CN114920824A

CN114920824A - TCR or antigen binding fragment thereof and uses thereof

Info

Publication number: CN114920824A
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: 2022-08-19
Anticipated expiration: 2042-05-27
Also published as: CN114920824B

Abstract

The invention belongs to the technical field of T cell immunotherapy drugs, and particularly relates to a T Cell Receptor (TCR) or an antigen binding fragment thereof and application thereof, wherein the TCR comprises a fragment encoded by a sequence shown as SEQ ID NO. 15. The TCR has strong specificity to 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 drugs, 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, the target selection of T cell immunotherapy targeting malignant solid tumors is evolving, from the original lineage antigens to viral antigens, while with the recent emergence of high throughput sequencing technologies, targeting tumor individuals is emerging as a target focused for the next generation of T cell therapy.

T cell therapy currently includes mainly TCR-T and CAR-T therapies. The CAR-T technology treatment has obvious effect in the treatment of leukemia, lymphoma and other hematological tumors, greatly improves the survival rate and the survival quality of patients, but the CAR-T treatment means greatly limits the application prospect of the CAR-T treatment means aiming at solid tumors due to limited specific targets at present. TCR-T technology differs from CAR-T cell technology in that TCRs are a characteristic marker of all T cell surfaces, binding non-covalently to CD3, forming the TCR-CD3 complex. In peripheral blood, 90% -95% of T cells express TCR, and the T cells of the TCR after genetic modification can perform specific recognition on antigen molecules on the surface of tumor cells, so as to generate immune response aiming at the tumor cells. Currently, among human cancers, KRAS gene is one of the most well-known oncogenes in oncology, and KRAS gene mutations (KRAS-G12C, KRAS-G12D, KRAS-G12V, 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 cancer, as well as biliary tract, cervical, bladder, and the like.

In view of the foregoing, there is a need for a TCR that specifically binds to KRAS mutant polypeptides and mediates T cell killing of KRAS mutant tumor cells.

Disclosure of Invention

One of the objectives of the present invention is to provide a TCR, which can specifically recognize the KRAS-G12D mutation site in a targeted manner, and specifically bind to a complex of KRAS-G12D mutated polypeptide and HLA, so as to stimulate T cell activation, mediate T cell to secrete cytokines such as IFN- γ, and further kill tumor cells expressing KRAS-G12D mutant genes. The TCR or the antigen binding fragment thereof has strong polypeptide specificity on KRAS-G12D mutation, and can mediate T cells to secrete a large amount of IFN-gamma cytokines to kill KRAS-G12D mutated tumor cells.

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

the first method comprises the following steps: the α chain variable region of the TCR may comprise the α chain CDR3 encoded by the sequence shown in seq id No. 3 and the β chain variable region of the TCR comprises the β chain CDR3 encoded by the sequence shown in seq id No. 6.

And the second method comprises the following steps: the TCR may comprise a fragment encoded by the sequence shown in SEQ ID NO. 15.

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

In a further aspect, the present invention provides a polynucleotide selected from the following two polynucleotides:

the first method comprises the following steps: can comprise the sequences shown as SEQ ID NO. 3 and SEQ ID NO. 6.

And the second method comprises the following steps: can comprise the sequence shown as SEQ ID NO. 15.

In a further aspect, the present invention provides an expression vector which may comprise either 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 level of IFN- γ and/or Granzyme-B cytokine secretion from T cells.

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

In a further aspect, the present invention provides a use of the above TCR or the above antigen-binding fragment or the above polynucleotide, or the above expression vector, or the above engineered cell in the preparation of a medicament for treating a disease caused by a mutation that carries KRAS-G12D.

The invention has the beneficial effects that: the TCR provided by the invention has strong specificity to 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 diagram of the results of specific flow cytometry detection of KT5 on KRAS-G12D mutant polypeptide in the examples.

FIG. 2 shows the condition of KT5 in the examples mediating the secretion of IFN-gamma cytokines from T cells.

FIG. 3 shows the KT5 in the examples mediating secretion of Granzyme-B cytokines by T cells.

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 has a significantly different meaning, the singular forms of expressions include the plural forms of expressions. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. 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, parts, elements, materials or combinations thereof may be present or may be added. As used herein, "/" can be interpreted as "and" or "depending on the circumstances.

As used herein, the term "antigen-binding fragment" refers to antigen-binding fragments and TCR analogs of TCRs, which typically include at least a portion of the antigen-binding or variable region of the parent TCR, such as 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 the TCR can specifically recognize a KRAS-G12D mutation site in a targeted mode, and is specifically combined with a complex of KRAS-G12D mutant polypeptide and HLA 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 the antigen binding fragment thereof has strong specificity to KRAS-G12D mutant polypeptide and can mediate a plurality of IFN-gamma cytokines secreted by T cells.

In one aspect, the invention provides a TCR comprising an α chain variable region and a β chain variable region, which TCR of the invention may be selected from the following TCRs:

a 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 the sequence shown in SEQ ID NO. 1 and/or an alpha chain CDR2 encoded by the sequence shown in SEQ ID NO. 2; the beta chain variable region may further comprise: a beta chain CDR1 encoded by the sequence shown in SEQ ID NO. 4 and/or a 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 the sequence shown in SEQ ID NO. 7, an alpha chain FR2 encoded by the sequence shown in SEQ ID NO. 8, an alpha chain FR3 encoded by the sequence shown in SEQ ID NO. 9 and an alpha chain FR4 encoded by the sequence shown in SEQ ID NO. 10; the beta chain variable region may further comprise: one or more of beta chain FR1 encoded by the sequence shown in SEQ ID NO. 11, beta chain FR2 encoded by the sequence shown in SEQ ID NO. 12, beta chain FR3 encoded by the sequence shown in SEQ ID NO. 13 and beta chain FR4 encoded by the sequence shown in SEQ ID NO. 14.

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

Specifically, FR (FR1, FR2, FR3, FR4) in the above TCR is a framework region for linking CDR regions, and is relatively stable. In addition to the above sequences, the FR regions in the first TCR may be sequences with identity of greater than or equal to 80%, such as greater than or equal to 80%, 85%, 90% or 95%, which may be murine or human (but not excluding other sources, such as animal sources, e.g., rabbit, pig, etc.), with the FR1, FR2, FR3 and FR 4. In some embodiments, the sequences of the FR and CDR regions of the α and β chains can be arranged according to FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 to form the α and β chain variable regions of the two TCRs, respectively, the sequence encoding the α chain variable region being shown in SEQ ID NO:16 and the sequence encoding the β chain variable region being shown in 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 further comprising an α chain constant region, and a β chain further comprising a β chain constant region, wherein the α chain constant region and the β chain constant region may be derived from murine, human or chimeric human-murine (but other sources are not excluded), and are regions in which mutations hardly occur.

A second TCR:

the second TCR comprises a fragment encoded by the sequence shown in SEQ ID NO. 15.

Specifically, in the second TCR, the fragment encoded by the sequence shown in 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 encoding TCR sequence can also be a sequence with identity of more than or equal to 80 percent with the sequence shown in SEQ ID NO. 15, such as a sequence with identity of more than or equal to 80 percent, 85 percent, 90 percent or 95 percent.

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

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

In a further aspect, the present invention provides a polynucleotide selected from the group consisting of:

a first polynucleotide:

the first polynucleotide comprises at least the sequence 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 the sequence shown in SEQ ID NO. 15.

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

Specifically, the polynucleotide may comprise (in addition to the polynucleotide) any other nucleotide encoding any of the above TCRs, such as a codon-optimized sequence comprising the sequence shown in seq id No. 3, a codon-optimized sequence comprising the sequence shown in seq id No. 6, or a codon-optimized sequence comprising the sequence shown in seq id No. 15.

Further, the expression vector is selected from any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, an adeno-associated viral 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-meidi virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV).

In still another aspect, the invention provides an engineered cell comprising the above expression vector. 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; or T cells, and the expression vector (loaded with the target gene) is transfected into the T cells to obtain TCR-T cells for recognizing and killing KRAS-G12D mutant polypeptides.

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

Specifically, pharmaceutically acceptable carriers and/or diluents refer to that the above-described TCR or the above-described antigen-binding fragment or the above-described polynucleotide or the above-described expression vector or the above-described engineered cell can be prepared into various desired dosage forms. Examples of the pharmaceutical composition include tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film-coated preparations, pellets, sublingual tablets, and ointments, which are oral preparations, and examples of the pharmaceutical composition include injections, suppositories, transdermal preparations, ointments, plasters, and external liquids, and those skilled in the art can select an appropriate dosage form according to the administration route and the administration subject.

Further, the medicine for improving the level of IFN-gamma and/or Granzyme-B cell factor secreted by T cells comprises cell medicines, protein medicines, ADC medicines or TCR and antigen combination medicines.

In another aspect, the present invention provides a use of the TCR or the antigen-binding fragment or the polynucleotide, or the expression vector, or the engineered cell, in the preparation of a reagent or a kit for detecting a tumor cell expressing a mutation of KRAS-G12D.

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

In a further aspect, the present invention provides a use of the above TCR or the above antigen-binding fragment or the above polynucleotide or the above expression vector or the above engineered cell in the preparation of a medicament for treating a disease caused by a mutation carrying KRAS-G12D.

Further, diseases caused by carrying the KRAS-G12D mutation include pancreatic cancer, colorectal cancer, endometrial cancer, lung cancer, bile duct cancer, cervical cancer or bladder cancer.

In particular, in the above application, the HLA-a1101 with stronger affinity is selected for the polypeptide that is HLA-restricted T cell epitope polypeptide of KRAS-G12D mutant, i.e. the polypeptide after KRAS-G12D mutation is presented by HLA-a1101 molecules, but the selection of other HLA molecules, such as other small molecules of HLA-a11 series, is not excluded.

Specifically, in the above application, the TCR is selected from any one of the above two TCRs; the polynucleotide may be any of the two polynucleotides described above. In the above applications, the TCR or antigen binding fragment or polynucleotide or expression vector or engineered cell may be added with adjuvants to form a drug, or may be used with other active drugs or detection reagents, such as existing chemotherapeutic drugs, e.g., alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer drugs, hormones, immunological agents, etc.; can also be used in combination with surgery. The specific condition is that the medicine is taken or combined according to the tumor condition.

Specifically, in the above applications, the polypeptides mutated in KRAS-G12D may be 9 polypeptides, 10 polypeptides or other number of polypeptides, respectively, and the amino acid sequence of 10 mutated KRAS-G12D polypeptides is shown in seq id No. 20.

For a better understanding of the present invention, the following further illustrates the contents of the present invention with reference to specific examples, but the contents of the present invention are not limited to the following examples.

In the following examples, it was verified that the wild-type polypeptide of KRAS-G12 (KRAS-G12 for short) has the sequence shown in SEQ ID NO: 18; verifying that the related KRAS-G12C mutant polypeptide (KRAS-G12C for short) has a sequence shown as SEQ ID NO. 19; verifying that the related KRAS-G12D mutant polypeptide (KRAS-G12D for short) has a sequence shown as SEQ ID NO. 20; the sequence of the related 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 _ RV1primer, Cb _ RV1primer) and the upstream primer was a primer containing an outer linker and a TCR signal peptide initiation 20bp sequence (AL primers, BL primers) (see in particular the example of content in Hamana H, Shitaoka K, Kishi H, Ozawa T, Muraguchi A.A node, Rapid and effective method of closing functional antigen-specific T-cell receptors from simple human and mouse T-cells Biochem Biophys Res Commun.2016Jun 10; 474(4): 709-714-doi: 10.1016/j.bb.2016.05.015.2016. 2016May 4.PMID: 27155153);

(2) second round PCR: taking the PCR product of the TCR obtained in the step (1) as a template, taking an upstream primer as an outer-layer joint primer (P2A-Cprimer), and taking a downstream primer as a specific primer (Ca _ RV2primer) of a section of TCR constant region at the upstream of the alpha chain constant region to obtain a second round PCR product of the TCR alpha chain; the upstream primer is an outer linker primer (BES-AP primer), the downstream primer is a specific primer (Cb _ RV2primer) of a TCR constant region upstream of a beta chain constant region, and a second round PCR product of the TCR beta chain is obtained (specifically, the second round PCR product is implemented according to the contents of the documents Hamana H, Shitaoka K, Kishi H, Ozawa T, Muraguchi A.A novel, Rapid and effective method of cloning functional anti-specific T-cell receptors from simple human and mouse T-cells, biochem Biophys Res Commun.2016Jun 10; 474(4):709-714 doi: 10.1016/j.bbrc.2016.05.015.Ep2016 May 4.PMID: 27155153);

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

(4) the target strip is subjected to first generation sequencing (Beijing Optimae company), and after TCR sequence analysis, a TCR clone which appears at high frequency in the target strip is selected and named as KT 5.

The amplification system used in the above-described procedures involving gene amplification, sequencing and analysis is shown in Table 1 below (specifically, it is described in the documents "Hamana H, Shitaoka K, Kishi H, Ozawa T, Muraguchi A.A novel, Rapid and effective methods of closing functional anti-genetic T-cell receptors from simple human and mouse T-cells. biochem Biophys Res Commun. Jun 10; 474 4: 709-714.doi: 10.1016/j.mb.2016.05.015. Epub.2016. PMID: 27155153).

TABLE 1 amplification System

In the following examples, human PBMC cells and human peripheral blood were both 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 KT5TCR alpha chain variable region refers to the fragment encoded by the alpha chain variable region genome of the first TCR consisting of the sequences shown in 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 in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4; the KT5TCR beta variable region refers to a fragment encoded by a beta variable region genome consisting of 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 in the first TCR according to the arrangement sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4; the full-length KT5TCR sequence refers to a TCR with a complete structure, wherein the whole structure is formed by connecting a KT5 alpha chain variable region and a beta chain variable region with other structures respectively, and the TCR is the second TCR (obtained by encoding sequences shown in SEQ ID NO: 15).

TABLE 2 nucleic acid or amino acid sequences of materials referred to in the examples

Material(s)	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 variable region (beta V region)	SEQIDNO:17
		Full-length KT5TCR sequence	SEQIDNO:15

In the following examples, the KRAS-G12, KRAS-G12V, KRAS-G12C and KRAS-G12D polypeptides were assigned to the Kinreis Biotechnology Co., Ltd for their synthesis.

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 QuickSwitch of MBL, Japan ^TM The Quant Tetramer Kit is prepared, and the reagent can be used for simply preparing a Tetramer reagent in a research room. The preparation process is as follows, respectively 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 QuickSwitchTetramer to obtain KRAS-G12/HLA-A1101 tetramer, KRAS-G12C/HLA-A1101 tetramer, KRAS-G12D/HLA-A1101 tetramer and KRAS-G12V/HLA-A1101 tetramer, respectively, and standing at room temperature for 4 hours to store at 4 ℃ for later use.

Example 1 specific TCR screening

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

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

(3) in vitro stimulation to expand KRAS-specific T cells: collecting peripheral blood of healthy people, detecting HLA gene subtype of the healthy people, and selecting an HLA-A1101 positive peripheral blood sample; separating mononuclear cells and initial T cells in HLA-A1101 positive peripheral blood, 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, then respectively adding the KRAS-G12D mutant polypeptide (the concentration is 10uM) into the DC cell culture solution, and placing the mixture into an incubator for standing and culturing for 16 hours; subsequently, the DC cells loaded with the mutant peptide were mixed with the original T cells, and the culture was continued for 3 weeks; detecting the content of specific T cells in the cultured T cell product by flow cytometry;

(4) take 1X 10 ⁷ The amplified T cells are centrifuged for 10min at 250G, the supernatant is discarded, 100ul of antibody staining solution (PBS containing 1ug/ml KRAS-G12D/HLA-A1101 tetramer and 1ug/ml APC-CD3 antibody) is added, and the mixture is incubated for 30min at room temperature; washed three times with 5ml PBS containing 0.5% BSA, centrifuged at 250g for 10 min; after resuspending the cells in 1ml PBS containing 0.5% BSA, single specific T cells were sorted using a flow cytometric sorting instrument;

(5) extracting total RNA from the sorted specific T cells, then carrying out RT-PCR amplification to obtain a TCR gene sequence, analyzing the TCR sequence structure through first-generation sequencing and IMGT database comparison to obtain a KT5 variable region which consists of an alpha chain variable region coded by a sequence shown as SEQIDNO 16 and a beta chain variable region coded by a sequence shown as SEQIDNO 17.

Example 2 verification of binding specificity of KT5

Connecting the alpha chain and beta chain variable regions (V regions) of KT5 with the constant region (C region) genes of the alpha chain and beta chain of 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; respectively constructing a KT5 lentiviral expression vector, namely a lentiviral expression vector pWPXL-KT5TCR, by taking a lentiviral expression plasmid pWPXL (purchased from Addegene company) as a framework vector; respectively cotransfecting the pWPXL-KT5TCR lentivirus expression vector, a lentivirus packaging plasmid pMD2.G and a psPAX2 plasmid into 293T cells according to the mass ratio of 1:0.5:1, and collecting virus supernatant after two days; jurkat cells were infected with viral supernatant, and after 24 hours of fluid change and 2-3 days of culture, Jurkat cells expressing KT5TCR were obtained for downstream experiments.

Mixing the Jurkat cells expressing KT5TCR and K562 cells expressing A1101 gene according to the cell number ratio of 1:1, respectively adding KRAS-G12, KRAS-G12C, KRAS-G12D and KRAS-G12V polypeptides with the final concentration of 10uM, and recovering the cells after 6 hours; then 50ul of cell stain containing PE-CD69 antibody is added, and incubation is carried out for 30min at room temperature; then washed three times with PBS containing 0.5% BSA, and centrifuged for 10min at 200-250 g; cells were then resuspended in PBS containing 0.5% BSA for downstream detection; jurkat cell activation analysis was performed using flow cytometry to examine the expression of CD69 in the Jurkat cell activation analysis.

The results of the Jurkat cell assay expressing KT5TCR are shown in FIG. 1, and the KT5TCR is specifically activated after stimulation by KRAS-G12D, and thus is a KRAS-G12D specific TCR.

Example 3 secretion level testing of IFN-. gamma.and Granzyme-B

KT5-T cells prepared using T cells from human PBMC, mixed 1:1 with A1101-K562 cells loaded with KRAS-G12, KRAS-G12C, KRAS-G12D, KRAS-G12V polypeptides, respectively, and added in a volume of 2X 10 to 100. mu.l ⁵ Cell number/well was added to 96-well plates in triplicate for each sample, where the PMA/Ionomycin (ION) group was diluted as working solution in advance by adding 1. mu.l of PMA/ionomycin mix (250X) per 250. mu.l of cell culture medium and served as positive stimulation control; t cells not infected with KT5 lentivirus (D1mock and D2mock) were added in parallel as negative controls; after 24h of incubation at 37 ℃, supernatants from 96-well plates were removed by centrifugation at 500G for 5min, and the supernatants were added to ELISA plates coated with anti-IFN- γ and anti-Granzyme-B antibodies, respectively, to detect the levels of anti-IFN- γ and anti-Granzyme-B antibodies in the supernatants, as shown in fig. 2 and 3, KT5-T cells secreted IFN- γ upon stimulation by KRAS-G12D, thus KT5 was a TCR specific for KRAS-G12D.

Sequence listing

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<213> Artificial Sequence (Artificial Sequence)

<400> 11

aatgctggtg tcactcagac cccaaaattc caggtcctga agacaggaca gagcatgaca 60

ctgcagtgtg cccaggat 78

<210> 12

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgtactggt atcgacaaga cccaggcatg ggactgaggc tgatttatta c 51

<210> 13

<211> 111

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<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

A TCR comprising an alpha chain variable region and a beta chain variable region; the alpha chain variable region comprises the alpha chain CDR3 encoded by the sequence shown in SEQ ID NO. 3 and the beta chain variable region comprises the beta chain CDR3 encoded by the sequence shown in SEQ ID NO. 6.
2. A TCR as claimed in claim 1 wherein the α chain variable region further comprises: an alpha chain CDR1 encoded by the sequence shown in SEQ ID NO. 1 and/or an alpha chain CDR2 encoded by the sequence shown in SEQ ID NO. 2; the beta chain variable region further comprises: 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.
3. A TCR as claimed in claim 1 or claim 2 wherein the α chain variable region further comprises: one or more of alpha chain FR1 encoded by the sequence shown in SEQ ID NO. 7, alpha chain FR2 encoded by the sequence shown in SEQ ID NO. 8, alpha chain FR3 encoded by the sequence shown in SEQ ID NO. 9 and alpha chain FR4 encoded by the sequence shown in SEQ ID NO. 10; the beta chain variable region further comprises: one or more of beta-chain FR1 coded by the sequence shown in SEQ ID NO. 11, beta-chain FR2 coded by the sequence shown in SEQ ID NO. 12, beta-chain FR3 coded by the sequence shown in SEQ ID NO. 13 and beta-chain FR4 coded by the sequence shown in SEQ ID NO. 14.
A TCR comprising a fragment encoded by the sequence shown in SEQ ID NO. 15.
5. An antigen-binding fragment comprising a TCR as claimed in any one of claims 1 to 4.
6. A polynucleotide comprising the sequence shown in SEQ ID NO. 3 and SEQ ID NO. 6.
7. The polynucleotide of claim 6, further comprising one or more of the sequence shown in SEQ ID NO. 1, the sequence shown in SEQ ID NO. 2, the sequence shown in SEQ ID NO. 4, the sequence shown in SEQ ID NO. 5, the sequence shown in SEQ ID NO. 7, the sequence shown in SEQ ID NO. 8, the sequence shown in SEQ ID NO. 9, the sequence shown in SEQ ID NO. 10, the sequence shown in SEQ ID NO. 11, the sequence shown in SEQ ID NO. 12, the sequence shown in SEQ ID NO. 13, and the sequence shown in SEQ ID NO. 14.
8. A polynucleotide comprising the sequence set forth in SEQ ID NO. 15.
9. An expression vector comprising the polynucleotide of any one of claims 6 to 8.
10. An engineered cell comprising the expression vector of claim 9.
11. A pharmaceutical composition comprising a TCR according to any one of claims 1 to 4 or an antigen-binding fragment according to claim 5 or a polynucleotide according to any one of claims 6 to 8 or an expression vector according to claim 9 or an engineered cell according to claim 10, and a pharmaceutically acceptable carrier and/or diluent.
12. Use of a TCR according to any one of claims 1 to 4 or an antigen-binding fragment of claim 5 or a polynucleotide according to any one of claims 6 to 8 or an expression vector of claim 9 or an engineered cell of claim 10 in the manufacture of a medicament for increasing the level of IFN- γ and/or Granzyme-B cytokine secretion by a T cell.
13. The use according to claim 12, wherein the agent for increasing the level of IFN- γ and/or Granzyme-B cytokine secretion from T cells comprises a proteinaceous agent, an ADC agent or a TCR in combination with an antigen.
14. Use of a TCR according to any one of claims 1 to 4 or an antigen-binding fragment of claim 5 or a polynucleotide of any one of claims 6 to 8 or an expression vector of claim 9 or an engineered cell of claim 10 in the preparation of a reagent or kit for detecting a tumor cell expressing a KRAS-G12D mutation.
15. Use of a TCR according to any one of claims 1 to 4 or an antigen-binding fragment of claim 5 or a polynucleotide of any one of claims 6 to 8 or an expression vector of claim 9 or an engineered cell of claim 10 in the manufacture of a medicament for the treatment of a disease caused by a mutation carrying KRAS-G12D.