TW202144403A - High-affinity TCR for recognizing hpv16 - Google Patents

Abstract

A T cell receptor (TCR), having the property of binding to a YMLDLQPET-HLA A0201 complex. The binding affinity of the TCR to the YMLDLQPET-HLA A0201 complex is at least 2 times that of a wild-type TCR to the YMLDLQPET-HLA A0201 complex. A fusion molecule of such TCR and a therapeutic agent is also provided. Such TCR can be used alone or in combination with a therapeutic agent to target tumor cells presenting the YMLDLQPET-HLA A0201 complex.

Description

A high-affinity TCR that recognizes HPV16

The present invention relates to the field of biotechnology, and more particularly to a T cell receptor (TCR) capable of recognizing polypeptides derived from HPV16 E7 protein. The present invention also relates to the preparation and use of said receptors.

Only two types of molecules are capable of recognizing antigens in a specific manner. One of them is immunoglobulin or antibody; the other is T cell receptor (TCR), which is a glycoprotein on the surface of the cell membrane in the form of heterodimers of α chain/β chain or γ chain/δ chain. The composition of the TCR repertoire of the immune system is generated in the thymus by V(D)J recombination followed by positive and negative selection. In the surrounding environment, TCR mediates the specific recognition of the major histocompatibility complex-peptide complex (pMHC) by T cells and is therefore crucial for the cellular immune function of the immune system.

TCR is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC), and this exogenous or endogenous peptide may be the only sign of abnormal cells. In the immune system, direct physical contact between T cells and antigen-presenting cells (APCs) is initiated through the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact. It causes a series of subsequent cell signaling and other physiological responses, so that T cells with different antigen specificities exert immune effects on their target cells.

The MHC class I and class II molecular ligands corresponding to TCR are also proteins of the immunoglobulin superfamily, but they are specific for the presentation of antigens. Different individuals have different MHCs, so that they can present different short-term proteins in a protein antigen. Peptides to the respective APC cell surfaces. The human MHC is often referred to as the HLA gene or HLA complex.

The HPV16 E7 gene is one of the early region genes of the human papillomavirus (HPV) genome, and the encoded E7 protein is a small acidic protein containing about 100 amino acids. High-risk HPV16 E7 protein is an important cause of cervical cancer, head and neck cancer, and anal cancer. After HPV16 E7 infection of cells, the newly synthesized antigens are processed into antigenic peptides in the cytoplasm, and combined with MHC (major histocompatibility complex) molecules to form complexes, which are presented on the cell surface. YMLDLQPET is a short peptide derived from the HPV16 E7 antigen and is a target for the treatment of HPV16 E7-related diseases.

Therefore, the YMLDLQPET-HLA A0201 complex provides a marker that TCR can target tumor cells. The TCR that can bind to YMLDLQPET-HLA A0201 complex has high application value for tumor therapy. For example, a TCR capable of targeting the tumor cell marker can be used to deliver cytotoxic or immunostimulatory agents to target cells, or be transfected into T cells, enabling T cells expressing the TCR to destroy tumor cells for Administer to patients during the course of treatment for adoptive immunotherapy. For the former purpose, the ideal TCR has a high affinity, so that the TCR can reside on the target cell for a long time. For the latter purpose, it is preferred to use a medium affinity TCR. Therefore, those skilled in the art are devoted to developing TCRs that can be used to target tumor cell markers for different purposes.

Summary of Invention

The purpose of the present invention is to provide a TCR with higher affinity for YMLDLQPET-HLA A0201 complex.

Another object of the present invention is to provide a preparation method of the above-mentioned type of TCR and the use of the above-mentioned type of TCR.

In a first aspect of the present invention, there is provided a T cell receptor (TCR), which has the activity of binding YMLDLQPET-HLA A0201 complex.

In a preferred embodiment of the present invention, the T cell receptor comprises a TCRα chain variable domain and a TCRβ chain variable domain, wherein it has the activity of binding to YMLDLQPET-HLA A0201 complex, and the α chain of the TCR can The variable domain comprises an amino acid sequence having at least 90% sequence homology with the sequence shown in SEQ ID NO: 1 and the TCR beta chain variable domain comprises at least 90% sequence with the sequence shown in SEQ ID NO: 2 Homologous amino acid sequences.

In another preferred embodiment, the affinity of the TCR to the YMLDLQPET-HLA A0201 complex is at least 2 times that of the wild-type TCR.

In another preferred embodiment, the α chain variable domain of the TCR comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% sequence homology.

In another preferred embodiment, the β chain variable domain of the TCR is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98%, 99% or 100% sequence homology.

In another preferred embodiment, the α-chain variable domain of the TCR comprises an amino acid sequence having at least 96% sequence homology with the sequence shown in SEQ ID NO: 1.

In another preferred embodiment, the β chain variable domain of the TCR comprises an amino acid sequence having at least 97% sequence homology with the sequence shown in SEQ ID NO: 2.

In another preferred embodiment, the reference sequences of the three CDR regions of the variable domain of the TCRα chain are as follows, CDR1α: ATGYPS CDR2α: ATKADDK CDR3α: ALYNQGGKLI, and CDR3α contains at least one of the following mutations: Residues before mutation mutated residue Position 4 N of CDR3α L or A or V Q at position 5 of CDR3α F or Y or W The 6th G of CDR3α N or S or D or A or V or T or H or M G at position 7 of CDR3α A or N or S or F K at position 8 of CDR3α V or R or L or I or M or Q or S or N

In another preferred embodiment, in the variable domain of the TCRα chain, the number of amino acid mutations in CDR3α is 2, 3 or 4.

In another preferred embodiment, the reference sequences of the three CDR regions of the variable domain of the TCRβ chain are as follows: CDR1α: ATGYPS CDR2α: ATKADDK CDR3α: ALYNQGGKLI, and CDR3α contains at least one of the following mutations: Residues before mutation mutated residue Position 4 N of CDR3α L Q at position 5 of CDR3α F or Y The 6th G of CDR3α N or S G at position 7 of CDR3α A or N or S K at position 8 of CDR3α V or R or L

In another preferred embodiment, the reference sequences of the three CDR regions of the variable domain of the TCRα chain are as follows, CDR1α: ATGYPS CDR2α: ATKADDK CDR3α: ALYNQGGKLI, and CDR3α contains at least one of the following mutations: Residues before mutation mutated residue Position 4 N of CDR3α L or A or V Q at position 5 of CDR3α F or Y or W The 6th G of CDR3α N or S or D or A or V or T or H or M G at position 7 of CDR3α A or N or S or F K at position 8 of CDR3α V or R or L or I or M or Q or S or N

And, the β chain variable domain of the TCR comprises an amino acid sequence having at least 97% sequence homology with the sequence shown in SEQ ID NO: 2.

In another preferred embodiment, the reference sequences of the three CDR regions of the variable domain of the TCRα chain are as follows: CDR1α: ATGYPS CDR2α: ATKADDK CDR3α: ALYNQGGKLI, and CDR3α contains at least one of the following mutations: Residues before mutation mutated residue Position 4 N of CDR3α L or A or V Q at position 5 of CDR3α F or Y or W The 6th G of CDR3α N or S or D or A or V or T or H or M G at position 7 of CDR3α A or N or S or F K at position 8 of CDR3α V or R or L or I or M or Q or S or N

And, the three CDR regions of the variable domain of the TCRβ chain are as follows: CDR1β: SGHTA CDR2β: FQGTGA and CDR3β:ASSLLAGSYEQY.

In another preferred embodiment, the reference sequences of the three CDR regions of the variable domain of the TCRα chain are as follows: CDR1α: ATGYPS CDR2α: ATKADDK CDR3α: ALYNQGGKLI, and CDR3α contains at least one of the following mutations: Residues before mutation mutated residue Position 4 N of CDR3α L or A or V Q at position 5 of CDR3α F or Y or W The 6th G of CDR3α N or S or D or A or V or T or H or M G at position 7 of CDR3α A or N or S or F K at position 8 of CDR3α V or R or L or I or M or Q or S or N

And, the amino acid sequence of the variable domain of the TCRβ chain is SEQ ID NO:2.

In another preferred embodiment, the reference sequences of the three CDR regions of the variable domain of the TCRβ chain are as follows, CDR1β: SGHTA CDR2β: FQGTGA CDR3β: ASSLLAGSYEQY, and CDR3β contains at least one of the following mutations: Residues before mutation mutated residue E at position 10 of CDR3β Y or M Q at position 11 of CDR3β V or L Y at position 12 of CDR3β S or E

In another preferred embodiment, the TCRβ chain variable domain comprises 3 CDR regions, and the amino acid sequences of the 3 CDR regions in the TCRβ chain variable domain are as follows: CDR1β: SGHTA CDR2β: FQGTGA and CDR3β: ASSLLAGSYEQY

In another preferred embodiment, the amino acid sequence of the variable domain of the TCRβ chain is SEQ ID NO: 2.

In another preferred embodiment, the variable domain of the TCRα chain comprises three CDR regions, wherein CDR1α is ATGYPS, and CDR2α is ATKADDK.

In another preferred embodiment, the TCRα chain variable domain comprises CDR1α, CDR2α and CDR3α, wherein the amino acid sequence of CDR1α is ATGYPS, the amino acid sequence of CDR2α is ATKADDK, and the amino acid sequence of CDR3α is: ALY [3αX1][3αX2][3αX3][3αX4][3αX5]LI, where [3αX1] is N or L or A or V, [3αX2] is Q or F or Y or W, and [3αX3] is G or N or S or D or A or V or T or H or M, [3αX4] is G or A or N or S or F, and [3αX5] is K or V or R or L or I or M or Q or S or N .

In another preferred embodiment, the TCRα chain variable domain comprises CDR1α, CDR2α and CDR3α, wherein the amino acid sequence of CDR1α is ATGYPS, the amino acid sequence of CDR2α is ATKADDK, and the amino acid sequence of CDR3α is: ALY [3αX1][3αX2][3αX3][3αX4][3αX5]LI, where [3αX1] is N or L or A or V.

In another preferred embodiment, the TCRα chain variable domain comprises CDR1α, CDR2α and CDR3α, wherein the amino acid sequence of CDR1α is ATGYPS, the amino acid sequence of CDR2α is ATKADDK, and the amino acid sequence of CDR3α is: ALY [3αX1][3αX2][3αX3][3αX4][3αX5]LI, where [3αX2] is Q or F or Y or W.

In another preferred embodiment, the TCRα chain variable domain comprises CDR1α, CDR2α and CDR3α, wherein the amino acid sequence of CDR1α is ATGYPS, the amino acid sequence of CDR2α is ATKADDK, and the amino acid sequence of CDR3α is: ALY [3αX1][3αX2][3αX3][3αX4][3αX5]LI, where [3αX3] is G or N or S or D or A or V or T or H or M.

In another preferred embodiment, the TCRα chain variable domain comprises CDR1α, CDR2α and CDR3α, wherein the amino acid sequence of CDR1α is ATGYPS, the amino acid sequence of CDR2α is ATKADDK, and the amino acid sequence of CDR3α is: ALY [3αX1][3αX2][3αX3][3αX4][3αX5]LI, where [3αX4] is G or A or N or S or F.

In another preferred embodiment, the TCRα chain variable domain comprises CDR1α, CDR2α and CDR3α, wherein the amino acid sequence of CDR1α is ATGYPS, the amino acid sequence of CDR2α is ATKADDK, and the amino acid sequence of CDR3α is: ALY [3αX1][3αX2][3αX3][3αX4][3αX5]LI, where [3αX5] is K or V or R or L or I or M or Q or S or N.

In another preferred embodiment, the amino acid sequence of the CDR3α is selected from the group consisting of: ALYLFNGKLI, ALYNYNAVLI, ALYNFSNRLI and ALYNFNSLLI.

In another preferred embodiment, the TCRβ chain variable domain comprises CDR1β, CDR2β and CDR3β, wherein the amino acid sequence of CDR1β is SGHTA, the amino acid sequence of CDR2β is FQGTGA, and the amino acid sequence of CDR3β is: ASSLLAGSY [3βX1][3βX2][3βX3], where [3βX1] is E or Y or M, [3βX2] is Q or V or L, and [3βX3] is Y or E or S.

In another preferred embodiment, the TCRβ chain variable domain comprises CDR1β, CDR2β and CDR3β, wherein the amino acid sequence of CDR1β is SGHTA, the amino acid sequence of CDR2β is FQGTGA, and the amino acid sequence of CDR3β is: ASSLLAGSY [3βX1][3βX2][3βX3], where [3βX1] is E or Y or M.

In another preferred embodiment, the TCRβ chain variable domain comprises CDR1β, CDR2β and CDR3β, wherein the amino acid sequence of CDR1β is SGHTA, the amino acid sequence of CDR2β is FQGTGA, and the amino acid sequence of CDR3β is: ASSLLAGSY [3βX1][3βX2][3βX3], where [3βX2] is Q or V or L.

In another preferred embodiment, the TCRβ chain variable domain comprises CDR1β, CDR2β and CDR3β, wherein the amino acid sequence of CDR1β is SGHTA, the amino acid sequence of CDR2β is FQGTGA, and the amino acid sequence of CDR3β is: ASSLLAGSY [3βX1][3βX2][3βX3], where [3βX3] is Y or E or S.

In another preferred embodiment, the TCR is mutated in the α chain variable domain shown in SEQ ID NO: 1, and the mutation is selected from N94L/A/V, Q95F/Y/W, G96N/S/D One or more of /A/V/T/H/M, G97A/N/S/F, K98V/R/L/I/M/Q/S/N, where the amino acid residues are numbered The numbering shown in SEQ ID NO: 1 is used.

In another preferred embodiment, the TCR is mutated in the β chain variable domain shown in SEQ ID NO: 2, and the mutation is selected from one or more of E102Y/M, Q103V/L, and Y104S/E group, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 2.

In another preference, the TCR has a CDR selected from the group consisting of: CDR number α-CDR1 α-CDR2 α-CDR3 β-CDR1 β-CDR2 β-CDR3 1 ATGYPS ATKADDK ALYLFNGKLI SGHTA FQGTGA ASSLLAGSYEQY 2 ATGYPS ATKADDK ALYNYNAVLI SGHTA FQGTGA ASSLLAGSYEQY 3 ATGYPS ATKADDK ALYNFSNRLI SGHTA FQGTGA ASSLLAGSYEQY 4 ATGYPS ATKADDK ALYNFNSLLI SGHTA FQGTGA ASSLLAGSYEQY 5 ATGYPS ATKADDK ALYNFNFVLI SGHTA FQGTGA ASSLLAGSYEQY 6 ATGYPS ATKADDK ALYLFSGKLI SGHTA FQGTGA ASSLLAGSYEQY 7 ATGYPS ATKADDK ALYNFDGILI SGHTA FQGTGA ASSLLAGSYEQY 8 ATGYPS ATKADDK ALYNFNFRLI SGHTA FQGTGA ASSLLAGSYEQY 9 ATGYPS ATKADDK ALYAFDGKLI SGHTA FQGTGA ASSLLAGSYEQY 10 ATGYPS ATKADDK ALYNFDFRLI SGHTA FQGTGA ASSLLAGSYEQY 11 ATGYPS ATKADDK ALYNFNFMLI SGHTA FQGTGA ASSLLAGSYEQY 12 ATGYPS ATKADDK ALYNFNFQLI SGHTA FQGTGA ASSLLAGSYEQY 13 ATGYPS ATKADDK ALYNFNFKLI SGHTA FQGTGA ASSLLAGSYEQY 14 ATGYPS ATKADDK ALYNFDGKLI SGHTA FQGTGA ASSLLAGSYEQY 15 ATGYPS ATKADDK ALYNFASKLI SGHTA FQGTGA ASSLLAGSYEQY 16 ATGYPS ATKADDK ALYNFVFKLI SGHTA FQGTGA ASSLLAGSYEQY 17 ATGYPS ATKADDK ALYNFNSKLI SGHTA FQGTGA ASSLLAGSYEQY 18 ATGYPS ATKADDK ALYNYDFRLI SGHTA FQGTGA ASSLLAGSYEQY 19 ATGYPS ATKADDK ALYNQGGKLI SGHTA FQGTGA ASSLLAGSYYVS 20 ATGYPS ATKADDK ALYNQGGKLI SGHTA FQGTGA ASSLLAGSYMQY twenty one ATGYPS ATKADDK ALYNQGGKLI SGHTA FQGTGA ASSLLAGSYYLE twenty two ATGYPS ATKADDK ALYNFSFVLI SGHTA FQGTGA ASSLLAGSYEQY twenty three ATGYPS ATKADDK ALYNWNFKLI SGHTA FQGTGA ASSLLAGSYEQY twenty four ATGYPS ATKADDK ALYVFTGKLI SGHTA FQGTGA ASSLLAGSYEQY 25 ATGYPS ATKADDK ALYLFDGKLI SGHTA FQGTGA ASSLLAGSYEQY 26 ATGYPS ATKADDK ALYNFDFVLI SGHTA FQGTGA ASSLLAGSYEQY 27 ATGYPS ATKADDK ALYNFHFRLI SGHTA FQGTGA ASSLLAGSYEQY 28 ATGYPS ATKADDK ALYNFDFSLI SGHTA FQGTGA ASSLLAGSYEQY 29 ATGYPS ATKADDK ALYNFSSVLI SGHTA FQGTGA ASSLLAGSYEQY 30 ATGYPS ATKADDK ALYNFNFNLI SGHTA FQGTGA ASSLLAGSYEQY 31 ATGYPS ATKADDK ALYNFMFKLI SGHTA FQGTGA ASSLLAGSYEQY 32 ATGYPS ATKADDK ALYNFDSKLI SGHTA FQGTGA ASSLLAGSYEQY 33 ATGYPS ATKADDK ALYNFSFKLI SGHTA FQGTGA ASSLLAGSYEQY 34 ATGYPS ATKADDK ALYNFNSILI SGHTA FQGTGA ASSLLAGSYEQY 35 ATGYPS ATKADDK ALYNFNAKLI SGHTA FQGTGA ASSLLAGSYEQY 36 ATGYPS ATKADDK ALYNFDAKLI SGHTA FQGTGA ASSLLAGSYEQY 37 ATGYPS ATKADDK ALYNFAFKLI SGHTA FQGTGA ASSLLAGSYEQY

In another preferred embodiment, the TCR is soluble.

In another preferred embodiment, the TCR is an αβ heterodimeric TCR, comprising the α chain TRAC constant region sequence and the β chain TRBC1 or TRBC2 constant region sequence.

In another preferred embodiment, the TCR comprises (i) all or part of the TCRα chain excluding its transmembrane domain, and (ii) all or part of the TCRβ chain excluding its transmembrane domain, wherein (i) and (ii) each comprise the variable domain and at least a portion of the constant domain of the TCR chain.

In another preferred embodiment, an artificial interchain disulfide bond is contained between the constant region of the α chain and the constant region of the β chain of the TCR.

In another preferred embodiment, the cysteine residue that forms an artificial interchain disulfide bond between the constant region of the TCRα and the β chain is substituted for one or more sets of addresses selected from the following: Thr48 in exon 1 of TRAC*01 and Ser57 in exon 1 of TRBC1*01 or TRBC2*01; Thr45 in exon 1 of TRAC*01 and Ser77 in exon 1 of TRBC1*01 or TRBC2*01; Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01; Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01; Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01; Arg53 in exon 1 of TRAC*01 and Ser54 in exon 1 of TRBC1*01 or TRBC2*01; Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01; and Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.

In another preferred embodiment, the amino acid sequence of the α chain variable domain of the TCR is selected from: SEQ ID NOs: 13-46; and/or the amino acid sequence of the β chain variable domain of the TCR is SEQ ID NO: 2.

In another preferred embodiment, the TCR is selected from the following group: TCR number Alpha chain variable domain sequence SEQ ID NO: Beta chain variable domain sequence SEQ ID NO: 1 13 2 2 14 2 3 15 2 4 16 2 5 17 2 6 18 2 7 19 2 8 20 2 9 twenty one 2 10 twenty two 2 11 twenty three 2 12 twenty four 2 13 25 2 14 26 2 15 27 2 16 28 2 17 29 2 18 30 2 19 1 47 20 1 48 twenty one 1 49 twenty two 31 2 twenty three 32 2 twenty four 33 2 25 34 2 26 35 2 27 36 2 28 37 2 29 38 2 30 39 2 31 40 2 32 41 2 33 42 2 34 43 2 35 44 2 36 45 2 37 46 2

In another preferred embodiment, the TCR is a single-chain TCR.

In another preferred embodiment, the TCR is a single-chain TCR composed 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 composed of a flexible short peptide sequence ( linker) connection.

In another preferred example, a conjugate is bound to the C- or N-terminus of the α chain and/or β chain of the TCR, preferably, the conjugate is a detectable label, a therapeutic agent, or a PK modification some or a combination of any of these substances.

In another preferred embodiment, the therapeutic agent bound to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the α or β chain of the TCR.

The second aspect of the present invention provides a multivalent TCR complex, comprising at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect of the present invention.

The third aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule described in the first aspect of the present invention or the multivalent TCR complex described in the second aspect of the present invention or a complementary sequence thereof .

The fourth aspect of the present invention provides a vector, the vector contains the nucleic acid molecule described in the third aspect of the present invention.

The fifth aspect of the present invention provides a host cell containing the vector of the fourth aspect of the present invention or incorporating the exogenous nucleic acid molecule of the third aspect of the present invention into a chromosome.

The sixth aspect of the present invention provides an isolated cell expressing the TCR of the first aspect of the present invention.

A seventh aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or The cell according to the sixth aspect of the present invention.

The eighth aspect of the present invention provides a method for treating a disease, comprising administering an appropriate amount of the TCR described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the present invention to an object in need of treatment. In the cell according to the sixth aspect, or the pharmaceutical composition according to the seventh aspect of the present invention, preferably, the disease is an HPV-positive tumor, and more preferably, the tumor is cervical cancer.

The ninth aspect of the present invention provides the use of the TCR described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the cell described in the sixth aspect of the present invention, for preparing and treating tumors Preferably, the disease is an HPV-positive tumor, more preferably, the tumor is cervical cancer.

A tenth aspect of the present invention provides a method for preparing the T cell receptor described in the first aspect of the present invention, comprising the steps of: (i) culturing the host cell described in the fifth aspect of the present invention, thereby expressing the T cell receptor described in the first aspect of the present invention; (ii) isolating or purifying the T cell receptor.

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 in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

detailed description

Through extensive and in-depth research, the present invention has obtained a high-affinity T cell receptor (TCR) that recognizes a YMLDLQPET short peptide (derived from HPV16 E7 protein), and the YMLDLQPET short peptide is characterized by a peptide-HLA A0201 complex. form is presented. The high-affinity TCR has 3 CDR regions in its alpha chain variable domain: CDR1α: ATGYPS CDR2α: ATKADDK CDR3α: mutated in ALYNQGGKLI; and/or in the 3 CDR regions of its β-chain variable domain: CDR1β: SGHTA CDR2β: FQGTGA CDR3β: Mutated in ASSLLAGSYEQY; and the affinity and/or binding half-life of the TCR of the present invention for the above-mentioned YMLDLQPET-HLA A0201 complex after mutation is at least 2 times that of the wild-type TCR.

Before the present invention is described, it is to be understood that this invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may 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, as the scope of the invention will be limited only by the scope of the appended claims.

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 in the present invention can be used in the practice or testing of the present invention, the preferred methods and materials are exemplified herein. Term T cell receptor (TCR)

TCRs can be described using the International Information System on Immunogenetics (IMGT). Native αβ heterodimeric TCRs have α and β chains. Broadly speaking, each chain contains a variable region, a linker region and a constant region, and the beta chain typically also 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 junctional region of the TCR is determined by the TRAJ and TRBJ of the unique IMGT, and the constant region of the TCR is determined by the TRAC and TRBC of the IMGT.

Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, chimerically incorporated in a framework sequence. In the IMGT nomenclature, the different numbers of TRAV and TRBV refer to different Vα types and Vβ types, respectively. In the IMGT system, the alpha chain constant domain has the following symbols: TRAC*01, where "TR" represents the T cell receptor gene; "A" represents the alpha chain gene; C represents the constant region; "*01" represents the allele Gene 1. The beta chain constant domain has the following symbols: TRBC1*01 or TRBC2*01, where "TR" denotes the T cell receptor gene; "B" denotes the beta chain gene; C denotes the constant region; "*01" denotes the allele 1. The constant region of the alpha chain is uniquely defined, and in the form of the beta chain, there are two possible constant region genes "C1" and "C2". Those skilled in the art can obtain the constant region gene sequences of TCRα and β chains through the public IMGT database.

The alpha and beta chains of a TCR are generally viewed as having two "domains" each, a variable domain and a constant domain. A variable domain consists of linked variable and linker regions. Thus, in the specification and scope of this application, "TCR alpha chain variable domain" refers to linked TRAV and TRAJ regions, and similarly, "TCR beta chain variable domain" refers to linked TRBV and TRBD/TRBJ regions. The three CDRs of the variable domain of the TCRα chain are CDR1α, CDR2α and CDR3α, respectively; the three CDRs of the variable domain of the TCRβ chain are CDR1β, CDR2β and CDR3β, respectively. The framework sequences of the TCR variable domains of the present invention may be of murine or human origin, preferably human. The constant domains of TCRs comprise an intracellular portion, a transmembrane region and an extracellular portion.

In the present invention, the amino acid sequences of the α and β chain variable domains of the wild-type TCR capable of binding to the YMLDLQPET-HLA A0201 complex are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, as shown in Figure 1a and Figure 1b . The amino acid sequence of α chain and the amino acid sequence of β chain of the soluble "reference TCR" in the present invention are SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in Figure 6a and Figure 6b. The α-chain extracellular amino acid sequence and the β-chain extracellular amino acid sequence of "wild-type TCR" described in the present invention are SEQ ID NO: 50 and SEQ ID NO: 51, respectively, as shown in Figure 9a and Figure 9b . The TCR sequences used in the present invention are of human origin. The amino acid sequence of the α chain and the amino acid sequence of the β chain of the "wild-type TCR" in the present invention are SEQ ID NO: 52 and SEQ ID NO: 53, respectively, as shown in Figures 10a and 10b. 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. Natural interchain disulfide bonds and artificial interchain disulfide bonds

There is a set of disulfide bonds between the Cα and Cβ chains in the near-membrane region of the native TCR, which are referred to as "native interchain disulfide bonds" in the present invention. In the present invention, the artificially introduced interchain covalent disulfide bond whose position is different from that of the natural interchain disulfide bond is referred to as "artificial interchain disulfide bond".

For the convenience of description, the position numbering of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention is numbered in sequence from the N-terminus to the C-terminus. For example, in TRBC1*01 or TRBC2*01, press The 60th amino acid in the sequence from the N-terminal to the C-terminal is P (proline), then in the present invention, it can be described as Pro60 of exon 1 of TRBC1*01 or TRBC2*01, or it can be Expressed as the 60th amino acid of exon 1 of TRBC1*01 or TRBC2*01, and in TRBC1*01 or TRBC2*01, the 61st amino acid in the order from N-terminal to C-terminal is Q (glutamic acid), it can be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1 in the present invention, or it can be described as TRBC1*01 or the 61st of TRBC2*01 exon 1 amino acid, and so on. In the present invention, the positions of the amino acid sequences of the variable regions TRAV and TRBV are numbered according to the positions listed in IMGT. For example, for a certain amino acid in TRAV, the position number listed in IMGT is 46, then it is described as the 46th amino acid of TRAV in the present invention, and so on. In the present invention, if the sequence position numbers of other amino acids have special instructions, they shall be specified according to the special instructions. tumor

The term "tumor" is meant to encompass all types of cancer cell growth or oncogenic processes, metastatic or malignantly transformed cells, tissues or organs, regardless of the type of pathology or stage of invasion. Examples of tumors include, without limitation, solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include: malignancies of various organ systems such as sarcomas, lung squamous cell carcinomas, and cancers. For example: infected prostate, lung, breast, lymph, gastrointestinal (eg: colon), and genitourinary tract (eg: kidney, epithelial cells), throat. Squamous cell carcinoma of the lung includes malignancies such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell carcinomas of the lungs, small bowel cancers and esophageal cancers. Metastatic lesions of the aforementioned cancers can likewise be treated and prevented with the methods and compositions of the present invention. Detailed description of the invention

It is well known that the α chain variable domain and the β chain variable domain of TCR each contain three CDRs, similar to the complementarity determining regions of antibodies. CDR3 interacts with antigenic short peptides, and CDR1 and CDR2 interact with HLA. Therefore, the CDRs of the TCR molecule determine its interaction with the antigenic short peptide-HLA complex. SEQ ID NO: 1 and SEQ ID NO: 2, the sequence was discovered by the inventor for the first time. It has the following CDR regions: Alpha chain variable domain CDRs CDR1α: ATGYPS CDR2α: ATKADDK CDR3α: ALYNQGGKLI and β-chain variable domain CDRs CDR1β: SGHTA CDR2β: FQGTGA and CDR3β:ASSLLAGSYEQY.

The present invention obtains a high-affinity TCR whose affinity with the YMLDLQPET-HLA A0201 complex is at least 2 times that of the wild-type TCR and the YMLDLQPET-HLA A0201 complex through mutation screening of the above-mentioned CDR region.

The present invention provides a T cell receptor (TCR) which has the activity of binding YMLDLQPET-HLA A0201 complex.

The T cell receptor includes a TCRα chain variable domain and a TCRβ chain variable domain, the TCRα chain variable domain includes 3 CDR regions, and the reference sequences of the 3 CDR regions of the TCRα chain variable domain are as follows, CDR1α : ATGYPS CDR2α: ATKADDK CDR3α: ALYNQGGKLI, and CDR3α contains at least one of the following mutations: Residues before mutation mutated residue Position 4 N of CDR3α L or A or V Q at position 5 of CDR3α F or Y or W The 6th G of CDR3α N or S or D or A or V or T or H or M G at position 7 of CDR3α A or N or S or F K at position 8 of CDR3α V or R or L or I or M or Q or S or N

And, the three CDR regions of the variable domain of the TCRβ chain are as follows: CDR1β: SGHTA CDR2β: FQGTGA and CDR3β:ASSLLAGSYEQY.

In more detail, the number of amino acid mutations in the CDR region of the TCRα chain may be 2, 3 or 4.

Further, the TCR of the present invention is an αβ heterodimeric TCR, and the α chain variable domain of the TCR comprises at least 85% of the amino acid sequence shown in SEQ ID NO: 1; preferably, at least 90%; more Preferably, at least 92%; more preferably, at least 94% (eg, can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) the amino acid sequence of sequence homology; And/or the β chain variable domain of described TCR comprises the amino acid sequence shown in SEQ ID NO: 2 with at least 90%, preferably, at least 92%; more preferably, at least 94% (e.g., can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology) amino acid sequence of sequence homology.

Further, the TCR of the present invention is a single-chain TCR, and the α-chain variable domain of the TCR comprises at least 85%, preferably at least 90%, of the amino acid sequence shown in SEQ ID NO: 3; more preferably , at least 92%; most preferably, at least 94% (eg, can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and/or the β chain variable domain of the TCR comprises at least 85% amino acid sequence with the amino acid sequence shown in SEQ ID NO:4 , preferably, at least 90%; more preferably, at least 92%; most preferably, at least 94%; (eg, can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% sequence homology) amino acid sequence of sequence homology.

In the present invention, the three CDRs of the wild-type TCRα chain variable domain SEQ ID NO: 1, namely CDR1, CDR2 and CDR3, are respectively located at positions 27-32, 50-56 and 91-100 of SEQ ID NO: 1 . Accordingly, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 1, 94N is the 4th N of CDR3α, 95Q is the 5th Q of CDR3α, 96G is the 6th G of CDR3α, 97G is the 7th G of CDR3α, and 98K is the 8th K of CDR3α.

The present invention provides a TCR having the property of binding to YMLDLQPET-HLA A0201 complex, and comprising an α chain variable domain and a β chain variable domain, characterized in that the TCR is in the α chain variable domain shown in SEQ ID NO: 1 A mutation occurs in the domain, and the amino acid residue address of the mutation includes one or more of 94N, 95Q, 96G, 97G and 98K, wherein the number of the amino acid residues is shown in SEQ ID NO: 1 Numbering.

Preferably, the mutated TCRα chain variable domain comprises one or more amino acid residues selected from the group consisting of: 94L or 94A or 94V; 95F or 95Y or 95W; 96N or 96S or 96D or 96A or 96V or 96T or 96H or 96M; 97A or 97N or 97S or 97F; 98V or 98R or 98L or 98I or 98M or 98Q or 98S or 98N, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:1.

More specifically, specific forms of the mutation in the alpha chain variable domain include N94L/A/V, Q95F/Y/W, G96N/S/D/A/V/T/H/M, G97A/N/S One or several groups of /F, K98V/R/L/I/M/Q/S/N.

In the present invention, the three CDRs of the wild-type TCR β chain variable domain SEQ ID NO: 2, namely CDR1, CDR2 and CDR3, are respectively located at positions 27-31, 49-54 and 93-104 of SEQ ID NO: 2 . Accordingly, the amino acid residue numbering adopts the numbering shown in SEQ ID NO: 2, 102E is the 10th E of CDR3β, 103Q is the 11th Q of CDR3β, and 104Y is the 12th Y of CDR3β.

The present invention provides a TCR with the property of binding YMLDLQPET-HLA A0201 complex, and comprising a β chain variable domain and a β chain variable domain, characterized in that the TCR is in the β chain variable shown in SEQ ID NO: 2 A mutation occurs in the domain, and the mutated amino acid residue addresses include one or more of 102E, 103Q, and 104Y, wherein the amino acid residue numbering adopts the numbering shown in SEQ ID NO: 2.

Preferably, the mutated TCRβ chain variable domain comprises one or more amino acid residues selected from the group consisting of 102Y or 102M, 103V or 103L, 104S or 104E, wherein the amino acid residues are numbered using The numbering shown in SEQ ID NO: 2.

More specifically, the specific form of the mutation in the beta chain variable domain includes one or several groups of E102Y/M, Q103V/L, Y104S/E.

According to the method of site-directed mutagenesis well known to those skilled in the art, Thr48 of TRAC*01 exon 1 of the wild-type TCR α chain constant region was mutated to cysteine, β chain constant region TRBC1*01 or TRBC2*01 exon 1 Ser57 is mutated to cysteine, that is, the reference TCR is obtained, and its amino acid sequence is SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in Figure 6a and Figure 6b, the cysteine residue after mutation Bases are shown in bold letters. The above cysteine substitutions enable the formation of artificial interchain disulfide bonds between the constant regions of the α and β chains of the reference TCR to form a more stable soluble TCR, allowing for easier evaluation of the TCR complex with YMLDLQPET-HLA A0201 binding affinity and/or binding half-life. It should be understood that the CDR regions of the TCR variable region determine the affinity between it and the pMHC complex, therefore, the cysteine substitutions in the TCR constant region described above have no effect on the binding affinity and/or binding half-life of the TCR. Therefore, in the present invention, the measured binding affinity between the reference TCR and the YMLDLQPET-HLA A0201 complex is regarded as the binding affinity between the wild-type TCR and the YMLDLQPET-HLA A0201 complex. Likewise, if the binding affinity between the TCR of the invention and the YMLDLQPET-HLA A0201 complex is measured to be at least 10 times the binding affinity between the reference TCR and the YMLDLQPET-HLA A0201 complex, it is equivalent to the TCR of the invention and the YMLDLQPET- The binding affinity between the HLA A0201 complexes was at least 10-fold higher than that between the wild-type TCR and the YMLDLQPET-HLA A0201 complex.

Binding affinity (inversely proportional to the dissociation equilibrium constant KD) and binding half-life (expressed as T1 _/2 ) can be determined by any suitable method, such as by surface plasmon resonance techniques. It will be appreciated that doubling the affinity of the TCR will result in a halving of the KD. T _{1/2 is} calculated as In2 divided by the dissociation rate (Koff). Therefore, _{doubling T 1/2} results in a halving of Koff. Preferably, the same assay protocol is used to measure the binding affinity or binding half-life of a given TCR several times, eg, 3 times or more, and the results are averaged. In a preferred embodiment, the surface plasmon resonance (BIAcore) method in the examples herein is used to detect the affinity of soluble TCR, and the conditions are: the temperature is 25°C, and the pH value is 7.1-7.5. This method detects that the dissociation equilibrium constant KD of the reference TCR to the YMLDLQPET-HLA A0201 complex is 6.31E-05M, that is, 63.1 μM. In the present invention, it is considered that the dissociation equilibrium constant KD of the wild-type TCR to the YMLDLQPET-HLA A0201 complex is also 63.1 μM. Since the doubling of the affinity of TCR will lead to the halving of KD, if the dissociation equilibrium constant KD of high-affinity TCR for YMLDLQPET-HLA A0201 complex is detected to be 6.31E-06M, that is, 6.31 μM, it means that the high-affinity TCR has a negative effect on YMLDLQPET- The affinity of the HLA A0201 complex was 10 times that of the wild-type TCR for the YMLDLQPET-HLA A0201 complex. Those skilled in the art are familiar with the conversion relationship between KD value units, namely 1M=106 μM, 1 μM=1000nM, 1nM=1000pM. In the present invention, the affinity of the TCR to the YMLDLQPET-HLA A0201 complex is at least 2 times that of the wild-type TCR; preferably, at least 5 times; more preferably, at least 10 times.

Mutagenesis can be performed using any suitable method, including but not limited to those based on polymerase chain reaction (PCR), restriction enzyme based cloning or ligation independent cloning (LIC) methods. These methods are detailed in many standard molecular biology textbooks. More details on polymerase chain reaction (PCR) mutagenesis and cloning by restriction enzymes can be found in Sambrook and Russell, (2001) Molecular Cloning - A Laboratory Manual (Third Edition) CSHL Press. More information on the LIC method can be found (Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6).

The method of generating the TCRs of the present invention can be, but is not limited to, screening TCRs with high affinity for the YMLDLQPET-HLA-A0201 complex from a diverse sequence library of phage particles displaying such TCRs, as described in the literature (Li, et al. (2005) Nature Biotech 23(3): 349-354).

It will be appreciated that either genes expressing wild-type TCR alpha and beta chain variable domain amino acids or genes expressing slightly modified wild-type TCR alpha and beta chain variable domain amino acids can be used to prepare template TCRs. The changes required to generate the high affinity TCRs of the invention are then introduced into the DNA encoding the variable domains of the template TCR.

The high-affinity TCR of the present invention comprises one of the alpha chain variable domain amino acid sequence of SEQ ID NO: 13-46 and/or the beta chain variable domain amino acid sequence of SEQ ID NO: 2. In the present invention, the amino acid sequences of the α-chain variable domain and the β-chain variable domain forming the heterodimeric TCR molecule are preferably selected from the following table 1: Table 1 TCR number Alpha chain variable domain sequence SEQ ID NO: Beta chain variable domain sequence SEQ ID NO: 1 13 2 2 14 2 3 15 2 4 16 2 5 17 2 6 18 2 7 19 2 8 20 2 9 twenty one 2 10 twenty two 2 11 twenty three 2 12 twenty four 2 13 25 2 14 26 2 15 27 2 16 28 2 17 29 2 18 30 2 19 1 47 20 1 48 twenty one 1 49 twenty two 31 2 twenty three 32 2 twenty four 33 2 25 34 2 26 35 2 27 36 2 28 37 2 29 38 2 30 39 2 31 40 2 32 41 2 33 42 2 34 43 2 35 44 2 36 45 2 37 46 2

For the purposes of the present invention, a TCR of the present invention is a portion having at least one variable domain of the TCRα and/or TCRβ chain. They usually contain both the TCRα chain variable domain and the TCRβ chain variable domain. They can be alpha beta heterodimers or single-chain forms or any other form that can be stably present. In adoptive immunotherapy, the full-length chain of αβ heterodimeric TCRs (comprising 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 combined with other molecules to prepare bifunctional polypeptides to target effector cells, in which case the TCR is preferably in a soluble form.

For stability, it is disclosed in the prior art that soluble and stable TCR molecules can be obtained by introducing artificial interchain disulfide bonds between the constant domains of the α and β chains of TCR, as described in the patent document PCT/CN2015/093806 . Thus, the TCR of the present invention may be one in which artificial interchain disulfide bonds are introduced between residues of its alpha and beta chain constant domains. Cysteine residues form artificial interchain disulfide bonds between the constant domains of the alpha and beta chains of the TCR. Cysteine residues can be substituted for other amino acid residues at appropriate addresses in native TCRs to form artificial interchain disulfide bonds. For example, substitution of Thr48 in exon 1 of TRAC*01 and substitution of Ser57 in exon 1 of TRBC1*01 or TRBC2*01 to form a disulfide bond. Other addresses for introducing cysteine residues to form disulfide bonds can also be: Thr45 of exon 1 of TRAC*01 and Ser77 of exon 1 of TRBC1*01 or TRBC2*01; exon 1 of TRAC*01 1 Tyr10 and TRBC1*01 or Ser17 of TRBC2*01 exon 1; Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon 1 Ser15 and Glu15 in exon 1 of TRBC1*01 or TRBC2*01; Arg53 in exon 1 of TRAC*01 and Ser54 in exon 1 of TRBC1*01 or TRBC2*01; Pro89 and exon 1 of TRAC*01 Ala19 of exon 1 of TRBC1*01 or TRBC2*01; or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01. That is, cysteine residues are substituted for any set of addresses in the constant domains of the alpha and beta chains described above. This can be achieved by truncation of up to 15, or up to 10, or up to 8 or less amino acids at the C-terminus of one or more of the TCR constant domains of the invention so that they do not include cysteine residues The purpose of deleting the natural interchain disulfide bond can also be achieved by mutating the cysteine residue that forms the natural interchain disulfide bond into another amino acid.

As described above, the TCRs of the present invention may comprise artificial interchain disulfide bonds introduced between residues of their alpha and beta chain constant domains. It should be noted that the TCRs of the invention may contain both a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bonds introduced above between the constant domains. The TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence can be linked through the native interchain disulfide bonds present in the TCR.

In addition, in terms of stability, patent document PCT/CN2016/077680 also discloses that the introduction of artificial interchain disulfide bonds between the α chain variable region and the β chain constant region of TCR can significantly improve the stability of TCR. Therefore, the high-affinity TCR of the present invention may also contain artificial interchain disulfide bonds between the variable region of the α chain and the constant region of the β chain. Specifically, 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 substituted for: the 46th amino acid of TRAV and TRBC1*01 or Amino acid position 60 of exon 1 of TRBC2*01; amino acid position 47 of TRAV and amino acid position 61 of exon 1 of TRBC1*01 or TRBC2*01; amino acid position 46 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01. Preferably, such a TCR may comprise (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 contain the variable domain and at least a part of the constant domain of the TCR chain, and the α chain and the β chain form a heterodimer. More preferably, such a TCR may contain an alpha chain variable domain and a beta chain variable domain and all or part of the beta chain constant domain except the transmembrane domain, but it does not contain the alpha chain constant domain, the alpha chain of the TCR. The chain variable domains form heterodimers with beta chains.

In terms of stability, on the other hand, the TCRs of the present invention also include TCRs with mutations in their hydrophobic core regions, and the mutations in these hydrophobic core regions are preferably mutations that can improve the stability of the TCRs of the present invention, such as those published in Publication No. Described in the patent document of WO2014/206304. Such TCRs can be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable domain amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or short peptide amino acid positions 3, 5, 7 from the bottom of the alpha chain J gene (TRAJ), and/or the 2nd, 4, and 6 last amino acid positions of the short peptide amino acid of the beta chain J gene (TRBJ) position, where the position number of the amino acid sequence is as listed in the International Information System on Immunogenetics (IMGT). Those skilled in the art are aware of the above-mentioned International Immunogenetic Information System, and can obtain the position numbers of amino acid residues of different TCRs in IMGT according to the database.

More specifically, in the present invention, the TCR in which the hydrophobic core region is mutated can be a highly stable single-chain TCR composed of a flexible peptide chain linking the variable domains of the α and β chains of the TCR. The CDR region of the TCR variable region determines its affinity with the short peptide-HLA complex, and the mutation of the hydrophobic core can make the TCR more stable, but does not affect its affinity with the short peptide-HLA complex. Affinity. It should be noted that the flexible peptide chain in the present invention can be any peptide chain suitable for linking the variable domains of the TCRα and β chains. The template chain constructed in Example 1 of the present invention for screening high-affinity TCR is the above-mentioned high-stability single-chain TCR containing a hydrophobic core mutation. Using a TCR with higher stability can more conveniently evaluate the affinity between the TCR and the YMLDLQPET-HLA-A0201 complex.

The CDR regions of the α-chain variable domain and the β-chain variable domain of the single-chain template TCR are identical to those of the wild-type TCR. That is, the three CDRs of the α chain variable domain are CDR1α: ATGYPS; CDR2α: ATKADDK; CDR3α: ALYNQGGKLI and the three CDRs of the β chain variable domain are CDR1β: SGHTA; CDR2β: FQGTGA; CDR3β: ASSLLAGSYEQY. The amino acid sequence (SEQ ID NO: 9) and nucleotide sequence (SEQ ID NO: 10) of the single-stranded template TCR are shown in Figures 5a and 5b, respectively. In this way, a single-chain TCR composed of α-chain variable domain and β-chain variable domain with high affinity for YMLDLQPET-HLA A0201 complex was screened out.

The αβ heterodimer with high affinity to the YMLDLQPET-HLA-A0201 complex of the present invention is obtained by transferring the CDR regions of the α and β chain variable domains of the screened high-affinity single-chain TCR to wild type The corresponding positions of the TCR alpha chain variable domain (SEQ ID NO: 1) and beta chain variable domain (SEQ ID NO: 2) were obtained.

The TCRs of the present invention may also be provided in the form of multivalent complexes. The multivalent TCR complexes of the present invention comprise two, three, four or more multimers formed by combining the TCRs of the present invention, for example, the tetramerization domain of p53 can be used to generate tetramers, or multiple A complex formed by combining a TCR of the present invention with another molecule. The TCR complexes of the present invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes with such applications.

The TCR of the present invention can be used alone, or can be combined with the conjugate in a covalent or other manner, preferably in a covalent manner. The conjugate includes a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the YMLDLQPET-HLA-A0201 complex), a therapeutic agent, a PK (protein kinase) modification moiety, or any of the above Combination binding or coupling of substances.

Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable products enzyme.

Therapeutic agents that can be bound or conjugated to the TCRs of the present invention include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. Biological toxicants (Chaudhary et al., 1989, Nature (Nature) 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines such as IL-2, etc. (Gillies et al., 1992, Proceedings of the National Academy of Sciences of the United States of America) (PNAS) 89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. Antibody Fc fragments ( Mosquera et al, 2005, The Journal Of Immunology 174, 4381); 5. Antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62, 319); 6. Gold nanoparticles / Nanorods (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 7. Viral particles (Peng et al., 2004, Gene therapy 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 9. Magnetic nanoparticles; 10. Prodrug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL); 11. chemotherapeutic agents (eg, cisplatin) or nanoparticles in any form, etc.

Antibodies or fragments thereof that bind to the TCR of the present invention include anti-T cell or NK-cell determining antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies, the binding of such antibodies or fragments thereof to TCR is capable of effecting effector cells. Oriented to better target cells. A preferred embodiment is that the TCR of the present invention binds to an anti-CD3 antibody or a functional fragment or variant of said anti-CD3 antibody. Specifically, the fusion molecule of TCR and anti-CD3 single-chain antibody of the present invention comprises one selected from the amino acid sequence of TCR α chain variable domain amino acid sequence SEQ ID NO: 13-46, and the amino acid sequence of TCR β chain variable domain amino acid sequence SEQ ID NO: 13-46 NO: 2.

The present invention also 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. DNA can be the coding or non-coding strand. For example, the nucleic acid sequences encoding the TCRs of the present invention may be identical or degenerate variants of the nucleic acid sequences shown in the figures of the present invention. To illustrate the meaning of "degenerate variant", as used herein, "degenerate variant" in the present invention refers to encoding a protein sequence having SEQ ID NO: 3, but having the same sequence as SEQ ID NO: 5. Different nucleic acid sequences.

The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can generally be obtained by, but not limited to, PCR amplification method, recombinant method or artificial synthesis method. At present, the DNA sequences encoding the TCRs of the present invention (or fragments thereof, or derivatives thereof) can be obtained completely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.

The present invention also relates to vectors comprising the nucleic acid molecules of the present invention, as well as host cells genetically engineered with the vectors or coding sequences of the present invention.

The present invention also includes isolated cells, particularly T cells, expressing the TCRs of the present invention. There are a number of methods suitable for transfection of T cells with DNA or RNA encoding the high affinity TCRs of the invention (eg, Robbins et al., (2008) J. Immunol. 180: 6116-6131). T cells expressing the high-affinity TCR of the present invention can be used for adoptive immunotherapy. Those skilled in the art are aware of many suitable methods for adoptive therapy (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308).

The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR of the present invention, or the TCR complex of the present invention, or a cell that exhibits the TCR of the present invention.

The present invention also provides a method of treating a disease, comprising administering an appropriate amount of a TCR of the present invention, or a TCR complex of the present invention, or a cell exhibiting a TCR of the present invention, or a pharmaceutical composition of the present invention to an object in need of treatment.

It should be understood that the name of amino acid in this paper adopts the international single English letter identification, and the three English letter abbreviations of the corresponding amino acid name are respectively: 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), Val (V);

In the present invention, both Pro60 or 60P represent proline at the 60th position. In addition, the expression of the specific form of the mutation in the present invention is such as "N93D" represents the substitution of N at the 93rd position by D, and similarly, "N93D/E" represents the substitution of the N at the 93rd position by D or by E. Others and so on.

In the art, substitution with amino acids with similar or similar properties usually does not change the function of the protein. The addition of one or several amino acids to the C-terminus and/or N-terminus also generally does not alter protein structure and function. Therefore, the TCRs of the present invention also include up to 5, preferably up to 3, more preferably up to 2, and optimally 1 amino acid (especially those located outside the CDR region) of the TCR of the present invention , is replaced by amino acids with similar or similar properties, and still retains its functional TCR.

The present invention also includes slightly modified TCRs of the present invention. Modified (generally without altering the primary structure) forms include: chemically derivatized forms of the TCRs of the present invention such as acetylated or carboxylated. Modifications also include glycosylation, such as those TCRs resulting from glycosylation modifications in the synthesis and processing of the TCRs of the invention or in further processing steps. This modification can be accomplished by exposing the TCR to enzymes that perform glycosylation, such as mammalian glycosylases or deglycosylases. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are TCRs that are modified to increase their resistance to proteolysis or to optimize solubility.

The TCRs, TCR complexes of the present invention, or TCR-transfected T cells of the present invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The TCRs, multivalent TCR complexes or cells of the present invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It may be provided in unit dosage form, usually in a sealed container, as part of a kit. Such kits, but not necessarily, include instructions for use. It may comprise a plurality of such unit dosage forms.

In addition, the TCRs of the present invention can be used alone, or in combination or conjugation with other therapeutic agents (eg, formulated in the same pharmaceutical composition).

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies detrimental to the individual receiving the composition and are not undue toxicity upon administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

Pharmaceutically acceptable carriers in therapeutic compositions can contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.

Generally, the therapeutic compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid carriers prior to injection can also be prepared.

Once formulated, the composition of the present invention can be administered by conventional routes, including (but not limited to): intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably gastrointestinal External includes subcutaneous, intramuscular or intravenous. The object to be prevented or treated may be an animal; especially a human.

When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be adopted according to the usage. Preferably, injections, oral preparations and the like can be exemplified.

These pharmaceutical compositions can be formulated according to conventional methods by mixing, diluting or dissolving, with occasional addition of suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in a conventional manner according to the dosage form.

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

When the pharmaceutical composition of the present invention is used for actual treatment, the TCR or the TCR complex of the present invention or the cells exhibiting the TCR of the present invention as an active ingredient can be used according to the weight, age, sex, symptom level of each patient to be treated And be reasonably determined, and ultimately by the physician to decide a reasonable dosage.

The main advantages of the present invention are: (1) The affinity and/or binding half-life of the high-affinity TCR of the present invention to the YMLDLQPET-HLA-A0201 complex is at least 2 times, preferably, at least 5 times, more than that of the wild-type TCR. Preferably, at least 10 times. (2) The high-affinity TCR of the present invention can specifically bind to the YMLDLQPET-HLA A0201, and the cells transfected with the high-affinity TCR of the present invention can be specifically activated and proliferated. (3) The effector cells transfected with the high-affinity TCR of the present invention have a strong poisoning effect on the target cells.

The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to conventional conditions, for example (people such as Sambrook and Russell, molecular colonization: laboratory manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL Press), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. Materials and methods

The experimental materials used in the examples of the present invention can be obtained from commercially available pipelines unless otherwise specified, wherein, E. coli DH5α was purchased from Tiangen, E. coli BL21 (DE3) was purchased from Tiangen, and E. coli Tuner (DE3) was purchased from Tiangen From Novagen, plastid pET28a was purchased from Novagen. Example 1 Generation of stable single-stranded TCR template strands with hydrophobic core mutations

The present invention utilizes the method of site-directed mutagenesis, according to the patent document WO2014/206304, to construct a stable single-chain TCR molecule composed of a flexible short peptide (linker) connecting TCRα and β chain variable domains. The acid and DNA sequences are SEQ ID NO: 9 and SEQ ID NO: 10, respectively, as shown in Figures 5a and 5b. And use the single-chain TCR molecule as a template to screen high-affinity TCR molecules. The amino acid sequences of the α variable domain (SEQ ID NO: 3) and the β variable domain (SEQ ID NO: 4) of the template chain are shown in Figures 2a and 2b; the corresponding DNA sequences are respectively SEQ ID NO : 5 and 6, as shown in Figures 3a and 3b; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are SEQ ID NOs: 7 and 8, respectively, as shown in Figures 4a and 4b.

The target gene carrying the template chain was cut with NcoI and NotI double restriction enzymes, and ligated with the pET28a vector cut with NcoI and NotI double restriction enzymes. The ligation product was transfected to E.coli DH5α, coated with LB culture plate containing kanamycin, and cultured overnight at 37°C by inversion. The positive clones were selected for PCR screening, and the positive recombinants were sequenced to confirm that the sequence was correct and then extracted. Recombinant plastids were transformed into E. coli BL21 (DE3) for expression. Example 2 Expression, renaturation and purification of the stable single-chain TCR constructed in Example 1

All the BL21 (DE 3) colonies containing the recombinant plastid pET28a-template chain prepared in Example 1 were inoculated into LB medium containing kanamycin, cultured at 37°C until OD600 was 0.6-0.8, and IPTG was added to the final concentration 0.5 mM, and continued to incubate for 4 h at 37 °C. Cell pellets were harvested by centrifugation at 5000 rpm for 15 min, lysed with Bugbuster Master Mix (Merck), inclusion bodies were recovered by centrifugation at 6000 rpm for 15 min, and washed with Bugbuster (Merck) to remove cell debris and membrane components, 6000 rpm Inclusion bodies were collected by centrifugation for 15 min. The inclusion bodies were dissolved in buffer (20 mM Tris-HCl pH 8.0, 8 M urea), and the insolubles were removed by high-speed centrifugation. The supernatant was quantified by BCA method, and then aliquoted and stored at -80 °C for future use.

To 5 mg of solubilized single-chain TCR inclusion body protein, add 2.5 mL buffer (6 M Gua-HCl, 50 mM Tris-HCl pH 8.1, 100 mM NaCl, 10 mM EDTA) followed by DTT to a final concentration of 10 mM, treated at 37°C for 30 min. 125 mL of renaturation buffer (100 mM Tris-HCl pH 8.1, 0.4 M L-arginine, 5 M urea, 2 mM EDTA, 6.5 mM β-mercapthoethylamine, 1.87 mM Cystamine) was added dropwise with a syringe after the above treatment. The single-chain TCR was stirred at 4 °C for 10 min, and then the renaturation solution was placed in a 4 kDa cellulose membrane dialysis bag, placed in 1 L of pre-cooled water, and slowly stirred at 4 °C overnight. After 17 hours, the dialysate was replaced with 1 L of pre-cooled buffer (20 mM Tris-HCl pH 8.0), and the dialysis was continued for 8 h at 4°C, and then the dialysate was replaced with the same fresh buffer and continued dialysis overnight. After 17 hours, the samples were filtered through a 0.45 µm filter, degassed in vacuo, passed through an anion exchange column (HiTrap Q HP, GE Healthcare), and purified with a linear gradient of 0-1 M NaCl in 20 mM Tris-HCl pH 8.0 The collected elution fractions were analyzed by SDS-PAGE. The fractions containing single-chain TCR were concentrated and further purified by gel filtration columns (Superdex 75 10/300, GE Healthcare). The target fractions were also subjected to SDS-PAGE. PAGE analysis.

The eluted fractions for BIAcore analysis were further tested for purity by gel filtration. The conditions are: chromatographic column Agilent Bio SEC-3 (300 A, φ7.8×300 mm), mobile phase is 150 mM phosphate buffer, flow rate is 0.5 mL/min, column temperature is 25 °C, UV detection wavelength is 214 nm . Example 3 Binding Characterization BIAcore Analysis

The binding activity of TCR molecules to YMLDLQPET-HLA-A0201 complex was detected using BIAcore T200 point-of-care analysis system. Anti-streptavidin antibody (GenScript) was added to coupling buffer (10 mM sodium acetate buffer, pH 4.77), and the antibody was immobilized on the surface of the wafer by flowing the antibody over a CM5 wafer previously activated with EDC and NHS , and finally blocked the unreacted activated surface with ethanolamine hydrochloric acid solution to complete the coupling process, and the coupling level was about 15,000 RU. The conditions are: the temperature is 25°C, and the pH value is 7.1-7.5.

Flow a low concentration of streptavidin over the antibody-coated wafer surface, then flow the YMLDLQPET-HLA-A0201 complex across the detection channel, the other channel as the reference channel, and 0.05 mM biotin at 10 µL/min flow through the wafer for 2 min, blocking the remaining binding sites of streptavidin. Affinity was determined using a single-loop kinetic assay, and TCR was diluted to several different concentrations in HEPES-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) at 30 The flow rate of µL/min was successively flowed across the surface of the wafer. The bonding time of each injection was 120 s, and the dissociation was 600 s after the last injection. The wafers were regenerated with 10 mM Gly-HCl pH 1.75 after each round of assays. Kinetic parameters were calculated using BIAcore Evaluation software.

The preparation process of the above-mentioned YMLDLQPET-HLA-A0201 complex is as follows:

a. Purification: Collect 100 ml of E. coli bacteria that induce heavy or light chain expression, centrifuge at 8000 g for 10 min at 4°C, wash the bacteria once with 10 ml PBS, and then use 5 ml BugBuster Master Mix Extraction Reagents (Merck ) Vigorously shake to resuspend the bacterial cells, and rotate at room temperature for 20 min, then centrifuge at 6000 g for 15 min at 4°C, discard the supernatant, and collect the inclusion bodies.

Resuspend the above inclusion bodies in 5 ml of BugBuster Master Mix and incubate at room temperature for 5 min; add 30 ml of 10-fold diluted BugBuster, mix well, and centrifuge at 6000 g at 4°C for 15 min; discard the supernatant and add 30 ml to dilute Resuspend the inclusion bodies with 10x BugBuster, mix well, centrifuge at 6000 g for 15 min at 4°C, repeat twice, add 30 ml of 20 mM Tris-HCl pH 8.0 to resuspend the inclusion bodies, mix well, centrifuge at 6000 g at 4°C for 15 min, Finally, the inclusion bodies were dissolved with 20 mM Tris-HCl 8M urea, the purity of inclusion bodies was detected by SDS-PAGE, and the concentration was determined by BCA kit.

b. Refolding: Dissolve the synthetic short peptide YMLDLQPET (Beijing Saibaisheng Gene Technology Co., Ltd.) in DMSO to a concentration of 20 mg/ml. Inclusion bodies of light and heavy chains were solubilized with 8 M urea, 20 mM Tris pH 8.0, 10 mM DTT, and further denatured by the addition of 3 M guanidine hydrochloride, 10 mM sodium acetate, 10 mM EDTA prior to renaturation. YMLDLQPET peptide was added at 25 mg/L (final concentration) 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 form Glutathione, 0.2 mM PMSF, cooled to 4°C), then 20 mg/L of light chain and 90 mg/L of heavy chain were added in sequence (final concentration, heavy chain was added in three times, 8 h/time), repeated The renaturation was carried out at 4°C for at least 3 days to complete, and SDS-PAGE was used to detect whether the renaturation was successful.

c. Post-renaturation purification: Dialyze against 10 volumes of 20 mM Tris pH 8.0 to replace the renaturation buffer, at least twice to reduce the ionic strength of the solution. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and loaded onto a HiTrap Q HP (GE) anion exchange column (5 ml bed volume). Proteins were eluted with a linear gradient of 0-400 mM NaCl prepared in 20 mM Tris pH 8.0 using an Akta purifier (GE), and pMHC was eluted at approximately 250 mM NaCl, and peak fractions were collected and detected by SDS-PAGE. purity.

d. Biotinylation: Purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while buffer exchanged to 20 mM Tris pH 8.0, followed by addition of biotinylation reagents 0.05 M Bicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 μM D-Biotin, 100 μg/ml BirA enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and the complete biotinylation was detected by SDS-PAGE.

e. Purify the biotinylated complex: Concentrate the biotinylated pMHC molecules to 1 ml with a Millipore ultrafiltration tube, purify the biotinylated pMHC by gel filtration chromatography, and use an Akta purifier (GE Universal Electric Corporation), pre-equilibrated HiPrepTM 16/60 S200 HR column (GE) with filtered PBS, loaded with 1 ml of concentrated biotinylated pMHC molecules, and eluted with PBS at a flow rate of 1 ml/min . Biotinylated pMHC molecules eluted as a single peak at about 55 ml. The fractions containing protein were combined, concentrated with Millipore ultrafiltration tube, the protein concentration was determined by BCA method (Thermo), and the biotinylated pMHC molecules were aliquoted and stored at -80°C by adding protease inhibitor cocktail (Roche). Example 4 Generation of High Affinity TCRs

Phage display technology is a means of generating TCR high-affinity variant sequence libraries for screening high-affinity variants. The TCR phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3): 349-354) was applied to the single-chain TCR template in Example 1. A sequence library of high-affinity TCRs is created and panned by mutating the CDR regions of the template strand. After several rounds of panning, the phage sequence library has specific binding to the corresponding antigen, and the single selection clones are selected and analyzed.

The CDR region of the screened high-affinity single-chain TCR was mutated into the corresponding address of the variable domain of αβ heterodimeric TCR, and its affinity with YMLDLQPET-HLA-A0201 complex was detected by BIAcore. The introduction of the high-affinity mutation points in the above-mentioned CDR regions adopts the method of site-directed mutagenesis well known to those skilled in the art. The amino acid sequences of the α-chain and β-chain variable domains of the wild-type TCR are shown in Figure 1a (SEQ ID NO: 1) and 1b (SEQ ID NO: 2), respectively.

It should be noted that in order to obtain a more stable soluble TCR for easier assessment of binding affinity and/or binding half-life between the TCR and the YMLDLQPET-HLA A0201 complex, the αβ heterodimeric TCR can be in the constant region of the α and β chains. A cysteine residue was introduced in the TCR to form an artificial interchain disulfide bond, and the amino acid sequences of the TCRα and β chains after the introduction of the cysteine residue in the present example were shown in Figure 6a (SEQ ID NO: 11) and 6b (SEQ ID NO: 12), the introduced cysteine residues are shown in bold letters.

The extracellular sequence genes for the TCR alpha and beta chains to be expressed were synthesized and inserted into the expressed TCR alpha and beta chains, respectively, by standard methods described in "Molecular Cloning a Laboratory Manual" (Third Edition, Sambrook and Russell). The vector pET28a+ (Novagene), the upstream and downstream breeding sites are NcoI and NotI, respectively. Mutations in the CDR regions are introduced by overlapping PCR (overlap PCR) well known to those skilled in the art. The insert was sequenced and confirmed to be correct. Example 5 Expression, renaturation and purification of high-affinity TCR

The expression vectors of TCR α and β chains were transformed into the expression bacteria BL21 (DE3) by chemical transformation, respectively. The bacteria were grown in LB medium and induced with a final concentration of 0.5 mM IPTG at OD600 = 0.6. The α and β chains of TCR expressed The resulting inclusion bodies were extracted with BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution. The inclusion bodies were finally dissolved in 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), and 10 mM ethylenediaminetetramine. Acetic acid (EDTA), 20 mM Tris (pH 8.1).

Solubilized TCR α and β chains were rapidly mixed in 5 M urea, 0.4 M arginine, 20 mM Tris (pH 8.1), 3.7 mM cystamine, 6.6 mM β-mercapoethylamine (4°C) at a mass ratio of 1:1 , the final concentration was 60 mg/mL. After mixing, the solution was dialyzed against 10 times the volume of deionized water (4°C). After 12 hours, the deionized water was exchanged for buffer (20 mM Tris, pH 8.0) and dialyzed at 4°C for 12 hours. The dialyzed solution was filtered through a 0.45 μM filter membrane and purified through an anion exchange column (HiTrap Q HP, 5 ml, GE Healthcare). The eluted peaks of TCR containing successfully renatured α and β dimers were confirmed by SDS-PAGE gel. 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 more than 90% determined by SDS-PAGE, and the concentration was determined by BCA method. Example 6 BIAcore analysis results

The method described in Example 3 was used to detect the affinity of the αβ heterodimeric TCR introduced with the high affinity CDR region to the complex of YMLDLQPET-HLA-A0201.

The present invention obtains high-affinity TCRα and β chain variable domain amino acid sequences, as shown in Fig. 7(1)-Fig. 7(34) and Fig. 8a-c, respectively. Since the CDR region of the TCR molecule determines its affinity with the corresponding pMHC complex, those skilled in the art can expect that the αβ heterodimeric TCR introduced with the high-affinity mutation point also has a high affinity for the YMLDLQPET-HLA-A0201 complex. The expression vector was constructed by the method described in Example 4, and the αβ heterodimeric TCR introduced with the high affinity mutation was expressed, renatured and purified by the method described in Example 5, and then the BIAcore T200 was used to determine its interaction with YMLDLQPET-HLA The affinity of the -A0201 complex is shown in Table 2 below. Table 2 TCR number Alpha chain variable domain sequence SEQ ID NO: Beta chain variable domain sequence SEQ ID NO: kD value 1 13 2 5.52E-06 2 14 2 5.35E-06 3 15 2 1.23E-05 4 16 2 2.16E-06 5 17 2 4.92E-06 6 18 2 7.19E-06 7 19 2 8.17E-06 8 20 2 8.87E-07 9 twenty one 2 3.04E-06 10 twenty two 2 1.24E-06 11 twenty three 2 2.20E-06 12 twenty four 2 1.89E-06 13 25 2 2.35E-07 14 26 2 1.63E-06 15 27 2 2.66E-06 16 28 2 8.81E-07 17 29 2 7.08E-07 18 30 2 2.73E-06 19 1 47 5.44E-06 20 1 48 1.21E-05 twenty one 1 49 1.08E-05 twenty two 31 2 2.94E-06 twenty three 32 2 2.16E-06 twenty four 33 2 2.25E-05 25 34 2 7.09E-06 26 35 2 3.46E-06 27 36 2 1.22E-05 28 37 2 1.55E-05 29 38 2 6.98E-06 30 39 2 4.50E-06 31 40 2 1.35E-06 32 41 2 7.50E-07 33 42 2 8.59E-07 34 43 2 3.10E-06 35 44 2 7.85E-07 36 45 2 6.03E-07 37 46 2 9.58E-07

As can be seen from Table 2 above, the affinity of the heterodimeric TCR is at least 2 times that of the wild-type TCR for the YMLDLQPET-HLA-A0201 complex. Example 7 Expression, renaturation and purification of fusions of anti- CD3 antibodies and high-affinity αβ heterodimeric TCRs

Fusion molecules were prepared by fusing anti-CD3 single chain antibody (scFv) to αβ heterodimeric TCR. The anti-CD3 scFv is fused to the β chain of TCR, the TCR β chain may comprise any of the above-mentioned high-affinity αβ heterodimeric β-chain variable domains, and the TCRα chain of the fusion molecule may comprise any of the above-mentioned high-affinity αβ heterodimers Alpha chain variable domains of dimeric TCRs. Construction of fusion molecule expression vector

1. Construction of α chain expression vector: The target gene of α chain carrying αβ heterodimeric TCR was cut with NcoI and NotI double restriction enzymes, and then ligated with the pET28a vector cut with NcoI and NotI double restriction enzymes. The ligation product was transformed into E.coli DH5α, spread on LB culture plate containing kanamycin, and incubated overnight at 37 °C upside down. The positive clones were selected for PCR screening, and the positive recombinants were sequenced to confirm that the sequence was correct and then extracted. Recombinant plastids were transformed into E. coli Tuner (DE3) for expression.

2. Construction of anti-CD3 (scFv)-β chain expression vector: Through the method of overlapping (overlap) PCR, primers were designed to connect the anti-CD3 scFv and the high-affinity heterodimeric TCRβ chain gene, and the connection in the middle was short. The peptide (linker) is GGGGS (SEQ ID NO: 31), and the gene fragment of the fusion protein of the anti-CD3 scFv and the high-affinity heterodimeric TCRβ chain is given the restriction endonuclease address NcoI (CCATGG (SEQ ID NO: 31). ID NO: 32)) and NotI (GCGGCCGC (SEQ ID NO: 33)). The PCR amplification product was cut with NcoI and NotI double restriction enzymes, and ligated with pET28a vector cut with NcoI and NotI double restriction enzymes. The ligation product was transformed into E.coli DH5α competent cells, coated with LB culture plate containing kanamycin, and cultured overnight at 37°C upside down. The positive clones were selected for PCR screening, and the positive recombinants were sequenced to confirm that the sequence was correct. The recombinant plastids were extracted and transformed into E. coli Tuner (DE3) competent cells for expression. Expression, renaturation and purification of fusion proteins

The expressing plastids were transformed into E. coli Tuner (DE3) competent cells, coated with LB plates (kanamycin 50 μg/mL) and incubated at 37 °C overnight. The next day, the selected clones were inoculated into 10 mL of LB liquid medium (kanamycin 50 μg/mL) for 2-3 h, then inoculated into 1 L of LB medium at a volume ratio of 1:100, and continued to culture until the OD600 was 0.5-0.8 , adding a final concentration of 1 mM IPTG to induce the expression of the target protein. After 4 hours of induction, cells were harvested by centrifugation at 6000 rpm for 10 min. The cells were washed once with PBS buffer, and the cells were aliquoted. The cells equivalent to 200 mL of bacterial culture were taken to lyse the bacteria with 5 mL of BugBuster Master Mix (Merck), and the inclusion bodies were collected by centrifugation at 6000 g for 15 min. Four detergent washes were then performed to remove cellular debris and membrane components. The inclusion bodies are then washed with a buffer such as PBS to remove detergents and salts. Finally, the inclusion bodies were dissolved in a buffer solution containing 6M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA), 20 mM Tris, pH 8.1, and the concentration of inclusion bodies was determined. It was frozen and stored at -80°C after being subpackaged.

Solubilized TCRα chain and anti-CD3 (scFv)-β chain were rapidly mixed at a mass ratio of 2:5 in 5 M urea (urea), 0.4 M L-arginine (L-arginine), 20 mM Tris pH 8.1 , 3.7 mM cystamine, 6.6 mM β-mercapoethylamine (4°C), the final concentrations of α chain and anti-CD3 (scFv)-β chain were 0.1 mg/mL and 0.25 mg/mL, respectively.

After mixing, the solution was dialyzed in 10 times the volume of deionized water (4 °C). After 12 hours, the deionized water was changed to buffer (10 mM Tris, pH 8.0), and the dialysis was continued at 4 °C for 12 hours. The solution after dialysis was filtered through a 0.45 μM filter membrane, and then purified through an anion exchange column (HiTrap Q HP 5 ml, GE healthcare). The eluted peaks containing successfully renatured TCR α chain and anti-CD3 (scFv)-β chain dimer were confirmed by SDS-PAGE gel. The TCR fusion molecules were then further purified by size exclusion chromatography (S-100 16/60, GE healthcare) and again by anion exchange column (HiTrap Q HP 5ml, GE healthcare). The purity of the purified TCR fusion molecule was determined by SDS-PAGE, and the concentration was determined by BCA method. Example 8 Activation function experiment of effector cells transfected with high-affinity TCR of the present invention for T2 cells carrying short peptides

This example verifies that the effector cells transfected with the high-affinity TCR of the present invention have a good specific activation effect on the target cells. The function and specificity of the high-affinity TCR of the present invention in cells are detected by the ELISPOT assay well known to those skilled in the art. Randomly select the high-affinity TCR of the present invention to transfect CD3+ T cells isolated from the blood of healthy volunteers as effector cells, and use the same volunteer to transfect wild-type TCR (WT-TCR) and other TCRs (A6) CD3+ T cells were used as a control group. The target cells used were T2 cells carrying the HPV16 E7 antigen short peptide YMLDLQPET, and T2 cells carrying other short peptides and empty T2 cells were used as control groups.

The following two TCR batches (I), (II) were tested successively:

(1) The high-affinity TCR and its numbering are learned from Table 2, which are respectively TCR1 (alpha chain variable domain SEQ ID NO: 13, beta chain variable domain SEQ ID NO: 2), TCR9 (alpha chain variable domain SEQ ID NO: 21, beta chain variable domain SEQ ID NO: 2), TCR3 (alpha chain variable domain SEQ ID NO: 15, beta chain variable domain SEQ ID NO: 2), TCR10 (alpha chain variable domain SEQ ID NO: 22, beta chain variable domain SEQ ID NO: 2), TCR11 (alpha chain variable domain SEQ ID NO: 23, beta chain variable domain SEQ ID NO: 2), TCR12 (alpha chain variable domain SEQ ID NO: 24, beta chain variable domain SEQ ID NO: 2), TCR14 (alpha chain variable domain SEQ ID NO: 26, beta chain variable domain SEQ ID NO: 2), TCR4 (alpha chain variable domain SEQ ID NO: 16, beta chain variable domain SEQ ID NO: 2), TCR15 (alpha chain variable domain SEQ ID NO: 27, beta chain variable domain SEQ ID NO: 2), TCR23 (alpha chain variable domain SEQ ID NO: 32, beta chain variable domain SEQ ID NO: 2), TCR2 (alpha chain variable domain SEQ ID NO: 14, beta chain variable domain SEQ ID NO: 2), TCR18 (alpha chain variable domain SEQ ID NO: 30, beta chain variable domain SEQ ID NO: 2), TCR19 (alpha chain variable domain SEQ ID NO: 1, beta chain variable domain SEQ ID NO: 47) and TCR21 (alpha chain variable domain SEQ ID NO: 1, beta chain variable domain SEQ ID NO: 49).

(II) The high-affinity TCR and its numbering are learned from Table 2, which are respectively TCR6 (alpha chain variable domain SEQ ID NO: 18, beta chain variable domain SEQ ID NO: 2), TCR16 (alpha chain variable domain SEQ ID NO: 28, beta chain variable domain SEQ ID NO: 2), TCR22 (alpha chain variable domain SEQ ID NO: 31, beta chain variable domain SEQ ID NO: 2), TCR7 (alpha chain variable domain SEQ ID NO: 19, beta chain variable domain SEQ ID NO: 2) and TCR5 (alpha chain variable domain SEQ ID NO: 17, beta chain variable domain SEQ ID NO: 2).

The following experimental steps were carried out for the above two batches: First, the ELISPOT culture plate was prepared. ELISPOT plates were coated with ethanol for activation, overnight at 4°C. On the first day of the experiment, remove the coating solution, wash the blocking solution, incubate at room temperature for two hours, remove the blocking solution, and add the components of the test to the ELISPOT culture plate: the target cells are 1*10 ⁴ cells/well, and the effect The number of cells was 2*10 ³ cells/well (calculated according to the positive rate of transfection), and two duplicate wells were set. The corresponding short peptides were added so that the final concentration of the short peptides in the ELISPOT plate was 1×10-6 M. Incubate overnight at constant temperature (37°C, 5% CO ₂ ). On the second day of the experiment, the plates were washed and subjected to secondary detection and color development, the plates were dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20).

The experimental results are shown in Figure 12a and Figure 12b, for the target cells carrying the HPV16 E7 antigen short peptide, the T cells transfected with the high-affinity TCR of the present invention are more significantly activated than the wild-type T cells transfected T cells transfected with other TCRs were not activated; meanwhile, T cells transfected with the high-affinity TCR of the present invention were not activated by T2 cells carrying other short peptides or empty. Example 9 Activation function experiment of effector cells transfected with high-affinity TCR of the present invention for tumor cell lines

This example also verifies that the effector cells transfected with the high-affinity TCR of the present invention have a very good specific activation effect on the target cells. The function and specificity of the high-affinity TCR of the present invention in cells are detected by the ELISPOT assay well known to those skilled in the art. The CD3+ T cells isolated from the blood of healthy volunteers transfected with the high-affinity TCR of the present invention were randomly selected as effector cells, and the CD3+ T cells transfected with other TCR (A6) from the same volunteer were used as the control group. Wherein the high-affinity TCR and its numbering are learned from Table 2, which are respectively TCR2 (alpha chain variable domain SEQ ID NO: 14, beta chain variable domain SEQ ID NO: 2), TCR6 (alpha chain variable domain SEQ ID NO: 2) NO: 18, β chain variable domain SEQ ID NO: 2), TCR16 (α chain variable domain SEQ ID NO: 28, β chain variable domain SEQ ID NO: 2), TCR5 (α chain variable domain SEQ ID NO: 2) NO: 17, beta chain variable domain SEQ ID NO: 2) and TCR8 (alpha chain variable domain SEQ ID NO: 20, beta chain variable domain SEQ ID NO: 2). Among the tumor cell lines used in this experiment, A375-E7 (overexpression of HPV16 E7) was a positive tumor cell line, while A375, HCCC9810, SK-MEL-1, and KATO-III cells were negative tumor cell lines as controls.

First prepare the ELISPOT culture dish. ELISPOT plates were coated with ethanol for activation, overnight at 4°C. On the first day of the experiment, remove the coating solution, wash the blocking solution, incubate at room temperature for two hours, remove the blocking solution, and add the components of the test to the ELISPOT culture plate in the following order: the target cells are 2*10 ⁴ cells/ Well, the number of effector cells is 2* ¹⁰³ /well (calculated according to the positive rate of transfection), and two duplicate wells are set. Incubate overnight at constant temperature (37°C, 5% CO ₂ ). On the second day of the experiment, the plates were washed and subjected to secondary detection and color development, the plates were dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20).

The experimental results are shown in Figure 13, the effector cells transfected with the high-affinity TCR of the present invention can be well activated by the positive tumor cell line, while for the negative target cells, the effector cells transfected with the high-affinity TCR of the present invention have basically no activation effect; Effector cells transfected with other TCRs cannot be activated. Example 10 Toxicity test of effector cells transfected with the high-affinity TCR of the present invention for T2 cells carrying a gradient of short peptides

In this example, the release of LDH is measured through non-radioactive cytotoxicity experiments well known to those skilled in the art, so as to verify the cytotoxicity of cells transfected with the TCR of the present invention. This assay is a colorimetric alternative to the 51Cr release cytotoxicity assay and quantifies lactate dehydrogenase (LDH) released after cell lysis. LDH released in the medium was detected using a 30 min coupled enzymatic reaction in which LDH converts a tetrazolium salt (INT) to red formazan. The amount of red product produced is proportional to the number of cells lysed. Visible absorbance data at 490 nm can be collected using a standard 96-well plate reader. Calculation formula: % cytotoxicity=100×(experimental-effector cell spontaneous-target cell spontaneous)/(target cell maximum-target cell spontaneous).

In the LDH experiment of this example, CD3+ T cells isolated from the blood of healthy volunteers were used to transfect the high-affinity TCR of the present invention as effector cells, and the same volunteer was used to transfect wild-type TCR (WT-TCR) and other TCR (A6) CD3+ T cells served as a control group. Wherein the high-affinity TCR and its numbering are learned from Table 2, which are respectively TCR1 (alpha chain variable domain SEQ ID NO: 13, beta chain variable domain SEQ ID NO: 2), TCR3 (alpha chain variable domain SEQ ID NO: 2) NO: 15, beta chain variable domain SEQ ID NO: 2), TCR4 (alpha chain variable domain SEQ ID NO: 16, beta chain variable domain SEQ ID NO: 2) and TCR2 (alpha chain variable domain SEQ ID NO: 2) NO: 14, beta chain variable domain SEQ ID NO: 2). The target cells were T2 cells carrying YMLDLQPET peptide, while other antigens and empty T2 cells were used as controls.

First prepare the LDH culture plate, first ^{add 3*10 4 cells/well of target cells and 3*10 4} cells/well of effector cells as corresponding, and then add HPV16 E7 antigen short peptide YMLDLQPET to the experimental group, and make it The final concentration of the short peptides in the ELISPOT well plate was 1×10-15 M to 1×10-8 M, a total of 8 gradients; other short peptides were added to the control group, and the final concentration of the short peptides was in order For 1×10-8 M to 1×10-6 M, a total of 3 gradients, and three replicate wells were set. Simultaneously set effector cell spontaneous wells, target cell spontaneous wells, target cell maximum wells, volume correction control wells and medium background control wells. Incubate overnight at constant temperature (37°C, 5% CO ₂ ). On the second day of the experiment, color development was detected, and the absorbance value was recorded at 490 nm with an enzyme label reader (Bioteck) after the reaction was terminated.

The experimental results are shown in Figure 14. For T2 cells carrying the HPV16 E7 antigen short peptide YMLDLQPET in a gradient, the effector cells transfected with the high-affinity TCR of the present invention have a strong poisoning function, and when the above-mentioned short peptide concentration is low. That is, it reacts, and the effector cells transfected with other TCRs have no cytotoxic effect from the beginning; meanwhile, the effector cells transfected with the high-affinity TCR of the present invention have no cytotoxic effect on the target cells carrying other short peptides. Example 11 Toxicity test (LDH test ) of effector cells transfected with high-affinity TCR of the present invention for tumor cell lines

In this example, the release of LDH is also measured by non-radioactive cytotoxicity experiments well known to those skilled in the art, so as to verify the cytotoxicity of cells transfected with the TCR of the present invention.

In the LDH experiment in this example, CD3+ T cells isolated from the blood of healthy volunteers were used to transfect the high-affinity TCR of the present invention as effector cells, and the same volunteer was used to transfect wild-type TCR (WT-TCR) and other CD3+ T cells of TCR (A6) served as a control group. Wherein the high-affinity TCR and its numbering are learned from Table 2, which are respectively TCR1 (alpha chain variable domain SEQ ID NO: 13, beta chain variable domain SEQ ID NO: 2), TCR3 (alpha chain variable domain SEQ ID NO: 2) NO: 15, beta chain variable domain SEQ ID NO: 2), TCR4 (alpha chain variable domain SEQ ID NO: 16, beta chain variable domain SEQ ID NO: 2) and TCR2 (alpha chain variable domain SEQ ID NO: 2) NO: 14, beta chain variable domain SEQ ID NO: 2). Among the target cells used in this experiment, CASKI was a positive cell line; while A375 and HCCC9810 were negative cell lines as controls.

First prepare the LDH culture plate, and add the components of the test to the culture plate in the following order: 25,000 cells/well of target cells and 20,000 cells/well of effector cells are added to the corresponding wells, and three duplicate wells are set. Simultaneously set effector cell spontaneous wells, target cell spontaneous wells, target cell maximum wells, volume correction control wells and medium background control wells. Incubate overnight at constant temperature (37°C, 5% CO ₂ ). On the second day of the experiment, color development was detected, and the absorbance value was recorded at 490 nm with an enzyme label reader (Bioteck) after the reaction was terminated.

The experimental results are shown in Figure 15. The cells transfected with wild-type TCR can kill 20% of the positive tumor cell lines. The cells transfected with the high-affinity TCR of the present invention have significantly enhanced killing effects on the positive tumor cell lines. The effector cells of other TCRs have basically no cytotoxic effect, and at the same time, the cells transfected with the high-affinity TCR of the present invention have basically no cytotoxic effect on target cells that do not express relevant antigens. Example 12 Verification of the killing function of effector cells transfected with the high-affinity TCR molecule of the present invention against tumor cell lines (IncuCyte experiment )

This example further verifies that the effector cells transfected with the high-affinity TCR of the present invention have a good specific killing effect on the target cells through the use of -IncuCyte experiments well known to those skilled in the art. IncuCyte is a functional analysis system that can automatically analyze images at different time points and quantify the number of apoptosis in real time through real-time microscopic photography in an incubator.

CD3+ T cells isolated from the blood of healthy volunteers were randomly selected for transfection with the TCR of the present invention as effector cells, and CD3+ T cells transfected with other TCR (A6) from the same volunteer were used as a control group. Described TCR and its numbering are learned from table 2, are respectively TCR1 (alpha chain variable domain SEQ ID NO: 13, beta chain variable domain SEQ ID NO: 2), TCR2 (alpha chain variable domain SEQ ID NO: 14 , β chain variable domain SEQ ID NO: 2). Among the target cell lines, A375-E7 (overexpression of HPV16 E7) was a positive cell line; HCCC9810 was a negative cell line as a control.

On the first day of the experiment, the target cells were treated with digestive enzymes and centrifuged; resuspended in complete medium of RPMI1640+10% FBS without phenol red, and the target cells were evenly plated in a 96-well plate: 2*10 ⁴ cells/ aperture; back 37 degrees, 5% CO ₂ incubator, the incubated overnight; a second 96-well plate culture medium was discarded and replaced with phenol red-free RPMI1640 + 10% FBS medium containing a dye caspase3 / 7 reagent of , so that the dye concentration is 2 drops/ml. Discarded old media, replacement of phenol red-free medium RPMI1640 + 10% FBS, and the effector cells 2 * ¹⁰⁴ cells / well (in terms of positive rate of transfection) in the experimental group was covered with the target cell Co-cultivation; put the culture plate into the IncuCyte ZooM, a real-time dynamic live cell imaging analyzer dedicated to Incucyte detection, and incubate for half an hour; start real-time observation and take pictures; use IncuCyte ZooM 2016A to process, analyze and export the test results.

The experimental results are shown in Figure 16a and Figure 16b, for positive tumor cell lines, the cells transfected with the high-affinity TCR of the present invention can achieve a strong and effective poisoning effect in a short period of time, while the effector cells transfected with other TCRs have basically no poisoning effect; At the same time, cells transfected with the high-affinity TCR of the present invention have basically no toxic effect on target cells that do not express relevant antigens.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended patent scope of the present application.

Figure 1a and Figure 1b show the wild-type TCRα and β chain variable domain amino acid sequences that can specifically bind to the YMLDLQPET-HLA A0201 complex, respectively.

Figure 2a and Figure 2b are respectively the amino acid sequence of the α variable domain and the amino acid sequence of the β chain variable domain of the single-chain template TCR constructed by the present invention.

Figure 3a and Figure 3b are respectively the DNA sequence of the alpha variable domain and the DNA sequence of the beta chain variable domain of the single-chain template TCR constructed by the present invention.

Figure 4a and Figure 4b are respectively the amino acid sequence and nucleotide sequence of the linker of the single-chain template TCR constructed by the present invention.

Figure 5a and Figure 5b are respectively the amino acid sequence and DNA sequence of the single-stranded template TCR constructed by the present invention.

Figures 6a and 6b show the amino acid sequences of the soluble reference TCR alpha and beta chains of the present invention, respectively.

Figures 7(1)-(34) show the alpha chain variable domain amino acid sequences of heterodimeric TCRs with high affinity for the YMLDLQPET-HLA A0201 complex, respectively, with mutated residues underlined.

Figures 8a-c show the β-chain variable domain amino acid sequences of heterodimeric TCRs with high affinity for the YMLDLQPET-HLA A0201 complex, respectively, with mutated residues underlined.

Figures 9a and 9b show the extracellular amino acid sequences of wild-type TCRα and β chains that can specifically bind to the YMLDLQPET-HLA A0201 complex, respectively.

Figures 10a and 10b show the amino acid sequences of wild-type TCR alpha and beta chains, respectively, that can specifically bind to the YMLDLQPET-HLA A0201 complex.

Figure 11 is a binding curve of a soluble reference TCR, wild-type TCR, to the YMLDLQPET-HLA A0201 complex.

Figure 12a and Figure 12b are the results of the activation function experiment of effector cells transfected with the high-affinity TCR of the present invention for T2 cells carrying short peptides.

Figure 13 shows the results of the activation function experiment of effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines.

Figure 14 shows the results of the poisoning function experiment on effector cells transfected with the high-affinity TCR of the present invention against T2 cells carrying short peptides.

FIG. 15 is the result of the poisoning function test (LDH test) of the effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines.

Fig. 16a and Fig. 16b are the results of the poisoning function experiment (incuCyte experiment) of effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines.

Claims (25)

A T cell receptor (TCR) comprising a TCRα chain variable domain and a TCRβ chain variable domain, characterized in that it has an activity of binding to the YMLDLQPET-HLA A0201 complex, and the α of the TCR The chain variable domain comprises an amino acid sequence with at least 90% sequence homology with the sequence shown in SEQ ID NO: 1 and the TCR beta chain variable domain comprises at least 90% with the sequence shown in SEQ ID NO: 2 Amino acid sequence of % sequence homology. The TCR of claim 1, wherein the affinity of the TCR to the YMLDLQPET-HLA A0201 complex is at least 2 times that of the wild-type TCR. The TCR of claim 1, wherein the TCR is soluble. The TCR according to claim 1, wherein the TCR is an αβ heterodimeric TCR, comprising the α chain TRAC constant region sequence and the β chain TRBC1 or TRBC2 constant region sequence. The TCR according to claim 1, wherein the TCR β chain variable domain comprises 3 CDR regions, and the amino acid sequences of the 3 CDR regions in the TCR β chain variable domain are as follows: CDR1β: SGHTA CDR2β: FQGTGA and CDR3β:ASSLLAGSYEQY. The TCR according to claim 1, wherein the amino acid sequence of the variable domain of the TCR beta chain is SEQ ID NO:2. The TCR according to claim 1, wherein the sequences of the three CDR regions of the variable domain of the TCRα chain are: CDR1α: ATGYPS CDR2α: ATKADDK CDR3α: ALYNQGGKLI, and three of the variable domains of the TCRβ chain The sequences of the CDR regions are: CDR1β: SGHTA CDR2β: FQGTGA CDR3β: ASSLLAGSYEQY; and the TCR contains at least one mutation in CDR3α; preferably, the mutation in CDR3α is selected from the group consisting of: Residues before mutation mutated residue Position 4 N of CDR3α L or A or V Q at position 5 of CDR3α F or Y or W The 6th G of CDR3α N or S or D or A or V or T or H or M G at position 7 of CDR3α A or N or S or F K at position 8 of CDR3α V or R or L or I or M or Q or S or N More preferably, the mutation in CDR3α is selected from the group consisting of: Residues before mutation mutated residue Position 4 N of CDR3α L Q at position 5 of CDR3α F or Y The 6th G of CDR3α N or S G at position 7 of CDR3α A or N or S K at position 8 of CDR3α V or R or L The TCR according to claim 7, wherein the number of amino acid mutations in the CDR3α is 2, 3 or 4. The TCR of claim 1, wherein the α-chain variable domain of the TCR comprises an amino acid sequence that has at least 96% sequence homology with the sequence shown in SEQ ID NO: 1; and/or The beta chain variable domain of the TCR comprises an amino acid sequence with at least 97% sequence homology to the sequence shown in SEQ ID NO:2. The TCR of claim 1, wherein the reference sequences of the three CDR regions of the variable domain of the TCRβ chain are as follows, CDR1β: SGHTA CDR2β: FQGTGA CDR3β: ASSLLAGSYEQY, and CDR3β contains at least one of the following mutations: Residues before mutation mutated residue E at position 10 of CDR3β Y or M Q at position 11 of CDR3β V or L Y at position 12 of CDR3β S or E. The TCR of claim 1, wherein the TCR has a CDR selected from the group consisting of: CDR number α-CDR1 α-CDR2 α-CDR3 β-CDR1 β-CDR2 β-CDR3 1 ATGYPS ATKADDK ALYLFNGKLI SGHTA FQGTGA ASSLLAGSYEQY 2 ATGYPS ATKADDK ALYNYNAVLI SGHTA FQGTGA ASSLLAGSYEQY 3 ATGYPS ATKADDK ALYNFSNRLI SGHTA FQGTGA ASSLLAGSYEQY 4 ATGYPS ATKADDK ALYNFNSLLI SGHTA FQGTGA ASSLLAGSYEQY 5 ATGYPS ATKADDK ALYNFNFVLI SGHTA FQGTGA ASSLLAGSYEQY 6 ATGYPS ATKADDK ALYLFSGKLI SGHTA FQGTGA ASSLLAGSYEQY 7 ATGYPS ATKADDK ALYNFDGILI SGHTA FQGTGA ASSLLAGSYEQY 8 ATGYPS ATKADDK ALYNFNFRLI SGHTA FQGTGA ASSLLAGSYEQY 9 ATGYPS ATKADDK ALYAFDGKLI SGHTA FQGTGA ASSLLAGSYEQY 10 ATGYPS ATKADDK ALYNFDFRLI SGHTA FQGTGA ASSLLAGSYEQY 11 ATGYPS ATKADDK ALYNFNFMLI SGHTA FQGTGA ASSLLAGSYEQY 12 ATGYPS ATKADDK ALYNFNFQLI SGHTA FQGTGA ASSLLAGSYEQY 13 ATGYPS ATKADDK ALYNFNFKLI SGHTA FQGTGA ASSLLAGSYEQY 14 ATGYPS ATKADDK ALYNFDGKLI SGHTA FQGTGA ASSLLAGSYEQY 15 ATGYPS ATKADDK ALYNFASKLI SGHTA FQGTGA ASSLLAGSYEQY 16 ATGYPS ATKADDK ALYNFVFKLI SGHTA FQGTGA ASSLLAGSYEQY 17 ATGYPS ATKADDK ALYNFNSKLI SGHTA FQGTGA ASSLLAGSYEQY 18 ATGYPS ATKADDK ALYNYDFRLI SGHTA FQGTGA ASSLLAGSYEQY 19 ATGYPS ATKADDK ALYNQGGKLI SGHTA FQGTGA ASSLLAGSYYVS 20 ATGYPS ATKADDK ALYNQGGKLI SGHTA FQGTGA ASSLLAGSYMQY twenty one ATGYPS ATKADDK ALYNQGGKLI SGHTA FQGTGA ASSLLAGSYYLE twenty two ATGYPS ATKADDK ALYNFSFVLI SGHTA FQGTGA ASSLLAGSYEQY twenty three ATGYPS ATKADDK ALYNWNFKLI SGHTA FQGTGA ASSLLAGSYEQY twenty four ATGYPS ATKADDK ALYVFTGKLI SGHTA FQGTGA ASSLLAGSYEQY 25 ATGYPS ATKADDK ALYLFDGKLI SGHTA FQGTGA ASSLLAGSYEQY 26 ATGYPS ATKADDK ALYNFDFVLI SGHTA FQGTGA ASSLLAGSYEQY 27 ATGYPS ATKADDK ALYNFHFRLI SGHTA FQGTGA ASSLLAGSYEQY 28 ATGYPS ATKADDK ALYNFDFSLI SGHTA FQGTGA ASSLLAGSYEQY 29 ATGYPS ATKADDK ALYNFSSVLI SGHTA FQGTGA ASSLLAGSYEQY 30 ATGYPS ATKADDK ALYNFNFNLI SGHTA FQGTGA ASSLLAGSYEQY 31 ATGYPS ATKADDK ALYNFMFKLI SGHTA FQGTGA ASSLLAGSYEQY 32 ATGYPS ATKADDK ALYNFDSKLI SGHTA FQGTGA ASSLLAGSYEQY 33 ATGYPS ATKADDK ALYNFSFKLI SGHTA FQGTGA ASSLLAGSYEQY 34 ATGYPS ATKADDK ALYNFNSILI SGHTA FQGTGA ASSLLAGSYEQY 35 ATGYPS ATKADDK ALYNFNAKLI SGHTA FQGTGA ASSLLAGSYEQY 36 ATGYPS ATKADDK ALYNFDAKLI SGHTA FQGTGA ASSLLAGSYEQY 37 ATGYPS ATKADDK ALYNFAFKLI SGHTA FQGTGA ASSLLAGSYEQY The TCR of claim 1, wherein the TCR comprises (i) all or part of the TCRα chain excluding its transmembrane domain, and (ii) all or part of the TCRβ excluding its transmembrane domain chain wherein (i) and (ii) both comprise the variable domain and at least a portion of the constant domain of the TCR chain. The TCR according to claim 1, wherein the TCR comprises an α-chain constant region and a β-chain constant region, and an artificial interchain disulfide bond is contained between the α-chain constant region and the β-chain constant region; preferably, Cysteine residues forming artificial interchain disulfide bonds between the constant regions of the TCR alpha and beta chains are substituted for one or more sets of addresses selected from: Thr48 in exon 1 of TRAC*01 and Ser57 in exon 1 of TRBC1*01 or TRBC2*01; Thr45 in exon 1 of TRAC*01 and Ser77 in exon 1 of TRBC1*01 or TRBC2*01; Tyr10 of exon 1 of TRAC*01 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01; Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01; Ser15 of exon 1 of TRAC*01 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01; Arg53 in exon 1 of TRAC*01 and Ser54 in exon 1 of TRBC1*01 or TRBC2*01; Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01; and Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01. The TCR according to claim 1, wherein the amino acid sequence of the α chain variable domain of the TCR is selected from: SEQ ID NOs: 1 and 13-46; and/or the β chain variable of the TCR Domain amino acid sequence is selected from SEQ ID NO:2 and 47-49; Preferably, described TCR is selected from following group: TCR number Alpha chain variable domain sequence SEQ ID NO: Beta chain variable domain sequence SEQ ID NO: 1 13 2 2 14 2 3 15 2 4 16 2 5 17 2 6 18 2 7 19 2 8 20 2 9 twenty one 2 10 twenty two 2 11 twenty three 2 12 twenty four 2 13 25 2 14 26 2 15 27 2 16 28 2 17 29 2 18 30 2 19 1 47 20 1 48 twenty one 1 49 twenty two 31 2 twenty three 32 2 twenty four 33 2 25 34 2 26 35 2 27 36 2 28 37 2 29 38 2 30 39 2 31 40 2 32 41 2 33 42 2 34 43 2 35 44 2 36 45 2 37 46 2. The TCR according to claim 1, wherein the TCR is a single-chain TCR; preferably, the TCR is a single-chain TCR composed of an α-chain variable domain and a β-chain variable domain, and the α-chain The variable domain and the beta chain variable domain are linked by a flexible short peptide sequence (linker). The TCR according to any one of the above claims, wherein the C- or N-terminus of the α chain and/or the β chain of the TCR is bound with a conjugate, preferably, the conjugate is detectable A marker or therapeutic agent; more preferably, the therapeutic agent is an anti-CD3 antibody. A multivalent TCR complex, characterized in that it comprises at least two TCR molecules, and at least one of the TCR molecules is the TCR described in any one of the above claims. A nucleic acid molecule, characterized in that the nucleic acid molecule comprises a nucleic acid sequence encoding the TCR described in claim 1 or a complementary sequence thereof. A vector, characterized in that the vector contains the nucleic acid molecule described in claim 18. A host cell, characterized in that the host cell contains the vector described in claim 19 or the nucleic acid molecule described in claim 18 that has been incorporated into a chromosome. An isolated cell, characterized in that the cell expresses the TCR of any one of claims 1-16. A pharmaceutical composition, characterized in that the composition contains a pharmaceutically acceptable carrier and the TCR described in any one of claim 1-16 or the TCR complex described in claim 17 or claim Cells described in 21. A method for treating a disease, comprising administering the TCR described in any one of claims 1-16, or the TCR complex described in claim 17, or the TCR complex described in claim 21 to an object in need of treatment The cells, or the pharmaceutical composition described in claim 22, preferably, the disease is an HPV-positive tumor. Use of the T cell receptor described in any one of claims 1-16, the TCR complex described in claim 17, or the cell described in claim 21, characterized in that, for the preparation of a medicament for treating tumors, Preferably, the tumor is an HPV positive tumor. A method for preparing the T cell receptor described in any one of claims 1-16, comprising the steps of: (i) culturing the host cell described in claim 20, thereby expressing the T cell receptor described in any one of claims 1-16; (ii) isolating or purifying the T cell receptor.

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