CN109776671B

CN109776671B - Isolated T cell receptor, modified cell thereof, encoding nucleic acid, expression vector, preparation method, pharmaceutical composition and application

Info

Publication number: CN109776671B
Application number: CN201711123690.XA
Authority: CN
Inventors: 侯亚非; 侯大炜
Original assignee: Hangzhou Converd Co Ltd
Current assignee: Hangzhou Converd Co Ltd
Priority date: 2017-11-14
Filing date: 2017-11-14
Publication date: 2022-05-27
Anticipated expiration: 2037-11-14
Also published as: CN109776671A; WO2019096115A1

Abstract

The invention provides isolated T cell receptors, modified cells thereof, encoding nucleic acids, expression vectors, methods of preparation, pharmaceutical compositions and uses. The isolated T Cell Receptor (TCR) comprises at least one of an alpha chain and a beta chain, both of which comprise a variable region and a constant region, wherein the T cell receptor is capable of specifically recognizing the antigen Her2/neu expressed by tumor cells, and wherein the amino acid sequence of the variable region of the alpha chain has at least 98% identity to the amino acid sequence set forth in SEQ ID NO:19 and the amino acid sequence of the variable region of the beta chain has at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 20. The TCR specifically recognizes tumor antigens while avoiding possible off-target toxic side effects. The immune cells modified by the TCR have obvious anti-tumor effect.

Description

Isolated T cell receptor, modified cell thereof, encoding nucleic acid, expression vector, preparation method, pharmaceutical composition and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an isolated T cell receptor, a modified cell thereof, a coding nucleic acid, an expression vector, a preparation method, a pharmaceutical composition and application.

Background

Her2/neu (ERBB2) is a transmembrane protein belonging to the EGFR family, and forms a dimer with other proteins of the family to regulate processes such as cell proliferation, differentiation and canceration through a series of intracellular signaling pathways (see the documents "Growth Factors, 2008; 26: 263", "Oncol biol. Phys, 2004; 58: 903"). The Her2/neu protein is overexpressed in cancer cells of various epithelial origins, such as breast cancer, stomach cancer, large intestine cancer, ovarian cancer, pancreatic cancer, lung cancer, esophageal cancer, bladder cancer, kidney cancer, etc. (see the literature "Trends in Molecular Med, 2013; 19: 677"), and is relatively uniformly expressed in cancer cells of primary and metastatic foci (see the literature "J Clin Oncol, 1998; 8: 103"), and thus, Her2/neu becomes an appropriate target for targeted therapy.

The survival period of a Her2/neu positive breast cancer patient can be prolonged remarkably by Herceptin of a humanized monoclonal antibody drug Herceptin targeting Her2/neu (see the literature 'N Engl J Med,2001,344: 783'), however, the clinical response rate of treating Her2 positive metastatic breast cancer by using Herceptin alone is only 11% to 26% (see the literature 'J Clin Oncol, 2002; 20: 7169'), which indicates that the curative effect of the Heceptin alone on most of the Her2 high-expression metastatic breast cancer is not ideal. Although chemotherapy in combination with Heceptin can improve clinical response rates, most breast cancer patients with Her2/neu over-expressed will develop resistance to Heceptin after one year (see document "J Clin Oncol, 2001; 19: 2587").

Patients with Her2/neu positive tumors develop endogenous antibodies and T-cell responses against Her2/neu antigen (see the literature "Cancer Res, 2005; 65: 650"), and thus, specific immunotherapy targeting Her2/neu antigen is a promising therapeutic approach. T cells specifically recognizing Her2/neu epitope polypeptide (epitope peptide)369-377 can be successfully separated from ovarian cancer ascites with Her2/neu high expression (see the literature J.exp.Med.1995; 181: 2109-2117). Tumor vaccines targeting the Her2/neu369-377 polypeptide antigen entered clinical trials, although clinical stage 1/2 showed that the vaccine induced specific T killer cells against the Her2/neu369-377 polypeptide antigen (see "Breast Care, 2016; 11: 116"), but clinical stage three did not achieve the pre-defined goal of extending patient survival (http:// www.onclive.com/web-exclusive/phase-iii-nellipipenemts-study-in-break-cancer-cultured-after-fertility-review). After adoptive transfer of in vitro cultured tumor-specific T cell therapies based on Chimeric Antigen Receptors (CARs) were developed, they entered clinical trials as the first CAR-T cell therapy targeting Her2/neu antigen against solid tumors, but were terminated by the death of the patient due to the generation of a strong Cytokine Release Syndrome (CRS) (see "Nature Med, 2016; 22: 26"). Severe cytokine storm and neurotoxicity are common toxic reactions in CAR-T therapy (see the literature "Blood, 2016; 127: 3321"), in part because of the possibility associated with unrestricted cell activation of this non-native T cell receptor of CARs (see the literature "Nat Ned, 2015; 21: 581"), or with autocrine secretion of cytokines without antigen stimulation (see the literature "Cancer immune Res, 2015; 3: 356").

TCR-T therapy by adoptive transfer of T cells genetically modified with a specific T cell receptor (i.e., TCR) is considered to be the most promising immunocytogene therapy for solid tumors (see "Adv Immunol.2016; 130: 279-94"). Among them, clinical studies of TCR-T therapy targeting the NY-ESO-1 antigen showed encouraging clinical therapeutic efficacy (see the literature "Nat Med. 2015Aug; 21(8): 914-. However, the number of specific TCRs currently known to target tumor antigens and efficiently recognize tumor cells is very limited, thus limiting the wide application of TCR-T therapy. In addition, although TCR-T therapy does not exhibit the severe cytokine storm toxicity exhibited by CAR-T therapy, if the target antigen is derived from self-proteins, low expression of the target antigen in normal tissue cells may result in a severe autoimmune response, i.e., a switch off target (on target off tumor) toxic response (Blood 2009; 114: 535-546). In addition, to obtain a high affinity TCR that efficiently recognizes tumor cells, a general strategy is to induce in vitro by genetic point mutations in the Complementarity Determining Regions (CDRs) on the TCR, or by induction from a pool of humanized mouse T cells that have not been screened for central tolerance mechanisms (see references "Front immunological. 2013; 4: 363"). However, such high affinity TCRs may cross-react with normal self-proteins to cause killing of normal tissue cells, i.e., severe or even fatal off-target (off-target) toxicity (see references "Curr Opin Immunol 2015; 33: 16-22", see references "Sci Transl Med.2013; 5(197):197ra 103"). Therefore, obtaining novel TCR genes that specifically target tumor antigens and efficiently recognize tumor cells while avoiding possible off-target toxic side effects is a major challenge to the successful development of TCR-T immune cell gene therapy.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides an isolated T cell receptor, a modified cell thereof, a coding nucleic acid, an expression vector, a preparation method, a pharmaceutical composition and application.

Specifically, the present invention provides:

(1) an isolated T cell receptor comprising at least one of an alpha chain and a beta chain, each comprising a variable region and a constant region, wherein said T cell receptor is capable of specifically recognizing the antigen Her2/neu expressed by tumor cells, and wherein the amino acid sequence of said variable region of said alpha chain has at least 98% identity to the amino acid sequence set forth in SEQ ID NO 19 and the amino acid sequence of said variable region of said beta chain has at least 98% identity to the amino acid sequence set forth in SEQ ID NO 20.

(2) The T cell receptor of (1), wherein said T cell receptor specifically recognizes an epitope polypeptide of said antigen Her2/neu presented by HLA-A2 molecule; preferably, the epitope polypeptide comprises Her2/neu369-377 as shown in SEQ ID NO: 18.

(3) The T cell receptor of (1), wherein the constant region of the alpha chain and/or the constant region of the beta chain is derived from a human; preferably, the constant region of the alpha chain is replaced in whole or in part by a homologous sequence derived from another species and/or the constant region of the beta chain is replaced in whole or in part by a homologous sequence derived from another species; more preferably, the other species is mouse.

(4) The T cell receptor of (1), wherein the constant region of the alpha chain is modified with one or more disulfide bonds and/or the constant region of the beta chain is modified with one or more disulfide bonds.

(5) The T cell receptor according to (1), wherein the amino acid sequence of the alpha chain is shown as SEQ ID NOs:2, 6 or 10, and the amino acid sequence of the beta chain is shown as SEQ ID NOs:4, 8 or 12.

(6) An isolated nucleic acid encoding a T cell receptor comprising a coding sequence for at least one of the alpha and beta chains of said T cell receptor, said alpha and beta chain coding sequences each comprising a variable region coding sequence and a constant region coding sequence, wherein said T cell receptor is capable of specifically recognizing the tumor cell expressed antigen Her2/neu and said alpha chain variable region coding sequence encodes an amino acid sequence having at least 98% identity to the amino acid sequence set forth in SEQ ID No. 19 and said beta chain variable region coding sequence encodes an amino acid sequence having at least 98% identity to the amino acid sequence set forth in SEQ ID No. 20.

(7) The nucleic acid according to (6), wherein the nucleic acid is DNA or RNA.

(8) The nucleic acid according to (6), wherein the alpha chain variable region encoding sequence is represented by SEQ ID NO:21 and the beta chain variable region encoding sequence is represented by SEQ ID NO: 22.

(9) The nucleic acid of (6), wherein said T cell receptor encoded by said nucleic acid is capable of specifically recognizing an epitope polypeptide of said antigen Her2/neu presented by HLA-A2 molecule; preferably, the epitope polypeptide comprises Her2/neu369-377 as shown in SEQ ID NO: 18.

(10) The nucleic acid of (6), wherein the alpha chain constant region coding sequence and/or the beta chain constant region coding sequence is derived from a human; preferably, the alpha chain constant region coding sequence is replaced in whole or in part by a homologous sequence from another species and/or the beta chain constant region coding sequence is replaced in whole or in part by a homologous sequence from another species; more preferably, the other species is mouse.

(11) The nucleic acid of (6), wherein the alpha chain constant region coding sequence comprises one or more disulfide bond coding sequences and/or the beta chain constant region coding sequence comprises one or more disulfide bond coding sequences.

(12) The nucleic acid according to (6), wherein the alpha chain coding sequence is represented by SEQ ID NOs:1, 5 or 9 and the beta chain coding sequence is represented by SEQ ID NOs:3, 7 or 11.

(13) The nucleic acid of any one of (6) - (11), wherein the alpha chain coding sequence and the beta chain coding sequence are linked by a coding sequence for a cleavable linker polypeptide.

(14) The nucleic acid according to (13), which has the sequence shown in SEQ ID NOs:13, 15 or 23.

(15) A recombinant expression vector comprising the nucleic acid according to any one of (6) to (14), and/or a complementary sequence thereof, operably linked to a promoter.

(16) A T cell receptor-modified cell whose surface is modified with the T cell receptor according to any one of (1) to (5), wherein the cell comprises an original T cell or a precursor cell thereof, an NKT cell, or a T cell line.

(17) A method for preparing a T cell receptor-modified cell according to (16), comprising the steps of:

1) providing a cell;

2) providing a nucleic acid encoding a T cell receptor according to any one of (1) - (5);

3) transfecting the nucleic acid into the cell.

(18) The method of (17), wherein the cells of step 1) are autologous or allogeneic.

(19) The method of (17), wherein the transfection comprises: transfection with a viral vector, preferably a gamma retroviral vector or a lentiviral vector; chemical means, preferably, the chemical means comprises means of lipofection; physical means, preferably, the physical means comprises electrotransfection means.

(20) The method according to (17), wherein the nucleic acid of step 2) is the nucleic acid according to any one of (6) to (14).

(21) Use of the T cell receptor-modified cell according to (16) for the preparation of a medicament for the treatment or prevention of tumors and/or cancers.

(22) The use of (21), wherein said tumor and/or cancer is antigen Her2/neu positive and is HLA-a2 positive.

(23) Use of the T cell receptor modified cell of (16) in the preparation of a medicament for detecting a tumor and/or cancer in a host.

(24) A pharmaceutical composition comprising the T cell receptor-modified cell according to (16) as an active ingredient, and a pharmaceutically acceptable excipient.

(25) The pharmaceutical composition of (24), wherein said pharmaceutical composition comprises a total dose per patient per course of treatment ranging from 1 x10³-1×10⁹One cell per Kg body weight of said T cell receptor modified cells.

(26) The pharmaceutical composition of (24), wherein the pharmaceutical composition is suitable for administration intraarterially, intravenously, subcutaneously, intradermally, intratumorally, intralymphatically, subarachnoid intracavity, intramedullally, intramuscularly and intraperitoneally.

(27) A method for treating tumors and/or cancers, comprising administering the T cell receptor modified cells according to (16) to a tumor and/or cancer patient.

(28) The method of (27), wherein said T cell receptor modified cells are administered at a total dose per patient per course of treatment ranging from 1 x10³-1×10⁹One cell/Kg body weight.

(29) The method of (27), wherein the T cell receptor modified cell is administered intra-arterially, intravenously, subcutaneously, intradermally, intratumorally, intralymphatically, subarachnoid intracavity, intramedullally, intramuscularly, and intraperitoneally.

(30) The method of (27), wherein said tumor and/or cancer is antigen Her2/neu positive and is HLA-a2 positive.

Compared with the prior art, the invention has the following advantages and positive effects:

the invention successfully induces T cell clone which has specificity to Her2/neu antigen epitope polypeptide (such as Her2/neu369-377 polypeptide) presented by HLA-A2 from peripheral blood of a healthy donor with positive HLA-A2, and screens T cell clone carrying natural TCR which specifically recognizes Her2/neu antigen epitope polypeptide (such as Her2/neu369-377 polypeptide), thereby obtaining the whole sequence of the TCR. The TCR belongs to CD8 molecule dependence, has medium affinity to Her2/neu epitope polypeptide (such as Her2/neu369-377 polypeptide), and can specifically recognize tumor cells which are positive to HLA-A2 and express Her2/neu antigen. In addition, T cell clones carrying this TCR were screened for central immune tolerance and entered the peripheral T cell pool. The killer T cells carrying the TCR exist in normal human peripheral blood and do not generate cross reaction to normal tissue cells which express Her2/neu protein in a trace amount. Thus, the present invention provides novel TCRs that specifically recognize tumor antigens while avoiding possible off-target toxic side effects.

In a further aspect of the invention, the constant region of the TCR is engineered (e.g. by disulphide bond modification or murine engineering) to further reduce or avoid the occurrence of mismatches with endogenous TCRs when expressed on immune cells.

Immune cells (e.g., naive T cells, precursor cells thereof, NKT cells, T cell lines) modified with the TCR specifically recognize various HLA-A2⁺And Her2/neu⁺The tumor cell strain has obvious anti-tumor effect. On the other hand, the TCR used to modify the immune cells does not cross-react with normal cells that express Her2/neu in minute quantities. Therefore, TCR-T therapy based on this TCR is expected to treat a variety of solid tumors.

When the immune cell modified by the TCR is used for treating tumors, cytokine storm and immune rejection caused by CAR-T treatment can be effectively avoided.

The TCR-modified immune cells of the invention are useful for treating HLA-A2⁺And Her2/neu⁺Provides a new choice for tumor patients and has good industrial application prospect.

Drawings

FIG. 1 shows HLA-A2 in example 1 of the present invention⁺Results of phenotypic and functional assays of Her2/neu369-377 polypeptide (Her2-E75) specific killer T cells induced in normal donor PBMC (specifically, #2 PBMC). FIG. 1A shows the results of flow cytometry analysis of PBMC cells stained with CD8-APC antibody and Her2-E75 pentamer-PE after two rounds of in vitro stimulation with Her2-E75 antigen polypeptide, and the right shows the results of polypeptide-stimulated cells on CD8⁺Pentameric polymers⁺The killer T cell population was FACS sorted to obtain T cell clones. The left panel shows control cells without polypeptide stimulation. The abscissa represents the fluorescence intensity of the expression of the CD8 molecule and the ordinate represents the fluorescence intensity of the conjugated Her2-E75 pentamer. FIG. 1B shows CD8⁺E75-tetramer⁺Phenotypic analysis of killer T cell clones by flow cytometry after staining with CD8-APC and Her2-E75 tetramer-PE, right panel showing CD8⁺Her2 tetramer⁺T cell clone Her2CTL 1B5 is a purified Her2-E75 polypeptide-specific CTL cell clone. The left panel shows control CTL cells without polypeptide stimulation. The abscissa represents the fluorescence intensity of the expression of the CD8 molecule and the ordinate represents the fluorescence intensity of the conjugated Her2-E75 tetramer. FIG. 1C shows the results of functional assays for T cell clones, T cell clone Her2CTL 1B5 (diagonal bars) or control CTL cells without peptide stimulation (dot bars) co-cultured with different concentrations of Her-E75 polypeptide presented by T2 cells, or with HLA-A2⁺Her2/neu⁺After co-culturing the colon cancer cell strain colo205, cell supernatants were taken for IFN-gamma ELISA analysis, and the results were shown as mean + -SD in each test group and control group. The abscissa represents the different experimental groups and the ordinate represents the concentration of IFN-. gamma.secreted by the T cells.

FIG. 2 shows the major functional fragments of two constructed lentiviral vectors carrying the Her2TCR-1B5TCR gene (shown in the figure as "pCDH-EF 1 α -Her2TCR- (PGK-GFP) vector" and "pCDH-EF 1 α -Her2TCR vector", respectively). The fragments shown above express both the TCR gene driven by the EF-1. alpha. promoter and the GFP gene driven by the PKG promoter, and the fragments shown below express only the TCR gene. The β chain and α chain of each TCR are linked by a coding sequence for a cleavable linker polypeptide (furin-F2A).

FIG. 3 shows the results of phenotypic and functional assays of T cell lines transfected with the Her2TCR-1B5TCR gene. J.rt3-T3.5T cell line (j.rt3) was transfected with lentiviral vectors encoding Her2TCR-1B5TCR and GFP and flow cytometric analysis was performed after Her2-E75 tetramer-PE staining. FIG. 3A shows GFP⁺Her2-E75 tetramer⁺The cell populations are Her2TCR-1B5TCR expressing cells and the percentages indicated are the ratio of each positive cell population to the total number of cells. The left diagram relates to a TCR with a disulfide bond structure added to the constant regions of an alpha chain and a beta chain (Her2TCR-1B5-dis), and the right diagram relates to a TCR with human alpha chain and beta chain constant regions replaced by homologous sequences of mouse constant regions (Her2TCR-1B 5-mC). The abscissa represents the fluorescence intensity of GFP molecule expression and the ordinate representsIndicating the fluorescence intensity of the bound Her2-E75 tetramer. FIG. 3B shows the expression of the constant regions of the TCR α and β chains in two T cell lines modified in different ways. In the figure, "Her 2TCR-1B 5-dis" means a TCR in which the constant regions of the alpha chain and the beta chain are each augmented by a disulfide bond structure; "Her 2TCR-1B 5-mC" refers to a TCR in which the constant regions of the human alpha and beta chains are replaced by homologous sequences of the mouse constant region. GFP (green fluorescent protein)⁺Her2-E75 tetramer⁺The cells are positive cells expressing Her2TCR-1B5TCR, and the ordinate represents the total GFP occupied by the TCR positive cells⁺Percentage of cells. The abscissa represents the different groups of T cell lines, wherein "Jurkat (TCR a + b +)" means Jurkat cells, both of which are expressing the alpha chain and beta chain, and "J.RT3 (TCRa + b-)" means J.RT3-T3.5 cells, which are derived from Jurkat cells, and the beta chain gene is deleted, the alpha chain is still expressed. FIG. 3C shows that T cell lines transfected with lentiviral vectors encoding the Her2TCR-1B5TCR gene can recognize the Her2-E75 polypeptide presented by T2 cells. The constant regions of the TCR α and β chains were modified in different ways, and T cell lines expressing TCR were cultured in mixed culture with T2 cells presenting varying concentrations of Her2-E75 polypeptide for 16 hours, stained with anti-CD 69-PE antibody and flow cytometric analysis was performed. In the figure, "J.RT3-Her 2-1B 5-dis" indicates J.RT3-T3.5 cells expressing Her2TCR-1B5-dis, "J.RT3-Her 2-1B 5-mC" indicates J.RT3-T3.5 cells expressing Her2TCR-1B5-mC, "Jurkat-Her 2-1B 5-dis" indicates Jurkat cells expressing Her2TCR-1B5-dis, and "Jurkat-Her 2-1B 5-mC" indicates Jurkat cells expressing Her2TCR-1B 5-mC. The abscissa indicates the concentration of Her2-E75 polypeptide presented by T2 cells. Ordinate is CD69⁺Total GFP of cells⁺Percentage of cells.

FIG. 4 shows the results of phenotypic and functional assays of Peripheral Blood Mononuclear Cells (PBMCs) transfected with the Her2TCR-1B5-mC TCR gene. FIG. 4A is the results of flow cytometry analysis of lentiviral vectors encoding Her2TCR-1B5-mC transfected with PBMC from two different donors, stained with Her2-E75 tetramer-PE and anti-CD 8-APC antibody. First, the lymphocyte population, Her2-E75 tetramer, was classified according to cell morphology and size⁺The cell population is Her2TCR-1B5TCR expressing cells. The abscissa indicates the fluorescence intensity of the expression of the CD8 molecule and the ordinate indicates the fluorescence of the bound Her2-E75 tetramerStrength. The percentages indicated are the ratio of each positive cell population to the number of lymphocytes sorted out. The left panel relates to peripheral blood mononuclear cells (#1PBMC) provided by one donor, and the right panel relates to PBMC (#2PBMC) provided by a different donor. CD8⁺Her2-E75 tetramer⁺The cells are Her2TCR-1B5-mC expressing killer T cells. CD8^-Her2-E75 tetramer⁺The cells may be Her2TCR-1B5-mC expressing CD4⁺Helper T cells. FIG. 4B shows that T cells expressing Her2TCR-1B5-mC can recognize Her2-E75 polypeptides presented by T2 cells. Two different donor PBMCs transfected with lentiviral vectors encoding Her2TCR-1B5-mC and GFP were separately cultured in mixture with T2 cells presenting varying concentration gradients of Her2-E75 polypeptide for 16 hours, and cell supernatants were taken for IFN-. gamma.ELISA analysis. The target cells in the control group are T2 cells presenting EBV virus antigen polypeptide LMP 2426-434 capable of binding HLA-A2 molecule. In the figure, "T2 + Her 2-E750.1. mu.g/ml" represents the T2 cell group presenting Her2-E75 polypeptide at 0.1. mu.g/ml, "T2 + Her 2-E750.01. mu.g/ml" represents the T2 cell group presenting Her2-E75 polypeptide at 0.01. mu.g/ml, "T2 + Her 2-E750.001. mu.g/ml" represents the T2 cell group presenting Her2-E75 polypeptide at 0.001. mu.g/ml, "T2 + EBV-LMP 1. mu.g/ml" represents the T2 cell group presenting EBV virus antigen polypeptide LMP 2426-434 at 1. mu.g/ml. The abscissa represents the PBMC groups of different donors and the ordinate represents the concentration of IFN- γ secreted by the T cells. Figure 4C shows the results of CD8 antibody blocking assay of T cell function. Wherein, when #2PBMC transfected by a lentiviral vector for coding Her2TCR-1B5-mC and GFP genes is co-cultured with antigen polypeptide presented by T2 cells, anti-human CD8 antibody is added to detect whether the function of the T cells for secreting IFN-gamma is inhibited. In the figure, "T2 + Her 2-E750.1. mu.g/ml" indicates the group of T2 cells presenting Her2-E75 polypeptide at 0.1. mu.g/ml without adding anti-human CD8 antibody, and "T2 + Her 2-E750.1. mu.g/ml + anti-CD 8" indicates the group of T2 cells presenting Her2-E75 polypeptide at 0.1. mu.g/ml with adding anti-human CD8 antibody. The abscissa represents the different experimental groups and the ordinate represents the concentration of IFN-. gamma.secreted by the T cells. In fig. 4B and 4C, each test group and control group was duplicate wells and the results are shown as mean ± SD.

FIG. 5 shows the results of functional assays of tumor cell lines recognized by Peripheral Blood Mononuclear Cells (PBMC) transfected with the Her2TCR-1B5-mC TCR gene. FIG. 5A shows the results of ELISA analysis of IFN-. gamma.from cell supernatants of #2PBMC transfected with lentiviral vectors encoding the Her2TCR-1B5-mC TCR gene after 16 h mixed culture with cells from different tumor cell lines. Each test group and control group were duplicate wells and the results are shown as mean ± SD. The abscissa represents the different experimental groups and the ordinate represents the concentration of IFN-. gamma.secreted by the T cells. FIG. 5B shows the results of the functional blockade assay performed after the anti-CD8 antibody (shown as "Colo 205+ anti-CD 8") or anti-HLA-ABC antibody (shown as "Colo 205+ anti-HLA-ABC") was added to the culture wells of the above transfected #2PBMC targeted Colo205 cells, respectively. The abscissa represents the different tumor cell line groups and the ordinate represents the concentration of IFN-. gamma.secreted by T cells. Colo205 and Coca-2 are HLA-A2 positive Her2/neu positive colon cancer cells, MAD-MB-231 is HLA-A2 positive Her2/neu positive breast cancer cells, H647 is HLA-A2 negative Her2/neu positive lung cancer cells, H1355 is HLA-A2 positive Her2/neu positive lung cancer cells, SK-OV-3 is HLA-A2 negative Her2/neu positive ovarian cancer cells, and Bjab is HLA-A2 positive Her2/neu negative lymphoma cells.

Detailed Description

The present invention is further described in the following description of the embodiments with reference to the drawings, which are not intended to limit the invention, and those skilled in the art may make various modifications or improvements based on the basic idea of the invention, but within the scope of the invention, unless departing from the basic idea of the invention.

In the present invention, the words "tumor", "cancer", "tumor cell", "cancer cell", "T cell receptor modification", "TCR variable region", "TCR constant region", "antigen", "epitope polypeptide", "homologous sequence", "coding", "antigen presentation", "recombinant DNA expression vector", "promoter", "complementary sequence", "transfection", "autologous", "heterologous", "specific recognition", "TCR-T therapy" encompass meanings commonly recognized in the art.

The Her2/neu antigen belongs to tumor-associated antigens, and the high-affinity T cell poly capable of recognizing the Her2/neu antigenThe numbers are cleared by central tolerance mechanisms to avoid causing possible autoimmune reactions (see literature "Immunol Rev.2016; 271(1): 127-40"). Therefore, it has become difficult to induce T cell clones from the peripheral blood T cell pool that specifically recognize Her2/neu antigen expressed by tumor cells. High affinity TCRs, which were induced from the peripheral blood of patients immunized with the Her2/neu369-377 polypeptide vaccine, which presented the Her2/neu369-377 polypeptide antigen using Dendritic cells (Dendritic cells), while recognizing very low amounts of exogenously loaded Her2/neu369-377 polypeptide, failed to recognize endogenously presented (endogenously presented) antigen polypeptide in tumor cells (see "cancer Res.1998; 58: 4902-4908"). This may be due to the difference in conformation (conformation) of the exogenously loaded polypeptide/HLA complex from the HLA/polypeptide complex naturally present inside the cell, or due to the fact that the Her2/neu369-377 polypeptide is located in the highly glycosylated region of the Her2 protein, the naturally-occurring Her2/neu369-377 polypeptide inside the cell may be glycosylated to cause the difference in TCR recognition conformation (see the references "Proc. Natl. Acad. Sci. USA 2003; 100: 15029-15034"). During the in vitro induction of T cells by the Her2/neu369-377 antigen polypeptide, the high-affinity T cell clone which can only recognize the exogenous antigen polypeptide is often subjected to dominant growth (dominant expansion), while the T cell clone which can specifically recognize the endogenous Her2/neu antigen polypeptide extracted by the cells is inhibited from growing (see the literature, "J Exp Med.2016Nov 14; 213(12): 2811-2829"), thereby increasing the difficulty of obtaining a functional TCR capable of recognizing tumor cells. After central tolerance mechanism screening, the TCRs for recognizing tumor cells mostly have medium affinity, and the functions of the TCRs are often dependent on the assistance of CD8 molecules. There are groups which induce allogeneic T cells (Allo-T cells) from HLA-A2-negative peripheral blood and specifically recognize HLA-A2-restricted Her2/neu369-377 antigen polypeptides, and after T cells are transfected with the obtained TCR gene, they can recognize not only Her2/neu369-377 antigen polypeptides extracted from tumor cells but also Her3 and Her4 antigen epitopes of the same family (see "Journal of Immunology,2008,180: 8135-8145"). However, there are existing generating needles for allogeneic allo-TCR based TCR therapyRisk of allogenic reactions (allo-reactions) to other normal self-protein epitopes (see the literature "int.j. cancer 2009; 125,649-655. Nat Immunol 2007; 8: 388-97"). Another group induced Her2/neu369-377 polypeptide-specific T cells from peripheral blood of tumor patients immunized with Her2/neu369-377 polypeptide vaccine, and paired alpha and beta chains from different T cells and screened for a high affinity TCR that recognized HLA-A2⁺Her2/neu⁺Of (2) (see the document "HUMAN GENE THERAPY 2014; 25: 730-. This TCR was not obtained from monoclonal T cells and therefore it was not possible to determine whether this TCR was a native TCR present in a pool of centrally tolerant screened T cells. The Her2/neu protein is also expressed in small amounts in important organs, i.e., myocardium, lung, esophagus, kidney, and bladder (see "oncogene.1990Jul; 5(7): 953-62"), and therefore TCR-T therapy based on high affinity Her2/neu antigen-specific TCR is at risk of off-target toxic response to normal tissues.

The Her2/neu protein is highly expressed by the tumor cells, so the quantity of the antigen polypeptide presented by HLA on the cell surface is correspondingly increased, and the difference of the quantity of the HLA/antigen polypeptide complex on the tumor cells and normal cells can be a window for specific T cells to distinguish normal tissues from tumor tissues. The present invention proposes to obtain natural TCR sequence from autologous T cell bank (auto-T cell reporter) and to express TCR on T cell in vitro, so that the obtained T cell expressing TCR can recognize Her2/neu antigen expressed by tumor cell.

In order to obtain TCRs which specifically recognize tumor antigens and at the same time avoid possible off-target toxic side effects, the present invention induces T-cell clones specific for the Her2/neu369 377 polypeptide presented by HLA-A2 from peripheral blood of a healthy donor positive for HLA-A2, and selects T-cell clones carrying natural TCRs with moderate affinity for the Her2/neu369 377 polypeptide therefrom. This is different from the strategy of inducing Her2/neu369-377 polypeptide-specific T cells from the peripheral blood of tumor patients immunized by the Her2/neu369-377 polypeptide vaccine (see the literature, "HUMAN GENE THERAPY 2014,25: 730-739"), which is considered by the invention that after immunization by the Her2/neu369-377 antigen polypeptide, a specific T cell clone aiming at the Her2/neu369-377 polypeptide will proliferate dominantly and thus cannot represent a specific T cell population which naturally exists in an in vivo T cell bank (reporteire) and can recognize the Her2/neu369-377 polypeptide antigen presented by target cells. The present invention also does not take The means of inducing polypeptide-specific T cells from HLA-A2-negative peripheral blood by other groups (see The Journal of Immunology,2010,184: 1617-1629), although allo-T cells recognizing The Her2/neu369-377 polypeptide antigen are more readily available from allogeneic PBMC, which also increases allogenic responses resulting from cross-recognition of other polypeptides presented by HLA-A2 molecules by T cells.

Based on the above concept, the present invention provides an isolated T cell receptor comprising at least one of an α chain and a β chain, both comprising a variable region and a constant region, characterized in that said T cell receptor is capable of specifically recognizing the antigen Her2/neu expressed by tumor cells, and the amino acid sequence of said variable region of said α chain has at least 98%, preferably at least 98.5%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID No. 19, and the amino acid sequence of said variable region of said β chain has at least 98%, preferably at least 98.5%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID No. 20, provided that the effects of the present invention are not significantly affected. It is also preferred that the amino acid sequence of the variable region of the alpha chain is shown in SEQ ID NO. 19 and the amino acid sequence of the variable region of the beta chain is shown in SEQ ID NO. 20.

The variable regions of the TCR alpha and beta chains, used to bind the antigen polypeptide/major histocompatibility complex (MHC I), each include three hypervariable regions or Complementarity Determining Regions (CDRs), namely CDR1, CDR2, CDR 3. Wherein the CDR3 region is important for specific recognition of an antigen polypeptide presented by an MHC molecule. The TCR alpha chain is formed by recombining different V and J gene segments, and the beta chain is formed by recombining different V, D and J gene segments. Formed by recombination and combination of specific gene segmentsThe corresponding CDR3 region, as well as The palindrome of The binding region and The randomly inserted nucleotides (palindrome and random nucleotide additions) contribute to The specificity of TCR recognition for antigen polypeptides (see "immunology: The animal system in health and disease.5)^theditin, Chapter 4, The generation of The Lymphocyte antigen receptors "). The MHC class I molecules include human HLA. The HLA includes: HLA-A, B, C.

More particularly, said T cell receptor is capable of specifically recognizing said epitope polypeptide of antigen Her2/neu presented by HLA-A2 molecule. The amino acid sequence of the antigen Her2/neu is shown as SEQ ID NO. 17. Preferably, the epitope polypeptide comprises Her2/neu369-377 as shown in SEQ ID NO: 18. HLA-a2 alleles expressed by HLA-a2 positive cells include HLA-a x 0201, 0202, 0203, 0204, 0205, 0206, and 0207. Preferably, the HLA-a2 molecule is HLA-a x 0201.

In one embodiment, the epitope polypeptide of antigen Her2/neu is the Her2/neu369-377 polypeptide (SEQ ID NO: 18). In other embodiments, the epitope polypeptide of antigen Her2/neu is an epitope polypeptide having 4-9 consecutive identical amino acids (e.g., 4, 5,6, 7, 8, or 9 consecutive identical amino acids) as the Her2/neu369-377 polypeptide, and these polypeptides are 8-11 amino acids in length. For example, in one embodiment, the epitope polypeptide of antigen Her2/neu is the Her2/neu 373-382 polypeptide (SEQ ID NO: 25).

Preferably, the maximum half-reactive polypeptide concentration for the T cell receptor to recognize the Her2/neu369-377 polypeptide is between 1.0-10ng/ml (e.g., between 3.0-8.0ng/ml, 5.0-7.0 ng/ml). In one embodiment of the invention, the maximum semi-reactive polypeptide concentration is about 6.9 ng/ml. The term "maximum half-reactive polypeptide concentration" refers to the concentration of polypeptide required to induce a T cell response that is 50% of the maximum. It has been reported that the maximum half-response polypeptide concentration of specific T cells against the Cytomegalovirus (CMV) antigen CMV pp65(495-503) polypeptide is between 0.1 and 1ng/ml, whereas this TCR is considered to have a high affinity for the CMV antigen polypeptide (see the literature "Journal of immunological methods 2007; 320: 119-131"). In the present invention, the T cell receptor has moderate affinity for the Her2/neu antigen, thereby avoiding off-target toxicity that may be associated with high affinity.

Exogenous TCR alpha and beta chains expressed by T cells may be mismatched with the alpha and beta chains of the T cell's own TCR, which not only dilutes the expression of the correctly paired exogenous TCR, but also makes the antigenic specificity of the mismatched TCR unclear, thus potentially risking recognition of the autoantigen, and therefore the constant regions of the TCR alpha and beta chains are preferably modified to reduce or avoid the mismatch.

In one embodiment, the constant region of the alpha chain and/or the constant region of the beta chain is derived from a human; preferably, the present inventors have found that the constant region of the alpha chain can be replaced, in whole or in part, by homologous sequences from other species, and/or the constant region of the beta chain can be replaced, in whole or in part, by homologous sequences from other species. More preferably, the other species is mouse. Such substitutions may increase the amount of TCR expression in the cell and may further improve the specificity of the Her2/neu antigen for cells modified by the TCR.

The constant region of the alpha chain may be modified with one or more disulfide bonds, and/or the constant region of the beta chain may be modified with one or more disulfide bonds, for example 1 or 2.

In a specific embodiment, two differently modified TCRs were prepared, Her2TCR-1B5-dis by adding a disulfide bond to the TCR constant region by point mutation, as described in the references Cancer res.2007apr 15; 67(8) 3898-903 ", which is incorporated herein by reference in its entirety. Her2TCR-1B5-mC is a replacement of the corresponding human TCR constant region sequence with a mouse TCR constant region sequence as described in the reference Eur.J.Immunol.200636: 3052-3059, which is incorporated herein by reference in its entirety.

In specific embodiments, the amino acid sequence of the alpha chain is set forth in SEQ ID NOs:2, 6, or 10, and the amino acid sequence of the beta chain is set forth in SEQ ID NOs:4, 8, or 12.

Wherein, for the alpha chain with the amino acid sequence shown as SEQ ID NO. 2, the sequence is the original human sequence; for the alpha chain with the amino acid sequence shown as SEQ ID NO. 6, 1 disulfide bond is modified in the constant region; for the alpha chain with the amino acid sequence shown in SEQ ID NO. 10, the constant region is replaced by a murine constant region.

Wherein, for the beta chain with the amino acid sequence shown as SEQ ID NO. 4, the sequence is the original human sequence; for the beta chain with the amino acid sequence shown as SEQ ID NO. 8,1 disulfide bond is modified in the constant region; for the beta chain with the amino acid sequence shown as SEQ ID NO. 12, the constant region is replaced by a murine constant region.

In a specific embodiment, the amino acid sequence of the α chain of the TCR is set forth in SEQ ID NO. 2 and the amino acid sequence of the β chain is set forth in SEQ ID NO. 4. In another embodiment, the amino acid sequence of the α chain of the TCR is set forth in SEQ ID NO. 6 and the amino acid sequence of the β chain is set forth in SEQ ID NO. 8. In yet another embodiment, the amino acid sequence of the α chain of the TCR is set forth in SEQ ID NO. 10 and the amino acid sequence of the β chain is set forth in SEQ ID NO. 12.

In other specific embodiments of the invention, the α chain of the TCR has an amino acid sequence resulting from the substitution, deletion, and/or addition of one or more amino acids in the amino acid sequence set forth in

SEQ ID NOs

2, 6, or 10; for example, the alpha chain has at least 90%, preferably at least 95%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NOs:2, 6 or 10.

In other specific embodiments of the invention, the β chain of the TCR has an amino acid sequence resulting from substitution, deletion, and/or addition of one or more amino acids in the amino acid sequence set forth in

SEQ ID NOs

4, 8, or 12; for example, the beta strand has at least 90%, preferably at least 95%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NOs:4, 8, or 12.

The α and/or β chains of the inventive TCR may also be terminally (e.g., C-terminally) linked to other functional sequences, such as functional region sequences of co-stimulatory signals CD28, 4-1BB, and/or CD3 zeta.

The invention also provides an isolated nucleic acid encoding a T cell receptor comprising a coding sequence for at least one of the alpha and beta chains of the T cell receptor, the alpha chain coding sequence and the beta chain coding sequence both comprise a variable region coding sequence and a constant region coding sequence, characterized in that the T cell receptor can specifically recognize the antigen Her2/neu expressed by tumor cells, and the alpha chain variable region encoding sequence encodes an amino acid sequence having an amino acid sequence identical to SEQ ID NO:19, is at least 98%, preferably at least 98.5%, more preferably at least 99%, the amino acid sequence coded by the beta-chain variable region coding sequence has the amino acid sequence similar to that of SEQ ID NO:20, is at least 98%, preferably at least 98.5%, more preferably at least 99%, as long as the effect of the present invention is not significantly affected. Also preferably, the alpha chain variable region encoding sequence encodes the amino acid sequence shown as SEQ ID NO 19 and the beta chain variable region encoding sequence encodes the amino acid sequence shown as SEQ ID NO 20.

The nucleic acid may be DNA or RNA.

Preferably, the alpha chain variable region encoding sequence is shown in SEQ ID NO:21 and the beta chain variable region encoding sequence is shown in SEQ ID NO: 22.

Further specifically, said T cell receptor encoded by said nucleic acid is capable of specifically recognizing an epitope polypeptide of said antigen Her2/neu presented by HLA-A2 molecule.

In one embodiment, the epitope polypeptide of antigen Her2/neu is the Her2/neu369-377 polypeptide (SEQ ID NO: 18). In other embodiments, the epitope polypeptide of antigen Her2/neu is an epitope polypeptide having 4-9 consecutive identical amino acids (e.g., 4, 5,6, 7, 8, or 9 consecutive identical amino acids) as the Her2/neu369-377 polypeptide, and these polypeptides are 8-10 amino acids in length. For example, in one embodiment, the epitope polypeptide of antigen Her2/neu is the Her2/neu 373-382 polypeptide (SEQ ID NO: 25).

Preferably, the maximum half-reactive polypeptide concentration for the T cell receptor recognition of the Her2/neu369-377 polypeptide encoded by the nucleic acid is between 1.0-10ng/ml (e.g., between 3.0-8.0ng/ml, 5.0-7.0 ng/ml). In one embodiment of the invention, the maximum semi-reactive polypeptide concentration is about 6.9 ng/ml. In this case, the T cell receptor has moderate affinity for the Her2/neu antigen, avoiding off-target toxicity that may be associated with high affinity.

In one embodiment, the constant region of the alpha chain and/or the constant region of the beta chain is derived from a human; preferably, the alpha chain constant region coding sequence is replaced in whole or in part by a homologous sequence from another species and/or the beta chain constant region coding sequence is replaced in whole or in part by a homologous sequence from another species. More preferably, the other species is mouse. Such substitutions may increase the amount of TCR expression in the cell and may further improve the specificity of the Her2/neu antigen for cells modified by the TCR.

The alpha chain constant region coding sequence may comprise one or more disulfide bond coding sequences, and/or the beta chain constant region coding sequence may comprise one or more disulfide bond coding sequences.

In specific embodiments, the alpha chain coding sequence is set forth in SEQ ID NOs:1, 5, or 9 and the beta chain coding sequence is set forth in SEQ ID NOs:3, 7, or 11.

Wherein, for the alpha chain with the coding sequence shown as SEQ ID NO. 1, the sequence is the original human sequence; for the alpha chain with the coding sequence shown as SEQ ID NO. 5, the alpha chain is modified with 1 disulfide bond in the constant region; for the alpha chain whose coding sequence is shown in SEQ ID NO. 9, the constant region was replaced with a murine constant region.

Wherein, for the beta chain with the coding sequence shown as SEQ ID NO. 3, the sequence is the original human sequence; for the beta chain with the coding sequence shown as SEQ ID NO. 7, the beta chain is modified with 1 disulfide bond in the constant region; for the beta-strand whose coding sequence is shown in SEQ ID NO. 11, the constant region was replaced with a murine constant region.

In a specific embodiment, the coding sequence for the α chain of the TCR is shown in SEQ ID NO. 1 and the coding sequence for the β chain is shown in SEQ ID NO. 3. In another embodiment, the coding sequence for the α chain of the TCR is shown in SEQ ID NO. 5 and the coding sequence for the β chain is shown in SEQ ID NO. 7. In yet another embodiment, the coding sequence for the α chain of the TCR is shown in SEQ ID NO 9 and the coding sequence for the β chain is shown in SEQ ID NO 11.

In another embodiment, the α chain coding sequence and the β chain coding sequence are linked by a coding sequence for a cleavable linker polypeptide, which increases the expression of the TCR in the cell. The term "cleavable linked polypeptide" means that the polypeptide functions as a link and can be cleaved by a specific enzyme, or that a nucleic acid sequence encoding the polypeptide is translated by ribosome skipping (ribosome skiping) so that the polypeptides linked by it are separated from each other. Examples of cleavable linker polypeptides are known in the art, such as the F2A polypeptide, the F2A polypeptide sequence including, but not limited to, the F2A polypeptide from picornavirus, and similar class 2A sequences from other viruses. In addition, the cleavable linker polypeptide also includes a standard four amino acid motif (cationic amino acid motif), i.e., the R-X- [ KR ] -R amino acid sequence, which is cleaved by Furin enzyme. The TCR encoded by this embodiment is a single chain chimeric T cell receptor in which upon completion of expression, the cleavable linker polypeptide that links the α and β chains is cleaved by a specific enzyme in the cell, thereby forming free α and β chains in equal amounts.

The α and β chains that make up the single-chain chimeric TCR may also be replaced, in whole or in part, by homologous sequences from other species, as described above, and/or modified (encoded) with one or more disulfide bonds.

In specific embodiments, the nucleic acid has the sequence set forth in SEQ ID NOs:13, 15, or 23.

Preferably, the nucleotide sequence of the nucleic acid is codon optimized to increase gene expression, protein translation efficiency, and protein expression, thereby enhancing the ability of the TCR to recognize an antigen. Codon optimization includes, but is not limited to, modification of the translation initiation region, alteration of mRNA structural segments, and use of different codons encoding the same amino acid.

In other embodiments, the sequence of the TCR-encoding nucleic acid described above can be mutated, including removal, insertion, and/or substitution of one or more amino acid codons, such that the function of the expressed TCR to recognize the Her2/neu antigen is unchanged or enhanced. For example, in one embodiment, conservative amino acid substitutions are made, including the substitution of one amino acid in the variable region of the α and/or β chains of the TCR with another amino acid that is similar in structural and/or chemical properties. The term "similar amino acids" refers to amino acid residues having similar properties of polarity, electrical charge, solubility, hydrophobicity, hydrophilicity, and the like. The mutated TCR still has the biological activity of recognizing the Her2/neu antigen polypeptide presented by the target cell. In another embodiment, TCR maturation (TCR mapping) modifications are performed, i.e., involving the removal, insertion and/or substitution of amino acids from the complementarity determining region 2(CDR2) and/or CDR3 regions in the variable regions of the α and/or β chains of the TCR described above, thereby altering the affinity of the TCR to bind the Her2/neu antigen.

The invention also provides an isolated mRNA transcribed from the DNA according to the invention.

The invention also provides a recombinant expression vector comprising a nucleic acid (e.g., DNA) according to the invention, and/or a complementary sequence thereof, operably linked to a promoter.

Preferably, in the recombinant expression vector, the DNA of the present invention is suitably operably linked to a promoter, an enhancer, a terminator and/or a polyA signal sequence.

The combination of the above-mentioned acting elements of the recombinant expression vector of the present invention can promote transcription and translation of DNA and enhance the stability of mRNA.

The basic backbone of a recombinant expression vector can be any known expression vector, including plasmids or viruses, viral vectors including, but not limited to, for example, retroviral vectors (the viral prototype is Moloney Murine Leukemia Virus (MMLV)) and lentiviral vectors (the viral prototype is human immunodeficiency type I virus (HIV)). Recombinant vectors expressing the TCRs of the invention can be obtained by recombinant DNA techniques conventional in the art.

In one embodiment, expression of the α chain and β chain genes on the recombinant expression vector can be driven by two different promoters, including various known types, such as strongly expressed, weakly expressed, inducible, tissue-specific, and differentiation-specific promoters. The promoter may be of viral or non-viral origin, such as the CMV promoter, the promoter on the LTR of MSCV, the EF 1-alpha promoter, and the PGK-1 promoter. The driving directions of the two promoters can be the same direction or opposite directions.

In another embodiment, the expression of the alpha and beta chain genes on the recombinant expression vector may be driven by the same promoter, for example in the case of a single-chain chimeric T cell receptor, the nucleotide sequence of the alpha chain and the nucleotide sequence of the beta chain are linked by a Furin-F2A polypeptide coding sequence.

In other embodiments, the recombinant expression vector may comprise coding sequences for other functional molecules in addition to the alpha and beta chain genes. One embodiment includes the expression of an autofluorescent protein (such as GFP or other fluorescent proteins) for in vivo follow-up imaging. Another embodiment includes expression of an inducible suicide gene system, such as inducible expression of the herpes simplex virus-thymidine kinase (HSV-TK) protein, or inducible expression of the Caspase 9(iCasp9) protein. Expression of these "safety-switch molecules" (safety-switch) may increase the safety of in vivo use of cells modified with the TCR genes of the invention. Another embodiment includes the expression of the human CD8 gene, including the expression of CD8 alpha chain and beta chain, alone or in combination, and the ability to specifically recognize Her2/neu antigen can be enhanced by the expression of CD8 molecules in cells modified with the TCR genes described herein, or by making CD8 negative T cells (e.g., CD4⁺T helper cells) gain the ability to specifically recognize Her2/neu antigen. Another embodiment includes expression of a human chemokine receptor gene, such as CCR2, which binds to a corresponding chemokine ligand highly expressed in tumor tissue, thereby increasing the homing of cells modified with the TCR gene of the invention into tumor tissue.

The invention also provides a T cell receptor-modified cell whose surface is modified with a T cell receptor according to the invention, wherein the cell comprises a T cell progenitor or precursor thereof, an NKT cell, or a T cell line.

The term "modification" in the "modification of T cell receptor" means that the T cell receptor of the present invention is expressed in a cell by gene transfection, that is, the T cell receptor is anchored to the cell membrane of the modified cell via a transmembrane region and has a function of recognizing an antigen polypeptide/MHC complex.

The invention also provides a method of making a T cell receptor modified cell according to the invention, comprising the steps of:

1) providing a cell;

2) providing a nucleic acid encoding a T cell receptor of the invention;

3) transfecting the nucleic acid into the cell.

The cells in step 1) can be derived from mammals, including human, dog, mouse, rat and transgenic animals thereof. The cells may be autologous or allogeneic. The allogeneic cells include cells from an isozygotic twin, allogeneic stem cells, genetically engineered allogeneic T cells.

The cells in step 1) comprise primitive T cells or precursor cells thereof, NKT cells or T cell lines. The term "naive T cells" refers to mature T cells in peripheral blood that have not been activated by the corresponding antigen. These cells can be isolated by methods known in the art. For example, T cells can be obtained from different tissue organs, including peripheral blood, bone marrow, lymphoid tissue, spleen, umbilical cord blood, tumor tissue. In one embodiment, the T cells may be derived from Hematopoietic Stem Cells (HSCs), including from bone marrow, peripheral blood, or cord blood, isolated by a stem cell marker molecule such as CD 34. In one embodiment, the T cell may be derived from induced pluripotent stem cells (iPS cells) by introducing a specific gene or a specific gene product into a somatic cell, and inducing differentiation into a T cell or a precursor cell thereof in vitro after the somatic cell is transformed into a stem cell. T cells can be obtained by a common method such as density gradient centrifugation, examples of which include Ficoll or Percoll density centrifugation. One embodiment is the use of apheresis or leukapheresis (leukaphe)resis) from peripheral blood. One embodiment is to label a specific cell population with an antibody and then separate it by magnetic beads (e.g., by magnetic bead separation)

System (Miltenyi Biotec)), or means of flow cytometric separation to obtain enriched CD8⁺Or CD4⁺T cells.

Preferably, the T cell precursor cells are hematopoietic stem cells. The encoding gene of the TCR provided by the invention can be directly introduced into hematopoietic stem cells, and then the hematopoietic stem cells are transferred into a body and further differentiated into mature T cells; the encoding gene may also be introduced into T cells differentiated into maturation from hematopoietic stem cells under specific conditions in vitro.

The cells can be resuspended in a cryopreservation solution and stored in liquid nitrogen. Common cryopreservation solutions include, but are not limited to, solutions comprising 10% (v/v) DMSO and 90% (v/v) human serum or fetal bovine serum. The cells were frozen at-80 ℃ at a reduced temperature of 1 ℃ per minute and then stored in the gas phase portion of a liquid nitrogen tank. Other methods for cryopreservation are to directly put the cells in the cryopreservation solution into-80 ℃ or liquid nitrogen for cryopreservation.

The nucleic acid in the step 2) is the nucleic acid according to the invention, and comprises the DNA and the RNA.

The transfection includes physical, biological and chemical means. The physical method is to introduce the TCR gene into cells in the form of DNA or RNA by calcium phosphate precipitation, liposome, microinjection, electroporation, gene gun, etc. There are commercially available Instruments including electrotransferases (e.g., Amaxa Nucleofector-II (Amaxa Biosystems, Germany), ECM 830(BTX) (Harvard Instruments, USA), Gene Pulser II (BioRad, USA), and Multipotator (Eppendort, Germany.) biologically by introducing TCR genes into cells via DNA or RNA vectors, retroviral vectors (e.g., gamma retroviral vectors) are common tools for transfecting and inserting foreign Gene fragments into animal cells (including human cells), and other viral vectors are derived from lentiviruses, poxviruses, herpesviruses, adenoviruses, and adenovirus-related viruses, etc. chemically, polynucleotides are introduced into cells, including colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, microbeads, micelles, and liposomes, whatever the TCR Gene is introduced into cells, whether the desired Gene is introduced into the target cell or not is analyzed by various detection methods, the detection method includes common molecular biological methods (such as Southern blot and Northern blot, RT-PCR, PCR and the like), or common biochemical methods (such as ELISA and Western blot), and the method mentioned in the present invention.

Preferably, the transfection is performed by a retroviral vector or a lentiviral vector.

The culture of the cells after transfection can be carried out by their respective conventional methods and conditions according to the actual application. For example, in vitro expansion can be achieved after co-activation of T cells by the TCR/CD3 complex on the surface, and a co-stimulatory molecule (e.g., CD 28). The stimuli (e.g., antibodies against TCR, CD3 or CD28) that activate TCR, CD3 and CD28 can be adsorbed onto the surface of the culture vessel, or co-cultured (e.g., magnetic beads), or can be added directly to the cell culture medium for co-culture. Another embodiment is the co-culture of T cells with feeder cells expressing a co-stimulatory molecule or corresponding ligand, including but not limited to HLA-A2, β 2-microglobulin, CD40, CD83, CD86, CD127, 4-1 BB.

According to a typical method for in vitro culture of mammalian cells, T cells are cultured and expanded under appropriate culture conditions. For example, cells can be passaged when they reach a confluent state (confluency) of 70% or more, typically by changing fresh culture medium for 2 to 3 days. When the number of cells reached a certain number, the cells were used directly, or frozen as described above. The in vitro culture time may be within 24 hours, or as long as 14 days or more. The frozen cells can be used for the next step after being thawed.

In one embodiment, the cells may be cultured in vitro for hours to 14 days, or any number of hours in between. T cell culture conditions include the use of basal media including, but not limited to, RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo. Other conditions required for cell survival and proliferation include, but are not limited toIn the use of serum (human or fetal bovine serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGF-beta and TNF-a, other culture additives (including amino acids, sodium pyruvate, vitamin C, 2-mercaptoethanol, growth hormones, growth factors). The cells may be subjected to appropriate culture conditions, e.g., the temperature may be at 37 ℃, 32 ℃, 30 ℃ or room temperature, and the atmospheric conditions may be, e.g., 5% CO₂Of the air of (2).

The invention also provides the use of a T cell receptor modified cell according to the invention for the preparation of a medicament for the treatment or prevention of a tumour and/or cancer.

The tumor and/or cancer is antigen Her2/neu positive and is HLA-a2 positive, including but not limited to breast, ovarian, gastric, esophageal, intestinal, pancreatic, bladder, renal, prostate, cervical, endometrial, salivary gland, skin, lung, bone, and brain cancer.

The invention also provides the use of a T cell receptor modified cell according to the invention in the manufacture of a medicament for detecting a tumour and/or cancer in a host.

In one embodiment of the present invention, a sample of tumor and/or cancer cells taken from a host may be contacted with the T cell receptor-modified cells of the present invention at a concentration that will determine whether the tumor and/or cancer is HLA-A2 positive or HLA-A2 negative, and whether the antigen Her2/neu is expressed, based on the extent of the reaction.

The invention also provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the T cell receptor modified cell as an active ingredient, and a pharmaceutically acceptable adjuvant.

The pharmaceutical composition preferably comprises a total dose per patient per course of treatment ranging from 1 × 10³-1×10⁹The T cell receptor modified cells per Kg body weight, including any number of cells between the two endpoints. Preferably, each course of treatment is 1-3 days, administered 1-3 times per day. Can be used for treating a patient according to actual conditions and needsOr multiple courses of treatment.

The medicinal auxiliary materials comprise medicinal or physiological carriers, excipients, diluents (including physiological saline and PBS solution), and various additives, including saccharides, lipids, polypeptides, amino acids, antioxidants, adjuvants, preservatives and the like.

The pharmaceutical compositions may be administered by a suitable route of administration, which is suitable for administration intraarterially, intravenously, subcutaneously, intradermally, intratumorally, intralymphatically, subarachnoid, intramedularly, intramuscularly and intraperitoneally.

The invention also provides a method of treating a tumor and/or cancer comprising administering to a patient having a tumor and/or cancer a T cell receptor modified cell according to the invention.

The tumor and/or cancer is positive for the antigen Her2/neu and is HLA-a2 positive, including but not limited to breast, ovarian, gastric, esophageal, intestinal, pancreatic, bladder, renal, prostate, cervical, endometrial, salivary gland, skin, lung, bone, and brain cancer.

The T cell receptor modified cells are preferably administered in a total dose ranging from 1X 10 per patient per course of treatment³-1×10⁹One cell/Kg body weight. Preferably, each course of treatment is 1-3 days, administered 1-3 times per day. The patient may be treated for one or more courses of treatment as is practical and desirable.

The T cell receptor modified cells may be administered by a suitable route of administration, which is suitable for administration via arterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, subarachnoid, intramedullary, intramuscular and intraperitoneal routes of administration.

The T cell receptor modified cells can eliminate tumor cells expressing Her2/neu antigen after entering into a treated object, and/or change the microenvironment of tumor tissues to induce other anti-tumor immune responses.

The invention also provides the use of the isolated T cell receptor to detect the proliferation or survival of the TCR-T cells in a patient treated with the TCR-modified T cells (i.e., TCR-T cells) to study drug metabolism and to understand the efficacy and toxicity of the TCR-T cells. Specifically, the TCR sequence can be used as a primer to detect the number of TCR-T cells carrying the TCR in vivo by PCR. The application requires a smaller amount of cells and is more sensitive than the method in which the fluorescently labeled HLA/polypeptide complex multimer is stained and then analyzed by flow cytometry.

The present invention will be further explained or illustrated below by way of examples, which should not be construed as limiting the scope of the invention.

Examples of the present invention

Unless otherwise indicated, the experimental procedures used in the following examples were performed using conventional experimental protocols, procedures, materials and conditions in the field of biotechnology.

Hereinafter, unless otherwise specified, the percentage concentration (%) of each agent refers to the volume percentage concentration (% (v/v)) of the agent.

Materials and methods

Cell lines: the cell line used for the preparation of lentiviral particles was 293T cells (ATCC CRL-3216). The T cell lines used to test TCR phenotype and function were Jurkat cells (clone E6-1, ATCC TIB-152) and J.RT3-T3.5 cells (ATCC TIB-153). The cell line used to present antigen polypeptides is T2 cells (174xCEM. T2, ATCC CRL-1992). Tumor cell lines for detecting TCR function are human colon cancer colo205 cells (ATCC CCL-222) and Caco-2 cells (ATCC HTB-37), human breast cancer MDA-MB-231 cells (ATCC HTB-26), human ovarian cancer SK-OV-3 cells (ATCC HTB-77), human lymphoma cells Bjab (ACC-757-DSMZ), human lung cancer H647 cells (ATCC CRL-5824) and H1355 cells (ATCC CRL-5865). The cell lines were maintained in RPMI-1640 complete medium (Lonza, cat #12-115F) to which 10% bovine serum FBS (ATCC 30-2020), 2mmol/L L-glutamic acid, 100. mu.g/ml penicillin and 100. mu.g/ml streptomycin were added.

Peripheral blood: human peripheral blood products from healthy donors used in the experiments were obtained from Pacific blood centers in san Francisco (#1PBMC and #2PBMC are the Trima residual cell fraction # R32334 and # R33941 from the Apheresis method collection kit, respectively).

In vitro induction of Her2/neu369-377 specific killer T Cells (CTL): mononuclear Cells (PBMC) were obtained after subjecting peripheral blood to Ficoll-Paque Premium (Sigma-Alorich Co., cat # GE-17-5442-02) density gradient centrifugation (. times.400 g) for 30 minutes. The cell matching type is determined to be HLA-A0201 by staining the cell with fluorescein FITC labeled anti-HLA-A2 antibody (Biolegend, cat #343303) to detect HLA-A2 phenotype, extracting RNA of positive cells after flow cytometry analysis, performing reverse transcription to cDNA, cloning the cDNA onto a vector, and performing HLA gene sequencing analysis. HLA-A2 positive PBMC cells were cultured in culture wells of a 24-well culture plate in the above RPMI-1640 complete medium. The Her2/neu369-377 polypeptide (Her2-E75, synthesized with Peptide2.0, 10. mu.g/ml in DMSO) was added at a concentration of 1. mu.g/ml to 2X 10E6/ml PBMC per well. Placing in 5% CO₂After culturing in an incubator at 37 ℃ for 16 to 24 hours, the following cytokines, human IL-2 (Peprotech, cat #200-02)100IU/ml, human IL-7(Peprotech, cat #200-07)5ng/ml, and human IL-15(Peprotech, cat #200-15)5ng/ml, were added. Cultured T cells were antigen restimulated for 10 to 14 days: 10e6 cultured cells per well were added to a 24-well plate, 2X 10e6 HLA-A2 positive autologous PBMC cells treated with 25. mu.g/ml mitomycin C (Santa Cruz Biotechnology, cat # SC-3514) for 2 hours were added as feeder cells, Her2/neu369-377 polypeptide was added to each well to a final concentration of 1. mu.g/ml, and IL-2100IU/ml, IL-75 ng/ml, IL-155 ng/ml were added after overnight incubation. After two rounds of antigen stimulation and restimulation as described above, expanded T cells were collected for phenotypic analysis and T cell cloning.

Flow cytometry analysis and single cell isolation: the T cell phenotype of the Her2/neu 369-377-specific TCR was analyzed by flow cytometry. The cells to be detected were collected in 1.5ml tubes (cell number about 10e 5), and 1ml of DPBS solution (2.7mM KCl, 1.5mM KH)₂PO₄，136.9mM NaCl,8.9mM Na₂HPO₄·7H₂O, pH 7.4) and placed in 100. mu.l of DPBS containing 1% calf serum, 5. mu.l of fluorescein APC-labeled anti-human CD8 antibody (Biolegend, cat #300912) and 10. mu.l of fluorescein PE-labeled Her2-E75/HLA-A2 tetramerThe bodies (Her2-E75 tetramer, MBL International Co., cat # T01014) or Her2-E75/HLA-A2 pentamer (Her2-E75 pentamer, Proimmunee, cat # F214-2A-D) were incubated on ice for 30 minutes, washed twice with DPBS solution and resuspended in 100. mu.l PBS solution for flow cytometry analysis. The flow cytometer was MACSQuant Analyzer 10(Miltenyi Biotec Co., Ltd.) and the results were analyzed by Flowjo software (Flowjo Co., Ltd.). T cell clones were obtained by single cell isolation using a flow cytometer (FACS sorter) and cultured. PBMCs stimulated with Her2/neu369-377 polypeptide antigen were stained with an APC-labeled anti-human CD8 antibody and a PE-labeled Her2-E75/HLA-A2 pentamer, followed by flow cytometric isolation (model: Sony cell sorter SH 800). Single CD8⁺Her2-E75/HLA-A2 pentamer⁺After the cells were sorted into individual culture wells of a 96-well culture plate, 10e5 cells per well of HLA-A2 positive autologous PBMC cells treated with mitomycin C for 2 hours at 25. mu.g/ml were added, and after overnight culture with Her2/neu369-377 polypeptide at 1. mu.g/ml, RPMI-1640 complete medium containing IL-2100IU/ml, IL-75 ng/ml and IL-155 ng/ml was added. And (4) replacing fresh culture solution containing the cytokine every 3-4 days, and observing whether T cell clone grows under a microscope. The expanded T cells are harvested, antigen restimulation is performed as described above to obtain sufficient numbers of cells, phenotypic or functional assays are performed, and RNA is extracted for cloning of the TCR gene.

And (3) detecting T cell functions: the function of the T cell line specifically TCR-modified by the Her2/neu369-377 polypeptide was examined by expression of CD69 on the surface of T cells. 10e 5T cells transfected with TCR genes and 10e 5T 2 cells were added to each well of a 96-well plate, and mixed culture was performed in 100. mu.l/well of RPMI-1640 complete medium, each test group being a duplicate well. Then adding Her2/neu369-377 polypeptide with different concentrations (1. mu.g/ml, 0.5. mu.g/ml, 0.1. mu.g/ml, 0.05. mu.g/ml, 0.01. mu.g/ml, 0.005. mu.g/ml, 0.001. mu.g/ml and 0. mu.g/ml respectively) and placing in 5% CO₂The cells were incubated overnight in an incubator at 37 ℃. Cells were collected and suspended in DPBS + 1% FBS solution, stained with APC-labeled anti-human CD69 antibody and subjected to flow cytometry. T cells expressing CD69 after stimulation with Her2/neu369-377 antigen are considered to carry and express Her2-neu369-377 specific TCRs. The function of the Her2/neu 369-377-specific CTL clones and TCR gene-transfected primary T cells in PBMCs was determined by measuring gamma interferon secreted in the cell supernatant after antigen stimulation. PBMC cells of Her2/neu 369-377-specific CTL clone or transfected TCR gene are mixed and cultured with target cells in a 96-well plate in 100 mu.l/well of RPMI-1640 complete culture medium, the target cells are T2 cells added with 10-fold dilution concentration of Her2/neu369-377 polypeptide or various tumor cell strain cells, and each test group is a multi-well. In the antibody function blocking assay, 10. mu.g/ml of anti-human CD8 antibody (Biolegend, cat #300912) or anti-HLA-ABC antibody (w6/32 clone, Biolegend, cat #311402) was added to the cell culture wells at the same time, and the cells were placed in 5% CO₂The cells were incubated overnight in an incubator at 37 ℃. Cell supernatants were collected at 18-24 hours and assayed for IFN- γ using a human IFN- γ ELISA Read-set-Go kit (eBioscience, cat # 88-7316).

Obtaining a monoclonal TCR gene: total RNA was purified from T cell clones using the Zymo Quick-RNA Microprep kit (Zymo Research, cat # R1050), and cDNA was obtained using the Smarter RACE 5 '/3' kit using this as a template (Takara Bio, USA, cat # 634858). PCR was performed with 5 ' -CDS primer and TCR beta chain 3 ' primer 5'-GCCTCTGGAATCCTTTCTCTTG-3' (SEQ ID NO:26) and alpha chain 3 ' primer 5'-TCAGCTGGACCACAGCCGCAG-3' (SEQ ID NO:27) to amplify TCR alpha and beta full sequence gene fragments, which were cloned into pRACE vector (Takara Bio, USA, cat #634858), respectively. The competent bacterium, Stellar (Takara Bio Inc., USA, cat #636763), was transformed and the plasmid was obtained and then sequenced.

Preparation of recombinant TCR Lentiviral expression vector: the viral vector for expressing the TCR is a replication-defective lentiviral vector comprising: GFP-expressing lentiviral vector pCDH-EF1 α -MCS- (PGK-GFP), available from System Biosciences (Cat # CD 811A-1); and a vector pCDH-EF1 alpha-MCS not expressing GFP, which is obtained by removing the PGK promoter and the GFP gene from the pCDH-EF1 alpha-MCS- (PGK-GFP) vector by a conventional technique in the art. Based on the obtained TCR Gene sequence, synthesizing TCR beta chain and alpha chain, F2A sequence which can be cut between the TCR beta chain and the alpha chain and the whole Gene sequence of the Furin enzyme cutting segment, linking to the multiple cloning site at the downstream of EF-1 alpha promoter of the carrier, and inserting the transcription sequence of TCR into TCR beta chain (without termination codon), Furin enzyme cutting segment, F2A segment and TCR alpha chain in turn (the method is shown in the literature' Gene ther.2008Nov; 15(21): 1411-. The vector expressing GFP is driven by the reverse PGK promoter. The vector not expressing GFP had the PGK promoter removed and the GFP fragment removed.

Preparation of recombinant TCR lentiviral particles: TCR lentiviral particles were obtained by transfecting 293T cells with Lipofectaine 3000 transfection reagent (Thermo Fisher Corp., cat # L3000001). 293T cells were prepared and transfected according to the manufacturer's instructions. Transfection was performed in 96-well plates by first preparing a liposome solution of the transfection plasmid using Opti-MEM 1 medium (Thermo Fisher, cat #51985091), adding the P3000reagent (P3000reagent) and the TCR lentiviral vector plasmid and the viral packaging plasmid of the pCDH system (SBI, cat # LV500A-1) to 250. mu.l of the medium according to the manufacturer's instructions, adding the Lipofectaine 3000reagent to 250. mu.l of the medium, mixing and incubating for 15 minutes, and adding 293T cell culture wells. 5% CO₂After culturing at 37 ℃ for 16 hours, the cells were cultured in DMEM medium containing 10% FBS (Thermo Fisher Co., cat #11965092) for 24 hours and 48 hours, and cell supernatants were collected, respectively, and after centrifugation at 2000g, virus particles obtained by filtration through a 0.4 μm filter were used for infecting the cells.

Recombinant TCR lentivirus transfects human T cells: frozen primary PBMC cells were thawed and cultured in RPMI-1640 complete medium for 24 hours, the dead cells were removed by Ficoll-Paque Premium density gradient centrifugation (. times.400 g) for 30 minutes, placed in 24-well plates and cultured at a cell concentration of 2 × 10e6/ml in 24-well plates treated with 2 μ g/ml anti-human CD3 antibody (Biolegend, OKT3 clone cat #317303) and 2 μ g/ml anti-human CD28 antibody (Biolegend, cat #302914) (100 μ l of DPBS solution containing the above-mentioned CD3 antibody and CD28 antibody was added to each well) for 24 hours, cultured for 48 hours, collected, resuspended in fresh recombinant TCR lentiviral particles and placed in 24-well plates, 4ng/ml polybrene (Sancta Cruz Biotechnology, cat # 134220) was added, centrifuged at 32 ℃ for 2 hours with 1000 g/ml IL-2100-75 IU/ml and IL-2100-75 ml/ml was added, The culture was continued after replacing half of the virus supernatant with the complete culture medium of IL-155 ng/ml RPMI-1640, and the culture medium containing the above cytokine was replaced every 3 days. Phenotypic and functional assays can be performed typically after 72 hours. Transfection of T cell lines was also performed as described above, and if the viral vector carries a GFP tag, GFP positive cells are typically observed under a fluorescent microscope 48 hours after transfection.

Example 1: induction of Her2/neu369-377 polypeptide specific killing T cells from peripheral blood of normal HLA-A2 positive donor

This example induced polypeptide-specific killer T cells from normal PBMC (#2PBMC) with low concentrations of Her2/neu369-377 polypeptide at 1. mu.g/ml by two in vitro stimulations, flow cytometry analysis and single cell isolation. The specific method is as described above. The results are as follows:

FIG. 1A right panel shows that 0.013% of lymphocytes are CD8 positive killer T cells that bind to the Her2/neu369 377/HLA-A2 pentamer (i.e., Her2-E75 pentamer), and control cells in the left panel that were not stimulated with the Her2 polypeptide did not show CD8 positive pentamer positive cells. The results indicate that the number of specific T cells recognizing the Her2/neu369-377 antigen polypeptide in the natural T cell bank is very small. Despite the small number, the T cells recognizing the Her2/neu369-377 polypeptide were clearly distinguished. In addition, according to the fluorescence intensity of the pentamer combined with Her2-E75, the positive cells comprise high-affinity T cells and low-affinity T cells. 453 CD8 positive pentamer positive cells were isolated by flow cytometry and cultured in monoclonals, and only one expanded T cell clone Her2CTL clone 1B5 (named Her2CTL 1B5) was obtained from the 453 isolated single T cells after two rounds of antigen polypeptide restimulation and cytokine amplification. FIG. 1B shows the right panel of this purified CD8⁺CTL clones bound Her2/neu 369-377/HLA-A2 tetramer (i.e., Her2-E75 tetramer). 5x10 per hole³Her2CTL 1B5 cell and 5x10 cell³Different concentrations of the Her2/neu369-377 antigen polypeptide presented by T2 cells (the Her2/neu369-377 antigen polypeptide is diluted 10 times from 1 mu g/ml so as to obtain different groups of final concentrations of 1 mu g/ml, 0.1 mu g/ml, 0.01 mu g/ml and 0.001 mu g/ml) mixedAfter incubation, IFN-gamma secreted by T cells in the supernatant was detected to determine the function of the T cell clone to specifically recognize the Her2/neu369-377 polypeptide. Tumor cell culture wells were filled with 5x10³After mixed culture of individual colo205 cells, IFN-gamma secreted by T cells in the supernatant was detected. The results in FIG. 1C show that T cell clone 1B5 was activated by antigenic polypeptide at a minimum concentration of 1ng/ml, and that the T cell recognition function was directly related (dose-dependent) to the concentration of antigenic polypeptide, indicating that this T cell clone specifically recognized the Her2/neu369-377 polypeptide presented by HLA-A2. More importantly, the T cell clone also identifies an HLA-A2 strain⁺Her2/neu⁺The colon cancer cell strain colo 205. Therefore, the T cell clone Her2TCR-1B5 not only can recognize Her-E75 polypeptide, but also can be activated by colo205 cells to secrete IFN-gamma.

Example 2: acquisition and characterization of the complete TCR sequence specific for the Her2/neu369-377 polypeptide

This example directly obtained from the Her2/neu369-377 polypeptide-specific CTL clone obtained in example 1 the complete TCR gene sequence comprising matching (i.e., the two chains together constitute a functional TCR recognizing the antigenic polypeptide) alpha and beta chains, encoding a TCR referred to as "Her 2TCR-1B 5". The amino acid sequence of the alpha chain of the TCR is shown as SEQ ID NO. 2, the coding sequence is shown as SEQ ID NO. 1, the amino acid sequence of the beta chain of the TCR is shown as SEQ ID NO. 4, and the coding sequence is shown as SEQ ID NO. 3. The TCR exists in a peripheral T cell bank of normal people positive to HLA-A2, and can not generate cross reaction on normal cells slightly expressing Her2/neu protein to cause autoimmune reaction. Specific detection methods are as described above. The results are as follows:

to test the antigenic specificity of the resultant TCR and its function, TCR α and β chain sequences were cloned into replication-defective lentiviral expression vectors. FIG. 2 shows a schematic diagram of the constructed TCR lentiviral vector fragment. The alpha chain and the beta chain are connected by a cleavable furin recognition fragment and a F2A polypeptide fragment. To follow the T cells transfected with the TCR lentiviral vector, the lentiviral vector (shown as "pCDH-EF 1 α -Her2TCR- (PGK-GFP) vector") was simultaneously expressing GFP, driven by a reverse PGK promoter (top panel of FIG. 2). Another expression vector (shown as "pCDH-EF 1. alpha. -Her2TCR vector" in the figure) (below FIG. 2) has the GFP and its promoter sequence removed. The vector length is reduced to increase the output of the lentivirus particles, and the mutual influence between two promoters is avoided, so that the expression amount of the TCR is increased.

The following sequences were ligated to the above-described vector expressing GFP and the vector not expressing GFP, respectively: i) the nucleotide sequences of TCR beta chain and alpha chain (SEQ ID NO:13) linked by a cleavable linking polypeptide with 1 disulfide bond added to the constant region (the corresponding TCR is Her2TCR-1B5-dis, the amino acid sequence is shown in SEQ ID NO: 14); ii) the nucleotide sequences of TCR beta chain and alpha chain (SEQ ID NO:15) which are linked by the cleavable connecting polypeptide and have constant regions replaced by mouse-derived sequences from human-derived sequences (the corresponding TCR is Her2TCR-1B5-mC, and the amino acid sequence is shown as SEQ ID NO: 16); iii) the original TCR beta and alpha chain nucleotide sequences (SEQ ID NO:23) linked by a cleavable linker polypeptide (the corresponding TCR is Her2TCR-1B 5-wt, the encoded amino acid sequence is shown in SEQ ID NO: 24); thus 6 recombinant lentiviral vectors were obtained:

1) her2TCR-1B5-dis recombinant lentiviral vector (carrying GFP);

2) her2TCR-1B5 dis w/o GFP recombinant lentiviral vector (not carrying GFP);

3) her2TCR-1B5-mC recombinant lentiviral vector (carrying GFP);

4) her2TCR-1B5-mC w/o GFP recombinant lentiviral vector (not carrying GFP).

5) Her2TCR-1B 5-wt recombinant lentiviral vector (carrying GFP);

6) her2TCR-1B 5-wt w/o GFP recombinant lentiviral vector (not carrying GFP).

After PCR amplification of each Her2TCR gene fragment, it was cloned downstream of the EF1- α promoter of each of the two lentiviral vectors described above (i.e., pCDH-EF1 α -MCS- (PGK-GFP) and pCDH-EF1 α -MCS): both the Her2TCR-1B5-dis and TCR-1B5-wt TCR fragments were amplified using 5 'primer 5'-AGAGCTAGCGAATTCAACATGGATACCTGGCTCGTATG-3'(SEQ ID NO:28) and 3' primer 5'-GTTGATTGTCGACGCCCTCAGCTGGACCACAGCCGCAG-3' (SEQ ID NO: 29). The TCR fragment of Her2TCR-1B5-mC was amplified using 5 'primer 5'-AGAGCTAGCGAATTCAACATGGATACCTGGCTCGTATG-3'(SEQ ID NO:30) and 3' primer 5'-GTTGATTGTCGACGCCCTCAACTGGACCACAGCCT-3' (SEQ ID NO: 31). PCR was carried out using Q5 high fidelity PCR kit (NEB, cat # M0543S) under 98 ℃ for 30 seconds, followed by 25 cycles of 98 ℃ for 10 seconds, 65 ℃ for 10 seconds, and 72 ℃ for 3 minutes. The obtained TCR fragment was cloned into the MCS region downstream of the EF 1a promoter of the pCDH-EF1 a-MCS- (PGK-GFP) vector or pCDH-EF1 a-MCS vector.

And preparing the constructed recombinant TCR lentiviral expression vector according to the method to obtain respective recombinant TCR lentiviral particles.

Two differently modified TCRs can be obtained in this way, Her2TCR-1B5-dis by adding a disulfide bond to the TCR constant region by point mutation, as described in Cancer res.2007apr 15; 67(8) 3898-903 ", which is incorporated herein by reference in its entirety. Her2TCR-1B5-mC is the replacement of the corresponding human TCR constant region sequence with a mouse TCR constant region sequence as described in the literature "Eur.J.Immunol.200636: 3052-3059", which is incorporated herein by reference in its entirety. FIG. 3A shows that a portion of GFP was present after transfection of T cell lines with lentiviruses carrying the Her2TCR-1B5TCR and GFP genes (i.e., the above-described Her2TCR-1B5-dis recombinant lentiviral vector and Her2TCR-1B5-mC recombinant lentiviral vector)⁺Cells could bind to Her2-E75 tetramer, suggesting that these lentivirus-transfected T cells could recognize Her2/neu369-377 polypeptide presented by HLA-A2. From the data obtained, 37.5% (13.2/(13.2+ 22). times.100%) of the transfected T cells (GFP) were calculated if the TCR constant region was increased by one disulfide bond⁺) The expressed TCR recognized Her2/neu polypeptide antigen (FIG. 3A, left panel). While the constant region of the TCR was replaced by the corresponding constant region of the mouse TCR, 60% (34.6/(34.6+ 23). times.100%) of the transfected T cells (GFP)⁺) The expressed TCR recognized Her2/neu polypeptide antigen (FIG. 3A, right panel). Compared with TCR obtained by modifying the exogenous TCR alpha chain and the exogenous TCR beta chain through disulfide bonds, hybrid TCR formed by replacing the exogenous TCR constant region with the corresponding mouse TCR constant region can further reduce the mismatching probability with the endogenous TCR. FIG. 3B shows that the constant region is conserved by mice regardless of whether J.RT-T3.5 cells expressing beta chain only or Jurkat cells expressing both alpha and beta chains are transfectedThe expression level of the exogenous TCR (Her2TCR-1B5-mC) obtained after the region sequence replacement is obviously higher than that of the TCR (Her2TCR-1B5-dis) with only one disulfide bond added in the constant region. FIG. 3C shows that T cell lines expressing exogenous Her2TCR-1B5 can be activated by Her2/neu369-377 polypeptide presented by T2 cells to express CD69, indicating that the TCR has the function of recognizing Her2/neu369-377 polypeptide antigen. The expression level of the exogenous TCR (Her2TCR-1B5-mC) obtained after the constant region is replaced by the mouse constant region sequence is increased, the specific recognition capability to the polypeptide antigen is obviously higher than that of the TCR (Her2TCR-1B5-dis) modified by the additional disulfide bond, and the exogenous TCR can be activated by the Her2/neu369-377 polypeptide with lower concentration. However, the minimum polypeptide concentration for activation of Her2TCR-1B5-mC expressing Jurkat cells was about 0.05. mu.g/ml, about 50 times higher than that for activation of Her2CTL 1B5 clones shown in FIG. 1C. This indicates that the Her2TCR-1B5TCR expressed by Jurkat cells has significantly lower ability (avidity) to recognize polypeptide antigens than CD8⁺Her2TCR-1B5TCR expressed on CTL. One reason for this is that Jurkat cells do not express the CD8 molecule, whereas CD8 plays an important ancillary role in the recognition function of Her2TCR-1B 5.

Example 3: normal peripheral blood T cells express specific TCR capable of recognizing Her2/neu369-377 polypeptide after Her2TCR-1B5-mC recombinant lentivirus transfection

To further verify whether the TCRs obtained by the present invention can be expressed in primary T cells and have the function of recognizing Her2/neu antigen polypeptide, peripheral blood T cells from two different normal donors, which are activated by CD3/CD28 antibody, are transfected by recombinant lentiviral particles carrying Her2TCR-1B5-mC gene (Her2TCR-1B5-mC recombinant lentiviral vector), and the cells are collected after 7 days for Her2-E75 tetramer staining. The specific method is as described above. The results are as follows:

FIG. 4A shows that lymphocytes from both donor peripheral blood mononuclear cells (PBMC #1 and PBMC #2, respectively) can bind to the Her2-E75 tetramer, indicating that Her2TCR-1B5-mC expressed by these cells can specifically recognize Her2/neu antigen polypeptide presented by HLA-A2. The results also show that most of the Her2-E75 tetramer positive cells (i.e., expressing Her2TCR-1B5-mC) were CD8⁺T killer cells, a small fraction of positive cellsIs a lymphocyte of CD8-, which is most likely CD4⁺The T helper cell of (1). CD8⁺The fluorescence intensity of CTL binding to Her2-E75 tetramer (# 1469 for Geom. mean in PBMC sample #2 and 1404 for Geom. mean in PBMC sample # 1) was also significantly greater than that of CD4⁺T cells (# 560 for Geom means in PBMC sample #2 and 504 for geom.means in PBMC sample # 1). If lentivirus infects CD8⁺And CD4⁺The transfection efficiency of T cells was the same, indicating that a portion was expressed in CD4⁺The exogenous Her2/neu 369-377-specific TCR on the cell was not able to bind the Her2-E75 tetramer, and even if it did, the affinity was lower than that expressed on CD8⁺Exogenous transfection of TCR on T cells. This further illustrates that transfected TCRs require the helper function of the CD8 molecule to bind effectively to the Her2/HLA-A2 complex.

After adding 10e5 TCR-transfected PBMC cells into each well of a 96-well plate and carrying out mixed culture with Her2/neu369-377 antigen polypeptide (Her2/neu 369-377 antigen polypeptide is diluted by 10 times from 0.1 mu g/ml so as to obtain different groups with final concentrations of 0.1 mu g/ml, 0.01 mu g/ml and 0.001 mu g/ml) presented by T2 cells (10 e5 per well), IFN-gamma secreted by T cells in supernatant is detected, so as to determine the function of the TCR-expressing PBMC cells for specifically recognizing the Her2/neu369-377 polypeptide. The target cells of the control group are T2 cells presenting 1 mu g/ml of EBV virus antigen polypeptide LMP 2426-434 capable of binding HLA-A2 molecules. FIG. 4B shows that PBMC expressing Her2TCR-1B5-mC can be activated by the Her2/neu369-377 antigen polypeptide presented by T2 cells to secrete IFN- γ, indicating that primary T cells expressing exogenous Her2TCR-1B5-mC can specifically recognize the Her2/neu369-377 polypeptide presented by HLA-A2 molecules. The ability to recognize antigenic polypeptides correlates with the amount of expression of exogenous TCR on T cells. It recognizes the largest half reaction (EC) of antigenic polypeptides₅₀) The polypeptide concentration was estimated to be about 6.9ng/ml (IC) by curve fitting₅₀Tool Kit, http:// www.ic50.tk /), although this response is less sensitive than the EC of the high affinity TCR recognizing foreign antigens such as viral antigens₅₀(EC₅₀About 10e-10M) (see documents "CANCER RESEARCH 1998, 58.4902-4908" and "HUMAN GENE THERAPY 2014,25: 730-Is within the TCR affinity range of (e.g., as described in the document "Eur J Immunol (2012)42: 3174-9").

FIG. 4C shows that the secretion of IFN-. gamma.by T cells was significantly inhibited by the addition of anti-human CD8 antibody when T cells were co-cultured with an antigen polypeptide presented by T2 cells (T2+ Her2-E75, i.e., Her2/neu369-377 polypeptide). This suggests that the CD8 molecule has a crucial role in the recognition of the Her2/neu369-377 antigen polypeptide by the exogenous TCR, and that the Her2TCR-1B5 of the invention is a CD8 function-dependent TCR.

Example 4: the Her2/neu369 377 polypeptide-specific TCR expressed by normal peripheral blood T cells after Her2TCR-1B5-mC recombinant lentivirus transfection can recognize HLA-A2⁺Her2/neu⁺Tumor cells

10e5 PBMCs transfected with Her2TCR-1B5-mC w/o GFP recombinant lentivirus (shown as #2 PBMCs transfected with Her2TCR-1B 5) or 10e 5TCR untransfected control #2 PBMCs were added to each well of a 96-well plate and cultured in admixture with 10e5 different tumor cell lines before the supernatant was assayed for secreted IFN-. gamma. The specific method is as described above. The results are as follows:

FIG. 5A shows that T cells expressing Her2TCR-1B5-mC can be both HLA-A2⁺Her2/neu⁺The tumor cell strain is activated and secretes IFN-gamma, and the tumor cell strain comprises colon cancer Colo205 cells, breast cancer MDA-MB-231 cells, colon cancer Caco-2 cells and lung cancer H1355 cells. And HLA-A2^-Her2/neu⁺SK-OV3 cells, lung cancer H647 cells, and HLA-A2 cells⁺Her2/neu^-The lymphoma Bjab cells in question did not activate T cells transfected with Her2TCR-1B5-mC TCR. It is shown that the Her2TCR-1B5-mC TCR can specifically recognize Her2/neu antigen presented by HLA-A2 on the surface of tumor cells. Control T cells from the same donor PBMC, cultured in parallel but not transfected with Her2TCR-1B5-mC, were not activated by the listed tumor cell lines, indicating that the response to tumor cells was not non-specific. FIG. 5B shows that T cells expressing Her2TCR-1B5-mC TCR (#2PBMC transfected with Her2TCR-1B 5) showed significant response to colo205 cells and recognition function was almost completely blocked by anti-CD8 antibodies and antibodies against HLA class molecules, further demonstrating recognition of tumor cellsThe effector cell of the Her2/neu antigen on the surface of the tumor cell is CD8⁺The specificity of the antigen recognition of killer T cells, which is dependent on the helper function of CD8, is consistent with the expression of Her2TCR-1B5-mC TCR when the Her2/neu369-377 polypeptide antigen presented by T2 cells is recognized.

Discussion of the related Art

The difference of response sensitivity of different tumor cell strains to specific T cells is probably related to the expression of different levels of Her2/neu antigen polypeptide/HLA-A2 complexes by the tumor cells and also related to the inhibition effect of the tumor cells on the different functions of the T cells. Although high affinity TCRs which specifically recognize the Her2/neu369-377 polypeptide can be obtained by in vitro induction of the Her2/neu369-377 polypeptide, these high affinity TCRs often fail to recognize the Her2/neu antigen presented by tumor cells (Cancer Res.1998; 58: 4902-4908. Cancer immunol.Immunother.2008; 57: 271-280). One reason may be that the configuration of the binding of the exogenous Her2/neu369-377 polypeptide to the HLA-A2 molecule differs from the configuration of the polypeptide/HLA complex presented in the cell (see the document "Journal of Immunology,2008,180: 8135-8145"). Another possible reason is that, the Her2/neu369-377 polypeptide acts as a mimotope (mimotope) antigen, and the induced specific TCR recognizes both the Her2/neu369-377 polypeptide and similar polypeptides presented by tumor cells, such as the Her2/neu 373 382 polypeptide (see the document "J Immunol.2013Jan 1; 190(1): 479-488), whereas the high-affinity TCR, although having high affinity for the Her2/neu369 377 polypeptide presented by HLA-A2, is not able to effectively recognize the corresponding mimotope polypeptide presented by tumor cells. The TCR specifically recognizing the Her2/neu369-377 polypeptide not only targets the Her2/neu369-377 polypeptide presented by tumor cells, but also can simultaneously recognize other mimotope polypeptides derived from Her2/neu presented by the tumor cells.

Since high affinity T cells that recognize self-antigens are mostly cleared by central tolerance mechanisms, TCRs that recognize Her2/neu antigens that occur naturally in the peripheral T cell repertoire are mostly of medium-low affinity. Another high affinity TCR that recognizes the CD8 function-independent form of tumor cells is derived from T cells specific for the Her2/neu 373-382 polypeptideThe multiple alpha and beta chains of the population are paired and then screened by functional detection (see the literature, "HUMAN GENE THERAPY 2024, 25: 730-739"; WO/2016/133779). Since it is not directly obtained from specific monoclonal T cells, it cannot be determined whether this TCR is present in the peripheral natural T cell pool. It is generally accepted that the therapeutic efficacy of adoptive transfer of high affinity T cells is superior to that of low affinity T cells targeting the same antigen (see the literature "Clin Exp Immunol (2015)180: 255-70"). However, high affinity TCRs themselves are prone to produce autoimmune responses that recognize self-antigens (see "Blood (2009)114: 535-46"), TCRs that have not been screened for central tolerance mechanisms also recognize normal tissues with low expression of antigen, or off-target toxicity that cross-reacts against other similar self-antigenic epitopes (see "Sci trans Med (2013)5:197ra103.Blood (2013)122: 863-71"). Another reason for selecting high affinity TCRs is that the function of these TCRs is independent of the helper function of CD8 and thus can be transfected with CD4⁺T cell to CD8⁺Adjuvant effects of killer T cell function. For the CD8 function-dependent TCR, the same purpose can be achieved by simultaneously expressing the TCR and the exogenous CD8 molecule through an expression vector.

In summary, the present invention provides a kit derived from HLA-A2⁺The Her2/neu 373-382 polypeptide specificity TCR alpha chain and beta chain complete sequence induced from the autologous peripheral T cell bank, the primary killer T cell expressing the TCR and the TCR with the modified constant region after transfection can recognize a plurality of HLA-A2⁺Her2/neu⁺The tumor cell of (2). Provides a new method and a new way for the development and clinical application of adoptive transfer of T cells modified by specific TCR to treat tumors.

SEQUENCE LISTING

<110> Hangzhou Kangwanda technology and technology of medicine

Synthetic immunization Co., Ltd (Synimmune, Inc.)

<120> isolated T cell receptor, modified cell thereof, encoding nucleic acid, expression vector, preparation method, pharmaceutical composition and application

<130> FI-173891-59:52/C

<160> 31

<170> PatentIn version 3.5

<210> 1

<211> 831

<212> DNA

<213> human (Homo sapiens)

<400> 1

atggcatgcc ctggcttcct gtgggcactt gtgatctcca cctgtcttga atttagcatg 60

gctcagacag tcactcagtc tcaaccagag atgtctgtgc aggaggcaga gaccgtgacc 120

ctgagctgca catatgacac cagtgagagt gattattatt tattctggta caagcagcct 180

cccagcaggc agatgattct cgttattcgc caagaagctt ataagcaaca gaatgcaaca 240

gagaatcgtt tctctgtgaa cttccagaaa gcagccaaat ccttcagtct caagatctca 300

gactcacagc tgggggatgc cgcgatgtat ttctgtgctt ataggctctt taataatgca 360

ggcaacatgc tcacctttgg agggggaaca aggttaatgg tcaaacccca tatccagaac 420

cctgaccctg ccgtgtacca gctgagagac tctaaatcca gtgacaagtc tgtctgccta 480

ttcaccgatt ttgattctca aacaaatgtg tcacaaagta aggattctga tgtgtatatc 540

acagacaaaa ccgtgctaga catgaggtct atggacttca agagcaacag tgctgtggcc 600

tggagcaaca aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa 660

gacaccttct tccccagccc agaaagttcc tgtgatgtca agctggtcga gaaaagcttt 720

gaaacagata cgaacctaaa ctttcaaaac ctgtcagtga ttgggttccg aatcctcctc 780

ctgaaagtgg ccgggtttaa tctgctcatg acgctgcggc tgtggtccag c 831

<210> 2

<211> 277

<212> PRT

<213> human (Homo sapiens)

<400> 2

Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr Cys Leu

1 5 10 15

Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser

20 25 30

Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser

35 40 45

Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln

50 55 60

Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr

65 70 75 80

Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser

85 90 95

Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys

100 105 110

Ala Tyr Arg Leu Phe Asn Asn Ala Gly Asn Met Leu Thr Phe Gly Gly

115 120 125

Gly Thr Arg Leu Met Val Lys Pro His Ile Gln Asn Pro Asp Pro Ala

130 135 140

Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu

145 150 155 160

Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser

165 170 175

Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp

180 185 190

Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala

195 200 205

Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe

210 215 220

Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe

225 230 235 240

Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe

245 250 255

Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu

260 265 270

Arg Leu Trp Ser Ser

275

<210> 3

<211> 939

<212> DNA

<213> human (Homo sapiens)

<400> 3

atggatacct ggctcgtatg ctgggcaatt tttagtctct tgaaagcagg actcacagaa 60

cctgaagtca cccagactcc cagccatcag gtcacacaga tgggacagga agtgatcttg 120

cgctgtgtcc ccatctctaa tcacttatac ttctattggt acagacaaat cttggggcag 180

aaagtcgagt ttctggtttc cttttataat aatgaaatct cagagaagtc tgaaatattc 240

gatgatcaat tctcagttga aaggcctgat ggatcaaatt tcactctgaa gatccggtcc 300

acaaagctgg aggactcagc catgtacttc tgtgccagca gtaccgattt ggtaggggtc 360

gaagaagctt tctttggaca aggcaccaga ctcacagttg tagaggacct gaacaaggtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tatgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc 900

gtgctgatgg ccatggtcaa gagaaaggat tccagaggc 939

<210> 4

<211> 313

<212> PRT

<213> human (Homo sapiens)

<400> 4

Met Asp Thr Trp Leu Val Cys Trp Ala Ile Phe Ser Leu Leu Lys Ala

1 5 10 15

Gly Leu Thr Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr

20 25 30

Gln Met Gly Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His

35 40 45

Leu Tyr Phe Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe

50 55 60

Leu Val Ser Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe

65 70 75 80

Asp Asp Gln Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu

85 90 95

Lys Ile Arg Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala

100 105 110

Ser Ser Thr Asp Leu Val Gly Val Glu Glu Ala Phe Phe Gly Gln Gly

115 120 125

Thr Arg Leu Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu

130 135 140

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

195 200 205

Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

210 215 220

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

225 230 235 240

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

245 250 255

Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr

260 265 270

Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly

275 280 285

Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala

290 295 300

Met Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 5

<211> 831

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 5

atggcatgcc ctggcttcct gtgggcactt gtgatctcca cctgtcttga atttagcatg 60

gctcagacag tcactcagtc tcaaccagag atgtctgtgc aggaggcaga gaccgtgacc 120

ctgagctgca catatgacac cagtgagagt gattattatt tattctggta caagcagcct 180

cccagcaggc agatgattct cgttattcgc caagaagctt ataagcaaca gaatgcaaca 240

gagaatcgtt tctctgtgaa cttccagaaa gcagccaaat ccttcagtct caagatctca 300

gactcacagc tgggggatgc cgcgatgtat ttctgtgctt ataggctctt taataatgca 360

ggcaacatgc tcacctttgg agggggaaca aggttaatgg tcaaacccca tatccagaac 420

cctgaccctg ccgtgtacca gctgagagac tctaaatcca gtgacaagtc tgtctgccta 480

ttcaccgatt ttgattctca aacaaatgtg tcacaaagta aggattctga tgtgtatatc 540

acagacaaat gcgtgctaga catgaggtct atggacttca agagcaacag tgctgtggcc 600

tggagcaaca aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa 660

gacaccttct tccccagccc agaaagttcc tgtgatgtca agctggtcga gaaaagcttt 720

gaaacagata cgaacctaaa ctttcaaaac ctgtcagtga ttgggttccg aatcctcctc 780

ctgaaagtgg ccgggtttaa tctgctcatg acgctgcggc tgtggtccag c 831

<210> 6

<211> 277

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 6

Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr Cys Leu

1 5 10 15

Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser

20 25 30

Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser

35 40 45

Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln

50 55 60

Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr

65 70 75 80

Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser

85 90 95

Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys

100 105 110

Ala Tyr Arg Leu Phe Asn Asn Ala Gly Asn Met Leu Thr Phe Gly Gly

115 120 125

Gly Thr Arg Leu Met Val Lys Pro His Ile Gln Asn Pro Asp Pro Ala

130 135 140

Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu

145 150 155 160

Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser

165 170 175

Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp

180 185 190

Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala

195 200 205

Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe

210 215 220

Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe

225 230 235 240

Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe

245 250 255

Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu

260 265 270

Arg Leu Trp Ser Ser

275

<210> 7

<211> 939

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 7

atggatacct ggctcgtatg ctgggcaatt tttagtctct tgaaagcagg actcacagaa 60

cctgaagtca cccagactcc cagccatcag gtcacacaga tgggacagga agtgatcttg 120

cgctgtgtcc ccatctctaa tcacttatac ttctattggt acagacaaat cttggggcag 180

aaagtcgagt ttctggtttc cttttataat aatgaaatct cagagaagtc tgaaatattc 240

gatgatcaat tctcagttga aaggcctgat ggatcaaatt tcactctgaa gatccggtcc 300

acaaagctgg aggactcagc catgtacttc tgtgccagca gtaccgattt ggtaggggtc 360

gaagaagctt tctttggaca aggcaccaga ctcacagttg tagaggacct gaacaaggtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tatgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc tgcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc 900

gtgctgatgg ccatggtcaa gagaaaggat tccagaggc 939

<210> 8

<211> 313

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 8

Met Asp Thr Trp Leu Val Cys Trp Ala Ile Phe Ser Leu Leu Lys Ala

1 5 10 15

Gly Leu Thr Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr

20 25 30

Gln Met Gly Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His

35 40 45

Leu Tyr Phe Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe

50 55 60

Leu Val Ser Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe

65 70 75 80

Asp Asp Gln Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu

85 90 95

Lys Ile Arg Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala

100 105 110

Ser Ser Thr Asp Leu Val Gly Val Glu Glu Ala Phe Phe Gly Gln Gly

115 120 125

Thr Arg Leu Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu

130 135 140

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr

180 185 190

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

195 200 205

Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

210 215 220

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

225 230 235 240

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

245 250 255

Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr

260 265 270

Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly

275 280 285

Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala

290 295 300

Met Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 9

<211> 822

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 9

atggcatgcc ctggcttcct gtgggcactt gtgatctcca cctgtcttga atttagcatg 60

gctcagacag tcactcagtc tcaaccagag atgtctgtgc aggaggcaga gaccgtgacc 120

ctgagctgca catatgacac cagtgagagt gattattatt tattctggta caagcagcct 180

cccagcaggc agatgattct cgttattcgc caagaagctt ataagcaaca gaatgcaaca 240

gagaatcgtt tctctgtgaa cttccagaaa gcagccaaat ccttcagtct caagatctca 300

gactcacagc tgggggatgc cgcgatgtat ttctgtgctt ataggctctt taataatgca 360

ggcaacatgc tcacctttgg agggggaaca aggttaatgg tcaaacccca tatccagaac 420

ccagaacctg ctgtgtacca gttaaaagat cctcggtctc aggacagcac cctctgcctg 480

ttcaccgact ttgactccca aatcaatgtg ccgaaaacca tggaatctgg aacgttcatc 540

actgacaaaa ctgtgctgga catgaaagct atggattcca agagcaatgg ggccattgcc 600

tggagcaacc agacaagctt cacctgccaa gatatcttca aagagaccaa cgccacctac 660

cccagttcag acgttccctg tgatgccacg ttgaccgaga aaagctttga aacagatatg 720

aacctaaact ttcaaaacct gtcagttatg ggactccgaa tcctcctgct gaaagtagcg 780

ggatttaacc tgctcatgac gctgaggctg tggtccagtt ga 822

<210> 10

<211> 273

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 10

Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr Cys Leu

1 5 10 15

Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser

20 25 30

Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser

35 40 45

Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln

50 55 60

Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr

65 70 75 80

Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser

85 90 95

Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys

100 105 110

Ala Tyr Arg Leu Phe Asn Asn Ala Gly Asn Met Leu Thr Phe Gly Gly

115 120 125

Gly Thr Arg Leu Met Val Lys Pro His Ile Gln Asn Pro Glu Pro Ala

130 135 140

Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu

145 150 155 160

Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser

165 170 175

Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp

180 185 190

Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr

195 200 205

Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp

210 215 220

Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met

225 230 235 240

Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu

245 250 255

Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser

260 265 270

Ser

<210> 11

<211> 921

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 11

atggatacct ggctcgtatg ctgggcaatt tttagtctct tgaaagcagg actcacagaa 60

cctgaagtca cccagactcc cagccatcag gtcacacaga tgggacagga agtgatcttg 120

cgctgtgtcc ccatctctaa tcacttatac ttctattggt acagacaaat cttggggcag 180

aaagtcgagt ttctggtttc cttttataat aatgaaatct cagagaagtc tgaaatattc 240

gatgatcaat tctcagttga aaggcctgat ggatcaaatt tcactctgaa gatccggtcc 300

acaaagctgg aggactcagc catgtacttc tgtgccagca gtaccgattt ggtaggggtc 360

gaagaagctt tctttggaca aggcaccaga ctcacagttg tagaggatct gagaaatgtg 420

actccaccca aggtctcctt gtttgagcca tcaaaagcag agattgcaaa caaacaaaag 480

gctaccctcg tgtgcttggc caggggcttc ttccctgacc acgtggagct gagctggtgg 540

gtgaatggca aggaggtcca cagtggggtc agcacggacc ctcaggccta caaggagagc 600

aattatagct actgcctgag cagccgcctg agggtctctg ctaccttctg gcacaatcct 660

cgcaaccact tccgctgcca agtgcagttc catgggcttt cagaggagga caagtggcca 720

gagggctcac ccaaacctgt cacacagaac atcagtgcag aggcctgggg ccgagcagac 780

tgtgggatta cctcagcatc ctatcaacaa ggggtcttgt ctgccaccat cctctatgag 840

atcctgctag ggaaagccac cctgtatgct gtgcttgtca gtacactggt ggtgatggct 900

atggtcaaaa gaaagaattc a 921

<210> 12

<211> 307

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 12

Met Asp Thr Trp Leu Val Cys Trp Ala Ile Phe Ser Leu Leu Lys Ala

1 5 10 15

Gly Leu Thr Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr

20 25 30

Gln Met Gly Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His

35 40 45

Leu Tyr Phe Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe

50 55 60

Leu Val Ser Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe

65 70 75 80

Asp Asp Gln Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu

85 90 95

Lys Ile Arg Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala

100 105 110

Ser Ser Thr Asp Leu Val Gly Val Glu Glu Ala Phe Phe Gly Gln Gly

115 120 125

Thr Arg Leu Thr Val Val Glu Asp Leu Arg Asn Val Thr Pro Pro Lys

130 135 140

Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser

195 200 205

Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe

210 215 220

Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro

225 230 235 240

Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp

245 250 255

Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val

260 265 270

Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu

275 280 285

Tyr Ala Val Leu Val Ser Thr Leu Val Val Met Ala Met Val Lys Arg

290 295 300

Lys Asn Ser

305

<210> 13

<211> 1866

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 13

atggatacct ggctcgtatg ctgggcaatt tttagtctct tgaaagcagg actcacagaa 60

cctgaagtca cccagactcc cagccatcag gtcacacaga tgggacagga agtgatcttg 120

cgctgtgtcc ccatctctaa tcacttatac ttctattggt acagacaaat cttggggcag 180

aaagtcgagt ttctggtttc cttttataat aatgaaatct cagagaagtc tgaaatattc 240

gatgatcaat tctcagttga aaggcctgat ggatcaaatt tcactctgaa gatccggtcc 300

acaaagctgg aggactcagc catgtacttc tgtgccagca gtaccgattt ggtaggggtc 360

gaagaagctt tctttggaca aggcaccaga ctcacagttg tagaggacct gaacaaggtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tatgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc tgcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc 900

gtgctgatgg ccatggtcaa gagaaaggat tccagaggcc gtgccaagcg atccggaagc 960

ggagcccctg taaagcagac tttgaatttt gaccttctca agttggcggg agacgtcgag 1020

tccaaccctg ggcccatggc atgccctggc ttcctgtggg cacttgtgat ctccacctgt 1080

cttgaattta gcatggctca gacagtcact cagtctcaac cagagatgtc tgtgcaggag 1140

gcagagaccg tgaccctgag ctgcacatat gacaccagtg agagtgatta ttatttattc 1200

tggtacaagc agcctcccag caggcagatg attctcgtta ttcgccaaga agcttataag 1260

caacagaatg caacagagaa tcgtttctct gtgaacttcc agaaagcagc caaatccttc 1320

agtctcaaga tctcagactc acagctgggg gatgccgcga tgtatttctg tgcttatagg 1380

ctctttaata atgcaggcaa catgctcacc tttggagggg gaacaaggtt aatggtcaaa 1440

ccccatatcc agaaccctga ccctgccgtg taccagctga gagactctaa atccagtgac 1500

aagtctgtct gcctattcac cgattttgat tctcaaacaa atgtgtcaca aagtaaggat 1560

tctgatgtgt atatcacaga caaatgcgtg ctagacatga ggtctatgga cttcaagagc 1620

aacagtgctg tggcctggag caacaaatct gactttgcat gtgcaaacgc cttcaacaac 1680

agcattattc cagaagacac cttcttcccc agcccagaaa gttcctgtga tgtcaagctg 1740

gtcgagaaaa gctttgaaac agatacgaac ctaaactttc aaaacctgtc agtgattggg 1800

ttccgaatcc tcctcctgaa agtggccggg tttaatctgc tcatgacgct gcggctgtgg 1860

tccagc 1866

<210> 14

<211> 622

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 14

Met Asp Thr Trp Leu Val Cys Trp Ala Ile Phe Ser Leu Leu Lys Ala

1 5 10 15

Gly Leu Thr Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr

20 25 30

Gln Met Gly Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His

35 40 45

Leu Tyr Phe Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe

50 55 60

Leu Val Ser Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe

65 70 75 80

Asp Asp Gln Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu

85 90 95

Lys Ile Arg Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala

100 105 110

Ser Ser Thr Asp Leu Val Gly Val Glu Glu Ala Phe Phe Gly Gln Gly

115 120 125

Thr Arg Leu Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu

130 135 140

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr

180 185 190

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

195 200 205

Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

210 215 220

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

225 230 235 240

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

245 250 255

Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr

260 265 270

Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly

275 280 285

Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala

290 295 300

Met Val Lys Arg Lys Asp Ser Arg Gly Arg Ala Lys Arg Ser Gly Ser

305 310 315 320

Gly Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala

325 330 335

Gly Asp Val Glu Ser Asn Pro Gly Pro Met Ala Cys Pro Gly Phe Leu

340 345 350

Trp Ala Leu Val Ile Ser Thr Cys Leu Glu Phe Ser Met Ala Gln Thr

355 360 365

Val Thr Gln Ser Gln Pro Glu Met Ser Val Gln Glu Ala Glu Thr Val

370 375 380

Thr Leu Ser Cys Thr Tyr Asp Thr Ser Glu Ser Asp Tyr Tyr Leu Phe

385 390 395 400

Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Ile Leu Val Ile Arg Gln

405 410 415

Glu Ala Tyr Lys Gln Gln Asn Ala Thr Glu Asn Arg Phe Ser Val Asn

420 425 430

Phe Gln Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile Ser Asp Ser Gln

435 440 445

Leu Gly Asp Ala Ala Met Tyr Phe Cys Ala Tyr Arg Leu Phe Asn Asn

450 455 460

Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys

465 470 475 480

Pro His Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

485 490 495

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

500 505 510

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

515 520 525

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

530 535 540

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

545 550 555 560

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys

565 570 575

Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn

580 585 590

Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val

595 600 605

Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

610 615 620

<210> 15

<211> 1836

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 15

atggatacct ggctcgtatg ctgggcaatt tttagtctct tgaaagcagg actcacagaa 60

cctgaagtca cccagactcc cagccatcag gtcacacaga tgggacagga agtgatcttg 120

cgctgtgtcc ccatctctaa tcacttatac ttctattggt acagacaaat cttggggcag 180

aaagtcgagt ttctggtttc cttttataat aatgaaatct cagagaagtc tgaaatattc 240

gatgatcaat tctcagttga aaggcctgat ggatcaaatt tcactctgaa gatccggtcc 300

acaaagctgg aggactcagc catgtacttc tgtgccagca gtaccgattt ggtaggggtc 360

gaagaagctt tctttggaca aggcaccaga ctcacagttg tagaggatct gagaaatgtg 420

actccaccca aggtctcctt gtttgagcca tcaaaagcag agattgcaaa caaacaaaag 480

gctaccctcg tgtgcttggc caggggcttc ttccctgacc acgtggagct gagctggtgg 540

gtgaatggca aggaggtcca cagtggggtc agcacggacc ctcaggccta caaggagagc 600

aattatagct actgcctgag cagccgcctg agggtctctg ctaccttctg gcacaatcct 660

cgcaaccact tccgctgcca agtgcagttc catgggcttt cagaggagga caagtggcca 720

gagggctcac ccaaacctgt cacacagaac atcagtgcag aggcctgggg ccgagcagac 780

tgtgggatta cctcagcatc ctatcaacaa ggggtcttgt ctgccaccat cctctatgag 840

atcctgctag ggaaagccac cctgtatgct gtgcttgtca gtacactggt ggtgatggct 900

atggtcaaaa gaaagaattc acgtgccaag cgatccggaa gcggagcccc tgtaaagcag 960

actttgaatt ttgaccttct caagttggcg ggagacgtcg agtccaaccc tgggcccatg 1020

gcatgccctg gcttcctgtg ggcacttgtg atctccacct gtcttgaatt tagcatggct 1080

cagacagtca ctcagtctca accagagatg tctgtgcagg aggcagagac cgtgaccctg 1140

agctgcacat atgacaccag tgagagtgat tattatttat tctggtacaa gcagcctccc 1200

agcaggcaga tgattctcgt tattcgccaa gaagcttata agcaacagaa tgcaacagag 1260

aatcgtttct ctgtgaactt ccagaaagca gccaaatcct tcagtctcaa gatctcagac 1320

tcacagctgg gggatgccgc gatgtatttc tgtgcttata ggctctttaa taatgcaggc 1380

aacatgctca cctttggagg gggaacaagg ttaatggtca aaccccatat ccagaaccca 1440

gaacctgctg tgtaccagtt aaaagatcct cggtctcagg acagcaccct ctgcctgttc 1500

accgactttg actcccaaat caatgtgccg aaaaccatgg aatctggaac gttcatcact 1560

gacaaaactg tgctggacat gaaagctatg gattccaaga gcaatggggc cattgcctgg 1620

agcaaccaga caagcttcac ctgccaagat atcttcaaag agaccaacgc cacctacccc 1680

agttcagacg ttccctgtga tgccacgttg accgagaaaa gctttgaaac agatatgaac 1740

ctaaactttc aaaacctgtc agttatggga ctccgaatcc tcctgctgaa agtagcggga 1800

tttaacctgc tcatgacgct gaggctgtgg tccagt 1836

<210> 16

<211> 612

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 16

Met Asp Thr Trp Leu Val Cys Trp Ala Ile Phe Ser Leu Leu Lys Ala

1 5 10 15

Gly Leu Thr Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr

20 25 30

Gln Met Gly Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His

35 40 45

Leu Tyr Phe Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe

50 55 60

Leu Val Ser Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe

65 70 75 80

Asp Asp Gln Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu

85 90 95

Lys Ile Arg Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala

100 105 110

Ser Ser Thr Asp Leu Val Gly Val Glu Glu Ala Phe Phe Gly Gln Gly

115 120 125

Thr Arg Leu Thr Val Val Glu Asp Leu Arg Asn Val Thr Pro Pro Lys

130 135 140

Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser

195 200 205

Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe

210 215 220

Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro

225 230 235 240

Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp

245 250 255

Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val

260 265 270

Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu

275 280 285

Tyr Ala Val Leu Val Ser Thr Leu Val Val Met Ala Met Val Lys Arg

290 295 300

Lys Asn Ser Arg Ala Lys Arg Ser Gly Ser Gly Ala Pro Val Lys Gln

305 310 315 320

Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn

325 330 335

Pro Gly Pro Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser

340 345 350

Thr Cys Leu Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro

355 360 365

Glu Met Ser Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr

370 375 380

Asp Thr Ser Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro

385 390 395 400

Ser Arg Gln Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln

405 410 415

Asn Ala Thr Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys

420 425 430

Ser Phe Ser Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met

435 440 445

Tyr Phe Cys Ala Tyr Arg Leu Phe Asn Asn Ala Gly Asn Met Leu Thr

450 455 460

Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro His Ile Gln Asn Pro

465 470 475 480

Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr

485 490 495

Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr

500 505 510

Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys

515 520 525

Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr

530 535 540

Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro

545 550 555 560

Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu

565 570 575

Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg

580 585 590

Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg

595 600 605

Leu Trp Ser Ser

610

<210> 17

<211> 1254

<212> PRT

<213> human (Homo sapiens)

<400> 17

Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu

1 5 10 15

Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys

20 25 30

Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His

35 40 45

Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr

50 55 60

Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val

65 70 75 80

Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu

85 90 95

Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr

100 105 110

Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro

115 120 125

Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser

130 135 140

Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln

145 150 155 160

Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn

165 170 175

Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys

180 185 190

His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser

195 200 205

Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys

210 215 220

Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys

225 230 235 240

Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu

245 250 255

His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val

260 265 270

Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg

275 280 285

Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu

290 295 300

Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln

305 310 315 320

Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys

325 330 335

Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu

340 345 350

Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys

355 360 365

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

370 375 380

Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe

385 390 395 400

Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro

405 410 415

Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg

420 425 430

Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu

435 440 445

Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly

450 455 460

Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val Thr Val Pro

465 470 475 480

Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr Ala

485 490 495

Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln

500 505 510

Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val

515 520 525

Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg

530 535 540

Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu

545 550 555 560

Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe

565 570 575

Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro

580 585 590

Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser

595 600 605

Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro

610 615 620

Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly

625 630 635 640

Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser Ala

645 650 655

Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly Ile

660 665 670

Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg Arg

675 680 685

Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Ala

690 695 700

Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu Arg

705 710 715 720

Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly

725 730 735

Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys

740 745 750

Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp

755 760 765

Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg Leu

770 775 780

Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu Met

785 790 795 800

Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg Leu

805 810 815

Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly Met

820 825 830

Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala Arg

835 840 845

Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe Gly

850 855 860

Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp Gly

865 870 875 880

Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg Arg

885 890 895

Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp

900 905 910

Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala Arg

915 920 925

Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro

930 935 940

Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile

945 950 955 960

Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe Ser

965 970 975

Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu Asp

980 985 990

Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu Leu

995 1000 1005

Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr Leu

1010 1015 1020

Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly Ala

1025 1030 1035

Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg Ser

1040 1045 1050

Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu Glu

1055 1060 1065

Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser Asp

1070 1075 1080

Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu Gln

1085 1090 1095

Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser Glu

1100 1105 1110

Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val Ala

1115 1120 1125

Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro Asp

1130 1135 1140

Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro Ala

1145 1150 1155

Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu Ser

1160 1165 1170

Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly Gly

1175 1180 1185

Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala Ala

1190 1195 1200

Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp Asn

1205 1210 1215

Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro Pro

1220 1225 1230

Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr Leu

1235 1240 1245

Gly Leu Asp Val Pro Val

1250

<210> 18

<211> 9

<212> PRT

<213> human (Homo sapiens)

<400> 18

Lys Ile Phe Gly Ser Leu Ala Phe Leu

1 5

<210> 19

<211> 137

<212> PRT

<213> human (Homo sapiens)

<400> 19

Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr Cys Leu

1 5 10 15

Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser

20 25 30

Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser

35 40 45

Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln

50 55 60

Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr

65 70 75 80

Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser

85 90 95

Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys

100 105 110

Ala Tyr Arg Leu Phe Asn Asn Ala Gly Asn Met Leu Thr Phe Gly Gly

115 120 125

Gly Thr Arg Leu Met Val Lys Pro His

130 135

<210> 20

<211> 134

<212> PRT

<213> human (Homo sapiens)

<400> 20

Met Asp Thr Trp Leu Val Cys Trp Ala Ile Phe Ser Leu Leu Lys Ala

1 5 10 15

Gly Leu Thr Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr

20 25 30

Gln Met Gly Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His

35 40 45

Leu Tyr Phe Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe

50 55 60

Leu Val Ser Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe

65 70 75 80

Asp Asp Gln Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu

85 90 95

Lys Ile Arg Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala

100 105 110

Ser Ser Thr Asp Leu Val Gly Val Glu Glu Ala Phe Phe Gly Gln Gly

115 120 125

Thr Arg Leu Thr Val Val

130

<210> 21

<211> 411

<212> DNA

<213> human (Homo sapiens)

<400> 21

atggcatgcc ctggcttcct gtgggcactt gtgatctcca cctgtcttga atttagcatg 60

gctcagacag tcactcagtc tcaaccagag atgtctgtgc aggaggcaga gaccgtgacc 120

ctgagctgca catatgacac cagtgagagt gattattatt tattctggta caagcagcct 180

cccagcaggc agatgattct cgttattcgc caagaagctt ataagcaaca gaatgcaaca 240

gagaatcgtt tctctgtgaa cttccagaaa gcagccaaat ccttcagtct caagatctca 300

gactcacagc tgggggatgc cgcgatgtat ttctgtgctt ataggctctt taataatgca 360

ggcaacatgc tcacctttgg agggggaaca aggttaatgg tcaaacccca t 411

<210> 22

<211> 402

<212> DNA

<213> human (Homo sapiens)

<400> 22

atggatacct ggctcgtatg ctgggcaatt tttagtctct tgaaagcagg actcacagaa 60

cctgaagtca cccagactcc cagccatcag gtcacacaga tgggacagga agtgatcttg 120

cgctgtgtcc ccatctctaa tcacttatac ttctattggt acagacaaat cttggggcag 180

aaagtcgagt ttctggtttc cttttataat aatgaaatct cagagaagtc tgaaatattc 240

gatgatcaat tctcagttga aaggcctgat ggatcaaatt tcactctgaa gatccggtcc 300

acaaagctgg aggactcagc catgtacttc tgtgccagca gtaccgattt ggtaggggtc 360

gaagaagctt tctttggaca aggcaccaga ctcacagttg ta 402

<210> 23

<211> 1866

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 23

atggatacct ggctcgtatg ctgggcaatt tttagtctct tgaaagcagg actcacagaa 60

cctgaagtca cccagactcc cagccatcag gtcacacaga tgggacagga agtgatcttg 120

cgctgtgtcc ccatctctaa tcacttatac ttctattggt acagacaaat cttggggcag 180

aaagtcgagt ttctggtttc cttttataat aatgaaatct cagagaagtc tgaaatattc 240

gatgatcaat tctcagttga aaggcctgat ggatcaaatt tcactctgaa gatccggtcc 300

acaaagctgg aggactcagc catgtacttc tgtgccagca gtaccgattt ggtaggggtc 360

gaagaagctt tctttggaca aggcaccaga ctcacagttg tagaggacct gaacaaggtg 420

ttcccacccg aggtcgctgt gtttgagcca tcagaagcag agatctccca cacccaaaag 480

gccacactgg tatgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg 540

gtgaatggga aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag 600

cccgccctca atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc 660

tggcagaacc cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat 720

gacgagtgga cccaggatag ggccaaaccc gtcacccaga tcgtcagcgc cgaggcctgg 780

ggtagagcag actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc 840

atcctctatg agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc 900

gtgctgatgg ccatggtcaa gagaaaggat tccagaggcc gtgccaagcg atccggaagc 960

ggagcccctg taaagcagac tttgaatttt gaccttctca agttggcggg agacgtcgag 1020

tccaaccctg ggcccatggc atgccctggc ttcctgtggg cacttgtgat ctccacctgt 1080

cttgaattta gcatggctca gacagtcact cagtctcaac cagagatgtc tgtgcaggag 1140

gcagagaccg tgaccctgag ctgcacatat gacaccagtg agagtgatta ttatttattc 1200

tggtacaagc agcctcccag caggcagatg attctcgtta ttcgccaaga agcttataag 1260

caacagaatg caacagagaa tcgtttctct gtgaacttcc agaaagcagc caaatccttc 1320

agtctcaaga tctcagactc acagctgggg gatgccgcga tgtatttctg tgcttatagg 1380

ctctttaata atgcaggcaa catgctcacc tttggagggg gaacaaggtt aatggtcaaa 1440

ccccatatcc agaaccctga ccctgccgtg taccagctga gagactctaa atccagtgac 1500

aagtctgtct gcctattcac cgattttgat tctcaaacaa atgtgtcaca aagtaaggat 1560

tctgatgtgt atatcacaga caaaaccgtg ctagacatga ggtctatgga cttcaagagc 1620

aacagtgctg tggcctggag caacaaatct gactttgcat gtgcaaacgc cttcaacaac 1680

agcattattc cagaagacac cttcttcccc agcccagaaa gttcctgtga tgtcaagctg 1740

gtcgagaaaa gctttgaaac agatacgaac ctaaactttc aaaacctgtc agtgattggg 1800

ttccgaatcc tcctcctgaa agtggccggg tttaatctgc tcatgacgct gcggctgtgg 1860

tccagc 1866

<210> 24

<211> 622

<212> PRT

<213> Artificial sequence (artificial sequence)

<400> 24

Met Asp Thr Trp Leu Val Cys Trp Ala Ile Phe Ser Leu Leu Lys Ala

1 5 10 15

Gly Leu Thr Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr

20 25 30

Gln Met Gly Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His

35 40 45

Leu Tyr Phe Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe

50 55 60

Leu Val Ser Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe

65 70 75 80

Asp Asp Gln Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu

85 90 95

Lys Ile Arg Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala

100 105 110

Ser Ser Thr Asp Leu Val Gly Val Glu Glu Ala Phe Phe Gly Gln Gly

115 120 125

Thr Arg Leu Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu

130 135 140

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

145 150 155 160

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr

195 200 205

Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

210 215 220

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

225 230 235 240

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

245 250 255

Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr

260 265 270

Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly

275 280 285

Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala

290 295 300

Met Val Lys Arg Lys Asp Ser Arg Gly Arg Ala Lys Arg Ser Gly Ser

305 310 315 320

Gly Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala

325 330 335

Gly Asp Val Glu Ser Asn Pro Gly Pro Met Ala Cys Pro Gly Phe Leu

340 345 350

Trp Ala Leu Val Ile Ser Thr Cys Leu Glu Phe Ser Met Ala Gln Thr

355 360 365

Val Thr Gln Ser Gln Pro Glu Met Ser Val Gln Glu Ala Glu Thr Val

370 375 380

Thr Leu Ser Cys Thr Tyr Asp Thr Ser Glu Ser Asp Tyr Tyr Leu Phe

385 390 395 400

Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Ile Leu Val Ile Arg Gln

405 410 415

Glu Ala Tyr Lys Gln Gln Asn Ala Thr Glu Asn Arg Phe Ser Val Asn

420 425 430

Phe Gln Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile Ser Asp Ser Gln

435 440 445

Leu Gly Asp Ala Ala Met Tyr Phe Cys Ala Tyr Arg Leu Phe Asn Asn

450 455 460

Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys

465 470 475 480

Pro His Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

485 490 495

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

500 505 510

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

515 520 525

Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

530 535 540

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

545 550 555 560

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys

565 570 575

Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn

580 585 590

Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val

595 600 605

Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

610 615 620

<210> 25

<211> 10

<212> PRT

<213> human (Homo sapiens)

<400> 25

Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp

1 5 10

<210> 26

<211> 22

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 26

gcctctggaa tcctttctct tg 22

<210> 27

<211> 21

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 27

tcagctggac cacagccgca g 21

<210> 28

<211> 38

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 28

agagctagcg aattcaacat ggatacctgg ctcgtatg 38

<210> 29

<211> 38

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 29

gttgattgtc gacgccctca gctggaccac agccgcag 38

<210> 30

<211> 38

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 30

agagctagcg aattcaacat ggatacctgg ctcgtatg 38

<210> 31

<211> 35

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 31

gttgattgtc gacgccctca actggaccac agcct 35

Claims

1. An isolated T cell receptor comprising an alpha chain and a beta chain, each comprising a variable region and a constant region, wherein said T cell receptor is capable of specifically recognizing the antigen Her2/neu expressed by tumor cells, and wherein the amino acid sequence of said variable region of said alpha chain is set forth in SEQ ID NO. 19 and the amino acid sequence of said variable region of said beta chain is set forth in SEQ ID NO. 20.

2. The T cell receptor of claim 1, wherein said T cell receptor is capable of specifically recognizing an epitope polypeptide of said antigen Her2/neu presented by HLA-a2 molecule.

3. The T cell receptor of claim 1, wherein the constant region of the alpha chain and/or the constant region of the beta chain is derived from a human.

4. The T cell receptor of claim 1, wherein the constant region of the alpha chain is modified with one or more disulfide bonds and/or the constant region of the beta chain is modified with one or more disulfide bonds.

5. The T cell receptor of claim 1, wherein the amino acid sequence of the alpha chain is set forth in SEQ ID No. 2 and the amino acid sequence of the beta chain is set forth in SEQ ID No. 4; or the amino acid sequence of the alpha chain is shown as SEQ ID NO. 6, and the amino acid sequence of the beta chain is shown as SEQ ID NO. 8; or the amino acid sequence of the alpha chain is shown as SEQ ID NO. 10, and the amino acid sequence of the beta chain is shown as SEQ ID NO. 12.

6. The T cell receptor of claim 2 wherein the epitope polypeptide comprises Her2/neu369-377 as set forth in SEQ ID NO: 18.

7. The T cell receptor of claim 3, wherein the constant region of the alpha chain is replaced, in whole or in part, by a homologous sequence derived from another species, and/or the constant region of the beta chain is replaced, in whole or in part, by a homologous sequence derived from another species.

8. The T cell receptor of claim 7, wherein the other species is a mouse.

9. An isolated nucleic acid encoding a T cell receptor comprising coding sequences for the alpha and beta chains of said T cell receptor, said alpha and beta chain coding sequences each comprising a variable region coding sequence and a constant region coding sequence, wherein said T cell receptor is capable of specifically recognizing the tumor cell-expressed antigen Her2/neu and wherein said alpha chain variable region coding sequence encodes the amino acid sequence set forth in SEQ ID NO 19 and said beta chain variable region coding sequence encodes the amino acid sequence set forth in SEQ ID NO 20.

10. The nucleic acid of claim 9, wherein the nucleic acid is DNA or RNA.

11. The nucleic acid of claim 9, wherein the alpha chain variable region encoding sequence is set forth in SEQ ID No. 21 and the beta chain variable region encoding sequence is set forth in SEQ ID No. 22.

12. The nucleic acid of claim 9, wherein said T cell receptor encoded by said nucleic acid is capable of specifically recognizing an epitope polypeptide of said antigen Her2/neu presented by HLA-a2 molecule.

13. The nucleic acid of claim 12, wherein the epitope polypeptide comprises Her2/neu369-377 as set forth in SEQ ID NO: 18.

14. The nucleic acid of claim 9, wherein the alpha chain constant region coding sequence and/or the beta chain constant region coding sequence is derived from a human.

15. The nucleic acid of claim 14, wherein the alpha chain constant region coding sequence is replaced in whole or in part by a homologous sequence from another species and/or the beta chain constant region coding sequence is replaced in whole or in part by a homologous sequence from another species.

16. The nucleic acid of claim 15, wherein the other species is a mouse.

17. The nucleic acid of claim 9, wherein the alpha chain constant region coding sequence comprises one or more disulfide bond coding sequences and/or the beta chain constant region coding sequence comprises one or more disulfide bond coding sequences.

18. The nucleic acid of claim 9, wherein the alpha chain coding sequence is set forth in SEQ ID No. 1, the beta chain coding sequence is set forth in SEQ ID No. 3; or the alpha chain coding sequence is shown as SEQ ID NO. 5, and the beta chain coding sequence is shown as SEQ ID NO. 7; or the alpha chain coding sequence is shown as SEQ ID NO. 9, and the beta chain coding sequence is shown as SEQ ID NO. 11.

19. The nucleic acid of any one of claims 9-17, wherein the alpha chain coding sequence and the beta chain coding sequence are linked by a coding sequence for a cleavable linker polypeptide.

20. The nucleic acid of claim 19, having the sequence set forth in SEQ ID NO 13, 15, or 23.

21. A recombinant expression vector comprising the nucleic acid of any one of claims 9-20, and/or a complementary sequence thereof, operably linked to a promoter.

22. A T cell receptor-modified cell, the surface of which is modified with the T cell receptor of any one of claims 1-8, wherein the cell comprises a naive T cell or a precursor cell thereof, an NKT cell, or a T cell line.

23. A method of making the T cell receptor modified cell of claim 22, comprising the steps of:

1) providing a cell;

2) providing a nucleic acid encoding a T cell receptor according to any one of claims 1-8;

3) transfecting the nucleic acid into the cell.

24. The method of claim 23, wherein the cells of step 1) are autologous or allogeneic.

25. The method of claim 23, wherein the means of transfection comprises: adopts a virus vector transfection mode, a chemical mode and a physical mode.

26. The method of claim 25, wherein the viral vector comprises a gammaretrovirus vector or a lentiviral vector.

27. The method of claim 25, wherein the chemical means comprises means of lipofection.

28. The method of claim 25, wherein the physical means comprises electrotransfection means.

29. The method according to claim 23, wherein the nucleic acid of step 2) is a nucleic acid according to any one of claims 9-20.

30. Use of a T cell receptor modified cell according to claim 22 for the preparation of a medicament for the treatment or prevention of a tumor and/or cancer, wherein said tumor and/or cancer is antigen Her2/neu positive and is HLA-a2 positive.

31. Use of a T cell receptor modified cell according to claim 22 in the manufacture of a medicament for detecting a tumor and/or cancer in a host.

32. A pharmaceutical composition comprising the T cell receptor-modified cell of claim 22 as an active ingredient, and a pharmaceutically acceptable excipient.

33. The pharmaceutical composition of claim 32, wherein the pharmaceutical composition comprises a total dose per patient per course of treatment ranging from 1 x10³-1×10⁹One cell per Kg body weight of said T cell receptor modified cells.

34. The pharmaceutical composition according to claim 32, wherein the pharmaceutical composition is suitable for administration intraarterially, intravenously, subcutaneously, intradermally, intratumorally, intralymphatically, subarachnoid intracavity, intramedullally, intramuscularly and intraperitoneally.