CN112687354B

CN112687354B - Polypeptide vaccine composition of kinase inhibitor aiming at targeted drugs PI3K, AKT and mTOR pathways and design method thereof

Info

Publication number: CN112687354B
Application number: CN202011331358.4A
Authority: CN
Inventors: 莫凡; 马治明; 林志伟; 韩宁; 陈荣昌; 周秀卿
Original assignee: Hangzhou Neoantigen Biotechnology Co ltd
Current assignee: Hangzhou Neoantigen Biotechnology Co ltd
Priority date: 2019-01-29
Filing date: 2019-01-29
Publication date: 2022-07-12
Anticipated expiration: 2039-01-29
Also published as: CN112687353A; CN112687352B; CN109887553A; CN112675297B; CN112687325A; CN112687352A; CN109887553B; CN112687325B; CN112687354A; CN112675297A; CN112687353B; CN112767998A

Abstract

The invention discloses a polypeptide vaccine of a kinase inhibitor aiming at targeted drugs PI3K, AKT and mTOR pathways 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 composition of kinase inhibitor aiming at targeted drugs PI3K, AKT and mTOR pathways and design method thereof

The present case is application number: 201910086371.9, title of the invention: a polypeptide vaccine aiming at a tumor targeted drug resistance site and a patent of a 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, 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 search for 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 new antigen is abnormal protein expressed on the surface of tumor cells caused by gene mutation, and is different from the conventional antigen target in that the new antigen is only expressed on the surface of the tumor cells and not expressed on the surface of normal cells, and can be recognized by the immune system and activate the immune system. In 2017, month 7 and day 13, the same day as Nature journal issued two successful cases of neoantigen-based personalized tumor vaccine for treating malignant melanoma. Professor Carmen Loquai in Germany and

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 sites leading to 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, where the polypeptide preparation can be obtained by chemical synthesis, by transcription and translation using nucleic acid molecules (such as DNA and RNA), or by expression using bacteria or virus as a vector. 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 for use, on one hand, the targeting drug can kill tumor cells carrying drug targets, on the other hand, the immune system of the organism activated by the polypeptide vaccine can effectively identify and recognize the drug-resistant tumor cells just before the drug-resistant tumor cells appearThe active immune system can maintain the killing effect on tumor cells in vivo for a long time, ensure the continuous inhibition on the drug resistance phenomenon and overcome the drug resistance of the targeted drug generated by drug resistance mutation to a certain extent. The market needs a set of screening and designing methods of the polypeptide vaccine aiming at the tumor targeted drug resistance site, can effectively reduce the occurrence probability of tumor resistance, has broad spectrum, shortens the time from analysis to treatment of the individualized polypeptide vaccine, 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 combination aiming at the kinase inhibitor of the targeting drugs PI3K, AKT and mTOR pathways, and the polypeptide vaccine of everolimus aims at the following mutations: the sequence of MTOR-F2108L and MTOR-F2108L is as follows: TQAWDLYYHVLRRISKQLPQ, respectively; the polypeptide vaccine of rapamycin is directed against the following mutations: the sequence of MTOR-F2108L and MTOR-F2108L is as follows: TQAWDLYYHVLRRISKQLPQ, respectively; the polypeptide vaccine of MEK inhibitor PD0325901 is directed to the following mutations: the sequences of MAP2K1-F129L and MAP2K1-F129L are as follows: HECNSPYIVGLYGAFYSDGEIKK; polypeptide vaccines of the C-MET inhibitor Savolitinib are directed against the following mutations: MET-D1246V, the sequence of MET-D1246V is: VKVADFGLARVMYDKEYYSVHK are provided.

The design method of the polypeptide vaccine combination of the kinase inhibitor aiming at the targeting drugs PI3K, AKT and mTOR pathways 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: truncating covering mutation sites for point mutationsThe method comprises the following steps of 1, dotting polypeptide sequences with 16 amino acids at the upstream and downstream, intercepting polypeptide sequences which extend forward by 16 lengths and extend backward to a stop codon as mutant polypeptides of a drug-resistant mutation site for frameshift mutation, simultaneously intercepting wild-type polypeptide sequences corresponding to corresponding positions, taking at least one database as a source to count high-frequency HLA typing and frequency, merging and de-duplicating the counted HLA to serve as candidate HLA typing for prediction, analyzing the binding affinity of the polypeptides corresponding to the mutation sites and HLA molecules by using various software, and classifying the affinity into three types by integrating the various software: strong affinity-SB, weak affinity-WB and no affinity, and compared with the relative wild type polypeptide, determine its affinity change, A type changes from no affinity to strong affinity, B type changes from no affinity to weak affinity, C type changes from weak affinity to strong affinity, D type changes to no change, the internal ordering considers A type is superior to C type to D type, use immunogenicity prediction tool to predict its immunogenicity, keep mutant polypeptide strong affinity, great affinity change and strong epitope of immunogenicity; 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;

AC: the size of the affinity change, A, B, C, D four different types of affinity changes are given different weights; f_hla: 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 drug-resistant Hedgehog signaling pathway antagonist virginia is generated aiming at SMO mutation, the drug-resistant ibrutinib is generated aiming at BTK mutation in the second class, some antiandrogen drugs such as abiraterone and the like aiming at AR drug-resistant mutation in the third class, some fenib drugs aiming at BRAF drug-resistant mutation in the fourth class, some tyrosine kinase inhibitors of fusion genes such as ALK and MET in the fifth class, tyrosine kinase inhibitors of EGFR pathway in the sixth class, and kinase inhibitors of everolimus and the like aiming at PI3K, AKT and mTOR pathways in the seventh class;

designing the corresponding vaccine polypeptide sequence.

The design method of the polypeptide vaccine composition aiming at the kinase inhibitors of the targeting drugs PI3K, AKT and mTOR pathways intercepts the drug-resistant mutant polypeptide sequence and predicts 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, counting high-frequency HLA typing and frequency by taking at least one database as a source, merging and de-duplicating the counted HLA, and taking the merged and de-duplicated HLA as a candidate HLA typing for prediction, wherein the database comprises: the public database and the clinical patient database are used for analyzing the binding affinity of the polypeptide corresponding to the mutation sites and HLA molecules 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, the various pieces of software including: the three types of software, namely netMHCpan, netMHC and Pickpockcket, change from no affinity to strong affinity in class A, change from no affinity to weak affinity in class B, change from weak affinity to strong affinity in class C, change from no change in class D, and internal ordering considers that class A is superior to class B over class D, and immunogenicity is predicted by using an immunogenicity prediction tool, and epitopes with strong affinity, large affinity change and strong immunogenicity of mutant polypeptides are reserved.

In the design method of the polypeptide vaccine composition of the kinase inhibitor for the targeted drugs PI3K, AKT and mTOR, cytoscape is used for making a network map of all the targeted drugs and the drug-resistant sites, the size of the drug-resistant sites represents the affinity score 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 classified into 7 types: the drug-resistant polypeptide is characterized by comprising a first Hedgehog signaling pathway antagonist vismodegib which generates drug resistance aiming at SMO mutation, a second drug-resistant ibrutinib which generates drug resistance aiming at BTK mutation, a third drug-resistant mutation anti-androgen drug such as abiraterone and the like aiming at AR, a fourth drug-resistant mutation phenanthrene drug, a fifth drug-resistant mutation tyrosine kinase inhibitor of fusion genes such as ALK and MET, a sixth drug-resistant EGFR pathway tyrosine kinase inhibitor and a seventh drug-resistant PI3K, AKT and mTOR pathway kinase inhibitors such as everolimus.

The invention has the advantages that:

the combination scheme of the targeted medicament and the polypeptide vaccine covers common targeted therapeutic medicaments; 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 remarkably prolong the effective action time of the targeted drugs.

Drawings

FIG. 1 is a graph of the interaction between 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 for each group of experiments 2-4 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 targeted therapeutic drug resistance mutation sites that were approved by FDA or CFDA or in clinical trial III/IV, and especially the less sample-size supported mutations for all candidate targeted drugs and resistance mutations, further looked up literature to determine their reliability, and determined truly fully reliable targeted drug resistance sites for downstream analysis, e.g., cosmic included a report confirming that the EGFR-T790M mutation is an austenitinib resistance site, but found through the look-up literature that T790M is not an austenitinib 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 of the polypeptide, 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. Target drug and drug-resistant site correlation and drugClustering objects: 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).

AC: the size of the affinity change, A, B, C, D four different types of affinity changes are given different weights; f_hla: 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 virginia for SMO mutation generation, the drug-resistant ibrutinib for BTK mutation generation, the drug-resistant epirubitinib for AR resistance mutation generation, the drug-resistant epirubicin for BRAF resistance mutation generation, the tyrosine kinase inhibitor for ALK, MET fusion gene, the tyrosine kinase inhibitor for EGFR pathway generation, and the kinase inhibitor for PI3K, AKT, mTOR pathway everolimus generation. 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

Table 2: database of clinical patients:

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: 7 product polypeptides are synthesized by Nanjing Jinslei Biotech Co., LtdThe purity is more than 90%, and the content of endotoxin is less 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 Single peptide ELISPOT test was performed separately according to groups, i.e., mice were diluted to 1-2 x 10 lymphocyte concentration⁶Laying 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 EGF, 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 using FITC-CD44 flow antibody to select positive Cell as subsequent transfected Cell marked as CD44⁺-AGS。

The purpose of the experiment is as follows: 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 polypeptides can be expressed and presented. a. Constructing a mutation 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 Jinsrui biological technology Co., Ltd, and the amino acid sequence of smo15 is SEQ ID: NO 5; the amino acid sequence of Smow7 is: (ii) SEQ ID: NO 6; wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences;

b. construction of a cell line capable of stably expressing the mutant polypeptide: gastric cancer cell line CD44⁺AGS by 2 x 10⁵Perwell was plated in 6-well plates and transfection was initiated when cells were 70-80% covered. 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 added^TMReagents, incubated at room temperature for 5min, and each with 5. mu.l Lipofectamine^TMLTX serum-free RPMI-1640100. mu.l was mixed and incubated at room temperature for 30 min. The liposome plasmid complex is respectively dripped into 2 parts of cells to be transfected containing 1000 μ l of serum-free RPMI-1640, gently shaken back and forth, kept stand for 6h, replaced by an RPMI-1640 culture medium containing 10% serum, and further cultured for 48h, and replaced by a 10% serum culture medium containing 700 μ G/mL G418 and 400 μ G/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. 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: i isbrutinib (ibrutinib) purchased from seleck Chemicals, human multiple myeloma cell line RPMI8226 (r)

CCL-155^TM) Purchased from ATCC and cultured in RPMI1640 medium plus 10% fetal bovine serum.

The purpose of the experiment is as follows: in order to verify that the T cells induced by the drug-resistant polypeptide of the present invention have killing activity, it is necessary to construct a stable transgenic cell line containing the specific mutation site of the present invention, which can express and present the polypeptide

a. Constructing a mutation site eukaryotic expression plasmid: the mini-gene capable of expressing all 5 mutated vaccine polypeptides 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: (ii) SEQ ID: NO 7; the amino acid sequence of BTKW2 is as follows: the amino acid sequence of SEQ ID: NO 8; wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences.

b. Construction of a cell line capable of stably expressing the mutant polypeptide: 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 added^TMReagents, incubated at room temperature for 5min, and each with 5. mu.l Lipofectamine^TMSerum-free RPMI-1640100 mu of LTXl the system 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 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: (

CRL-1435^TM) 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 of the present invention have killing activity, it is necessary to construct a set of stable transgenic cell lines containing the specific mutation sites of the present invention, which can express and present the polypeptide.

a. Constructing a mutation 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: (ii) SEQ ID: NO 9; the amino acid sequence of ARW2 is: the amino acid sequence of SEQ ID: NO 10; wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences.

b. Construction of a cell line capable of stably expressing the mutant polypeptide: 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 added^TMReagents, incubated at room temperature for 5min, and each with 5. mu.l Lipofectamine^TMLTX 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 (4) performing resistance screening for 10-14 days, after all cells in the control group die, growing a large amount of cells in the transfection group, digesting the cells, and planting the cells 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 AR4-pcDNA3.1(+) plasmid and the ARw2-pcDNA3.1(+) plasmid were named 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. Constructing a mutation 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 (+), and is named as 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: the amino acid sequence of SEQ ID: NO 11; MEw5 is the amino acid sequence: (ii) SEQ ID: NO 12; wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences.

b. Construction of a cell line capable of stably expressing the mutant polypeptide: 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 added^TMReagents, incubated at room temperature for 5min, and each with 5. mu.l Lipofectamine^TMLTX serum-free RPMI-1640100. mu.l was mixed and incubated at room temperature for 30 min. Dropping the liposome plasmid complex into 2 parts of cells to be transfected containing 1000 μ l of serum-free RPMI-1640, shaking gently, standing for 6 hr, changing to 10% serum-containing RPMI-1640 culture medium, culturing for 48 hr, and changing to 10% serum-containing 700 μ G/mL G418 and 400 μ G/mL hygromycin BThe culture medium is used 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 ME7-pcDNA3.1(+) plasmid and MEw5-pcDNA3.1(+) plasmid were named 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.

a. Constructing a mutation 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: (ii) SEQ ID: NO 13; the amino acid sequence of ALKw5 is: (ii) SEQ ID: NO 14; wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences.

a. 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 added^TMReagents, incubated at room temperature for 5min, and each with 5. mu.l Lipofectamine^TMLTX serum-free RPMI-1640100. mu.l was mixed and incubated at room temperature for 30 min. The liposome plasmid complex is respectively dripped into 2 parts of cells to be transfected containing 1000 μ l of serum-free RPMI-1640, gently shaken back and forth, kept stand for 6h, replaced by an RPMI-1640 culture medium containing 10% serum, and further cultured for 48h, and replaced by a 10% serum culture medium containing 700 μ G/mL G418 and 400 μ G/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.

The purpose of the experiment is as follows: in order to verify that the T cells induced by the drug-resistant polypeptide of the present invention have killing activity, it is necessary to construct a set of stable transgenic cell lines containing the specific mutation sites of the present invention, which can express and present the polypeptide.

a. Constructing a mutation site eukaryotic expression plasmid: in order to realize the expression of the mutant polypeptide, the invention is supposed to be over-expressed in the human chronic granulocytic leukemia cell line THP-1 by serially connecting each polypeptide gene (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 were connected by flexible connecting peptide GGSGGGGSGG, after codon optimization of the gene, GATATC (EcoR V) was introduced upstream and CTCGAG (Xho I) was introduced downstream, and they were 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 the ABL1 protein directly selects the 234-505 amino acid site, the FLT3 protein selects one protein near the 835 amino acid site, the EGFR protein selects 2 proteins near the 760 and 790 amino acid sites, the KIT protein 3 and the PDGFRA protein 1 wild-type polypeptides) and are cloned into a eukaryotic expression vector pcDNA3.1-hygro (+), and all gene segments of the wid8-hygro (+) are synthesized and constructed by Nanjing Kingsry Biotech limited company.

The amino acid sequence of Mut46-hy is: (ii) SEQ ID: NO 15; the amino acid sequence of Mut46-zeo is: the amino acid sequence of SEQ ID: NO 16; the amino acid sequence of Wid8-hy is as follows: the amino acid sequence of SEQ ID: NO 17; 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 a polypeptide capable of stably expressing a mutant polypeptideThe cell line of (1): 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 thereto^TMReagents were incubated at room temperature for 5min and then mixed with the solution containing 5. mu.l of Lipofectamine^TMLTX 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 being 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 procedure, cell lines constructed with mut46-hygro (+) plasmid and wid8-hygro (+) plasmid were designated 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 as described above, designated mut92(mut46-hygro (+), mut46-zeo (+)).

Example 7, construction of a seventh class of stable transgenic cell lines targeting specific mutation sites of kinase inhibitors such as everolimus for PI3K, AKT, mTOR pathway;

human embryonic kidney fibroblast strain HEK 293(

CRL-1573^TM) Purchased from ATCC, cultured in 10% FBS-DMEM medium.

a. Constructing a mutation site eukaryotic expression plasmid: the mini-gene capable of expressing all 3 mutated vaccine polypeptides 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), 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: (ii) SEQ ID: NO 19; the amino acid sequence of MEMw3 is: (ii) SEQ ID: NO 20; wherein amino acids 1-28 are signal peptide regions, represented in bold italics; the underlined sections are MITD sequences.

b. Construction of a cell line capable of stably expressing the mutant polypeptide: human embryonic kidney fibroblast strain HEK 293 with 2 x 10⁵Perwell in 6-well plates, transfection was initiated when cells were 70-80% covered. The MEM3-pcDNA3.1(+) plasmid and the MEMw3-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 added^TMReagents, incubated at room temperature for 5min, and each with 5. mu.l Lipofectamine^TMLTX serum-free RPMI-1640100. mu.l was mixed and incubated at room temperature for 30 min. The liposome plasmid complex is respectively dripped into 2 parts of cells to be transfected containing 1000 μ l of serum-free RPMI-1640, gently shaken back and forth, kept stand for 6h, replaced by an RPMI-1640 culture medium containing 10% serum, and further cultured for 48h, and replaced by a 10% serum culture medium containing 700 μ G/mL G418 and 400 μ G/mL hygromycin B for cell screening. And (4) performing resistance screening for 10-14 days, after all cells in the control group die, growing a large amount of cells in the transfection group, digesting the cells, and planting the cells into a 96-well plate by adopting a limiting dilution method. Monoclonal cells were selected under the microscope using a tide containing 700. mu.g/mL G418, 400. mu.g/mLThe culture medium of the mycin B is continuously cultured, and the liquid is 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 cell level of the 15 polypeptides in vitro can cause the killing effect of the 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. Target cells of smo15-ASG (containing smo15-pcDNA3.1(+)) and smow7-AGS (containing smow7-pcDNA3.1(+)) 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 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. 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. Target cells of smow7-AGS (containing smow7-pcDNA3.1(+)) were mixed with effector cells CTL according to the ratio of 1:5, 1:10 and 1:20, and added to a U-shaped 96-well plate in a volume of 200. mu.L per well as controls each of which was provided with three parallel control wells. The 96-well plate was incubated at 37 ℃ for 4 hours. 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 targeted 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. BTK5 (containing BTK5-pcDNA3.1(+)) and BTKw2 (containing BTKw2-pcDNA3.1(+)) target cells were labeled with CFSE under aseptic 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 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) × 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. Mixing BTK5 (containingBTK5-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 of which was provided with three parallel control wells. 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. The 96-well plate was incubated at 37 ℃ for 4 h. 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 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.

Experiments 2 to 3: in vitro cell killing experiment of polypeptide vaccine group of third type targeted drugs

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 aseptic 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) × 10⁶PermL, in 6-well plates, 3mL per well, with RPMI1640+ 10% FBS +1 × penicillin (100 μ g/mL) + streptomycin (100 μ g/mL) +1 × MEM non-evaporative 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. 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. 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). The 96-well plate was incubated at 37 ℃ for 4 hours. 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 7 polypeptides were verified to be able to induce a killing effect on tumor cells at the in vitro cell level.

The experimental method comprises the following steps: (1) the 5- (6) -Carboxy-fluoroscein succinimidyl ester (CFSE) dye was purchased from Invitrogen corporation. 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 polypeptide group 1 retained in example 4 was reducedMouse lymphocyte suspension, RPMI1640 medium heavy suspension, trypan blue staining count. 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. 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. 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). The 96-well plate was incubated at 37 ℃ for 4 hours. 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 percent.

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-fluoroescein succinimidyl ester(CFSE) dyes were 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 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 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. 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. 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). The 96-well plate was incubated at 37 ℃ for 4 h. 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, 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 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; after one week, the cells were resuspended and washed 2 times with PBS to prepare effector CTLs. 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. 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. The 96-well plate was incubated at 37 ℃ for 4 hours. 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. The target cells of MEM3 (containing MEM3-pcDNA3.1(+)) and MEMw3 (containing MEMw3-pcDNA3.1(+)) were marked with CFSE under sterile 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 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. MEM3 (containing MEM3-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 in a volume of 200. mu.L per well to prepare experimental groups each having three parallel control wells. 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. The 96-well plate was incubated at 37 ℃ for 4 hours. The 96-well plate was centrifuged to remove supernatant, the cell pellet was resuspended in 200. mu.L of precooled PBS, transferred to a flow-loading tube, and propidium iodide (Prodium iodide) was addede, PI) staining marker, concentration 1. mu.g/m L, staining for 3min, immediately flow-on-machine 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 combination of kinase inhibitor aiming at targeting drugs PI3K, AKT and mTOR pathways and design method thereof

<141> 2020-11-20

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 1

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> 2

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 2

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> 3

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 3

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> 4

<211> 24

<212> PRT

<213> Artificial Sequence

<400> 4

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> 5

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 5

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> 6

<211> 630

<212> PRT

<213> Artificial Sequence

<400> 6

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> 7

<211> 348

<212> PRT

<213> Artificial Sequence

<400> 7

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> 8

<211> 246

<212> PRT

<213> Artificial Sequence

<400> 8

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> 9

<211> 150

<212> PRT

<213> Artificial Sequence

<400> 9

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> 10

<211> 208

<212> PRT

<213> Artificial Sequence

<400> 10

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> 11

<211> 147

<212> PRT

<213> Artificial Sequence

<400> 11

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> 12

<211> 300

<212> PRT

<213> Artificial Sequence

<400> 12

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

290 295 300

<210> 13

<211> 238

<212> PRT

<213> Artificial Sequence

<400> 13

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> 14

<211> 390

<212> PRT

<213> Artificial Sequence

<400> 14

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 Ser Leu Gln Ser Leu Pro Arg Phe Ile Leu Met Glu Leu Met Ala

275 280 285

Gly Gly Asp Leu Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Phe

290 295 300

Leu Met Glu Ala Leu Ile Ile Ser Lys Val Asn His Gln Asn Ile Val

305 310 315 320

Arg Cys Ile Gly Lys Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly Ile

325 330 335

Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile Gly

340 345 350

Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys

355 360 365

Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser

370 375 380

Asp Val Ser Leu Thr Ala

385 390

<210> 15

<211> 247

<212> PRT

<213> Artificial Sequence

<400> 15

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

Asn Ile Thr Leu Ile Arg Gly Leu Gly His Gly Ala Phe Gly Glu Val

100 105 110

Tyr Glu Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Cys Pro Gly Pro

115 120 125

Gly Arg Val Ala Lys Ile Gly Asp Phe Gly Met Ala Arg Asp Ile Tyr

130 135 140

Arg Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Leu Gln Ser Leu

145 150 155 160

Pro Arg Phe Ile Leu Leu Glu Leu Met Ala Gly Gly Asp Leu Lys Ser

165 170 175

Phe Leu Arg Glu Thr Arg Gly Gly Ser Leu Gly Gly Gly Gly Ser Gly

180 185 190

Ile Val Gly Ile Val Ala Gly Leu Ala Val Leu Ala Val Val Val Ile

195 200 205

Gly Ala Val Val Ala Thr Val Met Cys Arg Arg Lys Ser Ser Gly Gly

210 215 220

Lys Gly Gly Ser Tyr Ser Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly

225 230 235 240

Ser Asp Val Ser Leu Thr Ala

245

<210> 16

<211> 1559

<212> PRT

<213> Artificial Sequence

<400> 16

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> 17

<211> 1511

<212> PRT

<213> Artificial Sequence

<400> 17

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> 18

<211> 625

<212> PRT

<213> Artificial Sequence

<400> 18

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> 19

<211> 180

<212> PRT

<213> Artificial Sequence

<400> 19

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 His Glu Cys Asn

50 55 60

Ser Pro Tyr Ile Val Gly Leu Tyr Gly Ala Phe Tyr Ser Asp Gly Glu

65 70 75 80

Ile Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Lys Val

85 90 95

Ala Asp Phe Gly Leu Ala Arg Val Met Tyr Asp Lys Glu Tyr Tyr Ser

100 105 110

Val His Lys 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> 20

<211> 180

<212> PRT

<213> Artificial Sequence

<400> 20

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 His Glu Cys Asn

50 55 60

Ser Pro Tyr Ile Val Gly Phe Tyr Gly Ala Phe Tyr Ser Asp Gly Glu

65 70 75 80

Ile Lys Lys Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Lys Val

85 90 95

Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu Tyr Tyr Ser

100 105 110

Val His Lys 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

Claims

1. The polypeptide vaccine combination of the drug resistant sites of kinase inhibitors everolimus, rapamycin and MEK inhibitor PD0325901 and C-MET inhibitor Savoltinib aiming at targeting drugs PI3K, AKT and mTOR pathways is characterized in that,

the polypeptide vaccine of the everolimus and rapamycin resistant site aims at the following mutations: the polypeptide vaccine of MTOR-F2108L, MEK inhibitor PD0325901 drug-resistant site aims at the following mutations: the polypeptide vaccine of MAP2K1-F129L, C-MET inhibitor Savolitinib drug-resistant site is directed against the following mutations: MET-D1246V;

the polypeptide vaccine is a peptide with the following sequence:

the sequence of MTOR-F2108L is: TQAWDLYYHVLRRISKQLPQ, respectively;

the sequence of MAP2K1-F129L is: HECNSPYIVGLYGAFYSDGEIKK, respectively;

the sequence of MET-D1246V is: VKVADFGLARVMYDKEYYSVHK, respectively;

the 3 mutant polypeptides are connected by flexible connecting peptide GGSGGGGSGG.