CN114920824B

CN114920824B - TCR or antigen binding fragment thereof and uses thereof

Info

Publication number: CN114920824B
Application number: CN202210591084.5A
Authority: CN
Inventors: 金艾顺; 韩晓建; 申美莹; 王君凡; 张静; 王易
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2022-05-27
Filing date: 2022-05-27
Publication date: 2023-12-05
Anticipated expiration: 2042-05-27
Also published as: CN114920824A

Abstract

The invention belongs to the technical field of T cell immunotherapy medicaments, and particularly relates to a T Cell Receptor (TCR) or an antigen binding fragment thereof and application thereof, wherein the TCR comprises a fragment obtained by coding a sequence shown as SEQ ID NO. 15. The TCR has strong specificity for KRAS-G12D mutation, and can mediate T cells to secrete a large amount of IFN-gamma cytokines and kill tumor cells with KRAS-G12D gene mutation.

Description

TCR or antigen binding fragment thereof and uses thereof

Technical Field

The invention belongs to the technical field of T cell immunotherapy medicaments, and particularly relates to a TCR or an antigen binding fragment thereof and application thereof.

Background

With the progress of research in the biomedical field, target selection of T cell immunotherapy targeting malignant solid tumors is evolving continuously, changing from initial lineage antigens to viral antigens, whereas with the advent of high throughput sequencing technology in recent years, targeted tumor individuals are mutated to targets focused for new generation T cell therapy.

T cell therapy currently includes mainly TCR-T and CAR-T therapeutic approaches. The existing CAR-T technology has remarkable effect in treating hematoma such as leukemia, lymphoma and the like, and greatly improves the survival rate and the survival quality of patients, but aiming at solid tumors, the CAR-T treatment means has limited application prospect due to limited specific targets at present. TCR-T technology differs from CAR-T cell technology in that TCRs are characteristic markers on the surface of all T cells, binding to CD3 non-covalently to form a TCR-CD3 complex. In peripheral blood, 90% -95% of T cells express TCR, and the T cells of the genetically modified TCR can specifically identify antigen molecules on the surface of tumor cells so as to generate immune response to the tumor cells. Currently, in human cancers, KRAS gene is one of the most well known oncogenes in the oncology field, KRAS gene mutations (KRAS-G12C mutation, KRAS-G12D mutation, KRAS-G12V mutation, etc.) occur in nearly 90% of pancreatic cancers, 30% -40% of colon cancers, 17% of endometrial cancers, 15% -20% of lung cancers including lobular lung cancers, as well as cholangiocarcinomas, cervical cancers, bladder cancers, etc.

With the foregoing in mind, there is a need for a TCR that specifically binds to KRAS mutant polypeptides and is capable of mediating T cell killing of KRAS mutant tumor cells.

Disclosure of Invention

One of the purposes of the invention is to provide a TCR which can specifically identify KRAS-G12D mutation sites in a targeted manner, specifically bind with a complex of KRAS-G12D mutated polypeptides and HLA, thereby stimulating T cell activation, mediating T cells to secrete cytokines such as IFN-gamma and the like, and further killing tumor cells expressing KRAS-G12D mutated genes. The TCR or antigen binding fragment thereof has strong specificity for KRAS-G12D mutant polypeptide, and can mediate T cells to secrete a large amount of IFN-gamma cytokines, so as to kill KRAS-G12D mutant tumor cells.

In one aspect the invention provides a TCR which can be selected from two TCRs:

first kind: the α chain variable region of the TCR may comprise an α chain CDR3 encoded by a sequence as set out in seq id No. 3, and the β chain variable region of the TCR comprises a β chain CDR3 encoded by a sequence as set out in seq id No. 6.

Second kind: the TCR may comprise a fragment encoded by a sequence as set forth in SEQ ID NO: 15.

In another aspect, the invention provides an antigen binding fragment that may comprise either of the two TCRs described above.

In yet another aspect, the invention provides a polynucleotide that can be selected from the group consisting of:

first kind: sequences shown as SEQ ID NO. 3 and SEQ ID NO. 6 can be contained.

Second kind: the sequence shown in SEQ ID NO. 15 may be contained.

In yet another aspect, the invention provides an expression vector which may comprise any one of the two polynucleotides described above.

In yet another aspect, the invention provides an engineered cell, which may comprise an expression vector as described above.

In a further aspect the invention provides a pharmaceutical composition which may comprise a TCR as defined above or an antigen-binding fragment as defined above or a polynucleotide as defined above or an expression vector as defined above or an engineered cell as defined above, and a pharmaceutically acceptable carrier and/or diluent.

In a further aspect, the invention provides the use of a TCR as defined above or an antigen-binding fragment as defined above or a polynucleotide as defined above or an expression vector as defined above or an engineered cell as defined above in the manufacture of a medicament for increasing the secretion of IFN- γ and/or Granzyme-B cytokine levels by a T cell.

In a further aspect, the invention provides the use of a TCR as defined above or an antigen-binding fragment as defined above or a polynucleotide as defined above or an expression vector as defined above or an engineered cell as defined above in the preparation of a tumour cell reagent or kit for detecting a mutation expressing KRAS-G12D.

In a further aspect, the invention provides the use of a TCR as defined above or an antigen-binding fragment as defined above or a polynucleotide as defined above or an expression vector as defined above or an engineered cell as defined above in the manufacture of a medicament for the treatment of a disease caused by a KRAS-G12D mutation.

The invention has the beneficial effects that: the TCR provided by the invention has strong specificity on KRAS-G12D mutant polypeptide, and can mediate T cells to secrete a large amount of IFN-gamma cytokines to kill KRAS-G12D mutant tumor cells.

Drawings

FIG. 1 is a graph showing the results of a flow cytometry detection of KRAS-G12D mutant polypeptide specificity by KT5 in the examples.

FIG. 2 shows the secretion of IFN-gamma cytokines by T cells mediated by KT5 in the examples.

FIG. 3 is a graph showing the secretion of Granzyme-B cytokines by T cells mediated by KT5 in the examples.

Detailed Description

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of a feature, number, operation, component, part, element, material, or combination. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, elements, materials or combinations thereof may be present or added. As used herein, "/" may be interpreted as "and" or "as appropriate.

In the present invention, the term "antigen binding fragment" refers to antigen binding fragments of TCRs and TCR analogs, which generally include at least a portion of the antigen binding or variable regions of the parent TCR, e.g., one or more CDRs. Fragments of the TCR retain at least some of the binding specificity of the parent TCR.

The invention aims to provide a TCR, and one of the aims of the invention is to provide a TCR which can specifically identify KRAS-G12D mutation site in a targeting manner and specifically bind with a complex of KRAS-G12D mutated polypeptide and HLA, so as to stimulate T cell activation, mediate T cells to secrete cytokines such as IFN-gamma and the like, and further kill tumor cells expressing KRAS-G12D mutation. The TCR or antigen binding fragment thereof has strong specificity for KRAS-G12D mutated polypeptides and is capable of mediating the secretion of large amounts of IFN-gamma cytokines by T cells.

In one aspect, the invention provides a TCR comprising an alpha chain variable region and a beta chain variable region, the TCR of the invention being selectable from:

first TCR:

in the first TCR, the alpha chain variable region comprises at least the alpha chain CDR3 encoded by the sequence shown in SEQ ID NO:3 and the beta chain variable region comprises at least the beta chain CDR3 encoded by the sequence shown in SEQ ID NO: 6.

Further, the α chain variable region in the first TCR may further comprise: an alpha chain CDR1 encoded by a sequence shown in SEQ ID NO. 1 and/or an alpha chain CDR2 encoded by a sequence shown in SEQ ID NO. 2; the β chain variable region may further comprise: the beta chain CDR1 encoded by the sequence shown in SEQ ID NO. 4 and/or the beta chain CDR2 encoded by the sequence shown in SEQ ID NO. 5.

Further, the α chain variable region in the first TCR may further comprise: one or more of an alpha chain FR1 encoded by a sequence shown in SEQ ID NO. 7, an alpha chain FR2 encoded by a sequence shown in SEQ ID NO. 8, an alpha chain FR3 encoded by a sequence shown in SEQ ID NO. 9 and an alpha chain FR4 encoded by a sequence shown in SEQ ID NO. 10; the β chain variable region may further comprise: one or more of a beta-strand FR1 encoded by a sequence shown as SEQ ID NO:11, a beta-strand FR2 encoded by a sequence shown as SEQ ID NO:12, a beta-strand FR3 encoded by a sequence shown as SEQ ID NO:13 and a beta-strand FR4 encoded by a sequence shown as SEQ ID NO: 14.

Specifically, CDR3 of the α chain and β chain in the above TCR belongs to a hypervariable region of a variable region, and when the TCR recognizes an MHC-antigen peptide complex, CDR3 directly binds to an antigen peptide, directly affects the recognition ability of the TCR to KRAS-G12D mutation. In the hypervariable regions of the α and β chains of the TCR, two hypervariable regions of CDR1 and CDR2 are also included, the CDR1 and CDR2 hypervariable regions being relatively stable with respect to CDR3, and when the TCR recognizes the MHC-antigen peptide complex, the two hypervariable regions of CDR1 and CDR2 recognize and bind to the side walls of the MHC molecule antigen binding groove.

Specifically, FR (FR 1, FR2, FR3, FR 4) in the above TCR is a framework region for connecting CDR regions, and is relatively stable. The FR regions in the first TCR may be selected from sequences other than those described above, such as sequences having greater than or equal to 80%, such as greater than or equal to 80%, 85%, 90%, or 95% sequence identity to the FR1, FR2, FR3, and FR4 sequences described above, and may be of murine or human origin (although other sources such as animal sources such as rabbit, pig, etc. are not excluded). In some embodiments, the sequences of the FR regions and CDR regions of the alpha and beta chains may be arranged according to FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 to form the alpha and beta chain variable regions of the two TCRs, respectively, with the sequence encoding the alpha chain variable region shown as SEQ ID NO:16 and the sequence encoding the beta chain variable region shown as SEQ ID NO: 17.

Specifically, the TCR comprises, in addition to the α chain variable region and the β chain variable region described above, an α chain constant region, and a β chain constant region, and the sources of the α chain constant region and the β chain constant region may be murine or human-murine chimeric (although other sources such as animal sources of rabbit origin, pig origin, and the like are not excluded), and a region in which almost no mutation occurs.

Second TCR:

the second TCR comprises a fragment encoded by a sequence as set forth in SEQ ID NO. 15.

Specifically, in the second TCR, the fragment encoded by the sequence shown as SEQ ID NO:15 comprises the alpha chain variable region and the beta chain variable region of the first TCR.

Specifically, in the second TCR, the coding TCR sequence may also be a sequence having 80% or more identity to the sequence shown in SEQ ID NO. 15, such as 80%, 85%, 90% or 95% or more identity.

Specifically, in the TCR described above, regarding the source of the variable and constant regions of the α chain or β chain, the source of the variable region of the α chain or β chain may be the same as or different from the source of the variable region of the α chain or β chain, from murine sources, from human sources (although other sources such as animal sources such as rabbit sources, pig sources, etc. are not excluded).

In a further aspect the invention provides an antigen binding fragment comprising either of the two TCRs described above.

a first polynucleotide:

the first polynucleotide at least comprises sequences shown as SEQ ID NO. 3 and SEQ ID NO. 6.

Further, the first polynucleotide may further comprise one or more of a sequence shown as SEQ ID NO. 1, a sequence shown as SEQ ID NO. 2, a sequence shown as SEQ ID NO. 4, a sequence shown as SEQ ID NO. 5, a sequence shown as SEQ ID NO. 7, a sequence shown as SEQ ID NO. 8, a sequence shown as SEQ ID NO. 9, a sequence shown as SEQ ID NO. 10, a sequence shown as SEQ ID NO. 11, a sequence shown as SEQ ID NO. 12, a sequence shown as SEQ ID NO. 13 and a sequence shown as SEQ ID NO. 14.

A second polynucleotide:

the second polynucleotide comprises a sequence as shown in SEQ ID NO. 15.

Specifically, the second polynucleotide comprises the sequence of the first polynucleotide, i.e., comprises the sequences shown as SEQ ID NO. 3 and SEQ ID NO. 6.

Specifically, the polynucleotides described above may also comprise any other nucleotide encoding any of the TCRs described above (in addition to the polynucleotides described above), such as a sequence codon optimized as shown in seq id No. 3, a sequence codon optimized as shown in seq id No. 6, and a sequence codon optimized as shown in seq id No. 15.

In a further aspect the invention provides an expression vector which may comprise any one of the two polynucleotides described above.

Further, the expression vector is selected from any one of a lentiviral expression vector, a retrovirus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a DNA vector, an RNA vector, and a plasmid.

In particular, the lentiviral vector may be selected from the group consisting of: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedivirus (VMV), caprine arthritis-encephalitis virus (CAEV), equine Infectious Anemia Virus (EIAV), feline Immunodeficiency Virus (FIV), bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV).

In a further aspect, the invention provides an engineered cell comprising the expression vector described above. Specifically, the engineering cell may be a host cell, and the expression vector is introduced into the host cell to encode to obtain the TCR polypeptide; t cells can also be used for identifying and killing KRAS-G12D mutant polypeptides by transfecting the T cells with the expression vector (loaded with the target gene).

In a further aspect the invention provides a pharmaceutical composition comprising a TCR as defined above or an antigen-binding fragment as defined above or a polynucleotide as defined above or an expression vector as defined above or an engineered cell as defined above, and a pharmaceutically acceptable carrier and/or diluent.

In particular, pharmaceutically acceptable carriers and/or diluents mean that the TCR described above or the antigen-binding fragment described above or the polynucleotide described above or the expression vector described above or the engineered cell described above can be prepared into various desired dosage forms. Examples of the "oral" formulation include tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film-coated tablets, pellets, sublingual tablets, and ointments, and examples of the "non-oral" formulation include injections, suppositories, transdermal agents, ointments, plasters, and external solutions, and those skilled in the art can select an appropriate formulation depending on the route of administration and the object to be administered.

Further, the drugs used for increasing the secretion of IFN-gamma and/or Granzyme-B cytokine levels by T cells comprise cell-based drugs, protein-based drugs, ADC drugs or TCR and antigen combination drugs.

The kit can be divided into various small boxes, and then various detection reagents are contained. The detection reagent and the detection kit can be indirectly or directly applied to various malignant tumors expressing KRAS-G12D mutation, such as pancreatic tumor, colorectal malignant tumor, endometrial malignant tumor, lung malignant tumor, bile duct malignant tumor cancer, cervical malignant tumor and the like.

Further, diseases caused by carrying KRAS-G12D mutations include pancreatic cancer, colorectal cancer, endometrial cancer, lung cancer, cholangiocarcinoma, cervical cancer, or bladder cancer.

In particular, in the above applications, HLA-A1101 with a high affinity is selected for HLA-restricted T cell epitope polypeptides of KRAS-G12D mutated polypeptides, i.e. the polypeptide after KRAS-G12D mutation is presented by HLA-A1101 molecules, but other HLA molecules, such as other small molecules of the HLA-A11 series, are not excluded.

Specifically, in the above application, the TCR selects either one of the two TCRs described above; polynucleotides either of the two polynucleotides described above is selected. In the above application, the TCR or antigen-binding fragment or polynucleotide or expression vector or engineering cell may be formulated with adjuvants, or may be combined with other active agents or detection agents, such as existing chemotherapeutic agents, e.g., alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer agents, hormones, immunological agents, etc.; can also be used in combination with surgery. The specific situation is to take medicine or combination medicine according to the tumor situation.

Specifically, in the application, the KRAS-G12D mutant polypeptides can be 9 polypeptides respectively, 10 polypeptides or other number of polypeptides, and the amino acid sequence of the 10 number of KRAS-G12D mutant polypeptides is shown as SEQ ID NO: 20.

For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.

In the following examples, the sequence of KRAS-G12 wild-type polypeptide (KRAS-G12 for short) is shown as SEQ ID NO. 18; verifying the related KRAS-G12C mutant polypeptide (KRAS-G12C for short), wherein the sequence is shown as SEQ ID NO. 19; verifying the related KRAS-G12D mutant polypeptide (KRAS-G12D for short), wherein the sequence is shown as SEQ ID NO. 20; the sequence of the KRAS-G12V mutant polypeptide (KRAS-G12V for short) is shown as SEQ ID NO. 21.

In the following examples, the gene amplification, sequencing and analysis methods involved were performed according to the following steps:

(1) RT-PCR: the downstream primer used was a TCR gene constant region specific primer (Ca-RV 1primer, cb-RV 1 primer) and the upstream primer was a primer containing an outer adaptor and a TCR signal peptide initiation 20bp sequence (AL primer, BL primer) (in particular according to the examples described in the documents Hamana H, shitaoka K, kishi H, ozawa T, muraguchi A.A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells, biochem Biophys Res Commun.2016Jun 10;474 (4): 709-714.Doi:10.1016/j. Bbrc.2016.05.015.Epub 2016May 4.PMID:27155153);

(2) Second round PCR: taking the PCR product of the TCR obtained in the step (1) as a template, wherein an upstream primer is an outer joint primer (P2A-Cprimer), and a downstream primer is a specific primer (Ca_RV2 primer) of a section of TCR constant region upstream of an alpha chain constant region to obtain a second round PCR product of the TCR alpha chain; the upstream primer is an outer adapter primer (BES-AP primer), the downstream primer is a specific primer (Cb_RV2 primer) of a TCR constant region at the upstream of the beta chain constant region, and the second round PCR product of the TCR beta chain is obtained (specifically, the PCR product is implemented according to the contents of documents Hamana H, shitaoka K, kishi H, ozawa T, muraguchi A.A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells.biochem Biophys Res Commun.2016Jun 10;474 (4): 709-714.doi:10.1016/j.bbrc.2016.05.015.Epub 2016May 4.PMID:27155153);

(3) Performing agarose gel electrophoresis on the amplified second round PCR amplification product containing TCR alpha chain and beta chain variable region genes to obtain TCR alpha chain or beta chain variable region target genes at 500bp-750bp positions;

(4) The target band was subjected to first generation sequencing (Beijing Optimaceae), TCR sequence analysis, and TCR clone, designated KT5, was selected from which high frequency occurred.

The amplification systems used in the above-described procedures involving the gene amplification, sequencing and analysis methods are shown in Table 1 below (specifically, according to the teachings of the documents Hamana H, shitaoka K, kishi H, ozawa T, muraguchi A.A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells. Biochem Biophys Res Commun.2016Jun 10;474 (4): 709-714.Doi:10.1016/j. Bbrc.2016.05.015.Epub 2016May 4.PMID:27155153).

TABLE 1 amplification System

In the examples below, both human PBMC cells and human peripheral blood were derived from volunteers.

In the following examples, the nucleic acid sequences or amino acid sequences of the materials involved are shown in Table 2 below, wherein the KT5TCR alpha chain variable region refers to a fragment of the first TCR encoded by the alpha chain variable region genome consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10 according to the arrangement order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4; the KT5TCR beta chain variable region refers to a fragment obtained by encoding a beta chain variable region genome of the first TCR, wherein the fragment is formed by sequences shown by SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 according to the arrangement sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4; the full length KT5TCR sequence refers to a complete structural TCR in which a KT5 alpha chain variable region and a beta chain variable region are respectively connected with other structures, and is the second TCR (obtained by encoding the sequence shown as SEQ ID NO: 15).

Table 2 examples relate to nucleic acid sequences or amino acid sequences of materials

Material	Nucleic acid sequence or amino acid sequence
		KRAS-WT	SEQIDNO:18
KRAS-G12C	SEQIDNO:19
		KRAS-G12D	SEQIDNO:20
KRAS-G12V	SEQIDNO:21
		KT5TCR alpha chain variable region (alpha V region)	SEQIDNO:16
KT5TCR beta chain variable region (beta V region)	SEQIDNO:17
		Full length KT5TCR sequences	SEQIDNO:15

In the following examples, KRAS-G12V, KRAS-G12C, KRAS-G12D polypeptides were synthesized by the company Jin Ruisi Biotechnology Co.

In the following examples, the tetramer of KRAS-G12/HLA-A1101, the tetramer of KRAS-G12C/HLA-A1101, the tetramer of KRAS-G12D/HLA-A1101 and the tetramer of KRAS-G12V/HLA-A1101 were all prepared using QuickSwitch from MBL Corp ^TM Quant Tetramer Kit, which can be prepared simply in a research laboratory. The preparation process is as follows, adding 1ul KRAS-G12 polypeptide and 1ul KRAS-G12V polypeptide displacer, 1ul KRAS-G12 polypeptide and 1ul KRAS-G12C polypeptide displacer, 1ul KRAS-G12 polypeptide and 1ul KRAS-G12D polypeptide displacer to 50ul QuickSwitchtetra mer respectively to obtain KRAS-G12/HLA-A1101 tetramer, KRAS-G12C/HLA-A1101 tetramer, KRAS-G12D/HLA-A1101 tetramer and KRAS-G12V/HLA-A1101 tetramer, standing at room temperature for 4 hours, and storing at 4deg.C for use.

EXAMPLE 1 specific TCR screening

(1) Predicting immunogenicity of KRAS-G12D mutant protein by predictive software (HLAthena), analyzing to obtain an antigen peptide sequence with immunogenicity, wherein the sequence of KRAS-G12D mutation is shown as SEQ ID NO: 20;

(2) Synthesizing related short peptide with purity of 85%, and dissolving synthesized polypeptide into 10mM solution by using DMSO;

(3) In vitro stimulation of expansion of KRAS-specific T cells: collecting peripheral blood of healthy people, detecting HLA gene subtype of the healthy people, and selecting a peripheral blood sample positive to HLA-A 1101; separating mononuclear cells and initial T cells in peripheral blood positive to HLA-A1101, adding 800U/ml IL-4 and 800U/ml GM-CSF into a mononuclear cell culture solution, culturing for 4 days to differentiate into DC cells, adding the KRAS-G12D mutant polypeptide (with the concentration of 10 uM) into the DC cell culture solution, and standing for 16 hours in a incubator; subsequently, the DC cells loaded with the mutant peptides were mixed with the initial T cells and cultured for 3 weeks; detecting the content of specific T cells of the cultured T cell product by flow cytometry;

(4) Taking 1×10 ⁷ After amplification of T cells of (2), centrifuging for 10min at 250g, discarding the supernatant, and adding 100ul of antibody dye solution(PBS containing 1ug/ml KRAS-G12D/HLA-A1101 tetramer, 1ug/ml APC-CD3 antibody), 30min incubation at room temperature; washing three times with 5ml PBS containing 0.5% BSA, and centrifuging at 250g for 10min; after resuspension of the cells with 1ml of PBS containing 0.5% bsa, single specific T cells were sorted using a flow cell sorting instrument;

(5) And extracting total RNA from the sorted specific T cells, performing RT-PCR amplification to obtain a TCR gene sequence, and analyzing the sequence structure of the TCR by means of first-generation sequencing and IMGT database comparison to obtain the composition of the KT5 variable region consisting of an alpha chain variable region coded by a sequence shown as SEQ ID NO:16 and a beta chain variable region coded by a sequence shown as SEQ ID NO: 17.

Example 2KT5 binding specificity validation

Connecting a KT5 alpha chain and beta chain variable region (V region) with a constant region (C region) gene of an alpha chain and a beta chain of the TCR, and then connecting the alpha chain and the beta chain to obtain a full-length KT5TCR sequence, wherein the full-length KT5TCR sequence gene is shown as SEQ ID NO. 15; the slow virus expression plasmid pWPXL (purchased from Addegene company) is taken as a skeleton vector to respectively construct KT5 slow virus expression vectors, namely slow virus expression vectors pWPXL-KT5TCR; respectively co-transfecting 293T cells with pWPXL-KT5TCR lentiviral expression vectors and lentiviral packaging plasmids pMD2.G and psPAX2 according to the mass ratio of 1:0.5:1, and collecting virus supernatant after two days; the Jurkat cells were infected with the virus supernatant, and the Jurkat cells expressing KT5TCR were obtained for downstream experiments after culturing for 2-3 days with a 24-hour change of the liquid after infection.

Mixing the Jurkat cells expressing KT5TCR and the K562 cells expressing A1101 gene in the cell number ratio of 1 to 1, adding KRAS-G12, KRAS-G12C, KRAS-G12D and KRAS-G12V polypeptide in the final concentration of 10uM, and recovering the cells after 6 hr; then adding 50ul of cell dye solution containing PE-CD69 antibody, and incubating for 30min at room temperature; then washing three times with PBS containing 0.5% BSA, centrifuging 200-250g for 10min; cells were then resuspended with PBS containing 0.5% bsa for downstream detection; the expression of CD69 was analyzed by measuring Jurkat cell activation by flow cytometry.

The results of Jurkat cell analysis of KT5 TCR-expressing are shown in FIG. 1, which shows that KT5TCR is specifically activated upon stimulation of KRAS-G12D, and thus is KRAS-G12D-specific TCR.

EXAMPLE 3 secretion level test of IFN-gamma and Granzyme-B

KT5-T cells prepared using T cells of human PBMC were mixed 1:1 with A1101-K562 cells loaded with KRAS-G12, KRAS-G12C, KRAS-G12D, KRAS-G12V polypeptide, respectively, and 2X 10 in a volume of 100. Mu.l ⁵ Cell number/well was added to 96-well plates, and three replicates were made for each sample, wherein the PMA/Ionomycin (ION) group was diluted into working fluid in advance by adding 1 μl PMA/ionomycin mix (250×) per 250 μl cell culture medium, and used as positive stimulation control; t cells not infected with KT5 lentivirus (D1 mock and D2 mock) were added in parallel as negative controls; after 24h incubation at 37℃the supernatant from the 96-well plate culture wells was removed and centrifuged at 500G for 5min to remove residual cells, the supernatant was added to ELISA plates coated with anti-IFN-gamma and anti-Granzyme-B antibodies, respectively, and the anti-IFN-gamma and anti-Granzyme-B antibody levels in the supernatant were detected, as shown in FIGS. 2 and 3, and KT5-T cells secreted IFN-gamma after KRAS-G12D stimulation, thus KT5 was the specific TCR for KRAS-G12D.

Sequence listing

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<400> 13

actgacaaag gagaagtccc caatggctac aatgtctcca gattaaacaa acgggagttc 60

tcgctcaggc tggagtcggc tgctccctcc cagacatctg tgtacttctg t 111

<210> 14

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ttcgggccgg gcaccaggct cacggtcaca 30

<210> 15

<211> 1401

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

ccccctaagg tgtccctgtt tgagccttct aaggccgaga tcgccaataa gcagaaggcc 60

accctggtgt gcctggcccg cggcttcttt ccagatcacg tggagctgtc ctggtgggtg 120

aacggcaagg aggtgcactc cggcgtgtct acagaccccc aggcctacaa ggagagcaat 180

tactcctatt gcctgagctc caggctgcgc gtgagcgcca ccttttggca caacccaagg 240

aatcacttcc gctgtcaggt gcagtttcac ggcctgtctg aggaggataa gtggccagag 300

ggcagcccaa agcctgtgac acagaacatc tccgccgagg cctggggaag ggcagactgt 360

ggcatcacca gcgcctccta tcaccagggc gtgctgagcg ccacaatcct gtacgagatc 420

ctgctgggca aggccaccct gtatgccgtg ctggtgtctg gcctggtgct gatggctatg 480

gtgaagaaga agaacagcag agccaaaaga agtggttctg gcgcgacgaa ttttagtttg 540

cttaagcaag ccggagatgt ggaggaaaat cctggaccga tgctgactgc cagcctgttg 600

agggcagtca tagcctccat ctgtgttgta tccagcatgg ctcagaaggt aactcaagcg 660

cagactgaaa tttctgtggt ggagaaggag gatgtgacct tggactgtgt gtatgaaacc 720

cgtgatacta cttattactt attctggtac aagcaaccac caagtggaga attggttttc 780

cttattcgtc ggaactcttt tgatgagcaa aatgaaataa gtggtcggta ttcttggaac 840

ttccagaaat ccaccagttc cttcaacttc accatcacag cctcacaagt cgtggactca 900

gcagtatact tctgtgctct gacctccccc gatagcaact atcagttaat ctggggcgct 960

gggaccaagc taattataaa gccagacatc cagaacccag agcccgccgt gtaccagctg 1020

aaggacccca gaagccagga tagcaccctg tgcctgttca ccgactttga ttctcagatc 1080

aatgtgccta agacaatgga gagcggcacc ttcatcacag acaagaccgt gctggatatg 1140

aaggctatgg actccaagtc taacggcgcc atcgcctggt ctaatcagac cagcttcacc 1200

tgccaggata tctttaagga gacaaatgcc acctatcctt cctctgacgt gccatgtgat 1260

gccaccctga cagagaagag cttcgagacc gacatgaacc tgaattttca gaacctgtcc 1320

gtgatgggcc tgagaatcct gctgctgaag gtggccggct tcaatctgct gatgacactg 1380

aggctgtgga gctcctgata a 1401

<210> 16

<211> 345

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gctcagaagg taactcaagc gcagactgaa atttctgtgg tggagaagga ggatgtgacc 60

ttggactgtg tgtatgaaac ccgtgatact acttattact tattctggta caagcaacca 120

ccaagtggag aattggtttt ccttattcgt cggaactctt ttgatgagca aaatgaaata 180

agtggtcggt attcttggaa cttccagaaa tccaccagtt ccttcaactt caccatcaca 240

gcctcacaag tcgtggactc agcagtatac ttctgtgctc tgacctcccc cgatagcaac 300

tatcagttaa tctggggcgc tgggaccaag ctaattataa agcca 345

<210> 17

<211> 336

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

aatgctggtg tcactcagac cccaaaattc caggtcctga agacaggaca gagcatgaca 60

ctgcagtgtg cccaggatat gaaccataac tccatgtact ggtatcgaca agacccaggc 120

atgggactga ggctgattta ttactcagct tctgagggta ccactgacaa aggagaagtc 180

cccaatggct acaatgtctc cagattaaac aaacgggagt tctcgctcag gctggagtcg 240

gctgctccct cccagacatc tgtgtacttc tgtgccagca gagtgaactg ggcttacgag 300

cagtacttcg ggccgggcac caggctcacg gtcaca 336

<210> 18

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 18

Val Val Val Gly Ala Gly Gly Val Gly Lys

1 5 10

<210> 19

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 19

Val Val Val Gly Ala Cys Gly Val Gly Lys

1 5 10

<210> 20

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 20

Val Val Val Gly Ala Asp Gly Val Gly Lys

1 5 10

<210> 21

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 21

Val Val Val Gly Ala Val Gly Val Gly Lys

1 5 10

Claims

1. A TC R targeting a complex of polypeptide-HLA-A 1101, characterized by comprising an alpha chain variable region and a beta chain variable region; the alpha chain variable region comprises an alpha chain CDR1 encoded by a sequence shown as SEQ ID NO. 1, an alpha chain CDR2 encoded by a sequence shown as SEQ ID NO. 2 and an alpha chain CDR3 encoded by a sequence shown as SEQ ID NO. 3, and the beta chain variable region comprises a beta chain CDR1 encoded by a sequence shown as SEQ ID NO. 4, a beta chain CDR2 encoded by a sequence shown as SEQ ID NO. 5 and a beta chain CDR3 encoded by a sequence shown as SEQ ID NO. 6; the polypeptide sequence is shown as SEQ ID NO. 20.

2. A TCR as claimed in claim 1 wherein the α chain variable region further comprises: one or more of an alpha chain FR1 encoded by a sequence shown in SEQ ID NO. 7, an alpha chain FR2 encoded by a sequence shown in SEQ ID NO. 8, an alpha chain FR3 encoded by a sequence shown in SEQ ID NO. 9 and an alpha chain FR4 encoded by a sequence shown in SEQ ID NO. 10; the beta chain variable region further comprises: one or more of a beta-strand FR1 encoded by a sequence shown as SEQ ID NO:11, a beta-strand FR2 encoded by a sequence shown as SEQ ID NO:12, a beta-strand FR3 encoded by a sequence shown as SEQ ID NO:13 and a beta-strand FR4 encoded by a sequence shown as SEQ ID NO: 14.

TCR characterized by comprising a fragment encoded by a sequence as shown in SEQ ID NO. 15.

4. An antigen binding fragment comprising a TCR as claimed in any one of claims 1 to 3.

5. A polynucleotide encoding a TCR according to claim 1, comprising sequences as set out in seq id No. 1, seq id No. 2, seq id No. 3, seq id No. 4, seq id No. 5 and seq id No. 6.

6. The polynucleotide according to claim 5, further comprising one or more of a sequence shown as SEQ ID NO. 7, a sequence shown as SEQ ID NO. 8, a sequence shown as SEQ ID NO. 9, a sequence shown as SEQ ID NO. 10, a sequence shown as SEQ ID NO. 11, a sequence shown as SEQ ID NO. 12, a sequence shown as SEQ ID NO. 13 and a sequence shown as SEQ ID NO. 14.

7. The polynucleotide encoding the TC R of claim 1, comprising a sequence as set forth in SEQ ID NO. 15.

8. An expression vector comprising a polynucleotide according to any one of claims 5 to 7.

9. An engineered cell comprising the expression vector of claim 8.

10. A pharmaceutical composition comprising the TC R of any one of claims 1 to 3 or the antigen binding fragment of claim 4 or the polynucleotide of any one of claims 5 to 7 or the expression vector of claim 8 or the engineered cell of claim 9, and a pharmaceutically acceptable carrier and/or diluent.

11. Use of a TCR according to any one of claims 1 to 3 or an antigen-binding fragment according to claim 4 or a polynucleotide according to any one of claims 5 to 7 or an expression vector according to claim 8 or an engineered cell according to claim 9 in the manufacture of a medicament for increasing the secretion of IFN- γ and/or Granzyme-B cytokine levels by T cells.

12. The use according to claim 11, wherein the medicament for increasing the level of IFN- γ and/or Granzyme-B cytokine secretion by T cells comprises a protein medicament, an ADC medicament or a combination of TC R and an antigen.

13. Use of a TCR according to any one of claims 1 to 3 or an antigen-binding fragment according to claim 4 or a polynucleotide according to any one of claims 5 to 7 or an expression vector according to claim 8 or an engineered cell according to claim 9 in the preparation of a tumour cell reagent or kit for detecting a mutation expressing KRAS-G12D.

14. Use of a TCR according to any one of claims 1 to 3 or an antigen-binding fragment according to claim 4 or a polynucleotide according to any one of claims 5 to 7 or an expression vector according to claim 8 or an engineered cell according to claim 9 in the manufacture of a medicament for the treatment of pancreatic cancer, colon cancer, endometrial cancer, lung cancer, cholangiocarcinoma, cervical cancer or bladder cancer.