T cell receptor targeting polypeptide with KRAS g12v mutation and use of t cell receptor

Abstract

Provided in the present invention are a T cell receptor targeting a polypeptide with a KRAS G12V mutation and the use of the T cell receptor. The T cell receptor molecule of the present invention specifically targets the KRAS G12V mutation, and the CDR3 sequence of the α chain variable region of the T cell receptor molecule contains CAVNPNTGNQFYF (SEQ ID NO: 1) or a mutant of SEQ ID NO: 1, and / or the CDR3 sequence of the β chain variable region contains CASSQDYGPQETQYF (SEQ ID NO: 2) or a mutant of SEQ ID NO: 2. Further provided in the present invention are a nucleic acid molecule encoding the T cell receptor molecule, a multivalent complex and dual-targeting protein molecule containing the T cell receptor molecule, a nucleic acid construct, a cell expressing the T cell receptor molecule, etc.

Description

T cell receptor targeting KRAS G12V mutant polypeptide and its use Technical Field

[0001] The present invention relates to a T cell receptor sequence targeting a KRAS G12V mutant polypeptide and its encoding nucleotide sequence. The present invention also relates to a TCR-T cell therapy method developed based on the T cell receptor, as well as a dual-targeted anti-tumor protein drug developed based on the T cell receptor. Background Art

[0002] Adoptive cellular immunotherapy (ACCT) is a newly developed, cutting-edge technology that has achieved unprecedented success in the treatment of hematologic malignancies. However, ACC technology faces numerous challenges in the treatment of solid tumors. Finding suitable targets and developing receptor molecules that specifically bind to these targets could open up new avenues for solid tumor treatment. The KRAS gene has garnered significant attention. KRAS, a key member of the Ras gene family (which includes NRAS, HRAS, and KRAS), is a murine sarcoma viral oncogene located on chromosome 12, approximately 35 kb in length, and encodes the KRAS protein. When KRAS mutates, it continuously binds to GTP, exhibiting tyrosine kinase activity and activating downstream signaling pathways, leading to uncontrolled cell proliferation and tumorigenesis. Studies have found that KRAS mutations are present in approximately 30% of tumors, including 90% of pancreatic cancers, 50% of colon cancers, and 25% of lung cancers.

[0003] KRAS mutations often occur at sites such as glycine 12, glycine 13, and glutamine 61. Mutations at glycine 12 and glycine 13 account for as much as 97%, mainly mutations such as G12C, G12D, G12V, G12R, and G13D. KRAS G12V mutations are expressed in approximately 30% of pancreatic cancer and 10% of colorectal cancer patients or non-small cell lung cancer. Moreover, RAS family members also share G12V hotspot mutations in different cancer types (such as NRAS in melanoma). Although KRAS mutations have been found in many tumors, it has been considered an undruggable target for many years because of the lack of a pocket on the KRAS surface that binds to small molecule inhibitors. Currently, only one KRAS G12C inhibitor, AMG 510, has been approved for marketing for the treatment of patients with advanced non-small cell lung cancer (NSCLC) who have previously received systemic treatment and carry the KRAS G12C mutation. However, KRAS G12C is rarely seen in other cancers (such as pancreatic cancer and colon cancer). Other highly abundant KRAS mutation types, such as G12D and G12V, also urgently need new treatments.

[0004] Targeting the high-abundance mutation of KRAS and combining it with adoptive immune cell therapy is the current breakthrough in this research direction. One of the adoptive immune cell therapies is called T cell receptor engineered T cell therapy (TCR-T), which has obvious advantages. TCR-T therapy utilizes the tumor-killing properties of T cells to transfer tumor-specific TCR genes into T cells, and mediates the specific recognition of tumor antigens by T cells through the receptors expressed by them, ultimately achieving the recognition and killing effect of tumors. TCR-T cell therapy has demonstrated good safety and efficacy in clinical trials at home and abroad for the treatment of refractory recurrent melanoma, synovial sarcoma, multiple myeloma, and lung cancer. Finding TCR receptors with high specificity and strong affinity is a key part and technical fortress of TCR-T technology. Summary of the Invention

[0005] In a first aspect, the present invention provides a T cell receptor (TCR) molecule, which specifically targets the KRAS G12V mutation, and the CDR3 sequence of the α chain variable region contains CAVNPNTGNQFYF (SEQ ID NO: 1) or a mutant of SEQ ID NO: 1, and / or the CDR3 sequence of the β chain variable region contains CASSQDYGPQETQYF (SEQ ID NO: 2) or a mutant of SEQ ID NO: 2.

[0006] A second aspect of the present invention provides a multivalent TCR complex, wherein the multivalent TCR complex comprises two or more TCR molecules according to any embodiment of the present invention.

[0007] The third aspect of the present invention provides a dual-targeting protein molecule that can simultaneously bind to tumor cells and immune cells, wherein the dual-targeting protein molecule includes a TCR molecule targeting the KRAS G12V mutation on the surface of tumor cells as described in any embodiment of the present invention and a single-chain antibody (scFv) for recruiting and redirecting immune cells to the periphery of tumor cells, wherein the signal peptide and transmembrane domain in the α chain variable region and the β chain variable region of the TCR molecule are deleted.

[0008] A fourth aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule or dual-targeting protein molecule according to any embodiment of the present invention or its complementary sequence;

[0009] The fifth aspect of the present invention provides a nucleic acid construct, wherein the nucleic acid construct comprises the nucleic acid molecule according to any embodiment of the present invention.

[0010] A sixth aspect of the present invention provides an isolated cell, wherein:

[0011] (1) containing the nucleic acid construct according to any embodiment of the present invention or a nucleic acid molecule according to any embodiment of the present invention integrated into a chromosome, and / or

[0012] (2) Expressing the TCR molecule described in any embodiment of the present invention or the dual-targeting protein molecule described in any embodiment of the present invention.

[0013] Preferably, the cells are immune effector cells, preferably T cells, NK cells and TIL cells.

[0014] In a seventh aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR molecule, TCR complex, dual-targeting protein molecule, nucleic acid molecule, recombinant expression vector or cell according to any embodiment of the present invention.

[0015] In an eighth aspect, the present invention provides the use of the TCR molecule, TCR complex, dual-targeting protein molecule, nucleic acid molecule, recombinant expression vector or cell according to any embodiment of the present invention in the preparation of a drug for treating or preventing a disease associated with the KRAS G12V mutant antigen in a patient.

[0016] The ninth aspect of the present invention provides a method for treating and / or preventing a disease associated with the KRAS G12V mutant antigen in a patient, comprising the step of adoptively transferring a vector of the present invention or a chromosome in which the nucleic acid molecule of the present invention is integrated, and / or a T cell expressing the TCR molecule described in any embodiment herein to the patient, or comprising the step of administering to the patient a dual-targeting protein molecule described in any embodiment of the present invention or a pharmaceutical composition containing the dual-targeting protein molecule.

[0017] The detailed description of the above aspects of the present invention is as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1: Schematic diagram of the pMSGV1_02-1 TCR vector.

[0019] Figure 2: Preparation of TCR-J reporter cells and in vitro antigen peptide activation detection results of Example 4.

[0020] FIG3 : Binding detection results of 02-1 TCR and polypeptide MHC complex in Example 6.

[0021] FIG4 : Specific detection results of the KRAS mutant antigen polypeptide of Example 7 on 02-1 TCR-T cell activation.

[0022] FIG5 : Sensitivity test results of the KRAS mutant antigen polypeptide of Example 7 on 02-1 TCR-T cell activation.

[0023] FIG6 : Detection results of the activation effect of KRAS mutant tumor cells on 02-1 TCR-T cells in Example 8.

[0024] FIG7 : Detection results of the killing effect of 02-1 TCR-T cells on KRAS mutant tumor cells in Example 9. DETAILED DESCRIPTION

[0025] The present invention has discovered a TCR molecule that specifically targets the KRAS G12V mutant antigen (especially the KRAS G12V mutant antigen shown in SEQ ID NO: 16). The TCR molecule can specifically bind to the KRAS G12V / HLA-A*11:01 complex on the surface of tumor cells, while normal non-cancerous cells are not recognized because they express unmutated wild-type KRAS protein. Therefore, the TCR molecule of the present invention has strong specificity, which reduces the toxic side effects of T cells expressing the TCR molecule after drug formation, does not damage normal non-cancerous cells, and has a wide range of applications in the treatment of tumors (such as pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer and prostate cancer, especially pancreatic cancer), thus completing the present invention.

[0026] The present invention will be described in detail below. It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form a preferred technical solution.

[0027] Definition of terms

[0028] Herein, TCR has the well-known meaning in the art. It is a glycoprotein on the cell membrane surface composed of a heterodimer of α / β or γ / δ chains, and is a characteristic marker on the surface of all T cells. The T cell receptor, together with the constant CD3 molecule, forms the T cell receptor complex. The TCR is the receptor for intracellular antigenic peptides presented by the major histocompatibility complex (MHC). The TCR of most T cells consists of a dimer of α and β peptide chains, while a few T cells have γ and δ peptide chains. Each subunit contains two extracellular domains: a variable region and a constant region. The constant region is located near the cell membrane and is connected to the transmembrane region, while the variable region is responsible for recognizing the peptide / MHC complex. The variable region contains three highly variable complementarity determining regions (CDRs): CDR1, CDR2, and CDR3. The most important CDR3 is responsible for direct binding to the peptide presented by the MHC. The CDR1 of the α and β subunits targets the N-terminus and C-terminus of the peptide, respectively. CDR2 is thought to be involved in MHC recognition. The β subunit has an additional CDR4, which is not usually involved in the recognition of peptide / MHC complexes but is involved in the action of superantigens.

[0029] In this context, the major histocompatibility complex (MHC) is a family of genes present in the genomes of most vertebrates and plays a role in antigen presentation and T cell activation. Human MHC glycoproteins, also known as human leukocyte antigens (HLA), include class I and class II MHC molecules. MHC molecules can present degraded fragments of intracellular proteins. For example, when a cell is infected with a virus, polypeptide fragments from the viral envelope can be presented on the cell surface via MHC molecules, allowing cytotoxic T cells (CD8+) to recognize and specifically kill the infected cell.

[0030] Herein, amino acid residues are described using the following abbreviations: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), valine (Val or V). In addition, in this specification, the amino acid sequence of a peptide is described in accordance with conventional methods with the amino terminus (hereinafter referred to as the N terminus) on the left and the carboxyl terminus (hereinafter referred to as the C terminus) on the right.

[0031] The term "targeting KRAS G12V mutant T cell receptor (KRAS G12V "-reactive T Cell Receptor)" is defined herein as a TCR molecule that is capable of binding to a KRAS G12V mutant polypeptide / MHC complex, thereby inducing T cell toxicity. In particular, the KRAS G12V mutant polypeptide includes but is not limited to the amino acid sequence shown in SEQ ID NO: 16, and the MHC is HLA-A*11:01.

[0032] The term "exogenous T cell receptor" (exogenous TCR) is defined herein as a recombinant TCR expressed in a cell by introducing an exogenous coding sequence. The TCRs targeting the KRAS G12V mutation provided herein are "exogenous T cell receptors" for human T cells that can be expressed in human T cells that naturally express an endogenous TCR that is insufficient to induce a cell or responder cell response to TCR ligand binding.

[0033] Herein, TCR-T cell therapy is the introduction of exogenous TCR genes into ordinary T cells so that the modified T cells can express TCRs that effectively recognize tumor cells, thereby guiding T cells to kill tumor cells. The therapy generally includes the step of administering to the patient T cells that express exogenous TCR genes after modification. The T cells are generally derived from the patient himself. Typically, T cells are obtained from the patient, modified in vitro to express exogenous TCR genes (such as the TCR genes described in any embodiment herein), and then returned to the patient.

[0034] Herein, the dual-targeting protein molecule is a class of artificial protein molecules designed based on the BiTE (Bi-specific T-cell engagers) strategy. One end of the protein is a high-affinity T cell receptor (TCR), which can target the KRAS G12V mutation on the surface of tumor cells; the other end is a single-chain antibody (scFv), which is used to recruit and redirect immune cells to the periphery of tumor cells. An exemplary single-chain antibody can be an anti-CD3 single-chain antibody, and an exemplary immune cell can be a T cell. The TCR first recognizes and binds to the polypeptide / MHC on the surface of the tumor cell. Then, the anti-CD3 antibody fragment recruits and redirects immune cells to the periphery of the tumor cell. In this way, the dual-targeting protein molecule builds a bridge between cancer cells and immune cells, forming an immune synapse, activating immune cells and releasing soluble granules, leading to cancer cell death.

[0035] Herein, sequence identity can be determined using methods well known in the art, for example, BLASTP can be used to determine the sequence identity of two aligned amino acid sequences. "Conservative substitutions" are known in the art as substitutions in which one or more amino acid residues are replaced by one or more amino acid residues having side chain R groups with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of a protein. Examples of groups of amino acids with side chains of similar chemical properties include: 1. aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2. aliphatic hydroxyl side chains: serine and threonine; 3. amide-containing side chains: asparagine and glutamine; 4. aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5. basic side chains: lysine, arginine, and histidine; 6. acidic side chains: aspartic acid and glutamic acid; and 7. sulfur-containing side chains: cysteine ​​and methionine. Amino acids can be classified according to the polarity of their side chain groups as follows: 1. non-polar amino acids (hydrophobic amino acids), including alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine; 2. polar amino acids (hydrophilic amino acids), including polar uncharged (neutral amino acids) such as glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, selenocysteine ​​and pyrrolysine, and polar positively charged amino acids (basic amino acids), including lysine, arginine and histidine; 3. polar negatively charged amino acids (acidic amino acids), including aspartic acid and glutamic acid.

[0036] Herein, immune cells refer to cells involved in or associated with immune responses, generally including lymphocytes, dendritic cells, monocytes / macrophages, granulocytes and mast cells, etc. Exemplary immune cells include T cells, natural killer cells (NK) and tumor infiltrating lymphocytes (TIL), etc., as well as their derived immune cells, such as stem cells such as hematopoietic stem cells (HSC) and induced pluripotent stem cells (iPS), etc.

[0037] T cell receptor (TCR)

[0038] The TCR molecule targeting the KRAS G12V mutation of the present invention is characterized in that the CDR3 sequence of its α chain variable region contains CAVNPNTGNQFYF (SEQ ID NO: 1) or a mutant of SEQ ID NO: 1, and / or the CDR3 sequence of its β chain variable region contains CASSQDYGPQETQYF (SEQ ID NO: 2) or a mutant of SEQ ID NO: 2. Preferably, compared with SEQ ID NO: 1, the mutant of SEQ ID NO: 1 has 1-5 (e.g., 1, 2, or 3) amino acid mutations, or has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, or preferably at least 98% sequence identity, and retains the binding activity of SEQ ID NO: 1 as the CDR3 of the TCR α chain variable region. Preferably, the mutant of SEQ ID NO: 2 has 1-5 (e.g., 1, 2, or 3) amino acid mutations, or has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, or preferably at least 98% sequence identity with SEQ ID NO: 2, and retains the binding activity of SEQ ID NO: 2 as the CDR3 of the TCR β chain variable region. The mutations contained in the mutants of SEQ ID NO: 1 and 2 can be selected from one or more of insertions, deletions, and substitutions. Preferably, the mutations are conservative mutations, such as conservative substitutions.

[0039] In some embodiments, the CDR1 sequence of the TCR α chain variable region of the present invention comprises SSSVPPY (SEQ ID NO: 3) or a mutant thereof, and the CDR2 sequence comprises KYTSAAT (SEQ ID NO: 4) or a mutant thereof. In some embodiments, the CDR1 sequence of the TCR β chain variable region of the present invention comprises LGHDTM (SEQ ID NO: 5) or a mutant thereof, and the CDR2 sequence comprises SYNNKE (SEQ ID NO: 6) or a mutant thereof. Each of the mutants of SEQ ID NOs: 3, 4, 5, and 6 may have one, two, or three amino acid mutations compared to the original sequence, including but not limited to one or more of insertions, deletions, and substitutions, and such mutations do not affect the biological function and activity of these CDR sequences in the TCR molecule. Preferred mutations are conservative mutations, such as conservative substitutions.

[0040] In some embodiments, the CDR1 sequence of the α chain variable region of the TCR molecule of the present invention is SSSVPPY (SEQ ID NO: 3), the CDR2 sequence is KYTSAAT (SEQ ID NO: 4), and the CDR3 sequence is CAVNPNTGNQFYF (SEQ ID NO: 1); and / or, the CDR1 sequence of the β chain variable region is LGHDTM (SEQ ID NO: 5), the CDR2 sequence is SYNNKE (SEQ ID NO: 6), and the CDR3 sequence is CASSQDYGPQETQYF (SEQ ID NO: 2).

[0041] The amino acid sequences of the CDR regions of the TCR molecules of the present invention can be embedded in any suitable framework structure to prepare a chimeric TCR. As long as the framework structure is compatible with the CDR regions of the TCR of the present invention, those skilled in the art can design or synthesize TCR molecules with corresponding functions based on the CDR regions disclosed in the present invention. Therefore, the TCR molecules of the present invention refer to TCR molecules comprising the above-mentioned α and / or β chain CDR region sequences and any suitable framework structure using the CDR region sequences of the present invention.

[0042] In some embodiments, the α chain variable region of a TCR molecule of the present invention comprises the amino acid sequence set forth in SEQ ID NO: 7, or comprises an amino acid sequence having one or more mutations compared to the amino acid sequence set forth in SEQ ID NO: 7, or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, preferably at least 95%, and more preferably at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7, or consists of said amino acid sequence. The number of mutated amino acid residues can be, for example, 1-15, such as 1-10, or 1-5 mutations; the mutation can be selected from one or more of insertions, deletions, and substitutions. The mutation can occur within any domain of SEQ ID NO: 7, including within its CDR and / or FR regions. In some embodiments, the mutation does not occur within the CDR1, CDR2, and CDR3 sequences contained in SEQ ID NO: 7. In some embodiments, the mutation occurs, for example, within the FR region of SEQ ID NO: 7. Preferably, the mutation is a conservative mutation, such as a conservative substitution.

[0043] In some embodiments, the beta chain variable region of a TCR molecule of the present invention comprises the amino acid sequence set forth in SEQ ID NO: 8, or comprises an amino acid sequence having one or more mutations compared to the amino acid sequence set forth in SEQ ID NO: 8, or comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, or consists of said amino acid sequence. The number of mutated amino acid residues can be, for example, 1-15, such as 1-10, or 1-5 mutations; the mutation can be selected from one or more of insertions, deletions, and substitutions. The mutation can occur within any domain of SEQ ID NO: 8, including within its CDR and / or FR regions. In some embodiments, the mutation does not occur within the sequences of CDR1, CDR2, and CDR3 contained in SEQ ID NO: 8. In some embodiments, the mutation occurs, for example, within the FR region of SEQ ID NO: 8. Preferably, the mutation is a conservative mutation, such as a conservative substitution.

[0044] It should be understood that the mutants of the TCR molecules of the present invention (i.e., mutants containing the α chain variable region and / or mutants containing the β chain variable region as described above) still retain the biological activity of the TCR molecules containing SEQ ID NO: 1 and SEQ ID NO: 2 (especially TCR molecules containing SEQ ID NO: 7 and 8) that depends on HLA-A*11:01 to specifically bind to the KRAS G12V mutation (especially the polypeptide shown in SEQ ID NO: 16).

[0045] In some embodiments, the TCR molecule of the present invention is a dimer composed of α and β chains, wherein the α chain comprises a variable region and a constant region, wherein the CDR1 sequence of the α chain variable region is SSSVPPY (SEQ ID NO: 3), the CDR2 sequence is KYTSAAT (SEQ ID NO: 4), and the CDR3 sequence is CAVNPNTGNQFYF (SEQ ID NO: 1), and the CDR1 sequence of the β chain variable region is LGHDTM (SEQ ID NO: 5), the CDR2 sequence is SYNNKE (SEQ ID NO: 6), and the CDR3 sequence is CASSQDYGPQETQYF (SEQ ID NO: 2). In some embodiments, the α chain variable region comprises the amino acid sequence of SEQ ID NO: 7 or a mutant sequence described above, and the β chain variable region comprises the amino acid sequence of SEQ ID NO: 8 or a mutant sequence described above. To further stabilize the formation of the TCR α and β chain dimer, a disulfide bond can be introduced between the α and β chains to form a dimer.

[0046] In some embodiments, the constant region of the TCR molecule of the present invention is a human constant region. The amino acid sequence of the human constant region can be obtained from a public database.

[0047] Studies have shown that replacing the constant region of a human TCR with the constant region of a mouse TCR can effectively prevent the rearrangement of exogenous T cell receptors with the body's own TCR, which can lead to off-target or even incorrect binding to the wrong target. Therefore, in some embodiments, the TCR molecules of the present invention contain a mouse α constant region and a mouse β constant region. The amino acid sequence of an exemplary mouse α constant region is shown in SEQ ID NO: 9, and the amino acid sequence of the β constant region is shown in SEQ ID NO: 10.

[0048] In some embodiments, the TCR of the present invention is provided in the form of a multivalent complex. The multivalent TCR complex of the present invention comprises a multimer formed by the association of two, three, four or more TCR molecules of the present invention.

[0049] Nucleic acid molecules

[0050] The present invention provides nucleic acid molecules encoding the α chain variable region, β chain variable region, α chain, β chain and TCR molecule described in any of the embodiments described herein.

[0051] The nucleotide sequence of the nucleic acid molecule of the present invention can be single-stranded or double-stranded, can be RNA or DNA, and may or may not contain introns. An exemplary sequence encoding the α chain variable region nucleic acid molecule of the present invention is shown in SEQ ID NO: 11. An exemplary polynucleotide sequence encoding the β chain variable region of the present invention is shown in SEQ ID NO: 12.

[0052] In some embodiments, the TCR molecule of the present invention comprises a human variable region and a mouse constant region, wherein the nucleic acid coding sequence of the α constant region of the mouse constant region may be as shown in SEQ ID NO: 17, and the nucleic acid coding sequence of the β constant region may be as shown in SEQ ID NO: 18.

[0053] It should be understood that due to the degeneracy of the genetic code, different nucleotide sequences can encode the same polypeptide. Therefore, the nucleic acid sequences encoding the α chain variable region, β chain variable region, α chain, β chain, and TCR molecule of the present invention may be identical to the nucleic acid sequences set forth herein or may be degenerate variants. For example, a "degenerate variant" of the present invention refers to a nucleic acid sequence encoding a protein sequence having SEQ ID NO: 7 or 8, but differing from the sequence of SEQ ID NO: 7 or 8.

[0054] To efficiently express TCRs in T cells, the nucleotide sequences of the present invention can be optimized using codon optimization methods. Different cells utilize different codons, and expression can be increased by altering the codons in the sequence based on the cell type. Codon usage tables for mammalian cells and various other organisms are well known to those skilled in the art.

[0055] The full-length sequence of the nucleic acid molecule of the present invention or its fragments can generally be obtained by, but not limited to, PCR amplification, recombinant methods, or artificial synthesis. Currently, DNA sequences encoding the TCR of the present invention (or its fragments, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors), mRNA, or cells known in the art. The DNA or mRNA can be either the coding strand or the non-coding strand.

[0056] Nucleic acid constructs

[0057] The present invention also includes nucleic acid constructs comprising the nucleic acid molecules described in any of the embodiments herein.

[0058] The nucleic acid construct herein can be an expression cassette comprising an operably linked promoter sequence, a nucleic acid molecule as described in any embodiment herein, and a poly A tail. The expression cassette can also contain other regulatory elements, such as enhancers, operably linked to the above elements.

[0059] In some embodiments, the nucleic acid construct is a vector. Herein, vectors include, but are not limited to, expression vectors and cloning vectors. An expression vector refers to a vector used to express the TCR of the present invention in vivo or in vitro, while a cloning vector refers to a vector used to prepare the nucleic acid molecule of the present invention. Expression vectors typically include expression control elements. Expression control elements are well known in the art and include, but are not limited to, promoters and enhancers. In some embodiments, the vector contains the expression cassette.

[0060] Herein, the expression vector can be a related vector based on a viral delivery system, including but not limited to adenoviral vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors; or a non-viral delivery system vector, including but not limited to transposon-based expression vectors, vectors based on gene editing methods, etc. Ideally, a suitable vector can transfer the TCR nucleic acid of the present invention into a cell, such as a T cell, so that the cell expresses a TCR specific for the KRAS G12V mutant antigen.

[0061] Dual targeting protein molecules

[0062] In some embodiments, the present invention provides a dual-targeting protein molecule that can simultaneously bind to tumor cells and immune cells (especially T cells). The dual-targeting protein molecule includes a TCR molecule that can target the KRAS G12V mutation on the surface of tumor cells as described in any embodiment herein and a single-chain antibody (scFv) for recruiting and redirecting immune cells to the periphery of tumor cells. The TCR molecule may include the α chain variable region and the α chain constant region of the TCR molecule and the β chain variable region and the β chain constant region, and the variable region and the constant region may be directly connected or may be connected by a flexible peptide chain. Typically, the signal peptide and transmembrane domain in the α chain variable region and the β chain variable region are deleted.

[0063] The single-chain antibody has various interesting monoclonal antibodies with the biological functions. An exemplary single-chain antibody can be an anti-CD3 single-chain antibody.

[0064] Typically, in this dual-targeting protein molecule, the α chain and β chain of the TCR molecule form a heterodimer, and the scFv is connected to the N-terminus of the β chain variable region of the TCR molecule. The scFv and the N-terminus of the β chain variable region can be directly connected or connected through a flexible peptide chain.

[0065] Herein, a flexible peptide chain can be any peptide chain without secondary structure. Suitable flexible peptide chains (linkers) are well known in the art and typically contain G and S. Exemplary flexible linkers include, but are not limited to, sequences of 2-30, such as 3-20 or 3-10 amino acid residues containing or consisting of G and S. The amino acid sequence of an exemplary linker is shown in SEQ ID NO: 19.

[0066] In some embodiments, to obtain a stable dual-targeting protein molecule, cysteine ​​mutations can be performed at appropriate sites in the constant regions of the α and β chains to introduce disulfide bonds to stabilize the dimer structure. It should be noted that the TCR molecules of the present invention can be linked by natural disulfide bonds present in the TCR, regardless of whether the constant regions contain or do not contain the artificial disulfide bonds introduced above.

[0067] cell

[0068] The present invention also relates to host cells genetically engineered using the vectors or nucleic acid molecules of the present invention. The term "host cell" refers to any type of cell that can contain a nucleic acid molecule or vector of the present invention or express a TCR molecule or dual-targeting protein molecule of the present invention. In some embodiments, the host cell is characterized by: containing a vector of the present invention or having a nucleic acid molecule of the present invention integrated into its chromosome, and / or expressing a TCR molecule and / or dual-targeting protein molecule described in any embodiment herein.

[0069] Host cells suitable for expressing the TCR of the present invention include, but are not limited to, prokaryotic cells and eukaryotic cells, such as Escherichia coli, yeast cells, insect cells, Chinese hamster ovary cells (CHO), African green monkey kidney cells (Vero cells), COS cells, HEK293 cells, etc. The host cell is preferably a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a primary T cell.

[0070] In some embodiments, the present invention particularly relates to immune cells, especially T cells, containing vectors or chromosomes of the present invention integrated with nucleic acid molecules of the present invention and / or expressing TCR molecules described in any embodiment herein. T cells can be any type of T cells and can be at any stage of development, including but not limited to: CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD4+T cells, CD8+T cells (e.g., cytotoxic T cells), memory T cells (e.g., central memory T cells and effector memory T cells), initial T cells, etc. More preferably, the T cells can be derived from CD8+T cells isolated from patients.

[0071] In some embodiments, the cells of the present invention can also be other types of immune cells, such as natural killer cells (NK), tumor infiltrating lymphocytes (TIL) and derived immune cells. Gene transfer to NK cells will not result in TCR expression on the cell surface because NK cells do not express CD3 molecules. However, when NK cells are induced to differentiate, or artificially constructed, the expression of CD3 molecules will activate TCR molecule expression in NK cells.

[0072] Pharmaceutical compositions and conjugates

[0073] The present invention also provides a pharmaceutical composition comprising T cells and a pharmaceutically acceptable carrier, wherein the T cells contain a vector expressing the TCR molecule described in any embodiment of the present invention or a nucleic acid molecule encoding the TCR molecule described in any embodiment of the present invention is integrated into their chromosomes, and / or express the TCR molecule described in any embodiment of the present invention.

[0074] In some embodiments, the pharmaceutical composition of the present invention contains the TCR molecule or dual-targeting protein molecule described in any embodiment herein and a pharmaceutically acceptable carrier.

[0075] Herein, a pharmaceutically acceptable carrier can be selected based on the specific active ingredient. For example, a pharmaceutically acceptable carrier in a pharmaceutical composition containing T cells can be any suitable carrier well known in the art for cell therapy. A pharmaceutically acceptable carrier in a pharmaceutical composition containing a TCR molecule or a dual-targeting protein molecule of the present invention can be a pharmaceutically acceptable carrier suitable for protein delivery.

[0076] Herein, the pharmaceutical composition can be administered by any appropriate route, such as parenteral, enteral, inhalation or intranasal.The pharmaceutical composition of the present invention can be prepared by methods well known in the art, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions.

[0077] The effective amount of the active ingredient in the pharmaceutical composition of the present invention, such as the T cell, TCR molecule, or dual-targeting protein molecule, depends on the disease or condition to be treated, the age and condition of the individual to be treated, and can be readily determined by those skilled in the art based on actual circumstances. Generally speaking, a suitable dosage range for the soluble TCR of the present invention can be between 25 ng / kg and 50 μg / kg.

[0078] The pharmaceutical compositions of the present invention can be used for various therapeutic purposes as described below.

[0079] In some embodiments, the present invention provides a coupling agent comprising a TCR molecule as described herein and a therapeutic agent or tracer covalently or otherwise bound to the TCR molecule for the treatment or diagnosis of a disease, particularly a tumor. The bound or coupled therapeutic agent includes, but is not limited to, radionuclides, chemotherapeutic agents, antibody Fc or scFv fragments, and nanoparticles. Tracers for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, magnetic materials for MRI (magnetic resonance imaging), contrast agents for CT (computerized tomography), and enzymes that can detect products.

[0080] Uses and treatment methods

[0081] The present invention also provides the use of the TCR molecules, dual-targeting protein molecules and cells (especially T cells) described in any embodiment herein in the preparation of a medicament for treating or preventing a disease associated with the KRAS G12V mutant antigen in a patient, as well as the TCR molecules, dual-targeting protein molecules and cells (especially T cells) described in any embodiment herein for treating or preventing a disease associated with the KRAS G12V mutant antigen.

[0082] The present invention also relates to a method for treating and / or preventing a disease associated with the KRAS G12V mutant antigen in a patient, comprising the step of adoptively transferring a vector of the present invention or a chromosome in which the nucleic acid molecule of the present invention is integrated, and / or a T cell expressing the TCR molecule described in any embodiment herein to the patient, or comprising the step of administering to the patient a dual-targeting protein molecule described in any embodiment of the present invention or a pharmaceutical composition containing the dual-targeting protein molecule.

[0083] Herein, the disease associated with the KRAS G12V mutant antigen is a tumor or cancer, which may include any one of the following: acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, In some embodiments, the disease is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, or prostate cancer. In some embodiments, the disease is pancreatic cancer.

[0084] Preferably, the patient's tumor cells or cancer cells carry KRAS G12V mutant antigen and HLA-A*11: 01. Preferably, the patient's tumor cells or cancer cells carry KRAS G12V mutant antigen including but not limited to the amino acid sequence shown in SEQ ID NO: 16.

[0085] Treatment can be carried out by isolating T cells from patients or volunteers suffering from diseases associated with the KRAS G12V mutant antigen, and modifying the T cells in vitro so that they contain the vector described in any embodiment herein or have integrated into their genome a TCR molecule capable of expressing any embodiment herein, so as to express the TCR molecule described in any embodiment herein, and then returning these genetically engineered cells to the patient for treatment.

[0086] In some embodiments, the T cells are derived from the patient. Therefore, in these embodiments, the treatment methods of the present invention further include: (1) isolating the patient's T cells, and (2) in vitro modifying the T cells to contain a vector described in any embodiment herein or to integrate into their genome a TCR molecule capable of expressing any embodiment herein, so as to express the TCR molecule described in any embodiment herein.

[0087] The mode of administration, timing, dosage, etc. can be determined by the physician based on the age, weight, general health condition, severity of the cancer being treated, etc. of each individual patient.

[0088] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention can be formulated into pharmaceutical compositions in combination with another pharmaceutically active agent or drug. The other pharmaceutically active agent or drug can be a chemotherapeutic agent, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like. It can also be a monoclonal antibody therapeutic drug, such as a targeted immune checkpoint antibody drug (CTLA-4, PD1, PD-L1 TIGIT LAG3, TIM3, etc.), or an immune regulatory element antibody drug (4-1BB, OX40, GITR, CD40, CD28, ICOS, CD47, etc.). In addition, it also includes other types of tumor therapeutic agents, such as oncolytic viruses and vaccines (including but not limited to mRNA, DNA, protein, protein subunits, cellular components or cells, etc.).

[0089] The following specific examples further illustrate the present invention. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (Molecular Cloning-A Laboratory Manual) (3rd edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise stated.

[0090] Example 1: Determination of TCR gene sequence targeting KRAS G12V mutation

[0091] Functional cell populations were isolated from peripheral blood mononuclear cell (PBMC) samples from patients with KRAS G12V mutation-bearing tumors (from the Department of General Surgery, Jinling Hospital, Affiliated to Nanjing University Medical School). TCR sequences were obtained through single-cell sequencing. After functional validation, the TCR No. 02-1 was shown to be able to specifically bind to the VVGAVGVGK / HLA-A*11:01 complex or the VVVGAVGVGK / HLA-A*11:01 complex. The amino acid sequence and coding sequence of its α variable region are shown in SEQ ID NO:7 and SEQ ID NO:11, respectively, and the amino acid sequence and coding sequence of its β variable region are shown in SEQ ID NO:8 and SEQ ID NO:12, respectively.

[0092] SEQ ID NO: 7

[0093]

[0094] (The sequences of CDR1, CDR2 and CDR3 are marked in bold and underlined, respectively).

[0095] SEQ ID NO: 8

[0096]

[0097] (The sequences of CDR1, CDR2 and CDR3 are marked in bold and underlined, respectively).

[0098] Example 2: Construction of vectors for high expression of TCR molecules

[0099] 1. Carrier information

[0100] The pMSGV1 vector was used to overexpress TCR molecules in T cells. The TCR nucleic acid sequence was optimized using human codons. The α chain variable region encoding sequence is shown in SEQ ID NO:11; the β chain variable region encoding sequence is shown in SEQ ID NO:12. Furthermore, TCR expression in T cells was achieved using a human-mouse hybrid approach. The amino acid sequence of the mouse α constant region is shown in SEQ ID NO:9, and the amino acid sequence of the β constant region is shown in SEQ ID NO:10. The SGSG-P2A sequence was used to concatenate the TCR α and β chains (amino acid sequence SEQ ID NO:20, nucleic acid sequence SEQ ID NO:21). The complete vector structure is shown in Figure 1. After vector construction, sequencing was performed to verify the identity of the vector. Sequencing primers are shown in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25.

[0101] 2. Plasmid Extraction

[0102] After sequencing was correct, the plasmid was extracted and purified using NucleoBond Xtra Maxi (MACHEREY-NAGEL). The concentration of the purified plasmid was calculated by measuring the absorbance at 259 nm using an ultraviolet spectrophotometer and stored at -20°C for subsequent experiments.

[0103] Example 3: Retroviral packaging

[0104] HEK293 cells were transiently transfected with retroviral packaging plasmids to prepare retroviral vectors, infect target cells, and generate TCR-T cells or reporter cells expressing 02-1 TCR. The specific operation process is as follows:

[0105] 1. Day 1: HEK293 cells were digested and cultured at 0.6 × 10 6 Cells were plated at 100 cells / ml, 5 ml of D10 culture medium (DMEM + 10% FBS) was added to the T25 flask, the cells were thoroughly mixed, and cultured at 37°C overnight.

[0106] Day 2: Transfect HEK293 cells when they reach approximately 90% confluency. Prepare the plasmid complex: 3 μg of pMSGV1-02-1 TCR, 1.9 μg of Gag-pol, and 0.75 μg of 10A1, in 300 μl of DMEM. Add 20 μl of EZ Trans Cell Transfection Reagent (Shanghai Liji Biotechnology) to 300 μl of DMEM. Add the PEI solution to the plasmid complex and vortex for 20 seconds. Gently add the mixture sideways to the HEK293 culture flask. Incubate at 37°C for 16 hours. Remove the culture medium and replace with fresh prewarmed culture medium.

[0107] 3. Day 4: 48 hours after transfection, collect the supernatant, filter it with a 0.45 μm filter, and store it in aliquots at -80°C.

[0108] The prepared retrovirus was named 02-1 TCR retrovirus.

[0109] Example 4: Preparation of TCR-J reporter cells and in vitro antigen peptide activation assay

[0110] The GFP reporter gene was knocked into the Nur77 locus of human T lymphocyte leukemia Jurkat cells, and CD8 and HLA-A*11:01 genes were stably transfected to construct a T cell activation reporter cell line (S1-1-1-CD8-A11). The 02-1 TCR retrovirus prepared in Example 3 was used to infect S1-1-1-CD8-A11 reporter cells to prepare 02-1 TCR-J cells, which were activated in vitro with KRAS mutant antigen polypeptides to detect TCR function. The 051 TCR published in CN117264043 was used as a control group. The specific operation process is as follows:

[0111] Preparation of 02-1 TCR-J reporter cells:

[0112] 1. Take S1-1-1-CD8-A11 reporter cells and adjust the cell density to 5×10 5 The cells were seeded into 24-well culture plates at a volume of 1 ml.

[0113] 2. Add 300 μl of 02-1 TCR retrovirus and 0.65 μl of polybrene (Santa Cruz) to each well. Centrifuge at 32°C, 2500 rpm for 90 min.

[0114] 3. After centrifugation, discard 800 μl of supernatant, add 800 μl of fresh R10 medium, and place the culture plate in a 37°C, 5% CO2 incubator for culture.

[0115] Antigen peptide activation function experiment:

[0116] 1. Two days after 02-1 TCR retrovirus infection of S1-1-1-CD8-A11 reporter cells, count the cells, centrifuge an appropriate amount, and resuspend in R10 medium to adjust the cell concentration to 2×10 6 / ml, 100 μl / well was added to a 96-well plate.

[0117] 2. Add KRAS G12V mutant 9mer polypeptide (SEQ ID NO: 15) to a final concentration of 1 μg / ml, mix well, and incubate at 37°C for approximately 4 hours.

[0118] 3. Transfer cells to flow cytometry tubes, wash with PBS, and discard the supernatant.

[0119] 4. Add 100 μl zombie green (BioLegend, 1:500 dilution) dye, incubate at room temperature in the dark for 10 min, wash with PBS, and discard the supernatant.

[0120] 5. Add 100 μl of mTCR antibody (1:300 dilution) and incubate at 4°C in the dark for 30 min. Wash with PBS, resuspend, and detect mTCR and GFP by flow cytometry.

[0121] The experimental results are shown in Figure 2. The results showed that the 02-1 TCR could be successfully constructed and expressed on S1-1-1-CD8-A11 reporter cells. In the presence of the KRAS G12V mutant 9mer peptide, the 02-1 TCR could recognize the KRAS G12V mutant 9mer peptide and activate the reporter cells.

[0122] Example 5: Preparation of 02-1 TCR-T cells

[0123] The 02-1 TCR retrovirus prepared in Example 3 was used to infect T cells from human peripheral blood mononuclear cells (PBMC) to prepare 02-1 TCR-T cells. The specific operation process is as follows:

[0124] 1. PBMC cells were separated using Ficoll separation medium (Tianjin Haoyang) and the cell density was adjusted to 2×10 6 / ml.

[0125] 2. The cells were seeded into culture plates at 1 ml / well, and 50 ng / ml anti-human CD3 antibody (Takara) was added, followed by 300 IU / ml interleukin-2 (Beijing Shuanglu), and the cells were stimulated and cultured for 48 hours before viral infection.

[0126] 3. One day after T cell activation culture, non-tissue treated culture plates were coated with Retronectin (Takara) diluted to a final concentration of 15 μg / ml in PBS. 300 μl was added to each well of a 24-well plate and incubated at 4°C overnight.

[0127] 4. After two days of T cell activation culture, remove the coated 24-well plate, discard the coating solution, add 1 ml of virus solution to each well, and centrifuge at 32°C, 2000g for 2 hours.

[0128] 5. Discard the supernatant and add 5×10 activated T cells to each well of the 24-well plate. 5The cells were cultured in 1 ml T cell culture medium supplemented with 300 IU / ml of IL-2. The cells were centrifuged at 32°C, 1000 g for 10 min.

[0129] 6. After centrifugation, place the culture plate in a 37°C, 5% CO2 incubator.

[0130] 7. After cell infection, add T cell culture medium containing IL-2 100 IU / ml in time to allow cells to expand.

[0131] Example 6: 02-1 Binding of TCR to Peptide MHC Complex

[0132] TCR specifically recognizes the antigen peptide presented by the MHC complex. By staining the 02-1 TCR-T cells prepared by fluorescently labeled peptide MHC tetramers, the binding of 02-1 TCR to the peptide MHC complex can be detected. The specific operation process is as follows:

[0133] 1. Take the prepared 02-1 TCR-T cells and 051 TCR-T cells (control group), wash them once with PBS, and discard the supernatant.

[0134] 2. Add 100 μl of mTCR antibody, CD8 antibody (1:300 dilution), and KRAS G12V mutation 9mer tetramer or KRAS G12V mutation 10mer tetramer (1:200 dilution), mix well, and incubate at 4°C in the dark for 30 min.

[0135] 3. Wash with PBS, resuspend, and detect by flow cytometry. The results are shown in Figure 3.

[0136] The results showed that 02-1 TCR could bind to both KRAS G12V mutant 9mer MHC complex and KRAS G12V mutant 10mer MHC complex.

[0137] Example 7: Activation of 02-1 TCR-T cells by KRAS mutant antigen peptides

[0138] After the TCR on the T cell membrane specifically recognizes the antigen peptide presented by the MHC complex, the T cell can be activated. By detecting the expression level of CD137 (4-1BB) on the T cell surface, the degree of T cell activation can be reflected. By comparing different antigen peptides or different antigen peptide concentrations, the specificity and sensitivity of TCR recognition can be compared. The specific operation process is as follows:

[0139] 1. Take the prepared 02-1 TCR-T cells prepared in Example 6 and the 051 TCR-T cells published in CN117264043 (control group), wash them once with PBS, and discard the supernatant.

[0140] 2. Resuspend the cells in X-VIVO15 medium containing 2% AB serum and adjust the cell concentration to 2×10 6 / ml, 100 μl / well was added to a 96-well plate.

[0141] 3. Four peptides: a KRAS wild-type 9mer peptide (SEQ ID NO: 13), a KRAS wild-type 10mer peptide (SEQ ID NO: 14), a KRAS G12V mutant 9mer peptide (SEQ ID NO: 15), and a KRAS G12V mutant 10mer peptide (SEQ ID NO: 16) were resuspended in X-VIVO 15 medium containing 2% AB serum at a final concentration of 10 ng / ml and added to a 96-well plate containing T cells at 100 μl / well. An anti-CD3 antibody (OKT3) was used as a positive control, and a blank solution containing no antigenic peptide was used as a negative control.

[0142] 4. After thorough mixing, place in a 5% CO2 incubator and incubate at 37°C for approximately 16 hours.

[0143] 5. Flow cytometry detection of mTCR+CD8+41BB+ cells.

[0144] The results are shown in Figure 4. The results show that both the KRAS G12V mutant 9mer and 10mer peptides can activate 02-1 TCR-T cells, while the KRAS wild-type 9mer and 10mer peptides cannot. The 02-1 TCR is specific for the KRAS G12V mutation.

[0145] 6. Four peptides, KRAS wild-type 9mer peptide (SEQ ID NO: 13), KRAS wild-type 10mer peptide (SEQ ID NO: 14), KRAS G12V mutant 9mer peptide (SEQ ID NO: 15), and KRAS G12V mutant 10mer peptide (SEQ ID NO: 16), were diluted in a 10-fold gradient and resuspended in X-VIVO15 medium containing 2% AB serum. 100 μl / well was added to a 96-well plate containing T cells, with a final concentration of 10 3 ng / ml to 10 -6 ng / ml.

[0146] 7. After thorough mixing, place in a 5% CO2 incubator and incubate at 37°C for approximately 16 hours.

[0147] 8. Flow cytometry detection of mTCR+CD8+41BB+ cells.

[0148] The results are shown in Figure 5. The results showed that both KRAS G12V mutant 9mer and 10mer peptides could activate 02-1 TCR-T cells, while KRAS wild-type 9mer and 10mer peptides could not. KRAS G12V mutant 9mer peptide activated 02-1 TCR-T ECs 50 =1.85ng / ml; KRAS G12V mutant 10mer peptide activates 02-1 TCR-T EC 50 =0.18ng / ml.

[0149] Example 8: Activation Effect of KRAS Mutant Tumor Cells on 02-1 TCR-T Cells

[0150] In tumor cells carrying the KRAS G12V mutation, KRAS G12V mutant antigen peptides are presented on the cell surface by MHC molecules and activate TCR-T cells upon contact. Various tumor cells carrying the KRAS G12V mutation were co-incubated with TCR-T cells to assess their activation effects on 02-1 TCR-T cells. The KRAS G12V mutation was stably expressed in the pancreatic cancer cell line PANC-1 using a retroviral vector to construct PANC-1-LUC-GFP-TMG cells. HLA-A*11:01 was stably expressed in SW620, CORL23, and CFPAC1 tumor cells naturally carrying the KRAS G12V mutation using a retroviral vector to construct SW620-LUC-GFP-A11, CORL23-LUC-GFP-A11, and CFPAC1-LUC-GFP-A11, respectively, for use in 02-1 TCR-T cell activation experiments. The specific operational procedures are as follows:

[0151] 1. Take the prepared 02-1 TCR-T cells, count them, take an appropriate amount and centrifuge, resuspend them in X-VIVO medium containing 2% AB serum, and adjust the cell concentration to 2×10 6 / mL, 100 μL / well was added to a 96-well plate.

[0152] 2. Take the target cells, count them, and adjust the concentration to 2×10 5 ~2×10 6 / mL, cells were suspended in X-VIVO medium containing 2% AB serum, 100 μL / well of T cells were added, CD3 antibody was used as a positive control, and blank medium was used as a negative control.

[0153] 3. After thorough mixing, place in a 5% CO2 incubator and incubate at 37°C for approximately 16 hours.

[0154] 4. Flow cytometry detection of mTCR+CD8+41BB+ cells.

[0155] The results are shown in Figure 6. The results showed that PANC-1-LUC-GFP-TMG, SW620-LUC-GFP-A11, CORL23-LUC-GFP-A11, and CFPAC1-LUC-GFP-A11 tumor cells carrying the KRAS G12V mutation could activate 02-1 TCR-T cells. However, PANC-1 tumor cells without the KRAS G12V mutation could not activate 02-1 TCR-T cells. Furthermore, SW620, CORL23, and CFPAC1 tumor cells carrying the KRAS G12V mutation but not HLA-A*11:01 also failed to activate 02-1 TCR-T cells. Activation of 02-1 TCR-T cells is specific for the KRAS G12V mutation and HLA-A*11:01.

[0156] Example 9:02-1 Killing effect of TCR-T cells on KRAS mutant tumor cells

[0157] After TCR-T recognizes the antigen peptide epitope presented by MHC on the surface of tumor cells, it is activated and can release effector cytokines such as IFN-γ, as well as cytotoxic molecules such as perforin and granzyme B, causing the death of tumor cells. The specific operation process is as follows:

[0158] 1. Take the prepared 02-1 TCR-T cells, count them, take an appropriate amount and centrifuge, resuspend them in X-VIVO medium containing 2% AB serum, and adjust the cell concentration to 2×10 6 / mL, 100 μL / well was added to a 96-well plate.

[0159] 2. Target cells were harvested, counted, and the concentration adjusted according to different effector-target ratios (E:Tratio). Cells were suspended in X-VIVO medium containing 2% AB serum, and T cells were added at 100 μL / well. 051 TCR-T cells were used as a control group.

[0160] 3. After thorough mixing, place in a 5% CO2 incubator and incubate at 37°C for approximately 16 hours.

[0161] 4. Add luciferase detection reagent and calculate the tumor cell killing ratio.

[0162] The results are shown in Figure 7. The results showed that tumor cells carrying KRAS G12V mutation, PANC-1-LUC-GFP-TMG, could be killed by 02-1 TCR-T cells at multiple different effector-target ratios.

Claims

1. A T cell receptor (TCR) molecule, characterized in that, The TCR molecule specifically targets the KRAS G12V mutation, and the CDR3 sequence of its alpha chain variable region contains CAVNPNTGNQFYF (SEQ ID NO: 1) or a mutant of SEQ ID NO: 1, and / or the CDR3 sequence of its beta chain variable region contains CASSQDYGPQETQYF (SEQ ID NO: 2) or a mutant of SEQ ID NO: 2; wherein, compared with SEQ ID NO: 1, the mutant of SEQ ID NO: 1 has 1-5 amino acid mutations or has at least 80% sequence identity, and retains the binding activity of SEQ ID NO: 1 as the CDR3 of the TCR alpha chain variable region; compared with SEQ ID NO: 2, the mutant of SEQ ID NO: 2 has 1-5 amino acid mutations or has at least 80% sequence identity, and retains the binding activity of SEQ ID NO: 2 as the CDR3 of the TCR beta chain variable region.

2. The TCR molecule according to claim 1, characterized in that, In the TCR molecule: The CDR1 sequence of the alpha chain variable region contains SSSVPPY (SEQ ID NO: 3) or a mutant thereof, and the CDR2 sequence contains KYTSAAT (SEQ ID NO: 4) or a mutant thereof; and / or The CDR1 sequence of the beta chain variable region contains LGHDTM (SEQ ID NO: 5) or a mutant thereof, and the CDR2 sequence contains SYNNKE (SEQ ID NO: 6) or a mutant thereof.

3. The TCR molecule according to claim 1, wherein The alpha chain variable region of the TCR molecule contains the amino acid sequence shown in SEQ ID NO: 7, or contains an amino acid sequence having one or more mutations compared with the amino acid sequence shown in SEQ ID NO: 7, or consists of the amino acid sequence; and / or The beta chain variable region of the TCR molecule contains the amino acid sequence shown in SEQ ID NO: 8, or contains an amino acid sequence having one or more mutations compared with the amino acid sequence shown in SEQ ID NO: 8, or consists of the amino acid sequence; wherein, the TCR molecule containing the mutation retains the biological activity of specifically targeting the KRAS G12V mutant polypeptide of the TCR molecule containing SEQ ID NO: 7 and SEQ ID NO:

8.

4. The TCR molecule according to any one of claims 1-3, characterized in that, The TCR molecule contains a murine constant region.

5. The TCR molecule according to claim 4, characterized in that, The amino acid sequence of the murine alpha constant region is as shown in SEQ ID NO: 9, and the amino acid sequence of the beta constant region is as shown in SEQ ID NO:

10.

6. A multivalent TCR complex, characterized in that, The multivalent TCR complex contains two or more TCR molecules according to any one of claims 1-5.

7. A dual-targeting protein molecule capable of simultaneously binding to tumor cells and immune cells, characterized in that, The dual-targeting protein molecule includes the TCR molecule targeting the KRAS G12V mutation on the surface of tumor cells according to any one of claims 1-5 and a single-chain antibody (scFv) for recruiting and redirecting immune cells to the periphery of tumor cells, wherein the signal peptides and transmembrane domains in the alpha chain variable region and beta chain variable region of the TCR molecule are deleted.

8. The dual-targeting protein molecule according to claim 7, wherein, The single-chain antibody is a single-chain antibody against CD3.

9. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleic acid sequence encoding the TCR molecule according to any one of claims 1-5 or the dual-targeting protein molecule according to claim 7 or 8, or a complementary sequence thereof.

10. The nucleic acid molecule according to claim 9, wherein The nucleic acid sequence of the nucleic acid molecule is selected from: SEQ ID NO: 11 and SEQ ID NO:

12.

11. A nucleic acid construct, characterized in that, The nucleic acid construct contains the nucleic acid molecule according to claim 9 or 10; Preferably, the nucleic acid construct is a vector, preferably an expression vector; preferably, the vector is a viral vector or a non-viral vector; more preferably, the vector is a retroviral vector.

12. An isolated cell, characterized in that, The cell: (1) contains the nucleic acid construct according to claim 11 or the nucleic acid molecule according to claim 9 or 10 is integrated into the chromosome, and / or (2) expresses the TCR molecule according to any one of claims 1-5 or the dual-targeting protein molecule according to claim 7 or 8; Preferably, the cell is an immune effector cell, preferably a T cell, an NK cell or a TIL cell.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains a pharmaceutically acceptable carrier and the TCR molecule according to any one of claims 1-5, the TCR complex according to claim 6, the dual-targeting protein molecule according to claim 7 or 8, the nucleic acid molecule according to claim 9 or 10, the recombinant expression vector according to claim 11 or the cell according to claim 12.

14. Use of the TCR molecule according to any one of claims 1-5, the TCR complex according to claim 6, the dual-targeting protein molecule according to claim 7 or 8, the nucleic acid molecule according to claim 9 or 10, the recombinant expression vector according to claim 11 or the cell according to claim 12 in the preparation of a drug for treating or preventing a disease associated with the KRAS G12V mutant antigen in a patient.

15. A method for treating or preventing a disease associated with the KRAS G12V mutant antigen in a patient, the method comprising the step of adoptive transfer of T cells containing the expression vector according to claim 11 or the nucleic acid molecule according to claim 9 or 10 integrated into the chromosome, and / or expressing the TCR molecule according to any one of claims 1-5 to the patient, or comprising the step of administering to the patient the dual-targeting protein molecule according to claim 7 or 8 or a pharmaceutical composition containing the dual-targeting protein molecule.

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