CN117659163A

CN117659163A - High-affinity T cell receptor for identifying AFP and application thereof

Info

Publication number: CN117659163A
Application number: CN202211093954.2A
Authority: CN
Inventors: 战凯; 黄金花; 刘敏; 张翠琼; 翁志明; 陈建君
Original assignee: Xiangxue Life Science Technology Guangdong Co ltd
Current assignee: Xiangxue Life Science Technology Guangdong Co ltd
Priority date: 2022-09-08
Filing date: 2022-09-08
Publication date: 2024-03-08

Abstract

The invention provides a high affinity T cell receptor recognizing AFP and application thereof, wherein the T cell receptor has the characteristic of binding to TSSELMAITR-HLA A A1101 complex. The invention also provides a multivalent T cell receptor complex, nucleic acid molecules encoding such T cell receptors, vectors comprising these nucleic acids, cells expressing such T cell receptors, and pharmaceutical compositions comprising the foregoing, which are useful for diagnosing, treating, and preventing AFP-positive diseases; the invention also provides a preparation method of the T cell receptor.

Description

High-affinity T cell receptor for identifying AFP and application thereof

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a high-affinity T cell receptor (T cell receptor, TCR) capable of recognizing AFP and application thereof, and more particularly relates to a T Cell Receptor (TCR) capable of recognizing polypeptide derived from AFP protein; the invention also relates to a preparation method and application of the T cell receptor.

Background

Only two types of molecules are able to recognize antigens in a specific manner. One of which is an immunoglobulin or antibody; the other is the T Cell Receptor (TCR), which is a glycoprotein on the surface of the cell membrane that exists as a heterodimer from the alpha/beta or gamma/delta chain. The composition of the TCR profile of the immune system is generated by V (D) J recombination in the thymus, followed by positive and negative selection. In the peripheral environment, TCRs mediate specific recognition of the major histocompatibility complex-peptide complex (pMHC) by T cells, and are therefore critical for cellular immune function of the immune system.

TCRs are the only receptors for specific antigenic peptides presented on the Major Histocompatibility Complex (MHC), and such foreign or endogenous peptides may be the only sign of abnormalities in cells. In the immune system, direct physical contact of T cells with Antigen Presenting Cells (APCs) is initiated by binding of antigen-specific TCRs to pMHC complexes, and then interaction of T cells with other cell membrane surface molecules of both APCs occurs, which causes a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells of different antigen specificities to exert immune effects on their target cells.

The MHC class I and class II molecular ligands corresponding to TCRs are also proteins of the immunoglobulin superfamily, but are specific for antigen presentation and different individuals have different MHC's, thereby being able to present different short peptides of one protein antigen to the respective APC cell surfaces. Human MHC is commonly referred to as an HLA gene or HLA complex.

AFP (alpha Fetoprotein), also known as alpha Fetoprotein, is a protein expressed during embryo development and is the main component of embryo serum. During development, AFP has relatively high expression levels in the yolk sac and liver, which are subsequently inhibited. In hepatocellular carcinoma, AFP expression is activated (Butterfield et al J immunol.,2001, apr 15;166 (8): 5300-8). AFP is degraded into small molecule polypeptides after intracellular production and is presented on the cell surface in association with MHC (major histocompatibility complex) molecules to form complexes. TSSELMAITR is a short peptide derived from an AFP antigen, a target for the treatment of AFP-related diseases.

Thus, the TSSELMAITR-HLA A1101 complex provides a marker for targeting tumor cells by TCR. The TCR capable of combining with the TSSELMAITR-HLA A1101 complex has high application value for treating tumors. For example, TCRs capable of targeting the tumor cell markers can be used to deliver cytotoxic or immunostimulatory agents to target cells, or be transformed into T cells, enabling T cells expressing the TCRs to destroy tumor cells for administration to a patient during a treatment process known as adoptive immunotherapy. For the former purpose, an ideal TCR is one that has a higher affinity, enabling the TCR to reside on the targeted cells for a long period of time. For the latter purpose, then, a TCR of moderate affinity is preferably used. Accordingly, those skilled in the art are working to develop TCRs that can be used to target tumor cell markers for different purposes.

Disclosure of Invention

In view of the shortcomings of the prior art, the present invention aims to provide a TCR with higher affinity for the TSSELMAITR-HLA a1101 complex. It is a further object of the present invention to provide a method of preparing a TCR of the type described above and the use of a TCR of the type described above.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

In a first aspect of the invention there is provided a T Cell Receptor (TCR) comprising an alpha chain variable domain and a beta chain variable domain, the T cell receptor having activity to bind to the TSSELMAITR-HLA a1101 complex;

and the amino acid sequence of the TCR a chain variable domain has at least 90% (e.g., may be 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, and the amino acid sequence of the TCR β chain variable domain has at least 90% (e.g., may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, etc.) sequence homology with the amino acid sequence shown in SEQ ID No. 2.

In a preferred embodiment, the amino acid sequence of the TCR α chain variable domain and the amino acid sequence of the TCR β chain variable domain are not identical to the amino acid sequence of the wild-type TCR α chain variable domain and the amino acid sequence of the wild-type TCR β chain variable domain.

In a further preferred embodiment, the amino acid sequence of the TCR α chain variable domain is not the amino acid sequence shown in SEQ ID No. 1, and/or the amino acid sequence of the TCR β chain variable domain is not the amino acid sequence shown in SEQ ID No. 2.

In another preferred embodiment, the α -chain variable domain of the TCR comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the sequence set forth in SEQ ID No. 1, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid residue insertions, deletions, substitutions or combinations thereof relative to the sequence set forth in SEQ ID No. 1.

In another preferred embodiment, the β -chain variable domain of the TCR is an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the sequence set forth in SEQ ID No. 2, or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid residue insertions, deletions, substitutions or combinations thereof relative to the sequence set forth in SEQ ID No. 2.

In another preferred embodiment, the amino acid sequence of the TCR alpha chain variable domain has at least 95% and not 100% sequence homology with the amino acid sequence set forth in SEQ ID NO. 1, and the amino acid sequence of the TCR beta chain variable domain has at least 95% and not 100% sequence homology with the amino acid sequence set forth in SEQ ID NO. 2.

In another preferred embodiment, the 3 CDRs of the tcra chain variable domain are:

CDR1α is: nsass qs;

CDR2α is: VYSSGN;

CDR3 a is: VVNPGSGNTGKLI.

In another preferred embodiment, the amino acid sequence of the TCR alpha chain variable domain is SEQ ID NO. 1.

In another preferred embodiment, the TCR is mutated in CDR3 β of the TCR β chain variable domain, and the number of mutations is 2-4 (e.g. may be 2, 3 or 4), preferably 3; more preferably, the mutation occurs at positions 9, 11 and 12 of CDR3β.

In another preferred embodiment, the reference sequence of the 3 CDR regions (complementarity determining regions) of the TCR alpha chain variable domain is as follows,

CDR1α：NSASQS

CDR2α：VYSSGN

CDR3 a: VVNPGSGNTGKLI, and CDR3α contains at least one mutation as shown in table 1:

TABLE 1

In another preferred embodiment, the reference sequence of the 3 CDR regions (complementarity determining regions) of the TCR β chain variable domain is as follows,

CDR1β：SGHTA

CDR2β：FQGNSA

CDR3 β: ASSLVFGSVWDTQY, and CDR3β contains at least one mutation as shown in table 2:

TABLE 2

Residues before mutation	Residues after mutation
		CDR3 beta position 9V	I
11 th D of CDR3 beta	A or S
		T at position 12 of CDR3 beta	E or Q
Position 14Y of CDR3 beta	F or H

In another preferred embodiment, the mutations are as shown in table 3:

TABLE 3 Table 3

Residues before mutation	Residues after mutation
		11 th D of CDR3 beta	A

In another preferred embodiment, the mutations are as shown in table 4:

TABLE 4 Table 4

Residues before mutation	Residues after mutation
		CDR3 beta position 9V	I
11 th D of CDR3 beta	A
		T at position 12 of CDR3 beta	E

In another preferred embodiment, the affinity of the TCR with the TSSELMAITR-HLA a1101 complex is at least 5 fold that of a wild-type TCR.

In another preferred embodiment, the TCR is mutated in the variable domain of the alpha chain as shown in SEQ ID NO. 1, said mutation being selected from one or more of the groups N91M/Y, P92R, G A/P/S, S94Q/R/G, G95V/R, N96M/P, T R/N and G98M/F, wherein the amino acid residue numbering is as shown in SEQ ID NO. 1.

In another preferred embodiment, the TCR is mutated in the β chain variable domain as set forth in SEQ ID NO. 2, said mutation being selected from one or more of the groups V101I, D103A/S, T104E/Q and Y106F/H, wherein the amino acid residue numbering is as set forth in SEQ ID NO. 2.

In another preferred embodiment, the TCR has CDRs selected from those shown in table 5:

TABLE 5

In another preferred embodiment, the TCR is soluble.

In another preferred embodiment, the TCR is an αβ heterodimeric TCR comprising 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) all or part of a TCR α chain other than its transmembrane domain, and (ii) all or part of a TCR β chain other than its transmembrane domain, wherein (i) and (ii) each comprise a variable domain and at least part of a constant domain of the TCR chain.

In another preferred embodiment, the TCR has an artificial interchain disulfide linkage between the α -chain constant region and the β -chain constant region.

In another preferred embodiment, the cysteine residues forming the artificial interchain disulfide bond between the constant regions of the TCR α and β chains are substituted at one or more sets of sites selected from the group consisting of:

thr48 of tranc x 01 exon 1 and Ser57 of TRBC1 x 01 or TRBC2 x 01 exon 1;

thr45 of tranc x 01 exon 1 and Ser77 of TRBC1 x 01 or TRBC2 x 01 exon 1;

tyr10 of TRAC x 01 exon 1 and Ser17 of TRBC1 x 01 or TRBC2 x 01 exon 1;

thr45 of TRAC x 01 exon 1 and Asp59 of TRBC1 x 01 or TRBC2 x 01 exon 1;

ser15 of TRAC x 01 exon 1 and Glu15 of TRBC1 x 01 or TRBC2 x 01 exon 1;

arg53 of TRAC x 01 exon 1 and Ser54 of TRBC1 x 01 or TRBC2 x 01 exon 1;

TRAC.01 exon 1 Pro89 and TRBC 1.01 or TRBC 2.01 exon 1 Ala19;

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

In another preferred embodiment, the alpha chain variable domain amino acid sequence of the TCR is selected from one of SEQ ID NO. 1, SEQ ID NO. 16-22; and/or the β chain variable domain amino acid sequence of the TCR is selected from one of SEQ ID NOs 2, 13-15, 23-25.

In another preferred embodiment, the TCR is selected from the group consisting of:

(1) The alpha chain variable domain sequence is SEQ ID NO. 1, and the beta chain variable domain sequence is SEQ ID NO. 13;

(2) The alpha chain variable domain sequence is SEQ ID NO. 1, and the beta chain variable domain sequence is SEQ ID NO. 14;

(3) The alpha chain variable domain sequence is SEQ ID NO. 1, and the beta chain variable domain sequence is SEQ ID NO. 15;

(4) The alpha chain variable domain sequence is SEQ ID NO. 16, and the beta chain variable domain sequence is SEQ ID NO. 2;

(5) The alpha chain variable domain sequence is SEQ ID NO. 17, and the beta chain variable domain sequence is SEQ ID NO. 2;

(6) The alpha chain variable domain sequence is SEQ ID NO. 18, and the beta chain variable domain sequence is SEQ ID NO. 2;

(7) The alpha chain variable domain sequence is SEQ ID NO. 19, and the beta chain variable domain sequence is SEQ ID NO. 2;

(8) The alpha chain variable domain sequence is SEQ ID NO. 20, and the beta chain variable domain sequence is SEQ ID NO. 2;

(9) The alpha chain variable domain sequence is SEQ ID NO. 21, and the beta chain variable domain sequence is SEQ ID NO. 2;

(10) The alpha chain variable domain sequence is SEQ ID NO. 22, and the beta chain variable domain sequence is SEQ ID NO. 2;

(11) The alpha chain variable domain sequence is SEQ ID NO. 1, and the beta chain variable domain sequence is SEQ ID NO. 23;

(12) The alpha chain variable domain sequence is SEQ ID NO. 1, and the beta chain variable domain sequence is SEQ ID NO. 24;

(13) The alpha chain variable domain sequence is SEQ ID NO. 1 and the beta chain variable domain sequence is SEQ ID NO. 25.

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

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

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

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

In another preferred embodiment, the TCR comprises an alpha chain constant region and a beta chain constant region, the alpha chain constant region being a murine constant region and/or the beta chain constant region being a murine constant region.

In another preferred embodiment, the C-or N-terminus of the alpha and/or beta chain of the TCR is conjugated to a conjugate.

Preferably, the conjugate comprises any one or a combination of at least two of a detectable label, a therapeutic agent, or a PK modifying moiety.

In another preferred embodiment, the therapeutic agent that binds to the TCR is an anti-CD 3 antibody that is linked to the C-or N-terminus of the alpha or beta chain of the TCR.

In a second aspect of the invention there is provided a multivalent T cell receptor complex comprising at least two T cell receptor molecules, and wherein at least one T cell receptor molecule is a T cell receptor according to the first aspect of the invention.

In a third aspect of the invention there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a T cell receptor according to the first aspect of the invention or a multivalent T cell receptor complex according to the second aspect of the invention, or a complement of a nucleotide sequence encoding a T cell receptor according to the first aspect of the invention or a multivalent T cell receptor complex according to the second aspect of the invention.

In a fourth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the third aspect of the invention.

In a fifth aspect of the invention there is provided a host cell comprising a vector according to the fourth aspect of the invention or having incorporated into its chromosome an exogenous nucleic acid molecule according to the third aspect of the invention.

In a sixth aspect of the invention there is provided an isolated cell expressing a T cell receptor according to the first aspect of the invention.

Preferably, the isolated cells comprise T cells, NK cells or NKT cells.

Most preferably, the isolated cells are T cells.

In another preferred embodiment, the isolated cell expresses the T cell receptor of the first aspect of the invention and also expresses an exogenous CD8 receptor.

Preferably, the CD8 receptor is CD8 a.

In a seventh aspect of the invention there is provided a pharmaceutical composition comprising any one or a combination of at least two of the T cell receptor of the first aspect of the invention, the multivalent T cell receptor complex of the second aspect of the invention or the isolated cell of the sixth aspect of the invention.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In an eighth aspect of the invention, there is provided a method of treating a disease comprising administering to a subject in need of treatment an appropriate amount of any one or a combination of at least two of the T cell receptor of the first aspect of the invention, the multivalent T cell receptor complex of the second aspect of the invention, the isolated cell of the sixth aspect of the invention or the pharmaceutical composition of the seventh aspect of the invention.

Preferably, the disease is an AFP-positive tumor.

More preferably, the tumor is liver cancer.

In a ninth aspect of the invention there is provided the use of a T cell receptor according to the first aspect of the invention, a multivalent T cell receptor complex according to the second aspect of the invention or an isolated cell according to the sixth aspect of the invention, the use comprising the manufacture of a medicament for the treatment of a tumour.

Preferably, the disease is an AFP-positive tumor.

More preferably, the tumor is liver cancer.

In a tenth aspect of the present invention, there is provided a method of preparing a T cell receptor according to the first aspect of the present invention, the method comprising the steps of:

(i) Culturing the host cell of the fifth aspect of the invention so as to express the T cell receptor of the first aspect of the invention;

(ii) Isolating or purifying the T cell receptor.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.

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

(1) The affinity and/or binding half-life of the high affinity TCRs of the invention for the TSSELMAITR-HLA-A1101 complex is at least 5-fold that of the wild-type TCR.

(2) The high affinity TCRs of the invention are capable of specifically binding to the TSSELMAITR-HLA a1101 cell, while cells transfected with the high affinity TCRs of the invention are capable of being specifically activated.

(3) Effector cells transfected with the high affinity TCRs of the present invention have strong specific killing effects.

Drawings

FIG. 1 is a graph showing the binding curves of a soluble reference TCR, i.e., a wild-type TCR, to a TSSELMAITR-HLA A A1101 complex.

FIG. 2 shows the results of an experiment on the activation function of effector cells transfected with the high affinity TCR of the invention against T2 cells loaded with a short peptide.

FIG. 3 shows the results of an experiment of the activation function of effector cells transfected with the high affinity TCR of the invention against a tumor cell line.

FIG. 4 shows the results of an LDH assay for killing function of effector cells transfected with high affinity TCRs of the invention against a tumor cell line.

Detailed Description

The present invention, through extensive and intensive studies, has resulted in a high affinity T Cell Receptor (TCR) which recognizes the TSSELMAITR short peptide (derived from AFP protein), which is presented in the form of a peptide-HLA a1101 complex. The high affinity TCR has 3 CDR regions in its alpha chain variable domain:

CDR1α：NSASQS SEQ ID NO:30

CDR2α：VYSSGN SEQ ID NO:31

CDR3 a: VVNPGSGNTGKLI SEQ ID mutation in NO. 32;

and/or 3 CDR regions in its β chain variable domain:

CDR1β：SGHTA SEQ ID NO:33

CDR2β：FQGNSA SEQ ID NO:34

CDR3 β: a mutation in ASSLVFGSVWDTQY SEQ ID NO: 35;

furthermore, the affinity and/or binding half-life of the inventive TCRs for the TSSELMAITR-HLA a1101 complex described above after mutation is at least 5-fold higher than that of wild-type TCRs.

Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the 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 described herein.

Terminology

T cell receptor (T cell receptor, TCR)

TCRs can be described using the international immunogenetic information system (IMGT). The native αβ heterodimeric TCR has an α chain and a β chain. In a broad sense, each chain comprises a variable region, a linking region and a constant region, and the β chain also typically contains a short polytropic region between the variable region and the linking region, but this polytropic region is often considered part of the linking region. The junction region of the TCR is determined by the unique TRAJ and TRBJ of IMGT, and the constant region of the TCR is determined by the TRAC and TRBC of IMGT.

Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are chimeric in framework sequences. In IMGT nomenclature, different numbers for TRAV and TRBV refer to different vα and vβ types, respectively. In IMGT systems, the α -chain constant domain has the following sign: TRAC x 01, wherein "TR" represents a T cell receptor gene; "A" represents an alpha chain gene; c represents a constant region; ".01" indicates allele 1. The β -strand constant domain has the following sign: TRBC1 x 01 or TRBC2 x 01, wherein "TR" represents a T cell receptor gene; "B" represents a beta-chain gene; c represents a constant region; ".01" indicates allele 1. The constant region of the alpha chain is uniquely defined, and in the form of the beta chain, there are two possible constant region genes "C1" and "C2". The constant region gene sequences for TCR alpha and beta chains can be obtained by those skilled in the art from the IMGT database disclosed.

The α and β chains of TCRs are generally considered to have two "domains" each, i.e., a variable domain and a constant domain. The variable domain is composed of a linked variable region and a linked region. Thus, in the description and claims of this application, a "TCR α chain variable domain" refers to the linked TRAV and TRAJ regions, and likewise, a "TCR β chain variable domain" refers to the linked TRBV and TRBD/TRBJ regions. The 3 CDRs of the TCR α chain variable domain are CDR1 α, CDR2 α and CDR3 α, respectively; the 3 CDRs of the TCR β chain variable domain are CDR1 β, CDR2 β and CDR3 β, respectively. The framework sequences of the TCR variable domains of the invention may be of murine or human origin, preferably of human origin. The constant domain of the TCR comprises an intracellular portion, a transmembrane region, and an extracellular portion.

In the present invention, the amino acid sequences of the alpha and beta chain variable domains of the wild type TCR capable of binding to the TSSELMAITR-HLA A A1101 complex are SEQ ID NO 1 and SEQ ID NO 2, respectively. The alpha chain amino acid sequence and the beta chain amino acid sequence of the soluble reference TCR are SEQ ID NO. 11 and SEQ ID NO. 12 respectively. The alpha chain extracellular amino acid sequence and the beta chain extracellular amino acid sequence of the wild TCR are SEQ ID NO. 26 and SEQ ID NO. 27 respectively. The TCR sequences used in the present invention are of human origin. The alpha chain amino acid sequence and the beta chain amino acid sequence of the wild-type TCR are SEQ ID NO. 28 and SEQ ID NO. 29 respectively. In the present invention, the terms "polypeptide of the invention", "TCR of the invention", "T cell receptor of the invention" are used interchangeably.

Natural inter-chain disulfide bonds and artificial inter-chain disulfide bonds

A set of disulfide bonds exist between the near membrane regions cα and cβ of a native TCR, referred to herein as "native interchain disulfide bonds". In the present invention, an inter-chain covalent disulfide bond, which is artificially introduced at a position different from that of a natural inter-chain disulfide bond, is referred to as an "artificial inter-chain disulfide bond".

For convenience of description, TRAC 01 and TRBC1 x 01 or TRBC2 x 01 amino acid sequence are numbered sequentially from N-terminal to C-terminal, for example, TRBC1 x 01 or TRBC2 x 01, and in order from N-terminal to C-terminal, the 60 th amino acid is P (proline), which may be described as TRBC1 x 01 or TRBC2 x 01 exon 1 Pro60, as TRBC1 x 01 or TRBC2 x 01 exon 1 60 th amino acid, as TRBC1 x 01 or TRBC2 x 01, and in order from N-terminal to C-terminal, the 61 th amino acid is Q (glutamine), which may be described as TRBC1 x 01 or TRBC2 x 01 exon 1 Gln61, and bc1 x 01 or TRBC2 x 01 exon 1 may be expressed as TRBC1 x 61, and so on. In the present invention, the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are according to the position numbers listed in IMGT. If an amino acid in TRAV is numbered 46 in IMGT, it is described in the present invention as TRAV amino acid 46, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described, and are specifically described.

Tumor(s)

The term "tumor" is meant to include all types of cancerous cell growth or oncogenic processes, metastatic tissue or malignant transformed cells, tissues or organs, regardless of the type of pathology or stage of infection. Examples of tumors include, without limitation: solid tumors, soft tissue tumors, and metastatic lesions. T cells transduced with the TCR of the invention can be used to treat diseases associated with target cells presenting the AFP antigen short peptide TSSELMAITR-HLA A A1101 complex, including but not limited to tumors, such as liver cancer.

Detailed Description

It is well known that the α -chain variable domain and β -chain variable domain of TCRs each contain 3 CDRs, similar to the complementarity determining regions of antibodies. CDR3 interacts with antigen oligopeptide and CDR1 and CDR2 interact with HLA. Thus, the CDRs of a TCR molecule determine its interaction with the antigen oligopeptide-HLA complex. The alpha chain variable domain amino acid sequence and the beta chain variable domain amino acid sequence of a wild-type TCR capable of binding to the antigen oligopeptide TSSELMAITR and HLA a1101 complex (i.e. TSSELMAITR-HLA a1101 complex) are SEQ ID No. 1 and SEQ ID No. 2, respectively, which sequences were first discovered by the inventors.

It has the following CDR regions:

alpha chain variable domain CDR:

CDR1α：NSASQS SEQ ID NO:30

CDR2α：VYSSGN SEQ ID NO:31

CDR3α：VVNPGSGNTGKLI SEQ ID NO:32

and a β chain variable domain CDR:

CDR1β：SGHTA SEQ ID NO:33

CDR2β：FQGNSA SEQ ID NO:34

CDR3β：ASSLVFGSVWDTQY SEQ ID NO:35

the invention obtains the high-affinity TCR with at least 5 times higher affinity with the TSSELMAITR-HLA A1101 complex than the wild-type TCR with TSSELMAITR-HLA A1101 complex through mutation screening of the CDR regions.

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

The 40 th position of SEQ ID NO. 1 is free C, and the person skilled in the art knows that the free C can generate mismatch to influence the protein renaturation effect, and the free C can be mutated into other amino acids, such as S, for better TCR protein renaturation. The sequence after affinity optimization is shown as SEQ ID NO. 16-22, and the mutation can be performed.

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

In the invention, 3 CDRs of the wild type TCR alpha chain variable domain SEQ ID NO:1, namely CDR1, CDR2 and CDR3, are respectively positioned at 27 th to 32 th positions, 50 th to 55 th positions and 89 th to 101 th positions of the SEQ ID NO: 1. Accordingly, the amino acid residue numbers are represented by SEQ ID NO. 1, 91N is the 3 rd N and 92P of CDR3 alpha is the 4 th P of CDR3 alpha, 93G is the 5 th G of CDR3 alpha, 94S is the 6 th S of CDR3 alpha, 95G is the 7 th G of CDR3 alpha, 96N is the 8 th N and 97T of CDR3 alpha is the 9 th T and 98G of CDR3 alpha.

In particular, specific forms of the mutation in the alpha chain variable domain include one or more of the groups N91M/Y, P92R, G A/P/S, S94Q/R/G, G95V/R, N96M/P, T97R/N, G98M/F.

In the present invention, the 3 CDRs of the wild type TCR.beta.chain variable domain SEQ ID NO. 2, namely CDR1, CDR2 and CDR3, are located at positions 27-31, 49-54 and 93-106 of SEQ ID NO. 2, respectively. Accordingly, the amino acid residue numbers are represented by SEQ ID NO. 2, 101V is the 9 th V of CDR3 beta, 103D is the 11 th D of CDR3 beta, 104T is the 12 th T of CDR3 beta, and 106Y is the 14 th Y of CDR3 beta.

In particular, specific forms of the mutation in the β chain variable domain include one or more of the groups of V101I, D A/S, T104E/Q, Y106F/H

It should be understood that, in this document, the amino acid names are identified by international single english letters, and the amino acid names corresponding to the amino acid names are abbreviated as "three english letters: ala (A), arg (R), asn (N), asp (D), cys (C), gln (Q), glu (E), gly (G), his (H), ile (I), leu (L), lys (K), met (M), phe (F), pro (P), ser (S), thr (T), trp (W), tyr (Y), val (V);

in the present invention, pro60 or 60P represents proline at position 60. In addition, the expression of the specific form of the mutation in the present invention, such as "N91M/Y" means that N at position 91 is substituted with M or Y, and so on.

According to the site-directed mutagenesis method well known to those skilled in the art, thr48 of wild-type TCR alpha chain constant region tran 01 exon 1 is mutated to cysteine, ser57 of beta chain constant region TRBC1 x 01 or TRBC2 x 01 exon 1 is mutated to cysteine, thus obtaining the reference TCR, the amino acid sequences of the soluble reference TCR alpha chain and the beta chain are SEQ ID No. 11 and SEQ ID No. 12, respectively, and the mutated cysteine residues are indicated in bold letters. Such cysteine substitutions enable the formation of artificial interchain disulfide bonds between the constant regions of the α and β chains of the reference TCR to form a more stable soluble TCR, thereby enabling more convenient assessment of the binding affinity and/or binding half-life between the TCR and the TSSELMAITR-HLA a1101 complex. It will be appreciated that the CDR regions of the TCR variable region determine their affinity to the pMHC complex and, therefore, the above-described cysteine substitutions of the TCR constant region do not have an effect on the binding affinity and/or binding half-life of the TCR. Thus, in the present invention, the binding affinity between the reference TCR and the TSSELMAITR-HLA A A1101 complex measured is considered to be the binding affinity between the wild-type TCR and the TSSELMAITR-HLA A A1101 complex. Likewise, if the binding affinity between the inventive TCR and the TSSELMAITR-HLA a1101 complex is measured to be at least 10 times greater than the binding affinity between the reference TCR and the TSSELMAITR-HLA a1101 complex, i.e. equivalent to at least 10 times greater than the binding affinity between the inventive TCR and the TSSELMAITR-HLA a1101 complex than between the wild-type TCR and the TSSELMAITR-HLA a1101 complex.

Binding affinity can be determined by any suitable method (equilibrium constant K with dissociation _D Inversely proportional) and binding half-life (expressed as T _1/2 ) Such as detection using surface plasmon resonance techniques. It will be appreciated that doubling the affinity of the TCR will result in K _D Halving. T (T) _1/2 Calculated as In2 divided by dissociation rate (K _off ). Thus T _1/2 Doubling can lead to K _off Halving. The binding affinity or binding half-life of a given TCR is preferably measured several times, e.g. 3 times or more, using the same assay protocol, and the results averaged. In a preferred embodiment, the affinity of the soluble TCR is detected using the surface plasmon resonance (BIAcore) method in the examples herein, provided that: the temperature is 25 ℃, and the PH value is 7.1-7.5. The method detects dissociation equilibrium constant K of the reference TCR pair TSSELMAITR-HLA A A1101 complex _D The dissociation equilibrium constant K of the wild-type TCR pair TSSELMAITR-HLA A A1101 complex is considered to be 2.891E-04M, namely 289.1. Mu.M _D Also 289.1. Mu.M. Since the affinity of TCR doubles, this will lead to K _D Halving, so if the dissociation equilibrium constant K of the high affinity TCR pair TSSELMAITR-HLA A A1101 complex is detected _D For 2.891E-05M, 28.91. Mu.M, this indicates that the affinity of the high affinity TCR for the TSSELMAITR-HLA A A1101 complex is 10 times greater than the affinity of the wild-type TCR for the TSSELMAITR-HLA A A1101 complex. K is well known to those skilled in the art _D Conversion relation between units of value, i.e. 1m=10 ⁶ μΜ,1 μΜ=1000 nm,1 nm=1000 pM. In the present invention, the affinity of the TCR with the TSSELMAITR-HLA a1101 complex is at least 5 fold that of the wild-type TCR.

Mutations may be made using any suitable method, 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 detail these methods. For more details on Polymerase Chain Reaction (PCR) mutagenesis and cloning in accordance with restriction enzymes see Sambrook and Russell, (2001) molecular cloning-laboratory Manual (Molecular Cloning-A Laboratory Manual) (third edition) CSHL Press. More information on LIC methods can be found (Rashtchian, (1995) Curr Opin Biotechnol (1): 30-6).

Methods of producing TCRs of the invention may be, but are not limited to, screening a diverse library of phage particles displaying such TCRs for TCRs having high affinity for the TSSELMAITR-HLA-A1101 complex, as described in the document (Li, et al (2005) Nature Biotech 23 (3): 349-354).

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

The high affinity TCR of the invention comprises an alpha chain variable domain amino acid sequence selected from one of SEQ ID NO. 1, SEQ ID NO. 16-22; and/or the β chain variable domain amino acid sequence of the TCR is selected from one of SEQ ID NOs 2, 13-15, 23-25. The amino acid sequences of the α chain variable domain and the β chain variable domain of the heterodimeric TCR molecules of the invention are preferably selected from table 6 below:

TABLE 6

For the purposes of the present invention, a TCR of the present invention is a portion having at least one TCR a and/or TCR β chain variable domain. They typically comprise both a TCR a chain variable domain and a TCR β chain variable domain. They may be αβ heterodimers or in single chain form or any other form that is stable. In adoptive immunotherapy, the full-length chain of the αβ heterodimeric TCR (comprising cytoplasmic and transmembrane domains) can be transfected. The TCRs of the invention may be used as targeting agents for delivery of therapeutic agents to antigen presenting cells or in combination with other molecules to produce bifunctional polypeptides to target effector cells, where the TCRs are preferably in soluble form.

For stability, the introduction of artificial interchain disulfide bonds between the α and β chain constant domains of TCRs has been disclosed in the prior art to enable soluble and stable TCR molecules to be obtained, as described in PCT/CN 2015/093806. Thus, the TCRs of the present invention may be TCRs that incorporate artificial interchain disulfide bonds between residues of their alpha and beta chain constant domains. Cysteine residues form artificial interchain disulfide bonds between the α and β chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at suitable sites in the native TCR to form artificial interchain disulfide bonds. For example, thr48 substituted for trac×01 exon 1 and Ser57 substituted for TRBC1×01 or TRBC2×01 exon 1 form disulfide bonds. Other sites for introducing cysteine residues to form disulfide bonds may also be:

Thr45 of tranc x 01 exon 1 and Ser77 of TRBC1 x 01 or TRBC2 x 01 exon 1;

tyr10 of TRAC x 01 exon 1 and Ser17 of TRBC1 x 01 or TRBC2 x 01 exon 1;

thr45 of TRAC x 01 exon 1 and Asp59 of TRBC1 x 01 or TRBC2 x 01 exon 1;

ser15 of TRAC x 01 exon 1 and Glu15 of TRBC1 x 01 or TRBC2 x 01 exon 1;

arg53 of TRAC x 01 exon 1 and Ser54 of TRBC1 x 01 or TRBC2 x 01 exon 1;

TRAC.01 exon 1 Pro89 and TRBC 1.01 or TRBC 2.01 exon 1 Ala19;

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

I.e., a cysteine residue replaces any of the set of sites in the constant domains of the alpha and beta chains described above. The deletion of the native interchain disulfide bond may be achieved by truncating up to 15, or up to 10, or up to 8 or less amino acids at one or more of the C-termini of the TCR constant domains of the present invention so that they do not include a cysteine residue, or by mutating the cysteine residue forming the native interchain disulfide bond to another amino acid.

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

In addition, for stability, patent document PCT/CN2016/077680 also discloses that the introduction of artificial inter-chain disulfide bonds between the α chain variable region and the β chain constant region of a TCR can provide a significant improvement in TCR stability. Thus, the high affinity TCRs of the present invention may also contain artificial interchain disulfide bonds between the α chain variable and β chain constant regions. Specifically, the cysteine residues that form the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR are substituted:

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

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

amino acid 46 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01;

or amino acid 47 of TRAV and amino acid 60 of TRBC1 x 01 or TRBC2 x 01 exon 1.

Preferably, such TCRs may comprise (i) all or part of the TCR a chain, except for its transmembrane domain, and (ii) all or part of the TCR β chain, except for its transmembrane domain, wherein (i) and (ii) each comprise a variable domain and at least part of a constant domain of the TCR chain, the a chain forming a heterodimer with the β chain.

More preferably, such TCRs may comprise an alpha chain variable domain and a beta chain variable domain and all or part of a beta chain constant domain other than the transmembrane domain, but they do not comprise an alpha chain constant domain, the alpha chain variable domain of the TCR forming a heterodimer with the beta chain.

For stability, on the other hand, the TCRs of the present invention also include TCRs having mutations in their hydrophobic core regions, preferably mutations that result in improved stability of the TCRs of the present invention, as described in the patent publication No. WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (α and/or β chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or the α chain J gene (TRAJ) short peptide amino acid position reciprocal 3,5,7, and/or the β chain J gene (TRBJ) short peptide amino acid position reciprocal 2,4,6, wherein the position numbers of the amino acid sequences are as listed in the international immunogenetic information system (IMGT). The person skilled in the art is aware of the above-mentioned international immunogenetic information system and can derive the position numbers of amino acid residues of different TCRs in IMGT from this database.

More specifically, the TCRs of the present invention in which the hydrophobic core region is mutated may be highly stable single chain TCRs formed by a flexible peptide chain linking the variable domains of the α and β chains of the TCRs. The CDR regions of the TCR variable region determine their affinity for the short peptide-HLA complex, and mutations in the hydrophobic core can make the TCR more stable, but do not affect their affinity for the short peptide-HLA complex. It should be noted that the flexible peptide chain of the present invention may be any peptide chain suitable for linking the variable domains of the TCR alpha and beta chains. The template strand for screening high affinity TCRs constructed in example 1 of the present invention is the high stability single chain TCR described above, which contains the hydrophobic core mutation. With a higher stability TCR, the affinity between the TCR and the TSSELMAITR-HLA-A1101 complex can be assessed more conveniently.

The CDR regions of the α chain variable domain and the β chain variable domain of the single chain template TCR are identical to those of the wild-type TCR. That is, the 3 CDRs of the α chain variable domain are CDR1α: nsass qs; CDR2 a: VYSSGN; CDR3 a: VVNPGSGNTGKLI; and the 3 CDRs of the β chain variable domain are CDR1β: SGHTA; CDR2 β: FQGNSA; CDR3 β: ASSLVFGSVWDTQY. The amino acid sequence of the single-chain template TCR is shown as SEQ ID NO. 9, and the nucleotide sequence of the single-chain template TCR is shown as SEQ ID NO. 10. Thus, a single chain TCR consisting of an alpha chain variable domain and a beta chain variable domain having high affinity for the TSSELMAITR-HLA A1101 complex was selected.

The αβ heterodimers of the invention with high affinity for the TSSELMAITR-HLA-A1101 complex were obtained by transferring the CDR regions of the α and β chain variable domains of the screened high affinity single chain TCRs to the corresponding positions of the wild type tcra chain variable domain (SEQ ID NO: 1) and the β chain variable domain (SEQ ID NO: 2).

The TCRs of the present invention may also be provided in the form of multivalent complexes. The multivalent TCR complexes of the invention comprise a multimer of two, three, four or more TCRs of the invention bound, e.g., a tetramer may be generated using the tetramer domain of p53, or a complex of a plurality of TCRs of the invention bound to another molecule. The TCR complexes of the invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, as well as to generate intermediates for other multivalent TCR complexes having such applications.

The TCRs of the present invention may be used alone or may be covalently or otherwise bound to the conjugate, preferably covalently. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the TSSELMAITR-HLA-A1101 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination or coupling of any of the above.

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

Therapeutic agents that may be conjugated or coupled to a TCR of the invention include, but are not limited to:

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

2. biotoxicity (Chaudhary et al, 1989, nature 339, 394; epel et al, 2002, cancer immunology and immunotherapy (Cancer Immunology and Immunotherapy) 51, 565);

3. cytokines such as IL-2 et al (Gillies et al, 1992, proc. Natl. Acad. Sci. USA (PNAS) 89, 1428; card et al, 2004, cancer immunology and immunotherapy (Cancer Immunology and Immunotherapy) 53, 345; halin et al, 2003, cancer Research (Cancer Research) 63, 3202);

4. Antibody Fc fragments (Mosquera et al, 2005, journal of immunology (The Journal Of Immunology) 174, 4381);

5. antibody scFv fragments (Zhu et al, 1995, J.cancer International (International Journal of Cancer) 62,319);

6. gold nanoparticles/nanorods (Lapotko et al, 2005, cancer communications (Cancer letters) 239, 36; huang et al, 2006, journal of American society of chemistry (Journal of the American Chemical Society) 128, 2115);

7. viral particles (Peng et al, 2004, gene therapy (Gene therapy) 11, 1234);

8. liposomes (Mamot et al 2005, cancer research 65, 11631);

9. nano magnetic particles;

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

11. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticle, and the like.

Antibodies or fragments thereof that bind to the TCRs of the invention include anti-T cell or NK-cell determining antibodies, such as anti-CD 3 or anti-CD 28 or anti-CD 16 antibodies, which bind to the TCRs to better target effector cells. A preferred embodiment is that the TCR of the invention binds to an anti-CD 3 antibody or a functional fragment or variant of said anti-CD 3 antibody. Specifically, the fusion molecule of the TCR and the anti-CD 3 single-chain antibody comprises a sequence selected from the TCR alpha chain variable domain amino acid sequences of SEQ ID NO. 1 and one of SEQ ID NO. 16-22; and/or the amino acid sequence of the beta chain variable domain of the TCR is one of SEQ ID NO. 2, SEQ ID NO. 13-15 and SEQ ID NO. 23-25.

The invention also relates to nucleic acid molecules encoding the TCRs of the invention. The nucleic acid molecules of the invention may be in the form of DNA or RNA. The DNA may be a coding strand or a non-coding strand. For example, the nucleic acid sequences encoding TCRs of the invention may be identical to or degenerate variants of the nucleic acid sequences set forth in the invention. By way of example, a "degenerate variant" as used herein refers to a nucleic acid sequence encoding a protein sequence having SEQ ID NO. 3, but differing from the sequence of SEQ ID NO. 5.

The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can be generally obtained by, but not limited to, PCR amplification, recombinant methods or artificial synthesis. At present, it is already possible to obtain the DNA sequence encoding the TCR of the invention (or a fragment or derivative thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.

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

The invention also includes isolated cells, particularly T cells, that express the TCRs of the invention. There are a number of methods suitable for T cell transfection with DNA or RNA encoding the high affinity TCR of the invention (e.g., robbins et al, (2008) J.Immunol.180:6116-6131). T cells expressing the high affinity TCRs of the invention can be used in adoptive immunotherapy. Those skilled in the art will be aware of many suitable methods of performing adoptive therapy (e.g., rosenberg et al, (2008) Nat Rev Cancer8 (4): 299-308).

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

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

In the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. The addition of one or several amino acids at the C-terminal and/or N-terminal will not generally alter the structure and function of the protein. Thus, the TCRs of the present invention also include TCRs in which up to 5, preferably up to 3, more preferably up to 2, most preferably 1 amino acid (especially amino acids located outside the CDR regions) of the inventive TCRs are replaced with amino acids of similar or similar nature and which are still capable of retaining their functionality.

The invention also includes TCRs that have been slightly modified from the TCRs of the invention. Modified (typically without altering the primary structure) forms include: chemically derivatized forms of TCRs of the invention are, for example, acetylated or carboxylated. Modifications also include glycosylation, such as those resulting from glycosylation modifications performed during the synthesis and processing or further processing steps of the TCRs of the present invention. Such modification may be accomplished by exposing the TCR to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). TCRs modified to improve their proteolytic resistance or to optimize solubility are also included.

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

In addition, the TCRs of the present invention may be used alone or in combination or coupling with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).

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

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

In general, the therapeutic compositions may be formulated as an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for incorporation into a solution or suspension, and a liquid carrier prior to injection.

Once formulated into the compositions of the present invention, they may 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 may be an animal; especially humans.

When the pharmaceutical composition of the present invention is used for actual treatment, various different dosage forms of the pharmaceutical composition can be employed according to the use condition. Preferably, injection, oral preparation and the like are exemplified.

These pharmaceutical compositions may be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents (isotonides), preservatives, wetting agents, emulsifying agents, dispersing agents, stabilizers and cosolvents, and the formulation process may be carried out in a conventional manner according to dosage forms.

The pharmaceutical compositions of the present invention may also be administered in the form of a slow release formulation. For example, the TCRs of the present invention may be incorporated into a pellet or microcapsule that is supported on a slow release polymer, which is then surgically implanted into the tissue to be treated. Examples of the slow release polymer include ethylene-vinyl acetate copolymer, polyhydroxymethacrylate (polyhydroxymethacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer, and the like, and preferably biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer.

When the pharmaceutical composition of the present invention is used for actual treatment, the inventive TCR or TCR complex or the cells presenting the inventive TCR as an active ingredient can be appropriately determined according to the weight, age, sex, degree of symptoms of each patient to be treated, and a reasonable amount is ultimately decided by a physician.

The invention has the main advantages that:

The amino acid sequence and nucleotide sequence related to the invention are shown as follows:

SEQ ID NO. 1 (variable domain amino acid sequence of wild type TCR. Alpha. -chain):

RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNPGSGNTGKLIFGQGTTLQVKP。

SEQ ID NO. 2 (variable domain amino acid sequence of wild type TCR. Beta. -chain):

GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSVWDTQYFGPGTRLTVL。

SEQ ID NO. 3 (amino acid sequence of the alpha chain variable domain of the single chain template TCR):

RKEVEQDPGPLNVPEGETVAINCTYSNSASQSFFWYRQDPGKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDVKPSDSATYFCVVNPGSGNTGKLIFGQGTTLQVKP。

SEQ ID NO. 4 (amino acid sequence of the β chain variable domain of the single chain template TCR):

GAGVSQSPSNLSVEKGKDVELRCDPISGHTALYWYRQRPGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRVQPEDSAVYFCASSLVFGSVWDTQYFGPGTRLTVD。

SEQ ID NO. 5 (DNA sequence of the alpha chain variable domain of the single-stranded template TCR):

cgtaaagaagttgaacaggaccctggcccgctgaatgttccggaaggtgaaaccgttgcaattaattgtacctatagcaatagcgcaagtcagagcttcttttggtatcgccaggaccctggtaaagaaccgaaactgctgatgagtgtgtatagtagcggcaatgaagatggtcgctttaccgcacagctgaatcgcgcaagccagtatattagcctgctgattcgcgatgtgaaaccgagtgatagtgccacctatttctgtgttgttaatcctggtagtggcaataccggtaaactgatcttcggtcagggtaccactctgcaggttaaaccg。

SEQ ID NO. 6 (DNA sequence of the β chain variable domain of the single-stranded template TCR):

ggtgcaggtgttagtcagagcccgagtaatctgagcgtggaaaagggcaaagatgttgaactgcgttgcgatccgattagtggccataccgcactgtattggtatcgtcagcgtccgggtcagggcctggaatttctgatctattttcagggcaatagcgccccggataaaagcggcctgccgagcgatcgttttagcgccgaacgcaccggtgaaagcgttagtaccctgaccattcagcgtgttcagccggaagatagtgccgtgtatttctgtgcaagtagtctggtgtttggcagtgtgtgggatacccagtattttggtcctggtactcgtctgaccgttgat。

SEQ ID NO. 7 (amino acid sequence of linker short peptide (linker) of single-chain template TCR):

GGGSEGGGSEGGGSEGGGSEGGTG。

SEQ ID NO. 8 (DNA sequence of the connecting short peptide (linker) of the single-stranded template TCR):

ggtggcggcagtgaaggtggcggtagtgaaggtggtggtagtgaaggcggtggcagcgaaggtggcaccggc。

SEQ ID NO 9 (amino acid sequence of single-stranded template TCR):

RKEVEQDPGPLNVPEGETVAINCTYSNSASQSFFWYRQDPGKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDVKPSDSATYFCVVNPGSGNTGKLIFGQGTTLQVKPGGGSEGGGSEGGGSEGGGSEGGTGGAGVSQSPSNLSVEKGKDVELRCDPISGHTALYWYRQRPGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRVQPEDSAVYFCASSLVFGSVWDTQYFGPGTRLTVD。

SEQ ID NO 10 (DNA sequence of single-stranded template TCR):

cgtaaagaagttgaacaggaccctggcccgctgaatgttccggaaggtgaaaccgttgcaattaattgtacctatagcaatagcgcaagtcagagcttcttttggtatcgccaggaccctggtaaagaaccgaaactgctgatgagtgtgtatagtagcggcaatgaagatggtcgctttaccgcacagctgaatcgcgcaagccagtatattagcctgctgattcgcgatgtgaaaccgagtgatagtgccacctatttctgtgttgttaatcctggtagtggcaataccggtaaactgatcttcggtcagggtaccactctgcaggttaaaccgggtggcggcagtgaaggtggcggtagtgaaggtggtggtagtgaaggcggtggcagcgaaggtggcaccggcggtgcaggtgttagtcagagcccgagtaatctgagcgtggaaaagggcaaagatgttgaactgcgttgcgatccgattagtggccataccgcactgtattggtatcgtcagcgtccgggtcagggcctggaatttctgatctattttcagggcaatagcgccccggataaaagcggcctgccgagcgatcgttttagcgccgaacgcaccggtgaaagcgttagtaccctgaccattcagcgtgttcagccggaagatagtgccgtgtatttctgtgcaagtagtctggtgtttggcagtgtgtgggatacccagtattttggtcctggtactcgtctgaccgttgat。

SEQ ID NO. 11 (amino acid sequence of soluble reference TCR. Alpha. -chain):

RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNPGSGNTGKLIFGQGTTLQVKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFCSPESS。

SEQ ID NO. 12 (amino acid sequence of soluble reference TCR. Beta. -chain):

GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSVWDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSECEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD。

SEQ ID NO. 13 (β chain variable domain amino acid sequence of heterodimeric TCR):

GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSIWAEQYFGPGTRLTVL。

SEQ ID NO. 14 (β chain variable domain amino acid sequence of heterodimeric TCR):

GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSIWAQQYFGPGTRLTVL。

SEQ ID NO. 15 (β chain variable domain amino acid sequence of heterodimeric TCR):

GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSVWAEQFFGPGTRLTVL。

SEQ ID NO. 16 (alpha chain variable domain amino acid sequence of heterodimeric TCR):

RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVMRAQGNTGKLIFGQGTTLQVKP。

SEQ ID NO. 17 (alpha chain variable domain amino acid sequence of heterodimeric TCR):

RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVYPPRGNTGKLIFGQGTTLQVKP。

SEQ ID NO. 18 (alpha chain variable domain amino acid sequence of heterodimeric TCR):

RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVYPARGNTGKLIFGQGTTLQVKP。

SEQ ID NO. 19 (alpha chain variable domain amino acid sequence of heterodimeric TCR):

RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVYPSRGNTGKLIFGQGTTLQVKP。

SEQ ID NO. 20 (alpha chain variable domain amino acid sequence of heterodimeric TCR):

RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVMRAGGNTGKLIFGQGTTLQVKP。

SEQ ID NO. 21 (alpha chain variable domain amino acid sequence of heterodimeric TCR):

RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNPGSVMRMKLIFGQGTTLQVKP。

SEQ ID NO. 22 (alpha chain variable domain amino acid sequence of heterodimeric TCR):

RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNPGSRPNFKLIFGQGTTLQVKP。

SEQ ID NO. 23 (β chain variable domain amino acid sequence of heterodimeric TCR):

GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSIWSEQYFGPGTRLTVL。

SEQ ID NO. 24 (β chain variable domain amino acid sequence of heterodimeric TCR):

GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSVWAEQHFGPGTRLTVL。

SEQ ID NO. 25 (β chain variable domain amino acid sequence of heterodimeric TCR):

GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSVWAEQYFGPGTRLTVL。

SEQ ID NO. 26 (extracellular amino acid sequence of wild-type TCR alpha chain):

RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNPGSGNTGKLIFGQGTTLQVKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS。

SEQ ID NO. 27 (extracellular amino acid sequence of wild-type TCR. Beta. -chain):

GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSVWDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD。

SEQ ID NO. 28 (amino acid sequence of wild type TCR alpha chain)

RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNPGSGNTGKLIFGQGTTLQVKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS。

SEQ ID NO. 29 (amino acid sequence of wild type TCR. Beta. Chain)

GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSLVFGSVWDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG。

SEQ ID NO. 30 (α chain variable domain CDR1 α of wild-type TCR): nsass qs.

SEQ ID NO. 31 (α chain variable domain CDR2 α of wild-type TCR): VYSSGN.

SEQ ID NO. 32 (α chain variable domain CDR3 α of wild-type TCR): VVNPGSGNTGKLI.

SEQ ID NO. 33 (β chain variable domain CDR1 β of wild type TCR): SGHTA.

SEQ ID NO 34 (β chain variable domain CDR2 β of wild-type TCR): FQGNSA.

SEQ ID NO. 35 (β chain variable domain CDR3 β of wild type TCR): ASSLVFGSVWDTQY.

The following specific examples further illustrate the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address specific conditions in the examples below, is generally followed by conventional conditions, for example those described in the laboratory Manual (Molecular Cloning-A Laboratory Manual) (third edition) (2001) CSHL Press, or by the manufacturer's recommendations (Sambrook and Russell et al, molecular cloning). Percentages and parts are by weight unless otherwise indicated.

Materials and methods

The experimental materials used in the examples of the present invention were commercially available from commercial sources, with E.coli DH 5. Alpha. From Tiangen, E.coli BL21 (DE 3) from Tiangen, E.coli Tuner (DE 3) from Novagen, and plasmid pET28a from Novagen, unless otherwise specified.

EXAMPLE 1 production of a stable Single chain TCR template chain with hydrophobic core mutation

The invention utilizes a site-directed mutagenesis method, and constructs a stable single-chain TCR molecule formed by connecting TCR alpha and beta chain variable domains by a flexible short peptide (linker) according to the patent document WO2014/206304, wherein the amino acid and DNA sequences are SEQ ID NO:9 and SEQ ID NO:10 respectively. And screening the high-affinity TCR molecules by taking the single-chain TCR molecules as templates. The alpha chain variable domain of the single-chain template TCR is shown as SEQ ID NO. 3, and the beta chain variable domain is shown as SEQ ID NO. 4; the corresponding DNA sequences are SEQ ID NO 5 and SEQ ID NO 6 respectively; the amino acid sequence and the DNA sequence of the flexible short peptide (linker) are SEQ ID NO. 7 and SEQ ID NO. 8 respectively.

The target gene carrying the template strand is subjected to double cleavage by NcoI and NotI, and is connected with a pET28a vector subjected to double cleavage by NcoI and NotI. The ligation product was transformed into E.coli DH 5. Alpha. And the ligation product was spread on LB plates containing kanamycin, incubated at 37℃overnight in an inverted position, positive clones were picked up for PCR screening, positive recombinants were sequenced, and after the correct sequence was confirmed, the recombinant plasmid was extracted and transformed into E.coli BL21 (DE 3) for expression.

EXAMPLE 2 expression, renaturation and purification of the stable single chain TCR constructed in example 1

BL21 (DE 3) colonies containing the recombinant plasmid pET28 a-template strand prepared in example 1 were all inoculated into LB medium containing kanamycin, and cultured at 37℃to OD ₆₀₀ IPTG was added to a final concentration of 0.5mM at 0.6-0.8 and incubation was continued for 4h at 37 ℃. Cell pellet was harvested by centrifugation at 5000rpm for 15min, cell pellet was lysed by Bugbuster Master Mix (Merck), inclusion bodies were recovered by centrifugation at 6000rpm for 15min, washed with Bugbuster (Merck) to remove cell debris and membrane components, and collected by centrifugation at 6000rpm for 15 min. The inclusion bodies were dissolved in buffer (20 mM Tris-HCl pH 8.0,8M urea), high-speed centrifuged to remove insoluble material, and the supernatant was quantified by BCA method and then sub-packaged and stored at-80℃for further use.

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

The eluted fractions for BIAcore analysis were further tested for purity by gel filtration. The conditions are as follows: the column Agilent Bio SEC-3 (300A,) The mobile phase is 150mM phosphate buffer solution, the flow rate is 0.5mL/min, the column temperature is 25 ℃, and the ultraviolet detection wavelength is 214nm.

Example 3 characterization in combination

BIAcore analysis

The binding activity of TCR molecules to the TSSELMAITR-HLA a1101 complex was detected using a BIAcore T200 real-time assay system. The coupling process was completed by adding an anti-streptavidin antibody (GenScript) to a coupling buffer (10 mM sodium acetate buffer, pH 4.77), then flowing the antibody through a CM5 chip previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally blocking the unreacted activated surface with an ethanolamine-HCl solution at a coupling level of about 15000RU. The conditions are as follows: the temperature is 25 ℃, and the PH value is 7.1-7.5.

The low concentration of streptavidin was flowed over the surface of the antibody-coated chip, then TSSELMAITR-HLA A A1101 complex was flowed over the detection channel, the other channel was used as a reference channel, and 0.05mM biotin was flowed over the chip at a flow rate of 10. Mu.L/min for 2min, blocking the remaining binding sites for streptavidin. The affinity was determined by single cycle kinetic analysis, the TCR was diluted to several different concentrations with HEPES-EP buffer (10mM HEPES,150mM NaCl,3mM EDTA,0.005%P20,pH7.4) and flowed sequentially over the chip surface at a flow rate of 30. Mu.L/min for a binding time of 120s each sample injection and allowed to dissociate for 600s after the end of the last sample injection. After each round of assay, the chip was regenerated with 10mM Gly-HCl pH 1.75. Kinetic parameters were calculated using BIAcore Evaluation software.

The preparation process of the TSSELMAITR-HLA A1101 compound is as follows:

a. purifying: collecting 100mL of E.coli bacterial liquid for inducing expression of heavy chains or light chains, centrifuging at 8000g at 4 ℃ for 10min, washing the bacterial body once by using 10mL of PBS, then severely shaking and re-suspending the bacterial body by using 5mL BugBuster Master Mix Extraction Reagents (Merck), rotating at room temperature for 20min, centrifuging at 6000g at 4 ℃ for 15min, discarding the supernatant, and collecting inclusion bodies.

The inclusion body is resuspended in 5mL BugBuster Master Mix and incubated for 5min at room temperature; adding 30mL of BugBuster diluted 10 times, mixing well, and centrifuging at 4 ℃ for 15min at 6000 g; the supernatant was discarded, the inclusion bodies were resuspended by adding 30mL of 10-fold diluted BugBuster, mixing, centrifuging at 4℃for 15min, repeating twice, adding 30mL of 20mM Tris-HCl pH 8.0, mixing, centrifuging at 4℃for 15min, finally dissolving the inclusion bodies with 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and measuring the concentration by BCA kit.

b. Renaturation: synthetic short peptide TSSELMAITR (Jiangsu St. Biotech Co., ltd.) was dissolved in DMSO to a concentration of 20 mg/mL. The inclusion bodies of the light and heavy chains were solubilized with a solution containing 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by adding 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. TSSELMAITR peptide was added to a 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 ℃) at 25mg/L (final concentration), followed by sequential addition of 20mg/L light chain and 90mg/L heavy chain (final concentration, three heavy chain additions, 8 h/time), renaturation was performed at 4℃for at least 3 days to completion, and SDS-PAGE was examined for success of renaturation.

c. Purifying after renaturation: the renaturation buffer was exchanged with 10 volumes of 20mM Tris pH 8.0 for dialysis, at least twice to sufficiently reduce the ionic strength of the solution. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and then loaded onto a HiTrap Q HP (GE general electric company) anion exchange column (5 mL bed volume). Using an Akta purifier (GE general electric), proteins were eluted with a linear gradient of 0-400mM NaCl in 20mM Tris pH 8.0, pMHC eluted at about 250mM NaCl, and fractions were collected for SDS-PAGE to check purity.

d. Biotinylation: purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while buffer was replaced with 20mM Tris pH 8.0, and then biotinylated reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. Mu.MD-Biotin, 100. Mu.g/ml BirA enzyme (GST-BirA), the mixture incubated overnight at room temperature and SDS-PAGE was performed to determine whether biotinylation was complete.

e. Purifying the biotinylated complex: biotinylated pMHC molecules were concentrated to 1mL using a Millipore ultrafiltration tube, biotinylated pMHC was purified using gel filtration chromatography, hiPrepTM 16/60s200 HR column (GE general electric company) was pre-equilibrated with filtered PBS using Akta purifier (GE general electric company), 1mL of concentrated biotinylated pMHC molecules were loaded, and then eluted with PBS at a flow rate of 1 mL/min. Biotinylated pMHC molecules appeared as a single peak elution at about 55 mL. The protein-containing fractions were pooled, concentrated by Millipore ultrafiltration tube, protein concentration was determined by BCA method (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80℃with the addition of protease inhibitor cocktail (Roche).

EXAMPLE 4 production of high affinity TCR

Phage display technology is one means of generating libraries 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. Libraries of high affinity TCRs were created by mutating the CDR regions of the template strand and panning was performed. Phage libraries after several rounds of panning all bind specifically to the corresponding antigen, from which the monoclonal was picked and analyzed.

CDR region mutations of the screened high affinity single chain TCRs were introduced into the corresponding sites of the variable domain of the αβ heterodimeric TCRs and their affinity to the TSSELMAITR-HLA a1101 complex was detected by BIAcore. The introduction of high affinity mutation points in the CDR regions is accomplished by site-directed mutagenesis methods well known to those skilled in the art. The amino acid sequences of the alpha chain and the beta chain variable domain of the wild TCR are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.

It should be noted that to obtain a more stable soluble TCR, in order to more conveniently assess the binding affinity and/or binding half-life between the TCR and the TSSELMAITR-HLA a1101 complex, the αβ heterodimeric TCR may be a TCR in which one cysteine residue is introduced in the constant regions of the α and β chains, respectively, to form an artificial interchain disulfide bond, the amino acid sequences of the TCR α and β chains following the introduction of the cysteine residue being shown in bold letters in SEQ ID NO:11 and SEQ ID NO:12, respectively.

Extracellular sequence genes of the α and β chains of the TCR to be expressed were synthesized and inserted into the expression vector pET28a+ (Novagene) by standard methods described in molecular cloning laboratory manual (Molecular Cloning a Laboratory Manual) (third edition, sambrook and Russell), upstream and downstream cloning sites were Nco I and Not I, respectively. Mutations in the CDR regions were introduced by overlapping PCR (overlap PCR), which is well known to those skilled in the art. The insert was confirmed by sequencing to be error-free.

EXAMPLE 5 expression, renaturation and purification of high affinity TCR

The expression vectors of TCR alpha and beta chains are respectively transformed into expression bacteria BL21 (DE 3) by a chemical transformation method, the bacteria are grown by LB culture solution, and the bacteria are grown on OD ₆₀₀ At 0.6, inclusion bodies formed after expression of the α and β chains of TCR were extracted by bugbaster Mix (Novagene) and washed repeatedly with bugbaster solution, and finally the inclusion bodies were dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediamine tetraacetic acid (EDTA), 20mM Tris (pH 8.1), induced with a final concentration of 0.5mM IPTG.

The TCR alpha and beta chains after dissolution were found to be 1:1 in mass ratio in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine,6.6mM beta-mercapoethylamine (4 ℃ C.) at a final concentration of 60mg/mL. After mixing the solution was dialyzed (4 ℃) in 10 volumes of deionized water, after 12 hours the deionized water was changed to buffer (20 mM Tris, pH 8.0) and dialysis was continued at 4℃for 12 hours. The dialyzed solution was filtered through a 0.45 μm filter and purified by an anion exchange column (HiTrap Q HP,5mL,GE Healthcare). The elution peak contains the successfully renatured alpha and beta dimer TCR as confirmed by SDS-PAGE gel. The 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 BCA.

EXAMPLE 6BIAcore analysis results

The affinity of the αβ heterodimeric TCR incorporating the high affinity CDR regions with the TSSELMAITR-HLA a1101 complex was examined using the method described in example 3.

The invention obtains new TCR alpha chain and beta chain variable domain amino acid sequences. Since the CDR regions of the TCR molecules determine their affinity for the corresponding pMHC complex, one skilled in the art would be able to expect that the αβ heterodimeric TCR incorporating the high affinity mutation point also has a high affinity for the TSSELMAITR-HLA a1101 complex. The expression vector was constructed using the method described in example 4, the above-described αβ heterodimeric TCR introduced with high affinity mutations was expressed, renatured and purified using the method described in example 5, and then its affinity to TSSELMAITR-HLA a1101 complex was determined using BIAcore T200, and fig. 1 is a binding curve of a soluble reference TCR, i.e., a wild-type TCR, to TSSELMAITR-HLA a1101 complex. The results of the high affinity mutated αβ heterodimeric TCR affinity assay are shown in table 7 below.

TABLE 7

From the above table, the affinity of the heterodimeric TCR was at least 5-fold that of the wild-type TCR for the TSSELMAITR-HLA a1101 complex.

Example 7 expression, renaturation and purification of fusions of anti-CD 3 antibodies with high affinity αβ heterodimeric TCRs

A single chain antibody (scFv) against CD3 was fused to an αβ heterodimeric TCR to prepare a fusion molecule. The anti-CD 3 scFv is fused to the β chain of a TCR, which may comprise the β chain variable domain of any of the above-described high affinity αβ heterodimeric TCRs, and the TCR α chain of the fusion molecule may comprise the α chain variable domain of any of the above-described high affinity αβ heterodimeric TCRs.

Construction of fusion molecule expression vector

1. Construction of an alpha chain expression vector: the target gene carrying the alpha chain of the alpha beta heterodimeric TCR is subjected to double cleavage by NcoI and NotI and is connected with a pET28a vector subjected to double cleavage by NcoI and NotI. The ligation product was transformed into E.coli DH 5. Alpha. And plated on LB plates containing kanamycin, incubated upside down at 37℃overnight, positive clones were picked up for PCR screening, positive recombinants were sequenced, and after the correct sequence was determined, the recombinant plasmid was extracted and transformed into E.coli Tuner (DE 3) for expression.

2. Construction of anti-CD 3 (scFv) -beta chain expression vector: primers were designed to link the anti-CD 3 scFv and the high affinity heterodimeric TCR β chain genes by overlap PCR, with the intermediate linking short peptide (linker) being GGGGS, and the gene fragments of the fusion proteins of the anti-CD 3 scFv and the high affinity heterodimeric TCR β chain bearing restriction enzyme sites NcoI (CCATGG) and NotI (GCGGCCGC). The PCR amplified product was digested with NcoI and NotI, and ligated to the NcoI and NotI digested pET28a vector. The ligation product was transformed into E.coli DH 5. Alpha. Competent cells, plated on LB plate containing kanamycin, cultured upside down at 37℃overnight, positive clones were picked up for PCR screening, positive recombinants were sequenced, and after determining that the sequence was correct, the extracted recombinant plasmid was transformed into E.coli Tuner (DE 3) competent cells for expression.

Expression, renaturation and purification of fusion proteins

The expression plasmids were transformed into E.coli Tuner (DE 3) competent cells, respectively, and the coated LB plates (kanamycin 50. Mu.g/mL) were incubated overnight at 37 ℃. The next day, the selected clone was inoculated into 10mL of LB liquid medium (kanamycin 50. Mu.g/mL) for 2-3h, inoculated into 1L of LB medium according to the volume ratio of 1:100, and continuously cultured until OD ₆₀₀ 0.5-0.8, and adding into the final productExpression of the protein of interest was induced at a concentration of 1mM IPTG. After 4h of induction, the cells were harvested by centrifugation at 6000rpm for 10 min. The cells were washed once with PBS buffer and were aliquoted, and cells corresponding to 200mL of the bacterial culture were lysed with 5mL BugBuster Master Mix (Merck) and the inclusion bodies were collected by centrifugation at 6000g for 15 min. Then 4 washing with detergent was performed to remove cell debris and membrane components. The inclusion bodies are then washed with a buffer such as PBS to remove detergents and salts. Finally, the inclusion bodies were solubilized with a buffer solution containing 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediamine tetraacetic acid (EDTA), 20mM Tris, pH 8.1, and the inclusion body concentration was measured, and the inclusion bodies were sub-packaged and then stored at-80℃under refrigeration.

The TCR alpha chain and anti-CD 3 (scFv) -beta chain after solubilization were combined at 2:5 in a mass ratio of 0.4. M L-arginine (L-arginine) in a solution containing 5M urea (4 ℃) at a final concentration of 0.1mg/mL for the alpha chain and 0.25mg/mL for the anti-CD 3 (scFv) -beta chain, respectively.

After mixing the solution was dialyzed (4 ℃) in 10 volumes of deionized water, after 12 hours the deionized water was changed to buffer (10 mM Tris, pH 8.0) and dialysis was continued at 4℃for 12 hours. The dialyzed solution was filtered through a 0.45 μm filter and purified by an anion exchange column (HiTrap Q HP 5mL,GE healthcare). The elution peak contains TCR for successfully renatured TCR alpha chain and anti-CD 3 (scFv) -beta chain dimers as confirmed by SDS-PAGE gels. The TCR fusion molecule was then further purified by size exclusion chromatography (S-100 16/60, GE healthcare), and again by anion exchange column (HiTrap Q HP 5ml,GE healthcare). The purity of the purified TCR fusion molecule is greater than 90% as determined by SDS-PAGE and the concentration is determined by BCA.

Example 8 experiment of activation function of effector cells transfected with high affinity TCR of the invention against T2 cells loaded with short peptide

IFN-gamma is a potent immunomodulator produced by activated T lymphocytes, and therefore this example demonstrates the activation function and antigen specificity of cells transfected with the high affinity TCR of the invention by detecting IFN-gamma numbers by ELISPOT experiments well known to those skilled in the art. High affinity of the inventionTCR (TCR numbering and its serial number is known from table 7) transfection to CD3 isolated from blood of healthy volunteers ⁺ T cells as effector cells and transfected with CD3 of other TCR (A6) or of wild-type TCR (WT-TCR) in the same volunteer ⁺ T cells served as controls. The target cells used were T2-A11 loaded with TSS45 antigen oligopeptide TSSELMAITR, other antigen oligopeptide loaded, or empty (referring to T2 cells transfected with HLA-A 1101).

Target cells 1X 10 for experiments ⁴ Individual cells/well, effector cells 2×10 ³ Individual cells/well (calculated as transfection positive rate).

The experimental procedure was as follows: firstly preparing an ELISPOT plate, adding target cells and effector cells into corresponding holes, then adding TSS45 antigen short peptide TSSELMAITR solution into an experimental group, adding other antigen short peptide solution into a control group, and enabling the final concentration of the short peptide to be 10 ^-6 M, adding an equal volume of culture medium into a blank group, and setting two compound holes. Incubation overnight (37 ℃,5% co) ₂ ). On day 2 of the experiment, the plates were washed and subjected to secondary detection and development, the plates were dried, and spots formed on the membrane were counted using an immunoblotter plate reader (ELISPOT READER system; AID 20).

FIG. 2 is a graph showing the results of an experiment of the activation function of effector cells transfected with the high affinity TCR of the invention against T2 cells loaded with a short peptide, which shows that effector cells transfected with the high affinity TCR of the invention have a more pronounced activating effect than effector cells transfected with a wild-type TCR, whereas effector cells transfected with other TCRs are substantially inactive against target cells loaded with the TSS45 antigen short peptide TSSELMAITR; meanwhile, effector cells transfected with the inventive TCRs are inactive against target cells loaded with other antigen oligopeptides or empty.

Example 9 experiment of activation function of effector cells transfected with high affinity TCR of the invention against tumor cell lines

This example again demonstrates the activation function and specificity of effector cells transfected with the high affinity TCRs of the present invention using tumor cell lines. Detection is likewise carried out by means of ELISPOT experiments which are known to the person skilled in the art. Transfection of the high affinity TCR of the invention into blood from healthy volunteersCD3 isolated in (C) ⁺ T cells as effector cells and transfected with CD3 of other TCR (A6) or of wild-type TCR (WT-TCR) in the same volunteer ⁺ T cells served as controls. The tumor cell lines used in the examples were SK-MEL-28, hepG2, SK-MEL-1, SK-MEL-5, SNU423, respectively. Wherein SK-MEL-28, SK-MEL-1, SNU423 were all purchased from Guangzhou Sakuku Biotechnology Co., ltd, hepG2 was purchased from the China academy of sciences cell bank, and SK-MEL-5 was purchased from ATCC. The test was performed:

the high affinity TCRs described in table 7 are known as TCR1, TCR2, TCR3, TCR4, respectively. The AFP positive tumor cell lines used in the batch are SK-MEL-28-AFP (AFP over-expression), hepG2-A11-B2M (simultaneously transferred into A1101 and B2M), and the negative cell lines are SK-MEL-28, SK-MEL-1, SK-MEL-5 and SNU423.

The following steps are carried out: first, an ELISPOT plate was prepared. ELISPOT plate ethanol activation coating, 4 ℃ overnight. On day 1 of the experiment, the coating was removed, the block was washed, incubated at room temperature for two hours, the block was removed, and the individual components of the experiment were added to the ELISPOT plate: target cells were 2X 10 ⁴ Per well, effector cells were 2×10 ³ Each well (calculated as positive rate of transfection) and two duplicate wells were set. Incubation overnight (37 ℃,5% co) ₂ ). On day 2 of the experiment, the plates were washed and subjected to secondary detection and development, the plates were dried, and spots formed on the membrane were counted using an immunoblotter plate reader (ELISPOT READER system; AID 20).

The experimental results are shown in FIG. 3, in which the effector cells transfected with the high affinity TCR of the invention have a significant activating effect against AFP positive tumor cell lines, while the effector cells transfected with other TCRs are substantially inactive; meanwhile, effector cells transfected with the high affinity TCRs of the present invention were inactive against AFP negative cell lines.

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

This example also demonstrates the killing function of cells transfected with TCRs of the invention by determining the release of LDH by non-radioactive cytotoxicity assays well known to those skilled in the art. Example LDH experiments were performed on blood from healthy volunteersIsolated CD3 ⁺ T cell transfection the high affinity TCR of the invention was used as effector cells and CD3 of other TCR (A6) transfected with the same volunteer ⁺ T cells served as controls. The tumor cell lines used in the examples were SK-MEL-28, hepG2, SK-MEL-1, SK-MEL-5, SNU423, respectively. Wherein SK-MEL-28, SK-MEL-1, SNU423 were all purchased from Guangzhou Sakuku Biotechnology Co., ltd, hepG2 was purchased from the China academy of sciences cell bank, and SK-MEL-5 was purchased from ATCC. Experiments were performed:

The high affinity TCRs described in table 7 are known as TCR1, TCR2, TCR3, TCR4, respectively. The AFP positive tumor cell lines used in the batch are SK-MEL-28-AFP (AFP over-expression), hepG2-A11-B2M (simultaneously transferred into A1101 and B2M), and the negative tumor cell lines are SK-MEL-28, SK-MEL-1, SK-MEL-5 and SNU423.

The experimental steps are as follows: LDH plates were first prepared and the individual components of the assay were added to the plates in the following order: target cells 3×10 ⁴ Individual cells/well, effector cells 6×10 ⁴ Individual cells/wells (calculated as transfection positivity) were added to the corresponding wells and three duplicate wells were set. And simultaneously setting an effector cell spontaneous pore, a target cell maximum pore, a volume correction control pore and a culture medium background control pore. Incubation overnight (37 ℃,5% co) ₂ ). On experiment day 2, the color development was detected and absorbance was recorded at 490nm with a microplate reader (biotek) after termination of the reaction.

The experimental results are shown in figure 4, in which the effector cells transfected with the high affinity TCRs of the present invention still exhibited strong killing efficacy against AFP positive tumor cell lines, while T cells transfected with other TCRs were substantially non-responsive, while T cells transfected with the high affinity TCRs of the present invention were substantially non-killing against negative tumor cell lines.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.

Claims

1. A T cell receptor comprising a TCR α chain variable domain and a TCR β chain variable domain, the T cell receptor having activity in binding to the TSSELMAITR-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 T cell receptor complex comprising at least two T cell receptor molecules, wherein at least one of the T cell receptor molecules is the T cell receptor of claim 1.

3. A nucleic acid molecule comprising a nucleic acid sequence encoding the T cell receptor of claim 1, or a complement of a nucleic acid sequence encoding the T cell receptor of claim 1.

4. A vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the vector of claim 4 or having incorporated into its chromosome the exogenous nucleic acid molecule of claim 3.

6. An isolated cell expressing the T cell receptor of claim 1;

preferably, the isolated cells also express exogenous CD8 receptor;

preferably, the CD8 receptor is CD8 a.

7. A pharmaceutical composition comprising any one or a combination of at least two of the T cell receptor of claim 1, the multivalent T cell receptor complex of claim 2, or the isolated cell of claim 6;

8. A method of treating a disease comprising administering to a subject in need thereof any one or a combination of at least two of the T cell receptor of claim 1, the multivalent T cell receptor complex of claim 2, the isolated cell of claim 6, or the pharmaceutical composition of claim 7;

Preferably, the disease is an AFP-positive tumor.

9. Use of the T cell receptor of claim 1, the multivalent T cell receptor complex of claim 2 or the isolated cell of claim 6, wherein the use comprises for the preparation of a medicament for treating a tumor;

preferably, the tumor is an AFP-positive tumor.

10. A method of preparing a T cell receptor according to claim 1, comprising the steps of:

(i) Culturing the host cell of claim 5, thereby expressing the T cell receptor of claim 1;

(ii) Isolating or purifying the T cell receptor.