CN109887553B - Polypeptide vaccine aiming at tumor targeted drug resistance site and design method thereof - Google Patents

Polypeptide vaccine aiming at tumor targeted drug resistance site and design method thereof Download PDF

Info

Publication number
CN109887553B
CN109887553B CN201910086371.9A CN201910086371A CN109887553B CN 109887553 B CN109887553 B CN 109887553B CN 201910086371 A CN201910086371 A CN 201910086371A CN 109887553 B CN109887553 B CN 109887553B
Authority
CN
China
Prior art keywords
gly
leu
lys
ser
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910086371.9A
Other languages
Chinese (zh)
Other versions
CN109887553A (en
Inventor
莫凡
马治明
林志伟
韩宁
陈荣昌
周秀卿
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hangzhou Neoantigen Biotechnology Co ltd
Original Assignee
Hangzhou Neoantigen Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to CN202011331358.4A priority Critical patent/CN112687354B/en
Priority to CN202011328452.4A priority patent/CN112767998A/en
Priority to CN201910086371.9A priority patent/CN109887553B/en
Priority to CN202011331275.5A priority patent/CN112687353B/en
Priority to CN202011328455.8A priority patent/CN112687352B/en
Application filed by Hangzhou Neoantigen Biotechnology Co ltd filed Critical Hangzhou Neoantigen Biotechnology Co ltd
Priority to CN202011331322.6A priority patent/CN112687325B/en
Priority to CN202011328459.6A priority patent/CN112675297B/en
Publication of CN109887553A publication Critical patent/CN109887553A/en
Application granted granted Critical
Publication of CN109887553B publication Critical patent/CN109887553B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/30—Detection of binding sites or motifs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/50—Mutagenesis
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/50—Molecular design, e.g. of drugs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/70—Machine learning, data mining or chemometrics
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a polypeptide vaccine aiming at tumor targeted drug resistant sites and a design method thereof, wherein the design method comprises the following steps: collecting drug resistance mutation data of the targeted drug; intercepting a drug-resistant mutant polypeptide sequence, and intercepting and predicting the affinity and immunogenicity of MHC molecules; the correlation between the targeted drug and the drug-resistant site and the clustering of the drug; artificially designing a corresponding vaccine polypeptide sequence; the targeted drug and polypeptide vaccine combination obtained by the design method can cover common targeted therapeutic drugs, can effectively reduce the occurrence probability of tumor drug resistance, prolong the effective action time of the targeted drug, increase the disease response rate of patients, has broad spectrum, shortens the time from analysis to treatment of individualized polypeptide vaccines, and reduces the medical cost.

Description

Polypeptide vaccine aiming at tumor targeted drug resistance site and design method thereof
Technical Field
The invention relates to the field of tumor vaccines, in particular to a polypeptide vaccine aiming at tumor targeted drug resistant sites and a design method thereof.
Background
In recent years, the progress of tumor-targeted therapy has entered a completely new era with the development of molecular biological techniques and further understanding of the pathogenesis from the cellular and molecular level. These fields are rapidly progressing and to date, many targeted drugs have played an extremely important and even fantastic role in the clinic. Molecular targeted therapy has become another important means for tumor therapy such as surgery, radiotherapy, chemotherapy and the like, and plays an increasingly important role in tumor therapy. The advantages over traditional therapies and chemotherapy are evident due to the high efficiency and low toxicity of targeted therapies. Targeted therapies have developed rapidly, and some have entered standard treatment protocols and specifications recognized by the international oncology community according to evidence-based medical principles. More and promising drugs are also under development in Haimaicha and early clinical trials.
The targeted medicine has become a great tool for treating tumors. However, with longer and longer treatment times, resistance occurs in most patients. As for the specific mechanism of drug resistance, there are currently 3 main types. First, drug resistance is generated by gene mutation. About 40% of the genes in the patients who are positive in the gene test result in new genes from the original genes, which results in insensitivity to the original drugs and thus drug resistance. Secondly, subtle cancer cells often make a 'trestle dark store' and go around to bend another way. This condition accounts for about 20% of patients with drug resistance. In addition to the above two resistance routes, the resistance mechanism of about 30% of patients remains unclear.
The "change" of target (resistance) is inevitable and almost all targeted drug therapies exhibit resistance, the root cause of which is due to heterogeneity and dynamic changes of cancer cells. At present, a targeting drug acts on a certain protein and a certain molecule of cancer cells, so that only one pathway of tumor growth can be inhibited. When one pathway is inhibited, tumor cells can self-seek a new 'birth pathway', other pathways are selected to synthesize substances required for self growth, and the molecular targeted drug loses the effect over time, so that drug resistance is generated, for example, after EGFR mutation patients take the targeted drug, 50% -60% of patients have drug resistance because of genetic mutation again, most of the mutations are T790M mutations, and at the moment, the patients can use third generation targeted drugs such as Ostinib. Most patients will have secondary drug resistance 8-14 months after receiving EGFR-TKI treatment, and how to solve the drug resistance problem of targeted drug treatment becomes a hotspot of research.
In recent years, the research of tumor polypeptide vaccines has been favored due to the discovery of tumor-associated antigens and tumor-specific antigens, as well as the intensive research and understanding of tumor-induced immune responses and tumor evasion immune surveillance mechanisms. The neoantigen is abnormal protein expressed on the surface of tumor cell caused by gene mutation, and is different from conventional antigen target pointThe antigen is expressed only on the surface of tumor cells and not on the surface of normal cells and can be recognized by and activate the immune system. 7/13.2017, the same day as the Nature journal issued two successful cases of treating malignant melanoma based on neoantigen's personalized tumor vaccine. Professor Carmen Loquai in Germany and
Figure BDA0001961892220000021
professor Tureci used the RNA corresponding to neoantigen as a vaccine to treat 13 patients in total. On the same day, the case of treating malignant melanoma was also successfully reported by the team led by the professor of cathine j.wu of harvard university, using the antigenic peptide corresponding to neoantigen as a vaccine.
The general design process of drug-resistant site polypeptide vaccine is to find out the mutation site leading to the drug resistance of the target drug, analyze the neoantigen generated by these mutations, and prepare the polypeptide vaccine capable of being specifically recognized by the histocompatibility complex (MHC) of the patient. The polypeptide vaccine is injected into a patient body to activate specific T cell response and immune storm, so that the patient's own immune system can effectively identify and kill the tumor cells with drug-resistant mutation.
The polypeptide vaccine and the targeting drug are combined, on one hand, the targeting drug can kill tumor cells carrying drug target spots, on the other hand, an organism immune system activated by the polypeptide vaccine can be effectively identified and cleared just before the drug-resistant tumor cells appear, so that the action time of the targeting drug is prolonged, the activated immune system can keep the killing effect on the tumor cells for a long time in vivo, the drug-resistant phenomenon is continuously inhibited, and the drug resistance of the targeting drug generated by drug-resistant mutation is overcome to a certain extent.
The market needs a set of polypeptide vaccine screening and designing methods aiming at tumor-targeted drug-resistant sites, can effectively reduce the occurrence probability of tumor drug resistance, has broad spectrum, shortens the time from analysis to treatment of individualized polypeptide vaccines, and reduces the medical cost; the present invention solves such problems.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a polypeptide vaccine aiming at a tumor targeted drug resistance site and a design method thereof, the provided targeted drug and polypeptide vaccine combination scheme covers common targeted therapeutic drugs, can effectively reduce the occurrence probability of tumor drug resistance, prolong the effective action time of the targeted drug, increase the disease response rate of patients, has broad spectrum, shortens the time from analysis to treatment of an individual polypeptide vaccine, and reduces the medical cost.
In order to achieve the above object, the present invention adopts the following technical solutions:
the polypeptide vaccine aiming at the tumor targeted drug resistance site is characterized in that aiming at the targeted drug, namely the vismodegib, the polypeptide vaccine combination aims at the following mutations: SMO-A459V, SMO-C469Y, SMO-D473G, SMO-D473H, SMO-D473Y, SMO-F460L, SMO-G497W, SMO-H231R, SMO-Q477E, SMO-T241M, SMO-V321A, SMO-V321M, SMO-W281L, SMO-W535L and SMO-W535R.
The polypeptide vaccine aiming at the tumor targeted drug resistance site is characterized in that aiming at the targeted drug ibrutinib, the polypeptide vaccine combination aims at the following mutations: BTK-C481F, BTK-C481R, BTK-C481S, BTK-C481Y and BTK-T316A.
The polypeptide vaccine aiming at the tumor targeted drug resistant site is characterized in that targeted drugs of abiraterone, enzalutamide, Flutamide and Ketoconazole are polymerized into one class,
the polypeptide vaccine combination of abiraterone was directed against the following mutations: AR-T878A, AR-T878S;
polypeptide vaccine combinations of enzalutamide are directed against the following mutations: AR-F877L, AR-T878A;
polypeptide vaccine combinations of flutamide are directed against the following mutations: AR-T878A, AR-T878S, AR-V716M;
the polypeptide vaccine of ketoconazole is directed against the following mutations: AR-T878A;
the polypeptide vaccine aiming at the tumor targeted drug resistance site is characterized in that targeted drugs such as dabrafenib, pembrolizumab, vemurafenib and semetinib are gathered into one class,
the polypeptide vaccine combination of dabrafenib is directed against the following mutations: MAP2K1-P124S, MAP2K2-Q60P, NRAS-G12D, NRAS-Q61R;
the polypeptide vaccine of pembrolizumab is directed against the following mutations: NRAS-Q61R;
the polypeptide vaccine combination of vemurafenib is directed against the following mutations: MAP2K1-G128V, MAP2K1-P124L, MAP2K1-Q56P, NRAS-Q61R;
the polypeptide vaccine of sematinib is directed against the following mutations: MAP2K 1-P124L.
The polypeptide vaccine aiming at the tumor targeted drug resistant site is characterized in that targeted drugs of Alranitinib, ceritinib and crizotinib are gathered into one class,
polypeptide vaccines for erlotinib are directed to the following mutations: ALK-G1202R;
polypeptide vaccine combinations of ceritinib are directed against the following mutations: ALK-D1203N, ALK-F1174V, ALK-G1123S, ALK-G1202R, ALK-G126 1269A, ALK-L1196M;
the polypeptide vaccine combination of crizotinib is directed against the following mutations: ALK-F1174L, ALK-F1174V, ALK-G1202R, ALK-G1269A, ALK-I1171T, ALK-L1196M, MET-D1246H, MET-D1246N.
Aiming at the polypeptide vaccine of the tumor targeted drug resistant site, targeted drugs Afatinib, bosutinib, dasatinib, erlotinib, gefitinib, lotinib, imatinib, nilotinib, Ostinib, sorafenib, sunitinib, tyrosine kinase inhibitor drugs, tevatinib and quinatinib are polymerized into one class,
the polypeptide vaccine of afatinib is directed against the following mutations: EGFR-T790M;
the polypeptide vaccine combination of bosutinib is directed against the following mutations: ABL1-F359V, ABL 1-V299L;
the polypeptide vaccine combination of dasatinib is directed against the following mutations: ABL1-E255K, ABL1-E255K, ABL1-F317L, ABL1-F359V, ABL1-T315A, ABL1-T315I, ABL 1-V299L;
the polypeptide vaccine of erlotinib is directed against the following mutations: EGFR-T790M;
the polypeptide vaccine combination of gefitinib is directed to the following mutations: EGFR-D761Y, EGFR-T790M;
lotinib is directed to the following mutations: EGFR-C797S;
the polypeptide vaccine combination of imatinib is directed to the following mutations: ABL1-A397P, ABL1-A399T, ABL1-A433T, ABL1-E255K, ABL1-E255V, ABL1-E275K, ABL1-E279A, ABL1-E279 1, ABL1-E279 1, ABL1-E282, ABL1-E292 1, ABL1-E352 1, ABL1-E352 1, ABL1-E352 1, ABL1-E355 1, ABL1-E355 1, ABL1-E355 1, ABL1-E450 1, ABL1-E450 36453-1, ABL 1-ABL 1, ABL 1-ABL 1-1, ABL 1-1, ABL 36453-1, ABL 1-ABL 1-1, ABL 1-36453-1, ABL 1-ABL 1, ABL 1-1, ABL 1-ABL 1-1, ABL 1-ABL 1, ABL 36, ABL1-P480L, ABL1-Q252E, ABL1-Q252H, ABL1-Q252K, ABL1-Q252R, ABL1-R328M, ABL M-S417M, ABL M-T277M, ABL M-T315M, ABL M-T315M, ABL M-V289M, ABL M-V299M, ABL M-V379M, ABL M-Y253M, ABL M-Y320, KIT-A829M, KIT-D816M, KIT-D816M, KIT-D816M, KIT-D36820, KIT-D M, KIT-D36709, KIT-D M, KIT-D36709, KIT-M, FRA M;
the polypeptide vaccine combination of nilotinib is directed against the following mutations: ABL1-E255K, ABL1-E255V, ABL1-H396R, ABL1-T315I, ABL1-T315V and KIT-N655T;
polypeptide vaccine combinations of ostinib are directed against the following mutations: EGFR-C797S, EGFR-G796D, EGFR-G796R, EGFR-G796S, EGFR-L792H, KIT-T790M;
the polypeptide vaccine combination of sorafenib was directed against the following mutations: FLT3-D835H, FLT 3-D835Y;
the polypeptide vaccine combination of sunitinib is directed against the following mutations: FLT3-D835Y, PDGFRA-D842V;
the polypeptide vaccine combination of the tyrosine kinase inhibitor drugs aims at the following mutations: ABL-A399, ABL-D325, ABL-E255, ABL-E255, ABL-E279, ABL-E282, ABL-E355, ABL-E453, ABL-F311, ABL-F317, ABL-F359, ABL-H396, ABL-H396, ABL-L298, ABL-L384, ABL-L387, ABL-L387, ABL-M244, ABL-M388, ABL-P310, ABL-Q252, ABL-Q252, ABL-T315, ABL-T315, ABL-T495, ABL-V289, ABL-V299, ABL-V338, ABL-V379, ABL-Y253, EGFR-T790;
the polypeptide vaccine of tevatinib is directed against the following mutations: EGFR-T790M;
the polypeptide vaccine combination of quinazatinib is directed against the following mutations: FLT3-D835Y, FLT3-D842V, FLT 3-D835Y.
Aiming at the polypeptide vaccine of the tumor targeted drug resistance site, targeted drugs of everolimus, rapamycin, MEK inhibitor PD0325901 and C-MET inhibitor Savolitinib are gathered into one class,
the polypeptide vaccine for everolimus is directed against the following mutations: MTOR-F2108L;
the polypeptide vaccine of rapamycin is directed against the following mutations: MTOR-F2108L;
the polypeptide vaccine of MEK inhibitor PD0325901 is directed to the following mutations: MAP2K 1-F129L;
polypeptide vaccines of the C-MET inhibitor Savolitinib are directed against the following mutations: MET-D1246V;
the design method of the polypeptide vaccine aiming at the tumor targeted drug resistance site comprises the following steps:
searching data of drug resistance mutation data of the targeted drug;
intercepting a drug-resistant mutant polypeptide sequence and predicting the affinity and immunogenicity of MHC molecules:
intercepting a polypeptide sequence covering 16 amino acids at the upstream and downstream of a mutation site for point mutation, intercepting a polypeptide sequence which extends forwards for 16 amino acids and extends backwards to a stop codon for frameshift mutation to serve as a mutant polypeptide of the drug-resistant mutation site, and intercepting a wild-type polypeptide sequence corresponding to a corresponding position at the same time,
counting the high-frequency HLA types and frequencies by using at least one database as sources, combining and de-duplicating the counted HLA to be used as candidate HLA types for prediction,
the binding affinity of the polypeptide corresponding to the mutation sites and HLA molecules is analyzed by using various types of software, and the affinity is divided into three types by combining the various types of software: strong affinity-SB, weak affinity-WB and no affinity, and determining changes in affinity compared to the relatively wild-type polypeptide,
class a changes from no affinity to strong affinity,
class B changes from no affinity to weak affinity,
class C changes from weak affinity to strong affinity,
the class D change is a no change,
internal ordering considers class a over class B over class C over class D,
the immunogenicity of the mutant polypeptide is predicted by using an immunogenicity prediction tool, and epitopes with strong affinity, large affinity change and strong immunogenicity of the mutant polypeptide are reserved;
the interrelation between the targeted drug and the drug-resistant site and the clustering of the drugs:
scoring the drug resistance site affinity:
comprehensively considering the number of epitopes with affinity of the sites and the change size of the affinity of each epitope, giving corresponding weight to A, B, C, D types of different affinity changes, giving weight according to the HLA frequency size corresponding to each epitope, and accumulating and summing the epitopes of the sites;
Figure BDA0001961892220000051
AC: the magnitude of the affinity change, A, B, C, D four different classes of affinity changes give different weights;
Fhla: a corresponding HLA frequency;
n: the number of each epitope of the locus;
the correlation between the targeted drugs and the drug-resistant sites is comprehensively analyzed, and the targeted drugs are clustered by combining the action mechanism of the targeted drugs, so that the targeted drugs are classified into 7 types:
the first Hedgehog signaling pathway antagonist, vismodegib, which is resistant to SMO mutation,
the second class generates drug-resistant ibrutinib against BTK mutations,
the third group aims at some antiandrogen drugs of AR drug resistant mutation, such as abiraterone and the like,
the fourth group is some phenanthrenib drugs aiming at BRAF drug-resistant mutation,
the fifth group is some tyrosine kinase inhibitors aiming at ALK, MET and other fusion genes,
a sixth class of tyrosine kinase inhibitors directed against the EGFR pathway,
the seventh kinase inhibitor such as everolimus for PI3K/AKT/mTOR pathway;
designing the corresponding vaccine polypeptide sequence.
The design method of the polypeptide vaccine aiming at the tumor targeted drug resistance site,
intercepting a drug-resistant mutant polypeptide sequence and predicting the affinity and immunogenicity of MHC molecules:
intercepting a polypeptide sequence covering 16 amino acids at the upstream and downstream of a mutation site for point mutation, intercepting a polypeptide sequence which extends forwards for 16 amino acids and extends backwards to a stop codon for frameshift mutation to serve as a mutant polypeptide of the drug-resistant mutation site, and intercepting a wild-type polypeptide sequence corresponding to a corresponding position at the same time,
counting high-frequency HLA types and frequencies by using at least one database as sources, combining and de-duplicating the counted HLA to be used as candidate HLA types for prediction, wherein the database comprises: a public database, a clinical patient database,
the binding affinity of the polypeptide corresponding to the mutation sites and HLA molecules is analyzed by using various types of software, and the affinity is divided into three types by combining the various types of software: strong affinity-SB, weak affinity-WB and no affinity and determining changes in affinity compared to a relatively wild-type polypeptide, comprising: three software of netMHCpan, netMHC and Pickpocket,
class a changes from no affinity to strong affinity,
class B changes from no affinity to weak affinity,
class C changes from weak affinity to strong affinity,
the class D change is a no change,
internal ordering considers class a over class B over class C over class D,
the immunogenicity of the mutant polypeptide is predicted by using an immunogenicity prediction tool, and epitopes with strong affinity, large affinity change and strong immunogenicity of the mutant polypeptide are reserved.
The design method of the polypeptide vaccine aiming at the tumor targeted drug resistance site,
using cytoscape to make a network map of all targeted drugs and drug-resistant sites, wherein the size of the drug-resistant sites represents the affinity score of the drug-resistant sites, and comprehensively analyzing the correlation between the targeted drugs and the drug-resistant sites and combining the action mechanism of the targeted drugs to cluster the targeted drugs so as to classify the targeted drugs into 7 types:
the first Hedgehog signaling pathway antagonist, vismodegib, which is resistant to SMO mutation,
the second class generates drug-resistant ibrutinib against BTK mutations,
the third group aims at some antiandrogen drugs of AR drug resistant mutation, such as abiraterone and the like,
the fourth group is some phenanthrenib drugs aiming at BRAF drug-resistant mutation,
the fifth group is some tyrosine kinase inhibitors aiming at ALK, MET and other fusion genes,
a sixth class of tyrosine kinase inhibitors directed against the EGFR pathway,
the seventh group is kinase inhibitors such as everolimus and the like aiming at the PI3K/AKT/mTOR pathway.
The invention has the advantages that:
the combination scheme of the targeted drug and the polypeptide vaccine provided by the invention covers common targeted therapeutic drugs; compared with single targeted therapy, the tumor drug resistance occurrence probability can be effectively reduced, and the effective action time of the targeted drug can be prolonged; the protocol we provide increases the disease response rate of patients compared to polypeptide vaccine alone;
the sequencing of genetic information of a patient and the special determination of HLA typing of the patient are not needed, the method has certain broad spectrum, the time from analysis to treatment of the individualized polypeptide vaccine is greatly shortened, and the medical cost is reduced; in addition, polypeptide vaccines also produce toxic and side effects;
the polypeptide vaccine can keep long-term tumor killing effect, the vaccine peptide is cut into short polypeptide by enzyme in vivo and MHC molecule is presented and is recognized by TCR, the organism can be stimulated to generate specific killing T cell and memory T cell, and the cell carrying mutation is recognized and eliminated, and the effect can be kept in vivo for a long time;
provides 31 polypeptide vaccines of targeted drugs, which basically cover common tumor targeted drugs;
the targeted drugs are classified into 7 categories according to mechanism and drug-resistant mutation clustering analysis, and the multi-target combination can effectively increase the killing effect of the polypeptide vaccine on tumor cells and obviously prolong the effective action time of the targeted drugs.
Drawings
FIG. 1 is a graph of the interaction of a targeted drug and a drug-resistant site of the present invention; the origin represents a targeted drug, the triangle represents a drug-resistant site, the size of the triangle represents the affinity score of the polypeptide corresponding to the site, and the large circle divides the network diagram into 7 parts which represent 7 products;
FIG. 2 is a graph of the number of ELISPOT spots for the negative controls of A-D and 137 peptides of experiment 1 of the present invention;
FIG. 3 is a graph showing the killing efficiency of effector T cells in each group of experiment 2-1 of the present invention;
FIG. 4 is a graph showing the killing efficiency of effector T cells in each group of experiments 2-2 of the present invention;
FIG. 5 is a graph showing the killing efficiency of effector T cells in each of the groups of experiments 2 to 3 of the present invention;
FIG. 6 is a graph showing the killing efficiency of effector T cells in each of the groups of experiments 2 to 4 of the present invention;
FIG. 7 is a graph showing the killing efficiency of effector T cells in each of the groups of experiments 2 to 5 of the present invention;
FIG. 8 is a graph showing the killing efficiency of effector T cells in each of the groups of experiments 2 to 6 of the present invention;
FIG. 9 is a graph showing the killing efficiency of effector T cells in each of the groups of experiments 2 to 7 of the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
The design method of the polypeptide vaccine aiming at the tumor targeted drug resistance site comprises the following steps:
1. collecting the drug resistance mutation data of the targeted drugs: the human cancer-associated somatic mutation catalog Cosmic (database official website https:// cancer. sanger. ac. uk/Cosmic) collates and notes reports of acquired resistance and primary resistance in clinical studies, including information of genes, mutations, drugs, cancer species and references. We downloaded all the drug resistance mutation sites of targeted therapeutic drugs approved by FDA or CFDA or in clinical trial stage III/IV, especially the mutations with less supported sample size for all candidate targeted drugs and drug resistance mutations, further searched literature to determine their reliability, and determined truly comprehensive and reliable targeted drug resistance sites for downstream analysis, for example cosmic included a report confirming that EGFR-T790M mutation is an austenitinib resistance site, but found through the searched literature that T790M is not an austenitib resistance site.
2. Intercepting a drug-resistant mutant polypeptide sequence and predicting MHC molecule affinity and immunogenicity:
2.1, intercepting a polypeptide sequence covering 16 amino acids at the upstream and downstream of a mutation site for point mutation, intercepting a polypeptide sequence which extends forwards for 16 amino acids and extends backwards to a stop codon for frameshift mutation to serve as a mutant polypeptide of the drug-resistant mutation site, and intercepting a wild-type polypeptide sequence corresponding to a corresponding position.
2.2, counting high-frequency HLA types and frequencies: the source of the high-frequency HLA mainly comprises two parts, namely statistics (IMGT/HLA and Chinese marrow bank) from public databases, combines information reported by recent literature, and summarizes common HLA allelic gene typing of Chinese population; secondly, a group of HLA types with higher occurrence frequency is counted from the database of clinical patients, and the specific database is shown in Table 2. The two groups of HLA were pooled and de-duplicated before being used as candidate HLA typing for prediction. It should be noted that the choice of the database is not limited, and this is only a preferred embodiment, and may also be a combination statistic of several other databases.
2.3, the binding affinities of the polypeptides corresponding to the mutation sites and the HLA molecules obtained in the step 2.2 are analyzed by using three software of netMHCpan, netMHC and Pickpocksocket, the three software are synthesized to divide the affinities into three classes (strong affinity-SB, weak affinity-WB and no affinity), and the affinity changes are determined by comparing with the relative wild-type polypeptides (no affinity is changed into strong affinity to define the class A change, no affinity is changed into weak affinity to define the class B change, weak affinity is changed into strong affinity to define the class C change, other no change is defined as the class D change, and the internal ordering considers that the class A is superior to the class C and is superior to the class D), in addition, an immunogenicity prediction tool provided by IEDB is used for predicting the immunogenicity, epitopes with strong affinity, large affinity change and strong immunogenicity of mutant polypeptides are reserved, and 137 drug-resistant mutations corresponding to 31 targeted drugs are obtained through co-screening.
3. The interrelation between the targeted drug and the drug-resistant site and the clustering of the drugs:
3.1 scoring the affinity of the drug-resistant site:
comprehensively considering the number of epitopes with affinity of the site and the change size of the affinity of each epitope (a, giving different weights to A, B, C, D four types of different affinity changes, b, giving weights according to the HLA frequency size corresponding to each epitope, and c, accumulating and summing the epitopes of the site).
Figure BDA0001961892220000091
AC: the magnitude of the affinity change, A, B, C, D four different classes of affinity changes give different weights;
Fhla: a corresponding HLA frequency;
n: the number of each epitope of the locus;
3.2 using cytoscape to make a network map (as shown in figure 1) of all the targeted drugs and the drug-resistant sites, wherein the size of the drug-resistant sites represents the affinity scores of the drug-resistant sites, and the mutual relation between the targeted drugs and the drug-resistant sites is comprehensively analyzed, and the targeted drugs are clustered by combining the action mechanism of the targeted drugs, so that the targeted drugs are divided into 7 types as shown in figure 1.
The drug-resistant Hedgehog signaling pathway antagonist Virungie is generated by aiming at SMO mutation in the first class, drug-resistant ibrutinib is generated by aiming at BTK mutation in the second class, a plurality of antiandrogen drugs such as abiraterone and the like are generated by aiming at AR resistance mutation in the third class, a plurality of phenanthrene drugs for BRAF resistance mutation in the fourth class, a plurality of tyrosine kinase inhibitors for ALK, MET and other fusion genes in the fifth class, a plurality of tyrosine kinase inhibitors for EGFR pathway in the sixth class, and a plurality of kinase inhibitors for everolimus and the like of PI3K/AKT/mTOR pathway in the seventh class.
Because the clustering of the targeted drugs comprehensively considers the interaction between the drug action mechanism and the drug-resistant sites, the targeted drugs with similar action mechanism and cross drug-resistant sites are clustered into a class, thereby maximally covering the drug-resistant sites with immunogenicity of the targeted drugs, and remarkably reducing the phenomenon of 'off-target' caused by the difference of HLA molecules of patients, so that the polypeptide vaccine can kill tumor cells with similar drug-resistant mutations in the maximum range, and the effectiveness of the polypeptide vaccine is remarkably improved compared with that of a single-drug vaccine.
4. Designing a polypeptide vaccine sequence: as an example, the corresponding vaccine polypeptide sequence can be designed manually, or the polypeptide vaccine sequence can be designed by inputting the vaccine design program iNeo-VaDes (V1.2) developed by us. As a polypeptide vaccine therapy aiming at targeted drug resistance, in the design process of vaccine polypeptide, the invention considers the distribution of antigen epitope, covers the epitope containing mutation sites as much as possible, and also considers the factors influencing the amino acid synthesis efficiency such as the length and the hydrophobic rate of the polypeptide and the factors influencing the safety of the polypeptide such as the toxicity and the homology of the polypeptide. The vaccine polypeptide combination of the present invention consists of polypeptide sequences comprising the following 137 mutations, as shown in table 1 below.
5. Preparing a vaccine: the polypeptide vaccine can be prepared by directly using chemical synthesis, can be obtained by transcription and translation by using nucleic acid molecules (such as DNA and RNA), or can be obtained by expression by using bacteria or virus as a vector. In this case we used chemical synthesis to obtain the polypeptide vaccine.
Table 1: targeted drug resistance mutation and polypeptide vaccine sequence
Figure BDA0001961892220000101
Figure BDA0001961892220000111
Figure BDA0001961892220000121
Figure BDA0001961892220000131
Figure BDA0001961892220000141
Table 2: database of clinical patients:
Figure BDA0001961892220000151
Figure BDA0001961892220000161
the invention provides a polypeptide vaccine of 31 targeted drugs, which basically covers common tumor targeted drugs and is used for experimental verification of the immunogenicity of each polypeptide by the following experiments:
experiment 1, determination of immunogenicity of polypeptides;
purpose of the experiment: through ELISpot experiments, the polypeptides corresponding to 7 products in the invention are verified to cause immune response in humanized mice.
The experimental method comprises the following steps:
experimental polypeptides: the 7 product polypeptides are synthesized by Nanjing Jinslei Biotechnology GmbH, the purity of the polypeptides is more than 90%, and the endotoxin content is lower than 0.5 EU/mg.
To detect the immune response of the polypeptide, an IFN- γ Enzyme Linked Immunosorbent (ELISPOT) assay is performed. The detailed experimental procedure is as follows: 24 humanized mice B-NSG (CD34+) were selected at 8 weeks of age and randomized into 8 groups of 3 mice each. After one week of adaptation, the samples were divided into polypeptide group 1 (corresponding to product 1), polypeptide group 2 (corresponding to product 2), polypeptide group 3 (corresponding to product 3) … … polypeptide group 7 (corresponding to product 7), and 7 groups and negative control group No. 8 were counted. CpG was used as adjuvant (0.2. mu.g/mouse), 50. mu.g of polypeptide was added to Freund's incomplete adjuvant (FIA, Sigma-Aldrich) 1:1, mixing and emulsifying for 30 minutes, mixing PBS with Freund's incomplete adjuvant 1:1 mixed emulsification for 30 minutes as a negative control, four times of subcutaneous immunization is carried out on the right chest of the back and the neck, the total dose is 0.5 mL/mouse, once every 1 week for three weeks, and 10 days after the third immunization, the spleen of the mouse is taken to prepare mouse lymphocyte suspension for ELISPOT detection.
In the ELISPOT detection result, the polypeptide with positive IFN-gamma result is determined as the positive candidate polypeptide. Experiment according to groups, the single peptide ELISPOT test is respectively carried out, namely, mouse lymphocyte is diluted to the concentration of 1-2 x 106Laying on 24-well plate, dividing into control group (DMSO with the same concentration as polypeptide) and corresponding single polypeptide group, numbering PHA positive control group (PHA lymphocyte is from negative control group), repeating each treatment 3 times (3 multiple wells), adding corresponding polypeptide (10 μ g/mL), pre-incubating for 72h, centrifuging, and adjusting cell concentration to 2 x 106The resulting spots were then visualized using IFN-. gamma.Elispot plates in accordance with the protocol of the IFN-. gamma.ELISPOT kit, and read using a CTL-ImmunoSpotoS 5 series enzyme-linked spot analyzer. The positive result of IFN-gamma indicates that antigen specific T cells are generated, the polypeptide can be regarded as capable of causing the immune reaction of the organism, and the number of spots reflects the intensity of the immunity.
The experimental results are as follows:
in order to further clarify the immune response of each individual polypeptide, ELISPOT experiments of the corresponding single peptide in each polypeptide group were performed, the number of spots generated by each polypeptide is shown in FIGS. 2A-D, each of which can cause immune response, but the spot difference generated by each polypeptide is relatively large, and varies from 10 spots to 200 spots, while the control group generates substantially no spots.
And (4) analyzing results: the polypeptides corresponding to 7 products in the invention can cause immune response in humanized mice.
Experiment II, the treatment and prevention effects of the drug-resistant polypeptide vaccine of the 7-class targeted drugs are verified;
in order to verify the treatment and prevention effects of the gastric cancer drug-resistant polypeptide vaccine, a set of stable transgenic cell line containing the specific mutation site in the invention needs to be constructed, and the experimental preparation of the following 7 examples is respectively carried out aiming at 7 types of targeted drugs; example 1, construction of stable transgenic cell lines of a first class of Hedgehog signaling pathway antagonist Vismodegib (Vismodegib) specific mutation sites for SMO mutation-conferring resistance:
vismodegib was purchased from LC Laboratories, gastric cancer cell line AGS was purchased from ATCC, FITC-CD44 was purchased from BD corporation (BD 555478). Preparation of cells: gastric carcinoma cell line AGS was cultured in DMEM containing 10% FBS,100U/mL penicilin and 100. mu.g/mL streptomycin, 2mmol/L l-glutamine for 48h, changed and supplemented with 20ng/mL of ofEGF, bFGF, N-2 (1X), and B27, and continued for 72h to promote spheroids ("sphenoid formation media"). Subjecting ellipsoid to Accutase (innovative Cell technologies) enzymolysis to form single Cell, and sorting out positive cells as subsequent transfected cells marked as CD44 with FITC-CD44 flow antibody+-AGS。
Purpose of the experiment: in order to verify that the T cells induced by the gastric cancer drug-resistant polypeptide have killing activity, a set of stable transfer cell lines containing the specific mutation sites in the invention needs to be constructed, and the stable transfer cell lines can express and present the polypeptides
a. Construction of mutant site eukaryotic expression plasmid
The mini-gene capable of expressing all 15 vaccine polypeptides mutated in the invention is obtained by adopting an artificial synthesis method and consists of the following parts: a signal peptide part (lysome-associated membrane glycoprotein 1, LAMP1), 15 mutant polypeptides and MHC class I trafficking domain (MITD), wherein the 15 mutant polypeptides are connected by a flexible connecting peptide GGSGGGGSGG, after codon optimization of the gene, GATATC (EcoR V) is introduced at the upstream, CTCGAG (Xho I) is introduced at the downstream, and the gene is cloned into a eukaryotic expression vector pcDNA3.1-hygro (+), namely smo15-pcDNA3.1 (+). The corresponding wild-type polypeptide was synthesized simultaneously as a control (7 wild-type polypeptides in total because the adjacent polypeptides could be assigned to one wild-type long peptide) and named smow7-pcDNA3.1 (+). All gene segments are synthesized and constructed by Nanjing Jinslei Biotechnology GmbH,
the amino acid sequence of smo15 is:
MAAPGSARRPLLLLLLLLLLGLMHCASALQGGSGGGGSGGMLRLGIFGFLVFGFVLITFSCKKKKKKGGSGGGGSGGAFGFVLITFSYHFYDFFNQAEKKKKKGGSGGGGSGGVLITFSCHFYGFFNQAEWERKKGGSGGGGSGGLRLGIFGFLALGFVLITFSCHKKKKKKGGSGGGGSGGYVLCQANVTIWLPTKQPIPDCKGGSGGGGSGGVLITFSCHFYHFFNQAEWERSKKKGGSGGGGSGGFTEAEHQDMRSYIAAFGAVTKKGGSGGGGSGGMFGTGIAMSTLVWTKATLLIWKKKKKKKKGGSGGGGSGGVLITFSCHFYYFFNQAEWERSKKKGGSGGGGSGGFSCHFYDFFNEAEWERSFRDYKKGGSGGGGSGGHSYIAAFGAVMGLCTLFTLAKKKKKKKKKGGSGGGGSGGTLSCVIIFVIAYYALMAGVVWKKKKKKKKKKGGSGGGGSGGNACFFVGSIGLLAQFMDGARKGGSGGGGSGGMFGTGIAMSTRVWTKATLLIWKKKKKKGGSGGGGSGGTLSCVIIFVIMYYALMAGVVWKKKKKKKKKKGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMC RRKSSGGKGGSYSQAASSDSAQGSDVSLTA；
the amino acid sequence of Smow7 is:
MAAPGSARRPLLLLLLLLLLGLMHCASALQMLRLRLGIFGFLAFGFVLITFSCHFYDFFNQAEWERSFRDGGSGGGGSGGYVLCQANVTIGLPTKQPIPDCKGGSGGGGSGGFTEAEHQDMHSYIAAFGAVTKKGGSGGGGSGGHSYIAAFGAVMGLCTLFTLAKKKKKKKKKGGSGGGGSGGTLSCVIIFVIVYYALMAGVVWKKKKKKKKKKGGSGGGGSGGNACFFVGSIGWLAQFMDGARKGGSGGGGSGGMFGTGIAMSTLVWTKATLLIWKKKKKKKGGSLGGGGSGIVG IVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA；
wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences;
b. construction of cell lines capable of stably expressing mutant Polypeptides
Gastric cancer cell line CD44+AGS by 2 x 105Planting in 6-well plate, and covering with 70-8 cellsTransfection was started at 0%. 2.5. mu.g of smo15-pcDNA3.1(+) plasmid and smow7-pcDNA3.1(+) plasmid 2 were diluted in 2 parts of 100. mu.l serum-free RPMI-1640 medium, and 2.5. mu.l PLUS was addedTMReagents, incubated at room temperature for 5min, and each with 5. mu.l LipofectamineTMLTX serum-free RPMI-1640100. mu.l was mixed and incubated at room temperature for 30 min. The liposome plasmid complex was dropped into 2 portions of cells to be transfected containing 1000. mu.l of serum-free RPMI-1640, gently shaken up and down, left to stand for 6 hours, and then replaced with 10% serum-containing RPMI-1640 medium, and after further culturing for 48 hours, the medium was replaced with 10% serum-containing medium containing 700. mu.g/mLG 418 and 400. mu.g/mL hygromycin B to perform cell selection. And (3) resistance screening for 10-14 days, after the cells of the control group are completely dead, the cells of the transfection group grow in a large amount, the cells are digested, and the cells are planted into a 96-well plate by adopting a limiting dilution method. Monoclonal cells were selected under the microscope and cultured in medium containing 700. mu.g/mL G418, 400. mu.g/mL hygromycin B, with the medium changed every other day. After further culturing for about 10 days, the monoclonal cells grow into a larger mass, digested, and transferred to a 24-well plate for culture. Simultaneously transfecting pcDNA3.1(+) -Hygro empty vector plasmid to carry out resistance screening by the same method, and taking the empty vector plasmid as a control cell and naming the empty vector plasmid as CD44+AGS (containing pcDNA3.1 (+)). In the above-described method, the cell lines constructed with smo15-pcDNA3.1(+) plasmid and smow7-pcDNA3.1(+) plasmid were designated smo15-ASG (containing smo15-pcDNA3.1(+)) and smow7-AGS (containing smow7-pcDNA3.1(+)), respectively.
Example 2 construction of a second class of stable transgenic cell lines that generate specific mutation sites of drug resistant Ibrutinib against BTK mutations:
ibrutinib (Ibrutinib) purchased from Selleck Chemicals, human multiple myeloma cell line RPMI8226 (C.) (
Figure BDA0001961892220000191
CCL-155TM) Purchased from ATCC and cultured in RPMI1640 medium plus 10% fetal bovine serum.
Purpose of the experiment: in order to verify that the T cells induced by the drug-resistant polypeptide have killing activity, a set of stable transfer cell lines containing the specific mutation sites in the invention needs to be constructed, and the stable transfer cell lines can express and present the polypeptide
a. Construction of mutant site eukaryotic expression plasmid
The mini-gene capable of expressing all 5 vaccine polypeptides mutated in the invention is obtained by adopting an artificial synthesis method and consists of the following parts: a signal peptide part (lysome-associated membrane glycoprotein 1, LAMP1), 5 mutant polypeptides and MHC class I trafficking domain (MITD), wherein the 5 mutant polypeptides are connected by a flexible connecting peptide GGSGGGGSGG, after codon optimization of the gene, GATATC (EcoR V) is introduced at the upstream, CTCGAG (Xho I) is introduced at the downstream, and the gene is cloned into a eukaryotic expression vector pcDNA3.1-hygro (+), namely BTK5-pcDNA3.1 (+). Meanwhile, the corresponding wild-type polypeptide was synthesized as a control and named BTKw2-pcDNA3.1(+) (since 4 of the polypeptides were SNP site-mutated, there were 2 wild-type polypeptides in total). All gene segments are synthesized and constructed by Nanjing Jinslei Biotechnology GmbH,
the amino acid sequence of BTK5 is:
MAAPGSARRPLLLLLLLLLLGLMHCASALQFIITEYMANGFLLNYLREMRHKKKGGSGGGGSGGIITEYMANGRLLNYLREMRHKGGSGGGGSGGVRDSSKAGKYAVSVFAKSTGDGGSGGGGSGGIITEYMANGSLLNYLREMRHKGGSGGGGSGGFIITEYMANGYLLNYLREMRHKKKGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKS SGGKGGSYSQAASSDSAQGSDVSLTA；
the amino acid sequence of BTKW2 is as follows:
MAAPGSARRPLLLLLLLLLLGLMHCASALQFIITEYMANGCLLNYLREMRHKKKGGSGGGGSGGVRDSSKTGKYAVSVFAKSTGDGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGS DVSLTA；
wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences.
b. Construction of cell lines capable of stably expressing mutant Polypeptides
Human multiple myeloma cell line RPMI8226 with 2 x 105Perwell in 6-well plates, transfection was initiated when cells were 70-80% covered. BTK5-pcDNA3.1(+) plasmid and BTKw2-pcDNA3.1(+) plasmid 2 were diluted in 2 parts of 100. mu.l serum-free RPMI-1640 medium, and 2.5. mu.l PLUS was addedTMReagents, incubated at room temperature for 5min, and each with 5. mu.l LipofectamineTMLTX serum-free RPMI-1640100. mu.l was mixed and incubated at room temperature for 30 min. The liposome plasmid complex was dropped into 2 portions of cells to be transfected containing 1000. mu.l of serum-free RPMI-1640, gently shaken up and down, left to stand for 6 hours, and then replaced with 10% serum-containing RPMI-1640 medium, and after further culturing for 48 hours, the medium was replaced with 10% serum-containing medium containing 700. mu.g/mLG 418 and 400. mu.g/mL hygromycin B to perform cell selection. And (3) resistance screening for 10-14 days, after the cells of the control group are completely dead, the cells of the transfection group grow in a large amount, the cells are digested, and the cells are planted into a 96-well plate by adopting a limiting dilution method. Monoclonal cells were selected under the microscope and cultured in medium containing 700. mu.g/mL G418, 400. mu.g/mL hygromycin B, with the medium changed every other day. After further culturing for about 10 days, the monoclonal cells grow into a larger mass, digested, and transferred to a 24-well plate for culture. In the above method, the cell lines constructed using the BTK5-pcDNA3.1(+) plasmid and the BTKw2-pcDNA3.1(+) plasmid were named BTK5 (containing BTK5-pcDNA3.1(+)) and BTKw2 (containing BTKw2-pcDNA3.1(+)), respectively. ,
example 3, construction of stable transgenic cell lines of a third class against specific mutation sites of some antiandrogen drugs such as abiraterone, etc. of AR-resistant mutations;
prostate cancer cell line PC-3: (
Figure BDA0001961892220000201
CRL-1435TM) Purchased from ATCC, cultured in F-12K medium plus 10% fetal bovine serum.
Purpose of the experiment: in order to verify that the T cells induced by the drug-resistant polypeptide have killing activity, a set of stable transfer cell lines containing the specific mutation sites in the invention needs to be constructed, and the stable transfer cell lines can express and present the polypeptide
a. Construction of mutant site eukaryotic expression plasmid
The mini-gene capable of expressing all 4 vaccine polypeptides mutated in the invention is obtained by adopting an artificial synthesis method and consists of the following parts: a signal peptide part (lysome-associated membrane glycoprotein 1, LAMP1), 4 mutant polypeptides and MHC class I trafficking domain (MITD), wherein the 4 mutant polypeptides are connected by a flexible connecting peptide GGSGGGGSGG, after codon optimization of the gene, GATATC (EcoR V) is introduced at the upstream, CTCGAG (Xho I) is introduced at the downstream, and the gene is cloned into a eukaryotic expression vector pcDNA3.1-hygro (+), namely AR4-pcDNA3.1 (+). Meanwhile, the corresponding wild-type polypeptide was synthesized as a control, which was named ARw2-pcDNA3.1(+) (since 4 of the polypeptides were SNP site-mutated, there were 2 wild-type polypeptides in total). All gene fragments were synthesized and constructed by Nanjing Kingsrei Biotech, Inc.
The amino acid sequence of AR4 is:
MAAPGSARRPLLLLLLLLLLGLMHCASALQPIARELHQFAFDLLIKSHMKKGGSGGGGSGGVQPIARELHQLTFDLLIKSHMKGGSGGGGSGGNELGERQLVHMVKWAKALPGFGGSGGGGSGGPIARELHQFSFDLLIKSHMGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA；
the amino acid sequence of ARW2 is:
MAAPGSARRPLLLLLLLLLLGLMHCASALQVQPIARELHQFTFDLLIKSHMGGSGGGGSGGNELGERQLVHMVKWAKALPGFGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVS LTA；
wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences.
b. Construction of cell lines capable of stably expressing mutant Polypeptides
Prostate cancer cell line PC-3 by 2 x 105Perwell in 6-well plates, transfection was initiated when cells were 70-80% covered. AR4-pcDNA3.1(+) plasmid and ARw2-pcDNA3.1(+) plasmid 2 were diluted in 2 portions of 100. mu.l serum-free RPMI-1640 medium, and 2.5. mu.l PLUS was addedTMReagents, incubated at room temperature for 5min, and each with 5. mu.l LipofectamineTMLTX serum-free RPMI-1640100. mu.l was mixed and incubated at room temperature for 30 min. The liposome plasmid complex is respectively dropped into 2 parts of cells to be transfected containing 1000 mul serum-free RPMI-1640, gently shaken back and forth, kept stand for 6h, replaced by an RPMI-1640 culture medium containing 10% serum, and continuously cultured for 48h, and then replaced by a 10% serum culture medium containing 700 mug/mL G418 and 400 mug/mL hygromycin B for cell screening. And (3) resistance screening for 10-14 days, after the cells of the control group are completely dead, the cells of the transfection group grow in a large amount, the cells are digested, and the cells are planted into a 96-well plate by adopting a limiting dilution method. Display deviceMonoclonal cells were picked under the microscope and cultured on medium containing 700. mu.g/mL G418, 400. mu.g/mL hygromycin B, with the media changed every other day. After further culturing for about 10 days, the monoclonal cells grow into a larger mass, digested, and transferred to a 24-well plate for culture. In the above method, the cell lines constructed using the AR4-pcDNA3.1(+) plasmid and the ARw2-pcDNA3.1(+) plasmid were designated AR4 (containing AR4-pcDNA3.1(+)) and ARw2 (containing ARw2-pcDNA3.1(+)), respectively.
Example 4, construction of a fourth class of stable transgenic cell lines for specific mutation sites of some of the fenib drugs against BRAF resistance mutations;
human melanoma cell line A378 was purchased from ATCC and cultured in DMEM medium plus 10% fetal bovine serum.
Purpose of the experiment: in order to verify that the T cells induced by the drug-resistant polypeptide have killing activity, a set of stable transfer cell lines containing the specific mutation sites in the invention needs to be constructed, and the stable transfer cell lines can express and present the polypeptide
a. Construction of mutant site eukaryotic expression plasmid
The mini-gene capable of expressing all 7 mutated vaccine polypeptides in the invention is obtained by an artificial synthesis method and consists of the following parts: a signal peptide part (lysome-associated membrane glycoprotein 1, LAMP1), 7 mutant polypeptides and MHC class I trafficking domain (MITD), wherein the 7 mutant polypeptides are connected by a flexible connecting peptide GGSGGGGSGG, after codon optimization of the gene, GATATC (EcoR V) is introduced at the upstream, CTCGAG (Xho I) is introduced at the downstream, and the gene is cloned into a eukaryotic expression vector pcDNA3.1-hygro (+), namely ME7-pcDNA3.1 (+). Meanwhile, a corresponding wild-type polypeptide is synthesized to be used as a control and is named as MEw5-pcDNA3.1(+) (wherein the MAP2K 1124 amino acid site mutant is derived from the same wild-type polypeptide and can be combined into a wild-type long peptide). All gene fragments were synthesized and constructed by Nanjing Kingsrei Biotech, Inc.
The amino acid sequence of ME7 is:
MAAPGSARRPLLLLLLLLLLGLMHCASALQLHECNSPYIVVFYGAFYSDGEKKGGSGGGGSGGYKLVVVGADGVGKSALGGSGGGGSGGCLLDILDTAGREEYSAMRDQYKKKGGSGGGGSGGRKRLEAFLTPKQKVGELKGGSGGGGSGGELQVLHECNSLYIVGFYGAFYKKKGGSGGGGSGGKKRLEAFLTPKAKVGELKGGSGGGGSGGLQVLHECNSSYIVGFYGAFYKKGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDV SLTA；
MEw5 is the amino acid sequence:
MAAPGSARRPLLLLLLLLLLGLMHCASALQYKLVVVGAGGVGKSALGGSGGGGSGGCLLDILDTAGQEEYSAMRDQYKKKGGSGGGGSGGRKRLEAFLTQKQKVGELKGGSGGGGSGGELQVLHECNSPYIVGFYGAFYSDGEKKGGSGGGGSGGKKRLEAFLTQKAKVGELKGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSY SQAASSDSAQGSDVSLTA；
wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences.
b. Construction of cell lines capable of stably expressing mutant Polypeptides
Human melanoma cell line a378 as 2 x 105Perwell in 6-well plates, transfection was initiated when cells were 70-80% covered. ME7-pcDNA3.1(+) plasmid and MEw5-pcDNA3.1(+) plasmid 2 were diluted in 2 parts of 100. mu.l serum-free RPMI-1640 medium, and 2.5. mu.l PLUS was addedTMReagents, incubated at room temperature for 5min, and each with 5. mu.l LipofectamineTMLTX serum-free RPMI-1640100. mu.l was mixed and incubated at room temperature for 30 min. The liposome plasmid complex is respectively dropped into 2 parts of cells to be transfected containing 1000 mul serum-free RPMI-1640, gently shaken back and forth, kept stand for 6h, replaced by an RPMI-1640 culture medium containing 10% serum, and continuously cultured for 48h, and then replaced by a 10% serum culture medium containing 700 mug/mL G418 and 400 mug/mL hygromycin B for cell screening. And (3) resistance screening for 10-14 days, after the cells of the control group are completely dead, the cells of the transfection group grow in a large amount, the cells are digested, and the cells are planted into a 96-well plate by adopting a limiting dilution method. Monoclonal cells were selected under the microscope and cultured in medium containing 700. mu.g/mL G418, 400. mu.g/mL hygromycin B, with the medium changed every other day. After further culturing for about 10 days, the monoclonal cells grow into a larger mass, digested, and transferred to a 24-well plate for culture. In the above method, the cell lines constructed with the ME7-pcDNA3.1(+) plasmid and the MEw5-pcDNA3.1(+) plasmid were designated ME7(ME7-pcDNA3.1(+)) and MEw5 (containing MEw5-pcDNA3.1(+)), respectively.
Example 5, construction of a fifth class of stable transgenic cell lines targeting specific mutation sites of some tyrosine kinase inhibitors of ALK, MET, etc. fusion genes;
human non-small cell lung carcinoma H2228(EML4-ALK variant 3a/b E6; A20) was purchased from ATCC and cultured in RPMI1640 medium plus 10% fetal bovine serum.
Purpose of the experiment: in order to verify that the T cells induced by the drug-resistant polypeptide have killing activity, a set of stable transfer cell lines containing the specific mutation sites in the invention needs to be constructed, and the stable transfer cell lines can express and present the polypeptide
a. Construction of mutant site eukaryotic expression plasmid
The mini-gene capable of expressing all 10 vaccine polypeptides mutated in the invention is obtained by adopting an artificial synthesis method and consists of the following parts: a signal peptide part (lysome-associated membrane glycoprotein 1, LAMP1), 10 mutant polypeptides and MHC class I trafficking domain (MITD), wherein the 10 mutant polypeptides are connected by a flexible connecting peptide GGSGGGGSGG, after codon optimization of the gene, GATATC (EcoR V) is introduced at the upstream, CTCGAG (Xho I) is introduced at the downstream, and the gene is cloned into a eukaryotic expression vector pcDNA3.1-hygro (+), namely ALK10-pcDNA3.1 (+). Meanwhile, a corresponding wild type polypeptide is synthesized to be used as a control and named ALKw5-pcDNA3.1(+) (wherein mutants near ALK 1171 amino acid site and 1174 amino acid site are derived from the same wild type polypeptide, mutants near ALK 1196 amino acid site, ALK 1202 amino acid site and 1203 amino acid site are derived from the same wild type polypeptide, MET 1246 amino acid site is derived from the same wild type polypeptide and can be combined into a wild type long peptide, and 5 wild type long peptides are calculated in total). All gene fragments were synthesized and constructed by Nanjing Kingsrei Biotech, Inc.
The amino acid sequence of ALK10 is:
MAAPGSARRPLLLLLLLLLLGLMHCASALQKVADFGLARHMYDKEYYSVHKGGSGGGGSGGNITLIRGLSHGAFGEVYEGGSGGGGSGGELDFLMEALITSKFNHQNIVRGGSGGGGSGGRFILLELMAGRDLKSFLRETRGGSGGGGSGGCPGPGRVAKIADFGMARDIYRGGSGGGGSGGFLMEALIISKLNHQNIVRCIGKGGSGGGGSGGKVADFGLARNMYDKEYYSVHGGSGGGGSGGILLELMAGGNLKSFLRETRGGSGGGGSGGSLQSLPRFILMELMAGGDLKGGSGGGGSGGFLMEALIISKVNHQNIVRCIGKGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSY SQAASSDSAQGSDVSLTA；
the amino acid sequence of ALKw5 is:
MAAPGSARRPLLLLLLLLLLGLMHCASALQKVADFGLARDMYDKEYYSVHKGGSGGGGSGGELDFLMEALIISKFNHQNIVRCIGKGGSGGGGSGGNITLIRGLGHGAFGEVYEGGSGGGGSGGCPGPGRVAKIGDFGMARDIYRGGSGGGGSGGSLQSLPRFILLELMAGGDLKSFLRETRGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRK SSGGKGGSYSQAASSDSAQGSDVSLTA；
wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences.
b. Construction of cell lines capable of stably expressing mutant Polypeptides
Human non-small cell lung carcinoma H2228 with 2 x 105Perwell in 6-well plates, transfection was initiated when cells were 70-80% covered. ALK10-pcDNA3.1(+) plasmid and ALKw5-pcDNA3.1(+) plasmid 2 were diluted in 2 parts of 100. mu.l serum-free RPMI-1640 medium, and 2.5. mu.l PLUS was addedTMReagents, incubated at room temperature for 5min, and mixed with a solution containing 5. mu.l LipofectamineTMLTX serum-free RPMI-1640100. mu.l was mixed and incubated at room temperature for 30 min. The liposome plasmid complex is respectively dropped into 2 parts of cells to be transfected containing 1000 mul serum-free RPMI-1640, gently shaken back and forth, kept stand for 6h, replaced by an RPMI-1640 culture medium containing 10% serum, and continuously cultured for 48h, and then replaced by a 10% serum culture medium containing 700 mug/mL G418 and 400 mug/mL hygromycin B for cell screening. And (3) resistance screening for 10-14 days, after the cells of the control group are completely dead, the cells of the transfection group grow in a large amount, the cells are digested, and the cells are planted into a 96-well plate by adopting a limiting dilution method. Monoclonal cells were selected under the microscope and cultured in medium containing 700. mu.g/mL G418, 400. mu.g/mL hygromycin B, with the medium changed every other day. After further culturing for about 10 days, the monoclonal cells grow into a larger mass, digested, and transferred to a 24-well plate for culture. In the above method, the cell lines constructed using ALK10-pcDNA3.1(+) plasmid and ALKw5-pcDNA3.1(+) plasmid were named ALK10(ALK10-pcDNA3.1(+)) and ALKw5 (containing ALKw5-pcDNA3.1(+)), respectively.
Example 6, construction of a sixth class of stable transgenic cell lines directed against specific mutation sites of tyrosine kinase inhibitors of the EGFR pathway;
the human chronic myelogenous leukemia cell line THP-1(ATCC TIB-202) was purchased from ATCC and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum.
Purpose of the experiment: in order to verify that the T cells induced by the drug-resistant polypeptide have killing activity, a set of stable transfer cell lines containing the specific mutation sites in the invention needs to be constructed, and the stable transfer cell lines can express and present the polypeptide
a. Construction of mutant site eukaryotic expression plasmid
In order to realize the expression of the mutant polypeptide, the invention is supposed to realize the overexpression in the human chronic myelogenous leukemia cell line THP-1 by serially connecting various polypeptide genes (namely mini-gene). However, because of the hot spot comparison of these mutations, 4 genes corresponding to these 92 polypeptides were divided into 2 groups, each group containing 46 genes, and cloned into eukaryotic expression vectors pcDNA3.1-hygro (+) and pcDNA3.1-zeo (+). Each vector was capable of expressing all 46 mutant mini-genes of the vaccine polypeptides of the present invention, consisting of: signal peptide part (lysome-associated membrane glycoprotein 1, LAMP1), 13 mutant polypeptides and MHC class I trafficking domain (MITD), 46 mutant polypeptides are connected by flexible connecting peptide GGSGGGGSGG, GATATC (EcoR V) is introduced at the upstream and CTCGAG (Xho I) is introduced at the downstream after codon optimization of the gene, and the mutant polypeptides are cloned into eukaryotic expression vector pcDNA3.1-hygro (+), named mut46-hygro (+) and mut46-zeo (+). Meanwhile, corresponding wild type polypeptides are synthesized to be used as a control and named as wid8-hygro (+) (wherein ABL1 protein directly selects 234-505 amino acid sites, FLT3 protein selects one near 835 amino acid sites, EGFR protein selects 2 near 760-790 amino acid sites, KIT protein 3 and PDGFRA protein 1 wild type polypeptides), the wid8-hygro (+) are cloned into a eukaryotic expression vector pcDNA3.1-hygro (+), and all gene segments of wid8-hygro (+) are synthesized and constructed by Nanjing Kingsley Biotech Co., Ltd.
The amino acid sequence of Mut46-hy is:
MAAPGSARRPLLLLLLLLLLGLMHCASALQTSPKANKEILYEAYVMASVDKGGSGGGGSGGICLTSTVQLIMQLMPFGCLLDKKKKKGGSGGGGSGGAPESLAYNKFYIKSDVWAFGVGGSGGGGSGGAVKTLKEDTMKVEEFLKEAAVKGGSGGGGSGGAVMKEIKHPNVVQLLGVCTRKGGSGGGGSGGCLVGENHLVKIADFGLSRLMTKGGSGGGGSGGCTREPPFYIIAEFMTYGNLLDKKGGSGGGGSGGQLITQLMPFDCLLDYVREHKKGGSGGGGSGGGENHLVKVADLGLSRLMGGSGGGGSGGIDLSQVYELLKKDYRMERGGSGGGGSGGITMKHKLGGGEYGEVYEGVWGGSGGGGSGGITMKHKLGGGHYGEVYEGVWKGGSGGGGSGGLTSTVQLITQHMPFGCLLDYVKKKKKGGSGGGGSGGCTREPPFYIINEFMTYGNLLDKKGGSGGGGSGGKEAAVMKEIRGGHPNLVQLLGVGGSGGGGSGGKHKLGGGQYGKVYEGVWKKYSGGSGGGGSGGKVADFGLSRMMTGDTYTAHAKKGGSGGGGSGGKWEMERTDITVKHKLGGGQYGGSGGGGSGGLAARNCLVGEYHLVKVADFKKKGGSGGGGSGGLLGVCTREPPLYIITEFMTYGKKKGGSGGGGSGGITMKHKLGGGMYGEVYEGVWKGGSGGGGSGGIDLSQVYELLLKDYRMERKGGSGGGGSGGLMTGDTYTAHPGAKFPIKWKKGGSGGGGSGGMTYGNLLDYLMECNRQEVNAVKKGGSGGGGSGGNCLVGENHLVRVADFGLSRLMGGSGGGGSGGREPPFYIITELMTYGNLLDYLKKGGSGGGGSGGRLMTGDTYTAPAGAKFPIKWKGGSGGGGSGGSAMEYLEKKNAIHRDLAARGGSGGGGSGGITMKHKLGGGRYGEVYEGVWKGGSGGGGSGGTLKEDTMEVEGFLKEAAVMKKKGGSGGGGSGGVEEFLKEAAFMKEIKHPNLVGGSGGGGSGGYMATQISSAMGYLEKKNFIHRGGSGGGGSGGYPGIDLSQVYALLEKDYRMERKGGSGGGGSGGVKICDFGLARFIMSDSNYVVRGGSGGGGSGGKICDFGLARAIKNDSNYVVKGGSGGGGSGGDFGLARDIKNVSNYVVKGNARGGSGGGGSGGCTIGGPTLVIIEYCCYGDLLNKKKGGSGGGGSGGSYLGNHMNIVTLLGACTIGGPKGGSGGGGSGGYPGIDLSQVYKLLEKDYRMERGGSGGGGSGGVKICDFGLARYIMSDSNYVVRGGSGGGGSGGTQISSAMEYLAKKNFIHRDGGSGGGGSGGWQWNPSDRPSSAEIHQAFETMKGGSGGGGSGGWAFGVLLWEITTYGMSPYPGIKKKGGSGGGGSGGVAVKTLKEDAMEVEEFLKEAKKKGGSGGGGSGGVKICDFGLARVIMHDSNYVSKGGSGGGGSGGDSNYVVKGNPRLPVKWMAGGSGGGGSGGKICDFGLARHIKNDSNYVVKGGSLGGGGSGIVGIVAGL AVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA；
the amino acid sequence of Mut46-zeo is:
MAAPGSARRPLLLLLLLLLLGLMHCASALQLITQLMPFGSLLDYVREHKDGGSGGGGSGGAVKTLKEDTMYVEEFLKEAAVKKGGSGGGGSGGCTREPPFYIIIEFMTYGNLLDKKGGSGGGGSGGDLSQVYELLGKDYRMERKGGSGGGGSGGDTYTAHAGTKFPIKWTAPEKKGGSGGGGSGGEFLKEAAVMKVIKHPNLVQLLKGGSGGGGSGGVQLITQLMPFRCLLDYVREHKKGGSGGGGSGGELMRACWQWNLSDRPSFAEIHGGSGGGGSGGESLAYNKFSIESDVWAFGVLLKKGGSGGGGSGGHLVKVADFGMSRLMTGDTYTKKGGSGGGGSGGIDLSQVYELLAKDYRMERKGGSGGGGSGGITMKHKLGGGEYGEVYEGVWGGSGGGGSGGCTREPPFYIIVEFMTYGNLLDKKAVKTLKEDTMVEEFLKEAKKGGSGGGGSGGKHKLGGGQYGVVYEGVWKKYSGGSGGGGSGGKTLKEDTMAVEEFLKEAAVKKGGSGGGGSGGKVADFGLSRLLTGDTYTAHAGKGGSGGGGSGGLLGVCTREPSFYIITEFMTYKKKKGGSGGGGSGGITMKHKLGGGKYGEVYEGVWKGGSGGGGSGGMTGDTYTAHARAKFPIKWTAPKKKGGSGGGGSGGPESLAYNKFSVKSDVWAFGVGGSGGGGSGGPFYIITEFMTCGNLLDYLRKKKGGSGGGGSGGQISSAMEYLGKKNFIHRDGGSGGGGSGGRECNRQEVNAFVLLYMATQIKGGSGGGGSGGRLMTGDTYTARAGAKFPIKWTKGGSGGGGSGGSAMEYLEKKNVIHRDLAARNGGSGGGGSGGTLKEDTMEVEKFLKEAAVMKKGGSGGGGSGGVEEFLKEAAAMKEIKHPNLVGGSGGGGSGGYMATQISSAMDYLEKKNFIHRGGSGGGGSGGYPGIDLSQVYGLLEKDYRMERKGGSGGGGSGGKICDFGLARHIMSDSNYVVRGGSGGGGSGGKICDFGLAREIKNDSNYVVKGGSGGGGSGGLARDIKNGSNYVVKGNGGSGGGGSGGCTIGGPTLVIEEYCCYGDLLNKKKGGSGGGGSGGLSYLGNHMNIANLLGACTIGKKGGSGGGGSGGVKICDFGLARVIMSDSNYVVRGGSGGGGSGGSFAEIHQAFERMFQESSISDEKKKGGSGGGGSGGSLTVAVKTLKKDTMEVEEFLKGGSGGGGSGGTEFMTYGNLLGYLRECNRQEVKGGSGGGGSGGTMKHKLGGGQFGEVYEGVWKKGGSGGGGSGGVKVADFGLSRFMTGDTYTAHAKKKGGSGGGGSGGVMKEIKHPNLLQLLGVCTREPGGSGGGGSGGEREALMSELEVLSYLGNHMNKKGGSGGGGSGGEAALYKNLLHFKESSCSDSTGGSGGGGSGGDFGLARDIKNYSNYVVKGNARGGSGGGGSGGFGLARDIKNDFNYVVKGNARGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA；
the amino acid sequence of Wid8-hy is as follows:
MAAPGSARRPLLLLLLLLLLGLMHCASALQKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSAMEYLEKKNFIHRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAGAKFPIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDLSQVYELLEKDYRMERPEGCPEKVYELMRACWQWNPSDRPSFAEIHQAFETMFQESSISDGGSGGGGSGGVKICDFGLARDIMSDSNYVVRGGSGGGGSGGTSPKANKEILDEAYVMASVDKGGSGGGGSGGICLTSTVQLITQLMPFGCLLDYVREHKGGSGGGGSGGVKICDFGLARDIMHDSNYVSKGGSGGGGSGGEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNGGSGGGGSGGEAALYKNLLHSKESSCSDSTGGSGGGGSGGKICDFGLARDIKNDSNYVVKGNARLPVKWMAGGSLGGGGSGIVGIVAGLAVLAVVVIGAVVATVMCRRKSS GGKGGSYSQAASSDSAQGSDVSLTA；
wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences.
Wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences.
b. Construction of cell lines capable of stably expressing mutant Polypeptides
Human chronic myelocytic leukemia cell line THP-1 with 2 x 105Perwell in 6-well plates, transfection was initiated when cells were 70-80% covered. Mu.l of 100. mu.l serum-free RPMI-1640 medium was diluted with mut46-hygro (+) plasmid and wid8-hygro (+) plasmid 2, respectively, and 2.5. mu.l PLUS was added theretoTMReagents, incubated at room temperature for 5min, and mixed with a solution containing 5. mu.l LipofectamineTMLTX serum-free RPMI-1640100. mu.l was mixed and incubated at room temperature for 30 min. The liposome plasmid complex is respectively dropped into 2 cells to be transfected containing 1000 mul serum-free RPMI-1640, gently shaken back and forth, kept stand for 6h, replaced by an RPMI-1640 culture medium containing 10% serum, continuously cultured for 48h, replaced by a 10% serum culture medium containing 700 mug/mL G418 and 400 mug/mL hygromycin B, and then cell screening is carried out. And (3) resistance screening for 10-14 days, after the cells of the control group are completely dead, the cells of the transfection group grow in a large amount, the cells are digested, and the cells are planted into a 96-well plate by adopting a limiting dilution method. Monoclonal cells were selected under the microscope and cultured in medium containing 700. mu.g/mL G418, 400. mu.g/mL hygromycin B, with the medium changed every other day. After further culturing for about 10 days, the monoclonal cells grow into a larger mass, digested, and transferred to a 24-well plate for culture. In the above method, the cell lines constructed with mut46-hygro (+) plasmid and wid8-hygro (+) plasmid were named mut46(mut46-hygro (+)) and wid8 (containing wid8-hygro (+)), respectively.
Mut46(mut46-hygro (+)) was reactivated and mut46-zeo (+) was introduced into the cell line and screened with 400. mu.g/mL zeo, designated mut92(mut46-hygro (+), mut46-zeo (+)) according to the transfection protocol described above.
Example 7, construction of a seventh class of stable transgenic cell lines targeting specific mutation sites of kinase inhibitors such as everolimus of the PI3K/AKT/mTOR pathway;
human embryonic kidney fibroblast strain HEK 293(
Figure BDA0001961892220000271
CRL-1573TM) Purchased from ATCC, cultured in 10% FBS-DMEM medium.
Purpose of the experiment: in order to verify that the T cells induced by the drug-resistant polypeptide have killing activity, a set of stable transfer cell lines containing the specific mutation sites in the invention needs to be constructed, and the stable transfer cell lines can express and present the polypeptide
a. Construction of mutant site eukaryotic expression plasmid
The mini-gene capable of expressing all 3 mutated vaccine polypeptides in the invention is obtained by an artificial synthesis method and consists of the following parts: a signal peptide part (lysome-associated membrane glycoprotein 1, LAMP1), 3 mutant polypeptides and MHC class I trafficking domain (MITD), wherein the 3 mutant polypeptides are connected by a flexible connecting peptide GGSGGGGSGG, after codon optimization of the gene, GATATC (EcoR V) is introduced at the upstream, CTCGAG (Xho I) is introduced at the downstream, and the gene is cloned into a eukaryotic expression vector pcDNA3.1-hygro (+), and is named as MEM3-pcDNA3.1 (+). The corresponding wild-type polypeptide was synthesized at the same time as a control and named MEMw3-pcDNA3.1 (+). All gene fragments were synthesized and constructed by Nanjing Kingsrei Biotech, Inc.
The amino acid sequence of MEM3 is:
MAAPGSARRPLLLLLLLLLLGLMHCASALQTQAWDLYYHVLRRISKQLPQGGSGGGGSGGHECNSPYIVGLYGAFYSDGEIKKGGSGGGGSGGVKVADFGLARVMYDKEYYSVHKGGSLGGGGSGIVGIVAGLAVLAVVVIGAV VATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA；
the amino acid sequence of MEMw3 is:
MAAPGSARRPLLLLLLLLLLGLMHCASALQTQAWDLYYHVFRRISKQLPQGGSGGGGSGGHECNSPYIVGFYGAFYSDGEIKKGGSGGGGSGGVKVADFGLARDMYDKEYYSVHKGGSLGGGGSGIVGIVAGLAVLAVVVIGAV VATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA；
wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences.
b. Construction of cell lines capable of stably expressing mutant Polypeptides
Human embryonic kidney fibroblast strain HEK 293 with 2 x 105Perwell in 6-well plates, transfection was initiated when cells were 70-80% covered. MEM3-pcDNA3.1(+) plasmid and MEMw3-pcDNA3.1(+) plasmid 2 were diluted in 2 portions of 100. mu.l serum-free RPMI-1640 medium, and 2.5. mu.l PLUS was addedTMReagents, incubated at room temperature for 5min, and mixed with the aqueous medium5μl Lipofectamine TMLTX serum-free RPMI-1640100. mu.l was mixed and incubated at room temperature for 30 min. The liposome plasmid complex was dropped into 2 portions of cells to be transfected containing 1000. mu.l of serum-free RPMI-1640, gently shaken up and down, left to stand for 6 hours, and then replaced with 10% serum-containing RPMI-1640 medium, and after further culturing for 48 hours, the medium was replaced with 10% serum-containing medium containing 700. mu.g/mLG 418 and 400. mu.g/mL hygromycin B to perform cell selection. And (3) resistance screening for 10-14 days, after the cells of the control group are completely dead, the cells of the transfection group grow in a large amount, the cells are digested, and the cells are planted into a 96-well plate by adopting a limiting dilution method. Monoclonal cells were selected under the microscope and cultured in medium containing 700. mu.g/mL G418, 400. mu.g/mL hygromycin B, with the medium changed every other day. After further culturing for about 10 days, the monoclonal cells grow into a larger mass, digested, and transferred to a 24-well plate for culture. In the above method, the cell lines constructed using the MEM3-pcDNA3.1(+) plasmid and the MEMw3-pcDNA3.1(+) plasmid were designated MEM3 (containing MEM3-pcDNA3.1(+)) and MEMw3 (containing MEMw3-pcDNA3.1(+)), respectively.
The lymphocyte suspensions from the mice of polypeptide group 1 obtained according to examples 1-7 above were subjected to in vitro cell killing experiments as follows:
experiment 2-1: in vitro cell killing experiment of the polypeptide vaccine group of the first targeted medicament;
purpose of the experiment: the results prove that the 15 polypeptides can cause the killing effect of tumor cells at the in vitro cell level.
The experimental method comprises the following steps:
(1)5- (6) -Carboxy-fluorochein succinimidyl ester (CFSE) dye was purchased from Invitrogen. The procedures were performed according to the kit instructions. Target cells of smo15-ASG (containing smo15-pcDNA3.1(+)) and smo 7-AGS (containing smo 7-pcDNA3.1(+)) were labeled with CFSE under sterile conditions as target cells for the experimental group and the control group, respectively.
(2) Killing experiment
a. Preparation of effector cells:
the lymphocyte suspension of the polypeptide group 1 mouse retained in example 1 was resuspended in RPMI1640 medium and counted by trypan blue staining.
b. Induction culture of effector cells CTL:
group 2 lymphocytes were diluted to a concentration of (1-2) × 106Each well was plated at 3mL in 6-well plates, and cultured in RPMI1640+ 10% FBS +1 XPenicillin (100. mu.g/mL) + streptomycin (100. mu.g/mL) +1 XPEM non-essential amino acids +1mM sodium sulfate +10mM HEPES buffer medium, and supplemented with 50U/mL rhIL 2. Adding 10 mu g/mL of corresponding antigen peptide pool group into each hole, culturing for 7 days, changing the liquid half a day every 3 days, and supplementing corresponding antigen peptide and rhIL 2; after one week, the cells were resuspended and washed 2 times with PBS to prepare effector CTLs.
1) Target cells of smo15-ASG (containing smo15-pcDNA3.1(+)) and effector cells CTL are mixed according to the cell number ratio of 1:5, 1:10 and 1:20, and added into a U-shaped 96-well plate, wherein each volume is 200 mu L, and the U-shaped 96-well plate is used as an experimental group, and three parallel control wells are arranged in each experimental group.
2) Target cells of smow7-AGS (containing smow7-pcDNA3.1(+)) and effector cells CTL were mixed at a cell number ratio of 1:5, 1:10 and 1:20, and added to a U-shaped 96-well plate at 200. mu.L per well volume to serve as controls, each of which was provided with three parallel control wells.
3) The 96-well plate was incubated at 37 ℃ for 4 hours.
4) The supernatant was centrifuged from a 96-well plate, the cell pellet was resuspended in 200. mu.L of precooled PBS, transferred to a flow-on tube, labeled with Propidium Iodide (PI) staining at a concentration of 1. mu.g/m L for 3min, and immediately subjected to flow-on detection.
The experimental results are as follows:
as shown in FIG. 3, the killing efficiency of the effector T cells induced by the experimental group (FIG. 3) is different from 40% to 80%, and the killing efficiency is obviously higher than that of the control group, which indicates that the mutant polypeptide group has a killing effect on the target cells. In the mutant polypeptide group, the killing effect of T cells is stronger and stronger along with the increase of the effective-target ratio, and when the effective-target ratio is 20:1, the killing efficiency of the mutant polypeptide group on target cells reaches over 75 percent.
Experiment 2-2: in vitro cell killing experiment of polypeptide vaccine group of second type targeting drug
Purpose of the experiment: the 5 polypeptides are verified to cause the killing effect of tumor cells at the in vitro cell level.
The experimental method comprises the following steps:
(1)5- (6) -Carboxy-fluorochein succinimidyl ester (CFSE) dye was purchased from Invitrogen. The procedures were performed according to the kit instructions. The target cells of BTK5 (containing BTK5-pcDNA3.1(+)) and BTKw2 (containing BTKw2-pcDNA3.1(+)) were marked with CFSE under aseptic conditions, and used as target cells for the experimental group and the control group, respectively.
(2) Killing experiment
a. Preparation of effector cells:
the lymphocyte suspension of the polypeptide group 1 mouse retained in example 2 was resuspended in RPMI1640 medium and counted by trypan blue staining.
b. Induction culture of effector cells CTL:
group 2 lymphocytes were diluted to a concentration of (1-2) × 106Per mL, spread on 6 well plates, 3mL per well, with
RPMI1640+ 10% FBS +1 XPenicillin (100. mu.g/mL) + streptomycin (100. mu.g/mL) +1 XPEM non-essential amino acids +1mM sodium sulfate +10mM HEPES buffer medium, supplemented with 50U/mL rhIL 2. Adding 10 mu g/mL of corresponding antigen peptide pool group into each hole, culturing for 7 days, changing the liquid half a day every 3 days, and supplementing corresponding antigen peptide and rhIL 2; after one week, the cells were resuspended and washed 2 times with PBS to prepare effector CTLs.
5) BTK5 (containing BTK5-pcDNA3.1(+)) target cells and effector cells CTL were mixed in a cell number ratio of 1:5, 1:10 and 1:20, respectively, and added to a U-shaped 96-well plate at a volume of 200. mu.L per well to serve as experimental groups each provided with three parallel control wells.
6) BTKw2 (containing BTKw2-pcDNA3.1(+)) target cells and effector cells CTL were mixed in a cell number ratio of 1:5, 1:10 and 1:20, respectively, and added to a U-shaped 96-well plate at a volume of 200. mu.L per well to serve as controls, each of which was provided with three parallel control wells.
7) The 96-well plate was incubated at 37 ℃ for 4 hours.
8) The supernatant was centrifuged from a 96-well plate, the cell pellet was resuspended in 200. mu.L of precooled PBS, transferred to a flow-on tube, labeled with Propidium Iodide (PI) staining at a concentration of 1. mu.g/m L for 3min, and immediately subjected to flow-on detection.
The experimental results are as follows:
as shown in FIG. 4, the killing efficiency of the effector T cells induced by the experimental group (FIG. 4) is different from 40% to 80%, and the killing efficiency is obviously higher than that of the control group, which indicates that the mutant polypeptide group has a killing effect on the target cells. In the mutant polypeptide group, the killing effect of T cells is stronger and stronger along with the increase of the effective-target ratio, and when the effective-target ratio is 20:1, the killing efficiency of the mutant polypeptide group on target cells reaches over 75 percent.
Experiment 2-3: in vitro cell killing experiment of polypeptide vaccine group of third type targeted drug
Purpose of the experiment: the in vitro cell level of the 4 polypeptides was verified to be capable of causing the killing effect of tumor cells.
The experimental method comprises the following steps:
(1)5- (6) -Carboxy-fluorochein succinimidyl ester (CFSE) dye was purchased from Invitrogen. The procedures were performed according to the kit instructions. The target cells of AR4 (containing AR4-pcDNA3.1(+)) and ARw2 (containing ARw2-pcDNA3.1(+)) were marked with CFSE under sterile conditions as target cells for the experimental group and the control group, respectively.
(2) Killing experiment
a. Preparation of effector cells:
the lymphocyte suspension of the polypeptide group 1 mouse retained in example 3 was resuspended in RPMI1640 medium and counted by trypan blue staining.
b. Induction culture of effector cells CTL:
group 2 lymphocytes were diluted to a concentration of (1-2) × 106Per mL, spread on 6 well plates, 3mL per well, with
RPMI1640+ 10% FBS +1 XPenicillin (100. mu.g/mL) + streptomycin (100. mu.g/mL) +1 XPEM non-essential amino acids +1mM sodium sulfate +10mM HEPES buffer medium, supplemented with 50U/mL rhIL 2. Adding 10 mu g/mL of corresponding antigen peptide pool group into each hole, culturing for 7 days, changing the liquid half a day every 3 days, and supplementing corresponding antigen peptide and rhIL 2; after one week, the cells were resuspended and washed 2 times with PBS to prepare effector CTLs.
9) Target cells of AR4 (containing AR4-pcDNA3.1(+)) and effector cells CTL were mixed at a cell number ratio of 1:5, 1:10 and 1:20, respectively, and added to a U-shaped 96-well plate at a volume of 200. mu.L per well to serve as experimental groups each provided with three parallel control wells.
10) ARw2 (containing ARw2-pcDNA3.1(+)) target cells were mixed with effector cells CTL at cell number ratios of 1:5, 1:10 and 1:20, respectively, and added to a U-shaped 96-well plate at a volume of 200. mu.L per well to serve as controls each of which was provided with three parallel control wells (deleted).
11) The 96-well plate was incubated at 37 ℃ for 4 hours.
12) The supernatant was centrifuged from a 96-well plate, the cell pellet was resuspended in 200. mu.L of precooled PBS, transferred to a flow-on tube, labeled with Propidium Iodide (PI) staining at a concentration of 1. mu.g/m L for 3min, and immediately subjected to flow-on detection.
The experimental results are as follows:
as shown in FIG. 5, the killing efficiency of the effector T cells induced by the experimental group (FIG. 5) is different from 40% to 90%, and the killing efficiency is obviously higher than that of the control group, which indicates that the mutant polypeptide group has a killing effect on the target cells. In the mutant polypeptide group, the killing effect of T cells is stronger and stronger along with the increase of the effective target ratio, and when the effective target ratio is 20:1, the killing efficiency of the mutant polypeptide group on target cells reaches 80 percent to obtain the target polypeptide group
Experiments 2 to 4: polypeptide vaccine group in vitro cell killing experiment of fourth type targeted drug
Purpose of the experiment: the results prove that the 7 polypeptides can cause the killing effect of tumor cells at the in vitro cell level.
The experimental method comprises the following steps:
(1)5- (6) -Carboxy-fluorochein succinimidyl ester (CFSE) dye was purchased from Invitrogen. The procedures were performed according to the kit instructions. CFSE was used to label ME7(ME7-pcDNA3.1(+)) and MEw5 (containing MEw5-pcDNA3.1(+)) target cells under sterile conditions as target cells for the experimental and control groups, respectively.
(2) Killing experiment
a. Preparation of effector cells:
the lymphocyte suspension of the polypeptide group 1 mouse retained in example 4 was resuspended in RPMI1640 medium and counted by trypan blue staining.
b. Induction culture of effector cells CTL:
group 2 lymphocytes were diluted to a concentration of (1-2) × 106Each well was plated at 3mL in 6-well plates, and cultured in RPMI1640+ 10% FBS +1 XPenicillin (100. mu.g/mL) + streptomycin (100. mu.g/mL) +1 XPEM non-essential amino acids +1mM sodium sulfate +10mM HEPES buffer medium, and supplemented with 50U/mL rhIL 2. Adding 10 mu g/mL of corresponding antigen peptide pool group into each hole, culturing for 7 days, changing the liquid half a day every 3 days, and supplementing corresponding antigen peptide and rhIL 2; after one week, the cells were resuspended and washed 2 times with PBS to prepare effector CTLs.
13) ME7(ME7-pcDNA3.1(+)) target cells were mixed with effector cells CTL at cell number ratios of 1:5, 1:10 and 1:20, respectively, and added to a U-shaped 96-well plate at a volume of 200. mu.L per well to serve as experimental groups each provided with three parallel control wells.
14) MEw5 (containing MEw5-pcDNA3.1(+)) target cells were mixed with effector cells CTL at cell number ratios of 1:5, 1:10 and 1:20, respectively, and added to a U-shaped 96-well plate at a volume of 200. mu.L per well to serve as controls each of which was provided with three parallel control wells (deleted).
15) The 96-well plate was incubated at 37 ℃ for 4 hours.
16) The supernatant was centrifuged from a 96-well plate, the cell pellet was resuspended in 200. mu.L of precooled PBS, transferred to a flow-on tube, labeled with Propidium Iodide (PI) staining at a concentration of 1. mu.g/m L for 3min, and immediately subjected to flow-on detection.
The experimental results are as follows:
as shown in FIG. 6, the killing efficiency of the effector T cells induced by the experimental group (FIG. 6) is different from 45% to 90%, and the killing efficiency is obviously higher than that of the control group, which indicates that the mutant polypeptide group has a killing effect on the target cells. In the mutant polypeptide group, the killing effect of T cells is stronger and stronger along with the increase of the effective-target ratio, and when the effective-target ratio is 20:1, the killing efficiency of the mutant polypeptide group on target cells reaches more than 85%.
Experiments 2 to 5: a polypeptide vaccine group in vitro cell killing experiment of a fifth type of targeted drug;
purpose of the experiment: it was verified that 10 polypeptides could cause killing effect of tumor cells at the in vitro cell level.
The experimental method comprises the following steps:
(1)5- (6) -Carboxy-fluorochein succinimidyl ester (CFSE) dye was purchased from Invitrogen. The procedures were performed according to the kit instructions. CFSE was used to label ALK10(ALK10-pcDNA3.1(+)) and ALKw5 (containing ALKw5-pcDNA3.1(+)) target cells under sterile conditions as target cells for experimental and control groups, respectively.
(2) Killing experiment
a. Preparation of effector cells:
the lymphocyte suspension of the polypeptide group 1 mouse retained in example 5 was resuspended in RPMI1640 medium and counted by trypan blue staining.
b. Induction culture of effector cells CTL:
group 2 lymphocytes were diluted to a concentration of (1-2) × 106Each well was plated at 3mL in 6-well plates, and cultured in RPMI1640+ 10% FBS +1 XPenicillin (100. mu.g/mL) + streptomycin (100. mu.g/mL) +1 XPEM non-essential amino acids +1mM sodium sulfate +10mM HEPES buffer medium, and supplemented with 50U/mL rhIL 2. Adding 10 mu g/mL of corresponding antigen peptide pool group into each hole, culturing for 7 days, changing the liquid half a day every 3 days, and supplementing corresponding antigen peptide and rhIL 2; after one week, the cells were resuspended and washed 2 times with PBS to prepare effector CTLs.
17) ALK10(ALK10-pcDNA3.1(+)) target cells were mixed with effector cells CTL at cell number ratios of 1:5, 1:10 and 1:20, respectively, and added to a U-shaped 96-well plate at a volume of 200. mu.L per well to serve as experimental groups each provided with three parallel control wells.
18) ALKw5 (containing ALKw5-pcDNA3.1(+)) target cells and effector cells CTL were mixed at a cell number ratio of 1:5, 1:10 and 1:20, and added to a U-shaped 96-well plate at 200. mu.L per well volume to serve as controls, each of which was provided with three parallel control wells (which may be deleted).
19) The 96-well plate was incubated at 37 ℃ for 4 hours.
20) The supernatant was centrifuged from a 96-well plate, the cell pellet was resuspended in 200. mu.L of precooled PBS, transferred to a flow-on tube, labeled with Propidium Iodide (PI) staining at a concentration of 1. mu.g/m L for 3min, and immediately subjected to flow-on detection.
The experimental results are as follows:
as shown in FIG. 7, the killing efficiency of the effector T cells induced by the experimental group (FIG. 7) is different from 40% to 90%, and the killing efficiency is obviously higher than that of the control group, which indicates that the mutant polypeptide group has a killing effect on the target cells. In the mutant polypeptide group, the killing effect of T cells is stronger and stronger along with the increase of the effective-target ratio, and when the effective-target ratio is 20:1, the killing efficiency of the mutant polypeptide group on target cells reaches more than 80%.
Experiments 2-6: a sixth group of targeted drug polypeptide vaccine group in vitro cell killing experiment;
purpose of the experiment: the in vitro cell level of 92 polypeptides was verified to be capable of causing killing effect of tumor cells.
The experimental method comprises the following steps:
(1)5- (6) -Carboxy-fluorochein succinimidyl ester (CFSE) dye was purchased from Invitrogen. The procedures were performed according to the kit instructions. The target cells of mut92(mut46-hygro (+), mut46-zeo (+)) and wid8 (containing wid8-hygro (+)) were labeled with CFSE under sterile conditions as target cells for the experimental and control groups, respectively.
(2) Killing experiment
a. Preparation of effector cells:
the lymphocyte suspension of the polypeptide group 1 mouse retained in example 6 was resuspended in RPMI1640 medium and counted by trypan blue staining.
b. Induction culture of effector cells CTL:
group 2 lymphocytes were diluted to a concentration of (1-2) × 106Each well was plated at 3mL in 6-well plates, and cultured in RPMI1640+ 10% FBS +1 XPenicillin (100. mu.g/mL) + streptomycin (100. mu.g/mL) +1 XPEM non-essential amino acids +1mM sodium sulfate +10mM HEPES buffer medium, and supplemented with 50U/mL rhIL 2. Adding 10 mu g/mL of corresponding antigen peptide pool group into each hole, culturing for 7 days, changing the liquid half a day every 3 days, and supplementing corresponding antigen peptide and rhIL 2; one week later the cells were resuspended with PBS is washed for 2 times to prepare effector cells CTL.
21) The mut92(mut46-hygro (+), mut46-zeo (+)) target cells were mixed with effector cells CTL at cell number ratios of 1:5, 1:10 and 1:20, respectively, and added to U-shaped 96-well plates in an amount of 200. mu.L per well, to serve as experimental groups each provided with three parallel control wells.
22) Wid8 (containing wid8-hygro (+)) target cells were mixed with effector cells CTL at cell number ratios of 1:5, 1:10 and 1:20, respectively, and added to a U-shaped 96-well plate at a volume of 200. mu.L per well to serve as controls, each of which was provided with three parallel control wells.
23) The 96-well plate was incubated at 37 ℃ for 4 hours.
24) The supernatant was centrifuged from a 96-well plate, the cell pellet was resuspended in 200. mu.L of precooled PBS, transferred to a flow-on tube, labeled with Propidium Iodide (PI) staining at a concentration of 1. mu.g/m L for 3min, and immediately subjected to flow-on detection.
The experimental results are as follows:
as shown in FIG. 8, the killing efficiency of the effector T cells induced by the experimental group (FIG. 8) was varied from 40% to 90%, and was significantly higher than that of the control group, indicating that the mutant polypeptide group had a killing effect on the target cells. In the mutant polypeptide group, the killing effect of T cells is stronger and stronger along with the increase of the effective-target ratio, and when the effective-target ratio is 20:1, the killing efficiency of the mutant polypeptide group on target cells reaches over 84 percent.
Experiments 2 to 7: a polypeptide vaccine group in vitro cell killing experiment of a seventh type of targeted drug;
purpose of the experiment: 3 polypeptides were shown to cause killing of tumor cells at the in vitro cell level.
The experimental method comprises the following steps:
(1)5- (6) -Carboxy-fluorochein succinimidyl ester (CFSE) dye was purchased from Invitrogen. The procedures were performed according to the kit instructions. Marking of MEM3 with CFSE (containing MEM3-pcDNA3.1(+)) under aseptic conditions
And MEMw3 (containing MEMw3-pcDNA3.1(+)) target cells, as target cells for experimental and control groups, respectively.
(2) Killing experiment
a. Preparation of effector cells:
the lymphocyte suspension of the polypeptide group 1 mouse retained in example 7 was resuspended in RPMI1640 medium and counted by trypan blue staining.
b. Induction culture of effector cells CTL:
group 2 lymphocytes were diluted to a concentration of (1-2) × 106Each well was plated at 3mL in 6-well plates, and cultured in RPMI1640+ 10% FBS +1 XPenicillin (100. mu.g/mL) + streptomycin (100. mu.g/mL) +1 XPEM non-essential amino acids +1mM sodium sulfate +10mM HEPES buffer medium, and supplemented with 50U/mL rhIL 2. Adding 10 mu g/mL of corresponding antigen peptide pool group into each hole, culturing for 7 days, changing the liquid half a day every 3 days, and supplementing corresponding antigen peptide and rhIL 2; after one week, the cells were resuspended and washed 2 times with PBS to prepare effector CTLs.
25) MEM3 (containing MEM3-pcDNA3.1(+)) target cells were mixed with effector cells CTL at cell number ratios of 1:5, 1:10 and 1:20, respectively, and added to a U-shaped 96-well plate at 200. mu.L per well volume to serve as experimental groups each provided with three parallel control wells.
26) The target cell of MEMw3 (containing MEMw3-pcDNA3.1(+)) and effector cell CTL were mixed at cell number ratios of 1:5, 1:10 and 1:20, and added to a U-shaped 96-well plate at a volume of 200. mu.L per well to serve as controls, each of which was provided with three parallel control wells.
27) The 96-well plate was incubated at 37 ℃ for 4 hours.
28) The supernatant was centrifuged from a 96-well plate, the cell pellet was resuspended in 200. mu.L of precooled PBS, transferred to a flow-on tube, labeled with Propidium Iodide (PI) staining at a concentration of 1. mu.g/m L for 3min, and immediately subjected to flow-on detection.
The experimental results are as follows:
as shown in FIG. 9, the killing efficiency of the effector T cells induced by the experimental group (FIG. 9) was varied from 40% to 80%, and was significantly higher than that of the control group, indicating that the mutant polypeptide group had a killing effect on the target cells. In the mutant polypeptide group, the killing effect of T cells is stronger and stronger along with the increase of the effective-target ratio, and when the effective-target ratio is 20:1, the killing efficiency of the mutant polypeptide group on target cells reaches more than 70%.
According to the 7 experiments, the design method of the invention enables the targeted drugs to be classified into 7 categories according to the mechanism and the drug-resistant mutation clustering analysis, and the multi-target combination can effectively increase the killing effect of the polypeptide vaccine on tumor cells and obviously prolong the effective action time of the targeted drugs.
The invention provides a polypeptide vaccine aiming at tumor targeted drug resistance sites and a design method thereof, the provided targeted drug and polypeptide vaccine combination scheme covers common targeted therapeutic drugs, can effectively reduce the occurrence probability of tumor drug resistance, prolong the effective action time of the targeted drug, increase the disease response rate of patients, has broad spectrum, shortens the time from analysis to treatment of individualized polypeptide vaccines, and reduces the medical cost.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Sequence listing
<110> Hangzhou Nianjin Biotechnology Co., Ltd
<120> polypeptide vaccine aiming at tumor targeted drug resistant site and design method thereof
<141> 2019-01-29
<160> 208
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> PRT
<213> Artificial Sequence
<400> 1
Met Leu Arg Leu Gly Ile Phe Gly Phe Leu Val Phe Gly Phe Val Leu
1 5 10 15
Ile Thr Phe Ser Cys Lys Lys Lys Lys Lys Lys
20 25
<210> 2
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 2
Ala Phe Gly Phe Val Leu Ile Thr Phe Ser Tyr His Phe Tyr Asp Phe
1 5 10 15
Phe Asn Gln Ala Glu Lys Lys Lys Lys Lys
20 25
<210> 3
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 3
Val Leu Ile Thr Phe Ser Cys His Phe Tyr Gly Phe Phe Asn Gln Ala
1 5 10 15
Glu Trp Glu Arg Lys Lys
20
<210> 4
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 4
Val Leu Ile Thr Phe Ser Cys His Phe Tyr His Phe Phe Asn Gln Ala
1 5 10 15
Glu Trp Glu Arg Ser Lys Lys Lys
20
<210> 5
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 5
Val Leu Ile Thr Phe Ser Cys His Phe Tyr Tyr Phe Phe Asn Gln Ala
1 5 10 15
Glu Trp Glu Arg Ser Lys Lys Lys
20
<210> 6
<211> 27
<212> PRT
<213> Artificial Sequence
<400> 6
Leu Arg Leu Gly Ile Phe Gly Phe Leu Ala Leu Gly Phe Val Leu Ile
1 5 10 15
Thr Phe Ser Cys His Lys Lys Lys Lys Lys Lys
20 25
<210> 7
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 7
Tyr Val Leu Cys Gln Ala Asn Val Thr Ile Trp Leu Pro Thr Lys Gln
1 5 10 15
Pro Ile Pro Asp Cys Lys
20
<210> 8
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 8
Phe Thr Glu Ala Glu His Gln Asp Met Arg Ser Tyr Ile Ala Ala Phe
1 5 10 15
Gly Ala Val Thr Lys Lys
20
<210> 9
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 9
Phe Ser Cys His Phe Tyr Asp Phe Phe Asn Glu Ala Glu Trp Glu Arg
1 5 10 15
Ser Phe Arg Asp Tyr Lys Lys
20
<210> 10
<211> 29
<212> PRT
<213> Artificial Sequence
<400> 10
His Ser Tyr Ile Ala Ala Phe Gly Ala Val Met Gly Leu Cys Thr Leu
1 5 10 15
Phe Thr Leu Ala Lys Lys Lys Lys Lys Lys Lys Lys Lys
20 25
<210> 11
<211> 31
<212> PRT
<213> Artificial Sequence
<400> 11
Thr Leu Ser Cys Val Ile Ile Phe Val Ile Ala Tyr Tyr Ala Leu Met
1 5 10 15
Ala Gly Val Val Trp Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
20 25 30
<210> 12
<211> 31
<212> PRT
<213> Artificial Sequence
<400> 12
Thr Leu Ser Cys Val Ile Ile Phe Val Ile Met Tyr Tyr Ala Leu Met
1 5 10 15
Ala Gly Val Val Trp Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
20 25 30
<210> 13
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 13
Asn Ala Cys Phe Phe Val Gly Ser Ile Gly Leu Leu Ala Gln Phe Met
1 5 10 15
Asp Gly Ala Arg Lys
20
<210> 14
<211> 29
<212> PRT
<213> Artificial Sequence
<400> 14
Met Phe Gly Thr Gly Ile Ala Met Ser Thr Leu Val Trp Thr Lys Ala
1 5 10 15
Thr Leu Leu Ile Trp Lys Lys Lys Lys Lys Lys Lys Lys
20 25
<210> 15
<211> 27
<212> PRT
<213> Artificial Sequence
<400> 15
Met Phe Gly Thr Gly Ile Ala Met Ser Thr Arg Val Trp Thr Lys Ala
1 5 10 15
Thr Leu Leu Ile Trp Lys Lys Lys Lys Lys Lys
20 25
<210> 16
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 16
Phe Ile Ile Thr Glu Tyr Met Ala Asn Gly Phe Leu Leu Asn Tyr Leu
1 5 10 15
Arg Glu Met Arg His Lys Lys Lys
20
<210> 17
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 17
Ile Ile Thr Glu Tyr Met Ala Asn Gly Arg Leu Leu Asn Tyr Leu Arg
1 5 10 15
Glu Met Arg His Lys
20
<210> 18
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 18
Ile Ile Thr Glu Tyr Met Ala Asn Gly Ser Leu Leu Asn Tyr Leu Arg
1 5 10 15
Glu Met Arg His Lys
20
<210> 19
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 19
Phe Ile Ile Thr Glu Tyr Met Ala Asn Gly Tyr Leu Leu Asn Tyr Leu
1 5 10 15
Arg Glu Met Arg His Lys Lys Lys
20
<210> 20
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 20
Val Arg Asp Ser Ser Lys Ala Gly Lys Tyr Ala Val Ser Val Phe Ala
1 5 10 15
Lys Ser Thr Gly Asp
20
<210> 21
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 21
Pro Ile Ala Arg Glu Leu His Gln Phe Ala Phe Asp Leu Leu Ile Lys
1 5 10 15
Ser His Met Lys Lys
20
<210> 22
<211> 19
<212> PRT
<213> Artificial Sequence
<400> 22
Pro Ile Ala Arg Glu Leu His Gln Phe Ser Phe Asp Leu Leu Ile Lys
1 5 10 15
Ser His Met
<210> 23
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 23
Val Gln Pro Ile Ala Arg Glu Leu His Gln Leu Thr Phe Asp Leu Leu
1 5 10 15
Ile Lys Ser His Met Lys
20
<210> 24
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 24
Pro Ile Ala Arg Glu Leu His Gln Phe Ala Phe Asp Leu Leu Ile Lys
1 5 10 15
Ser His Met Lys Lys
20
<210> 25
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 25
Pro Ile Ala Arg Glu Leu His Gln Phe Ala Phe Asp Leu Leu Ile Lys
1 5 10 15
Ser His Met Lys Lys
20
<210> 26
<211> 19
<212> PRT
<213> Artificial Sequence
<400> 26
Pro Ile Ala Arg Glu Leu His Gln Phe Ser Phe Asp Leu Leu Ile Lys
1 5 10 15
Ser His Met
<210> 27
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 27
Asn Glu Leu Gly Glu Arg Gln Leu Val His Met Val Lys Trp Ala Lys
1 5 10 15
Ala Leu Pro Gly Phe
20
<210> 28
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 28
Pro Ile Ala Arg Glu Leu His Gln Phe Ala Phe Asp Leu Leu Ile Lys
1 5 10 15
Ser His Met Lys Lys
20
<210> 29
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 29
Leu Gln Val Leu His Glu Cys Asn Ser Ser Tyr Ile Val Gly Phe Tyr
1 5 10 15
Gly Ala Phe Tyr Lys Lys
20
<210> 30
<211> 18
<212> PRT
<213> Artificial Sequence
<400> 30
Lys Lys Arg Leu Glu Ala Phe Leu Thr Pro Lys Ala Lys Val Gly Glu
1 5 10 15
Leu Lys
<210> 31
<211> 16
<212> PRT
<213> Artificial Sequence
<400> 31
Tyr Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys Ser Ala Leu
1 5 10 15
<210> 32
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 32
Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Arg Glu Glu Tyr Ser Ala
1 5 10 15
Met Arg Asp Gln Tyr Lys Lys Lys
20
<210> 33
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 33
Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Arg Glu Glu Tyr Ser Ala
1 5 10 15
Met Arg Asp Gln Tyr Lys Lys Lys
20
<210> 34
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 34
Leu His Glu Cys Asn Ser Pro Tyr Ile Val Val Phe Tyr Gly Ala Phe
1 5 10 15
Tyr Ser Asp Gly Glu Lys Lys
20
<210> 35
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 35
Glu Leu Gln Val Leu His Glu Cys Asn Ser Leu Tyr Ile Val Gly Phe
1 5 10 15
Tyr Gly Ala Phe Tyr Lys Lys Lys
20
<210> 36
<211> 18
<212> PRT
<213> Artificial Sequence
<400> 36
Arg Lys Arg Leu Glu Ala Phe Leu Thr Pro Lys Gln Lys Val Gly Glu
1 5 10 15
Leu Lys
<210> 37
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 37
Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Arg Glu Glu Tyr Ser Ala
1 5 10 15
Met Arg Asp Gln Tyr Lys Lys Lys
20
<210> 38
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 38
Glu Leu Gln Val Leu His Glu Cys Asn Ser Leu Tyr Ile Val Gly Phe
1 5 10 15
Tyr Gly Ala Phe Tyr Lys Lys Lys
20
<210> 39
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 39
Arg Phe Ile Leu Leu Glu Leu Met Ala Gly Arg Asp Leu Lys Ser Phe
1 5 10 15
Leu Arg Glu Thr Arg
20
<210> 40
<211> 19
<212> PRT
<213> Artificial Sequence
<400> 40
Ile Leu Leu Glu Leu Met Ala Gly Gly Asn Leu Lys Ser Phe Leu Arg
1 5 10 15
Glu Thr Arg
<210> 41
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 41
Phe Leu Met Glu Ala Leu Ile Ile Ser Lys Val Asn His Gln Asn Ile
1 5 10 15
Val Arg Cys Ile Gly Lys
20
<210> 42
<211> 18
<212> PRT
<213> Artificial Sequence
<400> 42
Asn Ile Thr Leu Ile Arg Gly Leu Ser His Gly Ala Phe Gly Glu Val
1 5 10 15
Tyr Glu
<210> 43
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 43
Arg Phe Ile Leu Leu Glu Leu Met Ala Gly Arg Asp Leu Lys Ser Phe
1 5 10 15
Leu Arg Glu Thr Arg
20
<210> 44
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 44
Cys Pro Gly Pro Gly Arg Val Ala Lys Ile Ala Asp Phe Gly Met Ala
1 5 10 15
Arg Asp Ile Tyr Arg
20
<210> 45
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 45
Ser Leu Gln Ser Leu Pro Arg Phe Ile Leu Met Glu Leu Met Ala Gly
1 5 10 15
Gly Asp Leu Lys
20
<210> 46
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 46
Phe Leu Met Glu Ala Leu Ile Ile Ser Lys Leu Asn His Gln Asn Ile
1 5 10 15
Val Arg Cys Ile Gly Lys
20
<210> 47
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 47
Phe Leu Met Glu Ala Leu Ile Ile Ser Lys Val Asn His Gln Asn Ile
1 5 10 15
Val Arg Cys Ile Gly Lys
20
<210> 48
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 48
Arg Phe Ile Leu Leu Glu Leu Met Ala Gly Arg Asp Leu Lys Ser Phe
1 5 10 15
Leu Arg Glu Thr Arg
20
<210> 49
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 49
Cys Pro Gly Pro Gly Arg Val Ala Lys Ile Ala Asp Phe Gly Met Ala
1 5 10 15
Arg Asp Ile Tyr Arg
20
<210> 50
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 50
Glu Leu Asp Phe Leu Met Glu Ala Leu Ile Thr Ser Lys Phe Asn His
1 5 10 15
Gln Asn Ile Val Arg
20
<210> 51
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 51
Ser Leu Gln Ser Leu Pro Arg Phe Ile Leu Met Glu Leu Met Ala Gly
1 5 10 15
Gly Asp Leu Lys
20
<210> 52
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 52
Lys Val Ala Asp Phe Gly Leu Ala Arg His Met Tyr Asp Lys Glu Tyr
1 5 10 15
Tyr Ser Val His Lys
20
<210> 53
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 53
Lys Val Ala Asp Phe Gly Leu Ala Arg Asn Met Tyr Asp Lys Glu Tyr
1 5 10 15
Tyr Ser Val His
20
<210> 54
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 54
Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe
1 5 10 15
Gly Cys Leu Leu Asp Lys Lys Lys Lys Lys
20 25
<210> 55
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 55
Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Val Ile His Arg Asp Leu
1 5 10 15
Ala Ala Arg Asn
20
<210> 56
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 56
Val Met Lys Glu Ile Lys His Pro Asn Leu Leu Gln Leu Leu Gly Val
1 5 10 15
Cys Thr Arg Glu Pro
20
<210> 57
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 57
Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Lys Val Tyr Glu Gly Val
1 5 10 15
Trp Lys Lys Tyr Ser
20
<210> 58
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 58
Arg Glu Pro Pro Phe Tyr Ile Ile Thr Glu Leu Met Thr Tyr Gly Asn
1 5 10 15
Leu Leu Asp Tyr Leu Lys Lys
20
<210> 59
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 59
Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Val Ile His Arg Asp Leu
1 5 10 15
Ala Ala Arg Asn
20
<210> 60
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 60
Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ala Glu Phe Met Thr Tyr
1 5 10 15
Gly Asn Leu Leu Asp Lys Lys
20
<210> 61
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 61
Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ile Glu Phe Met Thr Tyr
1 5 10 15
Gly Asn Leu Leu Asp Lys Lys
20
<210> 62
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 62
Val Met Lys Glu Ile Lys His Pro Asn Leu Leu Gln Leu Leu Gly Val
1 5 10 15
Cys Thr Arg Glu Pro
20
<210> 63
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 63
Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe
1 5 10 15
Gly Cys Leu Leu Asp Lys Lys Lys Lys Lys
20 25
<210> 64
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 64
Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Tyr Glu Ala Tyr Val Met
1 5 10 15
Ala Ser Val Asp Lys
20
<210> 65
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 65
Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe
1 5 10 15
Gly Cys Leu Leu Asp Lys Lys Lys Lys Lys
20 25
<210> 66
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 66
Leu Ile Thr Gln Leu Met Pro Phe Gly Ser Leu Leu Asp Tyr Val Arg
1 5 10 15
Glu His Lys Asp
20
<210> 67
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 67
Leu Met Thr Gly Asp Thr Tyr Thr Ala His Pro Gly Ala Lys Phe Pro
1 5 10 15
Ile Lys Trp Lys Lys
20
<210> 68
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 68
Asp Thr Tyr Thr Ala His Ala Gly Thr Lys Phe Pro Ile Lys Trp Thr
1 5 10 15
Ala Pro Glu Lys Lys
20
<210> 69
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 69
Trp Ala Phe Gly Val Leu Leu Trp Glu Ile Thr Thr Tyr Gly Met Ser
1 5 10 15
Pro Tyr Pro Gly Ile Lys Lys Lys
20
<210> 70
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 70
Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Lys Val Tyr Glu Gly Val
1 5 10 15
Trp Lys Lys Tyr Ser
20
<210> 71
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 71
Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Val Val Tyr Glu Gly Val
1 5 10 15
Trp Lys Lys Tyr Ser
20
<210> 72
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 72
Ser Leu Thr Val Ala Val Lys Thr Leu Lys Lys Asp Thr Met Glu Val
1 5 10 15
Glu Glu Phe Leu Lys
20
<210> 73
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 73
Lys Thr Leu Lys Glu Asp Thr Met Ala Val Glu Glu Phe Leu Lys Glu
1 5 10 15
Ala Ala Val Lys Lys
20
<210> 74
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 74
Ala Val Lys Thr Leu Lys Glu Asp Thr Met Lys Val Glu Glu Phe Leu
1 5 10 15
Lys Glu Ala Ala Val Lys
20
<210> 75
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 75
Ala Val Lys Thr Leu Lys Glu Asp Thr Met Tyr Val Glu Glu Phe Leu
1 5 10 15
Lys Glu Ala Ala Val Lys Lys
20
<210> 76
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 76
Ala Val Lys Thr Leu Lys Glu Asp Thr Met Glx Val Glu Glu Phe Leu
1 5 10 15
Lys Glu Ala Lys Lys
20
<210> 77
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 77
Thr Leu Lys Glu Asp Thr Met Glu Val Glu Gly Phe Leu Lys Glu Ala
1 5 10 15
Ala Val Met Lys Lys Lys
20
<210> 78
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 78
Glu Phe Leu Lys Glu Ala Ala Val Met Lys Val Ile Lys His Pro Asn
1 5 10 15
Leu Val Gln Leu Leu Lys
20
<210> 79
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 79
Tyr Met Ala Thr Gln Ile Ser Ser Ala Met Asp Tyr Leu Glu Lys Lys
1 5 10 15
Asn Phe Ile His Arg
20
<210> 80
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 80
Tyr Met Ala Thr Gln Ile Ser Ser Ala Met Gly Tyr Leu Glu Lys Lys
1 5 10 15
Asn Phe Ile His Arg
20
<210> 81
<211> 19
<212> PRT
<213> Artificial Sequence
<400> 81
Thr Gln Ile Ser Ser Ala Met Glu Tyr Leu Ala Lys Lys Asn Phe Ile
1 5 10 15
His Arg Asp
<210> 82
<211> 18
<212> PRT
<213> Artificial Sequence
<400> 82
Gln Ile Ser Ser Ala Met Glu Tyr Leu Gly Lys Lys Asn Phe Ile His
1 5 10 15
Arg Asp
<210> 83
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 83
Tyr Pro Gly Ile Asp Leu Ser Gln Val Tyr Ala Leu Leu Glu Lys Asp
1 5 10 15
Tyr Arg Met Glu Arg Lys
20
<210> 84
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 84
Tyr Pro Gly Ile Asp Leu Ser Gln Val Tyr Gly Leu Leu Glu Lys Asp
1 5 10 15
Tyr Arg Met Glu Arg Lys
20
<210> 85
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 85
Tyr Pro Gly Ile Asp Leu Ser Gln Val Tyr Lys Leu Leu Glu Lys Asp
1 5 10 15
Tyr Arg Met Glu Arg
20
<210> 86
<211> 19
<212> PRT
<213> Artificial Sequence
<400> 86
Ile Asp Leu Ser Gln Val Tyr Glu Leu Leu Ala Lys Asp Tyr Arg Met
1 5 10 15
Glu Arg Lys
<210> 87
<211> 18
<212> PRT
<213> Artificial Sequence
<400> 87
Asp Leu Ser Gln Val Tyr Glu Leu Leu Gly Lys Asp Tyr Arg Met Glu
1 5 10 15
Arg Lys
<210> 88
<211> 18
<212> PRT
<213> Artificial Sequence
<400> 88
Ile Asp Leu Ser Gln Val Tyr Glu Leu Leu Lys Lys Asp Tyr Arg Met
1 5 10 15
Glu Arg
<210> 89
<211> 19
<212> PRT
<213> Artificial Sequence
<400> 89
Ile Asp Leu Ser Gln Val Tyr Glu Leu Leu Leu Lys Asp Tyr Arg Met
1 5 10 15
Glu Arg Lys
<210> 90
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 90
Leu Leu Gly Val Cys Thr Arg Glu Pro Pro Leu Tyr Ile Ile Thr Glu
1 5 10 15
Phe Met Thr Tyr Gly Lys Lys Lys
20
<210> 91
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 91
Arg Glu Pro Pro Phe Tyr Ile Ile Thr Glu Leu Met Thr Tyr Gly Asn
1 5 10 15
Leu Leu Asp Tyr Leu Lys Lys
20
<210> 92
<211> 19
<212> PRT
<213> Artificial Sequence
<400> 92
Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Ala Ile His Arg Asp Leu
1 5 10 15
Ala Ala Arg
<210> 93
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 93
Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Val Ile His Arg Asp Leu
1 5 10 15
Ala Ala Arg Asn
20
<210> 94
<211> 17
<212> PRT
<213> Artificial Sequence
<400> 94
Gly Glu Asn His Leu Val Lys Val Ala Asp Leu Gly Leu Ser Arg Leu
1 5 10 15
Met
<210> 95
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 95
Trp Gln Trp Asn Pro Ser Asp Arg Pro Ser Ser Ala Glu Ile His Gln
1 5 10 15
Ala Phe Glu Thr Met Lys
20
<210> 96
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 96
Met Thr Gly Asp Thr Tyr Thr Ala His Ala Arg Ala Lys Phe Pro Ile
1 5 10 15
Lys Trp Thr Ala Pro Lys Lys Lys
20
<210> 97
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 97
Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Pro Ala Gly Ala Lys Phe
1 5 10 15
Pro Ile Lys Trp Lys
20
<210> 98
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 98
Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Arg Ala Gly Ala Lys Phe
1 5 10 15
Pro Ile Lys Trp Thr Lys
20
<210> 99
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 99
Pro Glu Ser Leu Ala Tyr Asn Lys Phe Ser Val Lys Ser Asp Val Trp
1 5 10 15
Ala Phe Gly Val
20
<210> 100
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 100
Lys Glu Ala Ala Val Met Lys Glu Ile Arg Gly Gly His Pro Asn Leu
1 5 10 15
Val Gln Leu Leu Gly Val
20
<210> 101
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 101
Asn Cys Leu Val Gly Glu Asn His Leu Val Arg Val Ala Asp Phe Gly
1 5 10 15
Leu Ser Arg Leu Met
20
<210> 102
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 102
Glu Ser Leu Ala Tyr Asn Lys Phe Ser Ile Glu Ser Asp Val Trp Ala
1 5 10 15
Phe Gly Val Leu Leu Lys Lys
20
<210> 103
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 103
Ala Val Met Lys Glu Ile Lys His Pro Asn Val Val Gln Leu Leu Gly
1 5 10 15
Val Cys Thr Arg Lys
20
<210> 104
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 104
His Leu Val Lys Val Ala Asp Phe Gly Met Ser Arg Leu Met Thr Gly
1 5 10 15
Asp Thr Tyr Thr Lys Lys
20
<210> 105
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 105
Val Lys Val Ala Asp Phe Gly Leu Ser Arg Phe Met Thr Gly Asp Thr
1 5 10 15
Tyr Thr Ala His Ala Lys Lys Lys
20
<210> 106
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 106
Lys Val Ala Asp Phe Gly Leu Ser Arg Met Met Thr Gly Asp Thr Tyr
1 5 10 15
Thr Ala His Ala Lys Lys
20
<210> 107
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 107
Lys Trp Glu Met Glu Arg Thr Asp Ile Thr Val Lys His Lys Leu Gly
1 5 10 15
Gly Gly Gln Tyr
20
<210> 108
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 108
Lys Val Ala Asp Phe Gly Leu Ser Arg Leu Leu Thr Gly Asp Thr Tyr
1 5 10 15
Thr Ala His Ala Gly Lys
20
<210> 109
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 109
Leu Ala Ala Arg Asn Cys Leu Val Gly Glu Tyr His Leu Val Lys Val
1 5 10 15
Ala Asp Phe Lys Lys Lys
20
<210> 110
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 110
Glu Leu Met Arg Ala Cys Trp Gln Trp Asn Leu Ser Asp Arg Pro Ser
1 5 10 15
Phe Ala Glu Ile His
20
<210> 111
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 111
Ile Thr Met Lys His Lys Leu Gly Gly Gly Glu Tyr Gly Glu Val Tyr
1 5 10 15
Glu Gly Val Trp
20
<210> 112
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 112
Ile Thr Met Lys His Lys Leu Gly Gly Gly His Tyr Gly Glu Val Tyr
1 5 10 15
Glu Gly Val Trp Lys
20
<210> 113
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 113
Ile Thr Met Lys His Lys Leu Gly Gly Gly Lys Tyr Gly Glu Val Tyr
1 5 10 15
Glu Gly Val Trp Lys
20
<210> 114
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 114
Ile Thr Met Lys His Lys Leu Gly Gly Gly Arg Tyr Gly Glu Val Tyr
1 5 10 15
Glu Gly Val Trp Lys
20
<210> 115
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 115
Met Thr Tyr Gly Asn Leu Leu Asp Tyr Leu Met Glu Cys Asn Arg Gln
1 5 10 15
Glu Val Asn Ala Val Lys Lys
20
<210> 116
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 116
Ala Pro Glu Ser Leu Ala Tyr Asn Lys Phe Tyr Ile Lys Ser Asp Val
1 5 10 15
Trp Ala Phe Gly Val
20
<210> 117
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 117
Val Ala Val Lys Thr Leu Lys Glu Asp Ala Met Glu Val Glu Glu Phe
1 5 10 15
Leu Lys Glu Ala Lys Lys Lys
20
<210> 118
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 118
Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ile Glu Phe Met Thr Tyr
1 5 10 15
Gly Asn Leu Leu Asp Lys Lys
20
<210> 119
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 119
Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Asn Glu Phe Met Thr Tyr
1 5 10 15
Gly Asn Leu Leu Asp Lys Lys
20
<210> 120
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 120
Val Glu Glu Phe Leu Lys Glu Ala Ala Phe Met Lys Glu Ile Lys His
1 5 10 15
Pro Asn Leu Val
20
<210> 121
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 121
Val Met Lys Glu Ile Lys His Pro Asn Leu Leu Gln Leu Leu Gly Val
1 5 10 15
Cys Thr Arg Glu Pro
20
<210> 122
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 122
Cys Leu Val Gly Glu Asn His Leu Val Lys Ile Ala Asp Phe Gly Leu
1 5 10 15
Ser Arg Leu Met Thr Lys
20
<210> 123
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 123
Thr Met Lys His Lys Leu Gly Gly Gly Gln Phe Gly Glu Val Tyr Glu
1 5 10 15
Gly Val Trp Lys Lys
20
<210> 124
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 124
Pro Phe Tyr Ile Ile Thr Glu Phe Met Thr Cys Gly Asn Leu Leu Asp
1 5 10 15
Tyr Leu Arg Lys Lys Lys
20
<210> 125
<211> 18
<212> PRT
<213> Artificial Sequence
<400> 125
Asp Ser Asn Tyr Val Val Lys Gly Asn Pro Arg Leu Pro Val Lys Trp
1 5 10 15
Met Ala
<210> 126
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 126
Lys Ile Cys Asp Phe Gly Leu Ala Arg Ala Ile Lys Asn Asp Ser Asn
1 5 10 15
Tyr Val Val Lys
20
<210> 127
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 127
Lys Ile Cys Asp Phe Gly Leu Ala Arg Glu Ile Lys Asn Asp Ser Asn
1 5 10 15
Tyr Val Val Lys
20
<210> 128
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 128
Lys Ile Cys Asp Phe Gly Leu Ala Arg His Ile Lys Asn Asp Ser Asn
1 5 10 15
Tyr Val Val Lys
20
<210> 129
<211> 16
<212> PRT
<213> Artificial Sequence
<400> 129
Leu Ala Arg Asp Ile Lys Asn Gly Ser Asn Tyr Val Val Lys Gly Asn
1 5 10 15
<210> 130
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 130
Asp Phe Gly Leu Ala Arg Asp Ile Lys Asn Val Ser Asn Tyr Val Val
1 5 10 15
Lys Gly Asn Ala Arg
20
<210> 131
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 131
Asp Phe Gly Leu Ala Arg Asp Ile Lys Asn Tyr Ser Asn Tyr Val Val
1 5 10 15
Lys Gly Asn Ala Arg
20
<210> 132
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 132
Glu Arg Glu Ala Leu Met Ser Glu Leu Glu Val Leu Ser Tyr Leu Gly
1 5 10 15
Asn His Met Asn Lys Lys
20
<210> 133
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 133
Glu Ala Ala Leu Tyr Lys Asn Leu Leu His Phe Lys Glu Ser Ser Cys
1 5 10 15
Ser Asp Ser Thr
20
<210> 134
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 134
Phe Gly Leu Ala Arg Asp Ile Lys Asn Asp Phe Asn Tyr Val Val Lys
1 5 10 15
Gly Asn Ala Arg
20
<210> 135
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 135
Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Glu Glu Tyr Cys Cys Tyr
1 5 10 15
Gly Asp Leu Leu Asn Lys Lys Lys
20
<210> 136
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 136
Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Ile Glu Tyr Cys Cys Tyr
1 5 10 15
Gly Asp Leu Leu Asn Lys Lys Lys
20
<210> 137
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 137
Leu Ser Tyr Leu Gly Asn His Met Asn Ile Ala Asn Leu Leu Gly Ala
1 5 10 15
Cys Thr Ile Gly Lys Lys
20
<210> 138
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 138
Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Val Ile Met His Asp Ser
1 5 10 15
Asn Tyr Val Ser Lys
20
<210> 139
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 139
Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Lys Val Tyr Glu Gly Val
1 5 10 15
Trp Lys Lys Tyr Ser
20
<210> 140
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 140
Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Val Val Tyr Glu Gly Val
1 5 10 15
Trp Lys Lys Tyr Ser
20
<210> 141
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 141
Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Arg Ala Gly Ala Lys Phe
1 5 10 15
Pro Ile Lys Trp Thr Lys
20
<210> 142
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 142
Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ile Glu Phe Met Thr Tyr
1 5 10 15
Gly Asn Leu Leu Asp Lys Lys
20
<210> 143
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 143
Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Val Glu Phe Met Thr Tyr
1 5 10 15
Gly Asn Leu Leu Asp Lys Lys
20
<210> 144
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 144
Ser Tyr Leu Gly Asn His Met Asn Ile Val Thr Leu Leu Gly Ala Cys
1 5 10 15
Thr Ile Gly Gly Pro Lys
20
<210> 145
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 145
Leu Ile Thr Gln Leu Met Pro Phe Gly Ser Leu Leu Asp Tyr Val Arg
1 5 10 15
Glu His Lys Asp
20
<210> 146
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 146
Gln Leu Ile Thr Gln Leu Met Pro Phe Asp Cys Leu Leu Asp Tyr Val
1 5 10 15
Arg Glu His Lys Lys
20
<210> 147
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 147
Val Gln Leu Ile Thr Gln Leu Met Pro Phe Arg Cys Leu Leu Asp Tyr
1 5 10 15
Val Arg Glu His Lys Lys
20
<210> 148
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 148
Gln Leu Ile Thr Gln Leu Met Pro Phe Ser Cys Leu Leu Asp Tyr Val
1 5 10 15
Arg Glu His Lys Lys
20
<210> 149
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 149
Leu Thr Ser Thr Val Gln Leu Ile Thr Gln His Met Pro Phe Gly Cys
1 5 10 15
Leu Leu Asp Tyr Val Lys Lys Lys Lys Lys
20 25
<210> 150
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 150
Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe
1 5 10 15
Gly Cys Leu Leu Asp Lys Lys Lys Lys Lys
20 25
<210> 151
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 151
Lys Ile Cys Asp Phe Gly Leu Ala Arg His Ile Met Ser Asp Ser Asn
1 5 10 15
Tyr Val Val Arg
20
<210> 152
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 152
Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Tyr Ile Met Ser Asp Ser
1 5 10 15
Asn Tyr Val Val Arg
20
<210> 153
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 153
Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Tyr Ile Met Ser Asp Ser
1 5 10 15
Asn Tyr Val Val Arg
20
<210> 154
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 154
Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Val Ile Met His Asp Ser
1 5 10 15
Asn Tyr Val Ser Lys
20
<210> 155
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 155
Asp Thr Tyr Thr Ala His Ala Gly Thr Lys Phe Pro Ile Lys Trp Thr
1 5 10 15
Ala Pro Glu Lys Lys
20
<210> 156
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 156
Thr Glu Phe Met Thr Tyr Gly Asn Leu Leu Gly Tyr Leu Arg Glu Cys
1 5 10 15
Asn Arg Gln Glu Val Lys
20
<210> 157
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 157
Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Lys Val Tyr Glu Gly Val
1 5 10 15
Trp Lys Lys Tyr Ser
20
<210> 158
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 158
Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Val Val Tyr Glu Gly Val
1 5 10 15
Trp Lys Lys Tyr Ser
20
<210> 159
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 159
Ala Val Lys Thr Leu Lys Glu Asp Thr Met Lys Val Glu Glu Phe Leu
1 5 10 15
Lys Glu Ala Ala Val Lys
20
<210> 160
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 160
Thr Leu Lys Glu Asp Thr Met Glu Val Glu Lys Phe Leu Lys Glu Ala
1 5 10 15
Ala Val Met Lys Lys
20
<210> 161
<211> 18
<212> PRT
<213> Artificial Sequence
<400> 161
Gln Ile Ser Ser Ala Met Glu Tyr Leu Gly Lys Lys Asn Phe Ile His
1 5 10 15
Arg Asp
<210> 162
<211> 18
<212> PRT
<213> Artificial Sequence
<400> 162
Ile Asp Leu Ser Gln Val Tyr Glu Leu Leu Lys Lys Asp Tyr Arg Met
1 5 10 15
Glu Arg
<210> 163
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 163
Leu Leu Gly Val Cys Thr Arg Glu Pro Pro Leu Tyr Ile Ile Thr Glu
1 5 10 15
Phe Met Thr Tyr Gly Lys Lys Lys
20
<210> 164
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 164
Arg Glu Pro Pro Phe Tyr Ile Ile Thr Glu Leu Met Thr Tyr Gly Asn
1 5 10 15
Leu Leu Asp Tyr Leu Lys Lys
20
<210> 165
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 165
Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Val Ile His Arg Asp Leu
1 5 10 15
Ala Ala Arg Asn
20
<210> 166
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 166
Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Pro Ala Gly Ala Lys Phe
1 5 10 15
Pro Ile Lys Trp Lys
20
<210> 167
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 167
Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Arg Ala Gly Ala Lys Phe
1 5 10 15
Pro Ile Lys Trp Thr Lys
20
<210> 168
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 168
Ala Val Met Lys Glu Ile Lys His Pro Asn Val Val Gln Leu Leu Gly
1 5 10 15
Val Cys Thr Arg Lys
20
<210> 169
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 169
His Leu Val Lys Val Ala Asp Phe Gly Met Ser Arg Leu Met Thr Gly
1 5 10 15
Asp Thr Tyr Thr Lys Lys
20
<210> 170
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 170
Val Lys Val Ala Asp Phe Gly Leu Ser Arg Phe Met Thr Gly Asp Thr
1 5 10 15
Tyr Thr Ala His Ala Lys Lys Lys
20
<210> 171
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 171
Lys Val Ala Asp Phe Gly Leu Ser Arg Met Met Thr Gly Asp Thr Tyr
1 5 10 15
Thr Ala His Ala Lys Lys
20
<210> 172
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 172
Lys Trp Glu Met Glu Arg Thr Asp Ile Thr Val Lys His Lys Leu Gly
1 5 10 15
Gly Gly Gln Tyr
20
<210> 173
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 173
Lys Val Ala Asp Phe Gly Leu Ser Arg Leu Leu Thr Gly Asp Thr Tyr
1 5 10 15
Thr Ala His Ala Gly Lys
20
<210> 174
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 174
Leu Leu Gly Val Cys Thr Arg Glu Pro Ser Phe Tyr Ile Ile Thr Glu
1 5 10 15
Phe Met Thr Tyr Lys Lys Lys Lys
20
<210> 175
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 175
Ile Thr Met Lys His Lys Leu Gly Gly Gly His Tyr Gly Glu Val Tyr
1 5 10 15
Glu Gly Val Trp Lys
20
<210> 176
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 176
Ile Thr Met Lys His Lys Leu Gly Gly Gly Met Tyr Gly Glu Val Tyr
1 5 10 15
Glu Gly Val Trp Lys
20
<210> 177
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 177
Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ala Glu Phe Met Thr Tyr
1 5 10 15
Gly Asn Leu Leu Asp Lys Lys
20
<210> 178
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 178
Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ile Glu Phe Met Thr Tyr
1 5 10 15
Gly Asn Leu Leu Asp Lys Lys
20
<210> 179
<211> 24
<212> PRT
<213> Artificial Sequence
<400> 179
Ser Phe Ala Glu Ile His Gln Ala Phe Glu Arg Met Phe Gln Glu Ser
1 5 10 15
Ser Ile Ser Asp Glu Lys Lys Lys
20
<210> 180
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 180
Val Glu Glu Phe Leu Lys Glu Ala Ala Ala Met Lys Glu Ile Lys His
1 5 10 15
Pro Asn Leu Val
20
<210> 181
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 181
Val Met Lys Glu Ile Lys His Pro Asn Leu Leu Gln Leu Leu Gly Val
1 5 10 15
Cys Thr Arg Glu Pro
20
<210> 182
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 182
Arg Glu Cys Asn Arg Gln Glu Val Asn Ala Phe Val Leu Leu Tyr Met
1 5 10 15
Ala Thr Gln Ile Lys
20
<210> 183
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 183
Cys Leu Val Gly Glu Asn His Leu Val Lys Ile Ala Asp Phe Gly Leu
1 5 10 15
Ser Arg Leu Met Thr Lys
20
<210> 184
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 184
Thr Met Lys His Lys Leu Gly Gly Gly Gln Phe Gly Glu Val Tyr Glu
1 5 10 15
Gly Val Trp Lys Lys
20
<210> 185
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 185
Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe
1 5 10 15
Gly Cys Leu Leu Asp Lys Lys Lys Lys Lys
20 25
<210> 186
<211> 26
<212> PRT
<213> Artificial Sequence
<400> 186
Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe
1 5 10 15
Gly Cys Leu Leu Asp Lys Lys Lys Lys Lys
20 25
<210> 187
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 187
Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Phe Ile Met Ser Asp Ser
1 5 10 15
Asn Tyr Val Val Arg
20
<210> 188
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 188
Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Val Ile Met Ser Asp Ser
1 5 10 15
Asn Tyr Val Val Arg
20
<210> 189
<211> 21
<212> PRT
<213> Artificial Sequence
<400> 189
Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Tyr Ile Met Ser Asp Ser
1 5 10 15
Asn Tyr Val Val Arg
20
<210> 190
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 190
Thr Gln Ala Trp Asp Leu Tyr Tyr His Val Leu Arg Arg Ile Ser Lys
1 5 10 15
Gln Leu Pro Gln
20
<210> 191
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 191
Thr Gln Ala Trp Asp Leu Tyr Tyr His Val Leu Arg Arg Ile Ser Lys
1 5 10 15
Gln Leu Pro Gln
20
<210> 192
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 192
His Glu Cys Asn Ser Pro Tyr Ile Val Gly Leu Tyr Gly Ala Phe Tyr
1 5 10 15
Ser Asp Gly Glu Ile Lys Lys
20
<210> 193
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 193
Val Lys Val Ala Asp Phe Gly Leu Ala Arg Val Met Tyr Asp Lys Glu
1 5 10 15
Tyr Tyr Ser Val His Lys
20
<210> 194
<211> 630
<212> PRT
<213> Artificial Sequence
<400> 194
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Gly Gly
20 25 30
Ser Gly Gly Gly Gly Ser Gly Gly Met Leu Arg Leu Gly Ile Phe Gly
35 40 45
Phe Leu Val Phe Gly Phe Val Leu Ile Thr Phe Ser Cys Lys Lys Lys
50 55 60
Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala Phe Gly
65 70 75 80
Phe Val Leu Ile Thr Phe Ser Tyr His Phe Tyr Asp Phe Phe Asn Gln
85 90 95
Ala Glu Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly
100 105 110
Gly Val Leu Ile Thr Phe Ser Cys His Phe Tyr Gly Phe Phe Asn Gln
115 120 125
Ala Glu Trp Glu Arg Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Leu Arg Leu Gly Ile Phe Gly Phe Leu Ala Leu Gly Phe Val Leu
145 150 155 160
Ile Thr Phe Ser Cys His Lys Lys Lys Lys Lys Lys Gly Gly Ser Gly
165 170 175
Gly Gly Gly Ser Gly Gly Tyr Val Leu Cys Gln Ala Asn Val Thr Ile
180 185 190
Trp Leu Pro Thr Lys Gln Pro Ile Pro Asp Cys Lys Gly Gly Ser Gly
195 200 205
Gly Gly Gly Ser Gly Gly Val Leu Ile Thr Phe Ser Cys His Phe Tyr
210 215 220
His Phe Phe Asn Gln Ala Glu Trp Glu Arg Ser Lys Lys Lys Gly Gly
225 230 235 240
Ser Gly Gly Gly Gly Ser Gly Gly Phe Thr Glu Ala Glu His Gln Asp
245 250 255
Met Arg Ser Tyr Ile Ala Ala Phe Gly Ala Val Thr Lys Lys Gly Gly
260 265 270
Ser Gly Gly Gly Gly Ser Gly Gly Met Phe Gly Thr Gly Ile Ala Met
275 280 285
Ser Thr Leu Val Trp Thr Lys Ala Thr Leu Leu Ile Trp Lys Lys Lys
290 295 300
Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val
305 310 315 320
Leu Ile Thr Phe Ser Cys His Phe Tyr Tyr Phe Phe Asn Gln Ala Glu
325 330 335
Trp Glu Arg Ser Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly
340 345 350
Gly Phe Ser Cys His Phe Tyr Asp Phe Phe Asn Glu Ala Glu Trp Glu
355 360 365
Arg Ser Phe Arg Asp Tyr Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser
370 375 380
Gly Gly His Ser Tyr Ile Ala Ala Phe Gly Ala Val Met Gly Leu Cys
385 390 395 400
Thr Leu Phe Thr Leu Ala Lys Lys Lys Lys Lys Lys Lys Lys Lys Gly
405 410 415
Gly Ser Gly Gly Gly Gly Ser Gly Gly Thr Leu Ser Cys Val Ile Ile
420 425 430
Phe Val Ile Ala Tyr Tyr Ala Leu Met Ala Gly Val Val Trp Lys Lys
435 440 445
Lys Lys Lys Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser
450 455 460
Gly Gly Asn Ala Cys Phe Phe Val Gly Ser Ile Gly Leu Leu Ala Gln
465 470 475 480
Phe Met Asp Gly Ala Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly
485 490 495
Gly Met Phe Gly Thr Gly Ile Ala Met Ser Thr Arg Val Trp Thr Lys
500 505 510
Ala Thr Leu Leu Ile Trp Lys Lys Lys Lys Lys Lys Gly Gly Ser Gly
515 520 525
Gly Gly Gly Ser Gly Gly Thr Leu Ser Cys Val Ile Ile Phe Val Ile
530 535 540
Met Tyr Tyr Ala Leu Met Ala Gly Val Val Trp Lys Lys Lys Lys Lys
545 550 555 560
Lys Lys Lys Lys Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile
565 570 575
Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly
580 585 590
Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys
595 600 605
Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser
610 615 620
Asp Val Ser Leu Thr Ala
625 630
<210> 195
<211> 348
<212> PRT
<213> Artificial Sequence
<400> 195
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Met Leu
20 25 30
Arg Leu Arg Leu Gly Ile Phe Gly Phe Leu Ala Phe Gly Phe Val Leu
35 40 45
Ile Thr Phe Ser Cys His Phe Tyr Asp Phe Phe Asn Gln Ala Glu Trp
50 55 60
Glu Arg Ser Phe Arg Asp Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
65 70 75 80
Tyr Val Leu Cys Gln Ala Asn Val Thr Ile Gly Leu Pro Thr Lys Gln
85 90 95
Pro Ile Pro Asp Cys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
100 105 110
Phe Thr Glu Ala Glu His Gln Asp Met His Ser Tyr Ile Ala Ala Phe
115 120 125
Gly Ala Val Thr Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
130 135 140
His Ser Tyr Ile Ala Ala Phe Gly Ala Val Met Gly Leu Cys Thr Leu
145 150 155 160
Phe Thr Leu Ala Lys Lys Lys Lys Lys Lys Lys Lys Lys Gly Gly Ser
165 170 175
Gly Gly Gly Gly Ser Gly Gly Thr Leu Ser Cys Val Ile Ile Phe Val
180 185 190
Ile Val Tyr Tyr Ala Leu Met Ala Gly Val Val Trp Lys Lys Lys Lys
195 200 205
Lys Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
210 215 220
Asn Ala Cys Phe Phe Val Gly Ser Ile Gly Trp Leu Ala Gln Phe Met
225 230 235 240
Asp Gly Ala Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Met
245 250 255
Phe Gly Thr Gly Ile Ala Met Ser Thr Leu Val Trp Thr Lys Ala Thr
260 265 270
Leu Leu Ile Trp Lys Lys Lys Lys Lys Lys Lys Gly Gly Ser Leu Gly
275 280 285
Gly Gly Gly Ser Gly Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu
290 295 300
Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val Met Cys Arg Arg
305 310 315 320
Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser
325 330 335
Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala
340 345
<210> 196
<211> 246
<212> PRT
<213> Artificial Sequence
<400> 196
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Phe Ile
20 25 30
Ile Thr Glu Tyr Met Ala Asn Gly Phe Leu Leu Asn Tyr Leu Arg Glu
35 40 45
Met Arg His Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
50 55 60
Ile Ile Thr Glu Tyr Met Ala Asn Gly Arg Leu Leu Asn Tyr Leu Arg
65 70 75 80
Glu Met Arg His Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val
85 90 95
Arg Asp Ser Ser Lys Ala Gly Lys Tyr Ala Val Ser Val Phe Ala Lys
100 105 110
Ser Thr Gly Asp Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Ile
115 120 125
Thr Glu Tyr Met Ala Asn Gly Ser Leu Leu Asn Tyr Leu Arg Glu Met
130 135 140
Arg His Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Phe Ile Ile
145 150 155 160
Thr Glu Tyr Met Ala Asn Gly Tyr Leu Leu Asn Tyr Leu Arg Glu Met
165 170 175
Arg His Lys Lys Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile
180 185 190
Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly
195 200 205
Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys
210 215 220
Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser
225 230 235 240
Asp Val Ser Leu Thr Ala
245
<210> 197
<211> 150
<212> PRT
<213> Artificial Sequence
<400> 197
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Phe Ile
20 25 30
Ile Thr Glu Tyr Met Ala Asn Gly Cys Leu Leu Asn Tyr Leu Arg Glu
35 40 45
Met Arg His Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
50 55 60
Val Arg Asp Ser Ser Lys Thr Gly Lys Tyr Ala Val Ser Val Phe Ala
65 70 75 80
Lys Ser Thr Gly Asp Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile
85 90 95
Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly
100 105 110
Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys
115 120 125
Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser
130 135 140
Asp Val Ser Leu Thr Ala
145 150
<210> 198
<211> 208
<212> PRT
<213> Artificial Sequence
<400> 198
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Pro Ile
20 25 30
Ala Arg Glu Leu His Gln Phe Ala Phe Asp Leu Leu Ile Lys Ser His
35 40 45
Met Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Gln Pro
50 55 60
Ile Ala Arg Glu Leu His Gln Leu Thr Phe Asp Leu Leu Ile Lys Ser
65 70 75 80
His Met Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asn Glu Leu
85 90 95
Gly Glu Arg Gln Leu Val His Met Val Lys Trp Ala Lys Ala Leu Pro
100 105 110
Gly Phe Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Pro Ile Ala Arg
115 120 125
Glu Leu His Gln Phe Ser Phe Asp Leu Leu Ile Lys Ser His Met Gly
130 135 140
Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile Val Gly Ile Val Ala Gly
145 150 155 160
Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val
165 170 175
Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln
180 185 190
Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala
195 200 205
<210> 199
<211> 147
<212> PRT
<213> Artificial Sequence
<400> 199
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Val Gln
20 25 30
Pro Ile Ala Arg Glu Leu His Gln Phe Thr Phe Asp Leu Leu Ile Lys
35 40 45
Ser His Met Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asn Glu Leu
50 55 60
Gly Glu Arg Gln Leu Val His Met Val Lys Trp Ala Lys Ala Leu Pro
65 70 75 80
Gly Phe Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile Val Gly Ile
85 90 95
Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val
100 105 110
Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser
115 120 125
Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser
130 135 140
Leu Thr Ala
145
<210> 200
<211> 340
<212> PRT
<213> Artificial Sequence
<400> 200
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Leu His
20 25 30
Glu Cys Asn Ser Pro Tyr Ile Val Val Phe Tyr Gly Ala Phe Tyr Ser
35 40 45
Asp Gly Glu Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr
50 55 60
Lys Leu Val Val Val Gly Ala Asp Gly Val Gly Lys Ser Ala Leu Gly
65 70 75 80
Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys Leu Leu Asp Ile Leu Asp
85 90 95
Thr Ala Gly Arg Glu Glu Tyr Ser Ala Met Arg Asp Gln Tyr Lys Lys
100 105 110
Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Arg Lys Arg Leu Glu
115 120 125
Ala Phe Leu Thr Pro Lys Gln Lys Val Gly Glu Leu Lys Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Glu Leu Gln Val Leu His Glu Cys Asn
145 150 155 160
Ser Leu Tyr Ile Val Gly Phe Tyr Gly Ala Phe Tyr Lys Lys Lys Gly
165 170 175
Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Lys Arg Leu Glu Ala Phe
180 185 190
Leu Thr Pro Lys Ala Lys Val Gly Glu Leu Lys Gly Gly Ser Gly Gly
195 200 205
Gly Gly Ser Gly Gly Leu Gln Val Leu His Glu Cys Asn Ser Ser Tyr
210 215 220
Ile Val Gly Phe Tyr Gly Ala Phe Tyr Lys Lys Gly Gly Ser Leu Gly
225 230 235 240
Gly Gly Gly Ser Gly Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu
245 250 255
Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val Met Cys Arg Arg
260 265 270
Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser
275 280 285
Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala Met Leu Arg Leu
290 295 300
Arg Leu Gly Ile Phe Gly Phe Leu Ala Phe Gly Phe Val Leu Ile Thr
305 310 315 320
Phe Ser Cys His Phe Tyr Asp Phe Phe Asn Gln Ala Glu Trp Glu Arg
325 330 335
Ser Phe Arg Asp
340
<210> 201
<211> 238
<212> PRT
<213> Artificial Sequence
<400> 201
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Tyr Lys
20 25 30
Leu Val Val Val Gly Ala Gly Gly Val Gly Lys Ser Ala Leu Gly Gly
35 40 45
Ser Gly Gly Gly Gly Ser Gly Gly Cys Leu Leu Asp Ile Leu Asp Thr
50 55 60
Ala Gly Gln Glu Glu Tyr Ser Ala Met Arg Asp Gln Tyr Lys Lys Lys
65 70 75 80
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Arg Lys Arg Leu Glu Ala
85 90 95
Phe Leu Thr Gln Lys Gln Lys Val Gly Glu Leu Lys Gly Gly Ser Gly
100 105 110
Gly Gly Gly Ser Gly Gly Glu Leu Gln Val Leu His Glu Cys Asn Ser
115 120 125
Pro Tyr Ile Val Gly Phe Tyr Gly Ala Phe Tyr Ser Asp Gly Glu Lys
130 135 140
Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Lys Arg Leu Glu
145 150 155 160
Ala Phe Leu Thr Gln Lys Ala Lys Val Gly Glu Leu Lys Gly Gly Ser
165 170 175
Leu Gly Gly Gly Gly Ser Gly Ile Val Gly Ile Val Ala Gly Leu Ala
180 185 190
Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr Val Met Cys
195 200 205
Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser Gln Ala Ala
210 215 220
Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr Ala
225 230 235
<210> 202
<211> 360
<212> PRT
<213> Artificial Sequence
<400> 202
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Lys Val
20 25 30
Ala Asp Phe Gly Leu Ala Arg His Met Tyr Asp Lys Glu Tyr Tyr Ser
35 40 45
Val His Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asn Ile Thr
50 55 60
Leu Ile Arg Gly Leu Ser His Gly Ala Phe Gly Glu Val Tyr Glu Gly
65 70 75 80
Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Leu Asp Phe Leu Met Glu
85 90 95
Ala Leu Ile Thr Ser Lys Phe Asn His Gln Asn Ile Val Arg Gly Gly
100 105 110
Ser Gly Gly Gly Gly Ser Gly Gly Arg Phe Ile Leu Leu Glu Leu Met
115 120 125
Ala Gly Arg Asp Leu Lys Ser Phe Leu Arg Glu Thr Arg Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Cys Pro Gly Pro Gly Arg Val Ala Lys
145 150 155 160
Ile Ala Asp Phe Gly Met Ala Arg Asp Ile Tyr Arg Gly Gly Ser Gly
165 170 175
Gly Gly Gly Ser Gly Gly Phe Leu Met Glu Ala Leu Ile Ile Ser Lys
180 185 190
Leu Asn His Gln Asn Ile Val Arg Cys Ile Gly Lys Gly Gly Ser Gly
195 200 205
Gly Gly Gly Ser Gly Gly Lys Val Ala Asp Phe Gly Leu Ala Arg Asn
210 215 220
Met Tyr Asp Lys Glu Tyr Tyr Ser Val His Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Gly Gly Ile Leu Leu Glu Leu Met Ala Gly Gly Asn Leu Lys
245 250 255
Ser Phe Leu Arg Glu Thr Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly
260 265 270
Gly Phe Leu Met Glu Ala Leu Ile Ile Ser Lys Val Asn His Gln Asn
275 280 285
Ile Val Arg Cys Ile Gly Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser
290 295 300
Gly Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val
305 310 315 320
Ile Gly Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly
325 330 335
Gly Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln
340 345 350
Gly Ser Asp Val Ser Leu Thr Ala
355 360
<210> 203
<211> 180
<212> PRT
<213> Artificial Sequence
<400> 203
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Lys Val
20 25 30
Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu Tyr Tyr Ser
35 40 45
Val His Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Leu Asp
50 55 60
Phe Leu Met Glu Ala Leu Ile Ile Ser Lys Phe Asn His Gln Asn Ile
65 70 75 80
Val Arg Cys Ile Gly Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
85 90 95
Ile Leu Leu Glu Leu Met Ala Gly Gly Asp Leu Lys Ser Phe Leu Arg
100 105 110
Glu Thr Arg Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile Val Gly
115 120 125
Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val
130 135 140
Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly
145 150 155 160
Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val
165 170 175
Ser Leu Thr Ala
180
<210> 204
<211> 1559
<212> PRT
<213> Artificial Sequence
<400> 204
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Thr Ser
20 25 30
Pro Lys Ala Asn Lys Glu Ile Leu Tyr Glu Ala Tyr Val Met Ala Ser
35 40 45
Val Asp Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Cys Leu
50 55 60
Thr Ser Thr Val Gln Leu Ile Met Gln Leu Met Pro Phe Gly Cys Leu
65 70 75 80
Leu Asp Lys Lys Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly
85 90 95
Gly Ala Pro Glu Ser Leu Ala Tyr Asn Lys Phe Tyr Ile Lys Ser Asp
100 105 110
Val Trp Ala Phe Gly Val Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Ala Val Lys Thr Leu Lys Glu Asp Thr Met Lys Val Glu Glu Phe Leu
130 135 140
Lys Glu Ala Ala Val Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
145 150 155 160
Ala Val Met Lys Glu Ile Lys His Pro Asn Val Val Gln Leu Leu Gly
165 170 175
Val Cys Thr Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys
180 185 190
Leu Val Gly Glu Asn His Leu Val Lys Ile Ala Asp Phe Gly Leu Ser
195 200 205
Arg Leu Met Thr Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys
210 215 220
Thr Arg Glu Pro Pro Phe Tyr Ile Ile Ala Glu Phe Met Thr Tyr Gly
225 230 235 240
Asn Leu Leu Asp Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
245 250 255
Gln Leu Ile Thr Gln Leu Met Pro Phe Asp Cys Leu Leu Asp Tyr Val
260 265 270
Arg Glu His Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Glu Asn His Leu Val Lys Val Ala Asp Leu Gly Leu Ser Arg Leu Met
290 295 300
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Asp Leu Ser Gln Val
305 310 315 320
Tyr Glu Leu Leu Lys Lys Asp Tyr Arg Met Glu Arg Gly Gly Ser Gly
325 330 335
Gly Gly Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu Gly Gly Gly
340 345 350
Glu Tyr Gly Glu Val Tyr Glu Gly Val Trp Gly Gly Ser Gly Gly Gly
355 360 365
Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu Gly Gly Gly His Tyr
370 375 380
Gly Glu Val Tyr Glu Gly Val Trp Lys Gly Gly Ser Gly Gly Gly Gly
385 390 395 400
Ser Gly Gly Leu Thr Ser Thr Val Gln Leu Ile Thr Gln His Met Pro
405 410 415
Phe Gly Cys Leu Leu Asp Tyr Val Lys Lys Lys Lys Lys Gly Gly Ser
420 425 430
Gly Gly Gly Gly Ser Gly Gly Cys Thr Arg Glu Pro Pro Phe Tyr Ile
435 440 445
Ile Asn Glu Phe Met Thr Tyr Gly Asn Leu Leu Asp Lys Lys Gly Gly
450 455 460
Ser Gly Gly Gly Gly Ser Gly Gly Lys Glu Ala Ala Val Met Lys Glu
465 470 475 480
Ile Arg Gly Gly His Pro Asn Leu Val Gln Leu Leu Gly Val Gly Gly
485 490 495
Ser Gly Gly Gly Gly Ser Gly Gly Lys His Lys Leu Gly Gly Gly Gln
500 505 510
Tyr Gly Lys Val Tyr Glu Gly Val Trp Lys Lys Tyr Ser Gly Gly Ser
515 520 525
Gly Gly Gly Gly Ser Gly Gly Lys Val Ala Asp Phe Gly Leu Ser Arg
530 535 540
Met Met Thr Gly Asp Thr Tyr Thr Ala His Ala Lys Lys Gly Gly Ser
545 550 555 560
Gly Gly Gly Gly Ser Gly Gly Lys Trp Glu Met Glu Arg Thr Asp Ile
565 570 575
Thr Val Lys His Lys Leu Gly Gly Gly Gln Tyr Gly Gly Ser Gly Gly
580 585 590
Gly Gly Ser Gly Gly Leu Ala Ala Arg Asn Cys Leu Val Gly Glu Tyr
595 600 605
His Leu Val Lys Val Ala Asp Phe Lys Lys Lys Gly Gly Ser Gly Gly
610 615 620
Gly Gly Ser Gly Gly Leu Leu Gly Val Cys Thr Arg Glu Pro Pro Leu
625 630 635 640
Tyr Ile Ile Thr Glu Phe Met Thr Tyr Gly Lys Lys Lys Gly Gly Ser
645 650 655
Gly Gly Gly Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu Gly Gly
660 665 670
Gly Met Tyr Gly Glu Val Tyr Glu Gly Val Trp Lys Gly Gly Ser Gly
675 680 685
Gly Gly Gly Ser Gly Gly Ile Asp Leu Ser Gln Val Tyr Glu Leu Leu
690 695 700
Leu Lys Asp Tyr Arg Met Glu Arg Lys Gly Gly Ser Gly Gly Gly Gly
705 710 715 720
Ser Gly Gly Leu Met Thr Gly Asp Thr Tyr Thr Ala His Pro Gly Ala
725 730 735
Lys Phe Pro Ile Lys Trp Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser
740 745 750
Gly Gly Met Thr Tyr Gly Asn Leu Leu Asp Tyr Leu Met Glu Cys Asn
755 760 765
Arg Gln Glu Val Asn Ala Val Lys Lys Gly Gly Ser Gly Gly Gly Gly
770 775 780
Ser Gly Gly Asn Cys Leu Val Gly Glu Asn His Leu Val Arg Val Ala
785 790 795 800
Asp Phe Gly Leu Ser Arg Leu Met Gly Gly Ser Gly Gly Gly Gly Ser
805 810 815
Gly Gly Arg Glu Pro Pro Phe Tyr Ile Ile Thr Glu Leu Met Thr Tyr
820 825 830
Gly Asn Leu Leu Asp Tyr Leu Lys Lys Gly Gly Ser Gly Gly Gly Gly
835 840 845
Ser Gly Gly Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Pro Ala Gly
850 855 860
Ala Lys Phe Pro Ile Lys Trp Lys Gly Gly Ser Gly Gly Gly Gly Ser
865 870 875 880
Gly Gly Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Ala Ile His Arg
885 890 895
Asp Leu Ala Ala Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile
900 905 910
Thr Met Lys His Lys Leu Gly Gly Gly Arg Tyr Gly Glu Val Tyr Glu
915 920 925
Gly Val Trp Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Thr Leu
930 935 940
Lys Glu Asp Thr Met Glu Val Glu Gly Phe Leu Lys Glu Ala Ala Val
945 950 955 960
Met Lys Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Glu
965 970 975
Glu Phe Leu Lys Glu Ala Ala Phe Met Lys Glu Ile Lys His Pro Asn
980 985 990
Leu Val Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Met Ala Thr
995 1000 1005
Gln Ile Ser Ser Ala Met Gly Tyr Leu Glu Lys Lys Asn Phe Ile His
1010 1015 1020
Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Pro Gly Ile Asp
1025 1030 1035 1040
Leu Ser Gln Val Tyr Ala Leu Leu Glu Lys Asp Tyr Arg Met Glu Arg
1045 1050 1055
Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Lys Ile Cys Asp
1060 1065 1070
Phe Gly Leu Ala Arg Phe Ile Met Ser Asp Ser Asn Tyr Val Val Arg
1075 1080 1085
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Ile Cys Asp Phe Gly
1090 1095 1100
Leu Ala Arg Ala Ile Lys Asn Asp Ser Asn Tyr Val Val Lys Gly Gly
1105 1110 1115 1120
Ser Gly Gly Gly Gly Ser Gly Gly Asp Phe Gly Leu Ala Arg Asp Ile
1125 1130 1135
Lys Asn Val Ser Asn Tyr Val Val Lys Gly Asn Ala Arg Gly Gly Ser
1140 1145 1150
Gly Gly Gly Gly Ser Gly Gly Cys Thr Ile Gly Gly Pro Thr Leu Val
1155 1160 1165
Ile Ile Glu Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Lys Lys Lys Gly
1170 1175 1180
Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Tyr Leu Gly Asn His Met
1185 1190 1195 1200
Asn Ile Val Thr Leu Leu Gly Ala Cys Thr Ile Gly Gly Pro Lys Gly
1205 1210 1215
Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Pro Gly Ile Asp Leu Ser
1220 1225 1230
Gln Val Tyr Lys Leu Leu Glu Lys Asp Tyr Arg Met Glu Arg Gly Gly
1235 1240 1245
Ser Gly Gly Gly Gly Ser Gly Gly Val Lys Ile Cys Asp Phe Gly Leu
1250 1255 1260
Ala Arg Tyr Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Gly Ser
1265 1270 1275 1280
Gly Gly Gly Gly Ser Gly Gly Thr Gln Ile Ser Ser Ala Met Glu Tyr
1285 1290 1295
Leu Ala Lys Lys Asn Phe Ile His Arg Asp Gly Gly Ser Gly Gly Gly
1300 1305 1310
Gly Ser Gly Gly Trp Gln Trp Asn Pro Ser Asp Arg Pro Ser Ser Ala
1315 1320 1325
Glu Ile His Gln Ala Phe Glu Thr Met Lys Gly Gly Ser Gly Gly Gly
1330 1335 1340
Gly Ser Gly Gly Trp Ala Phe Gly Val Leu Leu Trp Glu Ile Thr Thr
1345 1350 1355 1360
Tyr Gly Met Ser Pro Tyr Pro Gly Ile Lys Lys Lys Gly Gly Ser Gly
1365 1370 1375
Gly Gly Gly Ser Gly Gly Val Ala Val Lys Thr Leu Lys Glu Asp Ala
1380 1385 1390
Met Glu Val Glu Glu Phe Leu Lys Glu Ala Lys Lys Lys Gly Gly Ser
1395 1400 1405
Gly Gly Gly Gly Ser Gly Gly Val Lys Ile Cys Asp Phe Gly Leu Ala
1410 1415 1420
Arg Val Ile Met His Asp Ser Asn Tyr Val Ser Lys Gly Gly Ser Gly
1425 1430 1435 1440
Gly Gly Gly Ser Gly Gly Asp Ser Asn Tyr Val Val Lys Gly Asn Pro
1445 1450 1455
Arg Leu Pro Val Lys Trp Met Ala Gly Gly Ser Gly Gly Gly Gly Ser
1460 1465 1470
Gly Gly Lys Ile Cys Asp Phe Gly Leu Ala Arg His Ile Lys Asn Asp
1475 1480 1485
Ser Asn Tyr Val Val Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly
1490 1495 1500
Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile
1505 1510 1515 1520
Gly Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly
1525 1530 1535
Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly
1540 1545 1550
Ser Asp Val Ser Leu Thr Ala
1555
<210> 205
<211> 1511
<212> PRT
<213> Artificial Sequence
<400> 205
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Leu Ile
20 25 30
Thr Gln Leu Met Pro Phe Gly Ser Leu Leu Asp Tyr Val Arg Glu His
35 40 45
Lys Asp Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala Val Lys Thr
50 55 60
Leu Lys Glu Asp Thr Met Tyr Val Glu Glu Phe Leu Lys Glu Ala Ala
65 70 75 80
Val Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys Thr Arg
85 90 95
Glu Pro Pro Phe Tyr Ile Ile Ile Glu Phe Met Thr Tyr Gly Asn Leu
100 105 110
Leu Asp Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Leu
115 120 125
Ser Gln Val Tyr Glu Leu Leu Gly Lys Asp Tyr Arg Met Glu Arg Lys
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Asp Thr Tyr Thr Ala His
145 150 155 160
Ala Gly Thr Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Lys Lys Gly
165 170 175
Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Phe Leu Lys Glu Ala Ala
180 185 190
Val Met Lys Val Ile Lys His Pro Asn Leu Val Gln Leu Leu Lys Gly
195 200 205
Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Gln Leu Ile Thr Gln Leu
210 215 220
Met Pro Phe Arg Cys Leu Leu Asp Tyr Val Arg Glu His Lys Lys Gly
225 230 235 240
Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Leu Met Arg Ala Cys Trp
245 250 255
Gln Trp Asn Leu Ser Asp Arg Pro Ser Phe Ala Glu Ile His Gly Gly
260 265 270
Ser Gly Gly Gly Gly Ser Gly Gly Glu Ser Leu Ala Tyr Asn Lys Phe
275 280 285
Ser Ile Glu Ser Asp Val Trp Ala Phe Gly Val Leu Leu Lys Lys Gly
290 295 300
Gly Ser Gly Gly Gly Gly Ser Gly Gly His Leu Val Lys Val Ala Asp
305 310 315 320
Phe Gly Met Ser Arg Leu Met Thr Gly Asp Thr Tyr Thr Lys Lys Gly
325 330 335
Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Asp Leu Ser Gln Val Tyr
340 345 350
Glu Leu Leu Ala Lys Asp Tyr Arg Met Glu Arg Lys Gly Gly Ser Gly
355 360 365
Gly Gly Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu Gly Gly Gly
370 375 380
Glu Tyr Gly Glu Val Tyr Glu Gly Val Trp Gly Gly Ser Gly Gly Gly
385 390 395 400
Gly Ser Gly Gly Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Val Glu
405 410 415
Phe Met Thr Tyr Gly Asn Leu Leu Asp Lys Lys Ala Val Lys Thr Leu
420 425 430
Lys Glu Asp Thr Met Val Glu Glu Phe Leu Lys Glu Ala Lys Lys Gly
435 440 445
Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys His Lys Leu Gly Gly Gly
450 455 460
Gln Tyr Gly Val Val Tyr Glu Gly Val Trp Lys Lys Tyr Ser Gly Gly
465 470 475 480
Ser Gly Gly Gly Gly Ser Gly Gly Lys Thr Leu Lys Glu Asp Thr Met
485 490 495
Ala Val Glu Glu Phe Leu Lys Glu Ala Ala Val Lys Lys Gly Gly Ser
500 505 510
Gly Gly Gly Gly Ser Gly Gly Lys Val Ala Asp Phe Gly Leu Ser Arg
515 520 525
Leu Leu Thr Gly Asp Thr Tyr Thr Ala His Ala Gly Lys Gly Gly Ser
530 535 540
Gly Gly Gly Gly Ser Gly Gly Leu Leu Gly Val Cys Thr Arg Glu Pro
545 550 555 560
Ser Phe Tyr Ile Ile Thr Glu Phe Met Thr Tyr Lys Lys Lys Lys Gly
565 570 575
Gly Ser Gly Gly Gly Gly Ser Gly Gly Ile Thr Met Lys His Lys Leu
580 585 590
Gly Gly Gly Lys Tyr Gly Glu Val Tyr Glu Gly Val Trp Lys Gly Gly
595 600 605
Ser Gly Gly Gly Gly Ser Gly Gly Met Thr Gly Asp Thr Tyr Thr Ala
610 615 620
His Ala Arg Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Lys Lys Lys
625 630 635 640
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Pro Glu Ser Leu Ala Tyr
645 650 655
Asn Lys Phe Ser Val Lys Ser Asp Val Trp Ala Phe Gly Val Gly Gly
660 665 670
Ser Gly Gly Gly Gly Ser Gly Gly Pro Phe Tyr Ile Ile Thr Glu Phe
675 680 685
Met Thr Cys Gly Asn Leu Leu Asp Tyr Leu Arg Lys Lys Lys Gly Gly
690 695 700
Ser Gly Gly Gly Gly Ser Gly Gly Gln Ile Ser Ser Ala Met Glu Tyr
705 710 715 720
Leu Gly Lys Lys Asn Phe Ile His Arg Asp Gly Gly Ser Gly Gly Gly
725 730 735
Gly Ser Gly Gly Arg Glu Cys Asn Arg Gln Glu Val Asn Ala Phe Val
740 745 750
Leu Leu Tyr Met Ala Thr Gln Ile Lys Gly Gly Ser Gly Gly Gly Gly
755 760 765
Ser Gly Gly Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Arg Ala Gly
770 775 780
Ala Lys Phe Pro Ile Lys Trp Thr Lys Gly Gly Ser Gly Gly Gly Gly
785 790 795 800
Ser Gly Gly Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Val Ile His
805 810 815
Arg Asp Leu Ala Ala Arg Asn Gly Gly Ser Gly Gly Gly Gly Ser Gly
820 825 830
Gly Thr Leu Lys Glu Asp Thr Met Glu Val Glu Lys Phe Leu Lys Glu
835 840 845
Ala Ala Val Met Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
850 855 860
Val Glu Glu Phe Leu Lys Glu Ala Ala Ala Met Lys Glu Ile Lys His
865 870 875 880
Pro Asn Leu Val Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Met
885 890 895
Ala Thr Gln Ile Ser Ser Ala Met Asp Tyr Leu Glu Lys Lys Asn Phe
900 905 910
Ile His Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Pro Gly
915 920 925
Ile Asp Leu Ser Gln Val Tyr Gly Leu Leu Glu Lys Asp Tyr Arg Met
930 935 940
Glu Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Ile Cys
945 950 955 960
Asp Phe Gly Leu Ala Arg His Ile Met Ser Asp Ser Asn Tyr Val Val
965 970 975
Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Lys Ile Cys Asp Phe
980 985 990
Gly Leu Ala Arg Glu Ile Lys Asn Asp Ser Asn Tyr Val Val Lys Gly
995 1000 1005
Gly Ser Gly Gly Gly Gly Ser Gly Gly Leu Ala Arg Asp Ile Lys Asn
1010 1015 1020
Gly Ser Asn Tyr Val Val Lys Gly Asn Gly Gly Ser Gly Gly Gly Gly
1025 1030 1035 1040
Ser Gly Gly Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Glu Glu Tyr
1045 1050 1055
Cys Cys Tyr Gly Asp Leu Leu Asn Lys Lys Lys Gly Gly Ser Gly Gly
1060 1065 1070
Gly Gly Ser Gly Gly Leu Ser Tyr Leu Gly Asn His Met Asn Ile Ala
1075 1080 1085
Asn Leu Leu Gly Ala Cys Thr Ile Gly Lys Lys Gly Gly Ser Gly Gly
1090 1095 1100
Gly Gly Ser Gly Gly Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Val
1105 1110 1115 1120
Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Gly Ser Gly Gly Gly
1125 1130 1135
Gly Ser Gly Gly Ser Phe Ala Glu Ile His Gln Ala Phe Glu Arg Met
1140 1145 1150
Phe Gln Glu Ser Ser Ile Ser Asp Glu Lys Lys Lys Gly Gly Ser Gly
1155 1160 1165
Gly Gly Gly Ser Gly Gly Ser Leu Thr Val Ala Val Lys Thr Leu Lys
1170 1175 1180
Lys Asp Thr Met Glu Val Glu Glu Phe Leu Lys Gly Gly Ser Gly Gly
1185 1190 1195 1200
Gly Gly Ser Gly Gly Thr Glu Phe Met Thr Tyr Gly Asn Leu Leu Gly
1205 1210 1215
Tyr Leu Arg Glu Cys Asn Arg Gln Glu Val Lys Gly Gly Ser Gly Gly
1220 1225 1230
Gly Gly Ser Gly Gly Thr Met Lys His Lys Leu Gly Gly Gly Gln Phe
1235 1240 1245
Gly Glu Val Tyr Glu Gly Val Trp Lys Lys Gly Gly Ser Gly Gly Gly
1250 1255 1260
Gly Ser Gly Gly Val Lys Val Ala Asp Phe Gly Leu Ser Arg Phe Met
1265 1270 1275 1280
Thr Gly Asp Thr Tyr Thr Ala His Ala Lys Lys Lys Gly Gly Ser Gly
1285 1290 1295
Gly Gly Gly Ser Gly Gly Val Met Lys Glu Ile Lys His Pro Asn Leu
1300 1305 1310
Leu Gln Leu Leu Gly Val Cys Thr Arg Glu Pro Gly Gly Ser Gly Gly
1315 1320 1325
Gly Gly Ser Gly Gly Glu Arg Glu Ala Leu Met Ser Glu Leu Glu Val
1330 1335 1340
Leu Ser Tyr Leu Gly Asn His Met Asn Lys Lys Gly Gly Ser Gly Gly
1345 1350 1355 1360
Gly Gly Ser Gly Gly Glu Ala Ala Leu Tyr Lys Asn Leu Leu His Phe
1365 1370 1375
Lys Glu Ser Ser Cys Ser Asp Ser Thr Gly Gly Ser Gly Gly Gly Gly
1380 1385 1390
Ser Gly Gly Asp Phe Gly Leu Ala Arg Asp Ile Lys Asn Tyr Ser Asn
1395 1400 1405
Tyr Val Val Lys Gly Asn Ala Arg Gly Gly Ser Gly Gly Gly Gly Ser
1410 1415 1420
Gly Gly Phe Gly Leu Ala Arg Asp Ile Lys Asn Asp Phe Asn Tyr Val
1425 1430 1435 1440
Val Lys Gly Asn Ala Arg Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly
1445 1450 1455
Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile
1460 1465 1470
Gly Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly
1475 1480 1485
Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly
1490 1495 1500
Ser Asp Val Ser Leu Thr Ala
1505 1510
<210> 206
<211> 625
<212> PRT
<213> Artificial Sequence
<400> 206
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Lys Trp
20 25 30
Glu Met Glu Arg Thr Asp Ile Thr Met Lys His Lys Leu Gly Gly Gly
35 40 45
Gln Tyr Gly Glu Val Tyr Glu Gly Val Trp Lys Lys Tyr Ser Leu Thr
50 55 60
Val Ala Val Lys Thr Leu Lys Glu Asp Thr Met Glu Val Glu Glu Phe
65 70 75 80
Leu Lys Glu Ala Ala Val Met Lys Glu Ile Lys His Pro Asn Leu Val
85 90 95
Gln Leu Leu Gly Val Cys Thr Arg Glu Pro Pro Phe Tyr Ile Ile Thr
100 105 110
Glu Phe Met Thr Tyr Gly Asn Leu Leu Asp Tyr Leu Arg Glu Cys Asn
115 120 125
Arg Gln Glu Val Asn Ala Val Val Leu Leu Tyr Met Ala Thr Gln Ile
130 135 140
Ser Ser Ala Met Glu Tyr Leu Glu Lys Lys Asn Phe Ile His Arg Asp
145 150 155 160
Leu Ala Ala Arg Asn Cys Leu Val Gly Glu Asn His Leu Val Lys Val
165 170 175
Ala Asp Phe Gly Leu Ser Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala
180 185 190
His Ala Gly Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ser Leu
195 200 205
Ala Tyr Asn Lys Phe Ser Ile Lys Ser Asp Val Trp Ala Phe Gly Val
210 215 220
Leu Leu Trp Glu Ile Ala Thr Tyr Gly Met Ser Pro Tyr Pro Gly Ile
225 230 235 240
Asp Leu Ser Gln Val Tyr Glu Leu Leu Glu Lys Asp Tyr Arg Met Glu
245 250 255
Arg Pro Glu Gly Cys Pro Glu Lys Val Tyr Glu Leu Met Arg Ala Cys
260 265 270
Trp Gln Trp Asn Pro Ser Asp Arg Pro Ser Phe Ala Glu Ile His Gln
275 280 285
Ala Phe Glu Thr Met Phe Gln Glu Ser Ser Ile Ser Asp Gly Gly Ser
290 295 300
Gly Gly Gly Gly Ser Gly Gly Val Lys Ile Cys Asp Phe Gly Leu Ala
305 310 315 320
Arg Asp Ile Met Ser Asp Ser Asn Tyr Val Val Arg Gly Gly Ser Gly
325 330 335
Gly Gly Gly Ser Gly Gly Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu
340 345 350
Asp Glu Ala Tyr Val Met Ala Ser Val Asp Lys Gly Gly Ser Gly Gly
355 360 365
Gly Gly Ser Gly Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Thr
370 375 380
Gln Leu Met Pro Phe Gly Cys Leu Leu Asp Tyr Val Arg Glu His Lys
385 390 395 400
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Lys Ile Cys Asp Phe
405 410 415
Gly Leu Ala Arg Asp Ile Met His Asp Ser Asn Tyr Val Ser Lys Gly
420 425 430
Gly Ser Gly Gly Gly Gly Ser Gly Gly Glu Arg Glu Ala Leu Met Ser
435 440 445
Glu Leu Lys Val Leu Ser Tyr Leu Gly Asn His Met Asn Ile Val Asn
450 455 460
Leu Leu Gly Ala Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Thr Glu
465 470 475 480
Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Gly Gly Ser Gly Gly Gly Gly
485 490 495
Ser Gly Gly Glu Ala Ala Leu Tyr Lys Asn Leu Leu His Ser Lys Glu
500 505 510
Ser Ser Cys Ser Asp Ser Thr Gly Gly Ser Gly Gly Gly Gly Ser Gly
515 520 525
Gly Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Lys Asn Asp Ser
530 535 540
Asn Tyr Val Val Lys Gly Asn Ala Arg Leu Pro Val Lys Trp Met Ala
545 550 555 560
Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile Val Gly Ile Val Ala
565 570 575
Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr
580 585 590
Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser
595 600 605
Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr
610 615 620
Ala
625
<210> 207
<211> 244
<212> PRT
<213> Artificial Sequence
<400> 207
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Thr Gln
20 25 30
Ala Trp Asp Leu Tyr Tyr His Val Leu Arg Arg Ile Ser Lys Gln Leu
35 40 45
Pro Gln Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Met Lys Cys Lys
50 55 60
Asn Val Val Pro Leu Tyr Gly Leu Leu Leu Glu Met Leu Asp Ala His
65 70 75 80
Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly His Glu Cys Asn
85 90 95
Ser Pro Tyr Ile Val Gly Leu Tyr Gly Ala Phe Tyr Ser Asp Gly Glu
100 105 110
Ile Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Ser Met
115 120 125
Lys Cys Lys Asn Val Val Pro His Tyr Asp Leu Leu Leu Glu Met Leu
130 135 140
Asp Ala Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Lys Val
145 150 155 160
Ala Asp Phe Gly Leu Ala Arg Val Met Tyr Asp Lys Glu Tyr Tyr Ser
165 170 175
Val His Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile Val Gly
180 185 190
Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val
195 200 205
Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly
210 215 220
Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val
225 230 235 240
Ser Leu Thr Ala
<210> 208
<211> 214
<212> PRT
<213> Artificial Sequence
<400> 208
Met Ala Ala Pro Gly Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Leu Gln Thr Gln
20 25 30
Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Gln Leu
35 40 45
Pro Gln Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Tyr Ser Met Lys
50 55 60
Cys Lys Asn Val Val Pro Leu Tyr Asp Leu Leu Leu Glu Met Leu Asp
65 70 75 80
Ala His Arg Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly His Glu
85 90 95
Cys Asn Ser Pro Tyr Ile Val Gly Phe Tyr Gly Ala Phe Tyr Ser Asp
100 105 110
Gly Glu Ile Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val
115 120 125
Lys Val Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu Tyr
130 135 140
Tyr Ser Val His Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile
145 150 155 160
Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly
165 170 175
Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys
180 185 190
Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser
195 200 205
Asp Val Ser Leu Thr Ala
210

Claims (4)

1. The polypeptide vaccine aiming at the tumor targeted drug resistance site is characterized in that aiming at the targeted drug, namely the vismodegib, the polypeptide vaccine combination aims at the following mutations: SMO-A459V, SMO-C469Y, SMO-D473G, SMO-D473H, SMO-D473Y, SMO-F460L, SMO-G497W, SMO-H231R, SMO-Q477E, SMO-T241M, SMO-V321A, SMO-V321M, SMO-W281L, SMO-W535L and SMO-W535R;
the polypeptide vaccine comprises the following peptides:
the sequence of SMO-A459V is as follows: MLRLGIFGFLVFGFVLITFSCKKKKKK the flow of the air in the air conditioner,
the sequence of SMO-C469Y is: AFGFVLITFSYHFYDFFNQAEKKKKK the flow of the air in the air conditioner,
the sequence of SMO-D473G is: VLITFSCHFYGFFNQAEWERKK the flow of the air in the air conditioner,
the sequence of SMO-D473H is: VLITFSCHFYHFFNQAEWERSKKK the flow of the air in the air conditioner,
the sequence of SMO-D473Y is: VLITFSCHFYYFFNQAEWERSKKK the flow of the air in the air conditioner,
the sequence of SMO-F460L is: LRLGIFGFLALGFVLITFSCHKKKKKK the flow of the air in the air conditioner,
the sequence of SMO-G497W is: YVLCQANVTIWLPTKQPIPDCK the flow of the air in the air conditioner,
the sequence of SMO-H231R is: FTEAEHQDMRSYIAAFGAVTKK the flow of the air in the air conditioner,
the sequence of SMO-Q477E is: FSCHFYDFFNEAEWERSFRDYKK the flow of the air in the air conditioner,
the sequence of SMO-T241M is: HSYIAAFGAVMGLCTLFTLAKKKKKKKKK the flow of the air in the air conditioner,
the sequence of SMO-V321A is: TLSCVIIFVIAYYALMAGVVWKKKKKKKKKK the flow of the air in the air conditioner,
the sequence of SMO-V321M is: TLSCVIIFVIMYYALMAGVVWKKKKKKKKKK the flow of the air in the air conditioner,
the sequence of SMO-W281L is: NACFFVGSIGLLAQFMDGARK the flow of the air in the air conditioner,
the sequence of SMO-W535L is: MFGTGIAMSTLVWTKATLLIWKKKKKKKK the flow of the air in the air conditioner,
the sequence of SMO-W535R is: MFGTGIAMSTRVWTKATLLIWKKKKKK, respectively;
the 15 mutant polypeptides are connected by using a flexible connecting peptide GGSGGGGSGG.
2. The design method of the polypeptide vaccine aiming at the tumor targeted drug resistance site is characterized by comprising the following steps:
searching data of drug resistance mutation data of the targeted drug;
intercepting a drug-resistant mutant polypeptide sequence and predicting the affinity and immunogenicity of MHC molecules:
intercepting a polypeptide sequence covering 16 amino acids at the upstream and downstream of a mutation site for point mutation, intercepting a polypeptide sequence which extends forwards for 16 amino acids and extends backwards to a stop codon for frameshift mutation to serve as a mutant polypeptide of the drug-resistant mutation site, intercepting a wild-type polypeptide sequence corresponding to a corresponding position at the same time, counting high-frequency HLA typing and frequency by taking at least one database as a source, merging and de-duplicating the counted HLA to serve as candidate HLA typing for prediction,
the binding affinity of the polypeptide corresponding to the mutation sites and HLA molecules is analyzed by using various types of software, and the affinity is divided into three types by combining the various types of software: strong affinity-SB, weak affinity-WB and no affinity, and determining changes in affinity compared to the relatively wild-type polypeptide,
class a changes from no affinity to strong affinity,
class B changes from no affinity to weak affinity,
class C changes from weak affinity to strong affinity,
the class D change is a no change,
internal ordering considers class a over class B over class C over class D,
the immunogenicity of the mutant polypeptide is predicted by using an immunogenicity prediction tool, and epitopes with strong affinity, large affinity change and strong immunogenicity of the mutant polypeptide are reserved;
the interrelation between the targeted drug and the drug-resistant site and the clustering of the drugs:
scoring the drug resistance site affinity:
comprehensively considering the number of epitopes with affinity of the sites and the change size of the affinity of each epitope, giving corresponding weight to A, B, C, D types of different affinity changes, giving weight according to the HLA frequency size corresponding to each epitope, and accumulating and summing the epitopes of the sites;
Figure FDA0002812172130000021
AC: the magnitude of the affinity change, A, B, C, D four different classes of affinity changes give different weights;
Fhla: a corresponding HLA frequency;
n: the number of each epitope of the locus;
the correlation between the targeted drugs and the drug-resistant sites is comprehensively analyzed, and the targeted drugs are clustered by combining the action mechanism of the targeted drugs, so that the targeted drugs are classified into 7 types:
the first Hedgehog signaling pathway antagonist, vismodegib, which is resistant to SMO mutation,
the second class generates drug-resistant ibrutinib against BTK mutations,
the third group aims at some antiandrogen drugs of AR drug resistant mutation, such as abiraterone and the like,
the fourth group is some phenanthrenib drugs aiming at BRAF drug-resistant mutation,
the fifth group is some tyrosine kinase inhibitors aiming at ALK, MET and other fusion genes,
a sixth class of tyrosine kinase inhibitors directed against the EGFR pathway,
the seventh kinase inhibitor such as everolimus for PI3K/AKT/mTOR pathway;
designing the corresponding vaccine polypeptide sequence.
3. The method for designing a polypeptide vaccine against a tumor-targeted drug-resistant site according to claim 2,
intercepting a drug-resistant mutant polypeptide sequence and predicting the affinity and immunogenicity of MHC molecules:
intercepting a polypeptide sequence covering 16 amino acids at the upstream and downstream of a mutation site for point mutation, intercepting a polypeptide sequence which extends forwards for 16 amino acids and extends backwards to a stop codon for frameshift mutation to serve as a mutant polypeptide of the drug-resistant mutation site, and intercepting a wild-type polypeptide sequence corresponding to a corresponding position at the same time,
counting high-frequency HLA types and frequencies by using at least one database as sources, combining and de-duplicating the counted HLA to be used as candidate HLA types for prediction, wherein the database comprises: a public database, a clinical patient database,
the binding affinity of the polypeptide corresponding to the mutation sites and HLA molecules is analyzed by using various types of software, and the affinity is divided into three types by combining the various types of software: strong affinity-SB, weak affinity-WB and no affinity and determining changes in affinity compared to a relatively wild-type polypeptide, comprising: three software of netMHCpan, netMHC and Pickpocket,
class a changes from no affinity to strong affinity,
class B changes from no affinity to weak affinity,
class C changes from weak affinity to strong affinity,
the class D change is a no change,
internal ordering considers class a over class B over class C over class D,
the immunogenicity of the mutant polypeptide is predicted by using an immunogenicity prediction tool, and epitopes with strong affinity, large affinity change and strong immunogenicity of the mutant polypeptide are reserved.
4. The method for designing a polypeptide vaccine against a tumor-targeted drug-resistant site according to claim 2,
using cytoscape to make a network map of all targeted drugs and drug-resistant sites, wherein the size of the drug-resistant sites represents the affinity score of the drug-resistant sites, and comprehensively analyzing the correlation between the targeted drugs and the drug-resistant sites and combining the action mechanism of the targeted drugs to cluster the targeted drugs so as to classify the targeted drugs into 7 types:
the first Hedgehog signaling pathway antagonist, vismodegib, which is resistant to SMO mutation,
the second class generates drug-resistant ibrutinib against BTK mutations,
the third group aims at some antiandrogen drugs of AR drug resistant mutation, such as abiraterone and the like,
the fourth group is some phenanthrenib drugs aiming at BRAF drug-resistant mutation,
the fifth group is some tyrosine kinase inhibitors aiming at ALK, MET and other fusion genes,
a sixth class of tyrosine kinase inhibitors directed against the EGFR pathway,
the seventh group is kinase inhibitors such as everolimus and the like aiming at the PI3K/AKT/mTOR pathway.
CN201910086371.9A 2019-01-29 2019-01-29 Polypeptide vaccine aiming at tumor targeted drug resistance site and design method thereof Active CN109887553B (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
CN202011328452.4A CN112767998A (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition for targeted drugs such as dabrafenib, pembrolizumab, vemurafenib and semetinib and design method thereof
CN201910086371.9A CN109887553B (en) 2019-01-29 2019-01-29 Polypeptide vaccine aiming at tumor targeted drug resistance site and design method thereof
CN202011331275.5A CN112687353B (en) 2019-01-29 2019-01-29 Polypeptide vaccine combination aiming at tyrosine kinase inhibitor of targeted drug EGFR (epidermal growth factor receptor) pathway and design method thereof
CN202011328455.8A CN112687352B (en) 2019-01-29 2019-01-29 Polypeptide vaccine combination aiming at targeted drug ibrutinib and design method thereof
CN202011331358.4A CN112687354B (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition of kinase inhibitor aiming at targeted drugs PI3K, AKT and mTOR pathways and design method thereof
CN202011331322.6A CN112687325B (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition aiming at targeted drugs of alatinib, ceritinib and crizotinib and design method thereof
CN202011328459.6A CN112675297B (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition for targeted drugs abiraterone, enzalutamide, flutamide and ketoconazole and design method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910086371.9A CN109887553B (en) 2019-01-29 2019-01-29 Polypeptide vaccine aiming at tumor targeted drug resistance site and design method thereof

Related Child Applications (6)

Application Number Title Priority Date Filing Date
CN202011328459.6A Division CN112675297B (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition for targeted drugs abiraterone, enzalutamide, flutamide and ketoconazole and design method thereof
CN202011328455.8A Division CN112687352B (en) 2019-01-29 2019-01-29 Polypeptide vaccine combination aiming at targeted drug ibrutinib and design method thereof
CN202011331275.5A Division CN112687353B (en) 2019-01-29 2019-01-29 Polypeptide vaccine combination aiming at tyrosine kinase inhibitor of targeted drug EGFR (epidermal growth factor receptor) pathway and design method thereof
CN202011328452.4A Division CN112767998A (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition for targeted drugs such as dabrafenib, pembrolizumab, vemurafenib and semetinib and design method thereof
CN202011331358.4A Division CN112687354B (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition of kinase inhibitor aiming at targeted drugs PI3K, AKT and mTOR pathways and design method thereof
CN202011331322.6A Division CN112687325B (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition aiming at targeted drugs of alatinib, ceritinib and crizotinib and design method thereof

Publications (2)

Publication Number Publication Date
CN109887553A CN109887553A (en) 2019-06-14
CN109887553B true CN109887553B (en) 2021-01-26

Family

ID=66927249

Family Applications (7)

Application Number Title Priority Date Filing Date
CN202011331275.5A Active CN112687353B (en) 2019-01-29 2019-01-29 Polypeptide vaccine combination aiming at tyrosine kinase inhibitor of targeted drug EGFR (epidermal growth factor receptor) pathway and design method thereof
CN202011328452.4A Pending CN112767998A (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition for targeted drugs such as dabrafenib, pembrolizumab, vemurafenib and semetinib and design method thereof
CN202011328459.6A Active CN112675297B (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition for targeted drugs abiraterone, enzalutamide, flutamide and ketoconazole and design method thereof
CN202011328455.8A Active CN112687352B (en) 2019-01-29 2019-01-29 Polypeptide vaccine combination aiming at targeted drug ibrutinib and design method thereof
CN202011331322.6A Active CN112687325B (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition aiming at targeted drugs of alatinib, ceritinib and crizotinib and design method thereof
CN202011331358.4A Active CN112687354B (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition of kinase inhibitor aiming at targeted drugs PI3K, AKT and mTOR pathways and design method thereof
CN201910086371.9A Active CN109887553B (en) 2019-01-29 2019-01-29 Polypeptide vaccine aiming at tumor targeted drug resistance site and design method thereof

Family Applications Before (6)

Application Number Title Priority Date Filing Date
CN202011331275.5A Active CN112687353B (en) 2019-01-29 2019-01-29 Polypeptide vaccine combination aiming at tyrosine kinase inhibitor of targeted drug EGFR (epidermal growth factor receptor) pathway and design method thereof
CN202011328452.4A Pending CN112767998A (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition for targeted drugs such as dabrafenib, pembrolizumab, vemurafenib and semetinib and design method thereof
CN202011328459.6A Active CN112675297B (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition for targeted drugs abiraterone, enzalutamide, flutamide and ketoconazole and design method thereof
CN202011328455.8A Active CN112687352B (en) 2019-01-29 2019-01-29 Polypeptide vaccine combination aiming at targeted drug ibrutinib and design method thereof
CN202011331322.6A Active CN112687325B (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition aiming at targeted drugs of alatinib, ceritinib and crizotinib and design method thereof
CN202011331358.4A Active CN112687354B (en) 2019-01-29 2019-01-29 Polypeptide vaccine composition of kinase inhibitor aiming at targeted drugs PI3K, AKT and mTOR pathways and design method thereof

Country Status (1)

Country Link
CN (7) CN112687353B (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112402612B (en) * 2019-08-21 2023-05-26 苏州蓝马医疗技术有限公司 Method for improving therapeutic effect of tumor neoantigen vaccine immunotherapy
CN111171136A (en) * 2019-12-23 2020-05-19 维塔恩(广州)医药有限公司 tumor-associated gene PDGFR α mutation-associated antigen short peptide and application thereof
CN113292642B (en) * 2020-04-13 2023-02-28 倍而达药业（苏州）有限公司 Third-generation epidermal growth factor receptor inhibitor drug-resistant mutation neoantigen and application thereof
CN112501201A (en) * 2021-02-07 2021-03-16 无锡市人民医院 RNA vaccine for treating non-small cell lung cancer and construction method thereof

Family Cites Families (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4299990B2 (en) * 2001-05-24 2009-07-22 独立行政法人科学技術振興機構 New SMG-1
FR2857875A1 (en) * 2003-07-25 2005-01-28 Univ Reims Champagne Ardenne Immunogenic composition containing fatty acid-modified peptides from protein P-170, useful e.g. for reversing multidrug resistance of cancer cells
US20060153861A1 (en) * 2004-09-30 2006-07-13 Scheinberg David A BCR-ABL imatinib resistance-associated peptides and methods of use thereof
MX2009010066A (en) * 2007-03-19 2009-10-12 Globeimmune Inc Compositions and methods for targeted ablation of mutational escape of targeted therapies for cancer.
CN101648011B (en) * 2008-08-11 2012-06-13 同济大学附属上海市肺科医院 Tumor targeting recombinant DNA vaccine, preparation method thereof and application thereof
US9095592B2 (en) * 2008-11-07 2015-08-04 The Research Foundation For The State University Of New York Bruton's tyrosine kinase as anti-cancer drug target
BR112015001690A2 (en) * 2012-07-24 2017-11-07 Pharmacyclics Inc mutations associated with resistance to bruton tyrosine kinase inhibitors (btk)
CN103570818B (en) * 2012-07-27 2016-06-29 北京智飞绿竹生物制药有限公司 Tumor antigenic polypeptide and the purposes as tumor vaccine thereof
CN103040824B (en) * 2013-01-17 2015-05-27 四川大学 Signal channel inhibiting agent as well as preparation method and application thereof
EP3033088A4 (en) * 2013-08-12 2017-03-08 Tokai Pharmaceuticals, Inc. Biomarkers for treatment of neoplastic disorders using androgen-targeted therapies
CN103784950A (en) * 2014-01-22 2014-05-14 北京弘润源生物技术有限公司 Preparation method of breast cancer-specific epitope polypeptide-loaded dendritic cell vaccine and kit thereof
CA2954279C (en) * 2014-07-07 2023-11-14 Duke University Vaccines against an oncogenic isoform of esr1 and methods of using the same
WO2016115376A1 (en) * 2015-01-14 2016-07-21 The Regents Of The University Of California Detection and treatment of double drug resistant melanomas
JP2018512597A (en) * 2015-02-04 2018-05-17 ジェネンテック， インコーポレイテッド Mutant smoothened and method of using the same
AU2016260540B2 (en) * 2015-05-13 2021-01-07 Agenus Inc. Vaccines for treatment and prevention of cancer
MY190974A (en) * 2015-05-20 2022-05-25 Massachusetts Gen Hospital Shared neoantigens
GB201510771D0 (en) * 2015-06-19 2015-08-05 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy and methods for generating scaffolds for the use against pancreatic cancer
US11118233B2 (en) * 2016-05-18 2021-09-14 The University Of Chicago BTK mutation and ibrutinib resistance
CN107325172A (en) * 2017-07-06 2017-11-07 江苏迈健生物科技发展股份有限公司 Antigenic Peptide T790M 2 and its application in the medicine for preparing treatment non-small cell lung cancer
CN108785688B (en) * 2018-08-08 2022-02-25 厦门星际诺康细胞科技有限公司 Exosome with tumor cell targeting function and preparation method and application thereof

Also Published As

Publication number Publication date
CN112687354A (en) 2021-04-20
CN112687325A (en) 2021-04-20
CN112687352B (en) 2022-07-15
CN112687353B (en) 2023-08-18
CN112687353A (en) 2021-04-20
CN112687325B (en) 2024-01-26
CN112675297B (en) 2022-06-24
CN112767998A (en) 2021-05-07
CN112687354B (en) 2022-07-12
CN109887553A (en) 2019-06-14
CN112687352A (en) 2021-04-20
CN112675297A (en) 2021-04-20

Similar Documents

Publication Publication Date Title
CN109887553B (en) Polypeptide vaccine aiming at tumor targeted drug resistance site and design method thereof
Yu et al. MYD88 L265P mutation in lymphoid malignancies
Capietto et al. Mutation position is an important determinant for predicting cancer neoantigens
Song et al. Pharmacologic suppression of B7-H4 glycosylation restores antitumor immunity in immune-cold breast cancers
Kreiter et al. Mutant MHC class II epitopes drive therapeutic immune responses to cancer
CN110322925B (en) Method for predicting generation of neoantigen by fusion gene
CN109584966B (en) A kind of design method and cancer of pancreas general vaccines of tumour general vaccines
CN107847569A (en) Class φt cell receptor antibody with Fine specificity
CN104894068A (en) Method for preparing CAR-T cell by CRISPR/Cas9
Guo et al. Durable complete response to neoantigen-loaded dendritic-cell vaccine following anti-PD-1 therapy in metastatic gastric cancer
WO2020187016A1 (en) Robo1 car-nk cell carrying suicide gene, preparation method therefor and application thereof
Fidanza et al. Enhancing proteasomal processing improves survival for a peptide vaccine used to treat glioblastoma
CN109550044B (en) Universal polypeptide vaccine and application thereof in preparation of medicine for treating/preventing pancreatic cancer
CN112899233B (en) Method for constructing acute lymphocytic leukemia mouse model
CN111205361A (en) Interleukin 21 protein (IL21) mutant and application thereof
Lin et al. Prognostic impact of peripheral natural killer cells in primary central nervous system lymphoma
CN112646020B (en) Antigenic peptide based on histone H3K27M mutant peptide and application thereof
CN111116716B (en) Polypeptide specifically bound with myeloma cell high-expression antigen HLA-E and application thereof
Bushey et al. Antitumor immune mechanisms of the anti-complement factor H antibody GT103
CN115843317A (en) Novel T cell specificity and uses thereof
Abbott et al. Human EGFRvIII chimeric antigen receptor T cells demonstrate favorable safety profile and curative responses in orthotopic glioblastoma
CN114767841B (en) Composite nano vaccine and preparation method thereof, combined vaccine and preparation method thereof
CN111012782B (en) GPR84 inhibitor of G protein coupled receptor and application thereof
CN115724997A (en) anti-CD 97 chimeric antigen receptor, gene, expression vector, T cell and application
Lin et al. BCL2A1 neoepitopes-elicited cytotoxic T lymphocytes are a promising individualized immunotherapy of pancreatic cancer

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
CB03 Change of inventor or designer information
CB03 Change of inventor or designer information

Inventor after: Mo Fan

Inventor after: Ma Zhiming

Inventor after: Lin Zhiwei

Inventor after: Han Ning

Inventor after: Chen Rongchang

Inventor after: Zhou Xiuqing

Inventor before: Mo Fan

Inventor before: Ma Zhiming

Inventor before: Lin Zhiwei

Inventor before: Han Ning

Inventor before: Chen Rongchang

Inventor before: Zhou Xiuqing

Inventor before: Chen Shuqing

GR01 Patent grant
GR01 Patent grant