Screening of KRAS mutation-specific T cell receptors and their anti-tumor applications

Abstract

The present invention provides two types of specific T cell receptors (TCRs) that target epitopes of the G12V or G12C mutation in the KRAS gene, and their anti-tumor uses. Each of the two T cell receptors consists of two peptide chains, an α chain and a β chain. The present invention also provides antigen-binding fragments of the TCRs, nucleic acids encoding them, vectors containing the nucleic acids, host cells containing the vectors, and methods for producing TCRs or antigen-binding fragments thereof specific for the G12V mutation in KRAS. The specific TCRs and antigen-binding fragments of the present invention can be used as immunostimulants to stimulate the body's immune response, thereby generating anti-tumor effects against diseases such as tumors.

Description

[Technical Field]

[0001] The present invention relates to the pharmaceutical field, and specifically to a T cell receptor (TCR) or an antigen-binding fragment thereof that can specifically recognize antigen polypeptides of tumor KRAS gene G12V and G12C mutations.

[0002] Background technology In 2011, cancer surpassed heart disease to become the world's leading cause of death. In December 2013, the WHO announced that the number of new cancer cases worldwide exceeded 14 million per year, a significant increase from the 2008 statistic of 12.7 million cases. By 2020, cancer will cause 9.6 million deaths each year, and approximately $1 trillion will be spent on cancer treatment worldwide each year.

[0003] In the early 1980s, Allison and other researchers identified the genetic structure of the αβ T cell receptor (TCR), which is responsible for antigen recognition on the surface of T cells. In the late 1980s, Boone, Rosenberg, and Old discovered through their research that different tumor patients each have several tumor-specific antigens that can be recognized by T cells and specifically kill tumor cells. This rekindled hope for tumor immunotherapy, and a great deal of research has focused on the research and development of tumor therapeutic vaccines. In 2013, immune anti-cancer therapy was named one of the top 10 scientific and technological breakthroughs of the year by Science magazine.

[0004] In recent years, with the rapid development of stem cell biology, immunology, molecular technology, tissue engineering technology, etc., cellular immunotherapy, as a safe and effective therapeutic method, has become increasingly prominent in the treatment of tumors, etc. Currently, the research and development of new cell therapy technologies has become an important research field for solving tumor-related diseases.

[0005] Adoptive T cell therapy (ACT) is a highly personalized cancer treatment that can achieve antitumor effects by restoring a missing or weakened immune system in cancer patients. ACT therapy involves isolating immune-active cells from tumor patients, amplifying and functionally identifying them in vitro, and then injecting them back into the patient to either directly kill tumors or stimulate the body's immune response to kill tumor cells. A limiting factor in ACT therapy is the search for antigens that are expressed only in cancer tissues, not in normal, essential tissues.

[0006] Currently, ACT therapy can be realized by using T cell receptor engineered cells (TCR-T) and chimeric antigen receptor engineered T cells (CAR-T), which have shown good therapeutic effects on various cancers, including melanoma, cervical cancer, lymphoma, leukemia, cholangiocarcinoma, and neuroblastoma.

[0007] Currently, CAR-T has achieved significant breakthroughs in the treatment of diseases such as acute and chronic granular cell leukemia and lymphoma, significantly improving patient survival rates and quality of life. However, in research into the treatment of solid tumors, the therapeutic prospects of CAR-T cells are unclear due to the limited specific targets.

[0008] Unlike CAR-T cells, which target extracellular antigens using antibodies, TCRs are a characteristic marker on the surface of all T cells and bind non-covalently to CD3 to form a TCR-CD3 complex. TCRs are composed of two peptide chains, α and β, and belong to the immunoglobulin superfamily. Their antigen specificity is determined by the V region (CDR1, CDR2, and CDR3), with CDR3 directly determining the antigen-binding specificity of the TCR. In peripheral blood, 90–95% of T cells express TCRs. T cells with transgenic TCRs can specifically recognize antigen molecules on the surface of tumor cells and initiate an immune response against them.

[0009] TCR-T cell immunotherapy is a new cell therapy technology that has developed in recent years and is a typical "precision medicine" treatment technology. Currently, this technology shows promising therapeutic potential in the treatment of myeloma, melanoma, esophageal cancer, liver cancer, and other diseases. TCR-T cell immunotherapy was first used to treat HIV at the end of the 20th century, and recent research has demonstrated that autologous immune cells engineered based on TCRs specific for tumor antigens such as MART-1, MAGE-A4, NY-ESO-1, and WT-1 show good development prospects for the treatment of melanoma, esophageal cancer, multiple myeloma, and synovial cell sarcoma.

[0010] In particular, according to a 2015 report, 20 patients with stage I / II multiple myeloma received immunotherapy with NY-ESO-1-specific TCR-engineered cells, and 80% of the patients showed positive clinical therapeutic effects after receiving TCR-T therapy. Currently, TCR-T cell immunotherapy technology has become a hotspot in international oncology and infectious disease treatment research, with some technologies and products already in the preclinical or clinical research stage.

[0011] The protein encoded by the KRAS gene (Kirsten rat sarcoma virus oncogene homolog) is a small GTPase (SGTPase) belonging to the RAS protein superfamily and involved in intracellular signal transduction. The KRAS protein has 188 amino acids and a molecular weight of 21.6 kDa. It is a guanine nucleoside-binding protein with GTPase activity. In cells, KRAS protein converts between an inactive and an active state. When bound to guanine nucleoside diphosphate (GDP), KRAS is inactivated. When bound to guanine nucleoside triphosphate (GTP), KRAS is activated, and it can activate downstream signaling pathways, including the MAPK signaling pathway, PI3K signaling pathway, and Ral-GEFS signaling pathway. These signaling pathways play important roles in promoting cell survival, proliferation, and cytokine release.

[0012] In human cancers, KRAS is one of the most well-known oncogenes in oncology and was once considered a target for "unclear drugs." KRAS gene mutations 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 cholangiocarcinoma, cervical cancer, and bladder cancer. KRAS gene mutations account for 86% of all RAS gene mutations. Ninety-seven percent of KRAS gene mutations involve mutations at amino acid residues 12 or 13. The most common mutations are those in which amino acid 12 changes to asparagine (G12D), valine (G12V), cysteine ​​(G12C), or asparagine (G13D).

[0013] Structural studies have revealed that many of these genetic mutations disrupt the ability of KRAS to hydrolyze GTP. Several mutations in KRAS, including G12D, G12V, and G13D, disrupt GAP activity, allowing KRAS to bind GTP continuously, locking it in an active tyrosine kinase state and continuously activating downstream signaling pathways (e.g., PI3K, RAF-MEK-ERK (MAPK), RAL-GEF, etc.). Opening these downstream signaling pathways stimulates cell proliferation and migration, ultimately promoting tumorigenesis.

[0014] In recent years, covalent inhibitors have been developed for KRAS mutants. These target KRAS mutants through allosteric sites, reducing the affinity of KRAS mutants for GTP and "locking" their activity. Examples include Amgen's KRAS-G12C inhibitor AMG510 and MiratiTherapeutics' KRAS-G12C inhibitor MRTX1257, which is still in preclinical development. There are currently no relevant therapeutic agents for other KRAS mutations, and there is a lack of oncology drugs that utilize the body's immune mechanisms to detect and treat them.

[0015] Summary of the Invention In human cancers, KRAS gene mutations occur in nearly 90% of pancreatic cancers, 30-40% of colon cancers, 17% of endometrial cancers, and 15-20% of lung cancers. In 97% of KRAS gene mutations, mutations occur at amino acid residues 12 or 13. The most common mutations are G12D, G12V, G12C, and G13D. After mutation, KRAS is presented on the cell surface by MHC molecules in cells, where it is recognized by T cells, stimulating the T cell immune response and further eliminating tumor cells harboring KRAS mutations.

[0016] Specifically, when KRAS mutant polypeptide was used as an antigen, CD8 + It can generate a CTL (cytotoxic lymphocyte) response in vivo. Mutations occurring at several amino acid residues in the KRAS polypeptide can be presented by HLA molecules and recognized by T cells.

[0017] One embodiment of the present invention is to detect the KRAS-G12V variant of the tumor KRAS gene by a specific T cell receptor (TCR) single-cell screening technique. 8-16 (VVGA V GVGK) mutation (hereinafter also abbreviated as G12V, G12V mutation, or G12V mutation of the KRAS gene), KRAS-G12C 8-16 (VVGA C The method involves screening two types of TCRs that specifically target GVGK (hereinafter also referred to as G12C, G12C mutation, or G12C mutation of the KRAS gene).

[0018] One embodiment of the present invention provides specific T cell receptors and antigen-binding fragments thereof that target the epitope of the G12V or G12C mutation in the KRAS gene. Another embodiment of the present invention includes the use of the T cell receptors and antigen-binding fragments thereof in the manufacture of a medicament for treating tumors harboring the G12V and G12C mutations in the KRAS gene.

[0019] The present invention is based on the above-mentioned principle, and the TCR or antigen-binding fragment thereof specific to the KRAS mutant polypeptide of the present invention is a complex molecule of the KRAS G12V mutant polypeptide (VVGAVGVGK) and HLA-A11 and / or a complex molecule of the KRAS G12C mutant polypeptide (VVGACGVGK) and HLA-A11, or a complex molecule of the KRAS G12V mutant polypeptide (VVGACGVGK) and HLA-A11. 8-16 (VVGA V By specifically binding to the GVGK epitope and HLA-A03 complex molecule, it stimulates T cell activation, induces the secretion of cytokines such as IFN-γ from T cells, and kills tumor cells expressing KRAS mutant polypeptides (particularly tumor cells positive for the G12V and / or G12C mutations in the KRAS gene).

[0020] In the present invention, the expression "TCR specific for a KRAS mutant polypeptide" or "TCR specific for a mouse-derived KRAS mutant polypeptide" refers to a mouse-derived TCR directed against the HLA-A11-restricted CTL epitope polypeptide (whose sequence is VVGAVGVGK and / or VVGACGVGK) in the KRAS mutant polypeptide, and in a specific embodiment of the present invention is referred to as 1-2C TCR or 1-2C, 3-2E TCR or 3-2E.

[0021] The present application includes TCRs or derivatives that specifically bind to a complex molecule of the VVGAVGVGK and / or VVGACGVGK polypeptides mutated at amino acid position 12 derived from the KRAS mutant polypeptide and HLA-A11, and also includes antigen-specific TCR fragments that exhibit substantially the same function as the original TCR. "TCR fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of a TCR and a TCR analog, which typically contains at least a portion of the antigen-binding region or variable region of the parent TCR, for example, one or more CDRs. A TCR fragment maintains at least some of the binding specificity of the parent TCR.

[0022] When referring to a ligand / receptor, antibody / antigen, or other binding pair, "specific" binding refers to determining whether a binding reaction between the protein (e.g., VVGAVGVGK and / or VVGACGVGK polypeptide) and an HLA-A11 complex molecule occurs in a heterogeneous population of proteins and / or other biological reagents. Thus, under specified conditions, a particular ligand / antigen binds to a particular receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0023] The present invention also provides pharmaceutical compositions containing one or two TCRs or antigen-binding fragments thereof specific to the KRAS mutant polypeptides of the present invention. To produce the pharmaceutical compositions, the TCRs or antigen-binding fragments thereof specific to the KRAS mutant polypeptides or their antigen-binding fragments can be mixed with a pharmaceutical vector or excipient to produce various desired dosage forms. The pharmaceutical compositions of the present invention can be formulated in various dosage forms, such as oral tablets, powders, pills, powders, granules, fine granules, soft / hard capsules, film-coated tablets, pellets, sublingual tablets, and plasters, and parenteral injections, suppositories, transdermal preparations, ointments, plasters, and topical solutions. Those skilled in the art can select an appropriate dosage form depending on the route of administration and the intended recipient.

[0024] The dosage of the active ingredient of the pharmaceutical composition of the present invention varies depending on the subject, target organ, symptoms, administration method, etc., and can be determined based on the judgment of a physician, taking into consideration the type of dosage form, administration method, age and weight of the patient, symptoms of the patient, etc.

[0025] The pharmaceutical compositions of the present invention may also include other agents, including, but not limited to, cytotoxic agents, cell growth inhibitors, anti-angiogenic or antimetabolic agents, targeted tumor agents, immunostimulatory or immunomodulatory agents, or TCRs linked to cytotoxic agents, cell growth inhibitors, or other toxic agents.

[0026] Specifically, the present invention provides the following solutions:

[0027] 1. A T cell receptor (TCR) or an antigen-binding fragment thereof, The TCR or antigen-binding fragment thereof is KRAS-G12V 8-16 (VVGA V GVGK) epitope complex with HLA-A11, or KRAS-G12C 8-16 (VVGA C GVGK) epitope complex with HLA-A11, or KRAS-G12V 8-16 (VVGA V and a TCR capable of binding to a complex of a GVGK epitope and HLA-A03, and the TCR comprises an α chain variable region and a β chain variable region, and the TCR or antigen-binding fragment thereof comprises the following α chain complementarity-determining region and β chain complementarity-determining region: α chain complementarity determining region CDR1 set forth in SEQ ID NO:3; α chain complementarity determining region CDR2 set forth in SEQ ID NO:4; α chain complementarity determining region CDR3 set forth in SEQ ID NO: 5; β-chain complementarity-determining region CDR1 set forth in SEQ ID NO:8; β chain complementarity determining region CDR2 set forth in SEQ ID NO: 9; and β chain complementarity determining region CDR3 set forth in SEQ ID NO: 10; or α chain complementarity determining region CDR1 set forth in SEQ ID NO: 13; α chain complementarity determining region CDR2 set forth in SEQ ID NO: 14; α chain complementarity determining region CDR3 set forth in SEQ ID NO: 15; β-chain complementarity-determining region CDR1 set forth in SEQ ID NO: 18; β chain complementarity determining region CDR2 set forth in SEQ ID NO: 19; and β chain complementarity determining region CDR3 set forth in SEQ ID NO: 20.

[0028] 2, the α chain variable region set forth in SEQ ID NO: 2, and β-chain variable region set forth in SEQ ID NO:7; or an alpha chain variable region set forth in SEQ ID NO: 12, and β-chain variable region set forth in SEQ ID NO: 17; The T cell receptor (TCR) or antigen-binding fragment thereof according to Item 1, comprising:

[0029] 3. The TCR or antigen-binding fragment thereof according to item 1 or 2, wherein the TCR is a mouse-derived TCR, a human-mouse chimeric TCR, or a humanized TCR.

[0030] 4. A polynucleotide encoding the TCR or antigen-binding fragment thereof according to any one of items 1 to 3, which has one or more sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, and SEQ ID NO: 16.

[0031] 5. An expression vector comprising the polynucleotide according to Item 4, which is preferably a lentiviral vector.

[0032] 6. A host cell comprising the expression vector according to Item 5.

[0033] 7. A method for producing the TCR or antigen-binding fragment thereof according to any one of items 1 to 3, The method includes the following steps: 1) culturing the host cell according to item 6; and 2) recovering the TCR or antigen-binding fragment thereof according to any one of items 1 to 3 from the host cells or the culture medium thereof.

[0034] 8. A pharmaceutical composition comprising the TCR or antigen-binding fragment thereof according to any one of items 1 to 3 and a pharmaceutically acceptable carrier.

[0035] 9. Use of the TCR or antigen-binding fragment thereof according to any one of items 1 to 3 in the manufacture of a medicament for increasing the secretion level of IFN-γ cytokine from T cells, The pharmaceutical is, for example, a protein-based pharmaceutical, an ADC pharmaceutical, or a pharmaceutical combining a TCR and an antigen.

[0036] 10. The TCR or antigen-binding fragment thereof according to any one of items 1 to 3,8-16 (VVGA V GVGK) or KRAS-G12C 8-16 (VVGA C Use in the manufacture of a reagent for detecting tumor cells expressing a GVGK mutation, or in the manufacture of a reagent for detecting or diagnosing tumors, comprising: Preferably, the TCR or antigen-binding fragment thereof is KRAS-G12V 8-16 (VVGA V GVGK) / HLA-A11, or KRAS-G12C 8-16 (VVGA C specifically binds to GVGK) / HLA-A11, or The TCR or its antigen-binding fragment is an HLA molecule having antigen-binding properties similar to those of HLA-A11 or HLA-A03, such as KRAS-G12V 8-16 (VVGA V GVGK) or KRAS-G12C 8-16 (VVGA C 1. The method of claim 1, wherein the HLA molecule specifically binds to a HLA-A31, HLA-A33, HLA-A68, or HLA-A30 mutant polypeptide, and the HLA molecule is preferably HLA-A31, HLA-A33, HLA-A68, or HLA-A30.

[0037] 11. Use of the TCR or antigen-binding fragment thereof according to any one of items 1 to 3 in the manufacture of an antitumor drug for treating a patient with a tumor having the G12V and G12C mutations of the KRAS gene, The tumor is, for example, pancreatic cancer, colon cancer, or lung cancer, and the lung cancer is, for example, non-small cell lung cancer; Preferably, the G12V and G12C mutations of the KRAS gene are KRAS-G12V 8-16 (VVGA V GVGK) mutation or KRAS-G12C 8-16 (VVGA C GVGK) mutation, use.

[0038] Advantages of the Invention By using the TCR specific to the G12V or G12C mutation of the KRAS gene of the present invention to produce T lymphocytes (TCR-T) expressing the TCR, it is possible to effectively identify and kill tumor cells positive for the G12V or G12C mutation of the KRAS gene, and further to inhibit the growth of tumors, particularly solid tumors, and thereby achieve the effects of tumor treatment.

[0039] The two specific T cell receptors targeting the epitopes of the G12V or G12C mutation in the KRAS gene and the T cells expressing them have high infection efficiency and binding properties, and can be used to conduct early-stage pharmaceutical research and develop pharmaceuticals that target mutations associated with tumor growth and progression in test models, etc. Therefore, they can be used to diagnose and treat various tumors expressing KRAS gene mutations at various stages, particularly in the manufacture of pharmaceuticals for treating solid tumors with a high mutation frequency. [Brief explanation of the drawings]

[0040] [Figure 1] Results of molecular sieve chromatography and biotinylation level detection of different mutant KRAS polypeptides and HLA-A11 complex proteins. [Figure 2] Single-cell sorting of KRAS-G12V / HLA-A11 tetramer-specific T cells. Panel A shows the ELISPOT assay of specific T cells in splenocytes from mice immunized with KRAS-G12V polypeptide. "Mock" is the negative control (no stimulation), "KRAS-G12V8-16" is the polypeptide-stimulated detection channel, and "phorbol ester (PMA)" is the positive control. T1-T6 and TF1-TF6 indicate mouse numbers. Panel B shows the sorting of epitope-specific T cells in splenocytes from mice immunized with the polypeptide. WT is the negative control (non-immunized mouse). The abscissa indicates KRAS-G12V8-16 / HLA-A11 tetramer staining, and the ordinate indicates CD8 positive staining. KRAS-G12V8-16 / HLA-A11 tetramer-positive cells are indicated by the box in the figure. [Figure 3]Verification of the specific binding of 3-2E and 1-2C TCRs to KRAS-G12V8-16 / HLA-A11. (A) Flow cytometry results of a binding experiment in which 293T cells were transiently transfected with 3-2E or 1-2C TCR and then specifically bound to the KRAS-G12V8-16 / HLA-A11 tetramer. (B) Flow cytometry results of a binding experiment in which 293T cells were transiently transfected with 1-2C or 3-2E TCR and then specifically bound to the KRAS-G12V7-16 / HLA-A11 tetramer. [Figure 4] Cross-recognition of 1-2C and 3-2E TCRs with KRAS-G12V8-16 / HLA-A3. Panel A shows flow cytometry results of staining analysis of 293T cell tetramers expressing KRAS-G12V8-16 / HLA-A11 and 1-2C or 3-2E TCRs. Panel B shows flow cytometry results of staining analysis of 293T cell tetramers expressing KRAS-G12V8-16 / HLA-A03 and 1-2C or 3-2E TCRs. [Figure 5] Detection of infection efficiency of 1-2C or 3-2E TCR-T cells. The first row (horizontal) shows tetramer detection from PBMCs of two volunteers (D1 and D2) not infected with TCR lentivirus as a negative control. The second row (horizontal) and third row (horizontal) show TCR-T cells produced using PBMCs or CD8 T cells from volunteers D1 (row 2 (horizontal)) and D2 (row 3 (horizontal)), respectively. Flow cytometry staining was performed using the KRAS-G12V8-16 / HLA-A11 tetramer. The numbers in the quadrants indicate the positive rate of TCR expression. [Figure 6]Reaction of 1-2C or 3-2E TCR-T cells with different mutant KRAS polypeptides. Figures A and B show ELISPOT images and bar graphs of 1-2C or 3-2E TCR-T cells prepared using PBMC cells with KRAS-G12 wild-type and different mutant polypeptides. D1 and D2 indicate the volunteer numbers. Figure C shows ELISA bar graphs of IFN-γ levels produced after incubation of 1-2C or 3-2E TCR-T cells with KRAS-G12 wild-type and different mutant polypeptides. Incubation of TCR-T cells with medium serves as a negative control (mock), and PMA stimulation serves as a positive control. Figures D, E, and F show ELISPOT images of 1-2C or 3-2E TCR-T cells with KRAS-G12 wild-type and different mutant polypeptides, a bar graph of the results, and ELISA bar graphs of IFN-γ levels in these cells, respectively. [Figure 7] In vitro refolding and purification results of two types of TCR proteins, 1-2C and 3-2E. Figures A and B show the purification results of 1-2C TCR molecules after ion column and molecular sieve chromatography, respectively. Figures C and D show the purification results of 3-2E TCR molecules after ion column and molecular sieve chromatography, respectively. The small figures show the SDS-PAGE results of the peak proteins marked with asterisks. [Figure 8] Binding characteristics of 1-2C or 3-2E TCR with different mutant KRAS-G12 polypeptides and HLA-A11. Figures A and D show the affinity detection of 1-2C TCR with wild-type and different mutant KRAS-G12 polypeptides and HLA-A11 complex proteins, and Figures E and H show the affinity detection of 3-2E TCR with wild-type and different mutant KRAS-G12 polypeptides and HLA-A11 complex proteins. [Figure 9]Evaluation of the tumor-suppressing effect of 1-2C TCR-T cells in an NCG immunodeficient mouse tumor model. Figure A shows the flowchart of the mouse tumor-suppression experiment. PANC-1 tumor cells were inoculated on day 0 (D0), and TCR-T cells were injected intratumorally on day 7, followed by observation and measurement every 3–4 days. Figure B shows a comparison of the weight of isolated tumors at the end of the experiment between different treatment groups. Figure C shows the growth and comparison of tumor volume between different treatment groups. Each point represents the mean ± standard deviation of the tumor volume of each mouse in each group at that time point. Figures DG show the growth of tumor volume in a single mouse within each group. Statistical differences between groups were calculated by T test, where **: p<0.01, ***: p<0.001, ns, p>0.05.

[0041] Detailed Description of the Invention The present invention will further explain the technical solution of the present invention through specific embodiments and drawings, but those skilled in the art will understand that the following specific embodiments and examples are for the purpose of illustrating the present invention and should not limit the present invention in any way. Those skilled in the art will recognize that many modifications can be made to the present invention without departing from the spirit of the present invention, and such modifications will also fall within the scope of the present invention.

[0042] Unless otherwise specified, the experimental methods described below are all conventional experimental methods in this field, and the experimental materials used are all experimental materials that are readily available from commercial companies unless otherwise specified.

[0043] Example 1: Selection of T cells specific to KRAS-G12 mutant polypeptide and cloning of TCR genes In this example, we first synthesized HLA-A11-restricted epitope polypeptides predicted to have the KRAS-G12 mutation, immunized mice with these polypeptides, and then screened T cell responses using ELISPOT experiments to select mutant polypeptides with immunogenicity. At the same time, we produced tetramers of these KRAS mutant polypeptides and HLA-A11, and stained them with CD3 and CD8 antibodies to detect CD3 from spleen cells of immunized mice. + CD8 +The T cells were selected and sorted to obtain T cells specific to the KRAS-G12 mutant polypeptide.

[0044] 1. Prediction of HLA-restricted epitopes of KRAS-G12 mutant polypeptides Using the NetMHC-4.0 online prediction system, we predicted HLA-A11-restricted T cell epitopes for the KRAS-G12 mutant polypeptide, and found that the KRAS-G12V epitope has strong affinity for HLA-A11.

[0045] The contracted company (Sclight Biotechnology LLC) developed the KRAS-G12 wild-type polypeptide. 8-16 (described in SEQ ID NO: 34), G12V 8-16 (described in SEQ ID NO: 35), G12D 8-16 (described in SEQ ID NO: 36), G12C 8-16 (set forth in SEQ ID NO: 37) mutant polypeptide, KRAS-G12 wild-type polypeptide 7-16 (described in SEQ ID NO: 38), G12V 7-16 (described in SEQ ID NO: 39), G12D 7-16 (described in SEQ ID NO: 40), G12C 7-16 (described in SEQ ID NO: 41) was synthesized.

[0046] 2. Preparation of KRAS-G12V / HLA-A11 tetramer These polypeptides were evaluated and screened for their binding properties to HLA-A11, specific T cell responses, and specific TCRs.

[0047] Prokaryotic codon optimization was performed on β2m (β2-microglobulin, HLA-A11 light chain gene) (Uniprot: P61769) and HLA-A11 heavy chain gene (IMGT / HLA Acc No: HLA00043) using conventional methods. The resulting β2m nucleic acid sequence is set forth in SEQ ID NO: 48, the amino acid sequence encoded thereby is set forth in SEQ ID NO: 47, and the resulting HLA-A11 heavy chain gene is set forth in SEQ ID NO: 44, the amino acid sequence encoded thereby is set forth in SEQ ID NO: 43. For the HLA-A11 heavy chain gene, a sequence expressing a biotin-specific binding polypeptide (biotin-tag, the amino acid sequence of which is set forth in SEQ ID NO: 33) was added to the C-terminus of the heavy chain gene set forth in SEQ ID NO: 44.

[0048] The commissioned company synthesized these DNA sequences (Nanjing GenScript) and introduced the NdeI and XhoI restriction sites, respectively, with the NdeI restriction site located at the 5' end of the sequence and the XhoI restriction site located at the 3' end of the sequence. The NdeI and XhoI restriction sites were used to clone the synthesized DNA sequences of the β2m and HLA-A11 heavy chain genes into the expression vector pET-21a (Invitrogen), respectively, to construct the prokaryotic recombinant expression plasmids β2m-pET21a and HLA-A11-pET21a for the β2m and HLA-A11 heavy chain proteins.

[0049] The two expression plasmids were each introduced into E. coli BL21(DE3) competent cells (purchased from TIANDZ Biotech) by heat stimulation, and expression was induced by adding IPTG. The E. coli cells were then disrupted and homogenized to extract inclusion bodies, yielding inclusion body proteins of β2m and HLA-A11 heavy chain.

[0050] One ml of β2m inclusion bodies (30 mg / ml dissolved in a dissolution solution containing 6 M Gua-HCl, 50 mM Tris pH 8.0, 100 mM NaCl, 10 mM EDTA, and 10 mM DTT) was dissolved in 1 L of reconstitution solution (20 mM Tris-HCl, 400 mM L-arginine, 2 mM EDTA, GSH / GSSG) containing 5 mg of each of the KRAS-G12 wild-type polypeptides 8-16, G12V8-16, G12D8-16, and G12C8-16 mutant polypeptides prepared above, and KRAS-G12 wild-type polypeptides 7-16, G12V7-16, G12D7-16, and G12C7-16 polypeptides (synthesized by Sclight Biotechnology LLC). After slowly adding dropwise to a solution of β2m and HLA-A11 heavy chain (5 mM / 1 mM) for 1 hour, the HLA-A11 heavy chain inclusion body was slowly added dropwise to the above-mentioned reconstitution solution at a molar ratio of β2m:HLA-A11 heavy chain = 1:1 and reconstituted for more than 8 hours.

[0051] The reconstituted sample was concentrated through a 10 kDa filter using an ultrafiltration cup, and the sample was then transferred to a 20 mM Tris-Cl, 50 mM NaCl, pH 8.0 buffer solution. This was repeated twice. The sample was concentrated to approximately 20 ml and then added to 200 ml of a 20 mM Tris-Cl, 50 mM NaCl, pH 8.0 buffer solution. After further concentration to approximately 20 ml, the sample was again added to 20 mM Tris-Cl, 50 mM NaCl, pH 8.0 buffer to a final volume of approximately 10-20 ml. After removing the sample, the mixture was centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was transferred to an ultrafiltration tube and concentrated to approximately 0.5-1 ml. The KRAS-G12 wild-type or mutant polypeptide / HLA-A11 complex was purified through Superdex 200 molecular sieves (purchased from GE Healthcare). Based on the absorbance at 280 nm, the KRAS-G12 wild-type or mutant polypeptide / HLA-A11 complex protein peak (peak volume approximately 15.8 ml) was collected.

[0052] After purification through molecular sieves, the KRAS-G12 wild-type or mutant polypeptide / HLA-A11 complex protein sample was collected in an ultrafiltration concentration tube, concentrated to approximately 500 μL, and then centrifuged at 4°C to remove the precipitate, yielding the KRAS-G12 wild-type or mutant polypeptide / HLA-A11 complex protein sample.

[0053] (2) Biotinylation reaction Using the KRAS-G12V / HLA-A11 complex protein sample obtained in step (1), prepare the following biotinylation reaction systems (500 μl).

[0054] Biotinylation reaction system (purchased from AVIDITY): Total 500 μl 200 μl of 1 mg / ml KRAS-G12 wild-type and mutant polypeptide / HLA-A11 complex protein sample; Buffer A (N-bis(hydroxyethyl)glycine buffer) 50 μl, Buffer B (ATP, biotin) 50 μl, 200 μM biotin, 20 μl of Bir-A enzyme (3 mg / ml), 20 mM Tris-Cl, 50 mM NaCl, pH 8.0 (fill up to 500 μl).

[0055] After formulation, the mixture was mixed, placed on ice, and incubated overnight in a 4°C freezer.

[0056] The biotinylated sample was passed through a Superdex 200 molecular sieve to remove excess biotin and purify the biotinylated KRAS-G12 mutant polypeptide / HLA-A11 complex protein. The peak yield of the biotinylated KRAS-G12 wild-type or mutant polypeptide / HLA-A11 complex protein was approximately 15.8 mL (Figure 1).

[0057] Biotinylation efficiency detection: The biotinylated KRAS-G12 / HLA-A11 complex is concentrated to about 500 μl, sampled, and subjected to an SDS-PAGE shift test to verify the biotinylation effect.

[0058] Set up one sample and two controls: A. 8 μl of biotinylated KRAS-G12 / HLA-A11 complex sample + 2 μl of molecular sieve buffer; B. 8 μl of biotinylated KRAS-G12 / HLA-A11 complex sample + 2 μl of streptomycin (20 mg / ml); C. 2 μl of streptomycin + 8 μl of molecular sieve buffer.

[0059] The above three samples were incubated on ice for 30 minutes and then subjected to SDS-PAGE analysis. The results are shown in Figure 1.

[0060] As a result, the biotinylated KRAS-G12 / HLA-A11 complex can bind to streptavidin to form a polymer, resulting in delayed bending in SDS-PAGE. By comparing the SDS-PAGE bend grayscale ratio (bend grayscale after adding streptomycin to the KRAS-G12 / HLA-A11 complex / bend grayscale of the original KRAS-G12 / HLA-A11 complex) of each group of polypeptides before and after biotinylation, we determined that both wild-type and mutant KRAS-G12 polypeptides can be biotinylated efficiently (Figure 1).

[0061] (3) Production of KRAS-G12 wild-type and mutant polypeptide / HLA-A11-PE tetramers Biotinylated KRAS-G12 wild-type and mutant polypeptide / HLA-A11 complex molecules were concentrated by ultrafiltration, and then tetramerized with streptavidin-PE at a molar ratio of 1:5 (streptavidin-PE:KRAS-G12 wild-type or mutant polypeptide / HLA-A11 complex). Finally, the mixture was incubated overnight at 4°C to obtain KRAS-G12 wild-type or mutant polypeptide / HLA-A11 complex molecules. 8-16 (VVGA G GVGK) polypeptide, G12V 8-16 (VVGA V GVGK), G12D 8-16 (VVGA D GVGK), G12C 8-16 (VVGA C GVGK) mutant polypeptide, KRAS-G12 wild type 7-16 (VVVGA V GVGK), G12D 7-16 (VVVGA D GVGK), G12C 7-16 (VVVGA C GVGK) / HLA-A11-PE tetramer (for spare use) was obtained.

[0062] 3. HLA-A11 transgenic mice immunized with KRAS-G12V polypeptide In this step, KRAS-G12V 8-16 (VVGA V Using HLA-A11 transgenic mice (manufactured by Beijing Biocytogen) immunized with the mutant polypeptide (GVGK), KRAS-G12V was induced in the mice. 8-16 Induces the production of T cells specific for the mutant polypeptide and further inhibits KRAS-G12V 8-16 The specific TCR of the mutant polypeptide is obtained.

[0063] Specifically, chemically synthesized KRAS-G12V 8-16100 μg of mutant polypeptide (Sclight Biotechnology LLC) was dissolved in 100 μL of PBS and emulsified with an equal volume of Freund's complete adjuvant. HLA-A11 transgenic mice were immunized with the mixture of emulsified polypeptide and Freund's complete adjuvant via multiple subcutaneous injections into the dorsal region.

[0064] One week after the first immunization, the same method was used to detect KRAS-G12V 8-16 Immunoimmunization was performed by dissolving 100 μg of the mutant polypeptide in 100 μL of PBS, emulsifying it with an equal volume of Freund's incomplete adjuvant, and injecting it in the same manner. One week later, the mice were sacrificed, and their spleens were harvested and polished to obtain mouse splenocytes.

[0065] 4. KRAS-G12V 8-16 (VVGA V Selection of T cells specific for the GVGK / HLA-A11-PE tetramer and single-cell TCR gene amplification and sequencing After washing with PBS, resuspend the KRAS-G12V in step 1. 8-16 Approximately 1 x 10 spleen cells from mice immunized with the mutant polypeptide 7 The cells were centrifuged at 200-250 g for 10 minutes. The cells were washed three times with PBS containing 0.5% BSA and centrifuged at 200-250 g for 10 minutes. KRAS-G12V 8-16 (VVGA V Mouse spleen cells were incubated with a 1:1:1 molar ratio of GVGK / HLA-A11-PE tetramer (obtained by the tetramer production method described above), PerCP-Cy5-CD8 (purchased from BD), and FITC-CD3 fluorescent antibody (purchased from BD) for 20 minutes at 25°C. The cells were washed three times with PBS containing 0.5% BSA and centrifuged at 200-250 g for 10 minutes. The cells were resuspended in PBS containing 0.5% BSA.

[0066] The cells were then subjected to flow cytometry single cell sorting. Lymphocyte subpopulations were selected and CD3 + CD8 +Select T cells and screen for KRAS-G12V 8-16 (VVGA V GVGK) / HLA-A11-PE tetramer-positive CD8+ T cells were obtained (see Figure 2, where the square box indicates the tetramer-positive cells and their percentage).

[0067] Single positive cells were sorted into 96-well plates containing cell lysis solution (purchased from Tiangen Bio) and RNAse inhibitor (purchased from CW Bio). Then, KRAS-G12V in each well was isolated. 8-16 (VVGA V Total RNA was extracted from GVGK) / HLA-A11-PE tetramer-positive T cells, and 5'RACE TCR gene amplification was performed, as follows.

[0068] 5'RACE is divided into three steps: reverse transcription (RT-PCR), first PCR amplification, and second PCR amplification. The following procedure was performed using the Takara D315-FullRACE kit according to the manufacturer's instructions.

[0069] (1) RT-PCR: The downstream primer used was the TCR gene constant region-specific primer GSP1 (purchased from Takara), and the upstream primer was a target-switching primer (Takara) with an oligoguanine deoxyribonucleic acid (Oligo dG) at the 3' end.

[0070] (2) First PCR: Using the TCR cDNA obtained in (1) above as a template, the upstream primer was outer linker primer 1 (5' RACE outer primer, Takara), and the downstream primer was a specific primer for the TCR constant region upstream of the constant region GSP1 (purchased from Takara), to obtain the first PCR product of the TCR α chain or β chain.

[0071] (3) Second PCR: The first PCR product of the TCR α chain or β chain obtained in (2) above was used as a template. The upstream primer was inner linker primer 2 (5'RACE inner Primer, Takara), and the downstream primer was a TCR constant region-specific primer upstream of the constant region GSP2 (purchased from Takara, D315-FullRACE Kit). The second PCR product of the TCR α chain or β chain, respectively, was obtained.

[0072] The amplified second-round PCR products, containing the TCR α and β chain variable region genes, were subjected to agarose gel electrophoresis to identify the target TCR α and β chain variable region genes at 500 bp. The target bends were recovered and ligated into a T vector (pMD18T, Takara) using T4 ligase. DH5α cells (purchased from Tiangen Biotechnology) were then transformed with the ligated products, and monoclonal gene sequencing was performed (subcontracted to Ruiboxingke).

[0073] KRAS-G12V obtained through the above process 8-16 After performing single-cell TCR gene amplification sequencing on T cells specific to , and analyzing the results, the frequently occurring α-chain and β-chain combinations were selected as new TCRs, named 1-2C and 3-2E TCRs, which were further subjected to binding and functional verification.

[0074] Here, the 1-2C TCR comprises an α chain variable region set forth in SEQ ID NO: 2 and a β chain variable region set forth in SEQ ID NO: 7. The 3-2E TCR comprises an α chain variable region set forth in SEQ ID NO: 12 and a β chain variable region set forth in SEQ ID NO: 17.

[0075] Example 2. KRAS-G12V 8-16 Binding experiments of (VVGAVGVGK) / HLA-A11 tetramer with cells expressing 1-2C and 3-2E TCRs In this example, the inventors further demonstrated that the screened 1-2C and 3-2E TCRs were KRAS-G12V 8-16 (VVGAV In addition, in this example, 1-2C and 3-2E were confirmed to have specific recognition for KRAS-G12V (GVGK) / HLA-A11. 8-16 (VVGA V GVGK), as well as KRAS-G12V presented by HLA-A03. 8-16 (VVGA V It was also shown that it can bind to GVGK.

[0076] 1. Verification of binding specificity of 1-2C and 3-2E TCRs First, the α and β chain variable regions (V regions) of the 1-2C and 3-2E TCRs (1-2C of the α chain variable region The nucleic acid sequence is set forth in SEQ ID NO: 1, Nucleic acid of the beta chain variable region of 1-2C The sequence is set forth in SEQ ID NO: 6, 3-2E of the α chain variable region The nucleic acid sequence is SEQ ID NO: 11 is described in Nucleic acid of the β chain variable region of 3-2E The sequence is SEQ ID NO: 16 The 1-2C and 3-2E chimeric TCR α and β chain sequences were obtained by linking the α and β chain constant region (C region) genes of the human TCR (synthesized by Hongxun Biotechnology Co., Ltd.). The specific sequences of the chimeric sequences are shown in Table 1 below.

[0077] [Table 1] The α and β chains of the 1-2C and 3-2E TCRs were linked in between by a T2A sequence (the amino acid sequence of the T2A sequence is set forth in SEQ ID NO: 42), and the lentiviral expression plasmid pCDH (purchased from Invitrogen) was used as the starting plasmid to construct the 1-2C and 3-2E TCR lentiviral expression vectors, i.e., the lentiviral expression vector 1-2C-pCDH and the lentiviral expression vector 3-2E-pCDH, respectively.

[0078] HEK-293T cells (purchased from ATCC) were co-transfected with the 1-2C or 3-2E-pCDH lentiviral expression vector and the CD3-CD8-pCDH plasmid (purchased from Nanjing GenScript) expressing CD3 and CD8 at a 1:1 ratio. After 24 hours of co-transfection, the cells were centrifuged at 200-250 g for 10 minutes. After washing three times with PBS containing 0.5% BSA, the cells were centrifuged at 200-250 g for 10 minutes to obtain HEK-293T cells expressing the 1-2C or 3-2E TCR.

[0079] Thereafter, the present inventors further investigated the binding of 1-2C or 3-2E TCR to the KRAS-G12 wild-type polypeptide and other mutant polypeptides by using the KRAS-G12 wild-type polypeptide prepared above. 8-16 (VVGA G GVGK), G12V 8-16 (VVGA V GVGK), G12D 8-16 (VVGA D GVGK), G12C 8-16 (VVGA C The binding specificity was assessed by staining analysis of 293T cells expressing 1-2C or 3-2E TCR with a tetramer of the GVGK mutant polypeptide / HLA-A11.

[0080] Specifically, HEK-293T cells obtained by this cotransfection, KRAS-G12V produced above 8-16 (VVGA V The cells were co-incubated with a 1:1:1 molar ratio of GVGK / HLA-A11-PE tetramer and PerCP-Cy5-CD8 and FITC-CD3 antibodies (BD) for 30 minutes. The cells were washed three times with PBS containing 0.5% BSA and centrifuged at 200-250g for 10 minutes. The cells were resuspended in PBS containing 0.5% BSA to detect KRAS-G12V. 8-16 (VVGA V The frequency of GVGK / HLA-A11 positive T cells was analyzed using flow cytometry (shown in Figure 3A).

[0081] The first and second rows (horizontal direction) in Figure 3A are KRAS-G12V 8-16 (VVGA V Flow cytometry analysis of staining of HEK-293T cells co-transfected with KRAS-G12V / HLA-A11-PE tetramer. The results showed that 293T cells transfected with 1-2C or 3-2E TCRs showed KRAS-G12V expression. 8-16 (VVGA V The proportion of cells capable of binding to the GVGK) / HLA-A11-PE tetramer among CD8-positive cells was approximately 25.9%.

[0082] Detection of KRAS-G12 wild-type polypeptide and other mutant polypeptide / HLA-A11 tetramer binding specificities revealed that 293T cells expressing 1-2C or 3-2E TCRs were specifically targeted to KRAS-G12 wild-type polypeptide (VVGA) and other mutant polypeptide / HLA-A11 tetramers. G GVGK) or G12D 8-16 (VVGA D GVGK) and HLA-A11 cannot bind to the tetramer formed by G12C 8-16 (VVGA C We confirmed that the tetramer formed by GVGK and HLA-A11 can bind to each other (Fig. 3A).

[0083] As can be seen, the 1-2C or 3-2E TCR binds to G12V in cell-level binding experiments. 8-16 (VVGA V It can specifically bind to GVGK / HLA-A11 and also binds to G12C 8-16 (VVGA C It can also specifically bind to the GVGK / HLA-A11 complex.

[0084] 2. Detection of binding specificity between 1-2C and 3-2E TCR and KRAS-G12 10 peptide Considering that CD8 T cell epitope polypeptides are generally 9-10 amino acids in length and that all of the detected polypeptides were 9 amino acid polypeptides (9 peptides), the inventors considered that it was not possible to exclude the possibility that the 1-2C and 3-2E TCRs could bind to 10 amino acid polypeptides (10 peptides).

[0085] The motif of the HLA-A11 binding polypeptide allows for the forward shift of one amino acid at its N-terminus to still contain the T cell epitope, i.e., the KRAS-G12 wild-type polypeptide. 7-16 (set forth in SEQ ID NO: 38), G12V 7-16 (set forth in SEQ ID NO: 39), G12D 7-16 (set forth in SEQ ID NO: 40), G12C 7-16 In this example, using the same conditions and procedures as in Example 1, these 10-length polypeptides were further prepared into tetramers, and cell binding assays were performed with 293T cells expressing 1-2C or 3-2E TCR. Each procedure and condition was the same as in Example 1. 8-16 The results of flow cytometry are shown in Figure 3B.

[0086] As a result, according to the upper right quadrant of the flowchart, it was found that none of the tetramers prepared from the 10-peptide KRAS wild-type and mutant polypeptides could bind to 293T cells expressing 1-2C or 3-2E TCR. Therefore, the 1-2C or 3-2E TCR of the present invention is KRAS-G12V 8-16 (VVGA V GVGK) and KRAS-G12C 8-16 (VVGA C It was further demonstrated that the GVGK polypeptide epitope can be highly specifically recognized.

[0087] KRAS-G12V presented by 3.1-2C and 3-2E TCR and HLA-A03 8-16 (VVGAVGVGK) cross-identification detection HLA-A11 is known to belong to the HLA-A3 superfamily, which includes HLA-A03, HLA-A31, HLA-A33, and HLA-A68. HLA-A3 superfamily members share similar antigen presentation characteristics. The antigen presentation of HLA-A3 superfamily molecules is characterized by the C-terminus of the presented polypeptide, which typically contains lysine (K) or arginine (R). The two amino acids from the N-terminus and the C-terminus of the polypeptide are inserted into the polypeptide-binding chamber of the HLA molecule, exposing amino acids in the middle of the polypeptide for recognition by TCRs. Furthermore, HLA-A3 superfamily molecules, such as HLA-A03, HLA-A31, HLA-A33, and HLA-A68, have over 70% α1 and α2 helix conservatism, which allows them to interact with TCRs.

[0088] Therefore, the TCRs obtained by screening in Example 1 also bind to HLA-A3 superfamily molecules such as HLA-A03, HLA-A31, HLA-A33, and HLA-A68, and KRAS-G12V. 8-16 In this example, the compound can be used to distinguish between complexes formed with HLA-A03 and KRAS-G12V (VVGAVGVGK) or other mutant polypeptides. 8-16 Tetramers were prepared using (VVGAVGVGK) and the binding ability of the 1-2C and 3-2E TCRs screened above was detected by flow cytometry, and their specific binding ability to KRAS-G12 mutant polypeptides presented by other HLA-A3 superfamily molecules was confirmed.

[0089] Thus, in this example, the 1-2C and 3-2E TCRs and the KRAS-G12V presented by HLA-A03 8-16 The cross-discrimination ability of (VVGAVGVGK) was detected.

[0090] The HLA-A03 heavy chain gene used here is set forth in SEQ ID NO: 46, and the amino acid sequence it encodes is set forth in SEQ ID NO: 45. The remaining materials and procedures used are all the same as those used for HLA-A11.

[0091] In this example, HLA-A03 (IMGT / HLA Acc No: HLA00037) and KRAS-G12V were similarly identified using the method described in Example 1. 8-16 A tetrameric protein of (VVGAVGVGK) polypeptide was produced, and KRAS-G12V 8-16 293T cells expressing 1-2C or 3-2E TCRs were stained with (VVGAVGVGK) / HLA-A11 tetramers (Fig. 4).

[0092] As a result, KRAS-G12V 8-16 The (VVGAVGVGK) / HLA-A03 tetramer could significantly bind to 293T cells expressing the 1-2C or 3-2E TCR (Fig. 4B , upper right quadrant).

[0093] Thus, the 1-2C or 3-2E TCRs express KRAS-G12V, which is presented by HLA-A11. 8-16 (VVGAVGVGK) polypeptide, as well as KRAS-G12V presented by HLA-A03. 8-16 (VVGAVGVGK) polypeptide, suggesting that it may have broad utility among a wider range of people.

[0094] Examples 3.1-2C and 3-2E: Preparation of TCR-T cells and their reaction with different mutant KRAS polypeptides In this example, peripheral blood mononuclear cells (PBMCs) or CD8 T cells isolated from healthy volunteers on an HLA-A11 genetic background were transfected with the 1-2C or 3-2E TCR gene to generate TCR-T effector cells. 8-16 (VVGA G GVGK), G12V 8-16 (VVGA V GVGK), G12D 8-16 (VVGA D GVGK), G12C 8-16 (VVGA CThe GVGK mutant polypeptides were added to the above TCR-T effector cell system and co-cultured. The levels of FN-γ secreted by the effector cells in response to the interaction between the effector cells and target cells presenting wild-type and mutant KRAS polypeptides were measured, and the interaction between the 1-2C or 3-2E TCR and target cells expressing wild-type and mutant KRAS polypeptides / HLA-A11 was evaluated. The specific procedure was as follows.

[0095] 1. Production of lentiviruses expressing 1-2C and 3-2E TCRs The 1-2C and 3-2E TCR lentiviral expression plasmids (1-2C-pCDH and 3-2E-pCDH) from Example 2 and the lentiviral packaging plasmids PLP1, PLP2, and VSVG (purchased from Addgene) were mixed in a ratio of PLP1:PLP2:VSVG:TCR-pCDH = 20:13:5:20, and 20 μl of this mixture was diluted in 1.25 ml of DMEM medium to prepare a DNA solution. 20 μl of polyetherimide (PEI, 1 μg / μl) was added to 1.25 ml of DMEM, and the PEI / DMEM solution was added to the combined DNA solution. After incubation at room temperature for 15 minutes, the mixture was added to 293T cells (Cell Bank, Shanghai) cultured in 15 cm discs and mixed. After 6 hours, the culture medium was carefully removed, and 25 ml of fresh culture medium was added to continue culturing. After 72 hours, virus-containing supernatants, i.e., lentiviral supernatants expressing 1-2C or 3-2E TCR, were collected.

[0096] 2.1-2C and 3-2E TCR-T cell production and TCR expression efficiency detection Peripheral blood lymphocytes were collected from two healthy volunteers (D1 and D2) to obtain PBMCs. A portion of the PBMCs was then negatively selected and isolated from CD8 T cells using magnetic beads (Biolegend). Anti-CD3 / anti-CD28-coated microspheres (ThermoFisher) were added to PBMCs or CD8 T cells at a 1:1 ratio and cultured overnight. Then, 1-2C or 3-2E TCR lentivirus was added to PBMCs or CD8 T cells at a 1:1 volume ratio, mixed, and a virus infection hole was created as a control. The cells were then cultured at 37°C in a 5% CO2 incubator. After 24 hours, the medium was replaced with complete medium and cultured for up to 10 days.

[0097] The anti-CD3 / anti-CD28 microspheres were removed under magnetic field conditions and washed twice with the same medium as used for cell culture to obtain the 1-2C or 3-2E TCR-T effector cells of this example.

[0098] 1-2C or 3-2E TCR-T cells (cultured for up to 10 days) prepared using the above-mentioned PBMCs or CD8 T cells were cultured to express KRAS-G12V 8-16 (VVGA V Flow cytometry detection was performed as in Example 2 using GVGK) / HLA-A11 tetramer staining to confirm the expression of 1-2C or 3-2E TCR (Figure 5, upper right quadrant).

[0099] As a result, tetramer-positive T cells were detected in 1-2C and 3-2E TCR-T cells derived from volunteers D1 and D2, which were produced using PBMCs or CD8+ T cells, with different positive rates ranging from 0.26% to 2.38%. Of these, the positive rates in PBMCs were 1-2C: 0.26%, 0.77%, 3-2E: 1.03%, 0.55%, respectively, and in CD8+ T cells were 1-2C: 0.68%, 0.91%, 3-2E: 2.38%, 2.37%, respectively.

[0100] These TCR-T effector cells of the present invention are KRAS-G12V 8-16 (VVGA VIt was found that it can specifically bind to GVGK / HLA-A11.

[0101] 3.1-2C and 3-2E Detection of immune responses of TCR-T cells to KRAS wild-type and mutant polypeptides We then used different T cell detection methods (IFN-γ-MELISPOT and IFN-γ-ELISA) to detect the levels of IFN-γ secreted by 1-2C or 3-2E TCR-T cells in response to the interaction of target cells presenting wild-type and mutant KRAS polypeptides.

[0102] 1-2C or 3-2E TCR-T cells produced using PBMCs or CD8 T cells from two volunteers on days D1 and D2 were mixed at a 1:1 ratio with PBMCs from two volunteers on days D1 and D2, corresponding to their origins, to prepare antigen-presenting cells. 5 Cells were added at a cell number / well to pre-coated ELISPOT plates containing anti-IFN-γ antibodies, along with KRAS wild-type and mutant polypeptides (100 μl volume, 10 μg / ml). Cultures were continued for 18 hours at 37°C in a 100% humidified, 5% CO2 incubator. PMA / ionomycin (ION) (100 μl volume, 1 μg / ml) stimulation and 100 μl medium alone served as positive and negative controls, respectively. ELISPOT spot analysis was performed on specific T cells producing IFN-γ. The results are shown in Figures 6A-B and 6D.

[0103] Furthermore, 1-2C or 3-2E TCR-T cells, which were produced using PBMCs or CD8 T cells from two volunteers on days D1 and D2, were mixed with PBMCs from two volunteers on days D1 and D2 at a 1:1 ratio to prepare antigen-presenting cells. The prepared cell suspension was added to each well at a volume of 100 μl (2 × 10 5The cells were plated in a 96-well plate at 100 μL (100 cells / well), with triplicate replicates per well. The PMA / ionomycin (ION) group contained 1 μL of PMA / ionomycin mixture (250x) per 250 μL of cell culture medium, pre-diluted in the working solution. This served as a positive stimulation control. T cells not infected with 1-2C or 3-2E TCR lentivirus (D1 mock and D2 mock) were added in parallel as negative controls. After 20 h of incubation at 37°C, the supernatants from the 96-well plate culture wells were removed and centrifuged at 500 g for 5 minutes to remove remaining cells. The supernatants were then applied to an ELISA detection plate (BD) coated with anti-IFN-γ antibody (BD) to detect IFN-γ levels in the supernatants. The results are shown in Figure 6C and F.

[0104] ELISPOT experiments showed that 1-2C or 3-2E TCR-T cells generated using PBMCs from D1 and D2 volunteers were able to generate strong T cell immune responses against the KRAS-G12V mutant polypeptide and showed a certain level of cross-reactivity with the KRAS-G12C mutant polypeptide. On the other hand, no T cell responses were detected against the KRAS wild-type or KRAS-G12D mutant polypeptides (Figure 6A-B). 1-2C or 3-2E TCR-T cells generated using CD8 T cells from D1 and D2 volunteers generated T cell immune responses similar to those of TCR-T cells generated using PBMCs (Figure 6D-E).

[0105] ELISA experiments for IFN-γ levels showed that 1-2C or 3-2E TCR-T cells produced using PBMCs or CD8 T cells from D1 and D2 volunteers were both able to generate strong T cell immune responses against the KRAS-G12V mutant polypeptide, and IFN-γ levels were elevated, whereas no T cell responses were detected against the KRAS wild-type or KRAS-G12D mutant polypeptides (Fig. 6C and F).

[0106] Thus, 1-2C and 3-2E TCR T cells express KRAS-G12V 8-16 (VVGA VIt can specifically identify target cells that express KRAS-G12C (GVGK) / HLA-A11. 8-16 (VVGA C They have a certain degree of cross-reactivity with target cells expressing GVGK and are capable of specifically secreting the cytokine IFN-γ. Considering the potential cytotoxic effect of CD8+ T cells on target cells, the 1-2C and 3-2E TCR-T cells of the present invention are suggested to have potential target cell killing activity and tumor therapeutic value.

[0107] Example 4. Analysis of the binding characteristics of 1-2C and 3-2E TCRs to KRAS mutant polypeptide epitopes To accurately measure the binding characteristics and affinity of 1-2C and 3-2E TCRs with different mutant KRAS polypeptide / HLA-A11 complex proteins, the inventors further used surface plasma resonance (SPR) experiments to detect affinity at the protein level. The functional domain of 1-2C or 3-2E TCRs is the extracellular domain, and since the extracellular domain without the transmembrane domain is a soluble protein, the extracellular domain of 1-2C or 3-2E TCR was synthesized. The specific procedure is as follows:

[0108] 1. TCR Protein Expression and Purification The extracellular region genes of the 1-2C or 3-2E TCR α and β chains were optimized for prokaryotic codons, and the DNA sequences of the extracellular regions of the 1-2C and 3-2E TCR chimeras α and β chains were synthesized, respectively (1-2C: α chain, set forth in SEQ ID NO: 29; β chain, set forth in SEQ ID NO: 30; 3-2E: α chain, set forth in SEQ ID NO: 31; β chain, set forth in SEQ ID NO: 32). The 1-2C TCR contains an α chain variable region set forth in SEQ ID NO: 2 and a β chain variable region set forth in SEQ ID NO: 7. The 3-2E TCR contains an α chain variable region set forth in SEQ ID NO: 12 and a β chain variable region set forth in SEQ ID NO: 17. The NdeI and XhoI restriction enzyme recognition sites were introduced, respectively, with the NdeI restriction enzyme recognition site located at the 5' end of the sequence and the XhoI restriction enzyme recognition site located at the 3' end of the sequence. The synthesized DNA sequences of the extracellular domains of the 1-2C or 3-2E TCR α and β chains were cloned into the expression vector pET21a (Invitrogen) using the restriction enzyme recognition sites Nde I and Xho I, respectively, to construct prokaryotic recombinant expression plasmids for the extracellular domain proteins of the 1-2C or 3-2E TCR α and β chains.

[0109] Using the heat stimulation method, the expression plasmid was introduced into E. coli BL21 (DE3) competent cells, and expression was induced by adding IPTG to obtain the extracellular domain proteins of the 1-2C and 3-2E TCR α and β chains in the inclusion body state.

[0110] Six ml of inclusion bodies of the extracellular domains of the 1-2C or 3-2E TCR α and β chains (each inclusion body was dissolved at 30 mg / ml in a solution containing 6 M Gua-HCl, 50 mM Tris pH 8.0, 100 mM NaCl, 10 mM EDTA, and 10 mM DTT) was added dropwise at a 2:1 mass ratio to a reconstitution solution (5 M urea, 20 mM Tris-HCl, 400 mM L-arginine, 2 mM EDTA, and 5 mM GSH / GSSG / 1 mM GSH / GSSG) in two separate additions, 3 ml each time, with at least an 8-h interval between additions. The mixture was then concentrated using a concentrator cup (Millipore).

[0111] After concentration, the concentrates were dialyzed against 4 L of deionized water and 4 L of 10 mM Tris, pH 8.0, for 24 hours. Then, crude purification was performed using Source 15Q ion exchange chromatography, and the target protein was identified by SDS-PAGE.

[0112] Specifically, the target protein was concentrated using a concentration cup (Millipore) and then exchanged with a buffer solution of 20 mM Tris-HCl, 150 mM NaCl, pH 8.0. After concentration, the concentrate was purified using a Superdex 200 pg molecular sieve (GE Healthcare) to obtain approximately 2–3 mg of 1-2C or 3-2E TCR protein. The target protein was then detected by reducing (with dithiothreitol (DTT)) and non-reducing (without dithiothreitol (DTT)) SDS-PAGE (Figure 7).

[0113] As a result, 1-2C TCR eluted under 18.19 mS / cm conditions, and 3-2E TCR eluted under 21.19 mS / cm conditions. Molecular sieve chromatography of both TCRs revealed the target protein peak at 15 mL elution volume. SDS-PAGE analysis revealed that 1-2C and 3-2E are αβ heterodimers, with a bend size of approximately 52 kD in non-reducing SDS-PAGE without DTT. In reducing SDS-PAGE with DTT, the disulfide bond between the α and β chains was opened, resulting in bends of approximately 24 kD and 28 kD, respectively (Figure 7).

[0114] 2.SPR Detection Analysis The 1-2C and 3-2E TCR proteins, prepared by performing an in vitro refolding experiment using the same procedure as in Example 1, and the biotinylated KRAS wild-type and different mutant 9 peptide / HLA-A11 complex protein prepared in Example 1 were exchanged into SPR buffer (10 mM HEPES-HCl, 150 mM Na-Cl, 0.005% Tween-20, pH 7.4). The KRAS wild-type and different mutant 9 peptide / HLA-A11 complex proteins were diluted to 20 μg / ml and immobilized on an SA chip (GE Health). The 1-2C and 3-2E TCR proteins, diluted in gradients (0 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM), were then passed through each channel of the SA chip. Binding kinetic parameters were analyzed using BIA software, and affinity constants were calculated. The affinity of 1-2C and 3-2E TCRs to wild-type and KRAS wild-type and different mutant 9 peptide / HLA-A11 complex proteins was detected (FIG. 8).

[0115] As a result, as can be seen from the curves in Figure 8, the KRAS-G12V of the 1-2C TCR 8-16 (VVGA V The binding to KRAS-G12C / HLA-A11 was rapid on / fast off, with a binding affinity (KD) of 7.21 μM. 8-16 (VVGA C The binding affinity (KD) for KRAS-G12V / HLA-A11 was 46.2 μM. 8-16 significantly lower than the epitope.

[0116] On the other hand, 1-2C is KRAS wild-type KRAS-G12 8-16 (VVGA G GVGK) / HLA-A11 and KRAS-G12D 8-16 (VVGA D 3-2E TCR KRAS-G12V cannot bind to HLA-A11. 8-16 (VVGA V The binding affinity (KD) of 1-2C to KRAS-G12C / HLA-A11 was 35.9 μM, which was significantly lower than that of 1-2C. 8-16(VVGA C The binding affinity (KD) for KRAS-G12V / HLA-A11 was 50.5 μM. 8-16 is at the same level as

[0117] Similar to 1-2C, 3-2E TCR expresses KRAS wild-type KRAS-G12 8-16 (VVGA G GVGK) / HLA-A11 and KRAS-G12D 8-16 (VVGA D It cannot bind to GVGK / HLA-A11.

[0118] Therefore, as can be seen from the SPR results, the 1-2C TCR and 3-2E TCR are KRAS-G12V 8-16 (VVGA V It can specifically bind to HLA-A11 and bind to the 1-2C TCR and KRAS-G12C 8-16 (VVGA C GVGK) / HLA-A11 has some cross-reactivity, and 1-2C TCR and KRAS-G12C 8-16 (VVGA C The affinity of 1-2C and 3-2E TCRs for G12V and G12C mutations in KRAS / HLA-A11 is similar to that of KRAS-G12V. Therefore, it is speculated that the 1-2C and 3-2E TCRs have good binding properties and affinity, and that their use in antitumor therapy may induce IFN-γ production in tumor cells harboring the G12V and G12C mutations in the KRAS gene, leading to tumor cell killing and tumor treatment.

[0119] Example 5. Tumor-suppressive activity of 1-2C TCR-T cells in tumor mouse models In this example, the tumor-suppressing effect of 1-2C TCR-T cells was evaluated using an NCG immunodeficient mouse PANC-1 tumor model.

[0120] The steps of the TCR-T cell NCG mouse tumor suppression experiment include the following:

[0121] 1. Construction of NCG Mouse PANC-1 Tumor Model NCG mice were derived from Nanjing University-Nanjing Institute of Biomedicine. Each NCG mouse was subcutaneously inoculated with PANC-1 tumor cells (Beijing Union Cell Resource Center) carrying the KRAS-G12V mutant gene to establish a human immune system in the NCG mice: a) PANC-1 cell inoculation quantity: 4 × 10 6 Individual cells / 200μL / animal; b) Inoculation site: subcutaneous on the back.

[0122] 2.1-2C TCR-T cell therapy On day 7 after inoculation of NCG mice with PANC-1 tumor cells, the tumor volume was approximately 200 mm 3 The 1-2C TCR-T cells prepared from the PBMCs of two volunteers on days D1 and D2 in Example 3 were injected into the PANC-1 tumors of NCG mice via intratumoral multipoint injection. a) Inoculation quantity of 1-2C TCR-T cells: according to three doses: 1 × 10 7 , 1×10 6 , 1×10 5 cells / 200μL / animal; b) Inoculation site: intratumoral.

[0123] 3. Grouping and Processing: Approximately one week after tumor cell injection, mice with relatively uniform tumors were selected and grouped, and then treated with intratumoral injection of 1-2C TCR-T cells. In this example, TCR-free T cells (1 × 10 7 The injection group was the negative control, and each group consisted of 6 mice, of which 3 mice were treated with TCR-T cells on Days D1 and D2. The grouping information and treatments are as follows: [Table 2] After tumor formation, tumor size was measured every 3-4 days. 3 At this time, the experiment was terminated, the mice were sacrificed, and the tumors were isolated and weighed.

[0124] 4. Observation of treatment effect: 1) Tumor size detection: a) After injecting TCR-T cells, the diameter was measured using a caliper, the unit is mm, and the calculation formula is: v = 1 / 2 × a × b × b (a is the major axis, b is the minor axis); b) After one final observation, the experiment is terminated and the tumor tissue is isolated and weighed directly.

[0125] As a result, 1 × 10 7 High dose and 1 × 10 6 The tumor volume in the medium-dose 1-2C TCR-T cell treatment group was significantly smaller than that in the negative control group (T-test, p<0.01) (Figure 9C). 5 There was no significant difference in tumor volume between the low-dose group and the negative control group (T-test, p>0.05). At the end of the experiment, tumor weights in each group were analyzed, and the results showed that tumor weights in the high-dose group were significantly lower than those in the negative control group (Figure 9A). The results of this example demonstrate that 1-2C TCR-T cells can effectively suppress tumor growth, and the tumor-suppressing activity exhibited a significant dose-dependent effect on TCR-T cell quantity, making them potentially valuable for tumor treatment (Figure 9).

Claims

1. A T cell receptor (TCR) or an antigen-binding fragment thereof, the TCR comprises an α chain variable region and a β chain variable region; The TCR or antigen-binding fragment thereof is KRAS-G12V ８－１６ (VVGAVGVGK) epitope and HLA-A11 complex, or KRAS-G12C ８－１６ (VVGACGVGK) epitope complex with HLA-A11, or KRAS-G12V ８－１６ A T cell receptor (TCR) or antigen-binding fragment thereof capable of binding to a complex of the (VVGAVGVGK) epitope and HLA-A03, and comprising the following alpha chain complementarity determining regions (CDRs) and beta chain complementarity determining regions (CDRs): α chain complementarity determining region CDR1 set forth in SEQ ID NO:3; α chain complementarity determining region CDR2 set forth in SEQ ID NO: 4; α chain complementarity determining region CDR3 set forth in SEQ ID NO:5; β-chain complementarity-determining region CDR1 set forth in SEQ ID NO:8; β chain complementarity determining region CDR2 set forth in SEQ ID NO: 9; and β chain complementarity determining region CDR3 set forth in SEQ ID NO: 10; or α chain complementarity determining region CDR1 set forth in SEQ ID NO: 13; α chain complementarity determining region CDR2 set forth in SEQ ID NO: 14; α chain complementarity determining region CDR3 set forth in SEQ ID NO: 15; β chain complementarity determining region CDR1 set forth in SEQ ID NO: 18; β chain complementarity determining region CDR2 set forth in SEQ ID NO: 19; and β chain complementarity determining region CDR3 set forth in SEQ ID NO:

20.

2. an α chain variable region set forth in SEQ ID NO: 2, and β-chain variable region set forth in SEQ ID NO:7; or an α chain variable region set forth in SEQ ID NO: 12, and a β-chain variable region set forth in SEQ ID NO: 17; 2. The T cell receptor (TCR) or antigen-binding fragment thereof of claim 1, comprising:

3. The TCR or its antigen-binding fragment according to claim 1 or 2, wherein the TCR is a mouse-derived TCR, a human-mouse chimeric TCR, or a humanized TCR.

4. A polynucleotide encoding the TCR or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising 1) SEQ ID NO: 1 and SEQ ID NO: 6, or 2) SEQ ID NO: 11 and SEQ ID NO:

16.

5. A set of polynucleotides encoding a TCR or its antigen-binding fragment described in any one of claims 1 to 3, and consisting of 1) a polynucleotide comprising SEQ ID NO: 1 and a polynucleotide comprising SEQ ID NO: 6, or 2) a polynucleotide comprising SEQ ID NO: 11 and a polynucleotide comprising SEQ ID NO:

16.

6. An expression vector comprising the polynucleotide of claim 4, which is a lentiviral vector.

7. A set of expression vectors consisting of an expression vector containing one of the set of polynucleotides described in claim 5 and an expression vector containing the other of the polynucleotides, each expression vector being a lentiviral vector.

8. A host cell comprising an expression vector according to claim 6 or a set of expression vectors according to claim 7.

9. A method for producing the TCR or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising the steps of: 1) culturing the host cell of claim 8; and 2) recovering the TCR or antigen-binding fragment thereof according to any one of claims 1 to 3 from the host cell or its culture medium.

10. A pharmaceutical composition comprising the TCR or antigen-binding fragment thereof according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

11. Use of the TCR or antigen-binding fragment thereof according to any one of claims 1 to 3 in the manufacture of a medicament for increasing the level of IFN-γ cytokine secreted from T cells.

12. The use described in claim 11, wherein the pharmaceutical is a protein-based pharmaceutical, an ADC pharmaceutical, or a pharmaceutical combining a TCR and an antigen.

13. The TCR or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein KRAS-G12V ８－１６ (VVGAVGVGK) or KRAS-G12C ８－１６ (VVGACGVGK) mutation in the manufacture of a reagent for detecting tumor cells expressing the mutation, or for the manufacture of a reagent for detecting or diagnosing tumors.

14. The TCR or antigen-binding fragment thereof specifically binds to KRAS-G12V 8-16 (VVGAVGVGK) / HLA-A11, or KRAS-G12C 8-16 (VVGACGVGK) / HLA-A11, or KRAS-G12V 8-16 (VVGAVGVGK) epitope and HLA-A03; or The use according to claim 13, wherein the TCR or antigen-binding fragment thereof specifically binds to HLA-A31, HLA-A33, HLA-A68, or HLA-A30, and specifically binds to a KRAS-G12V 8-16 (VVGAVGVGK) or KRAS-G12C 8-16 (VVGACGVGK) mutant polypeptide.

15. Use of the TCR or antigen-binding fragment thereof according to any one of claims 1 to 3 in the manufacture of an antitumor drug for treating a patient with a tumor having the G12V and G12C mutations of the KRAS gene.

16. The use of claim 15, wherein the tumor is pancreatic cancer, colon cancer, or lung cancer.

17. The use described in claim 15, wherein the tumor is non-small cell lung cancer.

18. The use according to claim 15, wherein the G12V and G12C mutations in the KRAS gene are KRAS-G12V 8-16 (VVGAVGVGK) mutation and KRAS-G12C 8-16 (VVGACGVGK) mutation in the KRAS gene.

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