T cell receptor targeting KRAS G12V mutant polypeptide and application thereof

Abstract

The invention provides a T cell receptor targeting KRAS G12V mutant polypeptide and application of the T cell receptor. The T cell receptor molecule specifically targets KRAS G12V mutation, a CDR3 sequence of an alpha 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 a CDR3 sequence of a beta chain variable region of the T cell receptor molecule is a mutant containing CASSQNNEQFF (SEQ ID NO: 2) or SEQ ID NO: 2. The invention also provides a nucleic acid molecule for coding 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 for expressing the T cell receptor molecule and the like.

Description

Technical Field

[0001] The present invention relates to a T cell receptor sequence targeting the 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-target anti-tumor protein drug developed based on the T cell receptor. Background Art

[0002] Adoptive cellular immunotherapy is a newly developed cutting-edge technology that has achieved unprecedented success in the treatment of hematological malignancies. However, there are many difficulties in the treatment of solid tumors with adoptive cellular immunotherapy technology. Finding suitable targets and developing receptor molecules that specifically bind to these targets can open up a new situation for the treatment of solid tumors. Among them, the KRAS gene has come into the view of researchers. KRAS is one of the main members of the Ras gene family (including NRAS, HRAS, and KRAS), is a murine sarcoma virus oncogene, located on chromosome 12, about 35 kb in length, and encodes the KRAS protein. When KRAS mutates, it continuously binds to GTP, showing tyrosine kinase activity and activating downstream signaling pathways, thus causing uncontrolled cell proliferation and tumorigenesis. It is found that KRAS mutations exist in about 30% of tumors, including 90% of pancreatic cancers, 50% of colon cancers, and 25% of lung cancers.

[0003] KRAS mutations often occur at positions such as glycine at position 12, glycine at position 13, and glutamine at position 61. The mutations of glycine at position 12 and glycine at position 13 account for up to 97%, mainly including mutant types such as G12C, G12D, G12V, G12R, and G13D. The KRAS G12V mutation is expressed in about 30% of pancreatic cancer patients, 10% of colorectal cancer patients, or non-small cell lung cancer. Moreover, members of the RAS family also share the G12V hot spot mutation in different cancer types (such as NRAS in melanoma). Although KRAS mutations have been found in many tumors, due to the lack of a pocket for binding small molecule inhibitors on the surface of KRAS, it has been regarded as an undruggable target for many years. Currently, only one KRAS G12C inhibitor, AMG 510, has been approved for marketing, which is used to treat advanced non-small cell lung cancer (NSCLC) patients with KRAS G12C mutations who have received prior systemic treatment. However, the occurrence rate of KRAS G12C in other cancers (such as pancreatic cancer, colon cancer, etc.) is rare. For the remaining high-abundance KRAS mutation types, such as G12D and G12V, new treatment methods are also urgently needed.

[0004] Targeting KRAS high-abundance mutations and combining adoptive immunocyte therapy are the breakthrough points in current research in this direction. One of the adoptive immunocyte therapies is called T cell receptor engineered T cell therapy (TCR-T), which has obvious advantages. TCR-T therapy utilizes the tumor-killing characteristics of T cells, transfers tumor-specific TCR genes into T cells, and mediates the specific recognition of tumor antigens by T cells through the receptors expressed, ultimately achieving the recognition and killing effects on tumors. TCR-T cell therapy has demonstrated good safety and effectiveness in clinical trial studies for treating refractory and recurrent melanoma, synovial sarcoma, multiple myeloma, lung cancer, etc. at home and abroad. Finding TCR receptors with high specificity and strong affinity is the key part and technical stronghold of TCR-T technology. Summary of the Invention

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

[0006] In the second aspect of the present invention, a multivalent TCR complex is provided. The multivalent TCR complex comprises two or more TCR molecules according to any embodiment of the present invention.

[0007] In the third aspect of the present invention, a dual-targeting protein molecule capable of binding tumor cells and immune cells simultaneously is provided. The dual-targeting protein molecule comprises a TCR molecule targeting the KRAS G12V mutation on the surface of tumor cells according to any embodiment of the present invention and a single-chain antibody (scFv) for recruiting and redirecting immune cells to the periphery of tumor cells. Among them, the signal peptides and transmembrane domains in the variable regions of the α chain and β chain of the TCR molecule are deleted.

[0008] In the fourth aspect of the present invention, a nucleic acid molecule is provided. The nucleic acid molecule comprises a nucleic acid sequence encoding the TCR molecule or the dual-targeting protein molecule according to any embodiment of the present invention or its complementary sequence;

[0009] In the fifth aspect of the present invention, a nucleic acid construct is provided. The nucleic acid construct contains the nucleic acid molecule according to any embodiment of the present invention.

[0010] In the sixth aspect of the present invention, an isolated cell is provided. The cell:

[0011] (1) Comprising the nucleic acid construct described in any embodiment of the present invention or having integrated therein the nucleic acid molecule described in any embodiment of the present invention in 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 cell is an immune effector cell, preferably a T cell, an NK cell, or a TIL cell.

[0014] 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.

[0015] The 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.

[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, which includes the step of adoptive transfer of T cells containing the nucleic acid molecule integrated in the vector or chromosome of the present invention and / or expressing the TCR molecule described in any embodiment herein to the patient, or includes the step of administering to the patient the 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 described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Schematic diagram of the pMSGV1_02-2TCR vector.

[0019] Figure 2 : Binding experiment of two KRAS G12V mutant tetramers (9mer and 10mer, SEQ ID NO:15, SEQ ID NO:16) to 02-2TCR-J cells. After co-incubating the two KRAS G12V mutant tetramers with 02-2TCR-J cells, the binding of the tetramers to 02-2TCR-J cells was detected by a flow cytometer. The upper row from left to right is the flow cytometry gating analysis diagram after binding of the 9mer KRAS G12V mutant tetramer, and the lower row from left to right is the flow cytometry gating analysis diagram after binding of the 10mer KRAS G12V mutant tetramer.

[0020] Figure 3: 02-2 TCR-J cell polypeptide activation function experiment. After incubating wild-type KRAS (SEQ ID NO: 13, SEQ ID NO: 14) and G12V polypeptide (SEQ ID NO: 15, SEQ ID NO: 16) with 02-2 TCR-J cells at 37 °C for 4 h, the expression of mTCR and GFP in 02-2 TCR-J cells was detected after washing. Detailed implementation manners

[0021] The present invention discovers a TCR molecule that specifically targets the KRAS G12V mutant antigen (especially the KRAS G12V mutant antigen shown in SEQ ID NO: 16). This TCR molecule can specifically bind to the KRAS G12V / HLA-A*11:01 complex on the surface of tumor cells, and normal non-cancer cells are not recognized because they express the unmutated wild-type KRAS protein. Therefore, the TCR molecule of the present invention has strong specificity, which reduces the toxic and side effects after the T cells expressing this TCR molecule are made into drugs and does not damage normal non-cancer 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, prostate cancer, etc., especially pancreatic cancer), thus completing the present invention.

[0022] 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 examples) can be combined with each other to form a preferred technical solution.

[0023] Term Definition

[0024] In this article, TCR has the meaning well-known in the art and is a cell membrane surface glycoprotein existing in the form of a heterodimer of an α chain / β chain or a γ chain / δ chain, which 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. TCR is the receptor after the major histocompatibility complex (MHC) presents intracellular antigen peptides. The TCRs of most T cells are dimers composed of α and β peptide chains, and the TCRs of a few T cells are 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 connects to the transmembrane region, while the variable region is responsible for recognizing the polypeptide / MHC complex. The variable regions all contain 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 CDR1s of the α and β subunits act on the N-terminus and C-terminus of the polypeptide respectively. CDR2 is considered to be involved in recognizing MHC. The β subunit also has an additional CDR4, which usually does not participate in the recognition of the polypeptide / MHC complex but is related to the action of superantigens.

[0025] In this article, the major histocompatibility complex (MHC) is a gene family existing in the genomes of most vertebrates and is an antigen-presenting and T cell activation molecule. Among them, the MHC glycoprotein in humans is also called human leukocyte antigen (HLA for short). MHC molecules include class I and class II MHC molecules. MHC molecules can present the degradation fragments of intracellular proteins. For example, after a cell is infected with a virus, the polypeptide fragments of the corresponding virus outer membrane can be presented to the cell surface through MHC molecules for cytotoxic T cells (CD8+ cytotoxic T cells) to identify and specifically kill the cells infected with the virus.

[0026] In this text, amino acid residues are recorded using the following abbreviated symbols: 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 recorded in the conventional manner 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.

[0027] The term "T cell receptor targeting the KRAS G12V mutation (KRAS G12V -reactive T Cell Receptor)" is defined herein as a TCR molecule that can bind to the KRAS G12V mutant polypeptide / MHC complex, thereby inducing T cell cytotoxicity. 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.

[0028] 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 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, and the endogenous TCR naturally expressed by these T cells is not sufficient to induce a response of the cell or responding cells to TCR ligand binding.

[0029] In this text, TCR-T cell therapy involves introducing an exogenous TCR gene into ordinary T cells, enabling the modified T cells to express a TCR that can effectively recognize tumor cells, thereby guiding T cells to kill tumor cells. This therapy generally includes the step of administering to a patient T cells modified to express the exogenous TCR gene. These T cells are usually derived from the patient himself. Generally, the patient's T cells are obtained, modified in vitro to express the exogenous TCR gene (such as the TCR gene described in any embodiment herein), and then transfused back to the patient.

[0030] 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) that 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 a single-chain antibody against CD3, and an exemplary immune cell can be a T cell. The TCR first recognizes and binds to the polypeptide / 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, forms an immune synapse, activates immune cells and releases lytic granules, resulting in the death of cancer cells.

[0031] In this article, 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 substitution" is known in the art as a substitution in which one or more amino acid residues are replaced by one or more amino acid residues with side chain R groups having 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 similar chemical properties of side chains 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 the amino acid side chain groups as: 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.

[0032] In this article, immune cells refer to cells that participate in or are related to immune responses, and usually include lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. Exemplary immune cells include T cells, natural killer cells (NK), 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.

[0033] T cell receptor (TCR)

[0034] The characteristics of the TCR molecule targeting the KRAS G12V mutation of the present invention include that the CDR3 sequence of the variable region of its α chain contains CAIPPGGSGDKLTF (SEQ ID NO:1) or a mutant of SEQ ID NO:1, and / or the CDR3 sequence of the variable region of its β chain 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 the binding activity possessed by SEQ ID NO:1 as the CDR3 of the variable region of the TCR α chain. 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 possessed by SEQ ID NO:2 as the CDR3 of the variable region of the TCR β chain. The mutations contained in the mutants of SEQ ID NO:1 and 2 respectively can be selected from one or more of insertion, deletion and substitution. Preferably, the mutation is a conservative mutation, such as a conservative substitution.

[0035] 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 such mutations do not affect the biological functions and activities played by these CDR sequences in the TCR molecule. Preferred mutations are conservative mutations, such as conservative substitutions.

[0036] In some embodiments, the CDR1 sequence of the variable region of the α-chain 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 variable region of the β-chain 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).

[0037] The amino acid sequences of the CDR regions of the TCR molecule of the present invention can be embedded into 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 a TCR molecule with corresponding functions based on the CDR regions disclosed in the present invention. Therefore, the TCR molecule of the present invention refers to a TCR molecule that contains the above-mentioned α- and / or β-chain CDR region sequences and any suitable framework structure and adopts the CDR region sequences of the present invention.

[0038] In some embodiments, the variable region of the α-chain 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 as 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 as compared with the amino acid sequence shown 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 mutations can be selected from one or more of insertion, deletion, and substitution. The mutations can occur in any domain of SEQ ID NO:7, including in its CDR and / or FR regions. In some embodiments, the mutations do not occur in the sequences of CDR1, CDR2, and CDR3 contained in SEQ ID NO:7. In some embodiments, the mutations occur, for example, in the FR region of SEQ ID NO:7. Preferably, the mutations are conservative mutations, such as conservative substitutions.

[0039] 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 as 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 as compared with the amino acid sequence shown 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 mutations can be selected from one or more of insertion, deletion, and substitution. The mutations can occur in any domain of SEQ ID NO:8, including in its CDR and / or FR regions. In some embodiments, the mutations do not occur in the sequences of CDR1, CDR2, and CDR3 contained in SEQ ID NO:8. In some embodiments, the mutations occur, for example, in the FR region of SEQ ID NO:8. Preferably, the mutations are conservative mutations, such as conservative substitutions.

[0040] It should be understood that the mutants of the TCR molecule of the present invention (i.e., mutants containing the mutants of the α-chain variable region and / or β-chain variable region described above) still retain the biological activity of the TCR molecule containing SEQ ID NO:1 and SEQ ID NO:2 (especially the TCR molecule containing SEQ ID NO:7 and 8) that specifically binds to the KRAS G12V mutation (especially the polypeptide shown in SEQ ID NO:16) depending on HLA-A*11:01.

[0041] In some embodiments, the TCR molecule of the present invention is a dimer composed of an α chain and a β chain. The α chain includes a variable region and a constant region. The CDR1 sequence of the variable region of the α chain 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 variable region of the β chain 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 variable region of the α chain contains the amino acid sequence shown in SEQ ID NO: 7 or the mutant sequence described above, and the variable region of the β chain contains the amino acid sequence shown in SEQ ID NO: 8 or the 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 chain and the β chain to form a dimer.

[0042] 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 publicly available database.

[0043] Studies have shown that replacing the constant region of the human TCR with the constant region of the murine TCR can effectively avoid the rearrangement of exogenous T cell receptors in the human body and the autologous TCR, resulting in off-target or even binding to the wrong target problems. Therefore, in some embodiments, the TCR molecule of the present invention contains the murine α constant region and β constant region. The amino acid sequence of an exemplary murine 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.

[0044] 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.

[0045] Nucleic acid molecule

[0046] The present invention provides nucleic acid molecules encoding the variable region of the α chain, the variable region of the β chain, the α chain, the β chain, and the TCR molecule described in any of the above embodiments.

[0047] The nucleotide sequence of the nucleic acid molecule of the present invention can be single-stranded or double-stranded. The nucleic acid molecule can be RNA or DNA, and may or may not contain introns. An exemplary sequence of the nucleic acid molecule encoding the variable region of the α chain of the present invention is shown in SEQ ID NO: 11. An exemplary polynucleotide sequence encoding the variable region of the β chain of the present invention is shown in SEQ ID NO: 12.

[0048] In some embodiments, the TCR molecules of the present invention comprise human variable regions and murine constant regions. In the murine constant regions, the nucleic acid coding sequence of the α constant region can be as shown in SEQ ID NO: 17, and the nucleic acid coding sequence of the β constant region can be as shown in SEQ ID NO: 18.

[0049] 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 variable regions of the α chain, the variable regions of the β chain, the α chain, the β chain, and the TCR molecules of the present invention can be the same as or degenerate variants of the nucleic acid sequences shown in the present invention. Taking one example in the present invention, 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.

[0050] To efficiently express TCR in T cells, the nucleotide sequences of the present invention can be optimized by codon optimization methods. Different cells have different preferences in the use of specific codons, and the codons in the sequence can be changed according to the cell type to increase the expression level. Codon usage tables for mammalian cells and various other organisms are well known to those skilled in the art.

[0051] The full-length sequences or fragments of the nucleic acid molecules of the present invention can generally be obtained by, but not limited to, PCR amplification, recombination, or artificial synthesis methods. Currently, it is already possible to completely obtain the DNA sequence encoding the TCR (or its fragment, or its derivative) of the present invention by chemical synthesis. Then, this DNA sequence can be introduced into various existing DNA molecules (such as vectors), mRNA, or cells known in the art. The DNA or mRNA can be the coding strand or the non-coding strand.

[0052] Nucleic acid construct

[0053] The present invention also includes nucleic acid constructs containing the nucleic acid molecules described in any of the embodiments herein.

[0054] The nucleic acid constructs herein can be an expression cassette that contains a promoter sequence operably linked, the nucleic acid molecules described in any of the embodiments herein, and a polyA tail. Other regulatory elements operably linked to the above elements, such as enhancers, etc., can also be contained within the expression cassette.

[0055] In some embodiments, the nucleic acid construct is a vector. As used herein, vectors include, but are not limited to, expression vectors and cloning vectors. An expression vector refers to a vector used for expressing 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. Expression vectors generally include expression regulatory elements. Expression regulatory elements are well known in the art and include, but are not limited to, promoters and enhancers, etc. In some embodiments, the vector contains the expression cassette.

[0056] As used herein, the expression vector can be a vector related to a virus delivery system, including, but not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpesvirus 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 cells, such as T cells, so that the cells express a TCR specific for the KRAS G12V mutant antigen.

[0057] Dual-targeting protein molecule

[0058] 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 vicinity of tumor cells. The TCR molecule may include the variable region and constant region of the α chain of the TCR molecule and the variable region and constant region of the β chain. The variable region and the constant region may be directly connected or may be connected by a flexible peptide chain. Generally, the signal peptide and transmembrane domain in the variable region of the α chain and β chain are deleted.

[0059] The single-chain antibody has various monoclonal antibodies of interest with the biological function. An exemplary single-chain antibody can be a single-chain antibody against CD3.

[0060] Generally, 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 variable region of the β chain of the TCR molecule. The scFv and the N-terminus of the variable region of the β chain may be directly connected or may be connected through a flexible peptide chain.

[0061] As used 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 generally 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 2-30, such as 3-20 or 3-10 amino acid residues in length. An exemplary amino acid sequence of the linker is shown in SEQ ID NO:19.

[0062] In some embodiments, to obtain a stable dual-targeting protein molecule, cysteine mutations can be made 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 whether or not the constant regions contain the introduced artificial disulfide bonds described above, the TCR molecules of the present invention can be linked by the natural disulfide bonds present in the TCR.

[0063] Cell

[0064] The present invention also relates to host cells generated by genetic engineering using the vectors or nucleic acid molecules 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 having the nucleic acid molecule of the present invention integrated into the chromosome, and / or expressing the TCR molecule and / or dual-targeting protein molecule described in any embodiment herein.

[0065] 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 peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC). More preferably, the host cell is a primary T cell.

[0066] In some embodiments, the present invention particularly relates to immune cells, especially T cells, containing the vector of the present invention or having the nucleic acid molecule of the present invention integrated into the chromosome and / or expressing the TCR molecule described in any embodiment herein. The T cell can be any type of T cell and can be at any developmental stage, including but not limited to: CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD4+ T cells, CD8+ T cells (such as cytotoxic T cells), memory T cells (such as central memory T cells and effector memory T cells), naive T cells, etc. More preferably, the T cell can be derived from CD8+ T cells isolated from a patient.

[0067] 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. Transferring genes to NK cells will not result in the expression of TCR 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 initiate the expression of TCR molecules in NK cells.

[0068] Pharmaceutical composition and conjugate

[0069] The present invention also provides a pharmaceutical composition, which contains T cells and a pharmaceutically acceptable carrier. The T cells contain a vector expressing the TCR molecule described in any embodiment herein or a nucleic acid molecule encoding the TCR molecule described in any embodiment herein integrated into its chromosome, and / or express the TCR molecule described in any embodiment herein.

[0070] In some embodiments, the pharmaceutical composition of the present invention contains the TCR molecule or the dual-targeting protein molecule described in any embodiment herein and a pharmaceutically acceptable carrier.

[0071] Herein, the pharmaceutically acceptable carrier can be selected according to the specific active ingredient. For example, the pharmaceutically acceptable carrier in the pharmaceutical composition containing T cells can be various suitable carriers well known in the art for cell therapy. The pharmaceutically acceptable carrier in the pharmaceutical composition containing the TCR molecule or the dual-targeting protein molecule of the present invention can be a pharmaceutically acceptable carrier suitable for protein delivery.

[0072] Herein, the pharmaceutical composition can be administered by any appropriate route, such as parenteral, enteral, inhalation or intranasal routes. The pharmaceutical composition of the present invention can be prepared by methods well known in the art, such as by mixing the active ingredient with the carrier or excipient under sterile conditions.

[0073] The effective amount of the active ingredient such as the T cells, TCR molecule or dual-targeting protein molecule in the pharmaceutical composition of the present invention depends on the disease or disorder to be treated, the age and condition of the individual to be treated, etc., and can be easily determined by those skilled in the art according to the actual situation. Generally, the suitable dosage range of the soluble TCR of the present invention can be between 25 ng / kg and 50 μg / kg.

[0074] The pharmaceutical composition of the present invention can be used for various therapeutic uses described below.

[0075] In some embodiments, the present invention provides a conjugate, which contains the TCR molecule described herein and a therapeutic agent or tracer covalently or otherwise bound to the TCR molecule, for the treatment or diagnosis of diseases, especially tumors. The bound or conjugated therapeutic agents include but are not limited to: radionuclides, chemotherapeutic agents, antibody Fc or scFv fragments, and nanoparticles, etc. The 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 (computed tomography), and enzymes for detecting products, etc.

[0076] Use and treatment method

[0077] The present invention also provides the use of the TCR molecule, the dual-targeting protein molecule, and the cell (especially a T cell) 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 molecule, the dual-targeting protein molecule, and the cell (especially a T cell) described in any embodiment herein for treating or preventing a disease associated with the KRAS G12V mutant antigen.

[0078] 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 includes the step of adoptively transferring T cells containing the vector of the present invention or integrating the nucleic acid molecule of the present invention in the chromosome, and / or expressing the TCR molecule described in any embodiment herein to the patient, or includes the step of administering to the patient the dual-targeting protein molecule described in any embodiment herein or a pharmaceutical composition containing the dual-targeting protein molecule.

[0079] As used herein, the disease associated with the KRAS G12V mutant antigen is a tumor or cancer, and 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 cholangiocarcinoma, 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, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin 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, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral 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.

[0080] Preferably, the tumor cells or cancer cells of the patient carry the KRAS G12V mutant antigen and HLA-A*11:01. Preferably, the KRAS G12V mutant antigen carried by the tumor cells or cancer cells of the patient includes, but is not limited to, the amino acid sequence shown in SEQ ID NO:16.

[0081] T cells can be isolated from patients or volunteers suffering from diseases associated with the KRAS G12V mutant antigen, and the T cells can be genetically engineered in vitro to contain the vector described in any of the embodiments herein or to have integrated into their genomes a TCR molecule capable of expressing the TCR molecule described in any of the embodiments herein, so as to express the TCR molecule described in any of the embodiments herein. Subsequently, these genetically engineered cells can be transfused back into the patient's body for treatment.

[0082] In some embodiments, the T cells are derived from the patient himself / herself. Thus, in these embodiments, the treatment method of the present invention further includes: (1) isolating the patient's T cells, and (2) genetically engineering the T cells in vitro to contain the vector described in any of the embodiments herein or to have integrated into their genomes a TCR molecule capable of expressing the TCR molecule described in any of the embodiments herein, so as to express the TCR molecule described in any of the embodiments herein.

[0083] The mode, timing, dose, etc. of administration can be determined by a physician according to the age, weight, general health condition of each individual patient, the severity of the cancer being treated, etc.

[0084] 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. 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, etc. It can also be a monoclonal antibody-based 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 treatment reagents, such as oncolytic viruses and vaccines (including but not limited to mRNA, DNA, protein, protein subunit, cell component or cell, etc.).

[0085] 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 not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following examples, they are generally carried out under conventional conditions, such as the conditions described in (Sambrook and Russel1 et al., Molecular Cloning - A Laboratory Manual (Third Edition) (2001) CSHL Press), or 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 without special instructions.

[0086] Example 1: Determination of TCR gene sequences targeting KRAS G12V mutation

[0087] Functional cell populations were sorted from peripheral blood mononuclear cells (PBMCs) of tumor patients with KRAS G12V mutation (from the Department of General Surgery, Jinling Hospital Affiliated to Nanjing University School of Medicine), and TCR sequences were obtained through single-cell sequencing. After functional verification, TCR 02-2 was able to specifically bind to the VVGAVGVGK / HLA-A*11:01 complex. The amino acid sequences and coding sequences of its α variable region are shown as SEQ ID NO: 7 and SEQ ID NO: 11 respectively, and the amino acid sequences and coding sequences of its β variable region are shown as SEQ ID NO: 8 and SEQ ID NO: 12 respectively.

[0088] SEQ ID NO:7

[0089] (The sequences of CDR1, CDR2, and CDR3 are marked in bold and underlined in turn).

[0090] SEQ ID NO:8

[0091] (The sequences of CDR1, CDR2, and CDR3 are marked in bold and underlined in turn).

[0092] Example 2: Construction of vectors highly expressing TCR molecules

[0093] 1. Vector information

[0094] Overexpress the TCR molecule in T cells using the pMSGV1 vector. Use the human codon-optimized TCR nucleic acid sequence. The coding sequence of the variable region of the α chain is as shown in SEQ ID NO: 11; the coding sequence of the variable region of the β chain is as shown in SEQ ID NO: 12. In addition, the expression of TCR in T cells is completed in a human-mouse hybrid manner. The amino acid sequence of the α constant region of the mouse is as shown in SEQ ID NO: 9, and the amino acid sequence of the β constant region of the mouse is as shown in SEQ ID NO: 10. The TCR α chain and β chain are tandemly linked by the SGSG-P2A sequence (amino acid sequence SEQ ID NO: 20, nucleic acid sequence SEQ ID NO: 21). The complete vector structure is as Figure 1 shown. After the vector construction is completed, it is identified by sequencing. The sequencing primers are shown in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25.

[0095] 2. Plasmid extraction

[0096] After correct sequencing, use NucleoBond Xtra Maxi (MACHEREY-NAGEL) to extract and purify the plasmid. The concentration of the purified plasmid is measured by calculating the light absorption at 259 nm using an ultraviolet spectrophotometer and stored at -20 °C for subsequent experiments.

[0097] Example 3: Retrovirus packaging

[0098] 1. On the first day: Digest 293T cells and plate them at 0.6×10 6 cells / ml. Add 5 ml of D10 medium (DMEM + 10% FBS) to a T25 flask, mix the cells well, and culture them overnight at 37 °C.

[0099] 2. On the second day: Transfect when the confluence of 293T cells reaches about 90%. Prepare the plasmid complex. The amounts of various plasmids are as follows: 3 μg of pMSGV1-02-2TCR, 1.9 μg of Gag-pol, and 0.75 μg of 10A1. Add 300 μL of DMEM. Add 20 μL of EZ Trans cell transfection reagent (Shanghai Liji Biology) and 300 μL of DMEM. Add the PEI solution to the plasmid complex and vortex for 20 s. Gently add the mixture along the side to the 293T culture flask and culture at 37 °C for 16 h. Remove the medium and re-add pre-warmed fresh medium.

[0100] 3. On the fourth day: Collect the supernatant 48 h after transfection, filter it through a 0.45 μm filter, and aliquot and store it at -80 °C.

[0101] Example 4: Preparation of Retrovirus-Infected Reporter Cell S1-1-1-CD8-A11 (02-2TCR-J Cells)

[0102] 1. Take S1-1-1-CD8-A11 cells and adjust the cell density to 5×10 5 / mL with R10 medium (RPMI1640 + 10% FBS). Inoculate 1 ml of the cells into a 24-well culture plate, with a total of 2 wells inoculated.

[0103] 2. Add 300 μL of 02-2TCR virus and 0.65 μL of polybrene (Santa Cruz) to each well.

[0104] 3. Centrifuge at 32°C and 2500 rpm for 90 min.

[0105] 4. After centrifugation, discard 800 μL of the supernatant, supplement with 800 μL of fresh R10 medium, and place the culture plate in an incubator at 37°C and 5% CO2 for culture.

[0106] Example 5: Detection of TCR Expression in 02-2TCR-J Cells by Flow Cytometry

[0107] 1. Two days after infecting S1-1-1-CD8-A11 cells with 02-2TCR virus, pipette and mix the cells in the 24-well plate. Take 500 μL of the cells and transfer them to 2 flow tubes. Wash with PBS and discard the supernatant to obtain 02-2TCR-J cells.

[0108] 2. Add 100 μL of zombie green (BioLegend, diluted 1:500) dye to each tube, incubate in the dark at room temperature for 10 min, wash with PBS, and discard the supernatant.

[0109] 3. Add 100 μL of the antibody mixture (mTCR antibody diluted 1:300, CD8 antibody diluted 1:200) to each tube, incubate at 4°C in the dark for 30 min, add PBS for washing, and discard the supernatant.

[0110] 4. Add 100 μL of KRAS G12V 9mer tetramer or KRAS G12V 10mer tetramer (diluted 1:100) to each of the 2 tubes, incubate at 4°C in the dark for 30 min, wash with PBS, resuspend, and detect by flow cytometry.

[0111] The results are as Figure 2 shown. Figure 2 The upper row from left to right is the flow cytometry gating analysis diagram after binding with 9mer KRAS G12V mutant tetramer, and the lower row from left to right is the flow cytometry gating analysis diagram after binding with 10mer KRAS G12V mutant tetramer.

[0112] The experimental results showed that 02-2TCR-J cells could specifically recognize the 10mer G12V polypeptide, but not the 9mer G12V polypeptide.

[0113] Example 6: Polypeptide activation function experiment of 02-2TCR-J cells

[0114] The prepared 02-2TCR-J cells were counted, an appropriate amount was taken for centrifugation, resuspended in R10 medium, and the cell concentration was adjusted to 2×10 6 / mL, and 100 μL / well was added to a 96-well plate, with a total of 4 wells inoculated.

[0115] 2. Four kinds of polypeptides, namely KRAS G12V WT 9mer, KRAS G12V WT 10mer, KRAS G12V 9mer or KRAS G12V 10mer, were added to the wells at a final concentration of 1 μg / mL, mixed well, and incubated at 37°C for about 4 hours.

[0116] 3. The cells in the 4 wells were transferred to flow tubes, washed with PBS, and the supernatant was discarded.

[0117] 4. 100 μL of mTCR antibody (diluted 1:300) was added respectively, incubated at 4°C in the dark for 30 min, washed with PBS, resuspended, and mTCR and GFP were detected by flow cytometry.

[0118] The results are as Figure 3 shown. The experimental results showed that 02-2TCR-J cells could be activated by the 10mer G12V polypeptide, but not by the 9mer polypeptide.

Claims

1. A T cell receptor (TCR) molecule, characterized in that, The TCR molecule specifically targets the KRAS G12V mutation. 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 its 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 α-chain variable region of TCR; 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 β-chain variable region of TCR.

2. 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 a mutant thereof, and the CDR2 sequence contains FQNEAQ (SEQ ID NO:6) or a mutant thereof.

3. The TCR molecule according to claim 1, wherein The α-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 β-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; preferably, the amino acid sequence of the murine α constant region is as shown in SEQ ID NO:9, and the amino acid sequence of the β constant region is as shown in SEQ ID NO:

10.

5. A multivalent TCR complex, characterized in that, The multivalent TCR complex contains more than two TCR molecules according to any one of claims 1-3.

6. 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 1-4 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 α-chain variable region and β-chain variable region of the TCR molecule are deleted; Preferably, the single-chain antibody is an anti-CD3 single-chain antibody.

7. 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 1-3 or the dual-targeting protein molecule according to claim 6, or a complementary sequence thereof; Preferably, the nucleic acid sequence of the nucleic acid molecule is selected from: SEQ ID NO:11, SEQ ID NO:

12.

8. A nucleic acid construct, characterized in that, The nucleic acid construct contains the nucleic acid molecule according to claim 7; 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.

9. An isolated cell, characterized in that, The cell: (1) contains the nucleic acid construct according to claim 8 or the nucleic acid molecule according to claim 7 is integrated into the chromosome, and / or (2) expresses the TCR molecule according to any one of claims 1-4 or the dual-targeting protein molecule according to claim 6; Preferably, the cell is an immune effector cell, preferably a T cell, an NK cell or a TIL cell.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains a pharmaceutically acceptable carrier and the TCR molecule according to any one of claims 1-4, the TCR complex according to claim 5, the dual-targeting protein molecule according to claim 6, the nucleic acid molecule according to claim 7, the recombinant expression vector according to claim 8 or the cell according to claim 9.

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