WO2025140508A1 - T-cell receptor targeting kras g12v mutant polypeptide and use thereof - Google Patents

Abstract

The present invention provides a T-cell receptor targeting a KRAS G12V mutant polypeptide and a use thereof. A T-cell receptor molecule of the present invention specifically targets KRAS G12V mutation. The CDR3 sequence of the α-chain variable region of the T-cell receptor molecule is a mutant containing CAIPPGGSGDKLTF (SEQ ID NO: 1) or SEQ ID NO: 1, and/or the CDR3 sequence of the β-chain variable region is a mutant containing CASSQNNEQFF (SEQ ID NO: 2) or SEQ ID NO: 2. The present invention also provides a nucleic acid molecule encoding the T-cell receptor molecule, a multivalent complex and a dual-targeting protein molecule containing the T-cell receptor molecule, a nucleic acid construct, a cell expressing the T-cell receptor molecule, and the like.

Description

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

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. And a dual-targeted anti-tumor protein drug developed based on the T cell receptor.

Background Art

Adoptive cellular immunotherapy is a newly developed cutting-edge technology that has achieved unprecedented success in the treatment of hematological tumors. However, there are many difficulties in the treatment of solid tumors with adoptive cellular immunotherapy. Finding suitable targets and developing receptor molecules that specifically bind to these targets can open up new prospects for the treatment of solid tumors. Among them, the KRAS gene has entered the researchers' field of vision. KRAS is one of the main members of the Ras gene family (including NRAS, HRAS and KRAS). It is a murine sarcoma viral oncogene located on chromosome 12, about 35kb long, and encodes KRAS protein. When KRAS mutates, it continuously binds to GTP, exhibits tyrosine kinase activity, activates downstream signaling pathways, and causes uncontrolled cell proliferation and tumorigenesis. Studies have found that KRAS mutations exist in approximately 30% of tumors, including 90% of pancreatic cancer, 50% of colon cancer, and 25% of lung cancer.

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 about 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 regarded as an undruggable target for many years because of the lack of pockets on the surface of KRAS that bind 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 KRAS G12C mutations. However, KRAS G12C rarely appears in other cancers (such as pancreatic cancer, colon cancer, etc.). Other high-abundance KRAS mutation types, such as G12D and G12V, also urgently need to find new treatments.

Targeting KRAS high-abundance mutations and combining it with adoptive immune cell therapy is a 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 uses 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 they express, ultimately achieving the recognition and killing effect of tumors. TCR-T cell therapy has demonstrated good safety and effectiveness 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

The first aspect of 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 CAIPPGGSGDKLTF (SEQ ID NO: 1) or a mutant of SEQ ID NO: 1, and/or the CDR3 sequence of the β chain variable region contains CASSQNNEQFF (SEQ ID NO: 2) or a mutant of SEQ ID NO: 2.

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.

The third aspect of the present invention provides a dual-targeting protein molecule that can simultaneously bind to tumor cells and immune cells, the dual-targeting protein molecule comprising 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.

The fourth aspect of the present invention provides a nucleic acid molecule, which comprises a nucleic acid sequence encoding the TCR molecule or dual-targeting protein molecule described in any embodiment of the present invention or its complementary sequence.

The fifth aspect of the present invention provides a nucleic acid construct, wherein the nucleic acid construct comprises the nucleic acid molecule described in any embodiment of the present invention.

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

(1) containing the nucleic acid construct according to any embodiment of the present invention or a chromosome in which the nucleic acid molecule according to any embodiment of the present invention is integrated, and/or

(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.

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

The seventh aspect of the present invention provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR molecule, TCR complex, dual-targeting protein molecule, nucleic acid molecule, recombinant expression vector or cell described in any embodiment of the present invention.

An eighth aspect of the present invention provides the use of the TCR molecule, TCR complex, dual-targeting protein molecule, nucleic acid molecule, recombinant expression vector or cell described in 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.

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, which comprises 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 of the present invention to the patient, or comprises 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.

The detailed description of the above aspects of the present invention is as follows.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Schematic diagram of the pMSGV1_02-2 TCR vector.

Figure 2: Preparation of TCR-J reporter cells and peptide MHC tetramer staining results.

Figure 3: Results of in vitro antigen peptide activation assay of TCR-J reporter cells.

Figure 4: 02-2 Binding results of TCR and polypeptide MHC complex.

Figure 5: Specific detection results of KRAS mutant antigen peptide on 02-2 TCR-T cell activation.

Figure 6: Sensitivity test results of KRAS mutant antigen peptide on 02-2 TCR-T cell activation.

Figure 7: Detection results of the activation effect of KRAS mutant tumor cells on 02-2 TCR-T cells.

Figure 8:02-2 The killing effect of TCR-T cells on KRAS mutant tumor cells.

DETAILED DESCRIPTION

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, and 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, so that the toxic side effects of T cells expressing the TCR molecule after drug formation are reduced, and normal non-cancerous cells are not damaged. It 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), thereby completing the present invention.

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 embodiments) can be combined with each other to form a preferred technical solution.

Definition of terms

In this article, TCR has the well-known meaning in the art. It is a glycoprotein on the cell membrane surface that exists in the form of a heterodimer of α chain/β chain or γ chain/δ chain. It is a characteristic marker on the surface of all T cells. T cell receptors form a T cell receptor complex together with the constant CD3 molecule. TCR is a receptor after the major histocompatibility complex (MHC) presents intracellular antigen peptides. The TCR of most T cells is a dimer composed of α and β peptide chains, and the TCR of a few T cells is composed of γ and δ peptide chains. Each subunit contains two extracellular domains: a variable region and a constant region. The constant region is close to the cell membrane and is connected to the transmembrane region, while the variable region is responsible for recognizing the polypeptide/MHC complex. The variable region contains three highly variable complementarity determining regions (CDRs), namely CDR1, CDR2 and CDR3. The most important CDR3 is responsible for directly binding to the polypeptide presented by MHC. The CDR1 of the α subunit and the β subunit act on the N-terminus and C-terminus of the polypeptide, respectively. CDR2 is thought to be involved in the recognition of MHC. 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.

In this article, major histocompatibility complex (MHC) is a gene family present in most vertebrate genomes and is an antigen presentation and T cell activation molecule. Among them, human MHC glycoprotein is also called human leukocyte antigen (HLA). MHC molecules include class I and class II MHC molecules. MHC molecules can present degradation fragments of intracellular proteins. For example, after the cell is infected by a virus, the polypeptide fragments of the corresponding viral outer membrane can be presented to the cell surface through MHC molecules for cytotoxic T cells (CD8+cytotoxic T cells) to recognize and specifically kill cells infected by the virus.

Herein, amino acid residues are described by 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 the present specification, the amino acid sequence of a peptide is described in a conventional manner with the amino terminal (hereinafter referred to as N-terminus) located on the left and the carboxyl terminal (hereinafter referred to as C-terminus) located on the right.

The term "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.

The term "exogenous TCR" is defined herein as a recombinant TCR expressed in a cell by introducing an exogenous coding sequence. The TCR targeting the KRAS G12V mutation provided herein is an "exogenous T cell receptor" for human T cells, which can be expressed in human T cells whose natural expression of endogenous TCR is insufficient to induce a cell or responder cell response to TCR ligand binding.

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 giving the patient T cells that express exogenous TCR genes after modification. The T cells are usually from the patient himself. Typically, the T cells are obtained from the patient, and the T cells are modified in vitro to express exogenous TCR genes (such as the TCR genes described in any embodiment of the present invention), and then returned to the patient.

In this article, 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 vicinity 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. TCR first recognizes and binds to the peptide/MHC on the surface of tumor cells. Then, the anti-CD3 antibody fragment recruits and redirects immune cells to the vicinity of tumor cells. 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 particles, leading to cancer cell death.

Herein, sequence identity can be determined by 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 a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of the 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 into: 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.

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.

T cell receptor (TCR)

The characteristics of the TCR molecule targeting KRAS G12V mutation of the present invention include that the CDR3 sequence of its α chain variable region contains CAIPPGGSGDKLTF (SEQ ID NO: 1) or a mutant of SEQ ID NO: 1, and/or the CDR3 sequence of the β chain variable region contains CASSQNNEQFF (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 (such as 1, 2 or 3) amino acid mutations, or has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98% sequence identity, and retains SEQ ID NO: 1 as the binding activity of CDR3 of the TCR α chain variable region. Preferably, compared with SEQ ID NO: 2, the mutant of SEQ ID NO: 2 has 1-5 (such as 1, 2 or 3) amino acid mutations, or has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98% sequence identity, and retains the binding activity of SEQ ID NO: 2 as CDR3 of the variable region of the TCR β chain. The mutations contained in the respective 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.

In some embodiments, the CDR1 sequence of the variable region of the TCR α chain of the present invention comprises KTSINN (SEQ ID NO: 3) or a mutant thereof, and the CDR2 sequence comprises LIRSNE (SEQ ID NO: 4) or a mutant thereof. In some embodiments, the CDR1 sequence of the variable region of the TCR β chain of the present invention comprises SEHNRL (SEQ ID NO: 5) or a mutant thereof, and the CDR2 sequence comprises FQNEAQ (SEQ ID NO: 6) or a mutant thereof. Each mutant of SEQ ID NO: 3, 4, 5 and 6 may have 1, 2 or 3 amino acid mutations compared to itself, including but not limited to one or more of insertions, deletions and substitutions, and the mutation does not affect the biological functions and activities of these CDR sequences in the TCR molecule. Preferred mutations are conservative mutations, such as conservative substitutions.

In some embodiments, the CDR1 sequence of the α chain variable region of the TCR molecule of the present invention is KTSINN (SEQ ID NO: 3), the CDR2 sequence is LIRSNE (SEQ ID NO: 4), and the CDR3 sequence is CAIPPGGSGDKLTF (SEQ ID NO: 1); and/or, the CDR1 sequence of the β chain variable region is SEHNRL (SEQ ID NO: 5), the CDR2 sequence is FQNEAQ (SEQ ID NO: 6), and the CDR3 sequence is CASSQNNEQFF (SEQ ID NO: 2).

The CDR region amino acid sequence of the TCR molecule 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 region of the TCR of the present invention, those skilled in the art can design or synthesize a TCR molecule with corresponding functions based on the CDR region disclosed in the present invention. Therefore, the TCR molecule of the present invention refers to a TCR molecule comprising the above-mentioned α and/or β chain CDR region sequence and any suitable framework structure using the CDR region sequence of the present invention.

In some embodiments, the α chain variable region of the TCR molecule of the present invention 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 contains an amino acid sequence having at least 80%, at least 85%, at least 90%, preferably at least 95%, more preferably at least 98% sequence identity compared with the amino acid sequence shown in SEQ ID NO: 7, or consists of the amino acid sequence. The number of the 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 insertion, deletion and substitution. The mutation can occur in any domain of SEQ ID NO: 7, including in its CDR and/or FR region. In some embodiments, the mutation does not occur in the sequences of CDR1, CDR2 and CDR3 contained in SEQ ID NO: 7. In some embodiments, the mutation occurs in, for example, the FR region of SEQ ID NO: 7. Preferably, the mutation is a conservative mutation, such as a conservative substitution.

In some embodiments, the variable region of the β chain of the TCR molecule of the present invention 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 contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% sequence identity compared with the amino acid sequence shown in SEQ ID NO: 8, or consists of the amino acid sequence. The number of the 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 insertion, deletion and substitution. The mutation may occur in any domain of SEQ ID NO: 8, including in its CDR and/or FR region. In some embodiments, the mutation does not occur in the sequences of CDR1, CDR2 and CDR3 contained in SEQ ID NO: 8. In some embodiments, the mutation occurs in, for example, the FR region of SEQ ID NO: 8. Preferably, the mutation is a conservative mutation, such as a conservative substitution.

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).

In some embodiments, the TCR molecule of the present invention is a dimer composed of α and β chains, the α chain comprises a variable region and a constant region, the CDR1 sequence of the α chain variable region is KTSINN (SEQ ID NO: 3), the CDR2 sequence is LIRSNE (SEQ ID NO: 4), and the CDR3 sequence is CAIPPGGSGDKLTF (SEQ ID NO: 1), the CDR1 sequence of the β chain variable region is SEHNRL (SEQ ID NO: 5), the CDR2 sequence is FQNEAQ (SEQ ID NO: 6), and the CDR3 sequence is CASSQNNEQFF (SEQ ID NO: 2). In some embodiments, the α chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7 or the mutant sequence described above, and the β chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8 or the mutant sequence described above. In order to further stabilize the formation of TCRα and β chain dimers, a disulfide bond can be introduced between the α chain and the β chain to form a dimer.

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.

Studies have shown that replacing the constant region of human TCR with the constant region of mouse TCR can effectively avoid the rearrangement of exogenous T cell receptors with self-TCR in the human body, resulting in off-target or even binding to the wrong target. Therefore, in some embodiments, the TCR molecule of the present invention contains the α constant region and β constant region of mouse. The amino acid sequence of the 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.

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 polymer formed by combining two, three, four or more TCR molecules of the present invention.

Nucleic acid molecules

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.

The nucleotide sequence of the nucleic acid molecule of the present invention may be single-stranded or double-stranded, the nucleic acid molecule may 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.

In some embodiments, the TCR molecule of the present invention comprises a human variable region and a mouse constant region, in which the nucleic acid coding sequence of the α 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.

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 sequence encoding the α chain variable region, β chain variable region, α chain, β chain and TCR molecule of the present invention may be the same as the nucleic acid sequence shown in the present invention or a degenerate variant. Taking one of the examples in the present invention as an example, a "degenerate variant" refers to a nucleic acid sequence that encodes a protein sequence having SEQ ID NO: 7 or 8, but is different from the sequence of SEQ ID NO: 7 or 8.

In order to efficiently express TCR in T cells, the nucleotide sequence of the present invention can be optimized by codon optimization methods. Different cells have different specific codon usage, and the codons in the sequence can be changed to increase the expression amount according to the type of cell. The codon selection tables of mammalian cells and various other organisms are well known to those skilled in the art.

The full-length sequence of the nucleic acid molecule of the present invention or its fragment can usually be obtained by, but not limited to, PCR amplification, recombination or artificial synthesis. At present, the DNA sequence encoding the TCR of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The 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 a coding strand or a non-coding strand.

Nucleic acid constructs

The present invention also includes a nucleic acid construct comprising a nucleic acid molecule as described in any of the embodiments herein.

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

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, and a cloning vector refers to a vector used in the preparation of the nucleic acid molecule of the present invention. An expression vector generally includes an expression control element. 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.

Herein, the expression vector can be a related vector based on a viral delivery system, including but not limited to adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentivirus vectors and baculovirus 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.

Dual targeting protein molecules

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 α chain constant region of the TCR molecule and the β chain variable region and 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.

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.

Typically, in the 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 can be connected through a flexible peptide chain.

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

In some embodiments, in order 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 constant region contains or does not contain the artificial disulfide bonds introduced as described above, and the TCR molecules of the present invention can be connected by the natural disulfide bonds present in the TCR.

cell

The present invention also relates to host cells produced by genetic engineering using the vector or nucleic acid molecule of the present invention. The term "host cell" refers to any type of cell that can contain the nucleic acid molecule or vector of the present invention or express the TCR molecule or dual-targeting protein molecule of the present invention. In some embodiments, the characteristics of the host cell include: containing the vector of the present invention or the chromosome in which the nucleic acid molecule of the present invention is integrated, and/or expressing the TCR molecule and/or dual-targeting protein molecule described in any embodiment of the present invention.

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, HEK29 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.

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.

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 start the expression of TCR molecules in NK cells.

Pharmaceutical compositions and conjugates

The present invention also provides a pharmaceutical composition comprising a T cell and a pharmaceutically acceptable carrier, wherein the T cell contains 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 its chromosome, and/or expresses the TCR molecule described in any embodiment of the present invention.

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.

Herein, a pharmaceutically acceptable carrier can be selected according to the specific active ingredient. For example, a pharmaceutically acceptable carrier in a pharmaceutical composition containing T cells can be various suitable carriers suitable for cell therapy well known in the art. 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.

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.

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, etc., and can be easily determined by a person skilled in the art according to actual conditions. In general, the suitable dosage range of the soluble TCR of the present invention can be between 25 ng/kg and 50 μg/kg.

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

In some embodiments, the present invention provides a coupling agent, which contains 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, especially a tumor. The combined 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 x-ray tomography), and enzymes that can detect products.

Uses and treatment methods

The present invention also provides the use of the TCR molecules, dual-targeting protein molecules and cells (especially T cells) described in any embodiment of the present invention in the preparation of drugs for treating or preventing diseases associated with KRAS G12V mutant antigen in patients, as well as the TCR molecules, dual-targeting protein molecules and cells (especially T cells) described in any embodiment of the present invention for treating or preventing diseases associated with KRAS G12V mutant antigen.

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, which comprises the step of adoptively transferring a vector containing 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 of the present invention to the patient, or comprises 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.

In this article, the disease associated with the KRAS G12V mutant antigen is a tumor or cancer, which may include any 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 cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer. In some embodiments, the disease is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureter cancer, and bladder cancer. Preferred cancers are pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, or prostate cancer. In some embodiments, the disease is pancreatic cancer.

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

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

In some embodiments, the T cells are derived from the patient. Therefore, in these embodiments, the treatment method of the present invention further comprises: (1) isolating the patient's T cells, and (2) transforming the T cells in vitro so that they contain the vector described in any embodiment of the present invention or the genome is integrated with a TCR molecule capable of expressing any embodiment of the present invention, so as to express the TCR molecule described in any embodiment of the present invention.

The method, timing, and dosage of administration can be determined by the physician based on the age, weight, general health, and severity of the cancer being treated of each individual patient.

The TCR, polypeptide, protein, nucleic acid, recombinant expression vector and host cell (including its population) of the present invention can be formulated into a pharmaceutical composition in combination with another pharmaceutically active agent or drug. Another 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, etc. 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, proteins, protein subunits, cell components or cells, etc.).

The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as the conditions 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 channels unless otherwise specified.

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

Functional cell populations were sorted from peripheral blood mononuclear cell (PBMC) samples of tumor patients carrying KRAS G12V mutation (from the Department of General Surgery, Jinling Hospital Affiliated to Nanjing University Medical School), and TCR sequences were obtained by single-cell sequencing. After functional verification, TCR No. 02-2 was able to specifically bind to 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.

SEQ ID NO: 7

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

SEQ ID NO：8

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

Example 2: Construction of vector for high expression of TCR molecules

1. Carrier information

The pMSGV1 vector was used to overexpress TCR molecules in T cells. The TCR nucleic acid sequence was optimized using human codons. The coding sequence of the α chain variable region is shown in SEQ ID NO: 11; the coding sequence of the β chain variable region is shown in SEQ ID NO: 12. In addition, the expression of TCR in T cells was completed by human-mouse hybridization. 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 in series between the TCR α chain and β chain (amino acid sequence SEQ ID NO: 20, nucleic acid sequence SEQ ID NO: 21). The complete vector structure is shown in Figure 1. After the vector construction was completed, sequencing was performed for identification. The sequencing primers are shown in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25.

2. Plasmid Extraction

After sequencing was correct, NucleoBond Xtra Maxi (MACHEREY-NAGEL) was used to extract and purify the plasmid. The concentration of the purified plasmid was calculated by measuring the 259nm light absorption using an ultraviolet spectrophotometer and stored at -20°C for subsequent experiments.

Example 3: Retroviral packaging

HEK293 cells were transiently transfected with retroviral packaging plasmids to prepare retroviral vectors, infect target cells, and prepare TCR-T cells or reporter cells expressing 02-2 TCR. The specific operation process is as follows:

1. Day 1: HEK 293 cells were digested and plated at 0.6×10 ⁶ cells/ml. 5 ml of D10 medium (DMEM+10% FBS) was added to a T25 flask, the cells were thoroughly mixed, and cultured at 37°C overnight.

2. Day 2: HEK 293 cells were transfected when the confluence reached about 90%. Prepare plasmid complexes. The amounts of various plasmids were: pMSGV1-02-2 TCR 3μg, Gag-pol 1.9μg, 10A1 0.75μg, add 300μl DMEM. EZ Trans Cell Transfection Reagent (Shanghai Liji Biotechnology) 20μl, add 300μl DMEM. Add PEI solution to the plasmid complex and vortex for 20s. Gently add the mixture along the edge to the HEK 293 culture flask, culture at 37℃ for 16h, remove the culture medium, and re-add preheated fresh culture medium.

3. Day 4: 48 hours after transfection, collect the supernatant and filter it with a 0.45 μm filter, then divide it into aliquots and store it at -80°C.

The prepared retrovirus was named 02-2 TCR retrovirus.

Example 4: Preparation of TCR-J cells and in vitro antigen peptide activation detection

The GFP reporter gene was knocked into the Nur77 locus of human T lymphoid 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 S1-1-1-CD8-A11 reporter cells were infected with 02-2 TCR retrovirus to prepare 02-2 TCR-J cells. TCR specifically recognizes antigenic peptides presented by MHC complexes. The 02-2 TCR-J cells prepared by staining with fluorescently labeled peptide MHC tetramers can detect the binding of 02-2 TCR to peptide MHC complexes. The 051 TCR published in patent CN 117264043 was used as the control group. The specific operation process is as follows:

Preparation of 02-2 TCR-J reporter cells:

1. Take S1-1-1-CD8-A11 reporter cells and adjust the cell density to 5×10 ⁵ /ml using R10 medium (RPMI1640 + 10% FBS). Inoculate 1 ml of the cells into a 24-well culture plate.

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

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% CO ₂ incubator for culture.

Peptide MHC tetramer staining experiment:

1. Take the prepared 02-2 TCR-J cells and 051 TCR-J cells (control group), wash them once with PBS, and discard the supernatant.

2. Add 100 μl 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.

3. Wash with PBS, resuspend and detect by flow cytometry.

The results are shown in Figure 2. The results showed that 02-2 TCR could recognize KRAS G12V mutant 10mer peptide, but could not recognize KRAS G12V mutant 9mer MHC peptide.

Antigen peptide activation function experiment:

1.02-2 Two days after the TCR retrovirus infected S1-1-1-CD8-A11 reporter cells, count them, centrifuge an appropriate amount, resuspend in R10 medium, adjust the cell concentration to 2×10 ⁶ /ml, and add 100 μl/well to a 96-well plate.

2. Add 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), or KRAS G12V mutant 10mer peptide (SEQ ID NO: 16) to a final concentration of 1 μg/ml, mix well, and incubate at 37°C for about 4 hours.

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

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.

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

The experimental results are shown in Figure 3. The results showed that 02-2 TCR can recognize KRAS G12V mutant 10mer peptide and activate reporter cells, but does not recognize KRAS G12V mutant 9mer peptide, as well as KRAS wild-type 9mer and 10mer peptides.

Example 5: 02-2 Preparation of TCR-T cells

T cells from human peripheral blood mononuclear cells (PBMC) were infected with 02-2 TCR retrovirus to prepare 02-2 TCR-T cells. The specific operation process is as follows:

1. The obtained PBMC cells were separated with Ficoll separation solution (Tianjin Haoyang), and the cell density was adjusted to 2×10 ⁶ /ml with X-VIVO (LONZA) culture medium containing 5% AB serum.

2. Inoculate the cells into the culture plate at 1 ml/well, add 50 ng/ml anti-human CD3 antibody (Takara), then add 300 IU/ml interleukin 2 (Beijing Shuanglu), and stimulate viral infection after 48 hours of culture.

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 per well of a 24-well plate, and incubated at 4°C overnight for use.

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

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

6. After centrifugation, place the culture plate in a 37°C, 5% CO ₂ incubator.

7. After cell infection, add T cell culture medium containing IL-2 100IU/ml to allow cells to proliferate.

Example 6: 02-2 Binding of TCR to peptide MHC complex

TCR specifically recognizes the antigen peptide presented by the MHC complex. The binding of 02-2 TCR to the peptide MHC complex can be detected by preparing 02-2 TCR-T cells by staining with fluorescently labeled peptide MHC tetramers. The specific operation process is as follows:

1. Take the prepared 02-2 TCR-T cells and 051 TCR-T cells (control group), wash them once with PBS, and discard the supernatant.

2. Add 100 μl 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.

3. Wash with PBS, resuspend and detect by flow cytometry.

The results are shown in Figure 4. The results show that 02-2 TCR can bind to the KRAS G12V mutant 10mer MHC complex, but cannot bind to the KRAS G12V mutant 9mer MHC complex. 02-2 TCR only specifically recognizes the KRAS G12V mutant 10mer peptide (VVVGAVGVGK) presented by the HLA-A*11:01 complex.

Example 7: Activation of 02-2 TCR-T cells by KRAS mutant antigen peptide

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:

1. Take the prepared 02-2 TCR-T cells and 051TCR-T cells (control group), wash them once with PBS, and discard the supernatant.

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

3.4 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), KRAS G12V mutant 10mer peptide (SEQ ID NO: 16), were resuspended in X-VIVO15 medium containing 2% AB serum at a final concentration of 10 ng/ml, and 100 μl/well was added to a 96-well plate containing T cells. Anti-CD3 antibody (OKT3) was used as a positive control, and blank solvent without antigenic peptide was used as a negative control.

4. After thorough mixing, place in a 5% carbon dioxide incubator and incubate at 37°C for about 16 hours.

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

The results are shown in Figure 5. The results showed that 02-2 TCR-T cells can be activated by KRAS G12V 10mer peptide, but not by KRAS G12V 9mer peptide. 02-2 TCR has specific recognition of KRAS G12V mutant 10mer peptide.

6. 4 polypeptides, KRAS wild-type 9mer polypeptide (SEQ ID NO: 13), KRAS wild-type 10mer polypeptide (SEQ ID NO: 14), KRAS G12V mutant 9mer polypeptide (SEQ ID NO: 15), KRAS G12V mutant 10mer polypeptide (SEQ ID NO: 16), were diluted in a 10-fold gradient, resuspended in X-VIVO15 medium containing 2% AB serum, and 100 μl/well was added to a 96-well plate containing T cells, with a final concentration of 10 ² ng/ml to 10 ^-6 ng/ml.

7. After thorough mixing, place in a 5% carbon dioxide incubator and incubate at 37°C for about 16 hours.

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

The results are shown in Figure 6. The results show that 02-2 TCR-T cells can be activated by KRAS G12V 10mer peptide, but not by KRAS G12V 9mer peptide. 02-2 TCR has specific recognition of KRAS G12V mutant 10mer peptide. The EC ₅₀ of KRAS G12V mutant 10mer peptide to activate 02-2 TCR-T is 0.31ng/ml.

Example 8: Activation effect of KRAS mutant tumor cells on 02-2 TCR-T cells

In tumor cells carrying KRAS G12V mutation, KRAS G12V mutant antigen peptides are presented to the cell surface by MHC molecules and activate TCR-T cells when in contact with TCR-T cells. Select a variety of tumor cells carrying KRAS G12V mutations to co-incubate with TCR-T cells to detect the activation effect of 02-2 TCR-T cells. Stably express KRAS G12V mutations in the pancreatic cancer cell line PANC-1 using retroviral vectors to construct PANC-1-LUC-GFP-TMG cells. Stably express HLA-A*11:01 in tumor cells SW620, CORL23, and CFPAC1 that naturally carry KRAS G12V mutations using retroviral vectors to construct SW620-LUC-GFP-A11, CORL23-LUC-GFP-A11, and CFPAC1-LUC-GFP-A11, respectively, for the activation experiment of 02-2 TCR-T cells. The specific operation process is as follows:

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

2. Take the target cells, count them, adjust the concentration to 2×10 ⁵ ～2×10 ⁶ /ml, suspend the cells in X-VIVO medium containing 2% AB serum, add T cells at 100 μl/well, CD3 antibody as positive control, and blank medium as negative control.

3. After thorough mixing, place in a 5% carbon dioxide incubator and incubate at 37°C for about 16 hours.

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

The results are shown in Figure 7. The results showed that tumor cells PANC-1-LUC-GFP-TMG, SW620-LUC-GFP-A11, CORL23-LUC-GFP-A11 and CFPAC1-LUC-GFP-A11 carrying KRAS G12V mutations can all activate 02-2 TCR-T cells; while tumor cells PANC-1 without KRAS G12V mutations cannot activate 02-2 TCR-T cells; tumor cells SW620, CORL23 and CFPAC1 carrying KRAS G12V mutations but HLA typing is not HLA-A*11:01 cannot activate 02-2 TCR-T cells. The activation of 02-2 TCR-T is specific to KRAS G12V mutations and HLA-A*11:01.

Example 9: 02-2 The killing effect of TCR-T cells on KRAS mutant tumor cells

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:

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

2. Take the target cells, count them, adjust the concentration according to different effector-target ratios (E:Tratio), suspend the cells in X-VIVO medium containing 2% AB serum, and add T cells at 100 μl/well. 051TCR-T cells were used as the control group.

3. After thorough mixing, place in a 5% carbon dioxide incubator and incubate at 37°C for about 16 hours.

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

The results are shown in Figure 8. The results showed that tumor cells PANC-1-LUC-GFP-TMG carrying KRAS G12V mutation could be killed by 02-2 TCR-T cells at multiple effector-target ratios.

Claims (15)

A T cell receptor (TCR) molecule, characterized in that the TCR molecule specifically targets the KRAS G12V mutation, and the CDR3 sequence of its α chain variable region contains CAIPPGGSGDKLTF (SEQ ID NO: 1) or a mutant of SEQ ID NO: 1, and/or the CDR3 sequence of the β chain variable region contains CASSQNNEQFF (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 α 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 β chain variable region. The TCR molecule according to claim 1, wherein in the TCR molecule: The CDR1 sequence of the α chain variable region contains KTSINN (SEQ ID NO: 3) or a mutant thereof, and the CDR2 sequence contains LIRSNE (SEQ ID NO: 4) or a mutant thereof; and/or The CDR1 sequence of the β chain variable region contains SEHNRL (SEQ ID NO: 5) or its mutant, and the CDR2 sequence contains FQNEAQ (SEQ ID NO: 6) or its mutant. The TCR molecule according to claim 1, characterized in that The α chain variable region of the TCR molecule contains the amino acid sequence shown in SEQ ID NO: 7, or comprises an amino acid sequence having one or more mutations compared to the amino acid sequence shown in SEQ ID NO: 7, or consists of the amino acid sequence; and/or The variable region of the β chain 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 to the amino acid sequence shown in SEQ ID NO: 8, or consists of the amino acid sequence; Among them, the TCR molecule containing the mutation retains the biological activity of the TCR molecule containing SEQ ID NO: 7 and SEQ ID NO: 8 that specifically targets the KRAS G12V mutant polypeptide. The TCR molecule according to any one of claims 1 to 3, characterized in that the TCR molecule contains a mouse constant region. The TCR molecule as described in claim 4 is characterized in that 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. A multivalent TCR complex, characterized in that the multivalent TCR complex comprises two or more TCR molecules according to any one of claims 1-5. A dual-targeting protein molecule that can simultaneously bind to tumor cells and immune cells, characterized in that the dual-targeting protein molecule comprises a TCR molecule targeting the KRAS G12V mutation on the surface of tumor cells as described in 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 peptide and transmembrane domain in the α chain variable region and the β chain variable region of the TCR molecule are deleted. The dual-targeting protein molecule according to claim 7, characterized in that the single-chain antibody is an anti-CD3 single-chain antibody. 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 to 5 or the dual-targeting protein molecule according to claim 7 or 8, or its complementary sequence. The nucleic acid molecule as described in claim 9 is characterized in that the nucleic acid sequence of the nucleic acid molecule is selected from: SEQ ID NO: 11, SEQ ID NO: 12. 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. An isolated cell, characterized in that the cell: (1) containing the nucleic acid construct according to claim 11 or a chromosome in which the nucleic acid molecule according to claim 9 or 10 is integrated, and/or (2) expressing the TCR molecule described in any one of claims 1 to 5 or the dual-targeting protein molecule described in claim 7 or 8; Preferably, the cell is an immune effector cell, preferably a T cell, a NK cell or a TIL cell. 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 to 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 expression vector according to claim 11 or the cell according to claim 12. Use of the TCR molecule described in any one of claims 1-5, the TCR complex described in claim 6, the dual-targeting protein molecule described in claim 7 or 8, the nucleic acid molecule described in claim 9 or 10, the recombinant expression vector described in claim 11 or the cell described in claim 12 in the preparation of a drug for treating or preventing a disease associated with the KRAS G12V mutant antigen in a patient. A method for treating or preventing a disease associated with the KRAS G12V mutant antigen in a patient, the method comprising the step of adoptively transferring a T cell containing the expression vector of claim 11 or a chromosome in which the nucleic acid molecule of claim 9 or 10 is integrated, and/or expressing the TCR molecule of any one of claims 1-5 to the patient, or comprising the step of administering to the patient a dual-targeting protein molecule of claim 7 or 8 or a pharmaceutical composition containing the dual-targeting protein molecule.