675 680
685 Pro Gln Gly Thr Trp Ile Gly Glu Asp Ile Phe Pro Pro
Leu Leu Pro 690 695 700
Pro Thr Glu Gln Asp Leu Thr Lys Leu Leu Leu Glu Gly Gln Gly Glu 705
710 715 720 Ser Gly Gly Gly
Ser Leu Gly Ala Gln Pro Leu Leu Gln Pro Ser His 725
730 735 Tyr Gly Gln Ser Gly Ile Ser Met Ser
His Met Asp Leu Arg Ala Asn 740 745
750 Pro Ser Trp 755 41027PRTArtificial
SequenceSynthetic Polypeptide 410Gly Pro Pro Leu Lys Lys Leu Lys Gln Glu
Ala Thr Ser Lys Ser Gln 1 5 10
15 Ile Met Ser Leu Trp Gly Leu Val Ser Lys Met 20
25 4119PRTArtificial SequenceSynthetic
Polypeptide 411Pro Leu Lys Lys Leu Lys Gln Glu Ala 1 5
4129PRTArtificial SequenceSynthetic Polypeptide 412Leu Lys
Lys Leu Lys Gln Glu Ala Thr 1 5
4139PRTArtificial SequenceSynthetic Polypeptide 413Lys Lys Leu Lys Gln
Glu Ala Thr Ser 1 5 4149PRTArtificial
SequenceSynthetic Polypeptide 414Lys Leu Lys Gln Glu Ala Thr Ser Lys 1
5 4159PRTArtificial SequenceSynthetic
Polypeptide 415Leu Lys Gln Glu Ala Thr Ser Lys Ser 1 5
4169PRTArtificial SequenceSynthetic Polypeptide 416Lys Gln
Glu Ala Thr Ser Lys Ser Gln 1 5
4179PRTArtificial SequenceSynthetic Polypeptide 417Gln Glu Ala Thr Ser
Lys Ser Gln Ile 1 5 4189PRTArtificial
SequenceSynthetic Polypeptide 418Glu Ala Thr Ser Lys Ser Gln Ile Met 1
5 4199PRTArtificial SequenceSynthetic
Polypeptide 419Ala Thr Ser Lys Ser Gln Ile Met Ser 1 5
4209PRTArtificial SequenceSynthetic Polypeptide 420Thr Ser
Lys Ser Gln Ile Met Ser Leu 1 5
4219PRTArtificial SequenceSynthetic Polypeptide 421Ser Lys Ser Gln Ile
Met Ser Leu Trp 1 5 4229PRTArtificial
SequenceSynthetic Polypeptide 422Lys Ser Gln Ile Met Ser Leu Trp Gly 1
5 4239PRTArtificial SequenceSynthetic
Polypeptide 423Ser Gln Ile Met Ser Leu Trp Gly Leu 1 5
4249PRTArtificial SequenceSynthetic Polypeptide 424Gln Ile
Met Ser Leu Trp Gly Leu Val 1 5
4259PRTArtificial SequenceSynthetic Polypeptide 425Ile Met Ser Leu Trp
Gly Leu Val Ser 1 5 42627PRTArtificial
SequenceSynthetic Polypeptide 426Gly Pro Pro Leu Lys Lys Leu Lys Gln Glu
Ala Thr Ser Lys Ser Gln 1 5 10
15 Ile Met Ser Leu Trp Gly Leu Val Ser Lys Met 20
25 42720PRTArtificial SequenceSynthetic
Polypeptide 427Pro Cys Val Pro Ala Lys Pro Pro Leu Ala Gly Ser Pro Gln
Ile Glu 1 5 10 15
Asn Ile Gln Pro 20 42821PRTArtificial SequenceSynthetic
Polypeptide 428Pro Cys Val Pro Ala Lys Pro Pro Leu Ala Ala Glu Gln Met
Gly Lys 1 5 10 15
Asp Gly Arg Gly Tyr 20 4298PRTArtificial
SequenceSynthetic Polypeptide 429Ala Thr Ser Lys Ser Gln Ile Met 1
5 4308PRTArtificial SequenceSynthetic Polypeptide
430Glu Ala Thr Ser Lys Ser Gln Ile 1 5
4318PRTArtificial SequenceSynthetic Polypeptide 431Ile Met Ser Leu Trp
Gly Leu Val 1 5 4328PRTArtificial
SequenceSynthetic Polypeptide 432Lys Ser Gln Ile Met Ser Leu Trp 1
5 4338PRTArtificial SequenceSynthetic Polypeptide
433Gln Ile Met Ser Leu Trp Gly Leu 1 5
4348PRTArtificial SequenceSynthetic Polypeptide 434Ser Lys Ser Gln Ile
Met Ser Leu 1 5 4359PRTArtificial
SequenceSynthetic Polypeptide 435Glu Ala Thr Ser Lys Ser Gln Ile Met 1
5 4369PRTArtificial SequenceSynthetic
Polypeptide 436Lys Leu Lys Gln Glu Ala Thr Ser Lys 1 5
4379PRTArtificial SequenceSynthetic Polypeptide 437Gln Glu
Ala Thr Ser Lys Ser Gln Ile 1 5
4389PRTArtificial SequenceSynthetic Polypeptide 438Gln Ile Met Ser Leu
Trp Gly Leu Val 1 5 4399PRTArtificial
SequenceSynthetic Polypeptide 439Ser Gln Ile Met Ser Leu Trp Gly Leu 1
5 4409PRTArtificial SequenceSynthetic
Polypeptide 440Thr Ser Lys Ser Gln Ile Met Ser Leu 1 5
44110PRTArtificial SequenceSynthetic Polypeptide 441Ala Thr
Ser Lys Ser Gln Ile Met Ser Leu 1 5 10
44210PRTArtificial SequenceSynthetic Polypeptide 442Ile Met Ser Leu Trp
Gly Leu Val Ser Lys 1 5 10
44310PRTArtificial SequenceSynthetic Polypeptide 443Lys Lys Leu Lys Gln
Glu Ala Thr Ser Lys 1 5 10
44410PRTArtificial SequenceSynthetic Polypeptide 444Lys Gln Glu Ala Thr
Ser Lys Ser Gln Ile 1 5 10
44510PRTArtificial SequenceSynthetic Polypeptide 445Lys Ser Gln Ile Met
Ser Leu Trp Gly Leu 1 5 10
44610PRTArtificial SequenceSynthetic Polypeptide 446Gln Glu Ala Thr Ser
Lys Ser Gln Ile Met 1 5 10
44710PRTArtificial SequenceSynthetic Polypeptide 447Ser Gln Ile Met Ser
Leu Trp Gly Leu Val 1 5 10
44810PRTArtificial SequenceSynthetic Polypeptide 448Thr Ser Lys Ser Gln
Ile Met Ser Leu Trp 1 5 10
44911PRTArtificial SequenceSynthetic Polypeptide 449Ala Thr Ser Lys Ser
Gln Ile Met Ser Leu Trp 1 5 10
45011PRTArtificial SequenceSynthetic Polypeptide 450Glu Ala Thr Ser Lys
Ser Gln Ile Met Ser Leu 1 5 10
45111PRTArtificial SequenceSynthetic Polypeptide 451Ile Met Ser Leu Trp
Gly Leu Val Ser Lys Met 1 5 10
45211PRTArtificial SequenceSynthetic Polypeptide 452Lys Gln Glu Ala Thr
Ser Lys Ser Gln Ile Met 1 5 10
45311PRTArtificial SequenceSynthetic Polypeptide 453Lys Ser Gln Ile Met
Ser Leu Trp Gly Leu Val 1 5 10
45411PRTArtificial SequenceSynthetic Polypeptide 454Leu Lys Gln Glu Ala
Thr Ser Lys Ser Gln Ile 1 5 10
45511PRTArtificial SequenceSynthetic Polypeptide 455Gln Glu Ala Thr Ser
Lys Ser Gln Ile Met Ser 1 5 10
45611PRTArtificial SequenceSynthetic Polypeptide 456Gln Ile Met Ser Leu
Trp Gly Leu Val Ser Lys 1 5 10
45711PRTArtificial SequenceSynthetic Polypeptide 457Ser Gln Ile Met Ser
Leu Trp Gly Leu Val Ser 1 5 10
4588PRTArtificial SequenceSynthetic Polypeptide 458Pro Leu Ala Gly Ser
Pro Gln Ile 1 5 4599PRTArtificial
SequenceSynthetic Polypeptide 459Pro Pro Leu Ala Gly Ser Pro Gln Ile 1
5 46010PRTArtificial SequenceSynthetic
Polypeptide 460Lys Pro Pro Leu Ala Gly Ser Pro Gln Ile 1 5
10 46110PRTArtificial SequenceSynthetic Polypeptide
461Leu Ala Gly Ser Pro Gln Ile Glu Asn Ile 1 5
10 46211PRTArtificial SequenceSynthetic Polypeptide 462Gly Ser Pro
Gln Ile Glu Asn Ile Gln Pro Phe 1 5 10
46311PRTArtificial SequenceSynthetic Polypeptide 463Pro Leu Ala Gly Ser
Pro Gln Ile Glu Asn Ile 1 5 10
4648PRTArtificial SequenceSynthetic Polypeptide 464Ala Glu Gln Met Gly
Lys Asp Gly 1 5 4658PRTArtificial
SequenceSynthetic Polypeptide 465Leu Ala Ala Glu Gln Met Gly Lys 1
5 4668PRTArtificial SequenceSynthetic Polypeptide
466Pro Pro Leu Ala Ala Glu Gln Met 1 5
4679PRTArtificial SequenceSynthetic Polypeptide 467Lys Pro Pro Leu Ala
Ala Glu Gln Met 1 5 4689PRTArtificial
SequenceSynthetic Polypeptide 468Pro Leu Ala Ala Glu Gln Met Gly Lys 1
5 4699PRTArtificial SequenceSynthetic
Polypeptide 469Val Pro Ala Lys Pro Pro Leu Ala Ala 1 5
47011PRTArtificial SequenceSynthetic Polypeptide 470Ala Glu
Gln Met Gly Lys Asp Gly Arg Gly Tyr 1 5
10 47111PRTArtificial SequenceSynthetic Polypeptide 471Leu Ala Ala
Glu Gln Met Gly Lys Asp Gly Arg 1 5 10
47211PRTArtificial SequenceSynthetic Polypeptide 472Pro Ala Lys Pro Pro
Leu Ala Ala Glu Gln Met 1 5 10
47322PRTArtificial SequenceSynthetic Polypeptide 473Asp Gly Val Ser His
Cys Pro Gly Gly Glu Asp Glu Asn Arg Cys Gly 1 5
10 15 Ser Leu Ile Ser Cys Glu 20
47422PRTArtificial SequenceSynthetic Polypeptide 474Phe Leu Val
Gly Ala Ala Leu Ala Ala Gly Leu Leu Trp Lys Phe Arg 1 5
10 15 Ser Leu Ile Ser Cys Glu
20 47530PRTArtificial SequenceSynthetic Polypeptide 475Phe
Leu Val Gly Ala Ala Leu Ala Ala Gly Leu Leu Trp Lys Phe Arg 1
5 10 15 Thr Leu Leu Met Asn Ala
Val Trp Pro Lys Ala Gly Arg Trp 20 25
30 47620PRTArtificial SequenceSynthetic Polypeptide 476Met Ala
Leu Asn Ser Glu Ala Leu Ser Val Val Ser Glu Asp Gln Ser 1 5
10 15 Leu Phe Glu Cys
20 47718PRTArtificial SequenceSynthetic Polypeptide 477Met Ala Leu Asn
Ser Pro Ser Gly Ser Glu Gln Leu Val Asp Gly Leu 1 5
10 15 Ala Tyr 47818PRTArtificial
SequenceSynthetic Polypeptide 478Met Ala Leu Asn Ser Val Ile Pro Gly Ser
Leu Glu Thr Arg Gly Lys 1 5 10
15 Pro Cys 47922PRTArtificial SequenceSynthetic Polypeptide
479Val Cys Thr Gln Pro Lys Ser Pro Ser Gly Thr Val Cys Thr Ser Arg 1
5 10 15 Ser Leu Ile Ser
Cys Glu 20 48030PRTArtificial SequenceSynthetic
Polypeptide 480Val Cys Thr Gln Pro Lys Ser Pro Ser Gly Thr Val Cys Thr
Ser Ser 1 5 10 15
Tyr Ser Arg Ile Phe Gly Asp Pro Arg Lys Ala Val Leu Thr 20
25 30 48130PRTArtificial
SequenceSynthetic Polypeptide 481Ile His Thr Gln Pro Glu Val Ile Leu His
Gln Asn His Glu Glu Asp 1 5 10
15 Val Gln His Ile Lys Arg Arg Asp Ile Val Leu Lys Arg Glu
20 25 30 48230PRTArtificial
SequenceSynthetic Polypeptide 482Ile His Thr Gln Pro Glu Val Ile Leu His
Gln Asn His Glu Glu Gly 1 5 10
15 Pro Val Ala Val Ile Ser Gly Glu Glu Asp Ser Ala Ser Pro
20 25 30 48330PRTArtificial
SequenceSynthetic Polypeptide 483Val Ser Pro Pro Glu Glu His Ala Met Pro
Ile Gly Arg Ile Ala Asp 1 5 10
15 Val Gln His Ile Lys Arg Arg Asp Ile Val Leu Lys Arg Glu
20 25 30 48430PRTArtificial
SequenceSynthetic Polypeptide 484Val Ser Pro Pro Glu Glu His Ala Met Pro
Ile Gly Arg Ile Ala Asp 1 5 10
15 Val Gln His Ile Lys Arg Arg Asp Ile Val Leu Lys Arg Glu
20 25 30 485439PRTArtificial
SequenceSynthetic Polypeptide 485Met Ala Ser Asn Asp Tyr Thr Gln Gln Ala
Thr Gln Ser Tyr Gly Ala 1 5 10
15 Tyr Pro Thr Gln Pro Gly Gln Gly Tyr Ser Gln Gln Ser Ser Gln
Pro 20 25 30 Tyr
Gly Gln Gln Ser Tyr Ser Gly Tyr Ser Gln Ser Thr Asp Thr Ser 35
40 45 Gly Tyr Gly Gln Ser Ser
Tyr Ser Ser Tyr Gly Gln Ser Gln Asn Thr 50 55
60 Gly Tyr Gly Thr Gln Ser Thr Pro Gln Gly Tyr
Gly Ser Thr Gly Gly 65 70 75
80 Tyr Gly Ser Ser Gln Ser Ser Gln Ser Ser Tyr Gly Gln Gln Ser Ser
85 90 95 Tyr Pro
Gly Tyr Gly Gln Gln Pro Ala Pro Ser Ser Thr Ser Gly Ser 100
105 110 Tyr Gly Ser Ser Ser Gln Ser
Ser Ser Tyr Gly Gln Pro Gln Ser Gly 115 120
125 Ser Tyr Ser Gln Gln Pro Ser Tyr Gly Gly Gln Gln
Gln Ser Tyr Gly 130 135 140
Gln Gln Gln Ser Tyr Asn Pro Pro Gln Gly Tyr Gly Gln Gln Asn Gln 145
150 155 160 Tyr Asn Ser
Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Asn 165
170 175 Tyr Gly Gln Asp Gln Ser Ser Met
Ser Ser Gly Gly Gly Ser Gly Gly 180 185
190 Gly Tyr Gly Asn Gln Asp Gln Ser Gly Gly Gly Gly Ser
Gly Gly Tyr 195 200 205
Gly Gln Gln Asp Arg Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly Gly 210
215 220 Gly Gly Gly Gly
Gly Gly Gly Tyr Asn Arg Ser Ser Gly Gly Tyr Glu 225 230
235 240 Pro Arg Gly Arg Gly Gly Gly Arg Gly
Gly Arg Gly Gly Met Gly Gly 245 250
255 Ser Asp Arg Gly Gly Phe Asn Lys Phe Gly Gly Ser Gly Gln
Ile Gln 260 265 270
Leu Trp Gln Phe Leu Leu Glu Leu Leu Ser Asp Ser Ser Asn Ser Ser
275 280 285 Cys Ile Thr Trp
Glu Gly Thr Asn Gly Glu Phe Lys Met Thr Asp Pro 290
295 300 Asp Glu Val Ala Arg Arg Trp Gly
Glu Arg Lys Ser Lys Pro Asn Met 305 310
315 320 Asn Tyr Asp Lys Leu Ser Arg Ala Leu Arg Tyr Tyr
Tyr Asp Lys Asn 325 330
335 Ile Met Thr Lys Val His Gly Lys Arg Tyr Ala Tyr Lys Phe Asp Phe
340 345 350 His Gly Ile
Ala Gln Ala Leu Gln Pro His Pro Pro Glu Ser Ser Leu 355
360 365 Tyr Lys Tyr Pro Ser Asp Leu Pro
Tyr Met Gly Ser Tyr His Ala His 370 375
380 Pro Gln Lys Met Asn Phe Val Ala Pro His Pro Pro Ala
Leu Pro Val 385 390 395
400 Thr Ser Ser Ser Phe Phe Ala Ala Pro Asn Pro Tyr Trp Asn Ser Pro
405 410 415 Thr Gly Gly Ile
Tyr Pro Asn Thr Arg Leu Pro Thr Ser His Met Pro 420
425 430 Ser His Leu Gly Thr Tyr Tyr
435 48627PRTArtificial SequenceSynthetic Polypeptide
486Gly Pro Pro Leu Lys Lys Leu Lys Gln Glu Ala Thr Ser Lys Ser Gln 1
5 10 15 Ile Met Ser Leu
Trp Gly Leu Val Ser Lys Met 20 25
4878PRTArtificial SequenceSynthetic Polypeptide 487Gly Ser Gly Gln Ile
Gln Leu Trp 1 5 4888PRTArtificial
SequenceSynthetic Polypeptide 488Lys Phe Gly Gly Ser Gly Gln Ile 1
5 4899PRTArtificial SequenceSynthetic Polypeptide
489Phe Gly Gly Ser Gly Gln Ile Gln Leu 1 5
4909PRTArtificial SequenceSynthetic Polypeptide 490Gly Gly Ser Gly Gln
Ile Gln Leu Trp 1 5 49110PRTArtificial
SequenceSynthetic Polypeptide 491Lys Phe Gly Gly Ser Gly Gln Ile Gln Leu
1 5 10 49211PRTArtificial
SequenceSynthetic Polypeptide 492Lys Phe Gly Gly Ser Gly Gln Ile Gln Leu
Trp 1 5 10 49311PRTArtificial
SequenceSynthetic Polypeptide 493Asn Lys Phe Gly Gly Ser Gly Gln Ile Gln
Leu 1 5 10 494191PRTArtificial
SequenceSynthetic Polypeptide 494Met Ala Ser Asn Asp Tyr Thr Gln Gln Ala
Thr Gln Ser Tyr Gly Ala 1 5 10
15 Tyr Pro Thr Gln Pro Gly Gln Gly Tyr Ser Gln Gln Ser Ser Gln
Pro 20 25 30 Tyr
Gly Gln Gln Ser Tyr Ser Gly Tyr Ser Gln Ser Thr Asp Thr Ser 35
40 45 Gly Tyr Gly Gln Ser Ser
Tyr Ser Ser Tyr Gly Gln Ser Gln Asn Thr 50 55
60 Gly Tyr Gly Thr Gln Ser Thr Pro Gln Gly Tyr
Gly Ser Thr Gly Gly 65 70 75
80 Tyr Gly Ser Ser Gln Ser Ser Gln Ser Ser Tyr Gly Gln Gln Ser Ser
85 90 95 Tyr Pro
Gly Tyr Gly Gln Gln Pro Ala Pro Ser Ser Thr Ser Gly Ser 100
105 110 Tyr Gly Ser Ser Ser Gln Ser
Ser Ser Tyr Gly Gln Pro Gln Ser Gly 115 120
125 Ser Tyr Ser Gln Gln Pro Ser Tyr Gly Gly Gln Gln
Gln Ser Tyr Gly 130 135 140
Gln Gln Gln Ser Tyr Asn Pro Pro Gln Gly Tyr Gly Gln Gln Asn Gln 145
150 155 160 Tyr Asn Ser
Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Val Phe 165
170 175 Lys Lys Glu Val Tyr Leu His Thr
Ser Pro His Leu Lys Ala Asp 180 185
190 495283PRTArtificial SequenceSynthetic Polypeptide 495Met
Ala Ser Asn Asp Tyr Thr Gln Gln Ala Thr Gln Ser Tyr Gly Ala 1
5 10 15 Tyr Pro Thr Gln Pro Gly
Gln Gly Tyr Ser Gln Gln Ser Ser Gln Pro 20
25 30 Tyr Gly Gln Gln Ser Tyr Ser Gly Tyr Ser
Gln Ser Thr Asp Thr Ser 35 40
45 Gly Tyr Gly Gln Ser Ser Tyr Ser Ser Tyr Gly Gln Ser Gln
Asn Thr 50 55 60
Gly Tyr Gly Thr Gln Ser Thr Pro Gln Gly Tyr Gly Ser Thr Gly Gly 65
70 75 80 Tyr Gly Ser Ser Gln
Ser Ser Gln Ser Ser Tyr Gly Gln Gln Ser Ser 85
90 95 Tyr Pro Gly Tyr Gly Gln Gln Pro Ala Pro
Ser Ser Thr Ser Gly Ser 100 105
110 Tyr Gly Ser Ser Ser Gln Ser Ser Ser Tyr Gly Gln Pro Gln Ser
Gly 115 120 125 Ser
Tyr Ser Gln Gln Pro Ser Tyr Gly Gly Gln Gln Gln Ser Tyr Gly 130
135 140 Gln Gln Gln Ser Tyr Asn
Pro Pro Gln Gly Tyr Gly Gln Gln Asn Gln 145 150
155 160 Tyr Asn Ser Ser Ser Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Asn 165 170
175 Tyr Gly Gln Asp Gln Ser Ser Met Ser Ser Gly Gly Gly Ser Gly Gly
180 185 190 Gly Tyr
Gly Asn Gln Asp Gln Ser Gly Gly Gly Gly Ser Gly Gly Tyr 195
200 205 Gly Gln Gln Asp Arg Gly Gly
Arg Gly Arg Gly Gly Ser Gly Gly Gly 210 215
220 Gly Gly Gly Gly Gly Gly Gly Tyr Asn Arg Ser Ser
Gly Gly Tyr Glu 225 230 235
240 Pro Arg Gly Arg Gly Gly Gly Arg Gly Gly Arg Gly Gly Met Gly Gly
245 250 255 Ser Asp Arg
Gly Gly Phe Asn Lys Phe Gly Val Phe Lys Lys Glu Val 260
265 270 Tyr Leu His Thr Ser Pro His Leu
Lys Ala Asp 275 280
496294PRTArtificial SequenceSynthetic Polypeptide 496Met Ala Ser Asn Asp
Tyr Thr Gln Gln Ala Thr Gln Ser Tyr Gly Ala 1 5
10 15 Tyr Pro Thr Gln Pro Gly Gln Gly Tyr Ser
Gln Gln Ser Ser Gln Pro 20 25
30 Tyr Gly Gln Gln Ser Tyr Ser Gly Tyr Ser Gln Ser Thr Asp Thr
Ser 35 40 45 Gly
Tyr Gly Gln Ser Ser Tyr Ser Ser Tyr Gly Gln Ser Gln Asn Thr 50
55 60 Gly Tyr Gly Thr Gln Ser
Thr Pro Gln Gly Tyr Gly Ser Thr Gly Gly 65 70
75 80 Tyr Gly Ser Ser Gln Ser Ser Gln Ser Ser Tyr
Gly Gln Gln Ser Ser 85 90
95 Tyr Pro Gly Tyr Gly Gln Gln Pro Ala Pro Ser Ser Thr Ser Gly Ser
100 105 110 Tyr Gly
Ser Ser Ser Gln Ser Ser Ser Tyr Gly Gln Pro Gln Ser Gly 115
120 125 Ser Tyr Ser Gln Gln Pro Ser
Tyr Gly Gly Gln Gln Gln Ser Tyr Gly 130 135
140 Gln Gln Gln Ser Tyr Asn Pro Pro Gln Gly Tyr Gly
Gln Gln Asn Gln 145 150 155
160 Tyr Asn Ser Ser Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Asn
165 170 175 Tyr Gly Gln
Asp Gln Ser Ser Met Ser Ser Gly Gly Gly Ser Gly Gly 180
185 190 Gly Tyr Gly Asn Gln Asp Gln Ser
Gly Gly Gly Gly Ser Gly Gly Tyr 195 200
205 Gly Gln Gln Asp Arg Gly Gly Arg Gly Arg Gly Gly Ser
Gly Gly Gly 210 215 220
Gly Gly Gly Gly Gly Gly Gly Tyr Asn Arg Ser Ser Gly Gly Tyr Glu 225
230 235 240 Pro Arg Gly Arg
Gly Gly Gly Arg Gly Gly Arg Gly Gly Met Gly Gly 245
250 255 Ser Asp Arg Gly Gly Phe Asn Lys Phe
Gly Gly Pro Arg Asp Gln Gly 260 265
270 Ser Arg His Asp Ser Val Phe Lys Lys Glu Val Tyr Leu His
Thr Ser 275 280 285
Pro His Leu Lys Ala Asp 290 49727PRTArtificial
SequenceSynthetic Polypeptide 497Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly
Val Phe Lys Lys Glu Val 1 5 10
15 Tyr Leu His Thr Ser Pro His Leu Lys Ala Asp 20
25 4988PRTArtificial SequenceSynthetic
Polypeptide 498His Thr Ser Pro His Leu Lys Ala 1 5
4998PRTArtificial SequenceSynthetic Polypeptide 499Lys Glu Val Tyr
Leu His Thr Ser 1 5 5008PRTArtificial
SequenceSynthetic Polypeptide 500Val Phe Lys Lys Glu Val Tyr Leu 1
5 5018PRTArtificial SequenceSynthetic Polypeptide
501Val Tyr Leu His Thr Ser Pro His 1 5
5028PRTArtificial SequenceSynthetic Polypeptide 502Tyr Leu His Thr Ser
Pro His Leu 1 5 5039PRTArtificial
SequenceSynthetic Polypeptide 503Glu Val Tyr Leu His Thr Ser Pro His 1
5 5049PRTArtificial SequenceSynthetic
Polypeptide 504Gly Val Phe Lys Lys Glu Val Tyr Leu 1 5
5059PRTArtificial SequenceSynthetic Polypeptide 505Lys Glu
Val Tyr Leu His Thr Ser Pro 1 5
5069PRTArtificial SequenceSynthetic Polypeptide 506Val Tyr Leu His Thr
Ser Pro His Leu 1 5 5079PRTArtificial
SequenceSynthetic Polypeptide 507Tyr Leu His Thr Ser Pro His Leu Lys 1
5 50810PRTArtificial SequenceSynthetic
Polypeptide 508Glu Val Tyr Leu His Thr Ser Pro His Leu 1 5
10 50910PRTArtificial SequenceSynthetic Polypeptide
509Tyr Leu His Thr Ser Pro His Leu Lys Ala 1 5
10 51011PRTArtificial SequenceSynthetic Polypeptide 510Glu Val Tyr
Leu His Thr Ser Pro His Leu Lys 1 5 10
51111PRTArtificial SequenceSynthetic Polypeptide 511Lys Glu Val Tyr Leu
His Thr Ser Pro His Leu 1 5 10
5128PRTArtificial SequenceSynthetic Polypeptide 512Gly Val Phe Lys Lys
Glu Val Tyr 1 5 51327PRTArtificial
SequenceSynthetic Polypeptide 513Ser Asp Arg Gly Gly Phe Asn Lys Phe Gly
Val Phe Lys Lys Glu Val 1 5 10
15 Tyr Leu His Thr Ser Pro His Leu Lys Ala Asp 20
25 5148PRTArtificial SequenceSynthetic
Polypeptide 514Phe Asn Lys Phe Gly Val Phe Lys 1 5
5158PRTArtificial SequenceSynthetic Polypeptide 515Gly Phe Asn Lys
Phe Gly Val Phe 1 5 5168PRTArtificial
SequenceSynthetic Polypeptide 516Gly Val Phe Lys Lys Glu Val Tyr 1
5 5178PRTArtificial SequenceSynthetic Polypeptide
517His Thr Ser Pro His Leu Lys Ala 1 5
5188PRTArtificial SequenceSynthetic Polypeptide 518Lys Glu Val Tyr Leu
His Thr Ser 1 5 5198PRTArtificial
SequenceSynthetic Polypeptide 519Asn Lys Phe Gly Val Phe Lys Lys 1
5 5208PRTArtificial SequenceSynthetic Polypeptide
520Val Phe Lys Lys Glu Val Tyr Leu 1 5
5218PRTArtificial SequenceSynthetic Polypeptide 521Val Tyr Leu His Thr
Ser Pro His 1 5 5228PRTArtificial
SequenceSynthetic Polypeptide 522Tyr Leu His Thr Ser Pro His Leu 1
5 5239PRTArtificial SequenceSynthetic Polypeptide
523Glu Val Tyr Leu His Thr Ser Pro His 1 5
5249PRTArtificial SequenceSynthetic Polypeptide 524Phe Asn Lys Phe Gly
Val Phe Lys Lys 1 5 5259PRTArtificial
SequenceSynthetic Polypeptide 525Gly Phe Asn Lys Phe Gly Val Phe Lys 1
5 5269PRTArtificial SequenceSynthetic
Polypeptide 526Gly Gly Phe Asn Lys Phe Gly Val Phe 1 5
5279PRTArtificial SequenceSynthetic Polypeptide 527Gly Val
Phe Lys Lys Glu Val Tyr Leu 1 5
5289PRTArtificial SequenceSynthetic Polypeptide 528Lys Glu Val Tyr Leu
His Thr Ser Pro 1 5 5299PRTArtificial
SequenceSynthetic Polypeptide 529Val Tyr Leu His Thr Ser Pro His Leu 1
5 5309PRTArtificial SequenceSynthetic
Polypeptide 530Tyr Leu His Thr Ser Pro His Leu Lys 1 5
53110PRTArtificial SequenceSynthetic Polypeptide 531Glu Val
Tyr Leu His Thr Ser Pro His Leu 1 5 10
53210PRTArtificial SequenceSynthetic Polypeptide 532Phe Gly Val Phe Lys
Lys Glu Val Tyr Leu 1 5 10
53310PRTArtificial SequenceSynthetic Polypeptide 533Gly Phe Asn Lys Phe
Gly Val Phe Lys Lys 1 5 10
53410PRTArtificial SequenceSynthetic Polypeptide 534Tyr Leu His Thr Ser
Pro His Leu Lys Ala 1 5 10
53511PRTArtificial SequenceSynthetic Polypeptide 535Glu Val Tyr Leu His
Thr Ser Pro His Leu Lys 1 5 10
53611PRTArtificial SequenceSynthetic Polypeptide 536Phe Asn Lys Phe Gly
Val Phe Lys Lys Glu Val 1 5 10
53711PRTArtificial SequenceSynthetic Polypeptide 537Gly Gly Phe Asn Lys
Phe Gly Val Phe Lys Lys 1 5 10
53811PRTArtificial SequenceSynthetic Polypeptide 538Lys Glu Val Tyr Leu
His Thr Ser Pro His Leu 1 5 10
53911PRTArtificial SequenceSynthetic Polypeptide 539Lys Phe Gly Val Phe
Lys Lys Glu Val Tyr Leu 1 5 10
54011PRTArtificial SequenceSynthetic Polypeptide 540Asn Lys Phe Gly Val
Phe Lys Lys Glu Val Tyr 1 5 10
54111PRTArtificial SequenceSynthetic Polypeptide 541Arg Gly Gly Phe Asn
Lys Phe Gly Val Phe Lys 1 5 10
54227PRTArtificial SequenceSynthetic Polypeptide 542Pro Arg Asp Gln Gly
Ser Arg His Asp Ser Val Phe Lys Lys Glu Val 1 5
10 15 Tyr Leu His Thr Ser Pro His Leu Lys Ala
Asp 20 25 5438PRTArtificial
SequenceSynthetic Polypeptide 543Asp Ser Val Phe Lys Lys Glu Val 1
5 5448PRTArtificial SequenceSynthetic Polypeptide
544Gly Ser Arg His Asp Ser Val Phe 1 5
5458PRTArtificial SequenceSynthetic Polypeptide 545His Thr Ser Pro His
Leu Lys Ala 1 5 5468PRTArtificial
SequenceSynthetic Polypeptide 546Lys Glu Val Tyr Leu His Thr Ser 1
5 5478PRTArtificial SequenceSynthetic Polypeptide
547Ser Arg His Asp Ser Val Phe Lys 1 5
5488PRTArtificial SequenceSynthetic Polypeptide 548Ser Val Phe Lys Lys
Glu Val Tyr 1 5 5498PRTArtificial
SequenceSynthetic Polypeptide 549Val Phe Lys Lys Glu Val Tyr Leu 1
5 5508PRTArtificial SequenceSynthetic Polypeptide
550Val Tyr Leu His Thr Ser Pro His 1 5
5518PRTArtificial SequenceSynthetic Polypeptide 551Tyr Leu His Thr Ser
Pro His Leu 1 5 5529PRTArtificial
SequenceSynthetic Polypeptide 552Glu Val Tyr Leu His Thr Ser Pro His 1
5 5539PRTArtificial SequenceSynthetic
Polypeptide 553Gly Ser Arg His Asp Ser Val Phe Lys 1 5
5549PRTArtificial SequenceSynthetic Polypeptide 554Lys Glu
Val Tyr Leu His Thr Ser Pro 1 5
5559PRTArtificial SequenceSynthetic Polypeptide 555Gln Gly Ser Arg His
Asp Ser Val Phe 1 5 5569PRTArtificial
SequenceSynthetic Polypeptide 556Ser Arg His Asp Ser Val Phe Lys Lys 1
5 5579PRTArtificial SequenceSynthetic
Polypeptide 557Ser Val Phe Lys Lys Glu Val Tyr Leu 1 5
5589PRTArtificial SequenceSynthetic Polypeptide 558Val Tyr
Leu His Thr Ser Pro His Leu 1 5
5599PRTArtificial SequenceSynthetic Polypeptide 559Tyr Leu His Thr Ser
Pro His Leu Lys 1 5 56010PRTArtificial
SequenceSynthetic Polypeptide 560Asp Gln Gly Ser Arg His Asp Ser Val Phe
1 5 10 56110PRTArtificial
SequenceSynthetic Polypeptide 561Glu Val Tyr Leu His Thr Ser Pro His Leu
1 5 10 56210PRTArtificial
SequenceSynthetic Polypeptide 562Gly Ser Arg His Asp Ser Val Phe Lys Lys
1 5 10 56310PRTArtificial
SequenceSynthetic Polypeptide 563Arg His Asp Ser Val Phe Lys Lys Glu Val
1 5 10 56410PRTArtificial
SequenceSynthetic Polypeptide 564Tyr Leu His Thr Ser Pro His Leu Lys Ala
1 5 10 56511PRTArtificial
SequenceSynthetic Polypeptide 565Glu Val Tyr Leu His Thr Ser Pro His Leu
Lys 1 5 10 56611PRTArtificial
SequenceSynthetic Polypeptide 566Lys Glu Val Tyr Leu His Thr Ser Pro His
Leu 1 5 10 56711PRTArtificial
SequenceSynthetic Polypeptide 567Arg Asp Gln Gly Ser Arg His Asp Ser Val
Phe 1 5 10 56811PRTArtificial
SequenceSynthetic Polypeptide 568Ser Arg His Asp Ser Val Phe Lys Lys Glu
Val 1 5 10 56921PRTArtificial
SequenceSynthetic Polypeptide 569Ala Ser Glu Glu Thr Tyr Leu Ser His Leu
Glu Ala Leu Leu Leu Lys 1 5 10
15 Pro Phe Ser Gly Gln 20 57021PRTArtificial
SequenceSynthetic Polypeptide 570Glu Ile Thr Pro Arg Arg Gln Ser Met Thr
Val Lys Lys Gly Glu Lys 1 5 10
15 Pro Phe Ser Gly Gln 20 57130PRTArtificial
SequenceSynthetic Polypeptide 571Glu Ile Thr Pro Arg Arg Gln Ser Met Thr
Val Lys Lys Gly Glu Ser 1 5 10
15 Tyr Thr Phe Leu Ile Ser Ser Asp Tyr Glu Arg Ala Glu Trp
20 25 30 57230PRTArtificial
SequenceSynthetic Polypeptide 572Gly Val Ala Thr Asp Ile Gln Ala Leu Lys
Ala Ala Phe Asp Val Lys 1 5 10
15 Ala Leu Gln Arg Pro Val Ala Ser Asp Phe Glu Pro Gln Gly
20 25 30 57330PRTArtificial
SequenceSynthetic Polypeptide 573Gly Val Ala Thr Asp Ile Gln Ala Leu Lys
Ala Ala Phe Asp Val Arg 1 5 10
15 Pro Ser Pro Cys Glu Asp Arg His Trp Pro Gly Leu Ala Leu
20 25 30 57430PRTArtificial
SequenceSynthetic Polypeptide 574Lys Leu Gln Thr Val His Ser Ile Pro Leu
Thr Ile Asn Lys Glu Ala 1 5 10
15 Gly Ser Ile Glu Ala Leu Gln Arg Pro Val Ala Ser Asp Phe
20 25 30 57530PRTArtificial
SequenceSynthetic Polypeptide 575Lys Leu Gln Thr Val His Ser Ile Pro Leu
Thr Ile Asn Lys Glu Glu 1 5 10
15 Ala Leu Gln Arg Pro Val Ala Ser Asp Phe Glu Pro Gln Gly
20 25 30 57630PRTArtificial
SequenceSynthetic Polypeptide 576Lys Leu Gln Thr Val His Ser Ile Pro Leu
Thr Ile Asn Lys Glu Gly 1 5 10
15 Glu Lys Leu Arg Val Leu Gly Tyr Asn His Asn Gly Glu Trp
20 25 30 57721PRTArtificial
SequenceSynthetic Polypeptide 577Lys Thr Arg Val Tyr Arg Asp Thr Ala Glu
Pro Asn Trp Asn Glu Lys 1 5 10
15 Pro Phe Ser Gly Gln 20 57830PRTArtificial
SequenceSynthetic Polypeptide 578Lys Thr Arg Val Tyr Arg Asp Thr Ala Glu
Pro Asn Trp Asn Glu Leu 1 5 10
15 Asp Pro Gln Ala Leu Gln Asp Arg Asp Trp Gln Arg Thr Val
20 25 30 57930PRTArtificial
SequenceSynthetic Polypeptide 579Leu Asn Val Ile Val His Ser Ala Thr Gly
Phe Lys Gln Ser Ser Lys 1 5 10
15 Ala Leu Gln Arg Pro Val Ala Ser Asp Phe Glu Pro Gln Gly
20 25 30 58030PRTArtificial
SequenceSynthetic Polypeptide 580Leu Asn Val Ile Val His Ser Ala Thr Gly
Phe Lys Gln Ser Ser Ser 1 5 10
15 Glu Lys Leu Arg Val Leu Gly Tyr Asn His Asn Gly Glu Trp
20 25 30 58130PRTArtificial
SequenceSynthetic Polypeptide 581Gln Ile Trp Pro Asn Asp Gly Glu Gly Ala
Phe His Gly Asp Ala Glu 1 5 10
15 Ala Leu Gln Arg Pro Val Ala Ser Asp Phe Glu Pro Gln Gly
20 25 30 58230PRTArtificial
SequenceSynthetic Polypeptide 582Gln Ile Trp Pro Asn Asp Gly Glu Gly Ala
Phe His Gly Asp Ala Gly 1 5 10
15 Glu Lys Leu Arg Val Leu Gly Tyr Asn His Asn Gly Glu Trp
20 25 30 58330PRTArtificial
SequenceSynthetic Polypeptide 583Gln Trp Ser His Gln Gln Arg Val Gly Asp
Leu Phe Gln Lys Leu Asn 1 5 10
15 Leu Arg Ala Arg Ser Asn Lys Asp Ala Lys Asp Pro Thr Thr
20 25 30 58424PRTArtificial
SequenceSynthetic Polypeptide 584Arg Lys Gln Thr Gly Val Phe Gly Val Lys
Ile Ala Val Val Thr Lys 1 5 10
15 Ser Pro Ser Ala Ala Ser Ser Ile 20
58530PRTArtificial SequenceSynthetic Polypeptide 585Ser Asn Lys Asp
Ala Lys Asp Pro Thr Thr Lys Asn Ser Leu Glu Lys 1 5
10 15 Ala Leu Gln Arg Pro Val Ala Ser Asp
Phe Glu Pro Gln Gly 20 25
30 58624PRTArtificial SequenceSynthetic Polypeptide 586Ser Arg Asp Ala
Leu Val Ser Gly Ala Leu Glu Ser Thr Lys Ala Thr 1 5
10 15 Lys Ala Lys Pro Phe Ser Gly Gln
20 587498PRTArtificial SequenceSynthetic
Polypeptide 587Met Ala Ser Thr Asp Tyr Ser Thr Tyr Ser Gln Ala Ala Ala
Gln Gln 1 5 10 15
Gly Tyr Ser Ala Tyr Thr Ala Gln Pro Thr Gln Gly Tyr Ala Gln Thr
20 25 30 Thr Gln Ala Tyr Gly
Gln Gln Ser Tyr Gly Thr Tyr Gly Gln Pro Thr 35
40 45 Asp Val Ser Tyr Thr Gln Ala Gln Thr
Thr Ala Thr Tyr Gly Gln Thr 50 55
60 Ala Tyr Ala Thr Ser Tyr Gly Gln Pro Pro Thr Gly Tyr
Thr Thr Pro 65 70 75
80 Thr Ala Pro Gln Ala Tyr Ser Gln Pro Val Gln Gly Tyr Gly Thr Gly
85 90 95 Ala Tyr Asp Thr
Thr Thr Ala Thr Val Thr Thr Thr Gln Ala Ser Tyr 100
105 110 Ala Ala Gln Ser Ala Tyr Gly Thr Gln
Pro Ala Tyr Pro Ala Tyr Gly 115 120
125 Gln Gln Pro Ala Ala Thr Ala Pro Thr Arg Pro Gln Asp Gly
Asn Lys 130 135 140
Pro Thr Glu Thr Ser Gln Pro Gln Ser Ser Thr Gly Gly Tyr Asn Gln 145
150 155 160 Pro Ser Leu Gly Tyr
Gly Gln Ser Asn Tyr Ser Tyr Pro Gln Val Pro 165
170 175 Gly Ser Tyr Pro Met Gln Pro Val Thr Ala
Pro Pro Ser Tyr Pro Pro 180 185
190 Thr Ser Tyr Ser Ser Thr Gln Pro Thr Ser Tyr Asp Gln Ser Ser
Tyr 195 200 205 Ser
Gln Gln Asn Thr Tyr Gly Gln Pro Ser Ser Tyr Gly Gln Gln Ser 210
215 220 Ser Tyr Gly Gln Gln Ser
Ser Tyr Gly Gln Gln Pro Pro Thr Ser Tyr 225 230
235 240 Pro Pro Gln Thr Gly Ser Tyr Ser Gln Ala Pro
Ser Gln Tyr Ser Gln 245 250
255 Gln Ser Ser Ser Tyr Gly Gln Gln Asn Pro Ser Tyr Asp Ser Val Arg
260 265 270 Arg Gly
Ala Trp Gly Asn Asn Met Asn Ser Gly Leu Asn Lys Ser Pro 275
280 285 Pro Leu Gly Gly Ala Gln Thr
Ile Ser Lys Asn Thr Glu Gln Arg Pro 290 295
300 Gln Pro Asp Pro Tyr Gln Ile Leu Gly Pro Thr Ser
Ser Arg Leu Ala 305 310 315
320 Asn Pro Gly Ser Gly Gln Ile Gln Leu Trp Gln Phe Leu Leu Glu Leu
325 330 335 Leu Ser Asp
Ser Ala Asn Ala Ser Cys Ile Thr Trp Glu Gly Thr Asn 340
345 350 Gly Glu Phe Lys Met Thr Asp Pro
Asp Glu Val Ala Arg Arg Trp Gly 355 360
365 Glu Arg Lys Ser Lys Pro Asn Met Asn Tyr Asp Lys Leu
Ser Arg Ala 370 375 380
Leu Arg Tyr Tyr Tyr Asp Lys Asn Ile Met Thr Lys Val His Gly Lys 385
390 395 400 Arg Tyr Ala Tyr
Lys Phe Asp Phe His Gly Ile Ala Gln Ala Leu Gln 405
410 415 Pro His Pro Thr Glu Ser Ser Met Tyr
Lys Tyr Pro Ser Asp Ile Ser 420 425
430 Tyr Met Pro Ser Tyr His Ala His Gln Gln Lys Val Asn Phe
Val Pro 435 440 445
Pro His Pro Ser Ser Met Pro Val Thr Ser Ser Ser Phe Phe Gly Ala 450
455 460 Ala Ser Gln Tyr Trp
Thr Ser Pro Thr Gly Gly Ile Tyr Pro Asn Pro 465 470
475 480 Asn Val Pro Arg His Pro Asn Thr His Val
Pro Ser His Leu Gly Ser 485 490
495 Tyr Tyr 588520PRTArtificial SequenceSynthetic Polypeptide
588Met Ala Ser Thr Asp Tyr Ser Thr Tyr Ser Gln Ala Ala Ala Gln Gln 1
5 10 15 Gly Tyr Ser Ala
Tyr Thr Ala Gln Pro Thr Gln Gly Tyr Ala Gln Thr 20
25 30 Thr Gln Ala Tyr Gly Gln Gln Ser Tyr
Gly Thr Tyr Gly Gln Pro Thr 35 40
45 Asp Val Ser Tyr Thr Gln Ala Gln Thr Thr Ala Thr Tyr Gly
Gln Thr 50 55 60
Ala Tyr Ala Thr Ser Tyr Gly Gln Pro Pro Thr Gly Tyr Thr Thr Pro 65
70 75 80 Thr Ala Pro Gln Ala
Tyr Ser Gln Pro Val Gln Gly Tyr Gly Thr Gly 85
90 95 Ala Tyr Asp Thr Thr Thr Ala Thr Val Thr
Thr Thr Gln Ala Ser Tyr 100 105
110 Ala Ala Gln Ser Ala Tyr Gly Thr Gln Pro Ala Tyr Pro Ala Tyr
Gly 115 120 125 Gln
Gln Pro Ala Ala Thr Ala Pro Thr Arg Pro Gln Asp Gly Asn Lys 130
135 140 Pro Thr Glu Thr Ser Gln
Pro Gln Ser Ser Thr Gly Gly Tyr Asn Gln 145 150
155 160 Pro Ser Leu Gly Tyr Gly Gln Ser Asn Tyr Ser
Tyr Pro Gln Val Pro 165 170
175 Gly Ser Tyr Pro Met Gln Pro Val Thr Ala Pro Pro Ser Tyr Pro Pro
180 185 190 Thr Ser
Tyr Ser Ser Thr Gln Pro Thr Ser Tyr Asp Gln Ser Ser Tyr 195
200 205 Ser Gln Gln Asn Thr Tyr Gly
Gln Pro Ser Ser Tyr Gly Gln Gln Ser 210 215
220 Ser Tyr Gly Gln Gln Ser Ser Tyr Gly Gln Gln Pro
Pro Thr Ser Tyr 225 230 235
240 Pro Pro Gln Thr Gly Ser Tyr Ser Gln Ala Pro Ser Gln Tyr Ser Gln
245 250 255 Gln Ser Ser
Ser Tyr Gly Gln Gln Ser Ser Leu Leu Ala Tyr Asn Thr 260
265 270 Thr Ser His Thr Asp Gln Ser Ser
Arg Leu Ser Val Lys Glu Asp Pro 275 280
285 Ser Tyr Asp Ser Val Arg Arg Gly Ala Trp Gly Asn Asn
Met Asn Ser 290 295 300
Gly Leu Asn Lys Ser Pro Pro Leu Gly Gly Ala Gln Thr Ile Ser Lys 305
310 315 320 Asn Thr Glu Gln
Arg Pro Gln Pro Asp Pro Tyr Gln Ile Leu Gly Pro 325
330 335 Thr Ser Ser Arg Leu Ala Asn Pro Gly
Ser Gly Gln Ile Gln Leu Trp 340 345
350 Gln Phe Leu Leu Glu Leu Leu Ser Asp Ser Ala Asn Ala Ser
Cys Ile 355 360 365
Thr Trp Glu Gly Thr Asn Gly Glu Phe Lys Met Thr Asp Pro Asp Glu 370
375 380 Val Ala Arg Arg Trp
Gly Glu Arg Lys Ser Lys Pro Asn Met Asn Tyr 385 390
395 400 Asp Lys Leu Ser Arg Ala Leu Arg Tyr Tyr
Tyr Asp Lys Asn Ile Met 405 410
415 Thr Lys Val His Gly Lys Arg Tyr Ala Tyr Lys Phe Asp Phe His
Gly 420 425 430 Ile
Ala Gln Ala Leu Gln Pro His Pro Thr Glu Ser Ser Met Tyr Lys 435
440 445 Tyr Pro Ser Asp Ile Ser
Tyr Met Pro Ser Tyr His Ala His Gln Gln 450 455
460 Lys Val Asn Phe Val Pro Pro His Pro Ser Ser
Met Pro Val Thr Ser 465 470 475
480 Ser Ser Phe Phe Gly Ala Ala Ser Gln Tyr Trp Thr Ser Pro Thr Gly
485 490 495 Gly Ile
Tyr Pro Asn Pro Asn Val Pro Arg His Pro Asn Thr His Val 500
505 510 Pro Ser His Leu Gly Ser Tyr
Tyr 515 520 58920PRTArtificial SequenceSynthetic
Polypeptide 589Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Asn Pro Ser Tyr
Asp Ser 1 5 10 15
Val Arg Arg Gly 20 59020PRTArtificial SequenceSynthetic
Polypeptide 590Ser Gln Gln Ser Ser Ser Tyr Gly Gln Gln Ser Ser Leu Leu
Ala Tyr 1 5 10 15
Asn Thr Thr Ser 20 59110PRTArtificial SequenceSynthetic
Polypeptide 591Ser Ser Tyr Gly Gln Gln Asn Pro Ser Tyr 1 5
10 5929PRTArtificial SequenceSynthetic Polypeptide
592Ser Tyr Gly Gln Gln Asn Pro Ser Tyr 1 5
59310PRTArtificial SequenceSynthetic Polypeptide 593Ser Ser Tyr Gly Gln
Gln Asn Pro Ser Tyr 1 5 10
5948PRTArtificial SequenceSynthetic Polypeptide 594Tyr Gly Gln Gln Asn
Pro Ser Tyr 1 5 5958PRTArtificial
SequenceSynthetic Polypeptide 595Asn Pro Ser Tyr Asp Ser Val Arg 1
5 5969PRTArtificial SequenceSynthetic Polypeptide
596Gln Gln Asn Pro Ser Tyr Asp Ser Val 1 5
5979PRTArtificial SequenceSynthetic Polypeptide 597Asn Pro Ser Tyr Asp
Ser Val Arg Arg 1 5 5988PRTArtificial
SequenceSynthetic Polypeptide 598Tyr Gly Gln Gln Asn Pro Ser Tyr 1
5 59910PRTArtificial SequenceSynthetic Polypeptide
599Gln Gln Asn Pro Ser Tyr Asp Ser Val Arg 1 5
10 6009PRTArtificial SequenceSynthetic Polypeptide 600Gly Gln Gln
Ser Ser Leu Leu Ala Tyr 1 5
6018PRTArtificial SequenceSynthetic Polypeptide 601Gln Gln Ser Ser Leu
Leu Ala Tyr 1 5 60210PRTArtificial
SequenceSynthetic Polypeptide 602Ser Ser Ser Tyr Gly Gln Gln Ser Ser Leu
1 5 10 6039PRTArtificial
SequenceSynthetic Polypeptide 603Ser Ser Tyr Gly Gln Gln Ser Ser Leu 1
5 6049PRTArtificial SequenceSynthetic
Polypeptide 604Ser Ser Tyr Gly Gln Gln Ser Ser Leu 1 5
60510PRTArtificial SequenceSynthetic Polypeptide 605Ser Ser
Tyr Gly Gln Gln Ser Ser Leu Leu 1 5 10
6068PRTArtificial SequenceSynthetic Polypeptide 606Ser Tyr Gly Gln Gln
Ser Ser Leu 1 5 6079PRTArtificial
SequenceSynthetic Polypeptide 607Ser Tyr Gly Gln Gln Ser Ser Leu Leu 1
5 6088PRTArtificial SequenceSynthetic
Polypeptide 608Tyr Gly Gln Gln Ser Ser Leu Leu 1 5
60910PRTArtificial SequenceSynthetic Polypeptide 609Tyr Gly Gln Gln
Ser Ser Leu Leu Ala Tyr 1 5 10
61021PRTArtificial SequenceSynthetic Polypeptide 610His Ser Ala Thr Gly
Phe Lys Gln Ser Ser Lys Ala Leu Gln Arg Pro 1 5
10 15 Val Ala Ser Asp Phe 20
61120PRTArtificial SequenceSynthetic Polypeptide 611His Ser Ile Pro Leu
Thr Ile Asn Lys Glu Glu Ala Leu Gln Arg Pro 1 5
10 15 Val Ala Ser Asp 20
6128PRTArtificial SequenceSynthetic Polypeptide 612Ile Thr Tyr Thr Trp
Thr Arg Leu 1 5 6138PRTArtificial
SequenceSynthetic Polypeptide 613Val Gly Phe Asn Phe Arg Thr Leu 1
5 61420PRTArtificial SequenceSynthetic Polypeptide
614Pro Cys Val Pro Ala Lys Pro Pro Leu Ala Gly Ser Pro Gln Ile Glu 1
5 10 15 Asn Ile Gln Pro
20 61520PRTArtificial SequenceSynthetic Polypeptide 615Ser
Asp Arg Gly Gly Phe Asn Lys Phe Gly Gly Ser Gly Gln Ile Gln 1
5 10 15 Leu Trp Gln Phe
20
User Contributions:
Comment about this patent or add new information about this topic: