High-affinity TCR that recognizes the SSX2 antigen

Abstract

The present invention provides a T cell receptor (TCR) having the property of binding to the KASEKIFYV-HLA A0201 complex, wherein the binding affinity of the TCR to the KASEKIFYV-HLA A0201 complex is at least twice as high as the binding affinity of a wild-type TCR to the KASEKIFYV-HLA A0201 complex. The TCR can be used alone or in combination with a therapeutic agent to target tumor cells that present the KASEKIFYV-HLA A0201 complex.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, more specifically to a T cell receptor (TCR) capable of recognizing a polypeptide derived from the SSX2 protein. The present invention further relates to the preparation and use of said receptor. [Background technology]

[0002] Only two types of molecules can specifically recognize antigens: immunoglobulins or antibodies, and T cell receptors (TCRs), which are glycoproteins present on the cell membrane surface in the form of α / β or γ / δ chain heterodimers. The components of the immune system's TCR repertoire are generated in the thymus by V(D)J recombination followed by positive and negative selection. In the peripheral environment, TCRs mediate the specific recognition of T cells against major histocompatibility complex-peptide complexes (pMHC) and are therefore crucial for the cellular immune function of the immune system.

[0003] TCR is the only receptor for specific antigen peptides presented by major histocompatibility complex (MHC), and such exogenous or endogenous peptides may be the only sign of abnormal cells. In the immune system, the binding of antigen-specific TCR to pMHC complexes causes direct physical contact between T cells and antigen-presenting cells (APCs), which then interacts with other cell membrane surface molecules on both T cells and APCs, which triggers a series of subsequent cell signaling and other physiological responses, thus allowing T cells with different antigen specificities to exert immune effects on target cells.

[0004] The molecular ligands for TCRs, MHC I and II, are also proteins of the immunoglobulin superfamily, but they have specificity in antigen presentation. Different individuals have different MHCs, which allow them to present different short peptides of protein antigens on the surface of their APC cells. Human MHCs are usually called HLA genes or HLA complexes.

[0005] SSX2 is a synovial sarcoma X breakpoint protein, also known as HOM-MEL-40. SSX2 is one of 10 highly identical nucleic acid proteins in the SSX family. The SSX protein is a tumor-testis antigen and is expressed only in tumor cells and testicular blasts without MHC expression. SSX2 is expressed in a variety of human cancer cells, including, but not limited to, liver cancer, lung cancer, fibrosarcoma, breast cancer, colon cancer, and prostate cancer. After purification in cytoplasmic cells, SSX2 is degraded into small polypeptides, which bind to MHC (major histocompatibility complex) molecules to form complexes and are displayed on the cell surface. KASEKIFYV is a short peptide derived from the SSX2 antigen and is a target for the treatment of SSX2-related diseases.

[0006] Thus, the KASEKIFYV-HLA A0201 complex provides a marker by which TCRs can target tumor cells. TCRs capable of binding to the KASEKIFYV-HLA A0201 complex have high application value for tumor treatment. For example, TCRs capable of targeting tumor cell markers can be used to deliver cytotoxic or immunostimulatory agents to target cells or to transform T cells expressing the TCR so that they can destroy tumor cells, for administration to patients in a treatment process known as adoptive immunotherapy. For the former purpose, an ideal TCR would have a higher affinity, allowing the TCR to persist on target cells for a longer period of time. For the latter purpose, it is preferable to use a TCR with intermediate affinity. Therefore, those skilled in the art are devoting themselves to developing TCRs that can be used to target tumor markers for various purposes. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a TCR having higher affinity for the KASEKIFYV-HLA A0201 complex. Another object of the present invention is to provide a method for preparing TCRs of the above kind and uses of TCRs of the above kind. [Means for solving the problem]

[0008] A first aspect of the present invention provides a T cell receptor (TCR) that has the activity of binding to the KASEKIFYV-HLA A0201 complex. In another preferred embodiment, the T cell receptor (TCR) has an activity of binding to the KASEKIFYV-HLA A0201 complex, the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, the TCR α chain variable domain comprises three CDR regions, and the reference sequences of the three CDR regions of the TCR α chain variable domain are: CDR1α:DSSSTY, CDR2α:IFSNMDM, CDR3α:AEPNQAGTALI, containing at least one of the following mutations: [Table 1]

[0009] and / or the TCR β chain variable domain comprises three CDR regions, and the reference sequences of the three CDR regions of the TCR β chain variable domain are: CDR1β: MNHEY, CDR2β:SVGEGT, CDR3β: ASSSLEDPYEQY and contains at least one of the following mutations: [Table 2]

[0010] In another preferred embodiment, the affinity of the TCR for the KASEKIFYV-HLA A0201 complex is at least twice that of the wild-type TCR. In a preferred embodiment of the present invention, the affinity of the TCR for the KASEKIFYV-HLA A0201 complex is at least 2-fold, preferably at least 5-fold, more preferably at least 10-fold higher than that of the wild-type TCR.

[0011] In another preferred embodiment, the affinity of the TCR for the KASEKIFYV-HLA A0201 complex is at least 50 times, preferably at least 100 times, more preferably at least 1000 times, greater than that of the wild-type TCR. In another preferred embodiment, the affinity of the TCR for the KASEKIFYV-HLA A0201 complex is at least 10 times that of a wild-type TCR. 4 times, preferably at least 10 5 times, more preferably at least 5 x 10 5 It's double.

[0012] Specifically, the dissociation equilibrium constant of the TCR for the KASEKIFYV-HLA A0201 complex is K D ≦20 μM. In another preferred embodiment, the dissociation equilibrium constant of the TCR for the KASEKIFYV-HLA A0201 complex is 5 μM≦K D ≦10 μM, preferably 0.1 μM≦K D ≦1 μM, more preferably 1 nM≦K D ≦100 nM, more preferably 10 pM≦K D ≦100 pM.

[0013] In another preferred example, the TCR is an αβ heterodimeric TCR, and the TCR α chain variable domain comprises an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 94% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the amino acid sequence set forth in SEQ ID NO:1, and / or the TCR β chain variable domain comprises an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 94% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the amino acid sequence set forth in SEQ ID NO:2.

[0014] In another preferred embodiment, the mutations are made in one or more CDR regions of the α-chain and / or β-chain variable domains. In another preferred example, the number of mutations in the three CDR regions of the TCR α chain variable domain is 1 to 11, and / or the number of mutations in the three CDR regions of the TCR β chain variable domain is 1 to 8.

[0015] In another preferred example, the number of mutations in the CDR regions of the TCR α chain may be 3, 4, 5, 6, 7, 8, 9, 10 or 11. In another preferred example, the number of mutations in the CDR region of the TCR β chain may be one, two, three, four, five, six, seven or eight.

[0016] In another preferred example, the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, the TCR α chain variable domain comprises CDR1α, CDR2α and CDR3α, and the TCR β chain variable domain comprises CDR1β, CDR2β and CDR3β, wherein the amino acid sequence of CDR1β is MNHEY.

[0017] In another preferred example, the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, the TCR α chain variable domain comprises CDR1α, CDR2α and CDR3α, and the TCR β chain variable domain comprises CDR1β, CDR2β and CDR3β, wherein the amino acid sequence of the CDR3β is AS[3βX1][3βX2][3βX3][3βX4]DP[3βX5][3βX6][3βX7][3βX8], wherein [3βX1] is A or S, and / or Or [3βX2] is S or D, and / or [3βX3] is L or I or V, and / or [3βX4] is E or Q or T, and / or [3βX5] is Y or F, and / or [3βX6] is E or I or P or V, and / or [3βX7] is Q or K or L or M or V, and / or [3βX8] is Y or A or E or I or L or N or Q or R or S or T or V.

[0018] In another preferred example, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the TCR alpha chain variable domain comprises CDR1 alpha, CDR2 alpha and CDR3 alpha, and the TCR beta chain variable domain comprises CDR1 beta, CDR2 beta and CDR3 beta, wherein CDR3 beta is selected from the group consisting of ASSSLEDPFVKR, ASADVQDPYEQY, ASSSLEDPYEQY, ASADIQDPYEQY, ASSSLEDPYVMT, ASSSLEDPYIKY, ASSSLEDPYIVT, ASSSLEDPYVKI, ASSSLEDPYIKT, ASSSLEDPYILN, ASSSLEDPYILT, ASSDVTDPYEQY, ASSSLEDPYIMA, ASSSLEDPYVLE, ASSSLEDPYPML, ASSSLEDPYVVS, ASSSLEDPYVMV and ASSSVEDPYEQY.

[0019] In another preferred example, the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, the TCR α chain variable domain comprises CDR1α, CDR2α, and CDR3α, and the TCR β chain variable domain comprises CDR1β, CDR2β, and CDR3β, wherein the amino acid sequence of CDR2β is selected from SVGEGT, SLEVGT, HDWLGT, and SDWLGT. Preferably, the amino acid sequence of CDR2β is SVGEGT.

[0020] In another preferred example, the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, the TCR α chain variable domain comprises CDR1α, CDR2α and CDR3α, and the TCR β chain variable domain comprises CDR1β, CDR2β and CDR3β, wherein the amino acid sequence of CDR1α is selected from the group consisting of DSSSTY, DPWATY, DRMSTY, DTMSTY and DKMSTY.

[0021] In another preferred example, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the TCR alpha chain variable domain comprises CDR1 alpha, CDR2 alpha and CDR3 alpha, and the TCR beta chain variable domain comprises CDR1 beta, CDR2 beta and CDR3 beta, wherein the amino acid sequence of CDR2 alpha is selected from the group consisting of IFSNMDM, IFSYQST, IFSYMTE and IFSYQSE.

[0022] In another preferred example, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the TCR alpha chain variable domain comprises CDR1 alpha, CDR2 alpha and CDR3 alpha, and the TCR beta chain variable domain comprises CDR1 beta, CDR2 beta and CDR3 beta, wherein the amino acid sequence of CDR3 alpha is selected from the group consisting of AEPNQAGTALI, AEPNNSHSALI, AEPNASHSALI, AEPNKSHSALI, AEPNSSHSALI and EPNQSHSALI.

[0023] In another preferred example, the TCR alpha chain variable domain comprises CDR1 alpha, CDR2 alpha and CDR3 alpha, wherein the amino acid sequence of CDR1 alpha is DSSSTY, the amino acid sequence of CDR2 alpha is IFSNMDM and the amino acid sequence of CDR3 alpha is AEPNQAGTALI. In another preferred embodiment, the amino acid sequence of the TCR α chain variable domain is SEQ ID NO:1.

[0024] In another preferred embodiment, the mutations are made in one or more CDR regions of the α-chain and / or β-chain variable domains. In another preferred embodiment, the mutations occur in CDR1 and / or CDR2 and / or CDR3 of the α chain, and / or the mutations occur in CDR2 and / or CDR3 of the β chain.

[0025] In a preferred embodiment of the present invention, the T cell receptor (TCR) has an activity of binding to the KASEKIFYV-HLA A0201 complex, and comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the TCR is mutated in the alpha chain variable domain shown in SEQ ID NO: 1, and the mutated amino acid residue positions include one or more of 27S, 28S, 29S, 52N, 53M, 54D, 55M, 94Q, 95A, 96G, and 97T, wherein the amino acid residue numbers are adopted as the beta chain variable domain numbers shown in SEQ ID NO: 1, and / or the TCR is mutated in the alpha chain variable domain shown in SEQ ID NO: 1. Mutations are generated in the beta chain variable domain shown in SEQ ID NO:2, and the mutated amino acid residue positions include one or more of 49S, 50V, 51G, 52E, 94S, 95S, 96L, 97E, 100Y, 101E, 102Q, and 103Y, where the amino acid residue numbers are as shown in SEQ ID NO:2.

[0026] Preferably, the TCR alpha chain variable domain after mutation comprises one or more amino acid residues selected from the group consisting of 27K or 27P or 27R or 27T, 28M or 28W, 29A, 52Y, 53Q, 54S or 54T, 55E or T, 94A or 94D or 94E or 94K or 94N or 94R or 94S or 94T, 95S or 95V, 96H or 96Q or 96T and 97S, wherein the numbering of the amino acid residues is as shown in SEQ ID NO: The TCR β chain variable domain after adopting the numbering shown in SEQ ID NO:1 and / or mutation comprises one or more amino acid residues selected from the group consisting of 49H, 50D or 50L, 51E or 51W, 52L or 52V, 94A, 95D, 96I or 96V, 97Q or 97T, 100F, 101I or 101P or 101V, 102K or 102L or 102M or 102V and 103A or 103E or 103I or 103L or 103N or 103Q or 103R or 103S or 103T or 103V, wherein the numbering of the amino acid residues is adopted as shown in SEQ ID NO:2.

[0027] In another preferred embodiment, the TCR has a CDR selected from the following group: [Table 3-1] [Table 3-2]

[0028] In another preferred embodiment, the TCR is soluble. In another preferred embodiment, the TCR is an αβ heterodimeric TCR or a single chain TCR. In another preferred example, the TCR of the present invention is an αβ heterodimer TCR, and preferably, the TCR has an α chain constant region sequence TRAC*01 and a β chain constant region sequence TRBC1*01 or TRBC2*01.

[0029] In another preferred example, the TCR is an αβ heterodimeric TCR, and the TCR α chain variable domain comprises an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 94% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the amino acid sequence set forth in SEQ ID NO:1, and / or the TCR β chain variable domain comprises an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 94% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the amino acid sequence set forth in SEQ ID NO:2.

[0030] In another preferred example, the TCR comprises (i) all or a portion of the TCR alpha chain excluding its transmembrane domain, and (ii) all or a portion of the TCR beta chain excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least a portion of the constant domain of the TCR chain. In another preferred example, the TCR is an αβ heterodimer TCR, and an artificial interchain disulfide bond is contained between the α chain variable region and the β chain constant region of the TCR.

[0031] In another preferred embodiment, the cysteine ​​residue that forms an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR is the amino acid at position 46 of TRAV and the amino acid at position 60 of TRBC1*01 or TRBC2*01 exon 1; the amino acid at position 47 of TRAV and the amino acid at position 61 of TRBC1*01 or TRBC2*01 exon 1; the amino acid at position 46 of TRAV and the amino acid at position 61 of TRBC1*01 or TRBC2*01 exon 1; Alternatively, one or more sites selected from the group consisting of the amino acid at position 47 of TRAV and the amino acid at position 60 of exon 1 of TRBC1*01 or TRBC2*01 are substituted.

[0032] In another preferred example, a TCR containing an artificial interchain disulfide bond between the α chain variable region and the β chain constant region comprises an α chain variable domain and a β chain variable domain, and all or part of the β chain constant domain excluding the transmembrane domain, but it does not include the α chain constant domain, and the TCR α chain variable domain forms a heterodimer with the β chain.

[0033] In another preferred example, a TCR containing an artificial interchain disulfide bond between the α chain variable region and the β chain constant region comprises (i) all or a portion of the TCR α chain excluding its transmembrane domain, and (ii) all or a portion of the TCR β chain excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least a portion of the constant domain of the TCR chain.

[0034] In another preferred example, the TCR is an αβ heterodimeric TCR comprising (i) all or part of the TCR α chain excluding its transmembrane domain, and (ii) all or part of the TCR β chain excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain, and an artificial interchain disulfide bond is included between the α chain constant region and the β chain constant region. In another preferred example, an artificial interchain disulfide bond is contained between the α chain constant region and the β chain constant region of the TCR.

[0035] In another preferred embodiment, the cysteine ​​residue that forms an artificial interchain disulfide bond between the constant region of the α chain and the constant region of the β chain of the TCR is Thr48 in TRAC*01 exon 1 and Ser57 in TRBC1*01 or TRBC2*01 exon 1; Thr45 in TRAC*01 exon 1 and Ser77 in TRBC1*01 or TRBC2*01 exon 1; Tyr10 of TRAC*01 exon 1 and Ser17 of TRBC1*01 or TRBC2*01 exon 1; Thr45 in TRAC*01 exon 1 and Asp59 in TRBC1*01 or TRBC2*01 exon 1; Ser15 of TRAC*01 exon 1 and Glu15 of TRBC1*01 or TRBC2*01 exon 1; Arg53 in TRAC*01 exon 1 and Ser54 in TRBC1*01 or TRBC2*01 exon 1; Pro89 in TRAC*01 exon 1 and Ala19 in TRBC1*01 or TRBC2*01 exon 1; and Tyr10 of TRAC*01 exon 1 and Glu20 of TRBC1*01 or TRBC2*01 exon 1.

[0036] In another preferred example, the amino acid sequence of the TCR α chain variable domain is selected from SEQ ID NOs: 64 to 87, and / or the amino acid sequence of the TCR β chain variable domain is selected from SEQ ID NOs: 88 to 114.

[0037] In another preferred embodiment, the TCR is selected from the following group: [Table 4-1] [Table 4-2]

[0038] In another preferred embodiment, the TCR is a single chain TCR. In another preferred example, the TCR is a single-chain TCR consisting of an α chain variable domain and a β chain variable domain, and the α chain variable domain and the β chain variable domain are linked by a flexible short peptide sequence (linker).

[0039] In another preferred embodiment, mutations are generated in the hydrophobic core of the TCR α chain variable domain and / or β chain variable domain. In another preferred example, the TCR in which the hydrophobic core is mutated is a single-chain TCR composed of an α variable domain and a β variable domain, the α variable domain and the β variable domain being linked by a flexible short peptide sequence (linker).

[0040] In another preferred example, the TCR of the present invention is a single-chain TCR, wherein the TCR alpha chain variable domain comprises an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 92%, and most preferably at least 94% sequence identity (e.g., it may be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence shown in SEQ ID NO:3, and / or the TCR beta chain variable domain comprises an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 92%, and most preferably at least 94% sequence identity (e.g., it may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence shown in SEQ ID NO:4.

[0041] In another preferred example, the amino acid sequence of the TCR α chain variable domain is selected from SEQ ID NOs: 9 to 32, and / or the amino acid sequence of the TCR β chain variable domain is selected from SEQ ID NOs: 33 to 59.

[0042] In another preferred embodiment, the TCR is selected from the following group: [Table 5-1] [Table 5-2]

[0043] In another preferred example, the conjugate is attached to the C-terminus or N-terminus of the α chain and / or β chain of the TCR. In another preferred embodiment, the conjugate that binds to the TCR is a detectable marker, a therapeutic agent, a PK-modifying moiety, or any combination of these substances. In another preferred embodiment, the therapeutic agent that binds to the TCR is an anti-CD3 antibody linked to the C-terminus or N-terminus of the α or β chain of the TCR.

[0044] A second aspect of the present invention provides a multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR according to the first aspect of the invention.

[0045] A third aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding a TCR molecule according to the first aspect of the invention or a multivalent TCR complex according to the second aspect of the invention, or a complementary sequence thereof.

[0046] A fourth aspect of the present invention provides a vector, said vector comprising a nucleic acid molecule according to the third aspect of the invention.

[0047] A fifth aspect of the present invention provides a host cell, wherein the host cell comprises a vector according to the fourth aspect of the invention or has chromosomally integrated therein an exogenous nucleic acid molecule according to the third aspect of the invention.

[0048] A sixth aspect of the invention provides an isolated cell, said cell expressing a TCR according to the first aspect of the invention.

[0049] A seventh aspect of the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention.

[0050] An eighth aspect of the present invention provides a method of treating a disease comprising administering to a subject in need thereof an appropriate amount of a TCR according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention.

[0051] A ninth aspect of the present invention provides the use of a TCR according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention, for use in the preparation of a medicament for treating a tumour, preferably the tumour is an SSX2-positive tumour.

[0052] A tenth aspect of the present invention provides a TCR according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention, for use as a medicament for treating a tumour.

[0053] An eleventh aspect of the present invention is a method for manufacturing a semiconductor device comprising: (i) expressing a T cell receptor according to the first aspect of the invention by culturing a host cell according to the fifth aspect of the invention; (ii) isolating or purifying said T cell receptor. [Effects of the Invention]

[0054] 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 (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief explanation of the drawings]

[0055] [Figure 1a-1b] The amino acid sequences of the wild-type TCR α and β chain variable domains, each capable of specifically binding to the KASEKIFYV-HLA A0201 complex, are shown. [Figure 2a-2b] 1 shows the amino acid sequences of the single-chain template TCR α chain variable domain and β chain variable domain constructed according to the present invention, respectively. [Figure 3a-3b] 1 shows the DNA sequences of the single-chain template TCR α chain variable domain and β chain variable domain constructed according to the present invention, respectively.

[0056] [Figure 4a-4b] 1 shows the amino acid sequence and nucleotide sequence of the linker of the single-chain template TCR constructed according to the present invention. [Figures 5(1)-5(24)] The amino acid sequences of the single-chain TCR α chain variable domains with high affinity for the KASEKIFYV-HLA A0201 complex are shown, with mutated residues underlined. [Figure 6(1)-6(27)] The amino acid sequences of the single-chain TCR β chain variable domains with high affinity for the KASEKIFYV-HLA A0201 complex are shown, with mutated residues underlined.

[0057] [Figure 7a-7b] 1 shows the amino acid sequence and DNA sequence of the single-chain template TCR constructed according to the present invention. [Figure 8a-8b] 1 shows the amino acid sequences of the soluble reference TCR α and β chains of the present invention, respectively. [Figure 9(1)-9(24)] The amino acid sequences of the heterodimeric TCR α chain variable domains with high affinity for the KASEKIFYV-HLA A0201 complex are shown, with mutated residues underlined.

[0058] [Figures 10(1)-10(27)] The amino acid sequences of the heterodimeric TCR β chain variable domains with high affinity for the KASEKIFYV-HLA A0201 complex are shown, with mutated residues underlined. [Figure 11a-11b] The extracellular amino acid sequences of the wild-type TCR α and β chains, each capable of specifically binding to the KASEKIFYV-HLA A0201 complex, are shown. [Figure 12a-12b] The amino acid sequences of the wild-type TCR α and β chains that can specifically bind to the KASEKIFYV-HLA A0201 complex are shown.

[0059] [Figure 13] Binding curve of wild-type TCR to soluble reference TCR, i.e., KASEKIFYV-HLA A0201 complex. [Figure 14] 1 shows the results of an IFN-γ activation function experiment of effector cells transfected with the high affinity TCR of the present invention (target cells are tumor cell lines). [Figures 15a-15e] 1 shows the results of IFN-γ activation functional experiments of effector cells transfected with the high affinity TCR of the present invention (target cells are short peptide loaded T2).

[0060] [Figures 16a-16c] 1 shows the results of an IncuCyte killing function experiment of effector cells transfected with the high affinity TCR of the present invention. [Figure 17] 1 shows the results of an LDH killing function experiment of effector cells transfected with the high affinity TCR of the present invention. [Figures 18a-18d] 1 is a graph showing the results of an experiment redirecting fusion proteins to effector cells. DETAILED DESCRIPTION OF THE INVENTION

[0061] Through extensive and detailed research, the present invention has obtained a high-affinity T cell receptor (TCR) that recognizes KASEKIFYV short peptides (derived from AFP protein), which are presented in the form of peptide-HLA A0201 complexes. CDR1α:DSSSTY, CDR2α:IFSNMDM, CDR3α: mutations are generated in the three CDR regions of the α chain variable domain of AEPNQAGTALI, and / or CDR1β: MNHEY, CDR2β:SVGEGT, Mutations are generated in the three CDR regions of the β-chain variable domain of CDR3β:ASSSLEDPYEQY, and the affinity and / or binding half-life of the TCR of the present invention for the above KASEKIFYV-HLA A0201 complex after mutation is at least twice that of the wild-type TCR.

[0062] Before the present invention is described, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.

[0064] term T cell receptor (TCR) The International Immunogenetics Information System (IMGT) can be used to describe TCRs. Naturally occurring αβ heterodimeric TCRs have an α chain and a β chain. Roughly speaking, each chain contains a variable region, a linker region, and a constant region. The β chain usually contains a short variable region between the variable region and the linker region, but the variable region is often considered part of the linker region. The linker region of the TCR is determined via the unique IMGT TRAJ and TRBJ, and the constant region of the TCR is determined via the IMGT TRAC and TRBC.

[0065] Each variable region contains three CDRs (complementarity-determining regions), CDR1, CDR2, and CDR3, fitted to a framework sequence. In the IMGT nomenclature, the different numbers TRAV and TRBV refer to different Vα and Vβ types, respectively. In the IMGT system, the α chain constant domain is designated TRAC*01, where "TR" represents the T cell receptor gene, "A" represents the α chain gene, C represents the constant region, and "*01" represents allele 1. The β chain constant domain is designated TRBC1*01 or TRBC2*01, where "TR" represents the T cell receptor gene, "B" represents the β chain gene, C represents the constant region, and "*01" represents allele 1. The α chain constant region is uniquely determined, and in the β chain form, there are two possible constant region genes, "C1" and "C2." Those skilled in the art can obtain the constant region gene sequences of the TCR α and β chains through the publicly available IMGT database.

[0066] The α and β chains of TCRs are generally considered to have two "domains," a variable domain and a constant domain, respectively. The variable domain is composed of a linked variable region and a linker region. Thus, in the specification and claims of this application, the "TCR α chain variable domain" refers to the linked TRAV and TRAJ regions, and similarly, the "TCR β chain variable domain" refers to the linked TRBV and TRBD / TRBJ regions. The three CDRs of the TCR α chain variable domain are CDR1α, CDR2α, and CDR3α, respectively, and the three CDRs of the TCR β chain variable domain are CDR1β, CDR2β, and CDR3β, respectively. The framework sequences of the TCR variable domains of the present invention are derived from mice or humans, preferably humans. The constant domain of a TCR comprises an intracellular portion, a transmembrane region, and an extracellular portion. To obtain a soluble TCR and measure the affinity between the TCR and the KASEKIFYV-HLA A0201 complex, the TCRs of the present invention preferably do not comprise a transmembrane region. More preferably, the amino acid sequence of the TCR of the present invention refers to the extracellular amino acid sequence of the TCR.

[0067] The TCR sequences used in the present invention are of human origin. As shown in Figures 12a and 12b, the α-chain amino acid sequence and β-chain amino acid sequence of the "wild-type TCR" in the present invention are SEQ ID NO:117 and SEQ ID NO:118, respectively. As shown in Figures 8a and 8b, the α-chain amino acid sequence and β-chain amino acid sequence of the "reference TCR" in the present invention are SEQ ID NO:62 and SEQ ID NO:63, respectively. As shown in Figures 11a and 11b, the α-chain and β-chain extracellular amino acid sequences of the "wild-type TCR" in the present invention are SEQ ID NO:115 and SEQ ID NO:116, respectively. In the present invention, as shown in Figures 1a and 1b, the amino acid sequences of the α-chain and β-chain variable domains of the wild-type TCR capable of binding to the KASEKIFYV-HLA A0201 complex are SEQ ID NO:1 and SEQ ID NO:2, respectively. In the present invention, the terms "polypeptide of the present invention", "TCR of the present invention", and "T cell receptor of the present invention" are used interchangeably.

[0068] In a preferred embodiment of the present invention, the TCR according to the present invention comprises a TCR α chain variable domain and a TCR β chain variable domain, wherein the TCR α chain variable domain comprises CDR1α, CDR2α, and CDR3α, and the TCR β chain variable domain comprises CDR1β, CDR2β, and CDR3β.

[0069] In another preferred example, the CDR1α comprises the sequence D[1αX1][1αX2][1αX3]TY, where [1αX1], [1αX2], and [1αX3] are each independently selected from any naturally occurring amino acid residue. In another preferred example, the [1αX1] is S, K, P, R, or T.

[0070] In another preferred example, the [1αX2] is S, M, or W. In another preferred example, the [1αX3] is S or A. In another preferred example, the [1αX1] is S or K or P or R or T, [1αX2] is S or M or W, and [1αX3] is S or A.

[0071] In another preferred example, the CDR1α comprises a sequence selected from the group consisting of DSSSTY, DKMSTY, DPWATY, DRMSTY and DTMSTY. In another preferred example, the CDR2α comprises the sequence IFS[2αX1][2αX2][2αX3][2αX4], wherein [2αX1], [2αX2], [2αX3], and [2αX4] are each independently selected from any naturally occurring amino acid residue.

[0072] In another preferred example, the [2αX1] is N or Y. In another preferred example, the [2αX2] is M or Q. In another preferred example, the [2αX3] is D, S, or T. In another preferred example, the [2αX4] is M, E, or T.

[0073] In another preferred example, [2αX1] is N or Y, [2αX2] is M or Q, [2αX3] is D, S or T, and [2αX4] is M, E or T. In another preferred example, the CDR2α comprises a sequence selected from the group consisting of IFSNMDM, IFSYMTE, IFSYQSE and IFSYQST.

[0074] In another preferred example, the CDR3α comprises the sequence AEPN[3αX1][3αX2][3αX3][3αX4]ALI, wherein [3αX1], [3αX2], [3αX3] and [3αX4] are each independently selected from any naturally occurring amino acid residue. In another preferred example, the [3αX1] is Q or A or D or E or K or N or R or S or T. In another preferred example, the [3αX2] is A, S, or V.

[0075] In another preferred example, the [3αX3] is G, H, Q, or T. In another preferred example, the [3αX4] is T or S. In another preferred example, [3αX1] is T or F, [3αX2] is R or N, [3αX3] is G or H or Q or T, and [3αX4] is T or S.

[0076] In another preferred example, the CDR3α comprises a sequence selected from the group consisting of AEPNQAGTALI, AEPNNSHSALI, AEPNAAAHSALI, AEPNAASHSALI, AEPNSHSSALI, AEPNSAAHSALI, AEPNNNAHSALI, AEPNKSHSALI, AEPNQAHSALI, AEPNQSHSALI, AEPNRSHSALI, AEPNKAAHSALI, AEPNTSHSALI, AEPNDVTTALI, and AEPNESQSALI.

[0077] In a preferred embodiment of the present invention, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the TCR alpha chain variable domain comprises CDR1 alpha, CDR2 alpha, and CDR3 alpha, and the TCR beta chain variable domain comprises CDR1 beta, CDR2 beta, and CDR3 beta, wherein the CDR1 beta comprises the sequence MNHEY.

[0078] In another preferred example, the CDR2β comprises the sequence [2βX1][2βX2][2βX3][2βX4], wherein [2βX1], [2βX2], [2βX3] and [2βX4] are each independently selected from any naturally occurring amino acid residue.

[0079] In another preferred example, the [2βX1] is S or H. In another preferred example, the [2βX2] is V, D, or L. In another preferred example, the [2βX3] is G, E, or W.

[0080] In another preferred example, the [2βX4] is E, L, or V. In another preferred embodiment, the CDR2β comprises a sequence selected from the group consisting of SVGEGT, HDWLGT, SDWLGT and SLEVGT.

[0081] In another preferred example, the CDR3β comprises the sequence AS[3βX1][3βX2][3βX3][3βX4]DP[3βX5][3βX6][3βX7][3βX8], where [3βX1], [3βX2], [3βX3], [3βX4], [3βX5], [3βX6], [3βX7] and [3βX8] are each independently selected from any naturally occurring amino acid residue.

[0082] In another preferred example, the [3βX1] is A or S. In another preferred example, the [3βX2] is S or D. In another preferred example, the [3βX3] is L, I, or V. In another preferred example, the [3βX4] is E, Q, or T.

[0083] In another preferred example, the [3βX5] is Y or F. In another preferred example, the [3βX6] is E, I, P, or V. In another preferred example, the [3βX7] is Q, K, L, M, or V. In another preferred example, the [3βX8] is Y or A or E or I or L or N or Q or R or S or T or V.

[0084] In another preferred example, the CDR3β comprises a sequence selected from the group consisting of ASSSLEDPFVKR, ASADVQDPYEQY, ASSSLEDPYEQY, ASADIQDPYEQY, ASSSLEDPYVMT, ASSSLEDPYIKY, ASSSLEDPYIVT, ASSSLEDPYVKI, ASSSLEDPYIKT, ASSSLEDPYILN, ASSSLEDPYILT, ASSDVTDPYEQY, ASSSLEDPYIMA, ASSSLEDPYVLE, ASSSLEDPYPML, ASSSLEDPYVVS, ASSSLEDPYVMV and ASSSVEDPYEQY.

[0085] In another preferred example, the TCR alpha chain variable domain comprises CDR1α, CDR2α and CDR3α, wherein the amino acid sequence of CDR1α is VGISA, the amino acid sequence of CDR2α is LSSGK, and the amino acid sequence of CDR3α is AVETSYDKVI.

[0086] Natural and artificial interchain disulfide bonds A group of disulfide bonds exists between the Cα chain and the Cβ chain in the membrane proximal region of a native TCR, and in the present invention, these are referred to as "native interchain disulfide bonds." In the present invention, artificially introduced interchain covalent disulfide bonds whose positions are different from those of the native interchain disulfide bonds are referred to as "artificial interchain disulfide bonds."

[0087] For ease of explanation, the position numbers of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered in order from the N-terminus to the C-terminus. For example, in TRBC1*01 or TRBC2*01, if the amino acid at the 60th position in the order from the N-terminus to the C-terminus is P (proline), this can be expressed as Pro60 of TRBC1*01 or TRBC2*01 exon 1 in the present invention, and TRBC1*01 or TRBC2*01 exon 1 can be expressed as Pro60 of TRBC1*01 or TRBC2*01 exon 1 in the present invention. Alternatively, it may be expressed as the amino acid at position 60 in exon 1 of TRBC1*01 or TRBC2*01. For example, if the amino acid at position 61 in the sequential order from the N-terminus to the C-terminus of TRBC1*01 or TRBC2*01 is Q (glutamine), then in the present invention, it can be expressed as Gln61 in exon 1 of TRBC1*01 or TRBC2*01, or it can be expressed as the amino acid at position 61 in exon 1 of TRBC1*01 or TRBC2*01; other positions can be inferred by analogy. In the present invention, the position numbers of the amino acid sequences of TRAV and TRBV in the variable regions are numbered according to the position numbers listed in IMGT. For example, if the position number listed in IMGT for a specific amino acid in TRAV is 46, then in the present invention, it can be expressed as the amino acid at position 46 in TRAV; other positions can be inferred by analogy. In the present invention, if there are special instructions for the sequence position numbers of other amino acids, the special instructions shall be followed.

[0088] tumor The term "tumor" is intended to include all types of cancer cell proliferation or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of pathological type or stage of infection. Examples of tumors include, but are not limited to, solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignant tumors of various organ systems, such as sarcomas, lung squamous cell carcinomas, and carcinomas of the various organ systems, including prostate, lung, breast, lymphatic, gastrointestinal (e.g., colon), genitourinary (e.g., renal, epithelial), and pharyngeal. Lung squamous cell carcinomas include malignant tumors such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell lung cancers, small intestine cancers, and esophageal cancers. Metastatic lesions of the above cancers can be treated and prevented using the methods and compositions of the present invention.

[0089] Detailed Description of the Invention As is well known, the TCR α chain variable domain and β chain variable domain each contain three CDRs, which are similar to the complementarity-determining regions of an antibody. CDR3 interacts with a short antigen peptide, while CDR1 and CDR2 interact with HLA. Thus, the CDRs of a TCR molecule determine the interaction with a short antigen peptide-HLA complex. The amino acid sequences of the wild-type TCR α chain variable domain and β chain variable domain capable of binding to a short antigen peptide KASEKIFYV-HLA A0201 complex (i.e., the KASEKIFYV-HLA A0201 complex) are SEQ ID NO:1 and SEQ ID NO:2, respectively, and these sequences were first discovered by the present inventors.

[0090] Alpha chain variable domain CDR CDR1α:DSSSTY CDR2α:IFSNMDM CDR3α:AEPNQAGTALI β chain variable domain CDR CDR1β: MNHEY CDR2β:SVGEGT CDR3β: Has a CDR region such as ASSSLEDPYEQY.

[0091] By screening for mutations in the above CDR regions, the present invention obtained a high-affinity TCR whose affinity for the KASEKIFYV-HLA A0201 complex is at least twice that of the wild-type TCR for the KASEKIFYV-HLA A0201 complex. The present invention provides a T cell receptor (TCR) that has the activity of binding to the KASEKIFYV-HLA A0201 complex.

[0092] The T cell receptor comprises a TCR α chain variable domain and a TCR β chain variable domain, the TCR α chain variable domain comprises three CDR regions, and the reference sequences of the three CDR regions of the TCR α chain variable domain are: CDR1α:DSSSTY CDR2α:IFSNMDM CDR3α:AEPNQAGTALI, containing at least one of the following mutations: [Table 6]

[0093] and / or the TCR β chain variable domain comprises three CDR regions, and the reference sequences of the three CDR regions of the TCR β chain variable domain are: CDR1β:MNHEY CDR2β:SVGEGT CDR3β: ASSSLEDPYEQY and contains at least one of the following mutations: [Table 7]

[0094] In the present invention, the three CDRs of the wild-type TCR α chain variable domain of SEQ ID NO:1, namely CDR1, CDR2 and CDR3, are located at positions 26-31, 49-55 and 90-100 of SEQ ID NO:1, respectively. Accordingly, the amino acid residue numbers are taken from those shown in SEQ ID NO:1, where 27S is S at the second position of CDR1α, 28S is S at the third position of CDR1α, 29S is S at the third position of CDR1α, 52N is N at the fourth position of CDR2α, 53M is M at the fifth position of CDR2α, 54G is G at the sixth position of CDR2α, 55M is M at the seventh position of CDR2α, 94Q is Q at the fifth position of CDR3α, 95A is A at the sixth position of CDR3α, 96G is G at the seventh position of CDR3α, and 97T is T at the eighth position of CDR3α.

[0095] Similarly, in the present invention, the three CDRs of the wild-type TCR β chain variable domain of SEQ ID NO:2, namely, CDR1, CDR2 and CDR3, are located at positions 27 to 31, 49 to 54 and 92 to 103 of SEQ ID NO:2, respectively. Therefore, the amino acid residue numbers are those shown in SEQ ID NO:2, where 49S is S at the first position of CDR2β, 50V is V at the second position of CDR2β, 51G is G at the third position of CDR2β, 52E is E at the fourth position of CDR2β, 94S is S at the third position of CDR3β, 95S is S at the fourth position of CDR3β, 96L is L at the fifth position of CDR3β, 97E is E at the sixth position of CDR3β, 100Y is Y at the ninth position of CDR3β, 101E is E at the tenth position of CDR11β, 102Q is Q at the eleventh position of CDR3β, and 103Y is Y at the twelfth position of CDR3β.

[0096] Preferably, the TCR alpha chain variable domain after mutation comprises one or more amino acid residues selected from the group consisting of 27K or 27P or 27R or 27T, 28M or 28W, 29A, 52Y, 53Q, 54S or 54T, 55E or T, 94A or 94D or 94E or 94K or 94N or 94R or 94S or 94T, 95S or 95V, 96H or 96Q or 96T and 97S, wherein the numbering of the amino acid residues is as shown in SEQ ID NO: The TCR β chain variable domain after adopting the numbering shown in SEQ ID NO:1 and / or mutation comprises one or more amino acid residues selected from the group consisting of 49H, 50D or 50L, 51E or 51W, 52L or 52V, 94A, 95D, 96I or 96V, 97Q or 97T, 100F, 101I or 101P or 101V, 102K or 102L or 102M or 102V, 103A or 103E or 103I or 103L or 103N or 103Q or 103R or 103S or 103T or 103V, wherein the numbering of the amino acid residues is adopted as shown in SEQ ID NO:2.

[0097] More specifically, specific forms of the mutations in the α chain variable domain include one or more of the following groups: S27K / P / R / T, S28M / W, S29A, N52Y, M53Q, D54S / T, M55E / T, Q94A / D / E / K / N / R / S / T, A95S / V, G96H / Q / T, and T97S, and specific forms of the mutations in the β chain variable domain include one or more of the following groups: S49H, V50D / L, G51E / W, E52L / V, S94A, S95D, L96I / V, E97Q / T, Y100F, E101I / P / V, Q102K / L / M / V, and Y103A / E / I / L / N / Q / R / S / T / V.

[0098] More particularly, the number of mutations in the CDR region of the TCR α chain may be 3, 4, 5, 6, 7, 8, 9, 10 or 11, and / or the number of mutations in the CDR region of the TCR β chain may be 1, 2, 3, 4, 5, 6, 7 or 8.

[0099] Furthermore, the TCR of the present invention is an αβ heterodimeric TCR, wherein the TCR α chain variable domain comprises an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 94% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the amino acid sequence shown in SEQ ID NO:1, and / or the TCR β chain variable domain comprises an amino acid sequence having at least 90%, preferably at least 92%, more preferably at least 94% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) to the amino acid sequence shown in SEQ ID NO:2.

[0100] Furthermore, the TCR of the present invention is a single-chain TCR, wherein the TCR α chain variable domain comprises an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 92%, and most preferably at least 94% sequence identity (e.g., it may be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence shown in SEQ ID NO:3, and / or the TCR β chain variable domain comprises an amino acid sequence having at least 85%, preferably at least 90%, more preferably at least 92%, and most preferably at least 94% sequence identity (e.g., it may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence shown in SEQ ID NO:4.

[0101] Preferably, the TCR comprises (i) all or part of the TCR alpha chain excluding its transmembrane domain, and (ii) all or part of the TCR beta chain excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain.

[0102] Using site-directed mutagenesis techniques well known to those skilled in the art, the Thr48 mutation in exon 1 of the wild-type TCR α chain constant region TRAC*01 was mutated to cysteine, and the Ser57 mutation in exon 1 of the β chain constant region TRBC1*01 or TRBC2*01 was mutated to cysteine ​​to obtain a reference TCR, the amino acid sequences of which are shown in Figures 8a and 8b, respectively, with the mutated cysteine ​​residues indicated in bold. The cysteine ​​substitutions form an artificial interchain disulfide bond between the α and β chain constant regions of the reference TCR, resulting in a more stable and soluble TCR, which makes it easier to evaluate the binding affinity and / or binding half-life between the TCR and the KASEKIFYV-HLA A0201 complex. It should be understood that the CDR regions of the TCR variable region determine the affinity between the TCR and the pMHC complex, and therefore, the cysteine ​​substitutions in the TCR constant region do not affect the binding affinity and / or binding half-life of the TCR. Therefore, in the present invention, the measured binding affinity between a reference TCR and a KASEKIFYV-HLA A0201 complex is considered to be the binding affinity between a wild-type TCR and a KASEKIFYV-HLA A0201 complex. Similarly, if the binding affinity between a TCR of the present invention and a KASEKIFYV-HLA A0201 complex is measured to be at least 10-fold higher than the binding affinity between a reference TCR and a KASEKIFYV-HLA A0201 complex, the binding affinity between a TCR of the present invention and a KASEKIFYV-HLA A0201 complex is equivalent to at least 10-fold higher than the binding affinity between a wild-type TCR and a KASEKIFYV-HLA A0201 complex.

[0103] Binding affinity (dissociation equilibrium constant K) can be determined by any suitable method. D ) and binding half-life (T 1 / 2 For example, this can be detected using surface plasmon resonance technology. Doubling the affinity of the TCR results in a K D Please understand that T 1 / 2 is the dissociation rate of In2 (K off) is calculated as the sum of the two. 1 / 2 If we double it, K off is halved. Preferably, the same test protocol is used to detect the binding affinity or binding half-life of a given TCR a number of times, for example, three or more times, and the results are averaged. In a preferred embodiment, the affinity of soluble TCR is detected by the surface plasmon resonance (BIAcore) method described in the Examples herein, under conditions of a temperature of 25°C and a pH value of 7.1 to 7.5. The method involves determining the dissociation equilibrium constant K of the reference TCR with the KASEKIFYV-HLA A0201 complex. D The dissociation equilibrium constant K of the wild-type TCR for the KASEKIFYV-HLA A0201 complex was detected to be 3.01E-05M, which is 30.1 μM. D The affinity of the TCR is also considered to be 30.1 μM. Doubling the affinity of the TCR D is halved, the dissociation equilibrium constant K of the high-affinity TCR for the KASEKIFYV-HLA A0201 complex is D When the affinity of the high-affinity TCR for the KASEKIFYV-HLA A0201 complex is detected to be 3.01E-06M, which is 3.01 μM, the affinity of the high-affinity TCR for the KASEKIFYV-HLA A0201 complex is described as 10 times the affinity of the wild-type TCR for the KASEKIFYV-HLA A0201 complex. D Conversion relationship between units of value, i.e., 1M=10 6 Familiar with μM, 1 μM = 1000 nM, 1 nM = 1000 pM.

[0104] In a preferred embodiment of the present invention, the affinity of the TCR for the KASEKIFYV-HLA A0201 complex is at least 2-fold, preferably at least 5-fold, more preferably at least 10-fold higher than that of the wild-type TCR. In another preferred embodiment, the affinity of the TCR for the KASEKIFYV-HLA A0201 complex is at least 50 times, preferably at least 100 times, more preferably at least 500 times, greater than that of the wild-type TCR.

[0105] In another preferred embodiment, the affinity of the TCR for the KASEKIFYV-HLA A0201 complex is at least 10 times that of a wild-type TCR. 4 times, preferably at least 10 5 times, more preferably at least 5 x 10 5 It's double. In another preferred embodiment, the dissociation equilibrium constant of the TCR for the KASEKIFYV-HLA A0201 complex is 5 μM≦K D ≦10 μM, preferably 0.1 μM≦K D ≦1 μM, more preferably 1 nM≦K D ≦100 nM, more preferably 10 pM≦K D ≦100 pM.

[0106] Mutations can be made using any suitable method, including, but not limited to, polymerase chain reaction (PCR)-based, restriction enzyme-based cloning, or ligation-independent cloning (LIC) methods. Many standard molecular biology textbooks describe these methods in detail. For more details on polymerase chain reaction (PCR) mutagenesis and restriction enzyme-based cloning, see Sambrook and Russell, (2001) Molecular Cloning—A Laboratory Manual, 3rd Edition, CSHL Publishers. For more information on LIC methods, see (Rashtchian, (1995) Curr Opin Biotechnol 6(1):30-6).

[0107] Methods for generating the TCRs of the present invention include, but are not limited to, screening for TCRs with high affinity for the KASEKIFYV-HLA-A0201 complex from a diverse library of phage particles that display such TCRs, as described in, for example, Li, et al. (2005) Nature Biotech 23(3):349-354.

[0108] It will be appreciated that genes expressing wild-type TCR α and β chain variable domain amino acids or genes expressing slightly modified wild-type TCR α and β chain variable domain amino acids can both be used to prepare a template TCR, and DNA encoding the variable domains of that template TCR then introduces the changes necessary to generate the high affinity TCRs of the invention.

[0109] The high affinity TCRs of the present invention comprise one of the amino acid sequences of SEQ ID NOs:64-87 for the α chain variable domain and / or one of the amino acid sequences of SEQ ID NOs:88-114 for the β chain variable domain. Thus, a TCR α chain comprising the amino acid sequence of the wild-type TCR α chain variable domain (SEQ ID NO:1) can be combined with a TCR β chain comprising one of the amino acid sequences of SEQ ID NOs:88-114 to form a heterodimeric TCR or single-chain TCR molecule. Alternatively, a TCR β chain comprising the amino acid sequence of the wild-type TCR β chain variable domain (SEQ ID NO:2) can be combined with a TCR α chain comprising one of the amino acid sequences of SEQ ID NOs:64-87 to form a heterodimeric TCR or single-chain TCR molecule. Alternatively, a TCR α chain comprising one of the amino acid sequences of SEQ ID NOs: 64 to 87 of the TCR α chain variable domain is combined with a TCR β chain comprising one of the amino acid sequences of SEQ ID NOs: 88 to 114 of the TCR β chain variable domain to form a heterodimeric TCR or single-chain TCR molecule. In the present invention, the amino acid sequences of the α chain variable domain and the β chain variable domain that form the heterodimeric TCR molecule are preferably selected from Table 1 below.

[0110] [Table 8-1] [Table 8-2] [Table 8-3]

[0111] For the purposes of the present invention, the TCR of the present invention is a moiety having at least one TCR α and / or TCR β chain variable domain. Usually, they simultaneously comprise a TCR α chain variable domain and a TCR β chain variable domain. They may be in the form of an αβ heterodimer, a single chain, or other forms that can exist stably. In adoptive immunotherapy, the full-length chain of the αβ heterodimer TCR (including the cytoplasmic and transmembrane domains) can be transfected. The TCR of the present invention can be used as a targeting agent for delivering therapeutic agents to antigen-presenting cells, or can be combined with other molecules to prepare bifunctional polypeptides that target effector cells, in which case the TCR is preferably in a soluble form.

[0112] Regarding stability, the prior art discloses that soluble and stable TCR molecules can be obtained by introducing an artificial interchain disulfide bond between the TCR α and β chain constant domains, as described in patent document PCT / CN2015 / 093806. Therefore, the TCR of the present invention may be a TCR in which an artificial interchain disulfide bond has been introduced between residues in the constant domains of its α and β chains. A cysteine ​​residue forms an artificial interchain disulfide bond between the constant domains of the α and β chains of the TCR. The cysteine ​​residue can be substituted for another amino acid residue at an appropriate site in a native TCR to form an artificial interchain disulfide bond. For example, a disulfide bond can be formed by substituting Thr48 in TRAC*01 exon 1 for Ser57 in TRBC1*01 or TRBC2*01 exon 1. Further sites for introducing cysteine ​​residues to form disulfide bonds include Thr45 in TRAC*01 exon 1 and Ser77 in TRBC1*01 or TRBC2*01 exon 1, Tyr10 in TRAC*01 exon 1 and Ser17 in TRBC1*01 or TRBC2*01 exon 1, Thr45 in TRAC*01 exon 1 and Asp59 in TRBC1*01 or TRBC2*01 exon 1, and Asp10 in TRAC*01 exon 1. The cysteine ​​residues may be Ser15 and Glu15 in TRBC1*01 or TRBC2*01 exon 1, Arg53 in TRAC*01 exon 1 and Ser54 in TRBC1*01 or TRBC2*01 exon 1, Pro89 in TRAC*01 exon 1 and Ala19 in TRBC1*01 or TRBC2*01 exon 1, or Tyr10 in TRAC*01 exon 1 and Glu20 in TRBC1*01 or TRBC2*01 exon 1. That is, cysteine ​​residues are substituted at any group of sites in the α-chain and β-chain constant domains. Up to 15, 10, or 8 or fewer amino acids may be truncated at one or more C-termini of the TCR constant domains of the present invention to eliminate cysteine ​​residues, thereby achieving the goal of eliminating native interchain disulfide bonds, which can be achieved by mutating the cysteine ​​residues that form native interchain disulfide bonds to other amino acids.

[0113] As described above, the TCR of the present invention may comprise an artificial interchain disulfide bond introduced between residues of the constant domains of its α-chain and β-chain. It should be noted that the TCR of the present invention may comprise a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, regardless of whether or not the artificial interchain disulfide bond described above is introduced between the constant domains. The TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR may be linked by a natural interchain disulfide bond present in the TCR.

[0114] Furthermore, with regard to stability, patent document PCT / CN2016 / 077680 also discloses that the stability of TCR can be significantly improved by introducing an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR. Therefore, the high-affinity TCR of the present invention can include an artificial interchain disulfide bond between the chain variable region and the β chain constant region. Specifically, the cysteine ​​residues that form an artificial interchain disulfide bond between the α chain variable region and β chain constant region of the TCR substituted the amino acid at position 46 of TRAV and the amino acid at position 60 of TRBC1*01 or TRBC2*01 exon 1, the amino acid at position 47 of TRAV and the amino acid at position 61 of TRBC1*01 or TRBC2*01 exon 1, the amino acid at position 46 of TRAV and the amino acid at position 61 of TRBC1*01 or TRBC2*01 exon 1, or the amino acid at position 47 of TRAV and the amino acid at position 60 of TRBC1*01 or TRBC2*01 exon 1. Preferably, such a TCR may comprise (i) all or a portion of the TCR α chain excluding its transmembrane domain, and (ii) all or a portion of the TCR β chain excluding its transmembrane domain, where (i) and (ii) both comprise the variable domain and at least a portion of the constant domain of the TCR chain, and the α chain and β chain form a heterodimer. More preferably, such a TCR may comprise the α chain variable domain and β chain variable domain, and all or a portion of the β chain constant domain excluding the transmembrane domain, but not the α chain constant domain, and the TCR α chain variable domain and β chain form a heterodimer.

[0115] Regarding stability, the TCRs of the present invention include TCRs having mutations in their hydrophobic core regions, and these mutations in the hydrophobic core regions can preferably improve the stability of the TCRs of the present invention, as described, for example, in the patent document having publication number WO2014 / 206304. Such TCRs can be mutated at variable domain hydrophobic core positions at amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, and 94 of the (α and / or β chain) variable region, and / or at the penultimate, fifth, and seventh positions of a short peptide amino acid in the α chain J gene (TRAJ), and / or the penultimate, fourth, and sixth positions of a short peptide amino acid in the β chain J gene (TRBJ), where the position numbers of the amino acid sequences are those listed in the International Immunogenetics Information System (IMGT). Those skilled in the art can obtain the position numbers of amino acid residues of different TCRs in IMGT by knowing the International Immunogenetics Information System and following the database.

[0116] More specifically, in the present invention, a TCR with a mutation in the hydrophobic core region can be a stable, soluble single-chain TCR composed of a flexible peptide chain linking the variable domains of the TCR α and β chains. The CDR regions of the TCR variable region determine the affinity between short peptides and HLA complexes, and mutations in the hydrophobic core can make the TCR more stable without affecting the affinity between short peptides and HLA complexes. Note that the flexible peptide chain in the present invention can be any peptide chain suitable for linking the variable domains of the TCR α and β chains. The template chain for screening high-affinity TCRs constructed in Example 1 of the present invention is the highly stable single-chain TCR containing the hydrophobic core mutation. Using a more stable TCR, the affinity between the TCR and the KASEKIFYV-HLA-A0201 complex can be more easily evaluated.

[0117] The CDR regions of the single-chain template TCR α-chain variable domain and β-chain variable domain are identical to those of the wild-type TCR. That is, the three CDRs of the α-chain variable domain are CDR1α: DSSSTY, CDR2α: IFSNMDM, and CDR3α: AEPNQAGTALI, respectively, and the three CDRs of the β-chain variable domain are CDR1β: MNHEY, CDR2β: SVGEGT, and CDR3β: ASSSLEDPYEQY, respectively. The amino acid sequence (SEQ ID NO: 60) and nucleotide sequence (SEQ ID NO: 61) of the single-chain template TCR are shown in Figures 7a and 7b, respectively. In this way, single-chain TCRs consisting of the α-chain variable domain and the β-chain variable domain with high affinity for the KASEKIFYV-HLA A0201 complex are screened.

[0118] In the present invention, the three CDRs of the single-chain template TCR α chain variable domain SEQ ID NO:3, namely CDR1, CDR2 and CDR3, are located at positions 26-31, 49-55 and 90-100 of SEQ ID NO:3, respectively. Accordingly, the amino acid residue numbers are those shown in SEQ ID NO:3, where 27S is S at the second position of CDR1α, 28S is S at the third position of CDR1α, 29S is S at the third position of CDR1α, 52N is N at the fourth position of CDR2α, 53M is M at the fifth position of CDR2α, 54G is G at the sixth position of CDR2α, 55M is M at the seventh position of CDR2α, 94Q is Q at the fifth position of CDR3α, 95A is A at the sixth position of CDR3α, 96G is G at the seventh position of CDR3α, and 97T is T at the eighth position of CDR3α.

[0119] Similarly, in the present invention, the three CDRs of the single-chain template TCR β chain variable domain SEQ ID NO:4, i.e., CDR1, CDR2, and CDR3, are located at positions 27-31, 49-54, and 92-103 of SEQ ID NO:2, respectively. Therefore, the amino acid residue numbers are those shown in SEQ ID NO:4, where 49S is S at the first position of CDR2β, 50V is V at the second position of CDR2β, 51G is G at the third position of CDR2β, 52E is E at the fourth position of CDR2β, 94S is S at the third position of CDR3β, 95S is S at the fourth position of CDR3β, 96L is L at the fifth position of CDR3β, 97E is E at the sixth position of CDR3β, 100Y is Y at the ninth position of CDR3β, 101E is E at the tenth position of CDR11β, 102Q is Q at the eleventh position of CDR3β, and 103Y is Y at the twelfth position of CDR3β.

[0120] The αβ heterodimers of the present invention, which have high affinity for the KASEKIFYV-HLA-A0201 complex, can be obtained by transferring the CDR regions of the α and β chain variable domains of the screened high-affinity single-chain TCRs to the corresponding positions of the wild-type TCR α chain variable domain (SEQ ID NO: 1) and β chain variable domain (SEQ ID NO: 2). Another part can be obtained by artificially combining the CDR regions based on the mutation sites obtained by screening.

[0121] The high-affinity TCR of the present invention comprises one of the amino acid sequences SEQ ID NOs:9 to 32 for the α chain variable domain and / or one of the amino acid sequences SEQ ID NOs:33 to 59 for the β chain variable domain. Thus, the high-stability single-chain TCR α chain variable domain (SEQ ID NO:3) as a template chain can be combined with a TCR β chain variable domain having one of the amino acid sequences SEQ ID NOs:33 to 59 to form the single-chain TCR molecule. Alternatively, the high-stability single-chain TCR β chain variable domain (SEQ ID NO:4) as a template chain can be combined with a TCR α chain variable domain having one of the amino acid sequences SEQ ID NOs:9 to 32 to form the single-chain TCR molecule. Alternatively, the TCR α chain variable domain having one of the amino acid sequences SEQ ID NOs:9 to 32 can be combined with a TCR β chain variable domain having one of the amino acid sequences SEQ ID NOs:33 to 59 to form the single-chain TCR molecule. In the present invention, the amino acid sequences of the α chain variable domain and β chain variable domain of the high affinity single-chain TCR molecule are preferably selected from Table 2 below.

[0122] [Table 9-1] [Table 9-2]

[0123] The TCRs of the present invention can also be provided in the form of multivalent complexes. Multivalent TCR complexes of the present invention include polymers formed by binding two, three, four, or more TCRs of the present invention, for example, using the tetramerization domain of p53 to generate tetramers, or complexes formed by binding multiple TCRs of the present invention with another molecule. The TCR complexes of the present invention can be used to track or target cells that present specific antigens in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes for such uses.

[0124] The TCRs of the present invention can be used alone or can be covalently or otherwise linked, preferably covalently, to a conjugate comprising a detectable marker (for diagnostic purposes, where the TCR detects the presence of cells presenting the KASEKIFYV-HLA-A0201 complex), a therapeutic agent, a PK (protein kinase) modulating moiety, or any combination or coupling of these substances.

[0125] Detectable markers used for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized tomography) contrast agents, or enzymes capable of producing a detectable product.

[0126] Therapeutic agents that can bind or couple to the TCRs of the present invention include (1) radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539), (2) biological toxicities (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565), (3) cytokines such as IL-2 (Gillies et al., 1992, PNAS 89, 1428; Card et al., 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al., 2003, Cancer Research 63, 3202), and (4) antibody Fc fragments (Mosquera et al., 2005, The Journal of Immunology). Immunology 174, 4381), (5) antibody scFv fragments (Zhu et al., 1995, International Journal of Cancer 62, 319), (6) gold nanoparticles / nano rods (Lapotko et al., 2005, Cancer Letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115), (7) virus particles (Peng et al., 2004, Gene Therapy 11, 1234), (8) liposomes (Mamot et al., 2005, Cancer Research (Research) 65, 11631), (9) nanomagnetic particles, (10) prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), (11) chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0127] Antibodies or fragments thereof that bind to TCRs of the present invention include anti-T cell or NK cell determinant antibodies, such as anti-CD3, anti-CD28, or anti-CD16 antibodies, and the binding of these antibodies or fragments to TCRs can more appropriately target target cells to effector cells. In a preferred embodiment, the TCRs of the present invention bind to anti-CD3 antibodies or functional fragments or variants of said anti-CD3 antibodies. Specifically, fusion molecules of TCRs and anti-CD3 single-chain antibodies of the present invention comprise the amino acid sequence of a TCR α chain variable domain selected from the group consisting of SEQ ID NOs: 9 to 32, 64 to 87, and / or the amino acid sequence of a TCR β chain variable domain selected from the group consisting of SEQ ID NOs: 33 to 59, 88 to 114.

[0128] The present invention further relates to nucleic acid molecules encoding the TCRs of the present invention. The nucleic acid molecules of the present invention may be in the form of DNA or RNA. The DNA may be a coding strand or a non-coding strand. For example, the nucleic acid sequences encoding the TCRs of the present invention may be the same as the nucleic acid sequences shown in the figures of the present invention, or may be degenerate variants. To explain the meaning of "degenerate variant," as used herein, a "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein sequence having SEQ ID NO:60 but differs from the sequence of SEQ ID NO:61.

[0129] The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can be obtained typically, but not limited to, by PCR amplification, recombinant methods, or artificial synthesis. Currently, a DNA sequence encoding the TCR of the present invention (or a fragment thereof, or a derivative thereof) can be obtained entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, etc.) and cells known in the art.

[0130] The present invention also relates to vectors which include the nucleic acid molecules of the invention, and to host cells which are genetically engineered using the vectors or coding sequences of the invention.

[0131] The present invention further includes isolated cells, particularly T cells, expressing the TCRs of the present invention. Many methods are suitable for T cell transfection with DNA or RNA encoding the high-affinity TCRs of the present invention (e.g., Robbins et al., (2008) J. Immunol. 180:6116-6131). T cells expressing the high-affinity TCRs of the present invention can be used in adoptive immunotherapy. Those skilled in the art will be aware of many methods suitable for adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).

[0132] The present invention provides a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and a TCR of the present invention, or a TCR complex of the present invention, or a cell presenting a TCR of the present invention. The present invention further provides a method for treating a disease, comprising the step of administering to a subject in need of treatment an appropriate amount of a TCR of the present invention, or a TCR complex of the present invention, or a cell presenting a TCR of the present invention, or a pharmaceutical composition of the present invention.

[0133] The names of amino acids in this specification are represented by a single alphabetical letter in international use, and the corresponding three-letter abbreviations of the amino acid names are Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V), and in the present invention, Pro60 and 60P both represent proline at the 60th position. Furthermore, specific forms of the mutations in the present invention, such as "S27K / P / R / T", represent that S at position 27 is substituted with K, or with P, or with R, or with T; similarly, "S28M / W" represents that S at position 28 is substituted with M, or with W. Others can be inferred by such analogy.

[0134] In the art, substitution with amino acids having similar or similar properties usually does not change the function of the protein. Addition of one or more amino acids to the C-terminus and / or N-terminus usually does not change the structure and function of the protein. Therefore, the TCRs of the present invention include TCRs that still maintain their functionality even when up to five, preferably three, more preferably two, and most preferably one amino acid of the TCRs of the present invention (particularly the amino acids located below the CDR region) are substituted with amino acids having similar or similar properties.

[0135] The present invention further includes TCRs that have been slightly modified relative to the TCRs of the present invention. Modified forms (which generally do not alter the primary structure) include chemically derivatized forms of the TCRs of the present invention, such as acetylation or carboxylation. Modifications further include glycosylation, such as TCRs produced by glycosylation modification during the synthesis and processing of the TCRs of the present invention or in further processing steps. Such modifications can be achieved by exposing the TCR to a glycosylated enzyme (e.g., mammalian glycosylase or deglycosylase). Modified forms further include sequences with phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Further included are TCRs that have been modified to improve their proteolytic resistance or optimize their solubility.

[0136] The TCR, TCR complex, or TCR-transfected T cell of the present invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The TCR, multivalent TCR complex, or cell of the present invention is usually provided as part of a sterile pharmaceutical composition, which usually includes a pharmaceutically acceptable carrier. The pharmaceutical composition can be in any suitable form (depending on the patient's desired method of administration). It can be provided in a unit dosage form, usually in a sealed container, and can be provided as part of a kit. Such a kit (optionally) includes a manual. It can contain multiple unit dosage forms.

[0137] Furthermore, the TCRs of the present invention may be used alone or may be bound or coupled to other therapeutic agents and used together (eg, formulated into the same pharmaceutical composition).

[0138] Pharmaceutical compositions can contain pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to a carrier used in administering a therapeutic agent. This term refers to a pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and does not have excessive toxicity after administration. These carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.

[0139] Pharmaceutically acceptable carriers for therapeutic compositions can include liquids, such as water, saline, glycerol, ethanol, etc. Additionally, these carriers can contain auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like.

[0140] Typically, the therapeutic compositions can be prepared in an injectable form, for example, as liquid solutions or suspensions, or in solid forms suitable for solution in or suspension in liquid carriers prior to injection.

[0141] Once formulated, the compositions of the present invention can be administered by conventional routes, including, but not limited to, intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably parenteral, including subcutaneous, intramuscular, or intravenous. The subject to be prevented or treated can be an animal, particularly a human.

[0142] When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used depending on the conditions of use, and preferred examples include injections and oral preparations.

[0143] These pharmaceutical compositions can be formulated by mixing, diluting or dissolving based on conventional methods, and optionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and cosolvents, and the preparation process can be carried out in a usual manner according to the dosage form.

[0144] The pharmaceutical compositions of the present invention can be administered in the form of sustained-release formulations. For example, the TCRs of the present invention can be incorporated into a pill or microcapsules with a sustained-release polymer as a carrier, and the pill or microcapsules are then surgically implanted into the tissue to be treated. Examples of sustained-release polymers include ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, lactic acid-glycolic acid copolymers, etc., and preferably include biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.

[0145] When the pharmaceutical composition of the present invention is used in actual treatment, the TCR or TCR complex of the present invention or the cells presenting the TCR of the present invention as the active ingredient can be reasonably determined based on the weight, age, sex, severity of symptoms, etc. of each patient to be treated, and ultimately, a reasonable dosage will be determined by a physician.

[0146] The main advantages of the present invention are: (1) The affinity and / or binding half-life of the TCR of the present invention for the KASEKIFYV-HLA-A0201 complex is at least twice, preferably at least 10 times, that of the wild-type TCR. (2) The affinity and / or binding half-life of the TCR of the present invention for the KASEKIFYV-HLA-A0201 complex is at least 100 times, preferably at least 10 times, that of the wild-type TCR. 3 times, and more preferably 10 4 ~5×10 5 can be achieved twice as much. (3) Effector cells transduced with the high-affinity TCR of the present invention have a potent killing effect on target cells.

[0147] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without specific conditions are generally in accordance with general conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning - A Laboratory Manual (3rd Edition) (2001) CSHL Publishing) or conditions suggested by manufacturers. Unless otherwise specified, percentages and parts are calculated by weight.

[0148] material and method The experimental materials used in the examples of the present invention are available from commercial channels unless otherwise specified, where E. coli DH5α is purchased from Tiangen, E. coli BL21(DE3) is purchased from Tiangen, E. coli Tuner (DE3) is purchased from Novagen, and the plasmid pET28a is purchased from Novagen.

[0149] Example 1. Stability of Hydrophobic Core Mutations. Generation of Single TCR Template Strands. The present invention uses the site-directed mutagenesis method according to patent document WO2014 / 206304 to construct a stable single-chain TCR molecule consisting of a flexible short peptide (linker) connecting the TCR α and β chain variable domains. Its amino acid and DNA sequences are SEQ ID NO:60 and SEQ ID NO:61, respectively, as shown in Figures 7a and 7b. This single-chain TCR molecule is used as a template to screen for high-affinity TCR molecules. The amino acid sequences of the α variable domain (SEQ ID NO:3) and β variable domain (SEQ ID NO:4) of this template chain are as shown in Figures 2a and 2b, and their corresponding DNA sequences are SEQ ID NOs:5 and 6, respectively, as shown in Figures 3a and 3b. The amino acid and DNA sequences of the flexible short peptide (linker) are SEQ ID NOs:7 and 8, respectively, as shown in Figures 4a and 4b.

[0150] The target gene carrying the template strand was double-digested with NcoI and NotI and ligated into the pET28a vector, which had also been double-digested with NcoI and NotI. The ligation product was transformed into E. coli DH5α, coated on an LB plate containing kanamycin, and incubated overnight at 37°C in an inverted position. Positive clones were selected, PCR screened, and sequenced for positive recombinants. After confirming the correct sequence, the recombinant plasmid was extracted and transformed into E. coli BL21(DE3) for expression.

[0151] Example 2. Expression, renaturation and purification of stable single-chain TCRs constructed in Example 1 All of the BL21(DE3) colonies containing the recombinant plasmid pET28a-template strand prepared in Example 1 were inoculated into LB medium containing kanamycin and incubated at 37°C until OD 600The medium was cultured until the pH reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and the medium was then cultured at 37°C for 4 hours. The cell pellet was collected by centrifugation at 5000 rpm for 15 minutes, heat-dissociated with Bugbuster Master Mix (Merck), and centrifuged at 6000 rpm for 15 minutes to recover inclusion bodies. The inclusion bodies were washed with Bugbuster (Merck) to remove cell debris and membrane components, and centrifuged at 6000 rpm for 15 minutes to collect the inclusion bodies. The inclusion bodies were dissolved in a buffer (20 mM Tris-HCl pH 8.0, 8 M urea) and centrifuged at high speed to remove insoluble material. The supernatant was quantified by BCA, aliquoted, and stored at -80°C for later use.

[0152] 5 mg of dissolved single-chain TCR inclusion body protein was added to 2.5 mL of buffer (6 M Gua-HCl, 50 mM Tris-HCl pH 8.1, 100 mM NaCl, 10 mM EDTA), and DTT was added to a final concentration of 10 mM, followed by incubation at 37°C for 30 minutes. Using a syringe, the single-chain TCR after the above treatment was added dropwise to 125 mL of regeneration buffer (100 mM Tris-HCl pH 8.1, 0.4 M L-arginine, 5 M urea, 2 mM EDTA, 6.5 mM β-mercaptoethylamine, 1.87 mM cystamine), and the mixture was stirred at 4°C for 10 minutes. The regeneration solution was then incubated at 37°C for 30 minutes. DThe dialysis solution was placed in a cellulose membrane dialysis bag, which was then placed in 1 L of pre-chilled water and gently stirred overnight at 4°C. After 17 hours, the dialysate was replaced with 1 L of pre-chilled buffer (20 mM Tris-HCl, pH 8.0) and dialyzed for 8 hours at 4°C. The dialysate was then replaced with the same fresh buffer and dialyzed overnight. After 17 hours, the sample was filtered through a 0.45 μm filter membrane, degassed under vacuum, and passed through an anion exchange column (HiTrap Q HP, GE Healthcare). The protein was purified using a linear gradient elution of 0 to 1 M NaCl in 20 mM Tris-HCl, pH 8.0. The collected eluted components were analyzed by SDS-PAGE. The components containing the single-chain TCR were concentrated and further purified using a gel filtration column (Superdex 75 10 / 300, GE Healthcare). The target component was also analyzed by SDS-PAGE.

[0153] The purity of the eluted components used in BIAcore analysis was further tested by gel filtration under the following conditions: chromatography column Agilent Bio SEC-3 (300A, φ7.8 × 300 mm), mobile phase 150 mM phosphate buffer, flow rate 0.5 mL / min, column temperature 25°C, UV detection wavelength 214 nm.

[0154] Example 3. Characterization of binding BIAcore analysis The BIAcore T200 real-time analysis system was used to detect the binding activity of TCR molecules with the KASEKIFYV-HLA-A0201 complex. Anti-streptavidin antibodies (GenScript) were added to a coupling buffer (10 mM sodium acetate buffer, pH 4.77) and then flowed over a CM5 chip pre-activated with EDC and NHS to immobilize the antibody on the chip surface. Finally, unreacted activated surfaces were sealed with an ethanolamine hydrochloride solution to complete the coupling process, achieving a coupling level of approximately 15,000 RU. The conditions were as follows: temperature 25°C, pH 7.1-7.5.

[0155] A low concentration of streptavidin was flowed over the antibody-coated chip surface, followed by the KASEKIFYV-HLA-A0201 complex in the detection channel and another channel as the reference channel. 0.05 mM biotin was then flowed over the chip at a flow rate of 10 μL / min for 2 minutes to seal any remaining streptavidin binding sites. Affinity was measured using a single-cycle kinetic analysis method. TCRs were diluted to several different concentrations in HEPES-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) and sequentially flowed over the chip surface at a flow rate of 30 μL / min. The binding time for each injection was 120 seconds, with a 600-second dissociation period after the final injection. After each round, the chip was regenerated with 10 mM Gly-HCl, pH 1.75. Kinetic parameters were calculated using BIAcore Evaluation software.

[0156] The preparation process of the above KASEKIFYV-HLA-A0201 complex is as follows. Purification 100 mL of the E. coli solution inducing heavy or light chain expression was collected and centrifuged at 8000 g at 4°C for 10 minutes. The cells were then washed once with 10 mL of PBS and resuspended in 5 mL of BugBuster Master Mix Extraction Reagent (Merck) by vigorously shaking. The cells were then incubated at room temperature for 20 minutes with rotation, and then centrifuged at 6000 g at 4°C for 15 minutes. The supernatant was discarded and the inclusion bodies were collected.

[0157] The inclusion bodies were resuspended in 5 mL of BugBuster Master Mix and incubated at room temperature for 5 minutes with rotation. 30 mL of 10-fold diluted BugBuster was added, mixed evenly, and centrifuged at 6000 g and 4°C for 15 minutes. The supernatant was discarded, and 30 mL of 10-fold diluted BugBuster was added to resuspend the inclusion bodies. Mixed evenly, and centrifuged at 6000 g and 4°C for 15 minutes. This was repeated twice. 30 mL of 20 mM Tris-HCl pH 8.0 was added to resuspend the inclusion bodies, mixed evenly, and centrifuged at 6000 g and 4°C for 15 minutes. Finally, the inclusion bodies were dissolved in 20 mM Tris-HCl 8 M urea. The purity of the inclusion bodies was detected by SDS-PAGE, and the concentration was measured using a BCA kit.

[0158] b. Regeneration The synthesized short peptide KASEKIFYV (Beijing Saibaishen Gene Technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg / ml. The light and heavy chain inclusion bodies were dissolved in 8 M urea, 20 mM Tris pH 8.0, 10 mM DTT, and further denatured by adding 3 M guanidine hydrochloride, 10 mM sodium acetate, and 10 mM EDTA before renaturation. The KASEKIFYV peptide was added to renaturation buffer (0.4 M L-arginine, 100 mM Tris pH 8.3, 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, 0.2 mM PMSF, cooled to 4°C) at 25 mg / L (final concentration), followed by the addition of 20 mg / L light chain and 90 mg / L heavy chain (final concentrations; heavy chain was added in three portions, 8 hours each). Renaturation was completed after at least three days at 4°C, and the success of the renaturation was detected by SDS-PAGE.

[0159] c. Purification after regeneration The renaturation buffer was replaced with 10 volumes of 20 mM Tris pH 8.0 for dialysis. The buffer was replaced at least twice to sufficiently reduce the ionic strength of the solution. After dialysis, the protein solution was filtered through a 0.45 μM cellulose acetate filter membrane and then loaded onto a HiTrap Q HP (GE General Electric Company) anion exchange column (5 mL bed volume). The protein was eluted with a linear gradient of 0 to 400 mM NaCl prepared in 20 mM Tris pH 8.0 in an Äkta purifier (GE General Electric Company). pMHC elutes at approximately 250 mM NaCl. The peak components were collected and analyzed for purity by SDS-PAGE.

[0160] d. Biotinylation The purified pMHC molecules were concentrated using Millipore ultrafiltration tubes, and the buffer was exchanged for 20 mM Tris pH 8.0. Biotinylation reagent (0.05 M Bicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 μM D-biotin, and 100 μg / ml BirA enzyme (GST-BirA)) was then added. The mixture was incubated overnight at room temperature, and the completion of biotinylation was detected by SDS-PAGE.

[0161] e. Purification of the complex after biotinylation The biotinylated pMHC molecules were concentrated to 1 mL in Millipore ultrafiltration tubes and purified using gel filtration chromatography. An Äkta purifier (GE General Electric Company) was used. A HiPrep™ 16 / 60 S200HR column (GE General Electric Company) was pre-equilibrated with filtered PBS and loaded with 1 mL of concentrated biotinylated pMHC molecules. The column was then eluted with PBS at a flow rate of 1 mL / min. The biotinylated pMHC molecules eluted as a single peak at approximately 55 mL. The protein-containing fractions were combined and concentrated in Millipore ultrafiltration tubes. The protein concentration was measured using the BCA method (Thermo). A protease inhibitor cocktail (Roche) was added. The biotinylated pMHC molecules were aliquoted and stored at -80°C.

[0162] Example 4. Generation of high affinity single chain TCRs Phage display technology is a means for generating a TCR high-affinity mutant library and screening for high-affinity mutants. The TCR phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3):349-354) is applied to the single-chain TCR template in Example 1. A library of high-affinity TCRs is constructed by mutating the CDR regions of the template chain and then panned. After several rounds of panning, the phage library specifically binds to the corresponding antigen, and single clones are selected from the library and sequenced.

[0163] The BIAcore method of Example 3 was used to analyze the interaction between the TCR molecule and the KASEKIFYV-HLA-A0201 complex, and a high-affinity TCR whose affinity and / or binding half-life is at least twice that of the wild-type TCR was screened, i.e., the dissociation equilibrium constant K of the screened high-affinity TCR binding to the KASEKIFYV-HLA-A0201 complex was determined. D is the dissociation equilibrium constant K for wild-type TCR binding to the KASEKIFYV-HLA-A0201 complex.D The results are shown in Table 3 below. The K of the interaction between the reference TCR and the KASEKIFYV-HLA-A0201 complex detected using the above method. D The K value was 30.1 μM, and the interaction curve was as shown in FIG. 12, i.e., the K D The value is also 30.1 μM, or 3.01E-05M.

[0164] Specifically, using the numbering shown in SEQ ID NO:1, the α chain variable domains of these high affinity TCR mutants are mutated at amino acids at one or more of the following groups: 27S, 28S, 29S, 52N, 53M, 54D, 55M, 94Q, 95A, 96G, 97T; and / or using the numbering shown in SEQ ID NO:2, the β chain variable domains of these high affinity TCR mutants are mutated at one or more of the following groups: 49S, 50V, 51G, 52E, 94S, 95S, 96L, 97E, 100Y, 101E, 102Q, 103Y.

[0165] More specifically, using the numbering set forth in SEQ ID NO:1, these high affinity TCR alpha chain variable domains comprise one or more amino acid residues selected from the group consisting of 27K or 27P or 27R or 27T, 28M or 28W, 29A, 52Y, 53Q, 54S or 54T, 55E or T, 94A or 94D or 94E or 94K or 94N or 94R or 94S or 94T, 95S or 95V, 96H or 96Q or 96T and 97S, and / or Using the numbering shown in NO:2, these high affinity TCR β chain variable domains comprise one or more amino acid residues selected from the group consisting of 49H, 50D or 50L, 51E or 51W, 52L or 52V, 94A, 95D, 96I or 96V, 97Q or 97T, 100F, 101I or 101P or 101V, 102K or 102L or 102M or 102V, 103A or 103E or 103I or 103L or 103N or 103Q or 103R or 103S or 103T or 103V.

[0166] The specific amino acid sequences of the high-affinity single-chain TCR α-chain variable domains (SEQ ID NOs: 9-32) and β-chain variable domains (SEQ ID NOs: 32-59) are shown in Figures 5(1)-(24) and 6(1)-(27), respectively. [Table 10-1] [Table 10-2]

[0167] Example 5. Generation of high affinity αβ heterodimeric TCRs Mutations in the CDR regions of the high-affinity single-chain TCR screened in Example 4 were introduced into the corresponding sites in the variable domain of the αβ heterodimer TCR, and the affinity with the KASEKIFYV-HLA-A0201 complex was detected via BIAcore. High-affinity mutation sites in the CDR regions were introduced using site-directed mutagenesis, a method well known to those skilled in the art. The amino acid sequences of the α and β chain variable domains of the wild-type TCR are shown in Figures 1a (SEQ ID NO: 1) and 1b (SEQ ID NO: 2), respectively.

[0168] It should be noted that in order to obtain a more stable soluble TCR and more easily evaluate the binding affinity and / or binding half-life between the TCR and the KASEKIFYV-HLA A0201 complex, the αβ heterodimer TCR may be a TCR in which one cysteine ​​residue is introduced into each of the constant regions of the α chain and the β chain to form an artificial interchain disulfide bond. After the cysteine ​​residue is introduced in this example, the amino acid sequences of the TCR α chain and the β chain are as shown in Figure 8a (SEQ ID NO:62) and 8b (SEQ ID NO:63), respectively, and the introduced cysteine ​​residue is shown in bold.

[0169] The extracellular sequence genes of the TCR α and β chains to be expressed are synthesized by the standard method described in "Molecular Cloning a Laboratory Manual" (3rd edition, Sambrook and Russell), and then inserted into the expression vector pET28a+ (Novagene), respectively, with the upstream and downstream cloning sites being NcoI and NotI, respectively. Mutations in the CDR regions are introduced by overlap PCR, which is well known to those skilled in the art. The inserted fragments are confirmed by sequencing.

[0170] Example 6. Expression, Renaturation, and Purification of αβ Heterodimeric TCRs The expression vectors for the TCR α and β chains were transformed into the expression bacteria BL21(DE3) by chemical transformation, and the bacteria were grown in LB medium at OD . 600 If the pH is 0.6, the inclusion bodies formed after expression of the TCR α and β chains using BugBuster Mix (Novagene) were extracted and washed multiple times with BugBuster solution. Finally, the inclusion bodies were dissolved in 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA), and 20 mM Tris (pH 8.1).

[0171] The dissolved TCR α and β chains were rapidly mixed with 5 M urea, 0.4 M arginine, 20 mM Tris (pH 8.1), 3.7 mM cystamine, and 6.6 mM β-mercaptoethylamine (4°C) in a 1:1 mass ratio to a final concentration of 60 mg / mL. After mixing, the solution was dialyzed against 10 volumes of deionized water (4°C). After 12 hours, the deionized water was replaced with buffer (20 mM Tris, pH 8.0) and dialyzed for another 12 hours at 4°C. After dialysis, the solution was filtered through a 0.45 μM filter membrane and purified on an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare). The elution peak, containing successfully renatured α and β dimers of TCR, was confirmed by SDS-PAGE gel analysis. The TCR was then further purified by gel filtration chromatography (HiPrep 16 / 60, Sephacryl S-100 HR, GE Healthcare). The purity of the purified TCR was determined to be greater than 90% by SDS-PAGE, and the concentration was determined by the BCA method.

[0172] Example 7. BIAcore analysis results The method described in Example 3 is used to detect the affinity of the αβ heterodimer TCR with the high affinity CDR regions engineered into it to the KASEKIFYV-HLA-A0201 complex.

[0173] The CDR regions screened from the high-affinity single-chain TCR α and β chains were transferred to positions corresponding to SEQ ID NO:1 of the wild-type TCR α chain variable domain and SEQ ID NO:2 of the wild-type β chain variable domain, respectively, to form an αβ heterodimer TCR. Furthermore, the mutation sites in the CDR regions obtained by screening were artificially combined to form an αβ heterodimer TCR. The amino acid sequences of the resulting new TCR α and β chain variable domains are shown in Figures 9(1) to 9(24) and 10(1) to 9(27), respectively. Because the CDR regions of a TCR molecule determine its affinity with the corresponding pMHC complex, those skilled in the art can expect that the αβ heterodimer TCR with the introduced high-affinity mutation sites will also have high affinity for the KASEKIFYV-HLA-A0201 complex. As shown in Table 4 below, an expression vector was constructed using the method described in Example 5, and the αβ heterodimer TCR with the above-mentioned high-affinity mutations was expressed, refolded, and purified using the method described in Example 6, and then its affinity with the KASEKIFYV-HLA-A0201 complex was measured using a BIAcore T200.

[0174] [Table 11-1] [Table 11-2] [Table 11-3]

[0175] From Table 4 above, it can be seen that the αβ heterodimeric TCR with the mutations introduced into the CDR regions maintains high affinity for the KASEKIFYV-HLA-A0201 complex, which is at least twice as strong as the wild-type TCR for the KASEKIFYV-HLA-A0201 complex.

[0176] Example 8. Expression, renaturation, and purification of anti-CD3 antibody and high affinity single-chain TCR fusions The high-affinity single-chain TCR molecule of the present invention was fused with a single-chain anti-CD3 antibody (scFv) to construct a fusion molecule. Primers were designed to link the anti-CD3 antibody and high-affinity single-chain TCR molecule genes using overlap PCR. The central linker was designed to be GGGGS, and the gene fragment of the fusion molecule contained restriction endonuclease sites NcoI and NotI. The PCR-amplified product was doubly digested with NcoI and NotI and ligated into a pET28a vector doubly digested with NcoI and NotI. The ligated product was transformed into E. coli DH5α competent cells, coated on LB plates containing kanamycin, and grown overnight at 37°C in an upside-down position. Positive clones were selected and screened by PCR. The positive recombinants were sequenced to confirm the correct sequence. The recombinant plasmid was then extracted and transformed into E. coli BL21(DE3) competent cells for expression.

[0177] Expression of fusion proteins The expression plasmid containing the target gene was transformed into E. coli strain BL21(DE3), coated on an LB plate (kanamycin 50 μg / ml), and cultured overnight at 37 °C. The next day, the clone was selected and inoculated into 10 mL of LB liquid medium (kanamycin 50 μg / ml), cultured for 2-3 hours, and then inoculated into 1 L of LB medium (kanamycin 50 μg / ml) at a volume ratio of 1:100. The OD was then measured. 600The cells were cultured until the pH reached 0.5-0.8, and then expression of the target protein was induced using IPTG at a final concentration of 0.5 mM. After 4 hours of induction, the cells were harvested by centrifugation at 6000 rpm for 10 minutes. The cells were washed once with PBS buffer, aliquoted, and a selection of cells equivalent to a 200 mL bacterial culture was performed. The cells were then heat-lyzed with 5 mL of BugBuster Master Mix (Novagen) and centrifuged at 6000 g for 15 minutes to collect inclusion bodies. Next, the cells were washed four times with detergent to remove cell debris and membrane components. Next, the inclusion bodies were washed with a buffer, such as PBS, to remove detergent and salts. Finally, the inclusion bodies were dissolved in Tris buffer containing 8 M urea, the concentration of the inclusion bodies was measured, and the inclusion bodies were aliquoted and stored frozen at -80°C.

[0178] Fusion protein refolding Remove approximately 10 mg of inclusion bodies from a -80°C ultra-low temperature refrigerator and thaw. Add dithiothreitol (DTT) to a final concentration of 10 mM and incubate at 37°C for 30 minutes to 1 hour to ensure complete opening of disulfide bonds. Next, add the inclusion body sample solution dropwise to 200 mL of 4°C pre-chilled refolding buffer (100 mM Tris pH 8.1, 400 mM L-arginine, 2 mM EDTA, 5 M urea, 6.5 mM β-mercaptoethylamine, 1.87 mM cystamine) and stir slowly at 4°C for approximately 30 minutes. Dialyze the renatured solution against 8 volumes of pre-chilled HO for 16-20 hours. Dialyze twice again against 8 volumes of 10 mM Tris pH 8.0 and continue dialysis at 4°C for approximately 8 hours. After dialysis, filter the sample and purify as follows:

[0179] First-stage purification of fusion proteins The dialyzed refold (10 mM Tris pH 8.0) was subjected to gradient elution from 0 to 600 mM NaCl on an AKTA purifier (GE Healthcare) using a POROS HQ / 20 anion exchange chromatography prepacked column (Applied Biosystems). Each component was analyzed and combined by Coomassie Brilliant Blue-stained SDS-PAGE.

[0180] Second-stage purification of the fusion protein For this stage of purification, the sample solution purified and combined in the first stage is concentrated, and the fusion protein is purified using a Superdex 75 10 / 300GL gel filtration chromatography pre-packed column (GE Healthcare) pre-equilibrated with PBS buffer. The peak components are analyzed by SDS-PAGE stained with Coomassie Brilliant Blue and combined.

[0181] Example 9. Expression, Renaturation, and Purification of Fusions of Anti-CD3 Antibodies and High Affinity αβ Heterodimeric TCRs An anti-CD3 single-chain antibody (scFv) is fused to an αβ heterodimeric TCR to prepare a fusion molecule. The anti-CD3 scFv is fused to a TCR β chain, which can comprise any of the high-affinity αβ heterodimeric TCR β chain variable domains described above. The TCR α chain of the fusion molecule can comprise any of the high-affinity αβ heterodimeric TCR α chain variable domains described above.

[0182] Construction of fusion molecule expression vector 1. Construction of α-chain expression vector The target gene carrying the αβ heterodimer TCR α chain was double-digested with NcoI and NotI and ligated into the pET28a vector, which was also double-digested with NcoI and NotI. The ligated product was transformed into E. coli DH5α, coated on an LB plate containing kanamycin, and grown overnight at 37°C in an inverted position. Positive clones were selected and screened by PCR. The positive recombinants were sequenced to confirm the correct sequence. The recombinant plasmid was then extracted and transformed into E. coli Tuner (DE3) for expression.

[0183] 2. Construction of anti-CD3 (scFv) β chain expression vector Primers were designed to ligate the anti-CD3 scFv and high-affinity heterodimeric TCR β chain genes using overlap PCR. The central linker was GGGGS, and the gene fragment for the anti-CD3 scFv and high-affinity heterodimeric TCR β chain fusion protein contained the restriction endonuclease sites NcoI (CCATGG) and NotI (GCGGCCGC). The PCR-amplified product was double-digested with NcoI and NotI and ligated into a pET28a vector double-digested with NcoI and NotI. The ligated product was transformed into E. coli DH5α competent cells, coated on LB plates containing kanamycin, and grown overnight at 37°C in an upside-down position. Positive clones were selected and screened by PCR. The positive recombinants were sequenced to confirm the correct sequence. The recombinant plasmid was then extracted and transformed into E. coli Tuner (DE3) competent cells for expression.

[0184] Fusion protein expression, renaturation and purification Each expression plasmid was transformed into E. coli Tuner (DE3) competent cells, coated onto an LB plate (kanamycin 50 μg / mL), and cultured overnight at 37°C. The next day, clones were selected and inoculated into 10 mL of LB liquid medium (kanamycin 50 μg / mL), cultured for 2-3 hours, and then inoculated into 1 L of LB medium at a volume ratio of 1:100. The OD was then measured. 600The cells were cultured until the pH reached 0.5–0.8, and target protein expression was induced using a final concentration of 1 mM IPTG. After 4 hours of induction, the cells were harvested by centrifugation at 6,000 rpm for 10 minutes. The cells were washed once with PBS buffer, aliquoted, and a 200 mL bacterial culture equivalent was selected. The cells were then heat-lysed with 5 mL of BugBuster Master Mix (Merck) and centrifuged at 6,000 g for 15 minutes to collect inclusion bodies. Next, the cells were washed four times with detergent to remove cell debris and membrane components. The inclusion bodies were then washed with a buffer, such as PBS, to remove detergent and salts. Finally, the inclusion bodies were dissolved in a buffer solution containing 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA), and 20 mM Tris, pH 8.1. The concentration of the inclusion bodies was measured, aliquoted, and stored frozen at -80°C.

[0185] The dissolved TCR α chain and anti-CD3(scFv)-β chain were quickly mixed in a 2:5 mass ratio with 5 M urea, 0.4 M L-arginine, 20 mM Tris pH 8.1, 3.7 mM cystamine, and 6.6 mM β-mercaptoethylamine (4°C), to give final concentrations of 0.1 mg / mL and 0.25 mg / mL, respectively, of the α chain and anti-CD3(scFv)-β chain.

[0186] After mixing, the solution was dialyzed against 10 volumes of deionized water (4°C). After 12 hours, the deionized water was exchanged for buffer (10 mM Tris, pH 8.0) and dialyzed for another 12 hours at 4°C. The dialyzed solution was filtered through a 0.45 μM filter membrane and purified on an anion exchange column (HiTrap Q HP 5ml, GE Healthcare). The elution peak was confirmed to contain successfully refolded TCR α chain and anti-CD3 (scFv)-β chain dimer TCR by SDS-PAGE gel. The TCR fusion molecule was then further purified by size exclusion chromatography (S-100 16 / 60, GE Healthcare) and purified again by anion exchange column (HiTrap Q HP 5ml, GE Healthcare). The purity of the purified TCR fusion molecule was determined to be over 90% by SDS-PAGE, and the concentration was measured by the BCA method.

[0187] Example 10. Activation function experiments of effector cells transfected with the high affinity TCR of the present invention (target cells are tumor cell lines) This example verifies that effector cells transfected with the high-affinity TCR of the present invention have good specific activity against target cells. The function and specificity of the high-affinity TCR of the present invention in cells are detected by ELISPOT experiments.

[0188] Those skilled in the art are familiar with how to use ELISPOT experiments to detect cell function. CD3+ T cells isolated from the blood of healthy volunteers transfected with the TCR of the present invention are randomly selected as effector cells. The TCRs and their numbers can be seen from Table 4 and are respectively TCR4 (SEQ ID NO:1 for α chain variable domain, SEQ ID NO:91 for β chain variable domain), TCR25 (SEQ ID NO:1 for α chain variable domain, SEQ ID NO:98 for β chain variable domain), TCR5 (SEQ ID NO:1 for α chain variable domain, SEQ ID NO:92 for β chain variable domain), TCR6 (SEQ ID NO:1 for α chain variable domain, SEQ ID NO:93 for β chain variable domain), TCR7 (SEQ ID NO:1 for α chain variable domain, SEQ ID NO:94 for β chain variable domain), TCR10 (SEQ ID NO:1 for α chain variable domain, SEQ ID NO:97 for β chain variable domain) and TCR1 (SEQ ID NO:1 for α chain variable domain, SEQ ID NO:98 for β chain variable domain). The control effector cells are labeled as wild-type TCR (cells transfected with wild-type TCR) and A6 (cells transfected with other TCRs). The target cell lines are A375, K562-A2 (A2 overexpression), SW620-SSX2 (SSX2 overexpression), NCI-H1299-SSX2 (SSX2 overexpression), K562-A11 (A11 overexpression), and SW620 cells. Here, the target cell lines A375, K562-A2, and SW620-SSX2 are used as positive tumor cell lines, while NCI-H1299-SSX2, K562-A11, and SW620 are negative tumor cell lines and used as controls.

[0189] First, prepare an ELISPOT plate. Activate the ELISPOT plate with ethanol and coat it overnight at 4°C. On the first day of the experiment, remove the coating solution, wash, seal, and incubate at room temperature for 2 hours. Remove the sealing solution and add each component of the test to the ELISPOT plate, as follows: target cells at 2 x 10 4 cells / well, and effector cells were 103 Cells / well (calculated according to the transfected positive rate) were placed in two duplicate wells. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, wash the plate, and perform secondary detection and color development. Dry the plate and count the spots formed on the membrane using an immunospot plate reader (ELISPOT READER system, AID20 Company).

[0190] The experimental results are shown in Figure 14. In the case of positive target cell lines, the effector cells transfected with the high-affinity TCR of the present invention have very good specific activation effects, and their function is far superior to that of effector cells transfected with wild-type TCRs, while cells transfected with other TCRs show almost no activation.

[0191] Example 11. Activation function experiments of effector cells transfected with high affinity TCRs of the present invention (target cells are T2-loaded related short peptides) This example demonstrates in another aspect that effector cells transfected with the high-affinity TCR of the present invention have a very good specific activation effect on artificially prepared target cells. Those skilled in the art are familiar with methods for detecting the activation function of cells using ELISPOT experiments. CD8+ T cells isolated from the blood of healthy volunteers and transfected with the TCR of the present invention are randomly selected as effector cells. The TCRs and their numbers are known from Table 4 and are TCR7 (α chain variable domain SEQ ID NO:1, β chain variable domain SEQ ID NO:94), TCR8 (α chain variable domain SEQ ID NO:1, β chain variable domain SEQ ID NO:95), TCR26 (α chain variable domain SEQ ID NO:1, β chain variable domain SEQ ID NO:99), TCR9 (α chain variable domain SEQ ID NO:1, β chain variable domain SEQ ID NO:96), and TCR10 (α chain variable domain SEQ ID NO:1, β chain variable domain SEQ ID NO:97), respectively. The control effector cells are labeled as wild-type TCR (cells transfected with wild-type TCR) and A6 (cells transfected with other TCRs). The target cells in this example are T2 cells loaded with specific short peptides.

[0192] First, prepare the ELISPOT plate. Activate the ELISPOT plate with ethanol and coat it overnight at 4°C. On the first day of the experiment, remove the coating solution, wash, seal, and incubate at room temperature for 2 hours. Remove the sealing solution and add each component of the test to the ELISPOT plate, including: short peptides (final concentration of 1 x 10 in the ELISPOT well plate); ―13 g / ml ~ 1 × 10 ―8 (6 gradients in total, 2 x 10 µg / mL) and target cells 4 cells / well, and effector cells were 10 3Cells / well (calculated according to the transfected positive rate) are placed in two duplicate wells. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, wash the plate, and perform secondary detection and color development. Dry the plate, and count the spots formed on the membrane using an immunospot plate reader (ELISPOT READER system, AID20 Company).

[0193] The experimental results are shown in Figures 15(a) to 15(e), and show that effector cells transfected with the TCR of the present invention have a very strong activation effect on target cells loaded with specific short peptides, and their function is significantly superior to that of effector cells transfected with wild-type TCR, while effector cells transfected with other TCRs have no activation effect on target cells.

[0194] Example 12. IncuCyte killing function experiments with effector cells transfected with high affinity TCRs of the present invention This example verifies that effector cells transfected with the high affinity TCR of the present invention have good specific killing effect on target cells.

[0195] Those skilled in the art are familiar with the use of IncuCyte experiments to detect cell function, a non-invasive method for recording the real-time growth status of cells. PBL cells isolated from Australian fetal bovine serum (Gibco, #10099-141) transfected with the TCR of the present invention were randomly selected as effector cells. The TCRs and their numbers are found in Table 4 and are TCR2 (α-chain variable domain SEQ ID NO:1, β-chain variable domain SEQ ID NO:89) and TCR1 (α-chain variable domain SEQ ID NO:1, β-chain variable domain SEQ ID NO:88), respectively. Control effector cells are labeled as wild-type TCR (cells transfected with wild-type TCR) and A6 (cells transfected with other TCRs). The target cell lines are A375, SW620, and HCCC9810 cells. Here, the target cell line A375 is a positive tumor cell line, and SW620 and HCCC9810 are negative tumor cell lines and are used as controls.

[0196] Target cells were introduced, centrifuged, and resuspended in complete medium of phenol red-free RPMI 1640 + 10% FBS at 1 x 10 4 Count the cells / well, seed the target cells evenly into a 96-well plate, return to a 37°C, 5% CO2 incubator, and incubate overnight. The next day, discard the medium in the 96-well plate and replace it with RPMI1640 + 10% FBS medium without phenol red containing the dye caspase 3 / 7 reagent at a dye concentration of 2 drops / ml. Prepare the effector cells by centrifugation, discard the old medium, and replace it with new RPMI1640 + 10% FBS medium without phenol red. Add the effector cells (1 x 10 4The experimental group in which the transfected cells (cells / well, calculated according to the transfected positive rate) and target cells were plated were co-cultured, and for the control group containing only target cells, each well was supplemented with the same amount of complete medium. The plate was placed in Incucyte ZooM, a real-time dynamic live cell imaging analyzer dedicated to detection, and after 30 minutes of incubation, observation and photography were taken in real time. Incucyte ZooM 2016A was used to process the test results and analyze and derive the data.

[0197] As shown in the detection results in Figures 16a to 16c, cells transduced with the TCR of the present invention have a very strong killing effect on positive targets, and their function is significantly superior to that of effector cells transfected with wild-type TCRs. They have essentially no killing effect on negative target cells, and cells transduced with other TCRs have essentially no killing effect on positive target cells.

[0198] Example 13. Examination of the LDH killing function of effector cells transfected with the high affinity TCR of the present invention This example uses a non-radioactive cytotoxicity experiment to verify the killing function of cells transduced with the TCR of the present invention by measuring LDH release. This test is a colorimetric alternative to the 51Cr release cytotoxicity test, quantitatively measuring lactate dehydrogenase (LDH) released after cell pyrolysis. LDH released into the medium is detected using a 30-minute coupled enzymatic reaction, in which LDH can convert tetrazolium salt (INT) into red formazan. The amount of red product produced is proportional to the number of cells pyrolyzed. Visible light absorbance data at 490 nm can be collected using a standard 96-well plate reader.

[0199] Those skilled in the art are familiar with methods for detecting cell function using LDH release experiments. CD3+ T cells isolated from the blood of healthy volunteers transfected with the TCRs of the present invention were randomly selected as effector cells. The TCRs and their numbers are found in Table 4 and are TCR2 (α-chain variable domain SEQ ID NO:1, β-chain variable domain SEQ ID NO:89) and TCR1 (α-chain variable domain SEQ ID NO:1, β-chain variable domain SEQ ID NO:88), respectively. Control effector cells are labeled as wild-type TCR (cells transfected with wild-type TCR) and A6 (cells transfected with other TCRs). Target cell lines: HLF-A2 (A2 overexpression), HUH-1-A2 (A2 overexpression), HT1080-A2 (A2 overexpression), IM9, and HCCC9810 cells. Here, HLF-A2, HUH-1-A2 and HT1080-A2 are positive tumor cell lines, and IM9 and HCCC9810 are negative tumor cell lines and used as controls.

[0200] First, prepare the LDH plate. On the first day of the experiment, add the test components to the plate in the following order: 3 x 10 target cell line 4 cells / well, effector cells 3 x 10 4 Three duplicate wells were set up, with 10 cells per well (calculated according to the transfected positive rate). A spontaneous well for effector cells, a spontaneous well for target cells, a maximum well for target cells, a volume compensation control well, and a medium background control well were set up simultaneously. The well was incubated overnight (37°C, 5% CO2). On the second day of the experiment, color development was detected, and after the reaction was completed, the absorbance value at 490 nm was recorded using a microplate reader (Bioteck).

[0201] The experimental results are shown in Figure 17. Cells transduced with the TCR of the present invention have a very strong killing effect on positive target cells, which is much higher than that of cells transduced with wild-type TCR, while cells transduced with other TCRs have basically no killing effect on positive target cells.

[0202] Example 14. Functional experiment of the fusion protein of the high affinity TCR and anti-CD3 antibody of the present invention This example verifies that the high affinity TCR anti-CD3 antibody fusion protein of the present invention can redirect effector cells and has good activation effect.

[0203] Those skilled in the art are familiar with methods for detecting cell function using ELISPOT experiments. The effector cells used in the IFN-γ ELISPOT experiments in this example were CD8+ T cells isolated from the blood of healthy volunteers, and the target cell lines were T2, A375, U251, SW620-SSX2 (SSX2 overexpression), SW620, and K562-A11 (A11 overexpression) cells, where A375, U251, and SW620-SSX2 express the relevant antigen, and T2, SW620, and K562-A11 do not express the relevant antigen. High affinity TCRs of the present invention were randomly selected and fusion proteins were prepared as described in Example 8, designated Fusion Protein 1 (α chain SEQ ID NO:78, β chain SEQ ID NO:105), Fusion Protein 2 (α chain SEQ ID NO:79, β chain SEQ ID NO:105), Fusion Protein 3 (α chain SEQ ID NO:81, β chain SEQ ID NO:105) and Fusion Protein 4 (α chain SEQ ID NO:83, β chain SEQ ID NO:105), respectively.

[0204] First, prepare an ELISPOT plate. Activate the ELISPOT plate with ethanol and coat it overnight at 4°C. On the first day of the experiment, remove the coating solution, wash, seal, and incubate at room temperature for 2 hours. Remove the sealing solution and add each component of the test to the ELISPOT plate, as follows: fusion protein (final concentration in the ELISPOT well plate: 1 x 10 ―13 g / ml ~ 1 × 10 ―8 (µg / mL total of six gradients), target cell line (2 x 10 4 cells / well), effector cells (4 × 103 Effector cells (per well) were added to the corresponding wells, and two duplicate wells were placed. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, the plate was washed, and secondary detection and color development were performed. The plate was dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system, AID20 Company).

[0205] The experimental results are shown in Figures 18a to 18d. The high-affinity TCR and anti-CD3 antibody fusion protein of the present invention can fully recognize and bind to the specific short peptide loaded with T2, redirect effector cells, and have a very good activation effect. However, it is non-specific to T2 when loaded or does not react at all when not loaded.

[0206] All documents mentioned in this application are incorporated by reference in this application as if each document were incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art will be able to make various changes or modifications to the present invention, and these equivalents will also fall within the scope defined by the appended claims of this application.

Claims

1. a T cell receptor (TCR), having the activity of binding to the KASEKIFYV-HLA A0201 complex, and The TCR alpha chain variable domain comprises three CDRs, CDR1 alpha, CDR2 alpha, and CDR3 alpha, and the TCR beta chain variable domain comprises three CDR regions, CDR1 beta, CDR2 beta, and CDR3 beta, and the TCR alpha and beta chain variable domains are selected from the group consisting of: Table 1-1 Table 1-2 The TCR having a CDR selected from: Table 2-1 Table 2-2 is selected from The TCR.

2. 2. The TCR of claim 1, comprising: (i) all or a portion of a TCR alpha chain excluding its transmembrane domain; and (ii) all or a portion of a TCR beta chain excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least a portion of the constant domain of the TCR chain. The TCR.

3. An artificial interchain disulfide bond is included between the α chain constant region and the β chain constant region of the TCR, and a cysteine ​​residue that forms the artificial interchain disulfide bond between the α chain constant region and the β chain constant region of the TCR is Thr48 of TRAC*01 exon 1 and Ser57 of TRBC1*01 or TRBC2*01 exon 1, Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1, Tyr10 of TRAC*01 exon 1 and Ser17 of TRBC1*01 or TRBC2*01 exon 1, Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1, Ser15 of TRAC*01 exon 1 and Glu15 of TRBC1*01 or TRBC2*01 exon 1, Arg53 of TRAC*01 exon 1 and Ser54 of TRBC1*01 or TRBC2*01 exon 1, Pro89 in TRAC*01 exon 1 and Ala19 in TRBC1*01 or TRBC2*01 exon 1, and replacing one or more sites selected from the group consisting of Tyr10 of TRAC*01 exon 1 and Glu20 of TRBC1*01 or TRBC2*01 exon 1; The TCR of claim 2.

4. the amino acid sequence of the TCR alpha chain variable domain is selected from SEQ ID NOs: 1 and 64-87, and / or the amino acid sequence of the TCR beta chain variable domain is selected from SEQ ID NOs: 88-114; The TCR of claim 1.

5. It is a single-chain TCR, The TCR of claim 1.

6. 2. The TCR according to claim 1, which is a single-chain TCR consisting of an α chain variable domain and a β chain variable domain, wherein the α chain variable domain and the β chain variable domain are linked by a flexible short peptide sequence (linker). The TCR.

7. The TCR according to any one of claims 1 to 6, The TCR, wherein a conjugate is bound to the C-terminus or N-terminus of the α chain and / or β chain of the TCR.

8. The TCR of claim 7, wherein the conjugate is a detectable marker.

9. The TCR of claim 7, wherein the conjugate is an anti-CD3 antibody.

10. A multivalent TCR complex comprising at least two TCR molecules, wherein at least one TCR molecule is a TCR according to any one of claims 1 to 9.

11. A nucleic acid molecule comprising a nucleic acid sequence encoding the TCR of any one of claims 1 to 9.

12. A vector comprising the nucleic acid molecule of claim 11.

13. A host cell comprising the vector of claim 12 or having an exogenous nucleic acid molecule of claim 11.

14. An isolated cell expressing the TCR of any one of claims 1 to 9.

15. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1 to 9, or a TCR complex according to claim 10, or a cell according to claim 14.

16. Use of a TCR according to any one of claims 1 to 9, a TCR complex according to claim 10 or a cell according to claim 14, The use as described above for preparing a medicament for treating a tumor.

17. A drug for treating a tumor, comprising the TCR of any one of claims 1 to 9, the TCR complex of claim 10, or the cell of claim 14.

18. A method for preparing a T cell receptor according to any one of claims 1 to 9, comprising: (i) expressing the T cell receptor of any one of claims 1 to 9 by culturing a host cell of claim 13; and (ii) isolating or purifying said T cell receptor.

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