CN118812698A - A high-affinity T cell receptor for recognizing KRAS mutations and its application - Google Patents

Abstract

The present invention provides a T cell receptor (TCR), the TCR having the property of binding to the VVVGADGVGK-HLAA1101 complex. The present invention also provides a multivalent TCR complex, a nucleic acid molecule encoding the TCR, a vector comprising the nucleic acid molecule, a cell expressing the TCR, and a pharmaceutical composition comprising the aforementioned substances, and their use in the preparation of a drug for diagnosing, treating and preventing KRAS G12D-positive diseases; the present invention also provides a method for preparing the TCR.

Description

A high-affinity T cell receptor for recognizing KRAS mutations and its application

Technical Field

The present invention belongs to the field of biomedicine technology and relates to a high-affinity T cell receptor for recognizing KRAS G12D and its application; more specifically, it relates to a T cell receptor (T cell receptor, TCR) capable of recognizing a polypeptide derived from a KRAS G12D protein; the present invention also relates to a preparation method and application of the TCR.

Background Art

There are only two types of molecules that can recognize antigens in a specific manner. One is the immunoglobulin or antibody; the other is the T cell receptor (TCR), which is a glycoprotein on the cell membrane surface that exists as a heterodimer of α chain/β chain or γ chain/δ chain. The composition of the immune system's TCR repertoire is generated in the thymus by V(D)J recombination followed by positive and negative selection. In the peripheral environment, TCR mediates the specific recognition of T cells to the major histocompatibility complex-peptide complex (pMHC), and is therefore crucial to the cellular immunity function of the immune system.

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

The MHC class I and class II molecule ligands corresponding to TCR are also proteins of the immunoglobulin superfamily, but they are specific for antigen presentation. Different individuals have different MHCs, which can present different short peptides in a protein antigen to the surface of their respective APC cells. Human MHC is usually called HLA gene or HLA complex.

The KRAS gene (P21 gene) is a mouse sarcoma viral oncogene and a member of the ras gene family, encoding the KRAS protein. Once the KRAS gene mutates, it will continue to stimulate cell growth and lead to the occurrence of tumors. Among them, G12D is one of the common mutation sites, and the mutant is called KRAS G12D. KRAS G12D is expressed in a variety of human cancer cells, including but not limited to lung cancer, colorectal cancer, pancreatic cancer, gastric cancer, etc., as shown in the literature (FISHER GH et al (2001) Genes Dev 15 (24): 3249-3262; Brychta N et al (2016) Clin Chem 62 (11): 1482-1491; Ondrej Fiala et al (2016) Tumour Biol 37 (5): 6823-30; Ana S. Leal et al (2018) Curr Protoc Pharmacol 83 (1): e48, etc.) The short peptide VVVGADGVGK after KRAS G12D mutation is located at KRAS amino acids 7-16 and is a target for the treatment of related diseases. After KRAS G12D is produced in cells, it is degraded into small peptides and combined with MHC (major histocompatibility complex) molecules to form a complex that is presented on the cell surface. VVVGADGVGK is a short peptide derived from the KRAS G12D antigen. For the treatment of the above diseases, chemotherapy and radiotherapy can be used, but both will cause damage to the body's normal cells.

Therefore, the VVVGADGVGK-HLA A1101 complex provides a marker for TCR-targeted tumor cells. TCRs that can bind to the VVVGADGVGK-HLA A1101 complex have high application value for the treatment of tumors. For example, TCRs that can target the tumor cell marker can be used to deliver cytotoxic agents or immunostimulants to target cells, or be transformed into T cells so that T cells expressing the TCR can destroy tumor cells so as to be given to patients in a treatment process known as adoptive immunotherapy. For the former purpose, the ideal TCR has a higher affinity so that the TCR can reside on the targeted cells for a long time. For the latter purpose, it is preferred to use a TCR with a medium affinity. Therefore, those skilled in the art are committed to developing TCRs that can be used to target tumor cell markers to meet different purposes.

Summary of the invention

In view of the shortcomings of the prior art, the object of the present invention is to provide a TCR with high affinity for the VVVGADGVGK-HLA A1101 complex. Another object of the present invention is to provide a method for preparing the TCR and the use of the TCR.

In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

In a first aspect of the present invention, a T cell receptor (TCR), the TCR comprising a TCR α chain variable domain and a TCR β chain variable domain, the TCR having an activity of binding to a VVVGADGVGK-HLA A1101 complex;

And the amino acid sequence of the TCR α chain variable domain has at least 90% (for example, it can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, etc.) sequence homology with the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence of the TCR β chain variable domain has at least 90% (for example, it can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, etc.) sequence homology with the amino acid sequence shown in SEQ ID NO:2.

SEQ ID NO: 1:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQK STSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP.

SEQ ID NO:2:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT.

In a preferred example, the amino acid sequence of the TCRα chain variable domain and the amino acid sequence of the TCRβ chain variable domain are not the amino acid sequence of the wild-type TCRα chain variable domain and the amino acid sequence of the wild-type TCRβ chain variable domain at the same time.

In another preferred embodiment, the α chain variable domain of the TCR comprises an amino acid sequence that has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the sequence shown in SEQ ID NO:1, or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid residue mutations relative to the sequence shown in SEQ ID NO:1.

In another preferred embodiment, the β chain variable domain of the TCR is an amino acid sequence that has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with the sequence shown in SEQ ID NO:2, or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid residue mutations relative to the sequence shown in SEQ ID NO:2.

In another preferred embodiment, the amino acid sequence of the TCRα chain variable domain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence of the TCRβ chain variable domain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO:2.

In another preferred example, in the variable domain of the TCRβ chain, CDR1β is MNHEY, CDR2β is SVGEGT, and CDR3β is ASSYLWSYEQY.

In another preferred example, the TCR also has the activity of binding to the VVVGADGVGK-HLA A1101 complex.

In another preferred embodiment, the three CDRs of the TCRα chain variable domain are:

CDR1α: TRDTTYY SEQ ID NO:65;

CDR2α: RNSFDEQN SEQ ID NO: 66; and,

CDR3α:ALSEAGNDMR SEQ ID NO:67.

and contain at least one mutation listed in Table 1:

Table 1

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

In another preferred embodiment, the three CDRs of the TCRβ chain variable domain are:

CDR1β:MNHEY SEQ ID NO:116;

CDR2β: SVGEGT SEQ ID NO: 117; and,

CDR3β: ASSYLWSYEQY SEQ ID NO:118.

And contains at least one mutation in Table 2:

Table 2

In another preferred embodiment, the affinity of the TCR to the VVVGADGVGK-HLA A1101 complex is at least twice that of the wild-type TCR, for example, it can be 2, 2.5, 3, 3.5, 4, 4.5 or 5, etc.

In another preferred embodiment, the TCR has a mutation in the α chain variable domain shown in SEQ ID NO:1, and the mutation is selected from one or more groups of T30E/Q/D/M/S/V/Y, T31N/D, Y32F, R51Q, N52T/V/Q, F54Y/W, N99R, D100I/M/T/N/Q/L/S, M101T/L/Q/K/H and R102N/H/A/Q/K/S/T/D/V, wherein the amino acid residues are numbered as shown in SEQ ID NO:1.

In another preferred embodiment, the TCR undergoes a mutation in the β chain variable domain shown in SEQ ID NO: 2, and the mutation is selected from one or more groups of M27N, E30D, G51H, E52Q/N/K/T, G53E/H/D/N/Q/K/R/T, and T54H/S/I, wherein the amino acid residues are numbered as shown in SEQ ID NO: 2.

In another preferred embodiment, the TCR has a CDR selected from Table 3:

Table 3

The amino acid sequences of SEQ ID NO:68 to SEQ ID NO:115 are shown below:

SEQ ID NO:68: TRDTDYY

SEQ ID NO:69: TRDTNYY

SEQ ID NO:70: TRDQNYY

SEQ ID NO:71: TRDENFY

SEQ ID NO:72: TRDENYY

SEQ ID NO:73: ALSEAGRITN

SEQ ID NO:74: ALSEAGRMLH

SEQ ID NO:75: ALIEAGRTQA

SEQ ID NO:76: ALSEAGRNTQ

SEQ ID NO:77: ALSEAGRQLK

SEQ ID NO:78: ALSEAGRTTH

SEQ ID NO:79: ALSEAGRTKQ

SEQ ID NO:80: ALSEAGRNLA

SEQ ID NO:81: ALSEAGRNKS

SEQ ID NO:82: ALSEAGRLTH

SEQ ID NO:83: ALSEAGPLHS

SEQ ID NO:84: ALSEAGRQLS

SEQ ID NO:85: ALSEAGRMKK

SEQ ID NO:86: ALSEAGRQKT

SEQ ID NO:87: ALSEAGRLTA

SEQ ID NO:88: ALSEAGRLLT

SEQ ID NO:89: ALSEAGRQQT

SEQ ID NO:90: ALSEAGRSKN

SEQ ID NO:91: ALSEAGRNTD

SEQ ID NO:92: ALSEAGRQTV

SEQ ID NO:93: TRDDNYY

SEQ ID NO:94: TRDMNYY

SEQ ID NO:95: TRDSNYY

SEQ ID NO:96: TRDVNYY

SEQ ID NO:97: TRDYNYY

SEQ ID NO:98: QTSYDEQN

SEQ ID NO:99: QVSFDEQN

SEQ ID NO: 100: RQSWDEQN

SEQ ID NO: 101: RTSWDEQN

SEQ ID NO:102: SVHEGT

SEQ ID NO:103: SVGEET

SEQ ID NO:104: SVGEHT

SEQ ID NO:105: SVGEGH

SEQ ID NO:106: SVGEDT

SEQ ID NO:107: SVGENT

SEQ ID NO:108: SVGEQT

SEQ ID NO:109: SVGEKT

SEQ ID NO:110: SVGERT

SEQ ID NO:111: SVGQHS

SEQ ID NO:112: SVGNTI

SEQ ID NO:113: SVGKEI

SEQ ID NO:114: SVGTTT

SEQ ID NO:115: NNHDY

In another preferred embodiment, the TCR is soluble.

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

In another preferred embodiment, the TCR comprises (i) a TCR α chain variable domain and all or part of the TCR α chain constant region excluding the transmembrane domain; and (ii) a TCR β chain variable domain and all or part of the TCR β chain constant region excluding the transmembrane domain.

In another preferred embodiment, 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 of the TCR; preferably, the cysteine residues forming the artificial interchain disulfide bond between the constant regions of the TCRα and TCRβ chains are replaced by one or more groups of sites selected from the following:

Thr48 of exon 1 of TRAC*01 and Ser57 of exon 1 of TRBC1*01 or TRBC2*01;

Thr45 of exon 1 of TRAC*01 and Ser77 of 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 of exon 1 of TRAC*01 and Ser54 of 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,

or 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 one of SEQ ID NO: 1 and SEQ ID NO: 13-46; and/or the amino acid sequence of the β chain variable domain of the TCR is one of SEQ ID NO: 2 and SEQ ID NO: 47-60;

Preferably, the TCR is selected from the group consisting of:

(1) The α chain variable domain sequence is SEQ ID NO: 13, and the β chain variable domain sequence is SEQ ID NO: 2

(2) The α chain variable domain sequence is SEQ ID NO: 14, and the β chain variable domain sequence is SEQ ID NO: 2

(3) The α chain variable domain sequence is SEQ ID NO: 15, and the β chain variable domain sequence is SEQ ID NO: 2

(4) The α chain variable domain sequence is SEQ ID NO: 16, and the β chain variable domain sequence is SEQ ID NO: 2

(5) The α chain variable domain sequence is SEQ ID NO: 17, and the β chain variable domain sequence is SEQ ID NO: 2

(6) The α chain variable domain sequence is SEQ ID NO: 18, and the β chain variable domain sequence is SEQ ID NO: 2

(7) The α chain variable domain sequence is SEQ ID NO: 19, and the β chain variable domain sequence is SEQ ID NO: 2

(8) The α chain variable domain sequence is SEQ ID NO: 20, and the β chain variable domain sequence is SEQ ID NO: 2

(9) The α chain variable domain sequence is SEQ ID NO: 21, and the β chain variable domain sequence is SEQ ID NO: 2

(10) The α chain variable domain sequence is SEQ ID NO: 22, and the β chain variable domain sequence is SEQ ID NO: 2

(11) The α chain variable domain sequence is SEQ ID NO: 23, and the β chain variable domain sequence is SEQ ID NO: 2

(12) The α chain variable domain sequence is SEQ ID NO: 24, and the β chain variable domain sequence is SEQ ID NO: 2

(13) The α chain variable domain sequence is SEQ ID NO: 25, and the β chain variable domain sequence is SEQ ID NO: 2

(14) The α chain variable domain sequence is SEQ ID NO: 26, and the β chain variable domain sequence is SEQ ID NO: 2

(15) The α chain variable domain sequence is SEQ ID NO: 27, and the β chain variable domain sequence is SEQ ID NO: 2

(16) The α chain variable domain sequence is SEQ ID NO: 28, and the β chain variable domain sequence is SEQ ID NO: 2

(17) The α chain variable domain sequence is SEQ ID NO: 29, and the β chain variable domain sequence is SEQ ID NO: 2

(18) The α chain variable domain sequence is SEQ ID NO: 30, and the β chain variable domain sequence is SEQ ID NO: 2

(19) The α chain variable domain sequence is SEQ ID NO: 31, and the β chain variable domain sequence is SEQ ID NO: 2

(20) The α chain variable domain sequence is SEQ ID NO: 32, and the β chain variable domain sequence is SEQ ID NO: 2

(21) The α chain variable domain sequence is SEQ ID NO: 33, and the β chain variable domain sequence is SEQ ID NO: 2

(22) The α chain variable domain sequence is SEQ ID NO: 34, and the β chain variable domain sequence is SEQ ID NO: 2

(23) The α chain variable domain sequence is SEQ ID NO: 35, and the β chain variable domain sequence is SEQ ID NO: 2

(24) The α chain variable domain sequence is SEQ ID NO: 36, and the β chain variable domain sequence is SEQ ID NO: 2

(25) The α chain variable domain sequence is SEQ ID NO: 37, and the β chain variable domain sequence is SEQ ID NO: 2

(26) The α chain variable domain sequence is SEQ ID NO: 38, and the β chain variable domain sequence is SEQ ID NO: 2

(27) The α chain variable domain sequence is SEQ ID NO: 39, and the β chain variable domain sequence is SEQ ID NO: 2

(28) The α chain variable domain sequence is SEQ ID NO: 40, and the β chain variable domain sequence is SEQ ID NO: 2

(29) The α chain variable domain sequence is SEQ ID NO: 41, and the β chain variable domain sequence is SEQ ID NO: 2

(30) The α chain variable domain sequence is SEQ ID NO: 42, and the β chain variable domain sequence is SEQ ID NO: 2

(31) The α chain variable domain sequence is SEQ ID NO: 43, and the β chain variable domain sequence is SEQ ID NO: 2

(32) The α chain variable domain sequence is SEQ ID NO: 44, and the β chain variable domain sequence is SEQ ID NO: 2

(33) The α chain variable domain sequence is SEQ ID NO: 45, and the β chain variable domain sequence is SEQ ID NO: 2

(34) The α chain variable domain sequence is SEQ ID NO: 46, and the β chain variable domain sequence is SEQ ID NO: 2

(35) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 47

(36) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 48

(37) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 49

(38) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 50

(39) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 51

(40) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 52

(41) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 53

(42) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 54

(43) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 55

(44) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 56

(45) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 57

(46) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 58

(47) the α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 59, and,

(48) The α chain variable domain sequence is SEQ ID NO: 1, and the β chain variable domain sequence is SEQ ID NO: 60

In another preferred embodiment, the TCR is of human origin.

In another preferred embodiment, the TCR is isolated or purified.

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

Preferably, the TCR is a single-chain TCR consisting of an α chain variable domain and a β chain variable domain, and the α chain variable domain and the β chain variable domain are connected by a flexible short peptide sequence (linker).

In another preferred example, the TCR comprises an α chain constant region and a β chain constant region, the α chain constant region is a mouse constant region and/or the β chain constant region is a mouse constant region.

In another preferred example, the TCR comprises an α chain constant region and a β chain constant region, the α chain constant region is a mouse constant region and/or the β chain constant region is a mouse constant region.

In another preferred embodiment, 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 or a therapeutic agent; more preferably, the therapeutic agent is an anti-CD3 antibody.

A second aspect of the present invention provides a multivalent TCR complex, wherein the multivalent TCR complex comprises at least two TCRs, and at least one of the TCRs is the TCR described in the first aspect.

The third aspect of the present invention provides a nucleic acid molecule, which includes a nucleotide sequence encoding the TCR 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 of the nucleotide sequence encoding the TCR described in the first aspect of the present invention or the multivalent TCR complex described in the second aspect of the present invention.

The fourth aspect of the present invention provides a vector, wherein 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, wherein the host cell contains the vector described in the fourth aspect of the present invention, or the exogenous nucleic acid molecule described in the third aspect of the present invention is integrated into the chromosome of the host cell.

The sixth aspect of the present invention provides an isolated cell, wherein the isolated cell expresses the TCR described in the first aspect of the present invention.

Preferably, the isolated cells are T cells.

In another preferred embodiment, the cell expresses the TCR described in the first aspect of the present invention and also expresses exogenous CD8 receptor.

Preferably, the CD8 receptor is CD8α; more preferably, the isolated cell is a T cell.

The seventh aspect of the present invention provides a pharmaceutical composition, which comprises any one or a combination of at least two of the TCR described in the first aspect of the present invention, the multivalent TCR complex described in the second aspect of the present invention, and the isolated cells described in 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 cell described in the sixth aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention to a subject in need of treatment; preferably, the disease is a KRAS G12D-positive tumor, more preferably, lung cancer, colorectal cancer, pancreatic cancer, gastric cancer, malignant melanoma, human bile duct epithelial carcinoma cells, etc.

The ninth aspect of the present invention provides the use of the TCR described in the first aspect of the present invention, the multivalent TCR complex described in the second aspect of the present invention, or the isolated cell described in the sixth aspect of the present invention in preparing a specific binding reagent for a target protein.

In another preferred embodiment, the specific binding agent for the target protein is a diagnostic agent and a therapeutic agent for tumors expressing the target protein; preferably, the therapeutic agent is for KRAS G12D-positive tumors.

The tenth aspect of the present invention provides the use of the TCR described in the first aspect of the present invention, the multivalent TCR complex described in the second aspect of the present invention, or the isolated cells described in the sixth aspect of the present invention, wherein the use includes use in preparing drugs for treating tumors.

Preferably, the tumor includes a KRAS G12D-positive tumor, more preferably, lung cancer, colorectal cancer, pancreatic cancer, gastric cancer, malignant melanoma, human bile duct epithelial carcinoma cells, etc.

In an eleventh aspect of the present invention, there is provided a method for preparing the TCR according to the first aspect of the present invention, the preparation method comprising the following steps:

(i) culturing the host cell according to the fifth aspect of the present invention, wherein the host cell expresses the TCR according to the first aspect of the present invention; and,

(ii) isolating or purifying the TCR.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

The numerical range described in the present invention not only includes the point values listed above, but also includes any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The high-affinity TCR of the present invention can specifically bind to the VVVGADGVGK-HLA A1101, and cells transfected with the high-affinity TCR of the present invention can be specifically activated.

(2) Effector cells transfected with the high-affinity TCR of the present invention have a strong specific killing effect.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a binding curve of a soluble reference TCR, i.e., a wild-type TCR, and a VVVGADGVGK-HLAA1101 complex;

FIG2 is an activation function experimental result of the number of IFN-γ spots released by effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines;

FIG3 is an activation function experimental result of the number of granzyme spots released by effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines;

FIG4 is a result of an LDH experiment on the killing function of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines;

FIG5 is an ELISA test result of the killing function of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines;

FIG. 6a and FIG. 6b are the results of the IncuCyte experiment on the killing function of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines.

DETAILED DESCRIPTION

Through extensive and in-depth research, the present invention has obtained a high-affinity T cell receptor (TCR) that recognizes a short peptide VVVGADGVGK (derived from KRAS G12D protein), and the short peptide VVVGADGVGK is presented in the form of a peptide-HLA A1101 complex. The high-affinity TCR has three CDR regions in its α chain variable domain:

CDR1α：TRDTTYY SEQ ID NO:65

CDR2α: RNSFDEQN SEQ ID NO:66 and,

CDR3α: ALSEAGNDMR mutated in SEQ ID NO:67;

and/or in the three CDR regions of the variable domain of its beta chain:

CDR1β:MNHEY SEQ ID NO:116

CDR2β: SVGEGT SEQ ID NO: 117 and

CDR3β: ASSYLWSYEQY mutated in SEQ ID NO: 118;

Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terminology used herein is intended only to describe specific embodiments, and is not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

the term

T cell receptor (TCR)

The International Immunogenetics Information System (IMGT) can be used to describe TCRs. Natural αβ heterodimeric TCRs have α chains and β chains. Broadly speaking, each chain contains a variable region, a connecting region, and a constant region. The β chain usually also contains a short variable region between the variable region and the connecting region, but the variable region is often considered part of the connecting region. The connecting region of the TCR is determined by the unique IMGT TRAJ and TRBJ, and the constant region of the TCR is determined by IMGT TRAC and TRBC.

Each variable region contains three CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, embedded in the framework sequence. In the IMGT nomenclature, different numbers of TRAV and TRBV refer to different Vα types and Vβ types, respectively. In the IMGT system, the α chain constant domain has the following symbol: TRAC*01, where "TR" represents the T cell receptor gene; "A" represents the α chain gene; C represents the constant region; "*01" represents allele 1. The β chain constant domain has the following symbol: TRBC1*01 or TRBC2*01, where "TR" represents the T cell receptor gene; "B" represents the β chain gene; C represents the constant region; "*01" represents allele 1. The constant region of the α chain is uniquely determined, and in the form of the β 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 α and β chains of TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain. The variable domain is composed of a connected variable region and a connecting region. Therefore, in the specification and claims of the present application, "TCRα chain variable domain" refers to the connected TRAV and TRAJ regions, and similarly, "TCRβ chain variable domain" refers to the connected TRBV and TRBD/TRBJ regions. The three CDRs of the TCRα chain variable domain are CDR1α, CDR2α and CDR3α; the three CDRs of the TCRβ chain variable domain are CDR1β, CDR2β and CDR3β. The framework sequence of the TCR variable domain of the present invention can be of mouse or human origin, preferably of human origin. The constant domain of TCR comprises 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 VVVGADGVGK-HLA A1101 complex are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The amino acid sequence of the α chain and the amino acid sequence of the β chain of the soluble "reference TCR" described in the present invention are SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The extracellular amino acid sequence of the α chain and the extracellular amino acid sequence of the β chain of the "wild-type TCR" described in the present invention are SEQ ID NO: 61 and SEQ ID NO: 62, respectively. The TCR sequence used in the present invention is human. The amino acid sequence of the α chain and the amino acid sequence of the β chain of the "wild-type TCR" described in the present invention are SEQ ID NO: 63 and SEQ ID NO: 64, respectively. In the present invention, the terms "polypeptide of the present invention", "TCR of the present invention", and "T cell receptor of the present invention" are used interchangeably.

SEQ ID NO:61:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS.

SEQ ID NO:62:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFPGGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHF RCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD.

SEQ ID NO:63:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLV EKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS.

SEQ ID NO:64:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFPGGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHF RCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG.

Natural interchain disulfide bonds and artificial interchain disulfide bonds

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

For the convenience of description, the position numbers of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered in order from the N-terminus to the C-terminus. For example, in TRBC1*01 or TRBC2*01, the 60th amino acid in the order from the N-terminus to the C-terminus 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 in exon 1 of TRBC1*01 or TRBC2*01. For example, in TRBC1*01 or TRBC2*01, the 61st amino acid in the order from the N-terminus to the C-terminus is Q (glutamine), then in the present invention it can be described as Gln61 of exon 1 of TRBC1*01 or TRBC2*01, or it can be expressed as the 61st amino acid in exon 1 of TRBC1*01 or TRBC2*01, and so on. In the present invention, the position numbers of the amino acid sequences of variable regions TRAV and TRBV follow the position numbers listed in IMGT. For example, if a certain amino acid in TRAV is numbered 46 in IMGT, it will be described as the 46th amino acid in TRAV in the present invention, and so on. In the present invention, if there are special instructions for the sequence position numbers of other amino acids, they will be followed according to the special instructions.

Tumor

The term "tumor" refers to all types of cancer cell growth or carcinogenic processes, metastatic tissues or malignant transformed cells, tissues or organs, regardless of pathological type or stage of infection. Examples of tumors include, but are not limited to: solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include: malignant tumors of different organ systems, such as sarcomas, squamous cell carcinomas of the lung and carcinomas. For example: infected prostate, lung, breast, lymph, gastrointestinal (e.g., colon), and genitourinary tract (e.g., kidney, epithelial cells), pharynx.

DETAILED DESCRIPTION OF THE INVENTION

As is known to all, the α chain variable domain and β chain variable domain of TCR each contain 3 CDRs, similar to the complementary determining regions of antibodies. CDR3 interacts with the antigenic short peptide, and CDR1 and CDR2 interact with HLA. Therefore, the CDR of the TCR molecule determines its interaction with the antigenic short peptide-HLA complex. The amino acid sequence of the α chain variable domain and the amino acid sequence of the β chain variable domain of the wild-type TCR capable of binding to the antigenic short peptide VVVGADGVGK-HLA A1101 complex (i.e., VVVGADGVGK-HLA A1101 complex) are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the sequences are first discovered by the inventors. The TCR has the following CDR regions:

α chain variable domain CDR:

CDR1α: TRDTTYY SEQ ID NO:65;

CDR2α: RNSFDEQN SEQ ID NO: 66; and,

CDR3α: ALSEAGNDMR SEQ ID NO: 67;

and β chain variable domain CDRs:

CDR1β:MNHEY SEQ ID NO:116;

CDR2β: SVGEGT SEQ ID NO: 117; and,

CDR3β: ASSYLWSYEQY SEQ ID NO:118.

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

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

In the present invention, the three CDRs of the wild-type TCR α chain variable domain SEQ ID NO: 1, namely CDR1, CDR2 and CDR3, are located at positions 27-33, 51-58 and 93-102 of SEQ ID NO: 1, respectively. Accordingly, the amino acid residue numbering adopts the numbering shown in SEQ ID NO: 1, 30T is the 4th T of CDR1α, 31T is the 5th T of CDR1α, 32Y is the 6th Y of CDR1α, 51R is the 1st R of CDR2α, 52N is the 2nd N of CDR2α, 54F is the 4th F of CDR2α, 99N is the 7th N of CDR3α, 100D is the 8th D of CDR3α, 101M is the 9th M of CDR3α, and 102R is the 10th R of CDR3α.

Specifically, the specific forms of the mutations in the α chain variable domain include one or more of T30E/Q/D/M/S/V/Y, T31N/D, Y32F, R51Q, N52T/V/Q, F54Y/W, N99R, D100I/M/T/N/Q/L/S, M101T/L/Q/K/H and R102N/H/A/Q/K/S/T/D/V.

In the present invention, the three CDRs of the wild-type TCRβ chain variable domain SEQ ID NO: 2, namely CDR1, CDR2 and CDR3, are respectively located at positions 27-31, 49-54 and 92-102 of SEQ ID NO: 2. Accordingly, the amino acid residue numbering adopts the numbering shown in SEQ ID NO: 2, 27M is the first M of CDR1β, 30E is the fourth E of CDR1β, 51G is the third G of CDR2β, 52E is the fourth E of CDR2β, 53G is the fifth G of CDR2β, and 54T is the sixth T of CDR2β.

Specifically, the specific forms of the mutations in the β chain variable domain include one or more groups of M27N, E30D, G51H, E52Q/N/K/T, G53E/H/D/N/Q/K/R/T, and T54H/S/I.

It should be understood that the amino acid names herein are identified by internationally accepted single-letter English letters, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V).

In the present invention, Pro60 or 60P both represent proline at position 60. In addition, the specific form of the mutation in the present invention is expressed as "Y50I/M/L", which means that Y at position 50 is replaced by I, M or L, and the same applies to other forms.

According to the site-directed mutagenesis method well known to those skilled in the art, Thr48 of exon 1 of the wild-type TCR α chain constant region TRAC*01 is mutated to cysteine, and Ser57 of exon 1 of the β chain constant region TRBC1*01 or TRBC2*01 is mutated to cysteine, that is, a reference TCR is obtained, and the amino acid sequences of the reference TCR are SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and the mutated cysteine residues are represented by bold letters. The above cysteine substitution can form an artificial interchain disulfide bond between the constant regions of the α and β chains of the reference TCR to form a more stable soluble TCR, so that the binding affinity and/or binding half-life between the TCR and the VVVGADGVGK-HLA A1101 complex can be more conveniently evaluated. It should be understood that the CDR region of the TCR variable region determines its affinity with the pMHC complex, and therefore, the above cysteine substitution of the TCR constant region does not affect the binding affinity and/or binding half-life of the TCR. Therefore, in the present invention, the binding affinity between the reference TCR and the VVVGADGVGK-HLA A1101 complex is considered to be the binding affinity between the wild-type TCR and the VVVGADGVGK-HLAA1101 complex. Similarly, if the binding affinity between the TCR of the present invention and the VVVGADGVGK-HLA A1101 complex is at least 10 times the binding affinity between the reference TCR and the VVVGADGVGK-HLA A1101 complex, it is equivalent to the binding affinity between the TCR of the present invention and the VVVGADGVGK-HLA A1101 complex being at least 10 times the binding affinity between the wild-type TCR and the VVVGADGVGK-HLAA1101 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 detection using surface plasmon resonance technology. It should be understood that doubling the affinity of a TCR will result in a halving of the _KD . _T1/2 is calculated as In2 divided by the dissociation rate ( _Koff ). Therefore, doubling the T1 _/2 will result in a halving of the _Koff . Preferably, the same experimental protocol is used to detect the binding affinity or binding half-life of a given TCR several times, for example 3 times or more, and the results are averaged. In a preferred embodiment, the affinity of a soluble TCR is detected using the surface plasmon resonance (BIAcore) method in the examples herein, under the conditions of: temperature 25°C, pH 7.1-7.5. The method detected that the dissociation equilibrium constant K _D of the reference TCR for the VVVGADGVGK-HLAA1101 complex was 1.09E-05M, i.e., 10.90 μM. In the present invention, it is considered that the dissociation equilibrium constant K _D of the wild-type TCR for the VVVGADGVGK-HLA A1101 complex is also 10.90 μM. Since doubling the affinity of the TCR will result in a halving of the K _D , if the dissociation equilibrium constant K _D of the high-affinity TCR for the VVVGADGVGK-HLA A1101 complex is detected to be 1.09E-06M, i.e., 1.09 μM, it means that the affinity of the high-affinity TCR for the VVVGADGVGK-HLA A1101 complex is 10 times that of the wild-type TCR for the VVVGADGVGK-HLA A1101 complex. Those skilled in the art are familiar with the conversion relationship between _KD value units, that is, 1M=10 ⁶ μM, 1 μM=1000 nM, 1 nM=1000 pM.

Any suitable method may be used to perform mutations, including but not limited to those based on polymerase chain reaction (PCR), cloning based on restriction enzymes, or ligation-independent cloning (LIC) methods. Many standard molecular biology textbooks describe these methods in detail. More details on polymerase chain reaction (PCR) mutagenesis and cloning based on restriction enzymes can be found in Sambrook and Russell, (2001) Molecular Cloning-A Laboratory Manual (3rd Edition) CSHL Press. More information on the LIC method can be found in (Rashtchian, (1995) Curr Opin Biotechnol 6 (1): 30-6).

The method for producing the TCR of the present invention may be, but is not limited to, screening for a TCR with high affinity for the VVVGADGVGK-HLA A1101 complex from a diverse library of phage particles displaying such TCRs, as described in the literature (Li, et al (2005) Nature Biotech 23(3): 349-354).

It should be understood that genes expressing wild-type TCR α and β chain variable domain amino acids or genes expressing slightly modified wild-type TCR α and β chain variable domain amino acids can be used to prepare template TCRs. The changes required to generate the high-affinity TCR of the present invention are then introduced into the DNA encoding the variable domain of the template TCR.

The high-affinity TCR of the present invention comprises an α chain variable domain amino acid sequence of SEQ ID NO: 1, one of SEQ ID NO: 13-46; and/or the β chain variable domain amino acid sequence of the TCR is one of SEQ ID NO: 2, SEQ ID NO: 47-60. The amino acid sequences of the α chain variable domain and the β chain variable domain of the high-affinity TCR molecule of the present invention are preferably selected from the following Table 4:

Table 4

Mutated residues in SEQ ID NO: 13-SEQ ID NO: 60 are indicated by single underlining.

SEQ ID NO:13:

AQKVTQAQTEISVVEKEDVTLDCVYETRDT D YYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP.

SEQ ID NO:14:

AQKVTQAQTEISVVEKEDVTLDCVYETRDT N YYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP.

SEQ ID NO:15:

AQKVTQAQTEISVVEKEDVTLDCVYETRD QN YYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP.

SEQ ID NO:16:

AQKVTQAQTEISVVEKEDVTLDCVYETRD ENF YLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP.

SEQ ID NO:17:

AQKVTQAQTEISVVEKEDVTLDCVYETRD EN YYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP.

SEQ ID NO:18:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RITN FGAGTRLTVKP.

SEQ ID NO:19:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RMLH FGAGTRLTVKP.

SEQ ID NO:20:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCAL I EAG RTQA FGAGTRLTVKP.

SEQ ID NO:21:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RNTQ FGAGTRLTVKP.

SEQ ID NO:22:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RQLK FGAGTRLTVKP.

SEQ ID NO:23:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RTTH FGAGTRLTVKP.

SEQ ID NO:24:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RTKQ FGAGTRLTVKP.

SEQ ID NO:25:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RNLA FGAGTRLTVKP.

SEQ ID NO:26:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RNKS FGAGTRLTVKP.

SEQ ID NO:27:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RLTH FGAGTRLTVKP.

SEQ ID NO:28:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG PLHS FGAGTRLTVKP.

SEQ ID NO:29:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RQLS FGAGTRLTVKP.

SEQ ID NO:30:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RMKK FGAGTRLTVKP.

SEQ ID NO:31:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RQKT FGAGTRLTVKP.

SEQ ID NO:32:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RLTA FGAGTRLTVKP.

SEQ ID NO:33:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RLLT FGAGTRLTVKP.

SEQ ID NO:34:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RQQT FGAGTRLTVKP.

SEQ ID NO:35:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RSKN FGAGTRLTVKP.

SEQ ID NO:36:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RNTD FGAGTRLTVKP.

SEQ ID NO:37:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAG RQTV FGAGTRLTVKP.

SEQ ID NO:38:

AQKVTQAQTEISVVEKEDVTLDCVYETRD DN YYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP.

SEQ ID NO:39:

AQKVTQAQTEISVVEKEDVTLDCVYETRD MN YYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP.

SEQ ID NO:40:

AQKVTQAQTEISVVEKEDVTLDCVYETRD SN YYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP

SEQ ID NO:41:

AQKVTQAQTEISVVEKEDVTLDCVYETRD VN YYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP

SEQ ID NO:42:

AQKVTQAQTEISVVEKEDVTLDCVYETRD YN YYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP

SEQ ID NO:43:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIR QT S Y DEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP

SEQ ID NO:44:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIR QV SFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP

SEQ ID NO:45:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRR Q S W DEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP

SEQ ID NO:46:

AQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRR T S W DEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEAGNDMRFGAGTRLTVKP

SEQ ID NO:47:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSV H EGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

SEQ ID NO:48:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGE E TTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

SEQ ID NO:49:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGE H TTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

SEQ ID NO:50:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPMGLRLIHYSVGEG H TAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

SEQ ID NO:51:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGE D TTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

SEQ ID NO:52:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGE N TTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

SEQ ID NO:53:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGE Q TTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

SEQ ID NO:54:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGE K TTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

SEQ ID NO:55:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGE R TTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

SEQ ID NO:56:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPMGLRLIHYSVG QHS TAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

SEQ ID NO:57:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPMGLRLIHYSVG NTI TAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

SEQ ID NO:58:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPMGLRLIHYSVG KEI TAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

SEQ ID NO:59:

NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPMGLRLIHYSVG TT TTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

SEQ ID NO:60:

NAGVTQTPKFRVLKTGQSMTLLCAQD N NH D YMYWYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYLWSYEQYFGPGTRLTVT

For the purposes of the present invention, the TCR of the present invention is a part having at least one TCRα and/or TCRβ chain variable domain. They generally contain both TCRα chain variable domain and TCRβ chain variable domain. They can be αβ heterodimers or single chain forms or any other form that can be stably present. In adoptive immunotherapy, the full-length chain of αβ heterodimeric TCR (including 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 in combination with other molecules to prepare bifunctional polypeptides to direct effector cells, and at this time, the TCR is preferably in a soluble form.

For stability, the prior art discloses that the introduction of an artificial interchain disulfide bond between the α and β chain constant domains of the TCR can obtain a soluble and stable TCR molecule, as described in patent document PCT/CN2015/093806. Therefore, the TCR of the present invention may be a TCR that introduces an artificial interchain disulfide bond between the residues of its α and β chain constant domains. Cysteine residues form an artificial interchain disulfide bond between the α and β chain constant domains of the TCR. Cysteine residues can replace other amino acid residues at suitable sites in natural TCRs to form artificial interchain disulfide bonds. For example, replacing Thr48 of TRAC*01 exon 1 and replacing Ser57 of TRBC1*01 or TRBC2*01 exon 1 to form a disulfide bond. Other sites for introducing cysteine residues to form disulfide bonds may also be:

Thr45 of exon 1 of TRAC*01 and Ser77 of 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 of exon 1 of TRAC*01 and Ser54 of 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;

or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.

That is, cysteine residues replace any of the above-mentioned α and β chain constant domains. Up to 15, or up to 10, or up to 8 or fewer amino acids can be truncated at one or more C-termini of the TCR constant domain of the present invention so that it does not include cysteine residues to achieve the purpose of missing the natural interchain disulfide bond, and the above purpose can also be achieved by mutating the cysteine residues that form the natural interchain disulfide bond to another amino acid.

As described above, the TCR of the present invention may contain artificial interchain disulfide bonds introduced between residues of the constant domains of its α and β chains. It should be noted that the TCR of the present invention may contain a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR may be connected by a natural interchain disulfide bond present in the TCR.

In addition, regarding stability, patent document PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR can significantly improve the stability of the TCR. Therefore, the α chain variable region and the β chain constant region of the high-affinity TCR of the present invention may also contain an artificial interchain disulfide bond. Specifically, the cysteine residue that forms the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR is replaced by:

amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01;

amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01;

amino acid 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, the TCR may comprise (i) all or part of the TCR α chain excluding its transmembrane domain, and (ii) all or part of the TCR β chain excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least a portion of the constant domain of the TCR chain, and the α chain and the β chain form a heterodimer.

More preferably, the TCR may comprise an α chain variable domain and a β chain variable domain and all or part of the β chain constant domain excluding the transmembrane domain, but does not comprise an α chain constant domain, and the α chain variable domain of the TCR forms a heterodimer with the β chain.

For stability, on the other hand, the TCR of the present invention also includes a TCR that mutates in its hydrophobic core region, and the mutations in these hydrophobic core regions are preferably mutations that can improve the stability of the TCR of the present invention, as described in the patent document with publication number WO2014/206304. The TCR may mutate at the following variable domain hydrophobic core positions: (α and/or β chain) variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or α chain J gene (TRAJ) short peptide amino acid position penultimate 3, 5, 7, and/or β chain J gene (TRBJ) short peptide amino acid position penultimate 2, 4, 6, wherein the position numbering of the amino acid sequence is according to the position numbering listed in the International Immunogenetics Information System (IMGT). Those skilled in the art are aware of the above-mentioned International Immunogenetics Information System, and can obtain the position numbering of the amino acid residues of different TCRs in IMGT according to the database.

More specifically, the TCR in which the hydrophobic core region is mutated in the present invention can be a highly stable single-chain TCR composed of a flexible peptide chain connecting the variable domains of the α chain and the β chain of the TCR. The CDR region of the TCR variable region determines its affinity with the short peptide-HLA complex. The mutation of the hydrophobic core can make the TCR more stable, but it does not affect its affinity with the short peptide-HLA complex. It should be noted that the flexible peptide chain in the present invention can be any peptide chain suitable for connecting the variable domains of the TCR α and β chains. The template chain for screening high-affinity TCRs constructed in Example 1 of the present invention is the above-mentioned highly stable single-chain TCR containing a hydrophobic core mutation. Using a TCR with higher stability, it is more convenient to evaluate the affinity between the TCR and the VVVGADGVGK-HLA A1101 complex.

The CDR regions of the α chain variable domain and the β chain variable domain of the single-chain template TCR are exactly the same as the CDR regions of the wild-type TCR. That is, the three CDRs of the α chain variable domain are CDR1α: TRDTTYY, CDR2α: RNSFDEQN, CDR3α: ALSEAGNDMR, and the three CDRs of the β chain variable domain are CDR1β: MNHEY, CDR2β: SVGEGT, CDR3β: ASSYLWSYEQY. The amino acid sequence of the single-chain template TCR is shown in SEQ ID NO: 9, and the nucleotide sequence of the single-chain template TCR is shown in SEQ ID NO: 10. In this way, a single-chain TCR composed of an α chain variable domain and a β chain variable domain with high affinity for the VVVGADGVGK-HLA A1101 complex is screened out.

The αβ heterodimeric TCR (also referred to as high-affinity TCR) of the present invention having high affinity for the VVVGADGVGK-HLA A1101 complex is obtained by transferring the CDR regions of the α and β chain variable domains of the screened high-affinity single-chain TCR to the corresponding positions of the wild-type TCR α chain variable domain (SEQ ID NO: 1) and the β chain variable domain (SEQ ID NO: 2).

The TCR of the present invention can also be provided in the form of a multivalent complex. The multivalent TCR complex of the present invention comprises a polymer formed by combining two, three, four or more TCRs of the present invention, such as a tetramerization domain of p53 can be used to produce a tetramer, or a complex formed by combining multiple TCRs of the present invention with another molecule. The TCR complex of the present invention can be used to track or target cells presenting specific antigens in vitro or in vivo, and can also be used to produce intermediates of other multivalent TCR complexes with such applications.

The TCR of the present invention can be used alone or in combination with a conjugate, preferably covalently, wherein the conjugate includes a detectable marker (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the VVVGADGVGK-HLA A1101 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these substances.

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

Therapeutic agents that can be combined or coupled to the TCR of the present invention include, but are not limited to:

1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539);

2. Biological toxins (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565);

3. Cytokines such as IL-2 (Gillies et al., 1992, Proceedings of the National Academy of Sciences (PNAS) 89, 1428; Card et al., 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al., 2003, Cancer Research 63, 3202);

4. Antibody Fc fragment (Mosquera et al., 2005, The Journal of Immunology 174, 4381);

5. Antibody scFv fragment (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. Virus particles (Peng et al., 2004, Gene therapy 11, 1234);

8. Liposomes (Mamot et al., 2005, Cancer Research 65, 11631);

9.Nanomagnetic particles;

10. Prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL));

11. Chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, etc.

The antibodies or fragments thereof bound 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, and the binding of the above antibodies or fragments thereof to the TCR can direct effector cells to better target target cells. A preferred embodiment is that the TCR of the present invention is combined with an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody. Specifically, the fusion molecule of the TCR of the present invention and the anti-CD3 single-chain antibody includes a TCR α chain variable domain amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 13-46; and/or the amino acid sequence of the β chain variable domain of the TCR is SEQ ID NO: 2, SEQ ID NO: 47-60.

The present invention also relates to nucleic acid molecules encoding the TCR of the present invention. The nucleic acid molecules of the present invention can be in the form of DNA or RNA. The DNA can be a coding strand or a non-coding strand. For example, the nucleic acid sequence encoding the TCR of the present invention can be the same as the nucleic acid sequence shown in the present invention or a degenerate variant. To illustrate the meaning of "degenerate variant", as used herein, "degenerate variant" in the present invention refers to a nucleotide sequence that encodes a protein sequence having SEQ ID NO: 3, but is different from the sequence of SEQ ID NO: 5.

The full-length sequence of the nucleic acid molecule of the present invention or its fragment can usually be obtained by, but not limited to, PCR amplification, recombination or artificial synthesis. At present, the DNA sequence encoding the TCR of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The obtained DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

The present invention also relates to vectors comprising the nucleic acid molecules of the present invention, and host cells produced by genetic engineering using the vectors or coding sequences of the present invention.

The present invention also includes isolated cells, particularly T cells, expressing the TCR of the present invention. There are many methods suitable for transfecting T cells with DNA or RNA encoding the high-affinity TCR of the present invention (e.g., 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 will be aware of many suitable methods for adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8 (4): 299-308).

The present invention also provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR of the present invention, or the TCR complex of the present invention, or a cell presenting the TCR of the present invention.

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

In the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. Adding one or more amino acids at the C-terminus and/or N-terminus generally does not change the structure and function of the protein. Therefore, the TCR of the present invention also includes at most 5, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid (especially amino acids outside the CDR region) of the TCR of the present invention, which is replaced by amino acids with similar or similar properties and can still maintain its functionality.

The present invention also includes TCRs that are slightly modified from the TCRs of the present invention. Modifications (usually without changing the primary structure) include: chemical derivatization forms of the TCRs of the present invention such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the TCRs of the present invention or in further processing steps. This modification can be accomplished by exposing the TCR to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, or phosphothreonine). It also includes TCRs that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.

The TCR, TCR complex of the present invention or T cells transfected with the TCR of the present invention may be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The TCR, multivalent TCR complex or cell of the present invention is 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). The pharmaceutical composition may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such a kit (but not necessarily) includes instructions for use. It may include a plurality of such unit dosage forms.

In addition, the TCR of the present invention can be used alone or in combination or coupled with other therapeutic agents (such as 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 used for the administration of a therapeutic agent. The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies that are harmful to the individual receiving the composition, and there is no excessive toxicity after administration. These carriers 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, buffer, glucose, water, glycerol, ethanol, adjuvants and combinations thereof.

Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may also be present in these carriers.

Typically, therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid vehicles 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 parenteral including subcutaneous, intramuscular or intravenous. The subject to be prevented or treated can 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 used according to the usage conditions, preferably, injections, oral preparations, etc. can be cited.

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

The pharmaceutical composition of the present invention can also be administered in the form of a sustained release agent. For example, the TCR of the present invention can be incorporated into a pill or microcapsule with a sustained release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. As examples of sustained release polymers, ethylene-vinyl acetate copolymers, polyhydroxymethylacrylate (polyhydrometaacrylate), polyacrylamide, polyvinyl pyrrolidone, methylcellulose, lactic acid polymers or lactic acid-glycolic acid copolymers, etc., preferably biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers can be cited.

When the pharmaceutical composition of the present invention is used for actual treatment, the TCR or TCR complex of the present invention or the cell presenting the TCR of the present invention as the active ingredient can be reasonably determined based on the weight, age, gender, and symptom severity of each patient to be treated, and the reasonable dosage is ultimately determined by the physician.

The main advantages of the present invention are:

(1) The high-affinity TCR of the present invention can specifically bind to the VVVGADGVGK-HLA A1101, and cells transfected with the high-affinity TCR of the present invention can be specifically activated.

(2) Effector cells transfected with the high-affinity TCR of the present invention have a strong specific killing effect.

The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (Molecular Cloning-A Laboratory Manual) (3rd Edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

Materials and methods

Unless otherwise specified, the experimental materials used in the examples of the present invention can be obtained from commercial channels, among which E. coli DH5α was purchased from Tiangen, E. coli BL21 (DE3) was purchased from Tiangen, E. coli Tuner (DE3) was purchased from Novagen, and plasmid pET28a was purchased from Novagen.

Example 1 Stability of hydrophobic core mutations Generation of single-stranded TCR template chains

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 consisting of a flexible short peptide (linker) connecting the TCRα and β chain variable domains, and the amino acid and DNA sequences of the single-chain TCR molecule are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively. And the single-chain TCR molecule is used as a template for screening high-affinity TCR molecules. The amino acid sequence of the α chain variable domain of the template chain is shown in SEQ ID NO:3, and the amino acid sequence of the β chain variable domain is shown in SEQ ID NO:4; the corresponding nucleotide sequences are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively; the amino acid sequence and nucleotide sequence of the flexible short peptide (linker) are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

The target gene carrying the template strand was double-digested with NcoⅠ and NotⅠ, and then connected to the pET28a vector double-digested with NcoⅠ and NotⅠ. The ligation product was transformed into E.coli DH5α, coated with LB plates containing kanamycin, and inverted and cultured at 37℃ overnight. Positive clones were selected for PCR screening, and positive recombinants were sequenced. After confirming that the sequence was correct, the recombinant plasmid was extracted and transformed into E.coli BL21 (DE3) for expression.

SEQ ID NO:3:

AQKVTQSQTELSVVEGEDVTIDCVYETRDTTYYLFWYKQPPSGEPVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITDVQPNDSAVYFCALSEAGNDMRFGAGTRLTVKP.

SEQ ID NO:4:

NAGVTQTPKYLSVKTGQSVTLQCAQDMNHEYMYWYRQDPGQGLRLIYYSVGEGTTAKGEVPDRYNVSRLKKQNFLLGIESVTPSDTSVYFCASSYLWSYEQYFGPGTRLTVT.

SEQ ID NO:5:

gctcaaaaagttactcaaagccaaaccgagctgagcgtggttgagggtgaagacgtgaccatcgattgcgtttatgaaacccgtgacaccacctactacctgttctggtacaagcaaccgccgagcggcgagccggttttcctgatccgtcgtaacagctttgatgagcagaacgaaattagcggccgttatag ctggaactttcagaagagcaccagcagcttcaactttaccattaccgacgtgcagccgaacgatagcgcggtttacttctgcgcgctgagcgaagcgggtaacgacatgcgttttggtgcgggtacccgtctgaccgtgaaaccg.

SEQ ID NO:6:

aacgcgggcgttacccagaccccgaagtatctgagcgtgaaaaccggtcaaagcgttaccctgcagtgcgcgcaagacatgaaccacgagtacatgtattggtaccgtcaggacccgggtcaaggcctgcgtctgatctactatagcgtgggcgagggcaccacccgcgaaaggtgaagtgccggaccgttacaac gttagccgtctgaagaaacagaacttcctgctgggcattgagagcgtgaccccgagcgataccagcgtttatttctgcgcgagcagctacctgtggagctatgaacaatactttggtccgggcacccgtctgaccgttacc.

SEQ ID NO:7:

GGGSEGGGSEGGGSEGGGSEGGTG .

SEQ ID NO:8:

ggtggcggtagcgagggcggtggcagcgaaggtggcggtagcgagggcggtggcagcgaaggtggcac cggt .

SEQ ID NO:9:

AQKVTQSQTELSVVEGEDVTIDCVYETRDTTYYLFWYKQPPSGEPVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITDVQPNDSAVYFCALSEAGNDMRFGAGTRLTVKPGGGSEGGGSEGGGSEGGGSEGGTGNAGVTQTPKYLSVKTGQSVTLQCAQDMNHEYMYWYRQDPGQGLRLIYYSVGEGTTAKGEVPDRYNVSRLKK QNFLLGIESVTPSDTSVYFCASSYLWSYEQYFGPGTRLTVT.

SEQ ID NO:10:

gctcaaaaagttactcaaagccaaaccgagctgagcgtggttgagggtgaagacgtgaccatcgattgcgtttatgaaacccgtgacaccacctactacctgttctggtacaagcaaccgccgagcggcgagccggttttcctgatccgtcgtaacagctttgatgagcagaacgaaattagcggccgttatagct ggaactttcagaagagcaccagcagcttcaactttaccattaccgacgtgcagccgaacgatagcgcggtttacttctgcgcgctgagcgaagcgggtaacgacatgcgttttggtgcgggtacccgtctgaccgtgaaaccgggtggcggtagcgagggcggtggcagcgaaggtgg cggtagcgagggcggtggcagcgaaggtggcaccggtaacgcgggcgttacccagaccccgaagtatctgagcgtgaaaaccggtcaaagcgttaccctgcagtgcgcgcaagacatgaaccacgagtacatgtattggtaccgtcaggacccgggtcaaggcctgcgtctgatctactatagcgtgggcgagggc accaccgcgaaaggtgaagtgccggaccgttacaacgttagccgtctgaagaaacagaacttcctgctgggcattgagagcgtgaccccgagcgataccagcgtttatttctgcgcgagcagctacctgtggagctatgaacaatactttggtccgggcacccgtctgaccgttacc.

Example 2 Expression, renaturation and purification of the stable single-chain TCR constructed in Example 1

All BL21 (DE3) colonies containing the recombinant plasmid pET28a-template chain prepared in Example 1 were inoculated into LB medium containing kanamycin, cultured at 37°C until OD ₆₀₀ was 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and cultured at 37°C for 4 hours. The cell precipitate was harvested by centrifugation at 5000 rpm for 15 minutes, the cell precipitate was lysed with Bugbuster Master Mix (Merck), the inclusion bodies were recovered by centrifugation at 6000 rpm for 15 minutes, and then washed with Bugbuster (Merck) to remove cell debris and membrane components, and the inclusion bodies were collected by centrifugation at 6000 rpm for 15 minutes. The inclusion bodies were dissolved in a buffer (20 mM Tris-HCl pH 8.0, 8 M urea), insoluble matter was removed by high-speed centrifugation, and the supernatant was quantified by BCA method and packaged, and stored at -80°C for later use.

To 5 mg of dissolved single-chain TCR inclusion body protein, add 2.5 mL of buffer (6M Gua-HCl, 50 mM Tris-HCl pH 8.1, 100 mM NaCl, 10 mM EDTA), then add DTT to a final concentration of 10 mM, and treat at 37°C for 30 min. Use a syringe to add the above-treated single-chain TCR to 125 mL of refolding 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), stir at 4°C for 10 min, and then load the refolding solution into a cellulose membrane dialysis bag with a cutoff of 4 kDa, place the dialysis bag in 1 L of pre-cooled water, and stir slowly at 4°C overnight. After 17 hours, the dialysate was replaced with 1L pre-cooled buffer (20mM Tris-HCl pH 8.0), and the dialysis was continued for 8 hours at 4°C, and then the dialysate was replaced with the same fresh buffer and the dialysis was continued overnight. After 17 hours, the sample was filtered through a 0.45μm filter membrane, vacuum degassed, and passed through an anion exchange column (HiTrap Q HP, GE Healthcare). The protein was purified using a 0-1M NaCl linear gradient eluent prepared with 20mM Tris-HCl pH 8.0. The collected elution fractions were analyzed by SDS-PAGE, and the fractions containing the single-chain TCR were concentrated and further purified using a gel filtration column (Superdex 75 10/300, GE Healthcare). The target fraction was also analyzed by SDS-PAGE.

The eluted fractions used for BIAcore analysis were further tested for purity by gel filtration. The conditions were: chromatographic column Agilent Bio SEC-3 (300A, ), the mobile phase was 150 mM phosphate buffer, the flow rate was 0.5 mL/min, the column temperature was 25 °C, and the UV detection wavelength was 214 nm.

Example 3 Binding Characterization

BIAcore analysis

The binding activity of TCR molecules to the VVVGADGVGK-HLA A1101 complex was detected using the BIAcore T200 real-time analysis system. Anti-streptavidin antibody (GenScript) was added to the coupling buffer (10mM sodium acetate buffer, pH4.77), and then the antibody was passed through the CM5 chip pre-activated with EDC and NHS to fix the antibody on the chip surface. Finally, the unreacted activated surface was blocked with ethanolamine hydrochloric acid solution to complete the coupling process. The coupling level was about 15000RU. The conditions were: temperature 25°C, pH 7.1-7.5.

A low concentration of streptavidin was passed through the antibody-coated chip surface, and then the VVVGADGVGK-HLAA1101 complex was passed through the detection channel. The other channel was used as the reference channel, and 0.05mM biotin was passed through the chip at a flow rate of 10μL/min for 2min to block the remaining binding sites of streptavidin. The affinity was determined by single-cycle kinetic analysis. TCR was diluted to several different concentrations with HEPES-EP buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% P20 pH 7.4) and passed through the chip surface in sequence at a flow rate of 30μL/min. The binding time for each injection was 120s, and the dissociation was allowed for 600s after the last injection. After each round of measurement, the chip was regenerated with 10mM Gly-HCl at pH 1.75. The kinetic parameters were calculated using BIAcore Evaluation software.

The preparation process of the above VVVGADGVGK-HLA A1101 complex is as follows:

a. Purification: Collect 100 mL of E. coli culture induced to express heavy chain or light chain, centrifuge at 4°C and 8000 g for 10 min, wash the cells once with 10 mL PBS, then resuspend the cells with 5 mL BugBuster Master Mix Extraction Reagents (Merck) by vigorous shaking, incubate with rotation at room temperature for 20 min, then centrifuge at 4°C and 6000 g for 15 min, discard the supernatant, and collect the inclusion bodies.

The inclusion bodies were resuspended in 5 mL BugBuster Master Mix and incubated with rotation at room temperature for 5 min; 30 mL of 10-fold diluted BugBuster was added, mixed, and centrifuged at 4°C and 6000 g for 15 min; the supernatant was discarded, 30 mL of 10-fold diluted BugBuster was added to resuspend the inclusion bodies, mixed, and centrifuged at 4°C and 6000 g for 15 min, repeated twice, 30 mL of 20 mM Tris-HCl pH 8.0 was added to resuspend the inclusion bodies, mixed, and centrifuged at 4°C and 6000 g for 15 min, and finally 20 mM Tris-HCl and 8 M urea were used to dissolve the inclusion bodies, and the purity of the inclusion bodies was detected by SDS-PAGE and the concentration was measured by BCA kit.

b. Renaturation: The synthetic short peptide VVVGADGVGK (synthesized by Jiangsu GenScript Biotechnology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/mL. The inclusion bodies of the light chain and heavy chain were dissolved in a solution containing 8M urea, 20mM Tris pH 8.0, and 10mM DTT, and 3M guanidine hydrochloride, 10mM sodium acetate, and 10mM EDTA were added for further denaturation before renaturation. The VVVGADGVGK peptide was added to renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C) at 25mg/L (final concentration), and then 20mg/L of light chain and 90mg/L of heavy chain were added in sequence (final concentration, heavy chain was added three times, 8h/time), and renaturation was carried out at 4°C for at least 3 days until completion, and SDS-PAGE was used to detect whether renaturation was successful.

c. Purification after renaturation: Use 10 volumes of 20mM Tris pH 8.0 for dialysis to replace the renaturation buffer, and replace the buffer at least twice to fully reduce the ionic strength of the solution. After dialysis, filter the protein solution with a 0.45μm cellulose acetate filter and then load it onto a HiTrap Q HP (GE General Electric) anion exchange column (5mL bed volume). Use an Akta purifier (GE General Electric) to elute the protein with a 0-400mM NaCl linear gradient solution prepared with 20mM Tris pH 8.0. pMHC is eluted at about 250mM NaCl. Collect the peak components and detect the purity by SDS-PAGE.

d. Biotinylation: The purified pMHC molecules were concentrated using Millipore ultrafiltration tubes, and the buffer was replaced with 20 mM Tris pH 8.0. Then, biotinylation reagents 0.05 M Bicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 μM D-Biotin, and 100 μg/mL BirA enzyme (GST-BirA) were added. The mixture was incubated at room temperature overnight, and SDS-PAGE was used to detect whether the biotinylation was complete.

e. Purification of biotinylated complex: The biotinylated pMHC molecules were concentrated to 1 mL using Millipore ultrafiltration tubes, and the biotinylated pMHC was purified by gel filtration chromatography. The Akta purifier (GE General Electric Company) was used to pre-equilibrate the HiPrep ^TM 16/60S200 HR column (GE General Electric Company) with filtered PBS, and 1 mL of concentrated biotinylated pMHC molecules was loaded, and then eluted with PBS at a flow rate of 1 mL/min. The biotinylated pMHC molecules appeared as a single peak at about 55 mL. The protein-containing fractions were combined and concentrated using Millipore ultrafiltration tubes. The protein concentration was determined by the BCA method (Thermo), and the protease inhibitor cocktail (Roche) was added to the biotinylated pMHC molecules and stored at -80°C.

Example 4 Generation of high affinity TCR

Phage display technology is a means of generating a library of TCR high-affinity variants to screen for 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 library of high-affinity single-chain TCR was established by mutating the CDR region of the template chain and performing panning. After several rounds of panning, the phage library all had specific binding to the corresponding antigen, from which a single clone was picked and analyzed. The CDR regions of the α chain and β chain of the high-affinity single-chain TCR screened were as follows:

The CDR region mutation group of the screened high-affinity single-chain TCR, i.e., CDR numbers 1-48, was introduced one by one into the corresponding sites of the α and β chain variable domains of the wild-type TCR to obtain high-affinity TCRs, with TCR numbers corresponding to 1-48. And its affinity with the VVVGADGVGK-HLA A1101 complex was detected by BIAcore. The introduction of the high-affinity mutation points in the above CDR region adopts a site-directed mutagenesis method well known to those skilled in the art. The amino acid sequences of the α chain and β chain variable domains of the above wild-type TCR are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

It should be noted that a cysteine residue can be introduced into the constant region of the wild-type α and β chains to form an artificial interchain disulfide bond to obtain a more stable soluble TCR, so as to more conveniently evaluate the binding affinity and/or binding half-life between the TCR and the VVVGADGVGK-HLA A1101 complex. In this example, the amino acid sequences of the TCR α and β chains after the introduction of cysteine residues are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and the introduced cysteine residues are represented by bold and double underlined letters.

SEQ ID NO:11:

SEQ ID NO:12:

The extracellular sequence genes of the TCRα chain and TCRβ chain to be expressed were synthesized and inserted into the expression vector pET28a+ (Novagene) by the standard method described in Molecular Cloning a Laboratory Manual (3rd edition, Sambrook and Russell), and the upstream and downstream cloning sites were Nco I and Not I, respectively. Mutations in the CDR region were introduced by overlap PCR, which is well known to those skilled in the art. The inserted fragments were confirmed to be correct by sequencing.

Example 5 Expression, renaturation and purification of high affinity TCR

The expression vectors of TCR α chain and TCR β chain were transformed into the expression bacteria BL21 (DE3) by chemical transformation, and the bacteria were grown in LB culture medium and induced with a final concentration of 0.5 mM IPTG at OD ₆₀₀ = 0.6. The inclusion bodies formed after the expression of TCR α and β chains were extracted by BugBuster Mix (Novagene) and repeatedly washed with BugBuster solution. The inclusion bodies were finally dissolved in a solution containing 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA), and 20 mM Tris (pH 8.1).

The dissolved TCRα chain and TCRβ chain were quickly mixed in a solution containing 5 M urea, 0.4 M arginine, 20 mM Tris (pH 8.1), 3.7 mM cystamine, and 6.6 mMβ-mercapoethylamine (4℃) at a mass ratio of 1:1, with a final concentration of 60 mg/mL. After mixing, the solution was dialyzed in 10 volumes of deionized water (4℃). After 12 hours, the deionized water was replaced with buffer (20mM Tris, pH 8.0) and the dialysis was continued at 4℃ for 12 hours. After the dialysis was completed, the solution was filtered through a 0.45μm filter membrane and purified by an anion exchange column (HiTrap Q HP, 5mL, GE Healthcare). The elution peak contained TCR with successfully refolded α and β dimers, which was confirmed by SDS-PAGE gel. TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was greater than 90% as determined by SDS-PAGE, and the concentration was determined by the BCA method.

Example 6 BIAcore analysis results

The method described in Example 3 was used to detect the affinity of the wild-type TCR introduced with the high-affinity CDR region to the VVVGADGVGK-HLAA1101 complex.

The amino acid sequences of the novel TCR α chain and TCR β chain variable domains obtained by the present invention are shown in SEQ ID NO: 13-SEQ ID NO: 60. 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 wild-type TCR introduced with a high-affinity mutation point also has a high affinity for the VVVGADGVGK-HLA A1101 complex. The expression vector was constructed using the method described in Example 4, and the wild-type TCR introduced with the high-affinity mutation was expressed, renatured and purified using the method described in Example 5, and then its affinity with the VVVGADGVGK-HLA A1101 complex was determined using BIAcore T200. FIG1 is a binding curve of a soluble reference TCR, i.e., a wild-type TCR, and the VVVGADGVGK-HLA A1101 complex. The results of the high-affinity TCR affinity test are shown in Table 5 below:

Table 5

From the results in Table 5, it can be seen that the high-affinity TCR obtained in the present invention has improved affinity for the AQIPEKIQK-HLA A1101 complex.

Example 7 Expression, renaturation and purification of anti-CD3 antibody and high affinity TCR fusion

The anti-CD3 single-chain antibody (scFv) is fused with a high-affinity TCR to prepare a fusion molecule. The anti-CD3 scFv is fused with the β chain of the TCR, the TCR β chain can contain any of the above-mentioned high-affinity TCR β chain variable domains, and the TCR α chain of the fusion molecule can contain any of the above-mentioned high-affinity TCR α chain variable domains. The construction of the fusion molecule expression vector includes the following steps:

(1) Construction of α chain expression vector: The target gene of the α chain carrying high-affinity TCR was double-digested with NcoⅠ and NotⅠ, and ligated to the pET28a vector double-digested with NcoⅠ and NotⅠ. The ligation product was transformed into E.coli DH5α, spread on an LB plate containing kanamycin, and incubated at 37℃ overnight. Positive clones were selected for PCR screening, and positive recombinants were sequenced. After confirming that the sequence was correct, the recombinant plasmid was extracted and transformed into E.coli Tuner (DE3) for expression.

(2) Construction of anti-CD3 (scFv)-β chain expression vector: Through the overlap PCR method, primers were designed to connect the anti-CD3 scFv and high-affinity TCRβ chain genes, with the intermediate linker being GGGGS, and the gene fragment of the fusion protein of anti-CD3 scFv and high-affinity TCRβ chain was endowed with restriction endonuclease sites NcoⅠ (CCATGG) and NotⅠ (GCGGCCGC). The PCR amplification product was double-digested with NcoⅠ and NotⅠ, and ligated with the pET28a vector double-digested with NcoⅠ and NotⅠ. The ligation product was transformed into E.coli DH5α competent cells, coated with LB plates containing kanamycin, and inverted and cultured at 37℃ overnight. Positive clones were selected for PCR screening, and the positive recombinants were sequenced. After confirming that the sequence was correct, the recombinant plasmid was extracted and transformed into E.coli Tuner (DE3) competent cells for expression.

(3) Expression, renaturation and purification of fusion protein: The expression plasmids were transformed into E. coli Tuner (DE3) competent cells, coated with LB plates (kanamycin 50 μg/mL) and cultured at 37°C overnight. The next day, the clones were selected and inoculated into 10 mL LB liquid culture medium (kanamycin 50 μg/mL) and cultured for 2-3 h. They were inoculated into 1 L LB culture medium at a volume ratio of 1:100 and continued to be cultured until OD ₆₀₀ was 0.5-0.8. IPTG was added at a final concentration of 1 mM to induce the expression of the target protein. After 4 h of induction, the cells were harvested by centrifugation at 6000 rpm for 10 min. The cells were washed once with PBS buffer and aliquoted. The cells equivalent to 200 mL of bacterial culture were taken and lysed with 5 mL BugBuster Master Mix (Merck). The inclusion bodies were collected by centrifugation at 6000 g for 15 min. Then, four detergent washes were performed to remove cell debris and membrane components. Then, the inclusion bodies were washed with a buffer such as PBS to remove detergents and salts. Finally, the inclusion bodies were dissolved with a buffer solution containing 6 M guanidine hydrochloride, 10 mM dithiothreitol (DTT), 10 mM ethylenediaminetetraacetic acid (EDTA), and 20 mM Tris pH 8.1, and the inclusion body concentration was measured. The inclusion bodies were aliquoted and stored in a -80°C freezer.

The dissolved TCR α chain and anti-CD3 (scFv) -β chain were quickly mixed in a solution containing 5 M urea, 0.4 M L-arginine, 20 mM Tris pH 8.1, 3.7 mM cystamine, and 6.6 mM β-mercapoethylamine (4°C) at a mass ratio of 2:5. The final concentrations of the 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 volumes of deionized water (4°C), and after 12 hours, the deionized water was replaced with a buffer solution (10mM Tris, pH 8.0) and continued to be dialyzed at 4°C for 12 hours. After the dialysis was completed, the solution was filtered through a 0.45μm filter membrane and purified by an anion exchange column (HiTrap Q HP 5ml, GE healthcare). The elution peak contained TCRs with successfully renatured TCRα chains and anti-CD3 (scFv)-β chain dimers, which were confirmed by SDS-PAGE gel. The TCR fusion molecule was then further purified by size exclusion chromatography (S-100 16/60, GE healthcare) and purified again by an anion exchange column (HiTrap Q HP 5ml, GE healthcare). The purity of the purified TCR fusion molecule was greater than 90% by SDS-PAGE, and the concentration was determined by the BCA method.

Example 8 Activation function experiment of IFN-γ spot number release by effector cells transfected with high affinity TCR of the present invention for tumor cell lines

This example uses a tumor cell line to verify the activation function and specificity of the effector cells transfected with the high-affinity TCR of the present invention. The detection is also performed by the ELISPOT experiment well known to those skilled in the art. The high-affinity TCR of the present invention is transfected into CD3 ⁺ T cells isolated from the blood of healthy volunteers as effector cells, and CD3 ⁺ T cells transfected with other TCRs (A6) or not transfected with TCRs (NC) from the same volunteer are used as controls. The tumor cell lines used in the examples are SK-MEL28, HUCC-T1, SNU423, and Caki-2, respectively. Among them, SK-MEL28, HUCC-T1, SNU423, and Caki-2 were all purchased from Guangzhou Saiku Biotechnology Co., Ltd. The experiment was performed as follows:

The high affinity TCRs are TCR1, TCR2, TCR3, TCR4, and TCR5. The KRAS G12D positive tumor cell lines used are SK-MEL-28-KRAS G12D (KRAS G12D overexpression) and HUCC-T1-KRAS G12D (KRAS G12D overexpression), and the negative cell lines are SNU423, Caki-2, and effector cells only.

The following steps were performed: First, prepare the ELISPOT plate. The ELISPOT plate was activated and coated with ethanol and incubated at 4°C overnight. On the first day of the experiment, the coating solution was removed, the plate was washed and blocked, and incubated at room temperature for two hours. The blocking solution was removed, and the various components of the test were added to the ELISPOT plate: 2×10 ⁴ target cells/well, 1×10 ³ effector cells/well (calculated according to the positive rate of transfection), and two replicate wells were set. Incubate overnight (37°C, 5% CO ₂ ). On the second day of the experiment, the plate was washed and secondary detection and color development were performed. The plate was dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 company).

Figure 2 shows the activation function experimental results of effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines. The results of Figure 2 show that for KRAS G12D-positive tumor cell lines, effector cells transfected with the high-affinity TCR of the present invention exhibit very obvious activation effects, while effector cells transfected with other TCRs (A6) or untransfected TCRs (NC) are basically inactive; at the same time, effector cells transfected with the high-affinity TCR of the present invention are basically inactive for KRAS G12D-negative cell lines.

Example 9 Activation function experiment of the release of granzyme spots of effector cells transfected with the high affinity TCR of the present invention for tumor cell lines

This example uses tumor cell lines to verify the activation function and specificity of effector cells transfected with the high-affinity TCR of the present invention. The detection is also performed by the ELISPOT experiment familiar to those skilled in the art. The high-affinity TCR of the present invention was transfected into CD3 ⁺ T cells isolated from the blood of healthy volunteers as effector cells, and CD3 ⁺ T cells transfected with other TCRs (A6) from the same volunteer were used as controls. The tumor cell lines used in the examples are SK-MEL28, HUCC-T1, SNU423, Caki-2, and SK-MEL-5, respectively. Among them, SK-MEL28, HUCC-T1, SNU423, and Caki-2 were all purchased from Guangzhou Saiku Biotechnology Co., Ltd., and SK-MEL-5 was purchased from ATCC. The experiment was performed as follows:

The high affinity TCRs are TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, and TCR7. The KRAS G12D positive tumor cell lines used are SK-MEL-28-KRAS G12D (KRAS G12D overexpression), HUCC-T1-KRAS G12D (KRASG12D overexpression), and HUCC-T1, and the negative cell lines are SNU423, Caki-2, SK-MEL-5, SK-MEL-28, and effector cells only.

The following steps were performed: First, prepare the ELISPOT plate. The ELISPOT plate was activated and coated with ethanol and incubated at 4°C overnight. On the first day of the experiment, the coating solution was removed, the plate was washed and blocked, and incubated at room temperature for two hours. The blocking solution was removed, and the various components of the test were added to the ELISPOT plate: 2×10 ⁴ target cells/well, 1×10 ³ effector cells/well (calculated according to the positive rate of transfection), and two replicate wells were set. Incubate overnight (37°C, 5% CO ₂ ). On the second day of the experiment, the plate was washed and secondary detection and color development were performed. The plate was dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 company).

Figure 3 shows the activation function experimental results of effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines. The results of Figure 3 show that for KRAS G12D-positive tumor cell lines, effector cells transfected with the high-affinity TCR of the present invention exhibit very obvious activation effects, while effector cells transfected with other TCRs (A6) are basically inactive; at the same time, effector cells transfected with the high-affinity TCR of the present invention are basically inactive for KRAS G12D-negative cell lines.

Example 10 Killing function experiment of effector cells transfected with high affinity TCR of the present invention against tumor cell lines

Lactate dehydrogenase (LDH) is abundant in the cytoplasm and cannot pass through the cell membrane under normal circumstances. It can be released outside the cell when the cell is damaged or dead. At this time, the LDH activity in the cell culture fluid is proportional to the number of cell deaths. This example also measures the release of LDH by a non-radioactive cytotoxicity experiment well known to those skilled in the art, thereby verifying the killing function of cells transfected with the TCR of the present invention. In this example, the LDH experiment used CD3 ⁺ T cells isolated from the blood of healthy volunteers to transfect the high-affinity TCR of the present invention as effector cells, and CD3 ⁺ T cells transfected with other TCRs (A6) of the same volunteer as a control. The tumor cell lines used in the examples are SK-MEL28 and Caki-2, respectively. Among them, SK-MEL28 and Caki-2 were purchased from Guangzhou Saiku Biotechnology Co., Ltd. The following experiments were performed:

The high affinity TCRs are TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, and TCR7. The KRASG12D positive tumor cell line used is SK-MEL-28-KRAS G12D (KRAS G12D overexpression), and the negative tumor cell lines are SK-MEL28 and Caki-2.

Experimental steps: First, prepare the LDH plate, and add the components of the test to the plate in the following order: 3×10 ⁴ cells/well of target cells, 3×10 ⁴ cells/well of effector cells (calculated according to the transfection positive rate), add to the corresponding wells, and set up three replicate wells. At the same time, set up the effector cell spontaneous well, target cell spontaneous well, target cell maximum well, volume correction control well, and culture medium background control well. Incubate overnight (37°C, 5% CO ₂ ). On the second day of the experiment, detect the color development, and after terminating the reaction, use a microplate reader (Bioteck) to record the absorbance value at 490nm.

Figure 4 is the result of the LDH experiment on the killing function of the effector cells transfected with the high affinity TCR of the present invention against tumor cell lines. The results of Figure 4 show that the effector cells transfected with the high affinity TCR of the present invention have a significant strong killing effect against KRAS G12D positive tumor cell lines, while T cells transfected with other TCRs (A6) basically do not react. At the same time, T cells transfected with the high affinity TCR of the present invention have almost no killing effect on negative tumor cell lines.

Example 11 ELISA activation function experiment of effector cells transfected with high affinity TCR of the present invention for tumor cell lines

In order to verify the activation function and specificity of the effector cells transfected with the high-affinity TCR of the present invention, this example uses a tumor cell line for ELISA experiments. The high-affinity TCR of the present invention was transfected into CD3+T cells isolated from the blood of healthy volunteers as effector cells, and CD3+T cells transfected with other TCRs (A6) from the same volunteer were used as negative controls. The tumor cell lines used in the examples are SK-MEL-28 and Caki-2. Among them, SK-MEL28 and Caki-2 were purchased from Guangzhou Saiku Biotechnology Co., Ltd. The following experiments were performed:

The high affinity TCRs are TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, and TCR7. The KRASG12D positive tumor cell line used is SK-MEL-28-KRAS G12D (KRAS G12D overexpression), and the negative tumor cell lines are SK-MEL28, Caki-2, and effector cells only.

Experimental steps: Inoculate cells on the first day of the experiment: Inoculate the cell suspension of the tumor cell line and the TCR of the present invention into a U-shaped plate: 3×10 ⁴ target cells/well, 9×10 ⁴ effector cells/well (calculated according to the positive rate of transfection), and set three duplicate wells, incubate overnight (37°C, 5% CO ₂ ). IL-2 was coated on the ELISA plate with antibody and placed in a 4°C refrigerator overnight. On the second day of the experiment, remove the coating solution, wash and block, incubate at room temperature for two hours, and remove the blocking solution. Take the co-supernatant of the tumor cell line and the TCR of the present invention and add it to the ELISA plate, take the standard protein, dilute it at a 10-fold ratio, and add it to the ELISA plate, shake it at room temperature for two hours. Wash the plate, add biotin-labeled secondary antibody, shake it at room temperature for one hour, wash and add SA-HRP, shake it at room temperature for one hour, wash, add TMB to develop color for 5-10 minutes, and detect at a wavelength of 450nm after terminating the reaction.

Figure 5 is the result of an ELISA experiment on the killing function of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines. The results of Figure 5 show that the effector cells transfected with the high-affinity TCR of the present invention have a significant activation response against KRAS G12D-positive tumor cell lines, while T cells transfected with other TCRs (A6) basically do not react. At the same time, T cells transfected with the high-affinity TCR of the present invention have almost no killing effect on negative tumor cell lines.

Example 12 Killing function experiment of effector cells transfected with high affinity TCR of the present invention against tumor cell lines (IncuCyte experiment)

This example further verifies the specific killing effect and sensitivity of the effector cells transfected with the high-affinity TCR of the present invention on target cells through the IncuCyte experiment 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 apoptotic cells in real time by real-time microscopic photography in an incubator.

CD3+T cells isolated from the blood of healthy volunteers transfected with the TCR of the present invention were randomly selected as effector cells, and the experimental group in which the same volunteer was transfected with other TCRs (A6) or target cells only (Target cell only) was used as the control group. The high-affinity TCRs were TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, and TCR7. The positive tumor cell line used was SK-MEL-28-KRAS G12D (KRAS G12D overexpression); the negative tumor cell line was SNU423.

On the first day of the experiment, the target cells were digested and centrifuged; resuspended with phenol red-free RPMI1640 + 10% FBS complete medium, and the target cells were evenly spread in a 96-well plate: 2* ¹⁰⁴ cells/well; returned to the incubator at 37 degrees, 5% CO2, and incubated overnight; on the second day, the medium in the 96-well plate was discarded and replaced with phenol red-free RPMI1640 + 10% FBS medium containing the dye caspase3/7reagent, so that the dye concentration was 2 drops/ml. The old culture medium was discarded and replaced with new phenol red-free RPMI1640 + 10% FBS culture medium. The effector cells (1* ¹⁰⁴ cells/well) (calculated according to the positive rate of transfection) were co-incubated with the experimental group with target cells. The plate was placed in the real-time dynamic living cell imaging analyzer dedicated to Incucyte detection - IncuCyte ZooM. After incubation for half an hour, real-time observation and photography were started. The detection results were processed, analyzed and exported using IncuCyte ZooM 2016A.

Figures 6a and 6b are the results of the IncuCyte experiment on the killing function of effector cells transfected with the high-affinity TCR of the present invention against tumor cell lines. The results of Figures 6a and 6b show that for KRAS G12D-positive tumor cell lines, effector cells transfected with the high-affinity TCR of the present invention can have a significant strong killing effect in a short period of time, while T cells transfected with other TCRs basically do not react. At the same time, T cells transfected with the high-affinity TCR of the present invention have almost no killing effect on negative tumor cell lines.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings 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 defined by the claims attached to this application.

The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims (10)

1. A T Cell Receptor (TCR) comprising a TCR a chain variable domain and a TCR β chain variable domain, characterized in that it has activity of binding to VVVGADGVGK-HLA a1101 complex;

And the amino acid sequence of the TCR alpha chain variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO. 1, and the amino acid sequence of the TCR beta chain variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO. 2.

2. A multivalent TCR complex comprising at least two TCRs, wherein at least one of the TCRs is a TCR as claimed in claim 1.

3. A nucleic acid molecule comprising a nucleic acid sequence encoding the TCR of claim 1 or a complement thereof.

4. A carrier, characterized in that, the vector contains the nucleic acid molecule of claim 3.

5. A host cell comprising the vector of claim 4 or the nucleic acid molecule of claim 3 integrated into a chromosome.

6. An isolated cell, wherein the cell expresses the TCR of claim 1, preferably wherein the cell further expresses an exogenous CD8 receptor; more preferably, the CD8 receptor is CD8 a; preferably, the isolated cells are T cells.

7. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR as claimed in claim 1, or a TCR complex as claimed in claim 2, or a combination of any one or at least two of the cells as claimed in claim 6.

8. A method of treating a disease comprising administering to a subject in need thereof a TCR as claimed in claim 1, or a TCR complex as claimed in claim 2, or a cell as claimed in claim 6, or a pharmaceutical composition as claimed in claim 7; preferably, the disease is a KRAS G12D positive tumor, more preferably colorectal, pancreatic or gastric cancer.

9. Use of the T cell receptor of claim 1, the TCR complex of claim 2 or the cell of claim 6 for the manufacture of a medicament for the treatment of a tumour; preferably, the tumor is a KRAS G12D positive tumor, more preferably colorectal, pancreatic or gastric cancer.

10. A method of making the T cell receptor of claim 1, comprising the steps of:

(i) Culturing the cell of claim 5, thereby expressing the T cell receptor of claim 1; and, a step of, in the first embodiment,

(Ii) Isolating or purifying said T cell receptor.