CN109776671A

CN109776671A - Cell, code nucleic acid, expression vector, preparation method, pharmaceutical composition and the application of isolated T cell receptor, its modification

Info

Publication number: CN109776671A
Application number: CN201711123690.XA
Authority: CN
Inventors: 侯亚非; 侯大炜
Original assignee: SYNIMMUNE GmbH; Hangzhou Kang Wanda Medical Technology Co Ltd
Current assignee: Hangzhou Converd Co Ltd
Priority date: 2017-11-14
Filing date: 2017-11-14
Publication date: 2019-05-21
Anticipated expiration: 2037-11-14
Also published as: WO2019096115A1; CN109776671B

Abstract

The present invention provides isolated T cell receptors, cell, code nucleic acid, expression vector, preparation method, pharmaceutical composition and the application of its modification.The isolated T cell receptor (TCR) includes at least one of α chain and β chain, the α chain and β chain include variable region and constant region, it is characterized in that, the T cell receptor being capable of antigen Her2/neu expressed by specific recognition tumour cell, and the amino acid sequence of the variable region of the α chain has the consistency with amino acid sequence at least 98% shown in SEQ ID NO:19, and the amino acid sequence of the variable region of the β chain has the consistency with amino acid sequence at least 98% shown in SEQ ID NO:20.The TCR can specific recognition tumour antigen, while can avoid the possible toxicity that misses the target.There is significant antitumous effect with the immunocyte of this TCR modification.

Description

Cell, the code nucleic acid, expression vector, preparation of isolated T cell receptor, its modification Method, pharmaceutical composition and application

Technical field

The invention belongs to field of biotechnology, in particular to isolated T cell receptor, the cell of its modification, coding Nucleic acid, expression vector, preparation method, pharmaceutical composition and application.

Background technique

Her2/neu (ERBB2) is the other albumen formation dimers of a transmembrane protein and family for belonging to EGFR family And by a series of Cellular Signaling Transduction Mediated approach come regulating cell increment, the processes such as differentiation and canceration are (referring to document "Growth Factors,2008；26:263","Oncol Biol.Phys,2004；58:903").Her2/neu albumen is more It is over-expressed in the cancer cell of kind epithelial origin, such as breast cancer, gastric cancer, colorectal cancer, oophoroma, cancer of pancreas, lung cancer, the cancer of the esophagus, Bladder cancer, kidney etc. (referring to document " Trends in Molecular Med, 2013；19:677 "), and in primary tumor and turn Move expression in stove cancer cell it is relatively uniform (referring to document " J Clin Oncol, 1998；8:103 "), therefore, Her2/neu at For the appropriate target spot of targeted therapy.

The humanization monoclonal antibody medicine Herceptin of targeting Her2/neu can significantly extend the breast cancer of the Her2/neu positive Patient survival (referring to document " N Engl J Med, 2001,344:783 "), however be used alone Herceptin treat Her2 The clinical response rate of positive metastatic breast cancer there was only 11% to 26% (referring to document " J Clin Oncol, 2002；20: 7169 "), show to be applied alone Heceptin to the curative effect is unsatisfactory of the highly expressed metastatic breast cancer of most of Her2.Although Clinical response rate can be improved in Heceptin combined chemotherapy, but the breast cancer patients of most of Her2/neu overexpression are after one year Heceptin can be generated resistance (referring to document " J Clin Oncol, 2001；19:2587").

Her2/neu positive tumor patient's body can generate the endogenous antibody and t cell responses for Her2/neu antigen (referring to document " Cancer Res, 2005；65:650 "), thus, the specific active immunotherapy of targeting Her2/neu antigen becomes A kind of quite promising treatment means.Specific recognition Her2/neu antigen epitope polypeptide (epitope peptide) 369-377 T cell can be successfully separated from the highly expressed Ascite of Ovarian Cancer of Her2/neu (referring to document " J.Exp.Med.1995； 181:2109–2117").The tumor vaccine of targeting Her2/neu 369-377 polypeptide antigen enters clinical test, although clinic 1/ 2 phases showed that this vaccine can induce the specificity T killing cell for Her2/neu 369-377 polypeptide antigen (referring to document "Breast Care,2016；11:116 "), but clinical three phases do not reach extend patient's life cycle predeterminated target (http: // www.onclive.com/web-exclusives/phase-iii-nelipepimuts-study-in-breast-cancer- halted-after-futility-review).Transmission adopt through in vitro culture, is based on Chimeric antigen receptor (chimeric Antigen receptors, CARs) tumor specific T cells therapy developed after, as first for solid tumor, The CAR-T cell therapy of targeting Her2/neu antigen enters clinical test, but due to generating strong cytokine storm (cytokine release syndrome, CRS) cause patient dead and be terminated (referring to document " Nature Med, 2016； 22:26").Serious cytokine storm and neurotoxicity are toxic reactions common in CAR-T treatment (referring to document "Blood,2016；127:3321 "), partly cause may be living with this non-natural unrestricted cell of T cell receptor of CAR Change it is related (referring to document " Nat Ned, 2015；21:581 "), or have with the autocrine for the cell factor for being not required to antigenic stimulus Close (referring to document " Cancer Immunol Res, 2015；3:356").

Adopt and transfers the TCR-T therapy of the T cell by specific t-cell receptor (i.e. TCR) gene modification and be considered as For solid tumor most prospect immunocyte gene therapy (referring to document " Adv Immunol.2016；130:279-94"). Wherein, the clinical studies show for targeting the TCR-T therapy of NY-ESO-1 antigen goes out encouraging clinical therapeutic efficacy (referring to text Offer " Nat Med.2015Aug；21(8):914-921").However, the target tumor antigen that is currently known and effectively identifying tumour The quantity of the specificity TCR of cell is extremely limited, therefore limits the extensive use of TCR-T therapy.In addition, although TCR-T is treated There is not the serious cytokine storm toxicity shown in CAR-T therapy in method, but if target antigen derives from itself Albumen is possible to will lead to serious autoimmune response for the target antigen of low expression in normal tissue cell, i.e. switching is de- Target (or middle target takes off tumor) (on target off tumor) toxic reaction (Blood 2009；114:535-546).In addition, being The high-affinity TCR of the effective tumor cell of acquisition, general strategy are in vitro by the complementarity-determining region on TCR (complementarity determing regions, CDRs) carries out point mutation, or by from resistance to without maincenter It is induced by the humanization mouse T cell library that mechanism is screened (referring to document " Front Immunol.2013；4:363"). However, this high-affinity TCR may generate cross reaction to normal self-protein and normal tissue cell is caused to kill Wound effect, i.e., serious or even fatal (off-target) toxicity of missing the target is (referring to document " Curr OpinImmunol 2015； 33:16-22 ", referring to document " Sci Transl Med.2013；5(197):197ra103").Therefore, it obtains selectively targeted Tumour antigen and effective tumor cell, while the novel tcr gene for the toxicity that misses the target for avoiding being likely to occur, are successes The significant challenge that exploitation TCR-T immunocyte gene therapy faces.

Summary of the invention

For solve it is above-mentioned the problems of in the prior art, the present invention provides isolated T cell receptors, its modification Cell, code nucleic acid, expression vector, preparation method, pharmaceutical composition and application.

Specifically, the present invention provides:

(1) a kind of isolated T cell receptor, including at least one of α chain and β chain, the α chain and β chain include can Become area and constant region, which is characterized in that the T cell receptor being capable of antigen Her2/ expressed by specific recognition tumour cell Neu, and the amino acid sequence of the variable region of the α chain has with amino acid sequence shown in SEQ ID NO:19 at least The amino acid sequence of 98% consistency, the variable region of the β chain has and amino acid sequence shown in SEQ ID NO:20 The consistency of column at least 98%.

(2) T cell receptor according to (1), wherein the T cell receptor can specific recognition by HLA-A2 points The antigen epitope polypeptide for the antigen Her2/neu that son is offered；Preferably, the antigen epitope polypeptide includes such as SEQ Her2/neu 369-377 shown in ID NO:18.

(3) T cell receptor according to (1), wherein the perseverance of the constant region of the α chain and/or the β chain Area is determined from people；Preferably, the constant region of the α chain is entirely or partly derived from the homologous sequence of other species It is replaced and/or the constant region of the β chain is entirely or partly replaced by the homologous sequence from other species It changes；It is highly preferred that other species are mouse.

(4) T cell receptor according to (1), wherein the constant region of the α chain is modified with one or more two sulphur The constant region of key and/or the β chain is modified with one or more disulfide bond.

(5) T cell receptor according to (1), wherein the amino acid sequence of the α chain such as SEQ ID NOs:2,6 or 10 Shown, the amino acid sequence of the β chain is as shown in SEQ ID NOs:4,8 or 12.

(6) a kind of separation, encoding T cell receptor nucleic acid, in α chain and β chain comprising the T cell receptor at least The coded sequence of one, the α chain coding sequence and β chain coding sequence include variable region encoding sequences and constant region code sequence Column, which is characterized in that the T cell receptor is capable of the antigen Her2/neu of specific recognition tumor cells expression, and the α The amino acid sequence of chain variable region encoding sequences coding has and amino acid sequence at least 98% shown in SEQ ID NO:19 The amino acid sequence of consistency, the β chain variable region encoding sequences coding has and amino acid sequence shown in SEQ ID NO:20 The consistency of column at least 98%.

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

(8) nucleic acid according to (6), wherein the α chain variable region encoding sequences are as shown in SEQ ID NO:21, it is described β chain variable region encoding sequences are as shown in SEQ ID NO:22.

(9) nucleic acid according to (6), wherein being capable of specific recognition by the T cell receptor of the nucleic acid encode The antigen epitope polypeptide of the antigen Her2/neu offered by HLA-A2 molecule；Preferably, the antigen epitope polypeptide Including the Her2/neu 369-377 as shown in SEQ ID NO:18.

(10) nucleic acid according to (6), wherein the α chain constant region coded sequence and/or β chain constant region coding Sequence derives from people；Preferably, the α chain constant region coded sequence is entirely or partly derived from the homologous sequence of other species Column are replaced and/or the β chain constant region coded sequence is entirely or partly derived from the homologous sequence of other species It is replaced；It is highly preferred that other species are mouse.

(11) nucleic acid according to (6), wherein the α chain constant region coded sequence includes that one or more disulfide bond are compiled Code sequence and/or the β chain constant region coded sequence include one or more disulfide bond coded sequences.

(12) nucleic acid according to (6), wherein the α chain coding sequence is as shown in SEQ ID NOs:1,5 or 9, it is described β chain coding sequence is as shown in SEQ ID NOs:3,7 or 11.

(13) nucleic acid according to any one of (6)-(11), wherein the α chain coding sequence and the β chain encoding sequence It is connected between column by the coded sequence of cuttability connecting peptides.

(14) nucleic acid according to (13), sequence is as shown in SEQ ID NOs:13,15 or 23.

(15) a kind of recombinant expression carrier contains any one of effectively connect with promoter, basis (6)-(14) institute The nucleic acid stated and/or its complementary series.

(16) a kind of cell of T cell receptor modification, surface T cell described in any one of (1)-(5) of the cell Receptor modification, wherein the cell includes naive T cell or its precursor, NKT cell or T cell strain.

(17) a kind of method for the cell for preparing the modification of the T cell receptor according to (16), comprising the following steps:

1) cell is provided；

2) nucleic acid of coding T cell receptor according to any one of (1)-(5) is provided；

3) nucleic acid transfection is entered in the cell.

(18) method according to (17), wherein cell described in step 1) is from self or allosome.

(19) method according to (17), wherein the mode of the transfection include: using viral vectors transfection by the way of, Preferably, the viral vectors includes γ retroviral vector or slow virus carrier；Chemical mode, it is preferred that described Chemical mode includes by the way of liposome transfection；Physics mode, it is preferred that the physics mode includes electrotransfection side Formula.

(20) method according to (17), wherein according to nucleic acid described in step 2) described in any one of (6)-(14) Nucleic acid.

(21) cell of the modification of the T cell receptor according to (16) is in preparation for treating or preventing tumour and/or cancer Purposes in the drug of disease.

(22) purposes according to (21), wherein the tumour and/or cancer are antigen Her2/neu positive, and It is the HLA-A2 positive.

(23) cell of the modification of the T cell receptor according to (16) is preparing tumour and/or cancer for detecting host Purposes in the drug of disease.

(24) a kind of pharmaceutical composition, wherein the pharmaceutical composition includes the T according to (16) as active constituent The cell and pharmaceutically acceptable auxiliaries of cell receptor modification.

(25) pharmaceutical composition according to (24), wherein described pharmaceutical composition includes that each course for the treatment of of each patient is total Dosage range is 1 × 10³-1×10⁹The cell of a cell/Kg weight T cell receptor modification.

(26) pharmaceutical composition according to (24), wherein described pharmaceutical composition be suitable for through artery, vein, it is subcutaneous, In intradermal, tumor, in lymphatic vessel, in lymph node, in cavum subarachnoidale, in marrow, intramuscular and Intraperitoneal medication.

(27) a kind of method for treating tumour and/or cancer, including tumour and/or cancer patient are applied according to (16) institute The cell for the T cell receptor modification stated.

(28) method according to (27), wherein the administration dosage of the cell of T cell receptor modification is each trouble The each course for the treatment of total dose range of person is 1 × 10³-1×10⁹A cell/Kg weight.

(29) method according to (27), wherein the cell of T cell receptor modification by artery, vein, it is subcutaneous, In intradermal, tumor, in lymphatic vessel, in lymph node, in cavum subarachnoidale, in marrow, intramuscular and Intraperitoneal medication.

(30) method according to (27), wherein the tumour and/or cancer are antigen Her2/neu positive, and It is the HLA-A2 positive.

Compared with the prior art, the present invention has the following advantages and good effect:

The present invention successfully induces the Her2/neu offered to HLA-A2 from the healthy donors peripheral blood of the HLA-A2 positive Antigen epitope polypeptide (such as Her2/neu 369-377 polypeptide) has the T cell clone of specificity, and is screened out from it and carries spy The T cell clone of the natural TCR of opposite sex identification Her2/neu antigen epitope polypeptide (such as Her2/neu 369-377 polypeptide), in turn Obtain the TCR complete sequence.This TCR belongs to CD8 molecule dependence, to Her2/neu antigen epitope polypeptide (such as Her2/neu 369-377 polypeptide) there is medium compatibility, can specific recognition HLA-A2 it is positive and to express the tumour of Her2/neu antigen thin Born of the same parents.In addition, the T cell clone for carrying this TCR enters periphery T cell library after the screening of Central immunotolerance mechanism.Carry this The killer T cell of TCR is once present in normal human peripheral blood, does not produce to the normal tissue cell of trace expression Her2/neu albumen Raw cross reaction.Therefore, present invention obtains can specific recognition tumour antigen, while can be avoided missing the target of being likely to occur The novel TCR of toxicity.

Also the constant region of TCR is transformed in further invention (such as carry out disulfide bond modification or source of mouse Transformation) so that this TCR can be further reduced or avoid the generation with endogenous TCR mispairing when expressing on immunocyte.

It can be special with the immunocyte (such as naive T cell, its precursor, NKT cell, T cell strain) that this TCR is modified Property identification a variety of HLA-A2⁺And Her2/neu⁺Tumor cell line, have significant antitumous effect.On the other hand, for repairing The TCR for adoring immunocyte will not generate cross reaction to the normal cell of trace expression Her2/neu.Therefore, based on this TCR's TCR-T therapy is expected to treat a variety of solid tumors.

When the immune cell therapy tumour modified with TCR of the invention, it can effectively avoid caused when being treated using CAR-T Cytokine storm and immunological rejection.

The immunocyte of TCR modification of the invention is treatment HLA-A2⁺And Her2/neu⁺Tumor patient provides a kind of new Selection, have good industrial application prospect.

Detailed description of the invention

Fig. 1 is shown in the embodiment of the present invention 1 from HLA-A2⁺It is induced in Normal donor PBMC (specially #2PBMC) The phenotype and Function detection result of Her2/neu 369-377 polypeptide (Her2-E75) specific killing T cell.Figure 1A is warp After crossing two-wheeled Her2-E75 antigen polypeptide stimulated in vitro, PBMC cell is dyed through CD8-APC antibody and Her2-E75 pentamer-PE After carry out flow cytometric analysis results, right figure is the cell stimulated through polypeptide, to CD8⁺Pentamer⁺Killer T cell group carries out FACS Sorting, to obtain T cell clone.Left figure is the cellular control unit of no polypeptide stimulation.Abscissa indicates the glimmering of CD8 developed by molecule Luminous intensity, ordinate indicate the fluorescence intensity of the Her2-E75 pentamer combined.Figure 1B is CD8⁺E75-tetramer⁺Killer T is thin The phenotypic analysis of born of the same parents clone fluidic cell after CD8-APC the and Her2-E75 tetramer-PE dyeing, right figure show CD8⁺Her2 tetra- Aggressiveness⁺T cell clone Her2CTL 1B5 is the Her2-E75 polypeptid specificity CTL cell clone of purifying.Left figure is no polypeptide The control group CTL cell of stimulation.Abscissa indicates the fluorescence intensity of CD8 developed by molecule, and ordinate indicates the Her2-E75 combined The fluorescence intensity of the tetramer.Fig. 1 C be T cell cloning function testing result, T cell clone Her2CTL 1B5 (column with slant lines figure) or The control CTL cell (point column figure) that person does not have polypeptide to stimulate is co-cultured with the various concentration Her-E75 polypeptide offered by T2 cell, Or and HLA-A2⁺Her2/neu⁺Colorectal cancer cell lines colo205 co-culture after, take cell conditioned medium to carry out IFN-γ Elisa assay, each test group and control group are multiple holes, are as the result is shown average value ± SD.Abscissa indicates different experiments group Not, ordinate indicates the concentration of T cell secretion of gamma-IFN.

Fig. 2 shows the slow virus carriers of two kinds of constructed carrying Her2TCR-1B5TCR genes (to be shown respectively as in figure " pCDH-EF1 α-Her2TCR- (PGK-GFP) carrier ", " pCDH-EF1 α-Her2TCR carrier ") major function segment.Top The segment that shows while the tcr gene that EF-1 α promoter is driven and the GFP gene that PKG promoter is driven are expressed, lower section shows Segment out only expresses tcr gene.The β chain and α chain of each TCR is by the coded sequence (furin- of cuttability connecting peptides F2A it) is linked.

Fig. 3 shows the phenotype and Function detection result of the T cell strain transfected through Her2TCR-1B5TCR gene.With coding The slow virus carrier of Her2TCR-1B5TCR and GFP transfects J.RT3-T3.5T cell strain (J.RT3), through the Her2-E75 tetramer- Flow cytometry is carried out after PE dyeing.GFP shown in Fig. 3 A⁺The Her2-E75 tetramer⁺Cell mass is expression Her2TCR- The cell of 1B5TCR, shown percentage are the ratio that each positive cell group accounts for total cell number.The TCR that left figure is related to is α chain and β chain Constant region increase a disulfide bond pattern (Her2TCR-1B5-dis), the TCR that right figure is related to is the constant of people α chain and β chain Area (Her2TCR-1B5-mC) is replaced by the homologous sequence of murine constant region.Abscissa indicates that the fluorescence of GFP developed by molecule is strong Degree, ordinate indicate the fluorescence intensity of the Her2-E75 tetramer combined.Fig. 3 B shows the constant region of TCR α chain and β chain through difference The expression of the T cell strain at two kinds after mode is modified." Her2TCR-1B5-dis " refers to that α chain and the constant region of β chain respectively increase in figure The TCR of a disulfide bond pattern is added；" Her2TCR-1B5-mC " refers to the constant region of people α chain and β chain by the same of murine constant region The TCR that source sequence is replaced.GFP⁺The Her2-E75 tetramer⁺Cell is the positive cell for expressing Her2TCR-1B5TCR, ordinate For the total GFP of TCR positive cell Zhan⁺The percentage of cell.Abscissa indicates different T cell strain groups, wherein " Jurkat (TCR A+b+) " referring to Jurkat cell, α chain and β chain are expressed, and " J.RT3 (TCRa+b-) " refers to J.RT3-T3.5 cell, From Jurkat cell, and β chain gene lacks, and α chain still has expression.Fig. 3 C shows encoded Her2TCR-1B5TCR gene Slow virus carrier transfection T cell strain can identify the Her2-E75 polypeptide offered by T2 cell.The perseverance of TCR α chain and β chain Determine area to modify through different modes, express the T cell strain of TCR and offers the T2 cell co-cultivation of various concentration Her2-E75 polypeptide 16 hours, flow cytometry was carried out after being dyed with anti-CD 6 9-PE antibody." J.RT3-Her2-1B5-dis " indicates expression in figure The J.RT3-T3.5 cell of Her2TCR-1B5-dis, " J.RT3-Her2-1B5-mC " indicate expression Her2TCR-1B5-mC's J.RT3-T3.5 cell, " Jurkat-Her2-1B5-dis " indicate the Jurkat cell of expression Her2TCR-1B5-dis, " Jurkat-Her2-1B5-mC " indicates the Jurkat cell of expression Her2TCR-1B5-mC.Abscissa indicates that T2 cell is offered Her2-E75 polypeptide concentration.Ordinate is CD69⁺The total GFP of cell Zhan⁺The percentage of cell.

Fig. 4 shows the phenotype and function of the peripheral blood mononuclear cells (PBMC) transfected through Her2TCR-1B5-mC tcr gene It can testing result.Fig. 4 A is to encode the slow virus carrier transfection of Her2TCR-1B5-mC from the PBMC of two different donors, warp The Her2-E75 tetramer-PE and anti-CD8-APC antibody carry out the result of flow cytometry after dyeing.First according to cellular morphology Lymphocyte populations, the Her2-E75 tetramer are separated with size⁺Cell mass is the cell for expressing Her2TCR-1B5TCR.Abscissa table Show the fluorescence intensity of CD8 developed by molecule, ordinate indicates the fluorescence intensity of the Her2-E75 tetramer combined.Shown percentage is Each positive cell group accounts for the ratio of the lymphocyte number separated.Left figure is related to peripheral blood mononuclear cells provided by a donor (#1PBMC), right figure are related to the PBMC (#2PBMC) that another different donor provides.CD8⁺The Her2-E75 tetramer⁺Cell is table Up to the killer T cell of Her2TCR-1B5-mC.CD8^-The Her2-E75 tetramer⁺Cell may be expression Her2TCR-1B5-mC CD4⁺T helper cell.The T cell that Fig. 4 B shows expression Her2TCR-1B5-mC, which can be identified, is offered by T2 cell Her2-E75 polypeptide.Two difference donor PBMC difference of the slow virus carrier transfection of encoded Her2TCR-1B5-mC and GFP With offer T2 cell co-cultivation 16 hours of various concentration gradient Her2-E75 polypeptide, take cell conditioned medium to carry out IFN-γ Elisa assay.Target cell is the EBV virus antigen polypeptide LMP2 426-434 for offering to combine HLA-A2 molecule in control group T2 cell." 0.1 μ g/ml of T2+Her2-E75 " indicates to offer the T2 groups of cells of the Her2-E75 polypeptide of 0.1 μ g/ml in figure, " 0.01 μ g/ml of T2+Her2-E75 " indicates to offer the T2 groups of cells of the Her2-E75 polypeptide of 0.01 μ g/ml, " T2+Her2-E75 0.001 μ g/ml " indicates to offer the T2 groups of cells of the Her2-E75 polypeptide of 0.001 μ g/ml, " 1 μ g/ml of T2+EBV-LMP " expression Offer the T2 groups of cells of the EBV virus antigen polypeptide LMP2 426-434 of 1 μ g/ml.Abscissa indicates the PBMC group of different donors Not, ordinate indicates the concentration of the IFN-γ of T cell secretion.Fig. 4 C shows the CD8 antibody blocking test result of T cell function. Wherein, the antigen that the #2PBMC and T2 cell of the slow virus carrier transfection of encoded Her2TCR-1B5-mC and GFP gene offer Anti-human CD8 antibody is added in polypeptide when co-culturing, whether the function of detection T cell secretion of gamma-IFN is suppressed." T2+Her2- in figure 0.1 μ g/ml " of E75 indicate be not added anti-human CD8 antibody, offer the T2 groups of cells of the Her2-E75 polypeptide of 0.1 μ g/ml, " T2 0.1 μ g/ml+anti-CD8 " of+Her2-E75 indicate to be added anti-human CD8 antibody, offer the Her2-E75 polypeptide of 0.1 μ g/ml T2 groups of cells.Abscissa indicates different experiments group, and ordinate indicates the concentration of the IFN-γ of T cell secretion.Fig. 4 B and 4C In each test group and control group be multiple holes, be as the result is shown average value ± SD.

Fig. 5 shows thin (PBMC) tumor cell of the single core of the peripheral blood transfected through Her2TCR-1B5-mC tcr gene The Function detection result of strain.Fig. 5 A shows the slow virus carrier transfection #2PBMC of coding Her2 TCR-1B5-mC tcr gene, with Different tumor cell line cell co-cultivations take cell conditioned medium to carry out the elisa assay result of IFN-γ after 16 hours.Each test Group and control group are multiple holes, are as the result is shown average value ± SD.Abscissa indicates different experiments group, and ordinate indicates that T is thin The concentration of the IFN-γ of intracrine.Fig. 5 B is shown in the culture hole of the targeting Colo205 cell of the #2PBMC after above-mentioned transfection respectively (" colo205 is shown as in figure after anti-CD8 antibody (being shown as " colo205+anti-CD8 " in figure) or anti-HLA-ABC antibody is added + anti-HLA-ABC "), the function blocking test result of progress.Abscissa indicates different tumor cell line groups, ordinate table Show the concentration of the IFN-γ of T cell secretion.Colo205 and Coca-2 is HLA-A2 positive Her2/neu positive colon cancer cell, MAD-MB-231 is HLA-A2 positive Her2/neu positive breast cancer cells, and H647 is HLA-A2 feminine gender Her2/neu positive lung cancer Cell, H1355 are HLA-A2 positive Her2/neu positive lung cancer cells, and SK-OV-3 is HLA-A2 feminine gender Her2/neu positive ovum Nest cancer cell, Bjab are HLA-A2 positive Her2/neu feminine gender lymphoma cell.

Specific embodiment

Description below by way of specific embodiment and the invention will be further described referring to attached drawing, but it is pair that this, which is not, Limitation of the invention, those skilled in the art's basic thought according to the present invention, can make various modifications or improvements, but only Basic thought of the invention is not departed from, is all within the scope of the present invention.

In the present invention, word " tumour ", " cancer ", " tumour cell ", " cancer cell ", " T cell ", " T cell receptor ", " T cell receptor modification ", " variable region TCR ", " antigen ", " antigen epitope polypeptide ", " homologous sequence ", " are compiled " TCR constant region " Code ", " antigen is offered ", " recombinant dna expression vector ", " promoter ", " complementary series ", " transfection ", " self ", " allosome ", " spy Opposite sex identification ", " TCR-T therapy " cover the meaning that this field has been generally acknowledged that.

Her2/neu antigen belongs to tumor associated antigen, identifies the high-affinity T cell majority of Her2/neu antigen by Pivot tolerance mechanism is removed, to avoid cause possible autoimmune response (referring to document " Immunol Rev.2016；271 (1):127-40").Therefore, it is induced from periphery blood T cell library with Her2/ expressed by specific recognition tumour cell The T cell clone of neu antigen becomes very difficult.Offer Her2/neu using Dendritic Cells (Dendritic cell) 369-377 polypeptide antigen, and then the height induced from the peripheral blood in patients that Her2/neu 369-377 polypeptide vaccine was immunized Compatibility TCR is although (exogenously loaded) the Her2/neu 369-377 that can identify that very low amount external source is loaded is more Peptide, but endogenous (endogenously presented) antigen polypeptide offered of tumor cell is unable to (referring to document "CancerRes.1998；58:4902–4908").This may be polypeptide/HLA compound due to external source load in configuration (conformation) the HLA/ polypeptide complex offered naturally on cell interior is different and causes, or due to Her2/neu 369-377 polypeptide is located at Her2 albumen height saccharification area, the Her2/neu 369-377 that cell interior is deducted a percentage naturally Polypeptide may be glycosylated and cause the difference of TCR identification configuration (referring to document " Proc.Natl.Acad.Sci.USA 2003； 100:15029–15034").In vitro by being only capable of identifying during Her2/neu 369-377 antigen polypeptide inducing T cell The high-affinity T cell clone of external source Antigen polypeptide often obtains dominant growth (dominant expansion), and energy Specific recognition is suppressed by the T cell clonal growth for the endogenous Her2/neu antigen polypeptide that cell is deducted a percentage (referring to document "J Exp Med.2016Nov 14；213 (12): 2811-2829 "), thus increase the function of obtaining and can recognize tumour cell The difficulty of property TCR.After the screening of central tolerance mechanism, the TCR of these tumor cells belongs to medium compatibility, function more The booster action of CD8 molecule can be tended to rely on.There is research group to induce allogeneic T cells from the peripheral blood of HLA-A2 feminine gender (Allo-T cells), the Her2/neu 369-377 antigen polypeptide that can be limited with specific recognition HLA-A2, with the TCR of acquisition After gene transfecting T cells, the Her2/neu 369-377 antigen polypeptide that can be not only deducted a percentage with tumor cell can also intersect knowledge Not with Her3 the and Her4 epitope of family (referring to document " Journal of Immunology, 2008,180:8135- 8145").It generates however, the TCR-T therapy based on allosome allo-TCR exists for other normal self-protein epitopes The risk of simplified reaction (allo-reaction) is (referring to document " Int.J.Cancer 2009；125,649–655.Nat Immunol 2007；8:388–97").Another research group was immunized swollen from Her2/neu 369-377 polypeptide vaccine Her2/neu 369-377 polypeptid specificity T cell is induced in tumor peripheral blood in patients, and from different T cells Alpha and beta chain is matched and is filtered out a high-affinity TCR, and the T cell for transfecting this high-affinity TCR can recognize HLA-A2⁺Her2/neu⁺Kinds of tumor cells (referring to document " HUMAN GENE THERAPY 2014；25:730–739"). This TCR is not to obtain from monoclonal T cell, therefore not can determine that whether this TCR is to be present in screen by central tolerance T cell library natural TCR.Her2/neu albumen also has trace expression in cardiac muscle, lung, esophagus, kidney, bladder these important organs (referring to document " Oncogene.1990Jul；5 (7): 953-62 "), therefore it is based on high-affinity Her2/neu antigentic specificity The risk that the TCR-T therapy of TCR has normal tissue to generate the toxic reaction that misses the target.

Tumour cell height expresses Her2/neu albumen, therefore cell surface also can by the quantity of the HLA antigen polypeptide offered It increase accordingly, the difference of HLA/ antigen polypeptide complex populations can become specific T-cells on tumour cell and normal cell Distinguish normal and tumor tissues windows.Present invention proposition is obtained from Autologous T cells library (auto-T cell repertoire) The sequence of natural TCR is obtained, and then makes TCR expression in T cell in vitro, so that the T cell of obtained expression TCR can recognize Tumour cell increases the Her2/neu antigen of expression, is the key that the TCR-T therapy of successfully exploitation effectively low toxicity.

In order to obtain can specific recognition tumour antigen, while can be avoided the toxicity that misses the target being likely to occur TCR, the present invention induce the Her2/neu 369-377 offered to HLA-A2 more from the healthy donors peripheral blood of the HLA-A2 positive Peptide has the T cell clone of specificity, and is screened out from it and carries to Her2/neu 369-377 polypeptide with medium compatibility Natural TCR T cell clone.This is different from other research groups and was immunized from by Her2/neu 369-377 polypeptide vaccine Blood of cancer patients induction Her2/neu 369-377 polypeptid specificity T cell strategy (referring to document " HUMAN GENE THERAPY 2014,25:730-739 "), it is considered herein that after Her2/neu 369-377 antigen polypeptide is immune, for Specific T cells clone's meeting advantage proliferation of Her2/neu 369-377 polypeptide, thus internal T cell library cannot be represented (repertoire) specificity T for the Her2/neu 369-377 polypeptide antigen that the recognizable target cell of naturally occurring is offered in Cell mass.The present invention does not take other research groups inducing polypeptide specific T-cells from HLA-A2 feminine gender peripheral blood yet Mode (referring to document " The Journal of Immunology, 2010,184:1617-1629 "), although from allosome PBMC The allo-T cell of the identification Her2/neu 369-377 polypeptide antigen of high-affinity is more easily obtained, but this is also increased simultaneously T cell intersects simplified reaction caused by other polypeptides that identification is offered by HLA-A2 molecule.

Based on above-mentioned design, the present invention provides a kind of isolated T cell receptors, including at least one in α chain and β chain Person, the α chain and β chain include variable region and constant region, which is characterized in that the T cell receptor can specific recognition it is swollen Antigen Her2/neu expressed by oncocyte, and the amino acid sequence of the variable region of the α chain has and SEQ ID The consistency of amino acid sequence shown in NO:19 at least 98%, preferably at least 98.5%, more preferably at least 99%, the β chain The amino acid sequence of the variable region has and amino acid sequence at least 98% shown in SEQ ID NO:20, preferably at least 98.5%, more preferably at least 99% consistency, as long as not significantly affecting effect of the invention.It is also preferred that the α The amino acid sequence of the variable region of chain is as shown in SEQ ID NO:19, the amino acid sequence of the variable region of the β chain As shown in SEQ ID NO:20.

The variable region of TCR α chain and β chain is used to combine antigen polypeptide/major histocompatibility complex (MHC I), respectively It including three hypervariable regions or is complementary determining region (complementarity determining regions, CDRs), that is, CDR1,CDR2,CDR3.The antigen polypeptide that wherein specific recognition is offered in the region CDR3 by MHC molecule is most important.TCR α chain It is different V and J genetic fragment to recombinate, β chain is then different V, D and J genetic fragment and recombinates.Specific gene segment Recombination combines the nucleotide (palindromic for being formed by the corresponding region CDR3 and the bond area palindrome and radom insertion And random nucleotide additions) specificity that TCR identifies antigen polypeptide is formd (referring to document “Immunobiology:The immune system in health and disese.5^th editin,Chapter 4,The generation of Lymphocyte antigen receptors").The MHC I class molecule includes people HLA.The HLA It include: HLA-A, B, C.

Further specifically, the T cell receptor can specific recognition offered by HLA-A2 molecule it is described anti- The antigen epitope polypeptide of former Her2/neu.The amino acid sequence of antigen Her2/neu is as shown in SEQ ID NO:17.Preferably, The antigen epitope polypeptide includes the Her2/neu 369-377 as shown in SEQ ID NO:18.The expression of HLA-A2 positive cell HLA-A2 allele includes HLA-A*0201,0202,0203,0204,0205,0206 and 0207.Preferably, the HLA- A2 molecule is preferably HLA-A*0201.

In one embodiment, the antigen epitope polypeptide of the antigen Her2/neu is Her2/neu 369-377 polypeptide (SEQ ID NO:18).In other embodiments, the antigen epitope polypeptide of the antigen Her2/neu is and Her2/neu 369-377 polypeptide has the anti-of 4-9 continuous same amino acids (for example, 4,5,6,7,8 or 9 continuous same amino acids) Former epitope polypeptide, and the length of these polypeptides is 8-11 amino acid.For example, in one embodiment, the antigen The antigen epitope polypeptide of Her2/neu is Her2/neu 373-382 polypeptide (SEQ ID NO:25).

Preferably, the maximum half-reaction peptide concentration of the T cell receptor identification Her2/neu 369-377 polypeptide exists Between 1.0-10ng/ml (for example, between 3.0-8.0ng/ml, 5.0-7.0ng/ml).In one embodiment of the invention In, the maximum half-reaction peptide concentration is about 6.9ng/ml.Term " maximum half-reaction peptide concentration " refers to that inducing T cell is anti- The concentration of polypeptide needed for the 50% of maximum value should be reached.It is reported that being directed to cytomegalovirus (CMV) antigens c MV pp65 (495- 503) the maximum half-reaction peptide concentration of the specific T-cells of polypeptide is between 0.1-1ng/ml, and this TCR is more to CMV antigen Peptide is considered to have high-affinity (referring to document " Journal of Immunogical Methds 2007；320:119- 131").In the present invention, the T cell receptor has medium compatibility to Her2/neu antigen, to can avoid high-affinity Possible toxicity of missing the target.

The external source TCR α chain and β chain of T cell expression are possible to that mispairing occurs with the α chain of T cell TCR itself and β chain, not only The expression quantity of the external source TCR correctly matched can be diluted, the antigentic specificity of mispairing TCR is also indefinite, thus has identification itself anti- Former potential danger, therefore preferably modify the constant region of TCR α chain and β chain mispairing is reduced or avoided.

In one embodiment, the constant region of the α chain and/or the constant region of the β chain derive from people； Preferably, present invention discover that the constant region of the α chain can entirely or partly be derived from the homologous sequence of other species Column are replaced and/or the constant region of the β chain can entirely or partly be derived from the homologous sequence of other species Column are replaced.It is highly preferred that other species are mouse.The replacement can increase the expression quantity of TCR in cell, and can To further increase the cell modified by the TCR to the specificity of Her2/neu antigen.

The constant region of the α chain can be modified with the described of one or more disulfide bond and/or the β chain Constant region can be modified with one or more disulfide bond, such as 1 or 2.

In a particular embodiment, it is prepared for the TCR of two kinds of different modes modification, Her2 TCR-1B5-dis is to pass through Point mutation increases a disulfide bond in TCR constant region, and method is in document " Cancer Res.2007Apr 15；67(8):3898- Description in 903. ", full text is incorporated herein by reference.Her2TCR-1B5-mC is set with mouse TCR constant-region sequences Corresponding people TCR constant-region sequences are changed, method describes in document " Eur.J.Immunol.2006 36:3052-3059 ", Full text is incorporated herein by reference.

In specific embodiments, the amino acid sequence of the α chain is as shown in SEQ ID NOs:2,6 or 10, the β The amino acid sequence of chain is as shown in SEQ ID NOs:4,8 or 12.

Wherein, for amino acid sequence α chain as shown in SEQ ID NO:2, sequence is original source of people sequence；For Amino acid sequence α chain as shown in SEQ ID NO:6 is modified with 1 disulfide bond in constant region；Such as amino acid sequence α chain shown in SEQ ID NO:10, constant region replace with source of mouse constant region.

Wherein, for amino acid sequence β chain as shown in SEQ ID NO:4, sequence is original source of people sequence；For Amino acid sequence β chain as shown in SEQ ID NO:8 is modified with 1 disulfide bond in constant region；Such as amino acid sequence β chain shown in SEQ ID NO:12, constant region replace with source of mouse constant region.

In a specific embodiment, the amino acid sequence of the α chain of the TCR is as shown in SEQ ID NO:2, β chain Amino acid sequence is as shown in SEQ ID NO:4.In another embodiment, the amino acid sequence of the α chain of the TCR is such as Shown in SEQ ID NO:6, the amino acid sequence of β chain is as shown in SEQ ID NO:8.It is described in another specific embodiment The amino acid sequence of the α chain of TCR is as shown in SEQ ID NO:10, and the amino acid sequence of β chain is as shown in SEQ ID NO:12.

In the other specific embodiments of the present invention, the α chain of the TCR has shown in SEQ ID NOs:2,6 or 10 Amino acid sequence obtained from one or more amino acid is replaced, deletes, and/or added in amino acid sequence；For example, the α Chain has and amino acid sequence shown in SEQ ID NOs:2,6 or 10 at least 90%, preferably at least 95%, more preferably at least 99% Consistency.

In the other specific embodiments of the present invention, the β chain of the TCR has shown in SEQ ID NOs:4,8 or 12 Amino acid sequence obtained from one or more amino acid is replaced, deletes, and/or added in amino acid sequence；For example, the β Chain has and amino acid sequence shown in SEQ ID NOs:4,8 or 12 at least 90%, preferably at least 95%, more preferably at least 99% Consistency.

The α chain and/or β chain of TCR of the invention can also combine other functional sequences, example at end (such as C-terminal) Such as the function region sequence of costimulatory signal CD28,4-1BB and/or CD3zeta.

The present invention also provides a kind of separation, encoding T cell receptor nucleic acid, the α chain comprising the T cell receptor and The coded sequence of at least one of β chain, the α chain coding sequence and β chain coding sequence include variable region encoding sequences and Constant region coded sequence, which is characterized in that the T cell receptor is capable of the antigen Her2/ of specific recognition tumor cells expression Neu, and the amino acid sequence of α chain variable region encoding sequences coding has and amino acid sequence shown in SEQ ID NO:19 The consistency of column at least 98%, preferably at least 98.5%, more preferably at least 99%, the β chain variable region encoding sequences coding Amino acid sequence have with amino acid sequence at least 98% shown in SEQ ID NO:20, preferably at least 98.5%, more preferably extremely Few 99% consistency, as long as not significantly affecting effect of the invention.It is also preferred that α chain variable region code sequence Column coding amino acid sequence as shown in SEQ ID NO:19, β chain the variable region encoding sequences coding such as SEQ ID NO:20 Shown in amino acid sequence.

The nucleic acid can be DNA or RNA.

Preferably, the α chain variable region encoding sequences are as shown in SEQ ID NO:21, the β chain variable region encoding sequences As shown in SEQ ID NO:22.

Further specifically, by the T cell receptor of the nucleic acid encode can specific recognition by HLA-A2 molecule The antigen epitope polypeptide of the antigen Her2/neu offered.

In one embodiment, the antigen epitope polypeptide of the antigen Her2/neu is Her2/neu 369-377 polypeptide (SEQ ID NO:18).In other embodiments, the antigen epitope polypeptide of the antigen Her2/neu is and Her2/neu 369-377 polypeptide has the anti-of 4-9 continuous same amino acids (for example, 4,5,6,7,8 or 9 continuous same amino acids) Former epitope polypeptide, and the length of these polypeptides is 8-10 amino acid.For example, in one embodiment, the antigen The antigen epitope polypeptide of Her2/neu is Her2/neu 373-382 polypeptide (SEQ ID NO:25).

Preferably, by the nucleic acid encode the T cell receptor identification Her2/neu 369-377 polypeptide maximum half Peptide concentration is reacted between 1.0-10ng/ml (for example, between 3.0-8.0ng/ml, 5.0-7.0ng/ml).In the present invention An embodiment in, it is described maximum half-reaction peptide concentration be about 6.9ng/ml.In the case, the T cell receptor There is medium compatibility to Her2/neu antigen, can avoid the possible toxicity of missing the target of high-affinity.

In one embodiment, the constant region of the α chain and/or the constant region of the β chain derive from people； Preferably, the α chain constant region coded sequence is entirely or partly replaced by the homologous sequence from other species, and And/or β chain constant region coded sequence described in person is entirely or partly replaced by the homologous sequence from other species.It is more excellent Selection of land, other species are mouse.The replacement can increase the expression quantity of TCR in cell, and can be further improved Specificity of the cell modified by the TCR to Her2/neu antigen.

The α chain constant region coded sequence may include the coded sequence of one or more disulfide bond and/or described β chain constant region coded sequence may include the coded sequence of one or more disulfide bond.

In specific embodiments, the α chain coding sequence is as shown in SEQ ID NOs:1,5 or 9, the β chain encoding Sequence is as shown in SEQ ID NOs:3,7 or 11.

Wherein, for coded sequence α chain as shown in SEQ ID NO:1, sequence is original source of people sequence；For compiling Code sequence α chain as shown in SEQ ID NO:5, is modified with 1 disulfide bond in constant region；For coded sequence such as SEQ ID α chain shown in NO:9, constant region replace with source of mouse constant region.

Wherein, for coded sequence β chain as shown in SEQ ID NO:3, sequence is original source of people sequence；For compiling Code sequence β chain as shown in SEQ ID NO:7, is modified with 1 disulfide bond in constant region；For coded sequence such as SEQ ID β chain shown in NO:11, constant region replace with source of mouse constant region.

In a specific embodiment, the coded sequence of the α chain of the TCR is as shown in SEQ ID NO:1, the volume of β chain Code sequence is as shown in SEQ ID NO:3.In another embodiment, the coded sequence of the α chain of the TCR such as SEQ ID Shown in NO:5, the coded sequence of β chain is as shown in SEQ ID NO:7.In another specific embodiment, the α chain of the TCR Coded sequence is as shown in SEQ ID NO:9, and the coded sequence of β chain is as shown in SEQ ID NO:11.

In a further embodiment, it is connected between the α chain coding sequence and the β chain coding sequence by cuttability The coded sequence of polypeptide connects, and can increase the expression of TCR in the cell in this way.Term " cuttability connecting peptides " refers to this Polypeptide plays connection function, and can be cut by specific digestion, or encode this polypeptide nucleic acid sequence pass through ribosomes jump Jump mode (ribosome skipping) is translated, so that the polypeptide connected by it be made to be separated from each other.Cuttability connection is more The example of peptide is known in the art, such as F2A polypeptide, F2A polypeptide sequence include but is not limited to come from miRNA disease Poison F2A polypeptide and come from the similar 2A class sequence of other viruses.In addition, cuttability connecting peptides also include can quilt The tetramino acidic group sequence (canonical four amino acid motif) for the standard that Furin digestion is cut, i.e. R-X- [KR]-R Amino acid sequence.The encoded TCR of the embodiment is single-chain chimeric T cell receptor, which has expressed Cheng Hou, the cuttability connecting peptides for connecting α chain and β chain can be cut by the certain enzyme in cell, to form the free α of equivalent Chain and β chain.

The α chain and β chain for forming single-chain chimeric TCR can also be as described above, and constant region (and its corresponding coded sequence) is complete Portion is partly replaced by the homologous sequence from other species, and/or is modified with (coding) one or more two Sulfide linkage.

In specific embodiments, the sequence of the nucleic acid is as shown in SEQ ID NOs:13,15 or 23.

Preferably, the nucleotide sequence of the nucleic acid is carried out encoding sub- optimization to increase gene expression, protein translation effect Rate and protein expression, to enhance the ability of TCR identification antigen.Coding optimization includes but is not limited to translation starting region Modification changes mRNA structure fragment and the different codons using coding with monoamino-acid.

In other embodiments, the sequence of above-mentioned TCR code nucleic acid can be mutated, including removes, is inserted into And/or replace one or more amino acid codes so that the function of expressed TCR identification Her2/neu antigen it is constant or Enhancing.For example, in one embodiment, conservative amino acid replacement is carried out, including can be changed to above-mentioned TCR α chain and/or β chain An amino acid structure and/or another similar amino acid of chemical attribute in area are replaced.Term " similar amino Acid " refers to the amino acid residue with attributes such as similar polarity, electric load, solubility, hydrophobicity, hydrophilies.After mutation TCR still has the bioactivity for identifying the above-mentioned Her2/neu antigen polypeptide offered by target cell.In another embodiment, Carry out TCR maturity (TCR maturation) modification, that is, including the complementation in the variable region to above-mentioned TCR α chain and/or β chain Determine that the amino acid in area 2 (CDR2) and/or the region CDR3 is removed, is inserted into and/or replaces, to change TCR combination Her2/ The compatibility of neu antigen.

The present invention also provides a kind of separation, the mRNA that is transcribed by DNA according to the present invention.

The present invention also provides a kind of recombinant expression carrier, contain effectively connect with promoter it is described according to the present invention Nucleic acid (such as DNA) and/or its complementary series.

Preferably, in the recombinant expression carrier, DNA of the present invention suitably with promoter, enhancer, termination Son and/or polyA signal sequence effectively connect.

The combination of the above-mentioned functional element of recombinant expression carrier of the invention can promote the transcription and translation of DNA, and increase The stability of strong mRNA.

The basic framework of recombinant expression carrier can be any of expression vector, including plasmid or virus, and virus carries Body includes but is not limited to (for example) retroviral vector (Prototype is moloney murine leukemia virus (MMLV)) and slowly disease Poisonous carrier (Prototype is that human immune deficiency I type is viral (HIV)).The recombinant vector for expressing TCR of the present invention can lead to The recombinant DNA technology of this field routine is crossed to obtain.

In one embodiment, the expression of the α chain on recombinant expression carrier and β chain gene different can be opened by two Mover is driven, and promoter includes various known types, for example, strongly expressed, weak expression, derivable, tissue specificity And differentiation specificity promoter.Promoter can be viral source or non-viral source, such as CMV promoter, Promoter, EF1- α promoter and PGK-1 promoter on the LTR of MSCV.The driving direction of two promoters can be in the same direction It is also possible to reversed.

In another embodiment, the expression of the α chain and β chain gene on recombinant expression carrier can be by the same starting Son is driven, such as the case where encode single-chain chimeric T cell receptor, the nucleotide sequence of the nucleotide sequence of α chain and β chain by Furin-F2A polypeptid coding sequence is connected.

In other embodiments, recombinant expression carrier can also include other other than comprising α chain and β chain gene The coded sequence of functional molecular.One embodiment include expression autofluorescence albumen (such as GFP or other fluorescins) with In internal Tracking Imaging.Another embodiment includes expressing derivable suicide gene system, such as the simple blister of inducing expression Exanthema virus-thymidine kinase (HSV-TK) albumen or inducing expression Caspase 9 (iCasp9) albumen.Express this " safe shifting molecular " (safety-switch), which can increase the cell modified through tcr gene of the present invention in vivo, a bit makes Safety.Another embodiment includes expression people CD8 gene, including expresses CD8 α chain and β chain alone or in combination, through this The ability that its specific recognition Her2/neu antigen can be enhanced after the cell expression CD8 molecule of tcr gene modification is invented, Or make CD8 negative T cell (such as CD4⁺T helper cell) obtain specific recognition Her2/neu antigen ability.Another is implemented Scheme includes expression human chemokine receptor gene, such as CCR2, these chemokine receptors are in combination with table high in tumor tissues The corresponding chemokine ligand reached, so as to increase the cell modified through tcr gene of the present invention in tumor tissues It goes back to the nest.

The present invention also provides a kind of cell of T cell receptor modification, the T cell that the surface of the cell is described by the present invention Receptor modification, wherein the cell includes naive T cell or its precursor, NKT cell or T cell strain.

" modification " in " the T cell receptor modification " refers to, has cell expression by gene transfection of the present invention T cell receptor, that is, the T cell receptor is anchored on the cell membrane of modified cell by transmembrane region, and have identification Antigen polypeptide/MHC compound function.

The present invention also provides a kind of methods of cell for preparing T cell receptor modification according to the present invention, including Following steps:

1) cell is provided；

2) nucleic acid for encoding T cell receptor of the present invention is provided；

3) nucleic acid transfection is entered in the cell.

Cell described in step 1) can derive from mammal, including people, dog, mouse, rat and its transgenic animals. The cell can come from self or allosome.Variant cell includes cell from identical twin, allosome stem cell, through gene The allogeneic T cells of transformation.

Cell described in step 1) includes naive T cell or its precursor, NKT cell or T cell strain.Term is " original T cell (naive T cell) " refers to the mature T cells not yet activated by corresponding antigens in peripheral blood.These cells can lead to It is isolated to cross methods known in the art.For example, T cell can be obtained from different histoorgans, including peripheral blood, marrow, Lymphoid tissue, spleen, Cord blood, tumor tissues.In one embodiment, T cell be can come from candidate stem cell (HSCs), packet It includes from marrow, peripheral blood or Cord blood, acquisition is separated by stem cell labeling molecule such as CD34.One embodiment In, T cell can come from inductivity versatile stem cell (iPS cells), including specific gene or specific gene product are led Enter body cell, after making the somatic cell transformation stem cell, Differentiation Induction in vitro is at T cell or its precursor.T cell can lead to It crosses common method such as density-gradient centrifugation method and obtains, the example of density-gradient centrifugation method includes Ficoll or Percoll close Degree centrifugation.One embodiment is to utilize Apheresis (apheresis) or leucocyte removal method (leukapheresis) product of the T cell of enrichment is obtained from peripheral blood.One embodiment is to mark specific cells with antibody After group, by way of Beads enrichment (such asSystem (Miltenyi Biotec)) or fluidic cell separation Mode obtain the CD8 of enrichment⁺Or CD4⁺T cell.

Preferably, the T cell precursor is candidate stem cell.It can be straight by the encoding gene of TCR of the present invention Introducing candidate stem cell is connect, then transfers to internal, is further differentiated into as mature T cells；Encoding gene can also be introduced In vitro in the T cell under specified conditions by candidate stem cell differentiation and maturation.

The cell can be resuspended in freeze to be placed in liquid nitrogen in solution and save.The common solution that freezes includes but unlimited packet Containing 10% (v/v) DMSO and 90% (v/v) human serum or fetal calf serum.Cell is frozen with the condition for reducing by 1 DEG C of temperature per minute In -80 DEG C, it is then stored in the gas phase portion of liquid nitrogen container.Other cryopreservation methods be the cell for being placed in frozen stock solution is directly placed into- It is frozen in 80 DEG C or liquid nitrogen.

Nucleic acid described in step 2) is according to nucleic acid of the present invention, including the DNA and RNA.

The transfection includes physics mode, biological mode and chemical mode.Physics mode is by calcium phosphate precipitation, lipid The approach such as body, microinjection, electroporation, particle gun are imported tcr gene in the form of DNA or RNA into the cell.Existing business at present The instrument of change, including electrotransfer instrument (such as Amaxa Nucleofector-II (German Amaxa Biosystems company), ECM 830 (BTX) (U.S. Harvard Instruments), Gene Pulser II (BioRad company, the U.S.), Multiporator (German Eppendort company).Biological mode is tcr gene to be introduced intracellular, retrovirus by DNA or RNA carrier Carrier (such as γ retroviral vector) is to transfect and be inserted into exogenous genetic fragment to the normal of zooblast (including people's cell) With tool, other viral vectors derive from slow virus, poxvirus, herpes simplex virus, adenovirus and adeno-associated virus (AAV) Deng.Chemical mode be polynucleotides are introduced intracellular, including colloidal dispersion systems, such as macromolecular complexes, nanocapsules, Microsphere, microballon, micelle and liposome.No matter how tcr gene introduced cell, is analyzed with various detection methods Whether target gene introduces in target cell, the detection method include common molecular biology method (such as Southern print Mark and Northern trace, RT-PCR and PCR etc.) or common biochemical method (such as ELISA and Western print Mark) and the present invention mentioned by method.

Preferably, the transfection is carried out by retroviral vector or slow virus carrier.

The culture of the cell can be carried out according to practical application by its respective conventional method and condition after transfection.Example Such as, it after T cell is activated jointly by the TCR/CD3 complex and costimulatory molecules (such as CD28) on surface, can get external Amplification.The stimulant (such as antibody of anti-tcr, CD3 or CD28) of activation TCR, CD3 and CD28 can be adsorbed on culture vessel table Face or coculture (such as magnetic bead) surface, can also be directly added into co-incubation in cell culture fluid.Another embodiment party Case is by T cell and trophocyte co-incubation, and the trophocyte expresses costimulatory molecules or corresponding ligand, including But it is not limited to HLA-A2, β2-microglobulin, CD40, CD83, CD86, CD127,4-1BB.

According to the method for common mammalian cell in vitro culture, T cell training is cultivated under appropriate condition of culture And amplification.For example, cell can be passed on when reaching 70% or more Fusion Strain (confluence), renew within general 2 to 3 days fresh Culture solution.It directly uses when cell reaches certain amount, or is frozen as described above.The time of in vitro culture can be Within 24 hours, it can also be up to 14 days or longer.Freeze-stored cell can carry out next step application after thawing.

In one embodiment, cell can cultivate a few hours by 14 days in vitro, or between any hourage.T Cell culture condition includes using basic culture solution, including but not limited to RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F- 12, X-Vivo 15 and X-Vivo.Other cells survivals and be proliferated required for condition include but is not limited to use serum (people or Fetal calf serum), proleulzin (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL- 21, TGF-β and TNF-a, other culture additives (including amino acid, Sodium Pyruvate, vitamin C, 2 mercapto ethanol, growth swash Element, growth factor).Cell can be placed in condition of culture appropriate, for example, temperature can be at 37 DEG C, 32 DEG C, 30 DEG C or room temperature, And air conditions can be for (for example) containing 5%CO₂Air.

The present invention also provides the cell of T cell receptor according to the present invention modification in preparation for treating or preventing Purposes in the drug of tumour and/or cancer.

The tumour and/or cancer are antigen Her2/neu positive, and are the HLA-A2 positives, including but not limited to Breast cancer, oophoroma, gastric cancer, the cancer of the esophagus, intestinal cancer, cancer of pancreas, bladder cancer, kidney, prostate cancer, cervix cancer, endometrium Cancer, salivary-gland carcinoma, cutaneum carcinoma, lung cancer, osteocarcinoma and the cancer of the brain.

The present invention also provides the cell of T cell receptor according to the present invention modification in preparation for detecting host's Purposes in the drug of tumour and/or cancer.

It in one embodiment of the invention, can be by the sample of the tumour and/or cancer cell taken out from host and this The cell of the invention T cell receptor modification is contacted with a certain concentration, may determine that institute according to the extent of reaction of the two It states tumour and/or cancer is that HLA-A2 is positive or HLA-A2 is negative, and whether expresses antigen Her2/neu.

The present invention also provides a kind of pharmaceutical compositions, and wherein the pharmaceutical composition includes as active constituent according to this The cell and pharmaceutically acceptable auxiliaries of the invention T cell receptor modification.

It is 1 × 10 that described pharmaceutical composition, which preferably comprises each course for the treatment of total dose range of each patient,³-1×10⁹A cell/ The cell of the T cell receptor modification of Kg weight, including any amount of cell between two endpoints.Preferably, often A course for the treatment of 1-3 days, daily application 1-3 times.Can according to the actual situation with need to carry out one or more courses for the treatment of to patient to control It treats.

The pharmaceutically acceptable auxiliaries include medicinal or physiological carriers, excipient, diluent (including physiological saline, PBS solution), And various additives, including carbohydrate, lipid, polypeptide, amino acid, antioxidant, adjuvant, antistaling agent etc..

Described pharmaceutical composition can be administered by suitable administration route, be suitable for through artery, vein, subcutaneous, intradermal, tumor In interior, lymphatic vessel, in lymph node, in cavum subarachnoidale, in marrow, intramuscular and Intraperitoneal medication.

The present invention also provides a kind of methods for treating tumour and/or cancer, including apply to tumour and/or cancer patient The cell of T cell receptor modification according to the present invention.

It is 1 that the administration dosage of the cell of the T cell receptor modification, which is preferably each course for the treatment of total dose range of each patient, ×10³-1×10⁹A cell/Kg weight.Preferably, each course for the treatment of 1-3 days, daily application 1-3 times.It can be according to practical feelings Condition and the treatment for needing to carry out patient one or more courses for the treatment of.

The cell of the T cell receptor modification can be administered by suitable administration route, be suitable for through artery, vein, skin Under, in intradermal, tumor, in lymphatic vessel, in lymph node, in cavum subarachnoidale, in marrow, intramuscular and Intraperitoneal medication.

The cell of the T cell receptor modification can eliminate the swollen of expression Her2/neu antigen after entering in treatment object body Oncocyte, and/or change the microenvironment of tumor tissues and induce other anti tumor immune responses.

It is used to detect T cell (the i.e. TCR-T for receiving TCR modification the present invention also provides the isolated T cell receptor Cell) treatment patient's body the TCR-T cell proliferation or Survival application, to carry out drug metabolism study, With the curative effect and toxicity for understanding the TCR-T cell.Specifically, TCR sequence can be used as primer, detected by PCR method internal Carry the quantity of the TCR-T cell of this TCR.Flow cytometry is used with after the HLA/ polypeptide complex polymer dyeing of fluorescent marker The method analyzed is compared, and cell concentration required for the application is few, also more sensitive.

The contents of the present invention are further explained and described below by way of the mode of example, but these examples are understood not to Limitation to protection scope of the present invention.

Example

Below unless stated otherwise, otherwise in following example experimentally using the conventional real of bioengineering field Process, operation, material and condition is tested to carry out.

Below unless stated otherwise, otherwise the percentage concentration (%) of each reagent refers both to the concentration expressed in percentage by volume (% of the reagent (v/v))。

Material and method

Cell strain: the cell strain for being used to prepare lentiviral particle is 293T cell (ATCC CRL-3216).For detecting The T cell strain of TCR phenotype and function is Jurkat cell (clone E6-1, ATCC TIB-152) and J.RT3-T3.5 cell (ATCC TIB-153).Presenting cells strain for offering antigen polypeptide is T2 cell (174xCEM.T2, ATCC CRL- 1992).Tumor cell line for detecting TCR function is human colon carcinoma colo205 cell (ATCC CCL-222) and Caco-2 Cell (ATCC HTB-37), human breast carcinoma MDA-MB-231 cell (ATCC HTB-26), human ovarian cancer SK-OV-3 cell (ATCC HTB-77), human lymphoma cell Bjab (ACC-757-DSMZ), human lung cancer H647 cell (ATCC CRL-5824) and H1355 cell (ATCC CRL-5865).Cell strain maintains to train with RPMI-1640 complete medium (Lonza, cat#12-115F) It supports, is added in RPMI-1640 complete medium 10% calf serum FBS (ATCC 30-2020), 2mmol/L Pidolidone, 100 μ g/ml penicillin and 100 μ g/ml streptomysins.

Peripheral blood: the human peripheral product for testing health donors used is all from the Pacific blood in San Francisco The heart (Trima residual cell the component #R32334 and # of #1PBMC and #2PBMC respectively from Apheresis method collection kit R33941)。

External evoked Her2/neu 369-377 specific killing T cell (CTL): peripheral blood is through Ficoll-Paque Premium (Sigma-Alorich company, cat#GE-17-5442-02) density gradient centrifugation (× 400g) obtains after 30 minutes Mononuclearcell (PBMC).First with the anti-HLA-A2 antibody (Biolegend company, cat#343303) of fluorescein FITC label Dyeing detects the HLA-A2 phenotype of cell, and the RNA of positive cell is extracted after flow cytometry, and reverse transcription is cDNA and is cloned into On carrier, the analysis of HLA gene sequencing is carried out later, determines that cell distribution type is HLA-A*0201.The PBMC cell of the HLA-A2 positive The culture hole in 24- well culture plate is cultivated, culture solution is above-mentioned RPMI-1640 complete medium.Every 2 × 10e6/ml of hole PBMC is added Her2/neu 369-377 polypeptide (Her2-E75 is synthesized with Peptide2.0, and 10 μ g/ml are dissolved in DMSO), concentration For 1 μ g/ml.It is placed in 5%CO₂, following cell factor, human IL-2 is added behind incubator culture 16-24 hours under the conditions of 37 DEG C (Peprtech company, cat#200-02) 100IU/ml, hIL-7 (Peprotech company, cat#200-07) 5ng/ml, people IL-15 (Peprotech company, cat#200-15) 5ng/ml.Culture 10 to 14 days carries out antigen to the T cell of culture and pierces again Swash: 10e6 culture cell is added in every hole in 24- orifice plate, while being added 2 × 10e6 through 25 μ g/ml mitomycin Cs (Santa Cruz Biotechnology company, cat#SC-3514) handles the self PBMC cell of 2 hours HLA-A2 positives As trophocyte, the Her2/neu 369-377 polypeptide of final concentration of 1 μ g/ml is added in every hole, and IL-2 is added after overnight incubation 100IU/ml, IL-7 5ng/ml, IL-15 5ng/ml.After stimulating through the above-mentioned antigenic stimulus of two-wheeled and again, the T for collecting amplification is thin Born of the same parents carry out phenotypic analysis and T cell clone.

Flow cytometry and unicellular separation: the T cell phenotype of expression Her2/neu 369-377 specificity TCR is logical Overflow-type cell is analyzed.It collects detected cell and is placed in 1.5ml pipe (cell number is about 10e5), with 1ml DPBS Solution (2.7mM KCl, 1.5mM KH₂PO₄, 136.9mM NaCl, 8.9mM Na₂HPO₄·7H₂O, pH 7.4) it washes one time, lay equal stress on It is placed in 100 μ l to contain in the DPBS of 1% calf serum, the anti-human CD8 antibody (Biolegend of 5 μ l fluorescein APC label is added Company, cat#300912) and 10 μ l fluorescein PE label the Her2-E75/HLA-A2 tetramer (the Her2-E75 tetramer, MBL International Co company, cat#T01014) or Her2-E75/HLA-A2 pentamer (Her2-E75 pentamer, Proimmune company, cat#F214-2A-D), it is washed twice after being incubated for 30 minutes on ice with DPBS solution, is resuspended in 100 μ l PBS Solution carries out flow cytometry.Flow cytometer is MACSQuant Analyzer 10 (Miltenyi Biotec company), Interpretation of result is carried out with Flowjo software (Flowjo company).T cell clone is to utilize fluidic cell separator (FACS Sorter acquisition is cultivated after) carrying out unicellular separation.The PBMC stimulated Her2/neu369-377 polypeptide antigen is marked with APC Note anti-human CD8 antibody and PE label Her2-E75/HLA-A2 pentamer dyeing, then carry out fluidic cell separation (model: Sony cell sorter SH800).Single CD8⁺Her2-E75/HLA-A2 pentamer⁺Cell is sorted into 96- well culture plate Single culture hole after, be added through 25 μ g/ml mitomycin Cs handle 2 hours the HLA-A2 positive self PBMC cell, every hole 10e5 cell after 1 μ g/ml Her2/neu 369-377 polypeptide overnight incubation is added, is added and contains IL-2100IU/ml, IL- The RPMI-1640 complete culture solution of 7 5ng/ml, IL-15 5ng/ml.Renew within every 3-4 days the fresh training containing the cell factor Whether nutrient solution, microscopically observation have T cell clonal growth.The T cell of proliferation is collected, carry out antigen according to the above method stimulates again To obtain sufficient amount of cell, phenotype or Function detection are carried out, and extracts the clone that RNA carries out tcr gene.

T cell Function detection: the function of the T cell strain through Her2/neu 369-377 polypeptid specificity TCR modification is logical The expression of T cell surface C D69 is crossed to detect.In every hole of 96- orifice plate be added 10e5 transfect tcr gene T cell with And 10e5 T2 cell, it is mixed in the every hole RPMI-1640 complete medium of 100 μ l/, each test group is multiple holes. Add various concentration (respectively 1 μ g/ml, 0.5 μ g/ml, 0.1 μ g/ml, 0.05 μ g/ml, 0.01 μ g/ml, 0.005 μ g/ml, 0.001 μ g/ml and 0 μ g/ml) Her2/neu 369-377 polypeptide is placed on 5%CO₂, the incubator under the conditions of 37 DEG C trains overnight It supports.It collects cell and is suspended in DPBS+1%FBS solution, flowed after being dyed with the anti-human CD69 antibody that APC is marked Formula cell analysis.The T cell that CD69 is expressed after Her2/neu 369-377 antigenic stimulus is considered it and carries and express Her2/ The TCR of neu 369-377 specificity.Transfection tcr gene in Her2/neu 369-377 specific CTL clone and PBMC The function of primary T cells is the interferon by secreting in cell conditioned medium after detection antigenic stimulus to determine.Her2/neu The PBMC cell of 369-377 specific CTL clone or transfection tcr gene is respectively with target cell in 96 orifice plates, in 100 μ l/ It is mixed in every hole RPMI-1640 complete medium, target cell is the Her2/neu 369- that 10 times of diluted concentrations are added The T2 cell of 377 polypeptides or various tumor cell line cells, each test group are multiple holes.In antibody function blocking test, cell The anti-human CD8 antibody of 10 μ g/ml (Biolegend company, cat#300912) is added simultaneously in culture hole or anti-HLA-ABC is anti- Body (w6/32 clone, Biolegend company, cat#311402), cell is placed in 5%CO₂, the incubator under the conditions of 37 DEG C stays overnight Culture.18-24 hours collection cell conditioned mediums, and (eBioscience is public for employment IFN-γ ELISA Read-set-Go kit Department, cat#88-7316) IFN-γ in supernatant is detected.

Obtain monoclonal tcr gene: using Zymo Quick-RNA Microprep kit, (Zymo Research is public Department, cat#R1050) from T cell clone's purification total serum IgE, utilize 5 '/3 ' kit of Smarter RACE to obtain as template CDNA (Takara Bio company, the U.S., cat#634858).With 3 ' primer 5 ' of 5 '-CDS primers and TCR β chain- GCCTCTGGAATCCTTTCTCTTG-3 ' (SEQ ID NO:26) and 3 ' primer of α chain, 5 '-TCAGCTGGACCACAGCCGCAG- 3 ' (SEQ ID NO:27) carry out PCR, amplify TCR α and β complete sequence genetic fragment, and are cloned into pRACE carrier (beauty respectively State Takara Bio, cat#634858) on.Transformed competence colibacillus bacterium Stellar (Takara Bio company, the U.S., cat# 636763) it and after obtaining plasmid is sequenced.

Recombinate the preparation of TCR Lentiviral: for expressing the viral vectors of TCR as replication defect type slow virus load Body, comprising: it is public to be purchased from System Biosciences by the slow virus carrier pCDH-EF1 α-MCS- (PGK-GFP) for expressing GFP It takes charge of (Cat#CD811A-1)；And the carrier pCDH-EF1 α-MCS of GFP is not expressed, it is removed by using this field routine techniques PGK promoter and GFP gene on pCDH-EF1 α-MCS- (PGK-GFP) carrier and obtain.According to tcr gene sequence obtained Column, synthesize TCR β chain and α chain and between cleavable F2A sequence and Furin endonuclease bamhi complete genome sequence, and be linked to The multiple cloning sites in the EF-1 α promoter downstream of the carrier, be inserted into TCR transcription sequence be followed successively by TCR β chain (it is no terminate it is close Numeral), Furin endonuclease bamhi, F2A segment, (method is referring to document " Gene Ther.2008Nov for TCR α chain；15(21): 1411–1423").The carrier for expressing GFP is that the PGK promoter being reversed drives.The carrier for not expressing GFP then eliminates PGK promoter and GFP segment.

Recombinate the preparation of TCR lentiviral particle: TCR lentiviral particle is by 3000 transfection reagent of Lipofectaine (Thermo Fisher company, cat#L3000001) transfects 293T cell and obtains.It is thin to prepare 293T according to shop instruction Born of the same parents and transfection protocol.Transfection 96 well culture plates carry out, first with 1 culture solution of Opti-MEM (Thermo Fisher company, Cat#51985091 P3000 examination is added according to producer's explanation in the liposome solutions for) preparing transfected plasmids in 250 μ l culture solutions Viral packaging plasmid (SBI company, the cat# of agent (P3000reagent) and TCR slow virus carrier plasmid and pCDH system LV500A-1), 3000 reagent of Lipofectaine is in addition added in 250 μ l culture solutions, 293T is added after being incubated for 15 minutes in mixing Cell culture well.5%CO₂, cultivate 16 hours under the conditions of 37 DEG C, change DMEM culture solution (the Thermo Fisher containing 10%FBS Company, cat#11965092), cell conditioned medium is collected after continuing culture 24 hours and 48 hours respectively, after 2000g centrifugation, with 0.4 The virion obtained after the filtering of μm filter membrane is for infection cell.

Recombination TCR slow-virus transfection human T-cell: the primary PBMC cell frozen is cultivated after thawing in RPMI-1640 completely It cultivates 24 hours in liquid, through 30 minutes removal dead cells of Ficoll-Paque Premium density gradient centrifugation (× 400g), sets In with 2 μ g/ml anti-CD3antibodies (Biolegend company, OKT3 clone cat#317303) and the anti-human CD28 antibody of 2 μ g/ml (wherein every hole is added 100 μ l and contains above-mentioned CD3 antibody and CD28 antibody for (Biolegend company, cat#302914) processing DPBS solution) in 24 hours 24 orifice plate culture holes, cell concentration is 2 × 10e6/ml, and culture collected cell after 48 hours, weight It is suspended from fresh recombination TCR lentiviral particle supernatant and is placed in the hole of 24 orifice plates, 4ng/ml polybrene (Santa is added Cruz biotechnology company, cat#sc-134220) after, in 32 DEG C, 1000g is centrifuged 2 hours, with containing IL-2 100IU/ml, IL-7 5ng/ml, IL-15 5ng/ml RPMI-1640 complete culture solution displacement half viral supernatants after continue Culture, renews the fresh culture solution containing above-mentioned cell factor in every 3 days.Phenotype and Function detection can be carried out after general 72 hours.Turn Dye T cell strain is also carried out according to above-mentioned steps, 48 hours after general transfection can be if marked on viral vectors with GFP GFP positive cell is observed under fluorescence microscope.

Embodiment 1: it is killed from the normal donor peripheral blood of HLA-A2 positive induction Her2/neu 369-377 polypeptid specificity Hurt T cell

The present embodiment is with the low concentration Her2/neu 369-377 polypeptide of 1 μ g/ml by two-wheeled stimulated in vitro from normal Polypeptid specificity killer T cell is induced in PBMC (#2PBMC), and carries out flow cytometry and unicellular separation.Specific side Method is as described above.As a result as follows:

Figure 1A right figure shows 0.013% lymphocyte for combinable Her2/neu 369-377/HLA-A2 pentamer (i.e. Her2-E75 pentamer) CD8 positive killer T cell, the control cell not stimulated through Her2 polypeptide in left figure do not go out Existing CD8 positive pentamer positive cell.As a result illustrate in natural T cell library, identify Her2/neu 369-377 antigen polypeptide Specific T-cells quantity it is seldom.Although quantity is few, the T cell of this group of recognizable Her2/neu 369-377 polypeptides still can quilt It is clearly distinguished out.It again include high-affinity T in positive cell according further to the fluorescence intensity for combining Her2-E75 pentamer Cell and low compatibility T cell.Monoclonal is carried out after isolating 453 CD8 positive pentamer positive cells by fluidic cell Culture, stimulates again by two-wheeled antigen polypeptide and cell factor expands, and only obtains in the single T cell isolated from this 453 The T cell for obtaining a proliferation clones Her2 ctl clone 1B5 (referred to as Her2 CTL 1B5).Figure 1B right figure shows this purifying CD8⁺Ctl clone is in combination with the Her2/neu 369-377/HLA-A2 tetramer (i.e. the Her2-E75 tetramer).Every hole 5x10³It is a Her2 CTL 1B5 cell with by 5x10³The various concentration Her2/neu 369-377 antigen polypeptide (Her2/ that a T2 cell is offered Neu 369-377 antigen polypeptide carried out since 1 μ g/ml 10 times dilution, thus obtain final concentration of 1 μ g/ml, 0.1 μ g/ml, Different groups of 0.01 μ g/ml, 0.001 μ g/ml) mixed culture after, detect supernatant in T cell secretion IFN-γ, to determination The function of this T cell clone-specific identification Her2/neu 369-377 polypeptide.5x10 is added in hole in tumor cell culture³It is a After colo205 cell is mixed, the IFN-γ of T cell secretion in supernatant is detected.Fig. 1 C is the results show that T cell is cloned 1B5 can be activated by minimum concentration by the antigen polypeptide of 1ng/ml, and the concentration of the identification function of T cell and antigen polypeptide It is positively correlated and (there is dose dependent), illustrate that this T cell clone-specific identifies the Her2/neu offered by HLA-A2 369-377 polypeptide.Importantly, this T cell clone also identifies one plant of HLA-A2⁺Her2/neu⁺Colon cancer cell line colo205.Therefore, this T cell clone Her2 TCR-1B5 not only can recognize Her-E75 polypeptide, can also be by colo205 cell It activates and secretion of gamma-IFN.

The acquisition and identification of embodiment 2:Her2/neu 369-377 polypeptid specificity TCR complete sequence

The present embodiment is directly obtained from the Her2/neu 369-377 polypeptid specificity ctl clone obtained by embodiment 1 Comprising match (that is, two chains can collectively constitute identification antigen polypeptide functional TCR) α and β chain complete tcr gene sequence The TCR of column, coding is known as " Her2 TCR-1B5 ".The amino acid sequence of the α chain of the TCR is as shown in SEQ ID NO:2, coding Sequence is as shown in SEQ ID NO:1, and the amino acid sequence of the β chain of the TCR, as shown in SEQ ID NO:4, coded sequence is such as Shown in SEQ ID NO:3.This TCR is present in the periphery T cell library of HLA-A2 positive normal person, will not be to trace expression The normal cell of Her2/neu albumen generates cross reaction and leads to autoimmune response.Specific detection method is as described above. As a result as follows:

In order to detect the antigentic specificity and its function of obtained TCR, TCR α chain and β chain-ordering are cloned into replication defect type In Lentiviral.Fig. 2 shows constructed TCR slow virus carrier structure fragment schematic diagram.α chain and β chain are by that can cut off Furin identification segment and F2A polypeptide fragment be linked.In order to track the T cell transfected by TCR slow virus carrier, the slow disease Poisonous carrier (" pCDH-EF1 α-Her2TCR- (PGK-GFP) carrier " is shown as in figure) can express GFP simultaneously, be opened by reversed PGK Mover drives (above Fig. 2).Another expression vector (" pCDH-EF1 α-Her2 TCR carrier " is shown as in figure) (below Fig. 2) Eliminate GFP and its promoter sequence.By reducing carrier lengths to increase the output of lentiviral particle, in turn avoids two and open Influencing each other between mover, to increase the expression quantity of TCR.

Following sequence is respectively connected to the carrier of above-mentioned expression GFP and does not express the carrier of GFP: i) being connected by cuttability It is that polypeptide links, (right in the TCR β chain of constant region 1 disulfide bond of increase and the nucleotide sequence (SEQ ID NO:13) of α chain to connect The TCR answered is Her2 TCR-1B5-dis, and amino acid sequence is as shown in SEQ ID NO:14)；Ii) by cuttability connecting peptides Link, constant region is replaced with the TCR β chain of sequence of mouse source and the nucleotide sequence (SEQ ID NO:15) of α chain by source of people sequence (corresponding TCR is Her2TCR-1B5-mC, and amino acid sequence is as shown in SEQ ID NO:16)；Iii it) is connected by cuttability more The original TCR β chain of peptide linkage and α chain nucleotide sequence (SEQ ID NO:23) (corresponding TCR is Her2 TCR-1B5-wt, The amino acid sequence of coding is as shown in SEQ ID NO:24)；To obtain 6 kinds of recombined lentivirus vectors:

1) Her2 TCR-1B5-dis recombined lentivirus vector (carrying GFP)；

2) Her2 TCR-1B5-dis w/o GFP recombined lentivirus vector (not carrying GFP)；

3) Her2 TCR-1B5-mC recombined lentivirus vector (carrying GFP)；

4) Her2 TCR-1B5-mC w/o GFP recombined lentivirus vector (not carrying GFP).

5) Her2 TCR-1B5-wt recombined lentivirus vector (carrying GFP)；

6) Her2 TCR-1B5-wt w/o GFP recombined lentivirus vector (not carrying GFP).

After each Her2 tcr gene segment passes through PCR amplification, it is cloned into above two slow virus carrier (i.e. pCDH- respectively EF1 α-MCS- (PGK-GFP) and pCDH-EF1 α-MCS) EF1- α promoter downstream: Her2 TCR-1B5-dis and TCR- The TCR segment of 1B5-wt is with 5 ' primer, 5 '-AGAGCTAGCGAATTCAACATGGATACCTGGCTCGTATG-3 ' (SEQ ID NO:28) and 3 ' primer, 5 '-GTTGATTGTCGACGCCCTCAGCTGGACCACAGCCGCAG-3 ' (SEQ ID NO:29) expansion Increase and obtains.The TCR segment of Her2TCR-1B5-mC is with 5 ' primers 5 '- AGAGCTAGCGAATTCAACATGGATACCTGGCTCGTATG-3 ' (SEQ ID NO:30) and 3 ' primers 5 '- GTTGATTGTCGACGCCCTCAACTGGACCACAGCCT-3 ' (SEQ ID NO:31) is expanded and is obtained.PCR uses Q5 high-fidelity PCR kit (NEB, cat#M0543S), reaction condition were 98 DEG C after 30 seconds, carried out 98 DEG C of 25 circulations 10 seconds, 65 DEG C 10 seconds, 72 DEG C 3 minutes.The TCR segment of acquisition is cloned into pCDH-EF1 α-MCS- (PGK-GFP) carrier or pCDH-EF1 α-MCS carrier The region MCS in EF1 α promoter downstream.

Will the obtained recombination TCR Lentiviral of building by preceding method respective recombination TCR to be prepared sick slowly Malicious particle.

The TCR of two kinds of different modes modification can be obtained in the manner described above, and Her2 TCR-1B5-dis is prominent by putting Become and increase a disulfide bond in TCR constant region, method is in document " Cancer Res.2007Apr 15；67(8):3898-903." Middle description, full text is incorporated herein by reference.Her2 TCR-1B5-mC is to replace phase with mouse TCR constant-region sequences The people's TCR constant-region sequences answered, method description, full text in document " Eur.J.Immunol.2006 36:3052-3059 " Incorporated herein by reference.Fig. 3 A is shown, with the slow virus of carrying Her2TCR-1B5 TCR and GFP gene (that is, above-mentioned Her2 TCR-1B5-dis recombined lentivirus vector and Her2 TCR-1B5-mC recombined lentivirus vector) after transfecting T cells strain, A part of GFP⁺Cell can illustrate that these can identify quilt by the T cell of slow-virus transfection in conjunction with the Her2-E75 tetramer The Her2/neu 369-377 polypeptide that HLA-A2 molecule is offered.It is calculated by the data obtained, if TCR constant region increases by two sulphur Key, the T cell (GFP that 37.5% (13.2/ (13.2+22) × 100%) is transfected⁺) expressed by TCR can identify Her2/ Neu polypeptide antigen (Fig. 3 A left figure).And what TCR constant region was replaced by the corresponding constant region of mouse TCR, then have 60% (34.6/ (34.6+23) × 100%) T cell (GFP that is transfected⁺) expressed by TCR can identify Her2/neu polypeptide antigen (Fig. 3 A Right figure).Compared with the TCR obtained after external source TCR α chain and the modification of β chain warp disulfide bond, external source TCR constant region is by corresponding mouse TCR Constant region is replaced and the hybridization TCR that is formed can be further reduced mispairing probability with endogenous TCR.Fig. 3 B is shown, no matter turning Dye only expresses the J.RT-T3.5 cell of beta chain still while the Jurkat cell of express alpha chain and β chain, and constant region is by mouse perseverance The expression quantity for determining the external source TCR (Her2 TCR-1B5-mC) obtained after region sequence displacement, which is apparently higher than, only increases by one in constant region The TCR (Her2 TCR-1B5-dis) of a disulfide bond.Fig. 3 C shows, the T cell strain for expressing external source Her2 TCR-1B5 can be by The Her2/neu 369-377 polypeptide that T2 cell is offered activates and expresses CD69, illustrates that this TCR has identification Her2/neu The function of 369-377 polypeptide antigen.External source TCR (the Her2 TCR-1B5- that constant region is obtained after the sequence substitutions of murine constant region MC expression quantity) not only increases, and is also apparently higher than the TCR modified through additional disulfide bond to the specific recognition capability of polypeptide antigen (Her2 TCR-1B5-dis) can be activated by the Her2/neu 369-377 polypeptide of lower concentration.However, activation expression The minimum peptide concentration of the Jurkat cell of Her2 TCR-1B5-mC is about 0.05 μ g/ml, than the activation Her2 of Fig. 1 C display The peptide concentration of CTL 1B5 clone is about 50 times high.This illustrates the Her2 TCR-1B5 TCR Recognition polypeptide of Jurkat cell expression The ability (avidity) of antigen will be significantly lower than CD8⁺The Her2 TCR-1B5 TCR expressed on CTL.A possible reason It is that Jurkat cell does not express CD8 molecule, and CD8 plays important booster action to the identification function of Her2 TCR-1B5.

Embodiment 3: normal peripheral blood T cell is expressed after the transfection of Her2 TCR-1B5-mC recombinant slow virus and be can recognize The specificity TCR of Her2/neu 369-377 polypeptide

Can express and have in primary T cells to further verify present invention TCR obtained and identify Her2/neu The function of antigen polypeptide, with recombinant slow virus particle (the Her2 TCR-1B5-mC recombination for carrying Her2 TCR-1B5-mC gene Slow virus carrier) periphery blood T cells through CD3/CD28 antibody activation, from two different Normal donors are transfected, it is received after 7 days Collect cell and carries out Her2-E75 tetramer staining.Specific method is as described above.As a result as follows:

Fig. 4 A shows there is lymphocyte in two donor peripheral blood mononuclear cells (respectively #1PBMC and #2PBMC) It can illustrate that the Her2 TCR-1B5-mC of these cells expression can be with specific recognition by HLA- in conjunction with the Her2-E75 tetramer The Her2/neu antigen polypeptide that A2 offers.As a result it also shows, Her2-E75 tetramerpositive cell (i.e. expression Her2TCR-1B5- MC in), most of positive cells are CD8⁺T kills cell, and fraction positive cell is the lymphocyte of CD8-, this is likely to CD4⁺T helper cell.CD8⁺(Geom.Mean in #2PBMC sample is the fluorescence intensity of the CTL combination Her2-E75 tetramer Geom.Mean in 1469, #1PBMC samples is 1404) also significantly greater than CD4⁺T cell (the Geom in #2PBMC sample Mean is 504) Geom.Mean in 560, #1PBMC sample is.If slow-virus infection CD8⁺And CD4⁺The transfection of T cell is imitated Rate is the same, illustrates a part expression in CD4⁺External source Her2/neu 369-377 specificity TCR on cell can not combine The Her2-E75 tetramer, even with affinity is also below expression in CD8⁺External source in T cell transfects TCR.This is also further Illustrate that the TCR of transfection needs the miscellaneous function of CD8 molecule that could effectively combine Her2/HLA-A2 compound.

The PBMC cell of 10e5 transfection TCR is added in every hole of 96- orifice plate, with various concentration by T2 cell (every hole 10e5) (Her2/neu 369-377 antigen polypeptide is since 0.1 μ g/ml for the Her2/neu 369-377 antigen polypeptide offered 10 times of dilutions are carried out, to obtain final concentration of 0.1 μ g/ml, 0.01 μ g/ml, different groups of 0.001 μ g/ml) mixed culture Afterwards, the IFN-γ for detecting T cell secretion in supernatant, to determine that the PBMC cell-specific of this expression TCR identifies Her2/neu The function of 369-377 polypeptide.Control group target cell be offer 1 μ g/ml can in conjunction with HLA-A2 molecule EBV viral antigen it is more The T2 cell of peptide LMP2 426-434.Fig. 4 B shows, what the PBMC of expression Her2 TCR-1B5-mC can be offered by T2 cell Her2/neu 369-377 antigen polypeptide activates and secretion of gamma-IFN, illustrates the primary T for expressing external source Her2 TCR-1B5-mC The Her2/neu 369-377 polypeptide that cell can be offered with specific recognition by HLA-A2 molecule.Identify antigen polypeptide ability with Expression quantity of the external source TCR in T cell is related.It identifies the maximum half-reaction (half-maximum of antigen polypeptide Reaction, EC₅₀) peptide concentration through curve matching reckoning be about 6.9ng/ml (IC₅₀Tool Kit, http: // Www.ic50.tk/), although EC of this reaction susceptibility lower than high-affinity TCR of exogenous antigens such as identification viral antigens₅₀ (EC₅₀About 10e-10M) (referring to document " CANCER RESEARCH 1998,58.4902-4908 " and " HUMAN GENE THERAPY 2014,25:730-739 "), but still in the TCR affinity of recognizable kinds of tumor related antigen within the scope of (as described in document " Eur J Immunol (2012) 42:3174-9 ").

(T2+Her2-E75, i.e. Her2/neu 369-377 are more for the antigen polypeptide that Fig. 4 C display T cell and T2 cell are offered Peptide) co-culture when anti-human CD8 antibody is added after, the function of T cell secretion of gamma-IFN is suppressed significantly.This illustrates CD8 molecule Booster action is most important for external source TCR identification Her2/neu 369-377 antigen polypeptide, also shows of the present invention Her2TCR-1B5 is CD8 function dependent form TCR.

Embodiment 4: the Her2/ that normal peripheral blood T cell is expressed after the transfection of Her2 TCR-1B5-mC recombinant slow virus Neu 369-377 polypeptid specificity TCR can recognize HLA-A2⁺Her2/neu⁺Tumour cell

10e5 are added in every hole of 96- orifice plate after the transfection of Her2 TCR-1B5-mC w/o GFP recombinant slow virus PBMC (#2PBMC for transfection Her2TCR-1B5 as shown in the figure) or the 10e5 control #2PBMC for not transfecting TCR, with 10e5 different tumor cell line mixed culture, detects the IFN-γ secreted in supernatant later.Specific method is as described above. As a result as follows:

Fig. 5 A shows that the T cell of expression Her2 TCR-1B5-mC can be by HLA-A2⁺Her2/neu⁺Tumor cell line Simultaneously secretion of gamma-IFN is activated, tumor cell line includes colon cancer Colo205 cell, breast cancer MDA-MB-231 cell, colon Cancer Caco-2 cell and lung cancer H1355 cell.And HLA-A2^-Her2/neu⁺Oophoroma SK-OV3 cell, lung cancer H647 it is thin Born of the same parents and HLA-A2⁺Her2/neu^-Lymthoma Bjab cell cannot but activate transfection Her2 TCR-1B5-mC TCR T it is thin Born of the same parents.Illustrate that Her2 TCR-1B5-mC TCR can be resisted with the Her2/neu that specific recognition tumor cell surface is offered by HLA-A2 It is former.The same donor PBMC in source, parallel culture but the control group T cell without transfecting Her2 TCR-1B5-mC cannot be listed Tumor cell line is activated, and it is nonspecific for illustrating the reaction to tumour cell not.Fig. 5 B is shown, expresses Her2 TCR- The T cell (#2PBMC of transfection Her2 TCR-1B5) of 1B5-mC TCR goes out apparent reaction to colo205 cells show, and Identification function can almost be blocked by the antibody of anti-CD8 antibody and anti-HLA molecule, this further illustrates that identification is swollen The effector cell of oncocyte surface Her2/neu antigen is CD8⁺Killer T cell, specific antigen identification function depend on The miscellaneous function of CD8, Her2 TCR-1B5-mC when this her2/neu 369-377 polypeptide antigen also offered with identification T2 cell The performance of TCR is consistent.

It discusses

Different tumor cell lines may be with tumor cells expression different level to the reaction sensibility difference of specific T-cells Her2/neu antigen polypeptide/HLA-A2 complex is related, it is also possible to which the inhibition different to T cell function from tumour cell itself is made With related.Although the high-affinity TCR of specific recognition Her2/neu 369-377 polypeptide can pass through Her2/neu 369- 377 polypeptides are external evoked and obtain, but these high-affinities TCR tends not to the Her2/neu that tumor cell is offered and resists Original (Cancer Res.1998；58:4902–4908.Cancer Immunol.Immunother.2008；57:271–280).One A reason may be external source Her2/neu 369-377 polypeptide combination HLA-A2 molecular configuration with offered into the cell polypeptide/ The configuration of HLA compound is different (referring to document " Journal of Immunology, 2008,180:8135-8145 "). Another possible cause is that for Her2/neu 369-377 polypeptide as mimic epitope (mimotope) antigen, what is induced is special Property TCR both can recognize Her2/neu 369-377 polypeptide, also can recognize the similar polypeptide offered by tumour cell, such as Her2/ Neu 373-382 polypeptide is (referring to document " J Immunol.2013Jan 1；190 (1): 479-488 "), however high-affinity TCR Although there is high-affinity to the Her2/neu 369-377 polypeptide that HLA-A2 offers, cannot effectively identify accordingly by tumour The mimic epitope polypeptide that cell is offered.The TCR of specific recognition Her2/neu 369-377 polypeptide of the present invention, not only target The Her2/neu 369-377 polypeptide offered to tumour cell, it is also possible to while identification is derived from by what tumour cell was offered Other mimic epitope polypeptides of Her2/neu.

Since the high-affinity T cell of identification autoantigen is removed by central tolerance mechanism mostly, in periphery T cell library The TCR that can identify Her2/neu antigen of naturally occurring is mostly middle low compatibility.Another can be with tumor cell The high-affinity TCR of CD8 function independent form be from through the multiple of Her2/neu 373-382 polypeptid specificity T cell group After α chain and β chain are matched, by Function detection screening go out (referring to document " HUMAN GENE THERAPY 2024,25: 730–739"；WO/2016/133779).Due to being directly obtained from the monoclonal T cell of specificity, this TCR not can determine that With the presence or absence of in periphery nature T cell library.It is generally believed that adopting for high-affinity T cell transfers the curative effect for the treatment of and is better than target To the low compatibility T cell of same antigen (referring to document " Clin Exp Immunol (2015) 180:255-70 ").However, high Compatibility TCR itself be easy to produce identification autoantigen autoimmune reaction (referring to document " Blood (2009) 114: 535-46 "), the TCR not by the screening of central tolerance mechanism can also identify the normal tissue of antigen low expression, or be directed to it Its similar auto-antigen epitope generate cross reaction toxicity of missing the target (referring to document " Sci Transl Med (2013) 5: 197ra103.Blood(2013)122:863–71").Select high-affinity TCR another reason for be the function of these TCR not The miscellaneous function of CD8 is relied on, thus can be by transfecting CD4⁺T cell and obtain to CD8⁺The auxiliary of killer T cell function is made With.For CD8 function dependent form TCR, expressing TCR and external source CD8 molecule simultaneously by expression vector also be can achieve similarly Purpose.

In short, the present invention provides one kind from HLA-A2⁺Autologous peripheral T cells library in the Her2/neu that induces 373-382 polypeptid specificity TCR α chain and β chain complete sequence, express this TCR after transfecting and TCR that constant region is modified it is primary Killer T cell can identify a variety of HLA-A2⁺Her2/neu⁺Tumour cell.Adopt for exploitation and clinical application and transfers through spy The T cell of anisotropic TCR modification provides new method and approach to treat tumour.

SEQUENCE LISTING

<110>Hangzhou health Wanda Pharmaceutical Technology Co., Ltd

Synthesize immune limited liability company (Synimmune, Inc.)

<120>cell, the code nucleic acid, expression vector, preparation method, pharmaceutical composition of the T cell receptor, its modification that separate And application

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

<160> 31

<170> PatentIn version 3.5

<210> 1

<211> 831

<212> DNA

<213>people (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>people (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>people (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>people (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>people (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>people (Homo sapiens)

<400> 18

Lys Ile Phe Gly Ser Leu Ala Phe Leu

1 5

<210> 19

<211> 137

<212> PRT

<213>people (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>people (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>people (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>people (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>people (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. a kind of isolated T cell receptor, including at least one of α chain and β chain, the α chain and β chain include variable region and Constant region, which is characterized in that the T cell receptor can antigen Her2/neu expressed by specific recognition tumour cell, and And the amino acid sequence of the variable region of the α chain has and amino acid sequence at least 98% shown in SEQ ID NO:19 The amino acid sequence of consistency, the variable region of the β chain has with amino acid sequence shown in SEQ ID NO:20 at least 98% consistency.

2. T cell receptor according to claim 1, wherein the T cell receptor can specific recognition by HLA-A2 The antigen epitope polypeptide for the antigen Her2/neu that molecule is offered；Preferably, the antigen epitope polypeptide includes such as SEQ Her2/neu 369-377 shown in ID NO:18.

3. T cell receptor according to claim 1, wherein the constant region of the α chain and/or the β chain is described Constant region derives from people；Preferably, the constant region of the α chain is entirely or partly derived from the homologous sequence of other species Column are replaced and/or the constant region of the β chain is entirely or partly derived from the homologous sequence institute of other species Replacement；It is highly preferred that other species are mouse.

4. T cell receptor according to claim 1, wherein the constant region of the α chain is modified with one or more two The constant region of sulfide linkage and/or the β chain is modified with one or more disulfide bond.

5. T cell receptor according to claim 1, wherein the amino acid sequence of the α chain such as SEQ ID NOs:2,6 or Shown in 10, the amino acid sequence of the β chain is as shown in SEQ ID NOs:4,8 or 12.

6. a kind of separation, encoding T cell receptor nucleic acid, at least one of α chain and β chain comprising the T cell receptor Coded sequence, the α chain coding sequence and β chain coding sequence include variable region encoding sequences and constant region coded sequence, It is characterized in that, the T cell receptor is capable of the antigen Her2/neu of specific recognition tumor cells expression, and the α chain The amino acid sequence of variable region encoding sequences coding has one with amino acid sequence at least 98% shown in SEQ ID NO:19 The amino acid sequence of cause property, the β chain variable region encoding sequences coding has and amino acid sequence shown in SEQ ID NO:20 At least 98% consistency.

7. nucleic acid according to claim 6, wherein the nucleic acid is DNA or RNA.

8. nucleic acid according to claim 6, wherein the α chain variable region encoding sequences are as shown in SEQ ID NO:21, institute β chain variable region encoding sequences are stated as shown in SEQ ID NO:22.

9. nucleic acid according to claim 6, wherein being capable of specific recognition by the T cell receptor of the nucleic acid encode The antigen epitope polypeptide of the antigen Her2/neu offered by HLA-A2 molecule；Preferably, the antigen epitope polypeptide Including the Her2/neu 369-377 as shown in SEQ ID NO:18.

10. nucleic acid according to claim 6, wherein the α chain constant region coded sequence and/or the β chain constant region are compiled Code sequence derives from people；Preferably, the α chain constant region coded sequence is entirely or partly by from the homologous of other species Sequence is replaced and/or the β chain constant region coded sequence is entirely or partly derived from the homologous sequence of other species Column are replaced；It is highly preferred that other species are mouse.

11. nucleic acid according to claim 6, wherein the α chain constant region coded sequence includes one or more disulfide bond Coded sequence and/or the β chain constant region coded sequence include one or more disulfide bond coded sequences.

12. nucleic acid according to claim 6, wherein the α chain coding sequence is as shown in SEQ ID NOs:1,5 or 9, institute β chain coding sequence is stated as shown in SEQ ID NOs:3,7 or 11.

13. the nucleic acid according to any one of claim 6-11, wherein the α chain coding sequence and the β chain encoding sequence It is connected between column by the coded sequence of cuttability connecting peptides.

14. nucleic acid according to claim 13, sequence is as shown in SEQ ID NOs:13,15 or 23.

15. a kind of recombinant expression carrier, contain it is effectively being connect with promoter, according to any one of claim 6-14 Nucleic acid and/or its complementary series.

16. a kind of cell of T cell receptor modification, the surface of the cell by T cell of any of claims 1-5 by Body modification, wherein the cell includes naive T cell or its precursor, NKT cell or T cell strain.

17. a kind of method for the cell for preparing T cell receptor modification according to claim 16, comprising the following steps:

1) cell is provided；

2) nucleic acid for encoding T cell receptor according to any one of claims 1-5 is provided；

3) nucleic acid transfection is entered in the cell.

18. according to the method for claim 17, wherein cell described in step 1) comes from self or allosome.

19. according to the method for claim 17, wherein the mode of the transfection includes: the side transfected using viral vectors Formula, it is preferred that the viral vectors includes γ retroviral vector or slow virus carrier；Chemical mode, it is preferred that institute Stating chemical mode includes by the way of liposome transfection；Physics mode, it is preferred that the physics mode includes electrotransfection side Formula.

20. according to the method for claim 17, wherein nucleic acid described in step 2) is according to any in claim 6-14 Nucleic acid described in.

21. the cell of T cell receptor modification according to claim 16 is in preparation for treating or preventing tumour and/or cancer Purposes in the drug of disease.

22. purposes according to claim 21, wherein the tumour and/or cancer are antigen Her2/neu positive, and It and is the HLA-A2 positive.

23. the cell of T cell receptor modification according to claim 16 is preparing tumour and/or cancer for detecting host Purposes in the drug of disease.

24. a kind of pharmaceutical composition, wherein the pharmaceutical composition includes the T according to claim 16 as active constituent The cell and pharmaceutically acceptable auxiliaries of cell receptor modification.

25. pharmaceutical composition according to claim 24, wherein described pharmaceutical composition includes each course for the treatment of of each patient Total dose range is 1 × 10³-1×10⁹The cell of a cell/Kg weight T cell receptor modification.

26. pharmaceutical composition according to claim 24, wherein described pharmaceutical composition be suitable for through artery, vein, it is subcutaneous, In intradermal, tumor, in lymphatic vessel, in lymph node, in cavum subarachnoidale, in marrow, intramuscular and Intraperitoneal medication.

27. a kind of method for treating tumour and/or cancer, including to tumour and/or cancer patient's application according to claim 16 The cell of the T cell receptor modification.

28. according to the method for claim 27, wherein the administration dosage of the cell of T cell receptor modification is each trouble The each course for the treatment of total dose range of person is 1 × 10³-1×10⁹A cell/Kg weight.

29. according to the method for claim 27, wherein the cell of T cell receptor modification passes through artery, vein, skin Under, in intradermal, tumor, in lymphatic vessel, in lymph node, in cavum subarachnoidale, in marrow, intramuscular and Intraperitoneal medication.

30. according to the method for claim 27, wherein the tumour and/or cancer are antigen Her2/neu positive, and It and is the HLA-A2 positive.