CN108395479A - A kind of T cell receptor in relation to KRAS gene mutation - Google Patents

A kind of T cell receptor in relation to KRAS gene mutation Download PDF

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Publication number
CN108395479A
CN108395479A CN201710065966.7A CN201710065966A CN108395479A CN 108395479 A CN108395479 A CN 108395479A CN 201710065966 A CN201710065966 A CN 201710065966A CN 108395479 A CN108395479 A CN 108395479A
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chain
orderings
seq
ordering
amino acid
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CN201710065966.7A
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高军
吕垚
史军
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高军
吕垚
史军
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14Blood; Artificial blood
    • A61K35/17Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503Immunoglobulin superfamily
    • C07K14/7051T-cell receptor (TcR)-CD3 complex
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide

Abstract

The invention discloses a kind of T cell receptors, are specifically bound with 12 codon mutation G12D 26aa peptide fragments of KRAS genes, and the amino acid sequence of the peptide fragment is:MTEYKLVVVGADGVGKSALTIQLIQN, such as SEQ ID NO:Shown in 1;When the T cell receptor has identification HLA molecular antigen compounds, the 9 12aa antigen epitope polypeptides with higher affinity are as follows:VVVGADGVGK, such as SEQ ID NO:Shown in 48;KLVVVGADGVGK, such as SEQ ID NO:Shown in 49;The HLA molecules are HLA A0301;It discloses when identifying the antigenic compound that KRAS genes G12D mutation peptide fragments are formed with HLA A0301, the amino acid sequence of the α chains and β chains of T cell receptor.In addition, the invention also discloses encode the application of the α chains of above-mentioned T cell receptor and nucleotide sequence, the carrier comprising above-mentioned nucleotide sequence, the cell comprising above-mentioned nucleotide sequence and the above-mentioned carrier of β chains or above-mentioned cell in the drug for preparing prevention KRAS gene mutation relevant disease.

Description

A kind of T cell receptor in relation to KRAS gene mutation

Technical field

The present invention relates to a kind of T cell receptor (TCR) more particularly to a kind of T cell receptors in relation to KRAS gene mutation. The purposes to generate T cell is shifted the invention further relates to tcr gene and they are prominent with 12 codon of KRAS genes for preventing Become the pharmaceutical applications of relevant disease.

Background technology

KRAS is a kind of proto-oncogene, is about 35kb, is located at No. 12 chromosomes, is one of RAS gene family members, coding KRAS albumen.With the generation of tumour, rise in value, migration, diffusion and angiogenesis have relationship.

There are three types of KRAS genes (KRAS, p21) families and the relevant gene of human tumor --- H-ras, KRAS and N- Ras is respectively positioned on 11,12 and No. 1 chromosomes.The ras albumen also known as p21 genes of KRAS gene codes 21kD.In ras bases Because in, KRAS influences maximum to human cancer, it seems molecular switch:The path of regulating cell growth can be controlled when normal; When being abnormal, then lead to cell continued propagation, and prevents cell self-destruction.She participates in intracellular signal and transmits, when When KRAS gene mutation, which permanently activates, and cannot generate normal ras albumen, keeps Cellular Signaling Transduction Mediated disorderly, cell Proliferation out of control and canceration.

Research shows that about 30% human malignancies are related with RAS gene mutations, the product after ras mutation can be always In the state of activation.In leukaemia, lung cancer, in the carcinoma of the rectum and cancer of pancreas, KRAS mutation is very common, wherein in the carcinoma of the rectum 30%-35% patient has mutation.

The existence of itself and tumour cell is rised in value, and is migrated, and diffusion, angiogenesis has relationship.KRAS genes are divided into saltant type And wild type, common mutations site are 3 exons on No. 12 codons and No. 13 codons of 2 exon of KRAS genes No. 61 codons, wherein there is 7 mutantional hotspots:This 7 kinds mutation of G12C, G12R, G12S, G12V, G12D, G12A, G13V/D. Account for 90% or more.

KRAS genes (GenBank:NC_000012) mutation plays an important role in the occurrence and development of cancer of pancreas.It has been reported that It can detect that KRAS genes the 12nd, 13 or 61 codon point mutations in Pancreatic Adenocarcinoma, pancreatic juice or excrement, blood (Takahashi,et al.Gastrointestinal Endo 2005.61:76-9;Luigi L,et al.Oncogene 2002.21:4301-6;Tada M,et al.Cancer Res1993.53:2472-4;Lu XH,et al.Natl Med J China 2001.81:1050–3;N van Heek,et al.J.Clin.Pathol.2005.58:1315-1320).

Currently, there is not yet the T cell receptor in relation to KRAS gene mutation, exists to treating and/or preventing and KRAS genes The demand of the method for 12 codon mutation relevant diseases.

Invention content

One of the technical problem to be solved in the present invention is to provide a kind of T cell receptor in relation to KRAS gene mutation.

The second technical problem to be solved by the present invention is to provide the nucleotides sequence of the α chains and β chains that encode above-mentioned T cell receptor Row.

The third technical problem to be solved by the present invention is to provide the carrier for including above-mentioned nucleotide sequence.

The four of the technical problem to be solved in the present invention are to provide the cell for including above-mentioned nucleotide sequence.

The five of the technical problem to be solved in the present invention are to provide above-mentioned carrier or above-mentioned cell and are preparing prevention KRAS genes Application in the drug of mutation-related diseases.

Present inventors have developed the cell therapies of prevention KRAS gene mutation relevant disease comprising applies TCR Gene therapy specifically binds T cell to generate with 12 codon mutation G12D 26aa peptide fragments of KRAS genes.The invention of the present invention People assembles the T cell receptor to 12 codon mutation G12D 26aa peptide fragment specificity of KRAS genes, also constructs comprising TCR α With the retroviral vector of β genes, and use it for transduction human T-cell.The cell shows 12 password of expressing K RAS genes Son is mutated G12D 26aa peptide fragments specificity TCRs and shows functional antigentic specificity activity.

In order to solve the above-mentioned technical problem, the present invention is achieved through the following technical solutions:

In the first aspect of the present invention, a kind of T cell receptor is provided, with 12 codon mutation G12D of KRAS genes 26aa peptide fragments are specifically bound, and the amino acid sequence of the peptide fragment is:MTEYKLVVVGADGVGKSALTIQLIQN, such as SEQ ID NO:Shown in 1;When the T cell receptor has identification HLA molecular antigen compounds, the 9-12aa antigens with higher affinity Epitope polypeptide is as follows:VVVGADGVGK, such as SEQ ID NO:Shown in 48;KLVVVGADGVGK, such as SEQ ID NO:Shown in 49;Institute It is HLA-A0301 to state HLA molecules;When identifying the antigenic compound that KRAS genes G12D mutation peptide fragments are formed with HLA-A0301, The α chains of its T cell receptor and the sequence of β chains are as follows:

α chains use any one in following sequence:

α chain-orderings 1:Such as SEQ ID NO:Amino acid sequence shown in 2;

α chain-orderings 2:Such as SEQ ID NO:Amino acid sequence shown in 4;

α chain-orderings 3:Such as SEQ ID NO:Amino acid sequence shown in 6;

α chain-orderings 4:Such as SEQ ID NO:Amino acid sequence shown in 8;

α chain-orderings 5:Such as SEQ ID NO:Amino acid sequence shown in 10;

α chain-orderings 6:Such as SEQ ID NO:Amino acid sequence shown in 12;

α chain-orderings 7:Such as SEQ ID NO:Amino acid sequence shown in 14;

β chains use any one in following sequence:

β chain-orderings 1:Such as SEQ ID NO:Amino acid sequence shown in 16;

β chain-orderings 2:Such as SEQ ID NO:Amino acid sequence shown in 18;

β chain-orderings 3:Such as SEQ ID NO:Amino acid sequence shown in 20;

β chain-orderings 4:Such as SEQ ID NO:Amino acid sequence shown in 22;

β chain-orderings 5:Such as SEQ ID NO:Amino acid sequence shown in 24;

β chain-orderings 6:Such as SEQ ID NO:Amino acid sequence shown in 26;

β chain-orderings 7:Such as SEQ ID NO:Amino acid sequence shown in 28;

β chain-orderings 8:Such as SEQ ID NO:Amino acid sequence shown in 30;

β chain-orderings 9:Such as SEQ ID NO:Amino acid sequence shown in 32;

β chain-orderings 10:Such as SEQ ID NO:Amino acid sequence shown in 34;

β chain-orderings 11:Such as SEQ ID NO:Amino acid sequence shown in 36;

β chain-orderings 12:Such as SEQ ID NO:Amino acid sequence shown in 38;

β chain-orderings 13:Such as SEQ ID NO:Amino acid sequence shown in 40;

β chain-orderings 14:Such as SEQ ID NO:Amino acid sequence shown in 42;

β chain-orderings 15:Such as SEQ ID NO:Amino acid sequence shown in 44.

As currently preferred technical solution, in the antigen that identification KRAS G12D mutation peptide fragments are formed with HLA-A0301 When compound, the combination of the α chains and β chains of T cell receptor preferably it is following any one:

α chain-ordering 1+ β chain-orderings 1;α chain-ordering 1+ β chain-orderings 2;α chain-ordering 1+ β chain-orderings 3;α chain-ordering 2+ β chain sequences Row 1;α chain-ordering 2+ β chain-orderings 2;α chain-ordering 2+ β chain-orderings 3;α chain-ordering 3+ β chain-orderings 4;α chain-ordering 3+ β chain-orderings 5;α Chain-ordering 3+ β chain-orderings 6;α chain-ordering 3+ β chain-orderings 10;α chain-ordering 3+ β chain-orderings 11;α chain-ordering 3+ β chain-orderings 12;α chains Sequence 3+ β chain-orderings 13;α chain-ordering 3+ β chain-orderings 14;α chain-ordering 3+ β chain-orderings 15;α chain-ordering 4+ β chain-orderings 4;α chain sequences Arrange 4+ β chain-orderings 5;α chain-ordering 4+ β chain-orderings 6;α chain-ordering 4+ β chain-orderings 10;α chain-ordering 4+ β chain-orderings 11;α chain-orderings 4 + β chain-orderings 12;α chain-ordering 4+ β chain-orderings 13;α chain-ordering 4+ β chain-orderings 14;α chain-ordering 4+ β chain-orderings 15;α chain-orderings 5+ β chain-orderings 4;α chain-ordering 5+ β chain-orderings 5;α chain-ordering 5+ β chain-orderings 6;α chain-ordering 5+ β chain-orderings 10;α chain-ordering 5+ β chains Sequence 11;α chain-ordering 5+ β chain-orderings 12;α chain-ordering 5+ β chain-orderings 13;α chain-ordering 5+ β chain-orderings 14;α chain-ordering 5+ β chains Sequence 15;α chain-ordering 6+ β chain-orderings 7;α chain-ordering 6+ β chain-orderings 8;α chain-ordering 6+ β chain-orderings 9;α chain-ordering 7+ β chain-orderings 7;α chain-ordering 7+ β chain-orderings 8;α chain-ordering 7+ β chain-orderings 9.

As currently preferred technical solution, introduces and be mutated in the constant region of the α chains and β chains, additionally increase extracellular The disulfide bond in area.

As currently preferred technical solution, 2A or IRES sequences are introduced between α chains and β chains, make α chains and β chain structures It is built into an expression frame content, convenient for recombinantly expressing and assembling T cell receptor.

As currently preferred technical solution, the T cell receptor is preferably such as SEQ ID NO:Ammonia shown in 46 Base acid sequence.

In the second aspect of the present invention, the nucleotide of the α chains and β chains of the above-mentioned T cell receptor that coding is connected with each other is provided Sequence, the codon adopted are specific as follows by optimization:

Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 1:Shown in 3;

Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 2:Shown in 5;

Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 3:Shown in 7;

Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 4:Shown in 9;

Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 5:Shown in 11;

Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 6:Shown in 13;

Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 7:Shown in 15;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 1:Shown in 17;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 2:Shown in 19;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 3:Shown in 21;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 4:Shown in 23;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 5:Shown in 25;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 6:Shown in 27;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 7:Shown in 29;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 8:Shown in 31;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 9:Shown in 33;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 10:Shown in 35;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 11:Shown in 37;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 12:Shown in 39;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 13:Shown in 41;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 14:Shown in 43;

Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 15:Shown in 45.

As currently preferred technical solution, the nucleotide sequence is preferably such as SEQ ID NO:Sequence shown in 47 Row.

In the third aspect of the present invention, the carrier for including above-mentioned nucleotide sequence is provided.Such as the carrier can be reversed Record viral vectors.

In the fourth aspect of the present invention, the cell for including above-mentioned nucleotide sequence is provided.Preferably, the cell is that T is thin Born of the same parents or stem cell, such as:Cd4 t cell, cd8 t cell and corresponding dryness cell etc..The cell can be from the T for being isolated from subject It is cell-derived.

In the fifth aspect of the present invention, the carrier or the cell are provided and preparing prevention KRAS gene mutation phase Application in the drug of related disorders.Described KRAS gene mutation relevant disease, such as cancer of pancreas, colorectal cancer etc..

In the present invention, term " T cell receptor (T cell receptor, TCR) " be T cell surface specificity by Body is responsible for the antigen that identification is presented by major histocompatibility complex (MHC), different from B-cell receptor, does not identify Free antigen.Under normal conditions, lower affinity is possessed between T cell receptor and antigen, thus same antigen may be by not Same T cell receptor is identified that a certain receptor may also identify many kinds of antigens.

T cell receptor is heterodimer, is made of two different subunits.The receptor of 95% T cell is by α subunits It is constituted with β subunits, in addition 5% receptor is made of γ subunits and δ subunits.This than regular meeting because ontogeny or disease and Variation.

The specific binding of T cell receptor and the MHC polypeptides presented can cause a series of biochemical reactions, and by numerous Accessory receptor, enzyme and transcription factor activation T cell, promote its division and differentiation.

The constant domain of T cell receptor structural domain is made of short catenation sequence, and wherein cysteine residues form two Sulfide linkage generates connection between two chains.The T cell receptor of the present invention can have additional half in each of α chains and β chains Cystine amino so that the TCR includes two disulfide bond in constant region.It is introduced additionally in the constant domain of α and β chains Cysteine so that form additional disulfide bond, and enhance the pairing of the chain of introducing and reduce and matched with the mistake of wild type chain.

In the present invention, term " HLA " refers to HLA system, is the major histocompatibility complex of people (MHC) title, and include I class HLA antigens (A, B&C) and II class HLA antigens (DP, DQ&DR).

In the present invention, the nucleotide sequence is through codon optimization.Profit of the different cells in specific codon It uses and is different.This codon preference is corresponding with the relative abundance of specific tRNA in cell type.By changing sequence In codon, to by they it is special match at the relative abundance with corresponding tRNA, expression can be increased.

In the present invention, term " carrier " includes expression vector, can in vivo or in vitro/structure for expressing in vitro Body.Such as the carrier can be retroviral vector.Retrovirus is RNA virus, is had and lytic virus (lytic Virus) different life cycle.In this regard, retrovirus is the Infectious entities replicated by DNA intermediate.When inverse When Retroviral infection cell, genome is converted into DNA form by reverse transcriptase.The DNA copy serves as template and is used for It generates new rna gene group and assembles the virus-encoded proteinaceous needed for infectious virus particle.

In the present invention, term " cell " can be cd4 t cell, cd8 t cell and corresponding dryness cell etc..The cell The T cell detached from subject can be derived from.The T cell can be the mixed cellularity group detached from subject, such as periphery The part of blood lymphocyte (PBL) group.T cell in PBL groups can be activated by means commonly known in the art, such as be answered With anti-CD3 and CD28 antibody.T cell can be CD4+ T helper cell or CD8+ cytotoxic T cells.The cell can be in CD4+ In the mixing group of T helper cell/CD8+ cytotoxic T cells.Polyclonal activation, for example, using optionally with anti-CD28 antibody group The anti-CD 3 antibodies of conjunction will cause the proliferation of CD4+ and CD8+T cells, but can also cause the increasing of CD4+25+ regulatory T cells It grows.It is undesirable that tcr gene, which is transferred in regulatory T cells, because they may suppressors modification cell toxicant and auxiliary Help the antiviral activity of T cell.Therefore CD4+25+ groups can be removed before tcr gene transfer.The cell can be from waiting for adoptive turn It moves in the subject of the cell of the genetic modification and detaches.The cell can be by detaching T cell from subject, optionally activating and be somebody's turn to do T cell shifts tcr gene in vitro, and then by the cell of the adoptive transfer TCR- transductions immunization therapy subject and It is made.The cell can be or derived from stem cell, such as candidate stem cell (HSC).Gene transfer to HSC will not be caused TCR is expressed in cell surface, because stem cell does not express CD3 molecules.However, when stem cell is divided into the lymph migrated to thymus gland When precursor (lymphoid precursor), the startup of CD3 expression will cause in the surface expression of the thymocyte introducing TCR。

Compared with prior art, the present invention has the advantages that:

1. it has been demonstrated that the G12D mutation of KRAS genes are the key molecular events of kinds of tumors morbidity, it is prominent to target this molecule Detection, drug targeting treatment and the immune targeted therapy of change have significant precisely medical characteristic.Present invention discover that one kind can The TCR receptors of this mutant polypeptide epitope of targets identification, the thus technological progress in field bring promotion.

2. the neoantigen (Neo-antigen) that the mutator having now been found that generates focuses mostly in tumours such as melanomas Including multiple gene mutations, KRAS mutation generate Neo-antigen, only retrieve patent (international monopoly WO 2016/ 085904A1), in contrast, it is limited to HLA-A11.The TCR receptors that the present invention describes are HLA-A0301, are had with it aobvious The technology of work is distinguished and feature.

3. present invention T cell receptor to be protected with when HLA molecule forming composites with higher affinity Following epitope polypeptide:HLA-A0301:VVVGADGVGK, such as SEQ ID NO:Shown in 48;HLA-A0301:KLVVVGADGVGK, Such as SEQ ID NO:Shown in 49;For HLA-A0301, inventor passes through screening experiment, preferably up to the α chains and β of the T cell receptor Chain, it was unexpectedly found that best T cell receptor is preferably such as SEQ ID NO:Amino acid sequence shown in 46, such as SEQ ID NO:Nucleotide sequence shown in 47.

4. the present invention constructs a kind of retroviral vector of the T cell receptor in relation to KRAS gene mutation, TCR is generated The human T-cell of transduction, and by recombinantly expressing 9-12 epitope peptide recombinant human alpha chains and heterologous pair of β chains are mutated in combination with KRAS G12D The HLA-A0301CD8+T lymphocytes of chain TCR acceptor genes, flow cytomery confirm that the T for recombinantly expressing the invention is thin Born of the same parents' receptor can specific recognition target antigen, and with target killing tumour target cell ability, can further develop cell and exempt from Epidemic disease medicine.

Description of the drawings

The present invention is described in further detail with reference to the accompanying drawings and examples.

Fig. 1 is the structure schematic diagram of retroviral vector in the embodiment of the present invention 1;

Fig. 2 is the result schematic diagram in the embodiment of the present invention 2;

Fig. 3 is the result schematic diagram in the embodiment of the present invention 3.

Specific implementation mode

Following embodiment is only illustrative of the invention and is not intended to limit the scope of the invention.It is not specified in embodiment specific The experimental method of condition, usually according to normal condition, for example (,) Sambrook et al., molecular cloning:Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or built according to manufacturer The condition of view.

Embodiment 1 is in combination with KRAS G12D mutation 9-12 epitope peptide recombinant human alpha chains and β chain heteroduplex TCR acceptor genes The structure of retroviral vector

Additional cysteine (C) is introduced in the constant region of above-mentioned α chain-orderings 1 and β chain-orderings 1, sequence is as follows:Completely Amino acid sequence such as SEQ ID NO:Shown in 46, whole nucleotide sequence such as SEQ ID NO:Shown in 47.

After said gene sequence is synthesized, sequence in the gene mode is built into MP71 Retroviral Vectors referring to Fig.1. MP71 carrier frameworks (Linnemann, C etc., High-throughput identification of in document before Antigen-specific TCRs by TCR gene capture, Nature Medicine, 2013,19 (11) 1534- 1541) it has been described.The T cell that this carrier has been used for genetic modification in Germany is treated in the clinical test of HIV.

Embodiment 2 is carried in combination with KRAS genes G12D mutation 9-12 epitope peptide recombinant human alpha chains and β chain heteroduplexes TCR Acceptor gene Retroviral Transfer human T-cell

The combinable KRAS G12D mutation 9-12 epitope peptide recombinant human alpha chains and β chain heteroduplexes TCR that embodiment 1 is built Acceptor gene retrovirus, reference literature (Linnemann, C etc., High-throughput identification of Antigen-specificTCRs by TCR gene capture, Nature Medicine, 2013,19 (11) 1534- 1541) the PBMC cells of transfected with human HLA-A0301 are as experimental group (TCR1-1), using empty carrier transfection as control group (Non- Td).CD8+T lymphocytes can be with specific bond KRAS gene G12D mutable epitope peptides after being transfected using flow cytomery (SEQ ID NO:And the epitope peptide of HLA-A0301 molecules-MHC molecule compound pMHC 49).The results are shown in Figure 2, upper right as Cell number relative comparison group in limit, which has, to be dramatically increased, the results showed that, transfection recombinant expression people α chains and β chain heteroduplexes TCR The CD8+T cells of receptor can targets identification KRASG12D mutable epitopes peptide-MHC molecule (HLA-A0301) compound (pMHC).

Embodiment 3 is recombinantly expressed in combination with KRAS G12D mutation 9-12 epitope peptide recombinant human alpha chains and β chain heteroduplexes TCR The flow cytomery of the HLA-A0301CD8+T lymphocytes identification target cell of acceptor gene

The pancreas for carrying the G12D mutation of KRAS genes of the HLA-A0301+ and HLA-A0301- of original cuiture are chosen respectively Cancer cell line as target cell, reference literature (E etc., Targeting of cancer neoantigens with Donor-derived T cell receptorrepertoires, SCIENCE, 2016,352 (6291):1337-1341) with The CD8+T lymphocytes that tcr gene made from embodiment 2 transfects (transfect the PBMC cells of tcr gene through CD8+ in embodiment 2 Flow cytometer screening obtains) it is incubated jointly, the molecular marker of CD8+T lymphocyte activations after flow cytomery is incubated The expression of (IFN-γ and CD107a/b).The results are shown in Figure 3, after being incubated with HLA-A0301+ target cells, turns tcr gene CD8+T cells are after HLA-A0301+ target cells stimulate, right upper quadrant (while high expressing cell factor IFN-γ and lysosome membrane Glycoprotein a/b (CD107a/b)) CD8+ cells be significantly higher than control group, the results showed that, tcr gene transfection CD8+T lymphs Cell targets identification and can kill the tumour cell of HLA-A0301+, become the CTL cells of the cloning with target killing.

Sequence table

<110>Gao Jun, Lv's Yao, Shi Jun

<120>A kind of T cell receptor in relation to KRAS gene mutation

<130>HJ17-12750

<160>49

<170> PatentIn version 3.5

<210> 1

<211>26

<212>PRT

<213>Artificial sequence

<400> 1

MTEYKLVVVG ADGVGKSALT IQLIQN 26

<210> 2

<211>270

<212>PRT

<213>Artificial sequence

<400> 2

MRQVARVIVF LTLSTLSLAK TTQPISMDSY EGQEVNITCS HNNIATNDYI TWYQQFPSQG 60

PRFIIQGYKT KVTNEVASLF IPADRKSSTL SLPRVSLSDT AVYYCLVGDM DQAGTALIFG 120

KGTTLSVSSN IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKTVLD 180

MRSMDFKSNS AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESSCDVKLVE KSFETDTNLN 240

FQNLSVIGFR ILLLKVAGFN LLMTLRLWSS 270

<210> 3

<211>810

<212> DNA

<213>Artificial sequence

<400> 3

atgaggcaag tggcgagagt gatcgtgttc ctgaccctga gtactttgag ccttgctaag 60

accacccagc ccatctccat ggactcatat gaaggacaag aagtgaacat aacctgtagc 120

cacaacaaca ttgctacaaa tgattatatc acgtggtacc aacagtttcc cagccaagga 180

ccacgattta ttattcaagg atacaagaca aaagttacaa acgaagtggc ctccctgttt 240

atccctgccg acagaaagtc cagcactctg agcctgcccc gggtttccct gagcgacact 300

gctgtgtact actgcctcgt gggtgacatg gaccaggcag gaactgctct gatctttggg 360

aagggaacca ccttatcagt gagttccaat atccagaacc ctgaccctgc cgtgtaccag 420

ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 480

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 540

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 600

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 660

gaaagttcct gtgatgtcaa gctggtcgag aaaagctttg aaacagatac gaacctaaac 720

tttcaaaacc tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat 780

ctgctcatga cgctgcggct gtggtccagc 810

<210>4

<211>270

<212>PRT

<213>Artificial sequence

<400>4

MRQVARVIVF LTLSTLSLAK TTQPISMDSY EGQEVNITCS HNNIATNDYI TWYQQFPSQG 60

PRFIIQGYKT KVTNEVASLF IPADRKSSTL SLPRVSLSDT AVYYCLVGDM DQAGTALIFG 120

KGTHLSVSSN IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKTVLD 180

MRSMDFKSNS AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESSCDVKLVE KSFETDTNLN 240

FQNLSVIGFR ILLLKVAGFN LLMTLRLWSS 270

<210>5

<211>810

<212> DNA

<213>Artificial sequence

<400>5

atgaggcaag tggcgagagt gatcgtgttc ctgaccctga gtactttgag ccttgctaag 60

accacccagc ccatctccat ggactcatat gaaggacaag aagtgaacat aacctgtagc 120

cacaacaaca ttgctacaaa tgattatatc acgtggtacc aacagtttcc cagccaagga 180

ccacgattta ttattcaagg atacaagaca aaagttacaa acgaagtggc ctccctgttt 240

atccctgccg acagaaagtc cagcactctg agcctgcccc gggtttccct gagcgacact 300

gctgtgtact actgcctcgt gggtgacatg gaccaggcag gaactgctct gatctttggg 360

aagggaaccc acctatcagt gagttccaat atccagaacc ctgaccctgc cgtgtaccag 420

ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 480

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 540

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 600

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 660

gaaagttcct gtgatgtcaa gctggtcgag aaaagctttg aaacagatac gaacctaaac 720

tttcaaaacc tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat 780

ctgctcatga cgctgcggct gtggtccagc 810

<210>6

<211>273

<212>PRT

<213>Artificial sequence

<400>6

MKSLRVLLVI LWLQLSWVWS QQKEVEQNSG PLSVPEGAIA SLNCTYSDRG SQSFFWYRQY 60

SGKSPELIMF IYSNGDKEDG RFTAQLNKAS QYVSLLIRDS QPSDSATYLC AAAMDSSYKL 120

IFGSGTRLLV RPNIQNPDPA VYQLRDSKSS DKSVCLFTDF DSQTNVSQSK DSDVYITDKT 180

VLDMRSMDFK SNSAVAWSNK SDFACANAFN NSIIPEDTFF PSPESSCDVK LVEKSFETDT 240

NLNFQNLSVI GFRILLLKVA GFNLLMTLRL WSS 273

<210>7

<211>819

<212> DNA

<213>Artificial sequence

<400>7

atgaaatcct tgagagtttt actagtgatc ctgtggcttc agttgagctg ggtttggagc 60

caacagaagg aggtggagca gaattctgga cccctcagtg ttccagaggg agccattgcc 120

tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 180

tctgggaaaa gccctgagtt gataatgttc atatactcca atggtgacaa agaagatgga 240

aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 300

cagcccagtg attcagccac ctacctctgt gccgctgcca tggatagcag ctataaattg 360

atcttcggga gtgggaccag actgctggtc aggcctaata tccagaaccc tgaccctgcc 420

gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 480

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 540

gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 600

tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 660

cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 720

aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 780

gggtttaatc tgctcatgac gctgcggctg tggtccagc 819

<210>8

<211>273

<212>PRT

<213>Artificial sequence

<400>8

MKSLRVLLVI LWLQLSWVWS QQKEVEQNSG PLSVPEGAIA SLNCTYSDRG SQSFFWYRQY 60

SGKSPELIMS IYSNGDKEDG RFTAQLNKAS QYVSLLIRDS QPSDSATYLC AAAMDSSYKL 120

IFGSGTRLLV RPNIQNPDPA VYQLRDSKSS DKSVCLFTDF DSQTNVSQSK DSDVYITDKT 180

VLDMRSMDFK SNSAVAWSNK SDFACANAFN NSIIPEDTFF PSPESSCDVK LVEKSFETDT 240

NLNFQNLSVI GFRILLLKVA GFNLLMTLRL WSS 273

<210>9

<211>819

<212> DNA

<213>Artificial sequence

<400>9

atgaaatcct tgagagtttt actagtgatc ctgtggcttc agttgagctg ggtttggagc 60

caacagaagg aggtggagca gaattctgga cccctcagtg ttccagaggg agccattgcc 120

tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 180

tctgggaaaa gccctgagtt gataatgtcc atatactcca atggtgacaa agaagatgga 240

aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 300

cagcccagtg attcagccac ctacctctgt gccgctgcca tggatagcag ctataaattg 360

atcttcggga gtgggaccag actgctggtc aggcctaata tccagaaccc tgaccctgcc 420

gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 480

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 540

gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 600

tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 660

cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 720

aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 780

gggtttaatc tgctcatgac gctgcggctg tggtccagc 819

<210>10

<211>265

<212>PRT

<213>Artificial sequence

<400>10

MKSLRVLLVI LWLQLSWVWS QGPLSVPEGA IASLNCTYSD RVSQSFFWYR QYSGKSPELI 60

MSIYSNGDKE DGRFTAQLNK ASQYVSLLIR DSQPSDSATY LCAAAMDSSY KLIFGSGTRL 120

LVRPNIQNPD PAVYQLRDSK SSDKSVCLFT DFDSQTNVSQ SKDSDVYITD KTVLDMRSMD 180

FKSNSAVAWS NKSDFACANA FNNSIIPEDT FFPSPESSCD VKLVEKSFET DTNLNFQNLS 240

VIGFRILLLK VAGFNLLMTL RLWSS 265

<210>11

<211>795

<212> DNA

<213>Artificial sequence

<400>11

atgaaatcct tgagagtttt actagtgatc ctgtggcttc agttgagctg ggtttggagc 60

caaggacccc tcagtgttcc agagggagcc attgcctctc tcaactgcac ttacagtgac 120

cgagtttccc agtccttctt ctggtacaga caatattctg ggaaaagccc tgagttgata 180

atgtccatat actccaatgg tgacaaagaa gatggaaggt ttacagcaca gctcaataaa 240

gccagccagt atgtttctct gctcatcaga gactcccagc ccagtgattc agccacctac 300

ctctgtgccg ctgccatgga tagcagctat aaattgatct tcgggagtgg gaccagactg 360

ctggtcaggc ctaatatcca gaaccctgac cctgccgtgt accagctgag agactctaaa 420

tccagtgaca agtctgtctg cctattcacc gattttgatt ctcaaacaaa tgtgtcacaa 480

agtaaggatt ctgatgtgta tatcacagac aaaactgtgc tagacatgag gtctatggac 540

ttcaagagca acagtgctgt ggcctggagc aacaaatctg actttgcatg tgcaaacgcc 600

ttcaacaaca gcattattcc agaagacacc ttcttcccca gcccagaaag ttcctgtgat 660

gtcaagctgg tcgagaaaag ctttgaaaca gatacgaacc taaactttca aaacctgtca 720

gtgattgggt tccgaatcct cctcctgaaa gtggccgggt ttaatctgct catgacgctg 780

cggctgtggt ccagc 795

<210>12

<211>270

<212>PRT

<213>Artificial sequence

<400>12

MRQVARVIVF LTLSTLSLAK TTQPISMDSY EGQEVNITCS HNNIATNDYI TWYQQFPSQG 60

PRFIIQGYKT KVTNEVASLF IPADRKSSTL SLPRVSLSDT AVYYCLVGDR DQAGTALIFG 120

KGTTLSVSSN IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKTVLD 180

MRSMDFKSNS AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESSCDVKLVE KSFETDTNLN 240

FQNLSVIGFR ILLLKVAGFN LLMTLRLWSS 270

<210>13

<211>810

<212> DNA

<213>Artificial sequence

<400>13

atgaggcaag tggcgagagt gatcgtgttc ctgaccctga gtactttgag ccttgctaag 60

accacccagc ccatctccat ggactcatat gaaggacaag aagtgaacat aacctgtagc 120

cacaacaaca ttgctacaaa tgattatatc acgtggtacc aacagtttcc cagccaagga 180

ccacgattta ttattcaagg atacaagaca aaagttacaa acgaagtggc ctccctgttt 240

atccctgccg acagaaagtc cagcactctg agcctgcccc gggtttccct gagcgacact 300

gctgtgtact actgcctcgt gggtgacaga gaccaggcag gaactgctct gatctttggg 360

aagggaacca ccttatcagt gagttccaat atccagaacc ctgaccctgc cgtgtaccag 420

ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 480

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 540

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 600

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 660

gaaagttcct gtgatgtcaa gctggtcgag aaaagctttg aaacagatac gaacctaaac 720

tttcaaaacc tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat 780

ctgctcatga cgctgcggct gtggtccagc 810

<210>14

<211>270

<212>PRT

<213>Artificial sequence

<400>14

MRQVARVIVF LTLSTLSLAK TTQPISMDSY EGQEVNITCS HNNIATNDYI TWYQQFPSQG 60

PRFIIQGYKT KVTNEVASLF IPADRKSSTL SLPRVSLSDT AVYYCLVGDR DQAGTALIFG 120

KGTHLSVSSN IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKTVLD 180

MRSMDFKSNS AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESSCDVKLVE KSFETDTNLN 240

FQNLSVIGFR ILLLKVAGFN LLMTLRLWSS 270

<210>15

<211>810

<212> DNA

<213>Artificial sequence

<400>15

atgaggcaag tggcgagagt gatcgtgttc ctgaccctga gtactttgag ccttgctaag 60

accacccagc ccatctccat ggactcatat gaaggacaag aagtgaacat aacctgtagc 120

cacaacaaca ttgctacaaa tgattatatc acgtggtacc aacagtttcc cagccaagga 180

ccacgattta ttattcaagg atacaagaca aaagttacaa acgaagtggc ctccctgttt 240

atccctgccg acagaaagtc cagcactctg agcctgcccc gggtttccct gagcgacact 300

gctgtgtact actgcctcgt gggtgacaga gaccaggcag gaactgctct gatctttggg 360

aagggaaccc acctatcagt gagttccaat atccagaacc ctgaccctgc cgtgtaccag 420

ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 480

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 540

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 600

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 660

gaaagttcct gtgatgtcaa gctggtcgag aaaagctttg aaacagatac gaacctaaac 720

tttcaaaacc tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat 780

ctgctcatga cgctgcggct gtggtccagc 810

<210>16

<211>310

<212>PRT

<213>Artificial sequence

<400>16

MGPGLLCWAL LCLLGAGLVD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ 60

GPQFIFQYYE EEERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSLGEGRVD 120

GYTFGSGTRL TVVEDLNKVF PPEVAVFEPS EAEISHTQKA TLVCLATGFF PDHVELSWWV 180

NGKEVHSGVS TDPQPLKEQP ALNDSRYCLS SRLRVSATFW QNPRNHFRCQ VQFYGLSEND 240

EWTQDRAKPV TQIVSAEAWG RADCGFTSVS YQQGVLSATI LYEILLGKAT LYAVLVSALV 300

LMAMVKRKDF 310

<210>17

<211>930

<212> DNA

<213>Artificial sequence

<400>17

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct tgggcgaggg cagagtcgac 360

ggctacacct tcggttcggg gaccaggtta accgttgtag aggacctgaa caaggtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttcttc cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acggacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacccgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggctttac ctcggtgtcc taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcctgctagg gaaggccacc ctgtatgctg tgctggtcag cgcccttgtg 900

ttgatggcca tggtcaagag aaaggatttc 930

<210>18

<211>312

<212>PRT

<213>Artificial sequence

<400>18

MGPGLLCWAL LCLLGAGLVD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ 60

GPQFIFQYYE EEERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSLGEGRVD 120

GYTFGSGTRL TVVEDLKNVF PPEVAVFEPS EAEISHTQKA TLVCLATGFY PDHVELSWWV 180

NGKEVHSGVS TDPQPLKEQP ALNDSRYCLS SRLRVSATFW QNPRNHFRCQ VQFYGLSEND 240

EWTQDRAKPV TQIVSAEAWG RADCGFTSES YQQGVLSATI LYEILLGKAT LYAVLVSALV 300

LMAMVKRKDS RG 312

<210>19

<211>936

<212> DNA

<213>Artificial sequence

<400>19

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct tgggcgaggg cagagtcgac 360

ggctacacct tcggttcggg gaccaggtta accgttgtag aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacctgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 900

ctgatggcca tggtcaagag aaaggattcc agaggc 936

<210>20

<211>312

<212>PRT

<213>Artificial sequence

<400>20

MGPGLLCWAL LCLLGAGLVD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ 60

GPQFIFQYYE EEERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSLGEGRVD 120

GYTFGSGTRL TVVEDLKNVF PPEVAVFEPS EAEISHTQKA TLVCLATGFY PDHVELSWWV 180

NGKEVHSGVS TDPQPLKEQP ALNDSRYCLS SRLRVSATFW QNPRNHFRCQ VQFYGLSEND 240

EWTQDRAKPV TQIVSAEAWG RADCGFTSES YQQGVLSATI LYEILLGKAT LYAVLVSALV 300

LMAMVKRKDS RG 312

<210>21

<211>936

<212> DNA

<213>Artificial sequence

<400>21

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct tgggcgaggg cagagtcgac 360

ggctacacct tcggttcggg gaccaggtta accgttgtag aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtat gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacccgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 900

ctgatggcca tggtcaagag aaaggattcc agaggc 936

<210>22

<211>307

<212>PRT

<213>Artificial sequence

<400>22

MGTRLFFYVA LCLLWAGHRD AGITQSPRYK ITETGRQVTL MCHQTWSHSY MFWYRQDLGH 60

GLRLIYYSAA ADITDKGEVP DGYVVSRSKT ENFPLTLESA TRSQTSVYFC ASSDPGTEAF 120

FGQGTRLTVV EDLNKVFPPE VAVFEPSEAE ISHTQKATLV CLATGFFPDH VELSWWVNGK 180

EVHSGVSTDP QPLKEQPALN DSRYCLSSRL RVSATFWQNP RNHFRCQVQF YGLSENDEWT 240

QDRAKPVTQI VSAEAWGRAD CGFTSVSYQQ GVLSATILYE ILLGKATLYA VLVSALVLMA 300

MVKRKDF 307

<210>23

<211>921

<212> DNA

<213>Artificial sequence

<400>23

atgggcacca ggctcttctt ctatgtggcc ctttgtctgc tgtgggcagg acacagggat 60

gctggaatca cccagagccc aagatacaag atcacagaga caggaaggca ggtgaccttg 120

atgtgtcacc agacttggag ccacagctat atgttctggt atcgacaaga cctgggacat 180

gggctgaggc tgatctatta ctcagcagct gctgatatta cagataaagg agaagtcccc 240

gatggctatg ttgtctccag atccaagaca gagaatttcc ccctcactct ggagtcagct 300

acccgctccc agacatctgt gtatttctgc gccagcagtg accccggcac tgaagctttc 360

tttggacaag gcaccagact cacagttgta gaggacctga acaaggtgtt cccacccgag 420

gtcgctgtgt ttgagccatc agaagcagag atctcccaca cccaaaaggc cacactggtg 480

tgcctggcca caggcttctt ccccgaccac gtggagctga gctggtgggt gaatgggaag 540

gaggtgcaca gtggggtcag cacggacccg cagcccctca aggagcagcc cgccctcaat 600

gactccagat actgcctgag cagccgcctg agggtctcgg ccaccttctg gcagaacccc 660

cgcaaccact tccgctgtca agtccagttc tacgggctct cggagaatga cgagtggacc 720

caggataggg ccaaacccgt cacccagatc gtcagcgccg aggcctgggg tagagcagac 780

tgtggcttta cctcggtgtc ctaccagcaa ggggtcctgt ctgccaccat cctctatgag 840

atcctgctag ggaaggccac cctgtatgct gtgctggtca gcgcccttgt gttgatggcc 900

atggtcaaga gaaaggattt c 921

<210>24

<211>309

<212>PRT

<213>Artificial sequence

<400>24

MGTRLFFYVA LCLLWAGHRD AGITQSPRYK ITETGRQVTL MCHQTWSHSY MFWYRQDLGH 60

GLRLIYYSAA ADITDKGEVP DGYVVSRSKT ENFPLTLESA TRSQTSVYFC ASSDPGTEAF 120

FGQGTRLTVV EDLKNVFPPE VAVFEPSEAE ISHTQKATLV CLATGFYPDH VELSWWVNGK 180

EVHSGVSTDP QPLKEQPALN DSRYCLSSRL RVSATFWQNP RNHFRCQVQF YGLSENDEWT 240

QDRAKPVTQI VSAEAWGRAD CGFTSESYQQ GVLSATILYE ILLGKATLYA VLVSALVLMA 300

MVKRKDSRG 309

<210>25

<211>927

<212> DNA

<213>Artificial sequence

<400>25

atgggcacca ggctcttctt ctatgtggcc ctttgtctgc tgtgggcagg acacagggat 60

gctggaatca cccagagccc aagatacaag atcacagaga caggaaggca ggtgaccttg 120

atgtgtcacc agacttggag ccacagctat atgttctggt atcgacaaga cctgggacat 180

gggctgaggc tgatctatta ctcagcagct gctgatatta cagataaagg agaagtcccc 240

gatggctatg ttgtctccag atccaagaca gagaatttcc ccctcactct ggagtcagct 300

acccgctccc agacatctgt gtatttctgc gccagcagtg accccggcac tgaagctttc 360

tttggacaag gcaccagact cacagttgta gaggacctga aaaacgtgtt cccacccgag 420

gtcgctgtgt ttgagccatc agaagcagag atctcccaca cccaaaaggc cacactggtg 480

tgcctggcca caggcttcta ccccgaccac gtggagctga gctggtgggt gaatgggaag 540

gaggtgcaca gtggggtcag cacagacccg cagcccctca aggagcagcc cgccctcaat 600

gactccagat actgcctgag cagccgcctg agggtctcgg ccaccttctg gcagaacccc 660

cgcaaccact tccgctgtca agtccagttc tacgggctct cggagaatga cgagtggacc 720

caggataggg ccaaacctgt cacccagatc gtcagcgccg aggcctgggg tagagcagac 780

tgtggcttca cctccgagtc ttaccagcaa ggggtcctgt ctgccaccat cctctatgag 840

atcttgctag ggaaggccac cttgtatgcc gtgctggtca gtgccctcgt gctgatggcc 900

atggtcaaga gaaaggattc cagaggc 927

<210>26

<211>309

<212>PRT

<213>Artificial sequence

<400>26

MGTRLFFYVA LCLLWAGHRD AGITQSPRYK ITETGRQVTL MCHQTWSHSY MFWYRQDLGH 60

GLRLIYYSAA ADITDKGEVP DGYVVSRSKT ENFPLTLESA TRSQTSVYFC ASSDPGTEAF 120

FGQGTRLTVV EDLKNVFPPE VAVFEPSEAE ISHTQKATLV CLATGFYPDH VELSWWVNGK 180

EVHSGVSTDP QPLKEQPALN DSRYCLSSRL RVSATFWQNP RNHFRCQVQF YGLSENDEWT 240

QDRAKPVTQI VSAEAWGRAD CGFTSESYQQ GVLSATILYE ILLGKATLYA VLVSALVLMA 300

MVKRKDSRG 309

<210>27

<211>927

<212> DNA

<213>Artificial sequence

<400>27

atgggcacca ggctcttctt ctatgtggcc ctttgtctgc tgtgggcagg acacagggat 60

gctggaatca cccagagccc aagatacaag atcacagaga caggaaggca ggtgaccttg 120

atgtgtcacc agacttggag ccacagctat atgttctggt atcgacaaga cctgggacat 180

gggctgaggc tgatctatta ctcagcagct gctgatatta cagataaagg agaagtcccc 240

gatggctatg ttgtctccag atccaagaca gagaatttcc ccctcactct ggagtcagct 300

acccgctccc agacatctgt gtatttctgc gccagcagtg accccggcac tgaagctttc 360

tttggacaag gcaccagact cacagttgta gaggacctga aaaacgtgtt cccacccgag 420

gtcgctgtgt ttgagccatc agaagcagag atctcccaca cccaaaaggc cacactggta 480

tgcctggcca caggcttcta ccccgaccac gtggagctga gctggtgggt gaatgggaag 540

gaggtgcaca gtggggtcag cacagacccg cagcccctca aggagcagcc cgccctcaat 600

gactccagat actgcctgag cagccgcctg agggtctcgg ccaccttctg gcagaacccc 660

cgcaaccact tccgctgtca agtccagttc tacgggctct cggagaatga cgagtggacc 720

caggataggg ccaaacccgt cacccagatc gtcagcgccg aggcctgggg tagagcagac 780

tgtggcttca cctccgagtc ttaccagcaa ggggtcctgt ctgccaccat cctctatgag 840

atcttgctag ggaaggccac cttgtatgcc gtgctggtca gtgccctcgt gctgatggcc 900

atggtcaaga gaaaggattc cagaggc 927

<210>28

<211>310

<212>PRT

<213>Artificial sequence

<400>28

MGPGLLCWAL LCLLGAGLVD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ 60

GPQFIFQYYE EEERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSFGQSSNY 120

GYTFGSGTRL TVVEDLNKVF PPEVAVFEPS EAEISHTQKA TLVCLATGFF PDHVELSWWV 180

NGKEVHSGVS TDPQPLKEQP ALNDSRYCLS SRLRVSATFW QNPRNHFRCQ VQFYGLSEND 240

EWTQDRAKPV TQIVSAEAWG RADCGFTSVS YQQGVLSATI LYEILLGKAT LYAVLVSALV 300

LMAMVKRKDF 310

<210>29

<211>930

<212> DNA

<213>Artificial sequence

<400>29

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct tcggccagag cagcaactat 360

ggctacacct tcggttcggg gaccaggtta accgttgtag aggacctgaa caaggtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttcttc cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acggacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacccgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggctttac ctcggtgtcc taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcctgctagg gaaggccacc ctgtatgctg tgctggtcag cgcccttgtg 900

ttgatggcca tggtcaagag aaaggatttc 930

<210>30

<211>312

<212>PRT

<213>Artificial sequence

<400>30

MGPGLLCWAL LCLLGAGLVD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ 60

GPQFIFQYYE EEERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSFGQSSNY 120

GYTFGSGTRL TVVEDLKNVF PPEVAVFEPS EAEISHTQKA TLVCLATGFY PDHVELSWWV 180

NGKEVHSGVS TDPQPLKEQP ALNDSRYCLS SRLRVSATFW QNPRNHFRCQ VQFYGLSEND 240

EWTQDRAKPV TQIVSAEAWG RADCGFTSES YQQGVLSATI LYEILLGKAT LYAVLVSALV 300

LMAMVKRKDS RG 312

<210>31

<211>936

<212> DNA

<213>Artificial sequence

<400>31

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct tcggccagag cagcaactat 360

ggctacacct tcggttcggg gaccaggtta accgttgtag aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacctgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 900

ctgatggcca tggtcaagag aaaggattcc agaggc 936

<210>32

<211>312

<212>PRT

<213>Artificial sequence

<400>32

MGPGLLCWAL LCLLGAGLVD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ 60

GPQFIFQYYE EEERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSFGQSSNY 120

GYTFGSGTRL TVVEDLKNVF PPEVAVFEPS EAEISHTQKA TLVCLATGFY PDHVELSWWV 180

NGKEVHSGVS TDPQPLKEQP ALNDSRYCLS SRLRVSATFW QNPRNHFRCQ VQFYGLSEND 240

EWTQDRAKPV TQIVSAEAWG RADCGFTSES YQQGVLSATI LYEILLGKAT LYAVLVSALV 300

LMAMVKRKDS RG 312

<210>33

<211>936

<212> DNA

<213>Artificial sequence

<400>33

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct tcggccagag cagcaactat 360

ggctacacct tcggttcggg gaccaggtta accgttgtag aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtat gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacccgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 900

ctgatggcca tggtcaagag aaaggattcc agaggc 936

<210>34

<211>310

<212>PRT

<213>Artificial sequence

<400>34

MGPGLLCWAL LCLLGAGLVD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ 60

GPQFIFQYYE EEERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSLGRASNQ 120

PQHFGDGTRL SILEDLNKVF PPEVAVFEPS EAEISHTQKA TLVCLATGFF PDHVELSWWV 180

NGKEVHSGVS TDPQPLKEQP ALNDSRYCLS SRLRVSATFW QNPRNHFRCQ VQFYGLSEND 240

EWTQDRAKPV TQIVSAEAWG RADCGFTSVS YQQGVLSATI LYEILLGKAT LYAVLVSALV 300

LMAMVKRKDF 310

<210>35

<211>930

<212> DNA

<213>Artificial sequence

<400>35

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct tgggcagagc cagcaatcag 360

ccccagcatt ttggtgatgg gactcgactc tccatcctag aggacctgaa caaggtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttcttc cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acggacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacccgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggctttac ctcggtgtcc taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcctgctagg gaaggccacc ctgtatgctg tgctggtcag cgcccttgtg 900

ttgatggcca tggtcaagag aaaggatttc 930

<210>36

<211>312

<212>PRT

<213>Artificial sequence

<400>36

MGPGLLCWAL LCLLGAGLVD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ 60

GPQFIFQYYE EEERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSLGRASNQ 120

PQHFGDGTRL SILEDLKNVF PPEVAVFEPS EAEISHTQKA TLVCLATGFY PDHVELSWWV 180

NGKEVHSGVS TDPQPLKEQP ALNDSRYCLS SRLRVSATFW QNPRNHFRCQ VQFYGLSEND 240

EWTQDRAKPV TQIVSAEAWG RADCGFTSES YQQGVLSATI LYEILLGKAT LYAVLVSALV 300

LMAMVKRKDS RG 312

<210>37

<211>936

<212> DNA

<213>Artificial sequence

<400>37

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct tgggcagagc cagcaatcag 360

ccccagcatt ttggtgatgg gactcgactc tccatcctag aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacctgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 900

ctgatggcca tggtcaagag aaaggattcc agaggc 936

<210>38

<211>312

<212>PRT

<213>Artificial sequence

<400>38

MGPGLLCWAL LCLLGAGLVD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ 60

GPQFIFQYYE EEERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSLGRASNQ 120

PQHFGDGTRL SILEDLKNVF PPEVAVFEPS EAEISHTQKA TLVCLATGFY PDHVELSWWV 180

NGKEVHSGVS TDPQPLKEQP ALNDSRYCLS SRLRVSATFW QNPRNHFRCQ VQFYGLSEND 240

EWTQDRAKPV TQIVSAEAWG RADCGFTSES YQQGVLSATI LYEILLGKAT LYAVLVSALV 300

LMAMVKRKDS RG 312

<210>39

<211>936

<212> DNA

<213>Artificial sequence

<400>39

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct tgggcagagc cagcaatcag 360

ccccagcatt ttggtgatgg gactcgactc tccatcctag aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtat gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc acagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacccgtc acccagatcg tcagcgccga ggcctggggt 780

agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 900

ctgatggcca tggtcaagag aaaggattcc agaggc 936

<210>40

<211>309

<212>PRT

<213>Artificial sequence

<400>40

MGPGLLCWAL LCLLGAGLVD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ 60

GPQFIFQYYE EEERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSLGQTNYG 120

YTFGSGTRLT VVEDLNKVFP PEVAVFEPSE AEISHTQKAT LVCLATGFFP DHVELSWWVN 180

GKEVHSGVST DPQPLKEQPA LNDSRYCLSS RLRVSATFWQ NPRNHFRCQV QFYGLSENDE 240

WTQDRAKPVT QIVSAEAWGR ADCGFTSVSY QQGVLSATIL YEILLGKATL YAVLVSALVL 300

MAMVKRKDF 309

<210>41

<211>927

<212> DNA

<213>Artificial sequence

<400>41

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct tgggccagac caactatggc 360

tacaccttcg gttcggggac caggttaacc gttgtagagg acctgaacaa ggtgttccca 420

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 480

ctggtgtgcc tggccacagg cttcttcccc gaccacgtgg agctgagctg gtgggtgaat 540

gggaaggagg tgcacagtgg ggtcagcacg gacccgcagc ccctcaagga gcagcccgcc 600

ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 660

aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720

tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 780

gcagactgtg gctttacctc ggtgtcctac cagcaagggg tcctgtctgc caccatcctc 840

tatgagatcc tgctagggaa ggccaccctg tatgctgtgc tggtcagcgc ccttgtgttg 900

atggccatgg tcaagagaaa ggatttc 927

<210>42

<211>311

<212>PRT

<213>Artificial sequence

<400>42

MGPGLLCWAL LCLLGAGLVD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ 60

GPQFIFQYYE EEERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSLGQTNYG 120

YTFGSGTRLT VVEDLKNVFP PEVAVFEPSE AEISHTQKAT LVCLATGFYP DHVELSWWVN 180

GKEVHSGVST DPQPLKEQPA LNDSRYCLSS RLRVSATFWQ NPRNHFRCQV QFYGLSENDE 240

WTQDRAKPVT QIVSAEAWGR ADCGFTSESY QQGVLSATIL YEILLGKATL YAVLVSALVL 300

MAMVKRKDSR G 311

<210>43

<211>933

<212> DNA

<213>Artificial sequence

<400>43

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct tgggccagac caactatggc 360

tacaccttcg gttcggggac caggttaacc gttgtagagg acctgaaaaa cgtgttccca 420

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 480

ctggtgtgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 540

gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 600

ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 660

aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720

tggacccagg atagggccaa acctgtcacc cagatcgtca gcgccgaggc ctggggtaga 780

gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtctgc caccatcctc 840

tatgagatct tgctagggaa ggccaccttg tatgccgtgc tggtcagtgc cctcgtgctg 900

atggccatgg tcaagagaaa ggattccaga ggc 933

<210>44

<211>311

<212>PRT

<213>Artificial sequence

<400>44

MGPGLLCWAL LCLLGAGLVD AGVTQSPTHL IKTRGQQVTL RCSPKSGHDT VSWYQQALGQ 60

GPQFIFQYYE EEERQRGNFP DRFSGHQFPN YSSELNVNAL LLGDSALYLC ASSLGQTNYG 120

YTFGSGTRLT VVEDLKNVFP PEVAVFEPSE AEISHTQKAT LVCLATGFYP DHVELSWWVN 180

GKEVHSGVST DPQPLKEQPA LNDSRYCLSS RLRVSATFWQ NPRNHFRCQV QFYGLSENDE 240

WTQDRAKPVT QIVSAEAWGR ADCGFTSESY QQGVLSATIL YEILLGKATL YAVLVSALVL 300

MAMVKRKDSR G 311

<210>45

<211>933

<212> DNA

<213>Artificial sequence

<400>45

atgggccccg ggctcctctg ctgggcactg ctttgtctcc tgggagcagg cttagtggac 60

gctggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctc ctaagtctgg gcatgacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatgag gaggaagaga gacagagagg caacttccct 240

gatcgattct caggtcacca gttccctaac tatagctctg agctgaatgt gaacgccttg 300

ttgctggggg actcggccct ctatctctgt gccagcagct tgggccagac caactatggc 360

tacaccttcg gttcggggac caggttaacc gttgtagagg acctgaaaaa cgtgttccca 420

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 480

ctggtatgcc tggccacagg cttctacccc gaccacgtgg agctgagctg gtgggtgaat 540

gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 600

ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 660

aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720

tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 780

gcagactgtg gcttcacctc cgagtcttac cagcaagggg tcctgtctgc caccatcctc 840

tatgagatct tgctagggaa ggccaccttg tatgccgtgc tggtcagtgc cctcgtgctg 900

atggccatgg tcaagagaaa ggattccaga ggc 933

<210>46

<211>602

<212>PRT

<213>Artificial sequence

<400>46

MRSMDFKSNS AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESSCDVKLVE KSFETDTNLN 240

FQNLSVIGFR ILLLKVAGFN LLMTLRLWSS GSGATNFSLL KQAGDVEENP GPMGPGLLCW 300

ALLCLLGAGL VDAGVTQSPT HLIKTRGQQV TLRCSPKSGH DTVSWYQQAL GQGPQFIFQY 360

YEEEERQRGN FPDRFSGHQF PNYSSELNVN ALLLGDSALY LCASSLGEGR VDGYTFGSGT 420

RLTVVEDLNK VFPPEVAVFE PSEAEISHTQ KATLVCLATG FFPDHVELSW WVNGKEVHSG 480

VCTDPQPLKE QPALNDSRYC LSSRLRVSAT FWQNPRNHFR CQVQFYGLSE NDEWTQDRAK 540

PVTQIVSAEA WGRADCGFTS VSYQQGVLSA TILYEILLGK ATLYAVLVSA LVLMAMVKRK 600

DF 602

<210>47

<211>1806

<212> DNA

<213>Artificial sequence

<400>47

atgaggcaag tggcgagagt gatcgtgttc ctgaccctga gtactttgag ccttgctaag 60

accacccagc ccatctccat ggactcatat gaaggacaag aagtgaacat aacctgtagc 120

cacaacaaca ttgctacaaa tgattatatc acgtggtacc aacagtttcc cagccaagga 180

ccacgattta ttattcaagg atacaagaca aaagttacaa acgaagtggc ctccctgttt 240

atccctgccg acagaaagtc cagcactctg agcctgcccc gggtttccct gagcgacact 300

gctgtgtact actgcctcgt gggtgacatg gaccaggcag gaactgctct gatctttggg 360

aagggaacca ccttatcagt gagttccaat atccagaacc ctgaccctgc cgtgtaccag 420

ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 480

acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaatg cgtgctagac 540

atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacaa atctgacttt 600

gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 660

gaaagttcct gtgatgtcaa gctggtcgag aaaagctttg aaacagatac gaacctaaac 720

tttcaaaacc tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat 780

ctgctcatga cgctgcggct gtggtccagc ggcagcggcg ctaccaactt cagcctgctg 840

aagcaggccg gcgacgtgga ggaaaaccct ggccccatgg gccccgggct cctctgctgg 900

gcactgcttt gtctcctggg agcaggctta gtggacgctg gagtcaccca aagtcccaca 960

cacctgatca aaacgagagg acagcaagtg actctgagat gctctcctaa gtctgggcat 1020

gacactgtgt cctggtacca acaggccctg ggtcaggggc cccagtttat ctttcagtat 1080

tatgaggagg aagagagaca gagaggcaac ttccctgatc gattctcagg tcaccagttc 1140

cctaactata gctctgagct gaatgtgaac gccttgttgc tgggggactc ggccctctat 1200

ctctgtgcca gcagcttggg cgagggcaga gtcgacggct acaccttcgg ttcggggacc 1260

aggttaaccg ttgtagagga cctgaacaag gtgttcccac ccgaggtcgc tgtgtttgag 1320

ccatcagaag cagagatctc ccacacccaa aaggccacac tggtgtgcct ggccacaggc 1380

ttcttccccg accacgtgga gctgagctgg tgggtgaatg ggaaggaggt gcacagtggg 1440

gtctgcacgg acccgcagcc cctcaaggag cagcccgccc tcaatgactc cagatactgc 1500

ctgagcagcc gcctgagggt ctcggccacc ttctggcaga acccccgcaa ccacttccgc 1560

tgtcaagtcc agttctacgg gctctcggag aatgacgagt ggacccagga tagggccaaa 1620

cccgtcaccc agatcgtcag cgccgaggcc tggggtagag cagactgtgg ctttacctcg 1680

gtgtcctacc agcaaggggt cctgtctgcc accatcctct atgagatcct gctagggaag 1740

gccaccctgt atgctgtgct ggtcagcgcc cttgtgttga tggccatggt caagagaaag 1800

gatttc 1806

<210>48

<211>10

<212>PRT

<213>Artificial sequence

<400>48

VVVGADGVGK 10

<210>49

<211>12

<212>PRT

<213>Artificial sequence

<400>49

KLVVVGADGV GK 12

Claims (10)

1. a kind of T cell receptor, which is characterized in that itself and 12 codon mutation G12D 26aa peptide fragment specificity knots of KRAS genes It closes, the amino acid sequence of the peptide fragment is:MTEYKLVVVGADGVGKSALTIQLIQN, such as SEQ ID NO:Shown in 1;The T is thin When born of the same parents' receptor has identification HLA molecular antigen compounds, the 9-12aa antigen epitope polypeptides with higher affinity are as follows: VVVGADGVGK, such as SEQ ID NO:Shown in 48;KLVVVGADGVGK, such as SEQ ID NO:Shown in 49;The HLA molecules are HLA-A0301;When identifying the antigenic compound that KRAS genes G12D mutation peptide fragments are formed with HLA-A0301, T cell receptor α chains and β chains sequence it is as follows:
α chains use any one in following sequence:
α chain-orderings 1:Such as SEQ ID NO:Amino acid sequence shown in 2;
α chain-orderings 2:Such as SEQ ID NO:Amino acid sequence shown in 4;
α chain-orderings 3:Such as SEQ ID NO:Amino acid sequence shown in 6;
α chain-orderings 4:Such as SEQ ID NO:Amino acid sequence shown in 8;
α chain-orderings 5:Such as SEQ ID NO:Amino acid sequence shown in 10;
α chain-orderings 6:Such as SEQ ID NO:Amino acid sequence shown in 12;
α chain-orderings 7:Such as SEQ ID NO:Amino acid sequence shown in 14;
β chains use any one in following sequence:
β chain-orderings 1:Such as SEQ ID NO:Amino acid sequence shown in 16;
β chain-orderings 2:Such as SEQ ID NO:Amino acid sequence shown in 18;
β chain-orderings 3:Such as SEQ ID NO:Amino acid sequence shown in 20;
β chain-orderings 4:Such as SEQ ID NO:Amino acid sequence shown in 22;
β chain-orderings 5:Such as SEQ ID NO:Amino acid sequence shown in 24;
β chain-orderings 6:Such as SEQ ID NO:Amino acid sequence shown in 26;
β chain-orderings 7:Such as SEQ ID NO:Amino acid sequence shown in 28;
β chain-orderings 8:Such as SEQ ID NO:Amino acid sequence shown in 30;
β chain-orderings 9:Such as SEQ ID NO:Amino acid sequence shown in 32;
β chain-orderings 10:Such as SEQ ID NO:Amino acid sequence shown in 34;
β chain-orderings 11:Such as SEQ ID NO:Amino acid sequence shown in 36;
β chain-orderings 12:Such as SEQ ID NO:Amino acid sequence shown in 38;
β chain-orderings 13:Such as SEQ ID NO:Amino acid sequence shown in 40;
β chain-orderings 14:Such as SEQ ID NO:Amino acid sequence shown in 42;
β chain-orderings 15:Such as SEQ ID NO:Amino acid sequence shown in 44.
2. T cell receptor as described in claim 1, which is characterized in that in identification KRAS genes G12D mutation peptide fragments and HLA- When the antigenic compound that A0301 is formed, the α chains of T cell receptor and β chains be combined as it is following any one:
α chain-ordering 1+ β chain-orderings 1
α chain-ordering 1+ β chain-orderings 2
α chain-ordering 1+ β chain-orderings 3
α chain-ordering 2+ β chain-orderings 1
α chain-ordering 2+ β chain-orderings 2
α chain-ordering 2+ β chain-orderings 3
α chain-ordering 3+ β chain-orderings 4
α chain-ordering 3+ β chain-orderings 5
α chain-ordering 3+ β chain-orderings 6
α chain-ordering 3+ β chain-orderings 10
α chain-ordering 3+ β chain-orderings 11
α chain-ordering 3+ β chain-orderings 12
α chain-ordering 3+ β chain-orderings 13
α chain-ordering 3+ β chain-orderings 14
α chain-ordering 3+ β chain-orderings 15
α chain-ordering 4+ β chain-orderings 4
α chain-ordering 4+ β chain-orderings 5
α chain-ordering 4+ β chain-orderings 6
α chain-ordering 4+ β chain-orderings 10
α chain-ordering 4+ β chain-orderings 11
α chain-ordering 4+ β chain-orderings 12
α chain-ordering 4+ β chain-orderings 13
α chain-ordering 4+ β chain-orderings 14
α chain-ordering 4+ β chain-orderings 15
α chain-ordering 5+ β chain-orderings 4
α chain-ordering 5+ β chain-orderings 5
α chain-ordering 5+ β chain-orderings 6
α chain-ordering 5+ β chain-orderings 10
α chain-ordering 5+ β chain-orderings 11
α chain-ordering 5+ β chain-orderings 12
α chain-ordering 5+ β chain-orderings 13
α chain-ordering 5+ β chain-orderings 14
α chain-ordering 5+ β chain-orderings 15
α chain-ordering 6+ β chain-orderings 7
α chain-ordering 6+ β chain-orderings 8
α chain-ordering 6+ β chain-orderings 9
α chain-ordering 7+ β chain-orderings 7
α chain-ordering 7+ β chain-orderings 8
α chain-ordering 7+ β chain-orderings 9.
3. T cell receptor as claimed in claim 1 or 2, which is characterized in that introduced in the constant region of the α chains and β chains prominent Become, the additional disulfide bond for increasing extracellular region.
4. T cell receptor as claimed in claim 1 or 2, which is characterized in that introduce 2A or IRES sequences between α chains and β chains Row make α chains and β chain buildings enter an expression frame content, convenient for recombinantly expressing and assembling T cell receptor.
5. T cell receptor as claimed in claim 3, which is characterized in that it is such as SEQ ID NO:Amino acid sequence shown in 46 Row.
6. nucleotide sequence, α chains as claimed in claim 1 or 2 being connected with each other and as claimed in claim 1 or 2 are encoded β chains, the codon adopted is by optimization, it is characterised in that:
Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 1:Shown in 3;
Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 2:Shown in 5;
Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 3:Shown in 7;
Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 4:Shown in 9;
Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 5:Shown in 11;
Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 6:Shown in 13;
Encode the nucleotide sequence such as SEQ ID NO of the α chain-orderings 7:Shown in 15;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 1:Shown in 17;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 2:Shown in 19;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 3:Shown in 21;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 4:Shown in 23;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 5:Shown in 25;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 6:Shown in 27;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 7:Shown in 29;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 8:Shown in 31;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 9:Shown in 33;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 10:Shown in 35;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 11:Shown in 37;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 12:Shown in 39;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 13:Shown in 41;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 14:Shown in 43;
Encode the nucleotide sequence such as SEQ ID NO of the β chain-orderings 15:Shown in 45.
7. nucleotide sequence as claimed in claim 6, the codon adopted is by optimization, which is characterized in that it is such as SEQ ID NO:Sequence shown in 47.
8. including the carrier of the nucleotide sequence described in claim 6 or 7.
9. including the cell of the nucleotide sequence described in claim 6 or 7.
10. the cell described in carrier according to any one of claims 8 or claim 9 is preparing prevention KRAS gene mutation relevant disease Drug in application.
CN201710065966.7A 2017-02-06 2017-02-06 A kind of T cell receptor in relation to KRAS gene mutation CN108395479A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107223134A (en) * 2014-11-26 2017-09-29 美国卫生和人力服务部 The KRAS of anti-mutation φt cell receptor
CN110172089A (en) * 2019-06-11 2019-08-27 北京鼎成肽源生物技术有限公司 A kind of more antigen combinations of KRAS mutation, targeting KRAS mutation tumour CTL and its application

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107223134A (en) * 2014-11-26 2017-09-29 美国卫生和人力服务部 The KRAS of anti-mutation φt cell receptor
CN110172089A (en) * 2019-06-11 2019-08-27 北京鼎成肽源生物技术有限公司 A kind of more antigen combinations of KRAS mutation, targeting KRAS mutation tumour CTL and its application
CN110172089B (en) * 2019-06-11 2020-01-07 北京鼎成肽源生物技术有限公司 KRAS mutant multi-antigen combination, targeting KRAS mutant tumor CTL and application thereof

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