T cell receptors targeting KRAS G12V mutant polypeptides and uses thereof

Abstract

The present invention provides a T cell receptor targeting a KRAS G12V mutant polypeptide and its uses. The T cell receptor molecule of the present invention specifically targets the KRAS G12V mutation, the CDR3 sequence of its α-chain variable region contains CAVRDIEGAGNNRKLIW (SEQ ID NO:1) or a mutant of SEQ ID NO:1, and / or the CDR3 sequence of its β-chain variable region contains CASSEGQYSYEQYF (SEQ ID NO:2) or a mutant of SEQ ID NO:2. The present invention further provides a nucleic acid molecule encoding the T cell receptor molecule, a multivalent complex and a dual-target protein molecule containing the T cell receptor molecule, a nucleic acid construct, a cell expressing the T cell receptor molecule, etc.

Description

Detailed Description of the Invention

[0001] Technical Field The present invention relates to a T cell receptor sequence targeting a KRAS G12V mutant polypeptide and its coding 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.

[0002] Background Art Adoptive cellular immunotherapy is a newly developed cutting-edge technology that has achieved unprecedented success in the treatment of hematological malignancies. However, adoptive cellular immunotherapy has many difficulties in the treatment of solid tumors. Finding appropriate targets and developing receptor molecules that specifically bind to these targets can open up a new situation in 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. It is located on chromosome 12, about 35 kb in length, and is the murine sarcoma virus oncogene that encodes the KRAS protein. After KRAS mutates, it continuously binds to GTP, shows tyrosine kinase activity, activates downstream signaling pathways, thereby causing uncontrolled cell proliferation and tumorigenesis. According to research, KRAS mutations are present in about 30% of tumors (including 90% of pancreatic cancers, 50% of colorectal cancers, and 25% of lung cancers).

[0003] KRAS mutations always occur at sites 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 are as high as 97%, mainly in mutant forms such as G12C, G12D, G12V, G12R, G13D, etc. The KRAS G12V mutation is expressed in approximately 30% of pancreatic cancer, 10% of colorectal cancer patients, or non-small cell lung cancer. In addition, RAS family members, such as NRAS in melanoma, also share the G12V hot-spot mutation in different cancer types. Although KRAS mutations have been discovered in many tumors, for many years, due to the lack of a pocket on the KRAS surface that binds to small molecule inhibitors, it has not been considered a drug target. Currently, only one KRAS G12C inhibitor, AMG 510, has been approved and marketed, and it is used to treat advanced non-small cell lung cancer (NSCLC) patients with KRAS G12C mutations who have previously received systemic treatment. However, the probability of KRAS G12C appearing in other cancers, such as pancreatic cancer and colon cancer, is rare. There is also a need to find new treatment methods for the remaining high-abundance KRAS mutant forms, such as G12D and G12V.

[0004] Targeting the high-abundance mutations of KRAS and combining it with adoptive immunotherapy is currently a breakthrough point in research in this direction. One of the adoptive immunotherapies is called T cell receptor engineered T cell therapy (TCR-T), which has obvious advantages. The TCR-T therapy utilizes the tumor-killing characteristics of T cells, introduces tumor-specific TCR genes into T cells, and through the receptors expressed thereon, induces the specific recognition of tumor antigens by T cells, and finally realizes the recognition and killing effect of tumors. The TCR-T cell therapy has shown good safety and effectiveness in clinical trial studies for treating refractory and recurrent melanoma, synovial sarcoma, multiple myeloma, and lung cancer, etc. at home and abroad. Searching for TCR receptors with high specificity and high affinity is an important part and technical obstacle of TCR-T technology.

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

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

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

[0008] In a fourth aspect of the present invention, a nucleic acid molecule is provided, which comprises a nucleic acid sequence encoding the TCR molecule or dual-target protein molecule according to any one of the embodiments of the present invention or its complementary sequence;

[0009] In a fifth aspect of the present invention, a nucleic acid construct is provided, which comprises the nucleic acid molecule according to any one of the embodiments of the present invention.

[0010] In a sixth aspect of the present invention, an isolated cell is provided, wherein the cell: (1) contains the nucleic acid construct according to any one of the embodiments of the present invention, or the nucleic acid molecule according to any one of the embodiments of the present invention is integrated into the chromosome; and / or (2) It expresses the TCR molecule described in any one of the embodiments of the present invention or the dual-target protein molecule described in any one of the embodiments of the present invention. Preferably, the cell is an immune effector cell, preferably a T cell, an NK cell, and a TIL cell.

[0011] The seventh aspect of the present invention provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR molecule, TCR complex, dual-target protein molecule, nucleic acid molecule, recombinant expression vector, or cell described in any one of the embodiments of the present invention.

[0012] The eighth aspect of the present invention provides the application of the TCR molecule, TCR complex, dual-target protein molecule, nucleic acid molecule, recombinant expression vector, or cell described in any one of the embodiments of the present invention in the preparation of a drug for treating or preventing a disease related to the KRAS G12V mutant antigen of a patient.

[0013] The ninth aspect of the present invention provides a method for treating and / or preventing a disease related to the KRAS G12V mutant antigen of a patient, which includes the step of adoptively transferring to the patient a T cell that contains the vector of the present invention, or in which the nucleic acid molecule of the present invention is integrated into the chromosome, and / or that expresses the TCR molecule described in any one of the embodiments herein, or includes the step of administering to the patient the dual-target protein molecule described in any one of the embodiments of the present invention or a pharmaceutical composition containing the dual-target protein molecule.

[0014] The detailed description of each of the above aspects of the present invention is as follows.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

[0016] Specific Embodiments In the present invention, a TCR molecule that specifically targets the KRAS G12V mutant antigen (particularly, the KRAS G12V mutant antigen shown in SEQ ID NO:16) was found. The 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 wild-type KRAS protein without mutation. Therefore, the TCR molecule of the present invention has strong specificity, and the T cells expressing the TCR molecule can reduce the toxic side effects after becoming drugs, do not damage normal non-cancer cells, and have broad applications in the treatment of tumors such as pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, and prostate cancer, especially pancreatic cancer, leading to the present invention.

[0017] The present invention will be described in detail below. It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features (such as embodiments) specifically described below can be combined with each other to form preferred technologies.

[0018] Definition of Terms In this specification, TCR has the meaning known in the art and is a cell membrane surface glycoprotein in which the α-chain / β-chain or γ-chain / δ-chain exists in the form of a heterodimer and is a characteristic mark on the surface of all T cells. The T cell receptor forms a T cell receptor complex with the constant CD3 molecule. TCR is a receptor after the major histocompatibility complex (MHC) presents an intracellular antigen peptide. The TCRs of many T cells are dimers composed of α and β peptide chains, and the TCRs of a small number of 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 is connected to the transmembrane region, and the variable region is responsible for the recognition of the polypeptide / MHC complex. The variable region contains three highly variable complementarity determining regions (CDRs), CDR1, CDR2, and CDR3. The most important CDR3 binds directly to the polypeptide presented by the 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 considered to be involved in the recognition of MHC. The β subunit usually does not participate in the recognition of the polypeptide / MHC complex but has another CDR4 that is involved in the action of superantigens.

[0019] In this specification, the major histocompatibility complex (MHC) is a gene family present in most vertebrate genomes and is an antigen-presenting and T-cell activating molecule. Among them, the human MHC glycoprotein is also called human leukocyte antigen (abbreviated 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 with a virus, the corresponding polypeptide fragment of the virus outer membrane is presented on the cell surface through the MHC molecule, and cytotoxic cells (CD8+ cytotoxic T cells) can recognize the cell infected with the virus and specifically kill it.

[0020] In this specification, amino acid residues are represented by 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 described according to a conventional method such that the amino terminus (hereinafter referred to as the N-terminus) is located on the left side and the carboxyl terminus (hereinafter referred to as the C-terminus) is located on the right side.

[0021] The term "T cell receptor targeting the KRAS G12V mutation (KRAS G12V"exogenous TCR" (exogenous T Cell Receptor) is defined herein as a TCR molecule that binds to a KRAS G12V mutant polypeptide / MHC complex and can induce 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.

[0022] The term "exogenous TCR" (exogenous T cell receptor) is defined herein as a recombinant TCR that is expressed intracellularly by introducing an exogenous coding sequence. The TCR targeting the KRAS G12V mutation provided in this specification is an "exogenous TCR" for human-derived T cells, which can be expressed in human-derived T cells, and the endogenous TCR naturally expressed by the T cells is insufficient to induce a cellular or responder response to TCR ligand binding.

[0023] In this specification, TCR-T cell therapy usually involves introducing an exogenous TCR gene into normal T cells so that the modified T cells can express a TCR that can efficiently recognize tumor cells, and inducing the T cells to kill tumor cells. The therapy usually includes administering to a patient T cells modified to express an exogenous TCR gene. The T cells are usually derived from the patient himself. Usually, the T cells are obtained from the patient, modified in vitro, and then re-infused into the patient after expressing an exogenous TCR gene (for example, the TCR gene described in any one of the embodiments of the present invention).

[0024] In this specification, the bispecific protein molecule is an artificial protein molecule 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) used to recruit and redirect immune cells around tumor cells. An exemplary single-chain antibody may be a single-chain antibody against CD3, and an exemplary immune cell may be a T cell. The TCR first recognizes and binds to the polypeptide / MHC on the surface of tumor cells. Subsequently, the anti-CD3 antibody fragment recruits and redirects immune cells around tumor cells. In this way, the bispecific protein molecule bridges cancer cells and immune cells, forms an immune synapse, activates immune cells to release lytic particles, and causes cancer cells to die.

[0025] Here, the identity of the sequences can be determined by methods well known to those skilled in the art. For example, using BLASTP, the identity of the sequences of two aligned amino acid sequences can be determined. A "conservative substitution" is known to those skilled in the art to be a substitution in which one or more amino acid residues are replaced by one or more amino acid residues having side chain R groups with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the function and properties of the protein. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl group 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 as follows according to the polarity of the amino acid side chain groups: 1. Non-polar amino acids (hydrophobic amino acids) including alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine; 2. Polar, uncharged amino acids (neutral amino acids) such as glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, selenocysteine, pyrrolysine, etc., and polar, positively charged amino acids (basic amino acids) including lysine, arginine, and histidine, which are hydrophilic amino acids; 3. Polar, negatively charged amino acids (acidic amino acids) including aspartic acid and glutamic acid.

[0026] As used herein, an immune cell refers to a cell involved in or related to an immune response, including lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, and mast cells. Exemplary immune cells include T cells, natural killer cells (NK), and tumor-infiltrating lymphocytes (TIL), etc., and their derived immune cells such as stem cells (e.g., hematopoietic stem cells (HSC) and induced pluripotent stem cells (iPS)).

[0027] T cell receptor (TCR) The characteristics of the TCR molecule targeting the KRAS G12V mutation of the present invention include that the CDR3 sequence of its α-chain variable region contains CAVRDIEGAGNNRKLIW (SEQ ID NO: 1) or a mutant of SEQ ID NO: 1, and / or the CDR3 sequence of its β-chain variable region contains CASSEGQYSYEQYF (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 to 5 (for example, 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: 1 as the CDR3 of the TCR α-chain variable region. Preferably, compared with SEQ ID NO: 2, the mutant of SEQ ID NO: 2 has 1 to 5 (for example, 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 the CDR3 of the TCR β-chain variable region. The mutations included in the mutants of SEQ ID NO: 1 and 2 may be selected from one or more of insertion, deletion and substitution. Preferably, the mutation is a conservative mutation, for example, a conservative substitution.

[0028] In some embodiments, the CDR1 sequence of the variable region of the TCR α-chain of the present invention comprises SVSGNP (SEQ ID NO:3) or a mutant thereof, and the CDR2 sequence comprises YITGDN (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 SNHLY (SEQ ID NO:5) or a mutant thereof, and the CDR2 sequence comprises FYNNEI (SEQ ID NO:6) or a mutant thereof. Each of the mutants of SEQ ID NO:3, 4, 5, and 6 may have mutations of 1, 2, or 3 amino acids compared to themselves, and includes one or more of insertion, deletion, and substitution, but is not limited thereto, and the mutations do not affect the biological functions and activities performed by these CDR sequences in the TCR molecule. Preferred mutations are conservative mutations, such as conservative substitutions.

[0029] In some embodiments, the CDR1 sequence of the variable region of the α-chain of the TCR molecule of the present invention is SVSGNP (SEQ ID NO:3), the CDR2 sequence is YITGDN (SEQ ID NO:4), and the CDR3 sequence is CAVRDIEGAGNNRKLIW (SEQ ID NO:1); and / or, the CDR1 sequence of the variable region of the β-chain is SNHLY (SEQ ID NO:5), the CDR2 sequence is FYNNEI (SEQ ID NO:6), and the CDR3 sequence is CASSEGQYSYEQYF (SEQ ID NO:2).

[0030] By incorporating the amino acid sequences of the CDR regions of the TCR molecule of the present invention into any suitable framework structure, a chimeric TCR can be produced. As long as the framework structure is compatible with the CDR regions of the TCR of the present invention, those skilled in the art can design or synthesize TCR molecules having corresponding functions with the CDR regions disclosed in the present invention. Therefore, the TCR molecule of the present invention means a TCR molecule that includes the CDR region sequences of the above α and / or β chains and in which the CDR region sequences of the present invention are adopted in any suitable framework structure.

[0031] 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 compared to the amino acid sequence shown in SEQ ID NO:7, or has at least 80%, at least 85%, at least 90%, preferably at least 95%, more preferably at least 98% sequence identity compared to the amino acid sequence shown in SEQ ID NO:7, or consists of the amino acid sequence. The number of amino acid residues of the mutation may be, for example, 1-15, for example 1-10 or 1-5 mutations; the mutation may be selected from one or more of insertion, deletion and substitution. The mutation may occur in any domain of SEQ ID NO:7, including occurring in its CDR and / or FR regions. In some embodiments, the mutation does not occur in the sequences of CDR1, CDR2 and CDR3 included in SEQ ID NO:7. In some embodiments, the mutation occurs, for example, in the FR region of SEQ ID NO:7. Preferably, the mutation is a conservative mutation, for example a conservative substitution.

[0032] 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 to 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 to the amino acid sequence shown in SEQ ID NO:8, or consists of the amino acid sequence. The number of mutated amino acid residues may be, for example, 1-15, for example 1-10 or 1-5 mutations; the mutation may be selected from one or more of insertion, deletion and substitution. The mutation may occur in any domain of SEQ ID NO:8, including occurring in its CDR and / or FR regions. 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, for example, in the FR region of SEQ ID NO:8. Preferably, the mutation is a conservative mutation, for example a conservative substitution.

[0033] The mutant of the TCR molecule of the present invention (that is, the mutant containing the variable region of the α-chain and / or the mutant containing the variable region of the β-chain) retains the biological activity that the TCR molecule containing SEQ ID NO:1 and SEQ ID NO:2 (particularly the TCR molecule containing SEQ ID NO:7 and 8) specifically binds to the KRAS G12V mutation (particularly the polypeptide shown in SEQ ID NO:16) depending on HLA-A*11:01.

[0034] In some embodiments, the TCR molecule of the present invention is a dimer consisting of an α and a β chain. The α chain contains a variable region and a constant region. The CDR1 sequence of the α chain variable region is SVSGNP (SEQ ID NO:3), the CDR2 sequence is YITGDN (SEQ ID NO:4), the CDR3 sequence is CAVRDIEGAGNNRKLIW (SEQ ID NO:1). The CDR1 sequence of the β chain variable region is SNHLY (SEQ ID NO:5), the CDR2 sequence is FYNNEI (SEQ ID NO:6), and the CDR3 sequence is CASSEGQYSYEQYF (SEQ ID NO:2). In some embodiments, the α chain variable region contains the amino acid sequence shown in SEQ ID NO:7 or the above-mentioned mutant sequence, and the β chain variable region contains the amino acid sequence shown in SEQ ID NO:8 or the above-mentioned mutant sequence. To form a more stable dimer of the TCR α and β chains, a disulfide bond may be introduced between the α and β chains to form a dimer.

[0035] 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 publicly available databases.

[0036] According to a study, adopting the constant region of a mouse-derived TCR instead of the constant region of a human-derived TCR can effectively avoid the problem that the exogenous T cell receptor rearranges with the self-TCR in the human body and binds to off-target or even incorrect targets. Therefore, in some embodiments, the TCR molecule of the present invention contains the α and β constant regions of a mouse. The exemplary 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.

[0037] In some embodiments, the TCR molecule of the present invention is a TCR molecule excluding the signal peptide sequence and transmembrane sequence of the variable region. An exemplary amino acid sequence of such an α-chain variable region is shown in SEQ ID NO:19; an exemplary amino acid sequence of such a β-chain variable region is shown in SEQ ID NO:20. The TCR molecule may include the α-constant region and β-constant region of the human TCR molecule. The present invention also includes mutants of SEQ ID NO:19 and 20, that is, compared with SEQ ID NO:19 and 20, each of their mutants has one or more (for example, 1-15, for example, 1-10 or 1-5) mutations, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 98% sequence identity. The mutations of the mutants may occur in any one or more of the CDRs (for example, any one or more of the above CDR mutants), or in the FR region. The mutations may be selected from one or more of insertion, deletion and substitution. Preferably, the mutations are conservative mutations, for example, conservative substitutions.

[0038] 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 includes a multimer formed by binding two, three, four or more TCR molecules of the present invention to each other.

[0039] Nucleic acid molecule The present invention provides a nucleic acid molecule encoding the α-chain variable region, β-chain variable region, α-chain, β-chain and TCR molecule described in any one of the embodiments herein.

[0040] The nucleotide sequence of the nucleic acid molecule of the present invention may be single-stranded or double-stranded, and the nucleic acid molecule may be RNA or DNA, and may or may not contain introns. The sequence of the nucleic acid molecule encoding the exemplary α-chain variable region of the present invention is shown in SEQ ID NO: 11. The polynucleotide sequence encoding the exemplary β-chain variable region of the present invention is shown in SEQ ID NO: 12. In some embodiments, the α-chain variable region and the β-chain variable region of the present invention are modified, and the signal peptide sequence and the transmembrane sequence are deleted, and their exemplary coding sequences are shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

[0041] In some embodiments, the TCR molecule of the present invention contains a human variable region and a mouse constant region. In the mouse constant region, the nucleic acid coding sequence of the α constant region is shown in SEQ ID NO: 17, and the nucleic acid coding sequence of the β constant region is shown in SEQ ID NO: 18.

[0042] It should be understood that due to the degeneracy of the genetic code, different nucleotide sequences can encode the same polypeptide. Therefore, the nucleic acid sequences encoding the α-chain variable region, β-chain variable region, α-chain, β-chain and TCR molecule of the present invention may be the same as the nucleic acid sequences shown in the present invention, or may be degenerate variants. Taking an example of 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.

[0043] In order to efficiently express TCR in T cells, the nucleotide sequence of the present invention can be optimized using the codon optimization method. The specific codon usage varies depending on the cell, and the expression level can be increased by changing the codons in the sequence according to the cell type. The codon selection tables of mammalian cells and many other organisms are well known to those skilled in the art.

[0044] The full-length nucleotide sequence of the nucleic acid molecule of the present invention or a fragment thereof can usually be obtained by, but not limited to, PCR amplification, recombination, or chemical synthesis. At present, the DNA sequence encoding the TCR (or a fragment or derivative thereof) of the present invention can already be obtained by complete chemical synthesis. Subsequently, the DNA sequence may be introduced into each existing DNA molecule (such as a vector), mRNA, and cells known in the art. The DNA or mRNA may be a coding strand or a non-coding strand.

[0045] Nucleic acid construct The present invention further includes a nucleic acid construct containing the nucleic acid molecule described in any one of the embodiments herein.

[0046] The construct herein may be an expression cassette containing a promoter sequence operably linked, the nucleic acid molecule described in any one of the embodiments herein, and a polyA tail. The expression cassette may also include other control elements operably linked to the above elements, such as enhancers.

[0047] In some embodiments, the above nucleic acid construct is a vector. Here, the vector includes, but is not limited to, expression vectors and cloning vectors. An expression vector is a vector for expressing the TCR of the present invention in vivo or in vitro, and a cloning vector is a vector used for manufacturing the nucleic acid molecule of the present invention. Expression vectors usually contain expression control elements. Expression control elements are well known in the art and include, but are not limited to, promoters and enhancers. In some embodiments, the vector contains the above expression cassette.

[0048] In this specification, the expression vector may be a related vector based on a viral delivery system, including but not limited to an adenovirus vector, an adeno-associated virus (AAV) vector, a herpesvirus vector, a retrovirus vector, a lentivirus vector, a baculovirus vector; or it may be a vector based on a non-viral delivery system, including but not limited to an expression vector based on a transposon, a vector based on a gene editing method, etc. Ideally, the appropriate vector transfers the TCR nucleic acid of the present invention into cells, for example, into T cells, so that the cells can express a TCR specific for the KRAS G12V mutant antigen.

[0049] Dual-target protein molecule In some embodiments, the present invention provides a dual-target protein molecule that can bind to tumor cells and immune cells (especially T cells) simultaneously. The dual-target protein molecule includes a TCR molecule that targets the KRAS G12V mutation on the surface of tumor cells described in any one of the embodiments herein, and a single-chain antibody (scFv) for recruiting and redirecting immune cells around tumor cells. The TCR molecule may include a variable region and a constant region of the α-chain of the TCR molecule and a variable region and a constant region of the β-chain, and the variable region and the constant region may be directly connected or connected by a flexible peptide chain. Usually, the signal peptide and transmembrane domain in the variable regions of the α-chain and β-chain are deleted; an exemplary amino acid sequence of such a variable region of the α-chain is shown in SEQ ID NO:19; an exemplary amino acid sequence of such a variable region of the β-chain is shown in SEQ ID NO:20.

[0050] The single-chain antibody is various interesting monoclonal antibodies having the above biological functions. An exemplary single-chain antibody may be a single-chain antibody against CD3, for example, a single-chain antibody against CD3 derived from the UCHT1 clone (US7994289B2, SEQ ID NO:21).

[0051] Generally, for the double-target protein molecule, the α-chain and β-chain of the TCR molecule form a heterodimer, and the scFv is linked to the N-terminus of the variable region of the β-chain of the TCR molecule. The structural schematic diagram of the double-target protein molecule is shown in FIG. 7. The scFv and the N-terminus of the β-chain variable region may be directly connected or connected by a flexible peptide chain.

[0052] In this specification, the flexible peptide chain may 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 of 2-30, such as 3-20 or 3-10 amino acid residues containing or consisting of G and S. The amino acid sequence of an exemplary linker is shown in SEQ ID NO:22.

[0053] To efficiently express the modified α-chain variable region and β-chain variable region of the double-target protein molecule of the present invention in Escherichia coli, the present invention optimizes the coding sequences of the α-chain variable region and β-chain variable region using Escherichia coli codons to obtain the coding sequences of the α-chain variable region and β-chain variable region shown in SEQ ID NO:23 and SEQ ID NO:24.

[0054] In some embodiments, in order to obtain a stable double-target 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. The artificial disulfide bonds introduced as described above may or may not be included in the constant region, and any TCR molecule of the present invention may be bound by the natural disulfide bonds present in the TCR.

[0055] Cell The present invention further relates to host cells genetically produced using the vectors or nucleic acid molecules of the present invention. The term "host cell" refers to any type of cell that contains the nucleic acid molecule or vector of the present invention, or expresses the TCR molecule or dual-target 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 nucleic acid molecule of the present invention being integrated into the chromosome, and / or expressing the TCR molecule and / or dual-target protein molecule described in any one of the embodiments herein.

[0056] Host cells suitable for expressing the TCR of the present invention include, but are not limited to, prokaryotic cells and eukaryotic cells, such as, for example, 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.

[0057] In some embodiments, the present invention particularly relates to immune cells, particularly T cells, that contain the vector of the present invention, or the nucleic acid molecule of the present invention is integrated into the chromosome, and / or express the TCR molecule described in any one of the embodiments herein. The T cells can be of any type and at any stage of development, 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 cells can be derived from CD8+ T cells isolated from a patient.

[0058] In some embodiments, the cells of the present invention may be other types of immune cells, such as natural killer cells (NK), tumor infiltrating lymphocytes (TIL), and induced immune cells. Since NK cells do not express CD3 molecules, even if a gene is transferred into NK cells, TCR will not be expressed on the cell surface. However, when NK cells are induced to differentiate or artificially constructed, the expression of CD3 molecules initiates the expression of TCR molecules in NK cells.

[0059] Drug composition and conjugate The present invention further provides a pharmaceutical composition containing T cells and a pharmaceutically acceptable carrier, wherein the T cells contain a vector expressing the TCR molecule described in any one of the embodiments herein, or a nucleic acid molecule encoding the TCR molecule described in any one of the embodiments of the present invention is integrated into its chromosome, and / or express the TCR molecule described in any one of the embodiments herein.

[0060] In some embodiments, the pharmaceutical composition of the present invention contains the TCR molecule or dual-target protein molecule described in any one of the embodiments herein and a pharmaceutically acceptable carrier.

[0061] As used herein, the pharmaceutically acceptable carrier can be selected based on the specific active ingredient. For example, the pharmaceutically acceptable carrier in a pharmaceutical composition containing T cells can be any suitable carrier known in the art suitable for cell therapy. The pharmaceutically acceptable carrier in a pharmaceutical composition containing the TCR molecule or dual-target protein molecule of the present invention can be a pharmaceutically acceptable carrier suitable for protein delivery.

[0062] As used herein, the pharmaceutical composition can be administered by any suitable route, such as parenteral, enteral, inhalation, intranasal routes, etc. The pharmaceutical composition of the present invention can be manufactured by methods known to those skilled in the art, for example, by mixing the active ingredient with the carrier or excipient under sterile conditions.

[0063] The effective amount of the active ingredient of the pharmaceutical composition of the present invention, such as the aforementioned T cells, TCR molecules or bispecific protein molecules, 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 those skilled in the art according to the actual situation. Generally, the appropriate dosage range of the soluble TCR of the present invention is between 25 ng / kg and 50 μg / kg.

[0064] The pharmaceutical composition of the present invention can be used for various therapeutic applications described below.

[0065] In some embodiments, the present invention provides a conjugate comprising a TCR molecule described herein and a therapeutic agent or tracer covalently or otherwise bound to the TCR molecule, which is used for the treatment or diagnosis of diseases, particularly tumors. The bound or conjugated therapeutic agent includes, but is not limited to, radionuclides, chemotherapeutic agents, antibody Fc or scFv fragments, nanoparticles, etc. Tracers used for diagnostic purposes include, but are not limited to, fluorescent markers, luminescent markers, radioactive markers, magnetic substances for MRI (magnetic resonance imaging), contrast agents for CT (computed tomography), enzymes of detectable products, etc.

[0066] Use and Treatment Method The present invention further provides the application of a TCR molecule, a bispecific protein molecule and a cell (particularly a T cell) described in any one of the embodiments herein in the preparation of a drug for treating or preventing a disease related to the KRAS G12V mutant antigen of a patient, and a TCR molecule, a bispecific protein molecule and a cell (particularly a T cell) described in any one of the embodiments herein for treating or preventing a disease related to the KRAS G12V mutant antigen.

[0067] The present invention further relates to a method for treating and / or preventing a disease associated with a patient's KRAS G12V mutant antigen, which comprises the step of adoptively transferring into the patient T cells expressing a TCR molecule described in any one of the embodiments herein, or a double-target protein molecule described in any one of the embodiments herein or a pharmaceutical composition containing the double-target protein molecule, wherein the T cells contain the vector of the present invention or the nucleic acid molecule of the present invention is integrated into the chromosome.

[0068] As used herein, the disease associated with the KRAS G12V mutant antigen is a tumor or cancer, and may include any of the following: acute lymphoblastic leukemia, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, retinal 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, ureteral cancer, 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.

[0069] Preferably, the tumor cells or cancer cells of the patient have 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.

[0070] By isolating T cells from a patient or volunteer having a disease associated with a KRAS G12V mutant antigen, the T cells can be modified ex vivo, and in order to express a TCR molecule described in any one of the embodiments herein, the T cells are caused to contain a vector described in any one of the embodiments of the present invention, or a molecule capable of expressing a TCR described in any one of the embodiments of the present invention is integrated into the genome of the T cells, and then these genetically modified cells are returned to the patient's body for treatment.

[0071] 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 comprises: (1) isolating the patient's T cells; and (2) modifying the T cells ex vivo so as to contain a vector described in any one of the embodiments of the present invention or so that a molecule capable of expressing a TCR described in any one of the embodiments of the present invention is integrated into the genome in order to express a TCR molecule described in any one of the embodiments herein.

[0072] The mode of administration, timing, dosage, etc. can be determined by a physician according to the situation of each patient, such as age, weight, general health condition, severity of the cancer to be treated, etc.

[0073] The TCR, polypeptide, protein, nucleic acid, recombinant expression vector and host cell (including a population thereof) of the present invention can be combined with other drug active agents or drugs to prepare a drug composition. The other drug active agents or drugs may be chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine. They may also be monoclonal antibody-based therapeutic drugs such as antibody drugs targeting immune checkpoints (such as CTLA-4, PD1, PD-L1, TIGIT, LAG3, TIM3, etc.) and immune regulatory element antibody drugs (such as 4-1BB, OX40, GITR, CD40, CD28, ICOS, CD47, etc.). Also included are other types of tumor treatment reagents, for example, oncolytic viruses and vaccines (including, but not limited to, mRNA, DNA, protein, protein subunits, cell components or cells, etc.).

Example

[0074] Hereinafter, the present invention will be further described with reference to specific examples. It should be understood that these examples are not intended to limit the scope of the present invention, but are merely illustrative of the present invention. Experimental methods that do not specify specific conditions in the following examples are usually carried out according to the normal conditions described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (Third Edition) (2001) (Cold Spring Harbor Laboratory Press), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts refer to weight. Unless otherwise specified, percentages and parts refer to weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise stated.

[0075] Example 1: Determination of TCR gene sequences targeting KRAS G12V mutation A functional cell population was selected from tumor-infiltrating lymphocytes (TILs) of a tumor patient with the KRAS G12V mutation (derived from the Department of General Surgery, Jinling Hospital Affiliated to Nanjing University School of Medicine), sequenced as single cells, and TCR sequences were obtained. After functional verification, TCR No. 051 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 the β variable region are shown in SEQ ID NO:8 and SEQ ID NO:12, respectively.

[0076] [Table 1]

[0077] Example 2: Construction of vectors for highly expressed TCR molecules 1. Vector information Using the pMSGV1 vector, the TCR molecule was overexpressed in T cells. The TCR nucleic acid sequence was optimized using human-derived 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, TCR expression in T cells was performed in a human-mouse heterologous binding manner. 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 α-chain and β-chain of TCR are connected in series using the SGSG-P2A sequence (amino acid sequence SEQ ID NO:25, nucleic acid sequence SEQ ID NO:26). The complete vector structure is shown in Figure 1. After the construction of the vector was completed, sequencing identification was performed. Refer to SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30 for the sequencing primers.

[0078] 2. Plasmid extraction After the sequencing was performed correctly, the plasmid was extracted and purified using NucleoBond Xtra Maxi (MACHEREY-NAGEL). The purified plasmid was measured for light absorption at 259 nm using an ultraviolet spectrophotometer to calculate the plasmid concentration, stored at -20°C, and used in subsequent experiments.

[0079] Example 3: Retrovirus packaging 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 culture flask, mix the cells well, and culture them overnight at 37°C. 2. Day 2: When the 293T cell confluence reached about 90%, transfection was performed. Plasmid complexes were prepared, and the amounts of each plasmid were pMSGV1-051 TCR 3μg, Gag-pol 1.9μg, and 10A1 0.75μg, respectively, and 300μL of DMEM was added. 20μL of EZ Trans Cell Transfection Reagent (Shanghai Liji Biology) and 300μL of DMEM were added. The PEI solution was added to the plasmid complexes and vortexed for 20s. The mixture was gently placed into a 293T culture flask along the edge and cultured at 37℃ for 16 hours, the medium was removed, and pre-warmed fresh medium was added again. 3. Day 4: After 48 hours of transfection, the supernatant was collected, filtered through a 0.45 μm filter, and then stored in small aliquots at -80 degrees.

[0080] Example 4: Infection of human T cells with retroviruses 1. The obtained PBMC cells were separated using Ficoll separation solution (Tianjin ●Yang) (● is Sansuinikei Page) and cultured at a cell density of 2 × 10 6 The cells were seeded onto a culture plate at 1 ml / well, and anti-human CD3 antibody (Takara) was added at 50 ng / ml, and interleukin 2 (Beijing Shuanglu) was added at 300 IU / ml. After 48 hours of stimulation and culture, the cells were infected with the virus. 2. The day after T cell activation culture, retronectin (Takara) diluted in PBS to a final concentration of 15 μg / ml was coated on a 24-well non-tissue treated culture plate at 300 μL per well overnight at 4°C in preparation for use. 3. After 2 days of T cell activation culture, the coated 24-well plate was removed, the coating solution was aspirated and discarded, and the virus solution was added to the wells, 1 ml of the virus solution was added per well, and the plate was centrifuged at 32°C and 2000 g for 2 hours.

[0081] 4. Discard the supernatant and inoculate each well of a 24-well plate with 5 × 10 activated T cells. 5Add in an amount of 1 ml, and the medium was the T cell medium supplemented with 300 IU / ml of IL-2. Centrifuge at 32 °C and 1000 g for 10 min. 5. After centrifugation was completed, the culture plates were cultured in a 37 °C, 5% CO2 incubator. 6. After cell infection, the T cell culture medium containing 100 IU / ml of IL-2 was supplemented in a timely manner to amplify the cells.

[0082] Example 5: Detection of TCR expression of T lymphocytes after infection by flow cytometry 1. The TCR-T cells and NT cells (control group) after infection were each collected by centrifugation, washed once with PBS, then the supernatant was discarded, the corresponding mTCR, CD8 antibody, and G12V 10mer tetramer were added, and after 30 min at 4 °C in the dark, they were washed with PBS, resuspended, and detected by flow cytometry. The results are shown in Figure 2. 2. The prepared TCR-T cells were counted, an appropriate amount was taken for centrifugation, and resuspended in X-VIVO medium containing 2% AB serum, and the cell concentration was adjusted to 2×10 6 / mL, and placed in a 96-well plate at 100 μL / well. 3. Four types of polypeptides (see SEQ ID NO: 13-16 for the sequences) were diluted in a 10-fold gradient, a total of 11 gradients, suspended in X-VIVO medium containing 2% AB serum, and added to the T cells at 100 μL / well, and the final concentration was 10 4 ng / ml to 10 -6 ng / ml.

[0083] 4. After thorough mixing, place in an incubator with 5% carbon dioxide and incubate at 37 °C for about 16 hours. 5. Detect mTCR+CD8+41BB+ cells by flow cytometry, and the results are shown in Figure 3. 6. Calculate the reaction EC 50 value of TCR-T and G12V 10mer. The results are shown in Figure 4, and the EC 50 was 0.9 ng / ml.

[0084] Example 6: Detection of TCR-T activation by different tumor cells 1. Count the prepared TCR-T cells, take an appropriate amount, perform centrifugation, resuspend in X-VIVO medium containing 2% AB serum, and adjust the cell concentration to 2×10 6 / mL. Place 100 μL / well into a 96-well plate. 2. Collect 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 and add them to the T cells at 100 μL / well. Use the CD3 antibody as the positive control and the blank medium as the negative control. 3. After thorough mixing, place in an incubator with 5% carbon dioxide and incubate at 37 °C for about 16 hours. 4. Detect mTCR+CD8+41BB+ cells by flow cytometry, and the results are shown in Figure 5.

[0085] Example 7: Experiment on the inhibition of pancreatic cancer cell growth in mice by TCR-T 1. For each 6- to 8-week-old female M-NSG mouse, inject 1×10 6 PANC1-luc-TMG cells subcutaneously to construct a pancreatic cancer tumor model. 2. Seven days later, randomly divide the mice into three groups: (1) TCR-T (A1101), (2) TCR-T (C0102), and (3) the untreated group. Inject 1×10 7 cells (recognizing HLA-A*11:01) intravenously into the mice in group (1), and inject 1×10 7 cells of TCR-T recognizing HLA-C*01:02 intravenously into the mice in group (2). Groups (2) and (3) were used as controls. Measure the tumor size weekly, and within two months after administering TCR-T, statistically analyze the tumor sizes of the mice in the three groups. The results are shown in Figure 6. As a result, it was found that compared with the two control groups, 051 TCR-T has an obvious effect of suppressing the growth of pancreatic cancer tumors.

[0086] Example 8: Expression, folding, and purification of the TCR-ICE protein molecule Based on recombinant DNA technology, after connecting the TCR sequence and the anti-CD3 single-chain antibody sequence of the present invention in series, a bifunctional protein molecule capable of coupling killer T cells and tumor cells was prepared. This was named TCR-ICE. The TCR-ICE molecule contains two chains, TCR-α and TCR-β, in the TCR sequence of the present invention (the sequences are shown in SEQ ID NO:19 and SEQ ID NO:20). However, the one in which the anti-CD3 antibody sequence (the sequence is shown in SEQ ID NO:21) is linked to the N-terminus of the β chain (Figure 7) was produced by an in vitro recombinant expression, folding, and purification method.

[0087] The sequences of the α and β chains of the 051-TCR-ICE molecule optimized for E. coli codons are shown in SEQ ID NO:23 and SEQ ID NO:24. After synthesizing cDNA, this was cloned into the pET21α vector. The 0TCR-ICE-α and 051-TCR-ICE-β-αCD3 expression vectors were each transformed into the E. coli strain BL21(DE3), and the E. coli was cultured and grown in LB medium, OD 600When it reached 0.8, IPTG with a final concentration of 1 mM was added to induce expression. After culturing at 37°C for 3 hours, bacterial inclusion bodies were collected. The inclusion bodies were washed twice with deionized water and then dissolved in buffer 20 mM Tris-Cl (pH 8.0), 6 M guanidine hydrochloride, 0.5 mM ethylenediaminetetraacetic acid (EDTA). After dissolution, TCR-ICE-α and TCR-ICE-β-αCD3 were mixed in equal amounts and rapidly diluted to a final concentration of 10 mg / ml with folding buffer (20 mM Tris-Cl (pH 8.0), 5 M urea, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 0.4 M arginine, 0.5 mM oxidized glutathione, 5 mM reduced glutathione), and folded overnight at 4°C. After centrifuging the product after overnight, the supernatant was dialyzed with 10 mM Tris-Cl (pH 8.0) buffer for 24 hours and repeated twice. The dialyzed product was purified for the target protein using an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare). Then, the product was further purified by gel filtration chromatography (Superdex 200 10 / 300 GL). The purity of the purified TCR-ICE product was identified by SDS-PAGE, and the results of SDS-PAGE are shown in Figure 8.

[0088] Example 9: Activation of T cells by TCR-ICE protein molecules Normally, in peripheral blood lymphocytes, when T cells are activated, they show the characteristic of highly expressing 4-1BB and OX40, and at the same time secrete gamma interferon. In this experiment, 4-1BB / OX40 and gamma interferon were used as indicators of T cell activation to detect the function of the TCR-ICE protein molecule prepared in Example 8. The effector cells are peripheral blood lymphocytes collected from healthy donors. The target cell lines are K562-TMG-A11 (expressing HLA-A1101 and KRAS G12V and K562, CFPAC1-A11 (expressing HLA-A1101, and the cells have KRAS G12V(with mutations), such as the CFPAC1 cell line. Among them, K562-TMG-A11 and CFPAN1-A11 are experimental cell lines, and K562 and CFPAN1 are negative control cell lines. Reagents related to this analysis include cell culture medium RPMI-1640, 10% FBS, D-PBS, and a human-derived γ-interferon ELISA detection kit. Antibodies: 4-1BB-APC, OX40-PE, and CD3-APC-Cy7. After co-culturing effector cells and target cells at a ratio of 4:1 overnight, the expression of 4-1BB and OX40 on the surface of effector cells was detected using a flow cytometer.

[0089] As a result of the experiment, as shown in Figure 9, the TCR-ICE protein molecule can efficiently activate T cells, and the activation effect shows concentration dependence. At the same time, for the supernatant of the co-culture of effector cells and target cells, the γ-interferon content in it was detected using a human-derived γ-interferon ELISA test kit. As a result, as shown in Figure 10, the target cell lines K562-TMG-A11 and CFPAN1-A11 alone cannot activate T cells, indicating that T cells secrete γ-interferon only in the presence of the TCR-ICE molecule. Similarly, the activation of T cells by TCR-ICE to secrete γ-interferon also shows concentration dependence. K562 and CFPAN1, which are target cells as negative controls, were unable to stimulate T cells. In the experimental group without adding TCR-ICE, after co-culturing effector cells and K562-TMG-A11, T cells could not be stimulated to secrete γ-interferon.

[0090] Example 10: Tumor growth inhibitory effect by TCR-ICE The inhibitory effect of TCR-ICE on the growth of mouse tumors was studied. A tumor model of CFPAC1-luc-GFP-A11 pancreatic cancer mice was constructed. 1×10 6 CFPAC1-luc-GFP-A11 cells were subcutaneously injected into 2 female M-NSG mice aged 6-8 weeks. After 40 days, 5×10 6Peripheral blood lymphocytes from healthy donors were injected at the cell / mouse dosage. After 24 hours, TCR-ICE prepared in Example 8 was administered at a dosage of 0.1 mg / kg, and a PBS solution was injected as a negative control. Thereafter, on days 2, 3, 4, and 5, TCR-ICE was continuously injected daily. The time of the last injection of TCR-ICE was recorded as Day0, and thereafter, the tumor size was recorded every 7 days.

[0091] The results are shown in Figure 11. The TCR-ICE injection group showed an effect of suppressing tumor growth from day 28. Thereafter, the growth of the in vivo tumors of the mice in the control group was obvious, and the tumors of the mice in the TCR-ICE injection group gradually decreased. TCR-ICE was injected for about 40 days, and the tumor shrank to the level before injection and then continued to shrink, indicating that TCR-ICE mediates tumor killing by T cells in the mouse body.

Claims

1. The T cell receptor (TCR) molecule specifically targets the KRAS G12V mutation, and the CDR3 sequence of its α-chain variable region contains CAVRDIEGAGNNRKLIW (SEQ ID NO: 1) or a mutant of SEQ ID NO: 1, and / or the CDR3 sequence of the β-chain variable region contains CASSQEYSYEQYF (SEQ ID NO: 2) or a mutant of SEQ ID NO: 2; provided that compared with SEQ ID NO: 1, the mutant of SEQ ID NO: 1 has 1 to 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 to 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; Preferably, in the TCR molecule: The CDR1 sequence of the α-chain variable region contains SVSGNP (SEQ ID NO: 3) or a mutant thereof, and the CDR2 sequence contains YITGDN (SEQ ID NO: 4) or a mutant thereof; and / or The CDR1 sequence of the β-chain variable region contains SNHLY (SEQ ID NO: 5) or a mutant thereof, and the CDR2 sequence contains FYNNEI (SEQ ID NO: 6) or a mutant thereof; A T cell receptor (TCR) molecule characterized by the above.

2. The α-chain variable region of the TCR molecule contains the amino acid sequence shown in SEQ ID NO: 7, or includes 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 includes 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; However, the TCR molecule containing the mutation retains the biological activity of the TCR molecule containing SEQ ID NO: 7 and SEQ ID NO: 8 to specifically target the KRAS G12V mutant polypeptide The TCR molecule according to claim 1, characterized in that...

3. The variable region of the α-chain of the TCR molecule contains the amino acid sequence shown in SEQ ID NO: 19, or includes an amino acid sequence having one or more mutations as compared with the amino acid sequence shown in SEQ ID NO: 19, 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: 20, or includes an amino acid sequence having one or more mutations as compared with the amino acid sequence shown in SEQ ID NO: 20, or consists of the amino acid sequence; However, the TCR molecule containing the mutation retains the biological activity that the TCR molecule containing SEQ ID NO: 19 and SEQ ID NO: 20 specifically targets the KRAS G12V mutant polypeptide. The TCR molecule according to claim 1, characterized in that...

4. The TCR molecule contains a constant region of a mouse; preferably, the amino acid sequence of the α-constant region of the mouse 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 TCR molecule according to any one of claims 1 to 2.

5. A multivalent TCR complex, characterized by containing the TCR molecule according to any one of claims 1 to 4.

6. The dual-target protein molecule includes a TCR molecule that targets the KRAS G12V mutation on the surface of tumor cells according to any one of claims 1 to 4, and a single-chain antibody (scFv) for recruiting and redirecting immune cells around tumor cells. However, the signal peptide and transmembrane domain in the variable region of the α-chain and β-chain of the TCR molecule are deleted; Preferably, the amino acid sequence of the variable region of the α-chain of the TCR molecule is shown in SEQ ID NO: 19, and the amino acid sequence of the variable region of the β-chain is shown in SEQ ID NO: 20; Preferably, the single-chain antibody is a single-chain antibody against CD3. A dual-target protein molecule capable of simultaneously binding tumor cells and immune cells, characterized by...

7. The nucleic acid molecule includes a nucleic acid sequence encoding the TCR molecule according to any one of claims 1 to 4 or the dual-target protein molecule according to claim 6, or its complementary sequence; Preferably, the nucleic acid sequence of the nucleic acid molecule is selected from SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 23 and SEQ ID NO: 24 A nucleic acid molecule characterized by the above. **Claim 8** 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 A nucleic acid construct characterized by the above. **Claim 9** The cell is (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 to 4 or the dual-target 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 An isolated cell characterized by the above. **Claim 10** A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR molecule according to any one of claims 1 to 4, the TCR complex according to claim 5, the dual-target 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.

Images