阅读说明：本技术 一种识别ssx2的高亲和力t细胞受体 (High-affinity T cell receptor for recognizing SSX2 ) 是由 黄金花 于 2019-09-05 设计创作，主要内容包括：本发明提供了一种识别SSX2的高亲和力T细胞受体(TCR),其具有结合KASEKIFYV-HLA A0201复合物的特性；并且所述TCR对所述KASEKIFYV-HLA A0201复合物的结合亲和力是野生型TCR对KASEKIFYV-HLA A0201复合物的结合亲和力的至少2倍。本发明还提供了此类TCR与治疗剂的融合分子。此类TCR可以单独使用,也可与治疗剂联用,以靶向呈递KASEKIFYV-HLA A0201复合物肿瘤细胞。(The present invention provides a high affinity T Cell Receptor (TCR) that recognizes SSX2, having the property of binding to the KASEKIFYV-HLA a0201 complex; and the binding affinity of said TCR to said KASEKIFYV-HLA A0201 complex is at least 2-fold greater than the binding affinity of a wild-type TCR to the KASEKIFYV-HLA A0201 complex. The invention also provides fusion molecules of such TCRs with therapeutic agents. Such TCRs can be used alone or in combination with therapeutic agents to target tumor cells presenting KASEKIFYV-HLA a0201 complex.)

1. A T Cell Receptor (TCR) which has binding activity to the KASEKIFYV-HLA a0201 complex and the α chain variable domain of the TCR comprises an amino acid sequence having at least 90% sequence homology to the amino acid sequence set forth in SEQ ID No. 1; and/or the β chain variable domain of the TCR comprises an amino acid sequence having at least 90% sequence homology with the amino acid sequence set forth in SEQ ID NO. 2.

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

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

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

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

6. An isolated cell expressing a TCR as claimed in claim 1; preferably, the cell is a T cell.

7. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to claim 1, or a TCR complex according to claim 2, or a cell according to claim 6.

8. Use of the T cell receptor of claim 1, the TCR complex of claim 2 or the cell of claim 6 for the preparation of a medicament for the treatment of a tumor; preferably, the tumor is an SSX2 positive tumor.

9. The T cell receptor of claim 1, the TCR complex of claim 2, or the cell of claim 6, for use as a medicament for treating a tumor; preferably, the tumor is an SSX2 positive tumor.

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

(i) culturing the host cell of claim 5 so as to express the T cell receptor of claim 1;

(ii) isolating or purifying said T cell receptor.

Technical Field

The present invention relates to the field of biotechnology, and more specifically to T Cell Receptors (TCRs) capable of recognizing polypeptides derived from SSX2 protein. The invention also relates to the preparation and use of said receptors.

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 cell membrane surface glycoprotein that exists as a heterodimer from the α chain/β chain or the γ chain/δ chain. The composition of the TCR repertoire of the immune system is produced by v (d) J recombination in the thymus, followed by positive and negative selection. In the peripheral environment, TCRs mediate the specific recognition of the major histocompatibility complex-peptide complex (pMHC) by T cells, and are therefore critical to the 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 exogenous or endogenous peptides may be the only signs of cellular abnormalities. In the immune system, direct physical contact between T cells and Antigen Presenting Cells (APCs) is initiated by the binding of antigen-specific TCRs to pMHC complexes, and then other cell membrane surface molecules of both T cells and APCs interact, which causes a series of subsequent cell signaling and other physiological responses, thereby allowing T cells of different antigen specificities to exert immune effects on their target cells.

The MHC class I and II molecular ligands corresponding to the TCR are also proteins of the immunoglobulin superfamily but are specific for presentation of antigens, with different individuals having different MHC, and thus are capable of presenting different short peptides of one protein antigen to the cell surface of the respective APC. Human MHC is commonly referred to as an HLA gene or HLA complex.

SSX2 is a synovial sarcoma X breakpoint, also known as HOM-MEL-40. SSX2 is one of ten highly homologous nucleic acid proteins of the SSX family. SSX proteins are tumor testis antigens and are expressed only in tumor cells and in testicular germ cells that do not have MHC expression. SSX2 is expressed in a variety of human cancer cells including, but not limited to, liver cancer, lung cancer, fibrosarcoma, breast cancer, colon cancer, prostate cancer. SSX2 is degraded into small polypeptides after intracellular production and is presented to the cell surface as a complex with MHC (major histocompatibility complex) molecules. KASEKIFYV is a short peptide derived from the SSX2 antigen, and is a target for the treatment of SSX 2-related diseases.

Thus, the KASEKIFYV-HLA a0201 complex provides a marker that the TCR can target tumor cells. The TCR capable of binding the KASEKIFYV-HLA A0201 complex has high application value for tumor treatment. For example, TCRs capable of targeting the tumor cell marker can be used to deliver cytotoxic or immunostimulatory agents to target cells, or transformed into T cells, enabling T cells expressing the TCR to destroy tumor cells for administration to patients in a therapeutic process known as adoptive immunotherapy. For the former purpose, the ideal TCR is of higher affinity, enabling the TCR to reside on the targeted cell for a long period of time. For the latter purpose, it is preferred to use a medium affinity TCR. Accordingly, those skilled in the art are working to develop TCRs that target tumor cell markers that can be used to meet different objectives.

Disclosure of Invention

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

It is a further object of the present invention to provide a method for preparing a TCR of the above type and uses thereof.

In a first aspect of the invention, there is provided a T Cell Receptor (TCR) having binding activity to the KASEKIFYV-HLA A0201 complex.

In another preferred embodiment, the α chain variable domain of the TCR comprises at least 90% of the amino acid sequence set forth in SEQ ID NO 1; preferably, at least 92%; more preferably, at least 94% (e.g., can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) of sequence homology of amino acid sequence; and/or the β chain variable domain of the TCR comprises at least 90%, preferably at least 92%, of the amino acid sequence set forth 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% sequence homology) of sequence homology.

In another preferred embodiment, the TCR is represented in SEQ ID NO:2, and the mutation is selected from one or more of D96V, E98I, L99P, L100E/I/N/P/V, F101D/H/N/Y, Y102A/K/N/P/V, N103A/D/E/P, E104L/V, Q105P/T, F106H, wherein the numbering of the amino acid residues is that shown in SEQ ID NO: 2.

In another preferred embodiment, the TCR α chain variable domain comprises 3 CDR regions wherein the amino acid sequence of CDR3 α is: AYRSGIIQGAQKLV are provided.

In another preferred embodiment, the TCR α chain variable domain comprises 3 CDR regions, the sequence of the 3 CDR regions being:

CDR1α：TSESDYY

CDR2α：QEAYKQQN

CDR3α：AYRSGIIQGAQKLV

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

In another preferred embodiment, the TCR β chain variable domain comprises 3 CDR regions, wherein the amino acid sequence of CDR1 β is PRHDT; and CDR2 β is: FYEKMQ.

In another preferred embodiment, the CDR3 β of the TCR β chain variable domain is selected from the group consisting of: ASSSDRIPPYYNEQF, ASSSDRELNDAPEQF, ASSSVRELNYVDEQF, and ASSSDRELLFYNEQF.

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

in another preferred embodiment, the α chain variable domain amino acid sequence of the TCR is selected from the group consisting of: 1, SEQ ID NO; and/or the beta chain variable domain amino acid sequence of the TCR is selected from: 13-24 of SEQ ID NO.

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

in another preferred embodiment, the TCR has at least 2-fold greater affinity for the KASEKIFYV-HLA a0201 complex than a wild-type TCR.

In a preferred embodiment of the invention, the TCR has at least 2-fold greater affinity for the KASEKIFYV-HLA a0201 complex than a wild-type TCR; preferably, at least 5 times; more preferably, at least 10 times.

In another preferred embodiment, the TCR has at least 50-fold greater affinity for the KASEKIFYV-HLA a0201 complex than a wild-type TCR; preferably, at least 100 times; more preferably, at least 300 times.

Specifically, the dissociation equilibrium constant KD of the TCR to the KASEKIFYV-HLA A0201 complex is less than or equal to 50 mu M;

in another preferred embodiment, the TCR has a dissociation equilibrium constant for the KASEKIFYV-HLA A0201 complex of 1 μ M ≦ KD ≦ 50 μ M.

In another preferred embodiment, the TCR is soluble.

In another preferred embodiment, the TCR is an α β heterodimeric TCR. Preferably, the TCR has an alpha chain constant region sequence TRAC 01 and a beta chain constant region sequence TRBC 101 or TRBC2 01.

In another preferred embodiment, the TCR comprises (i) all or part of a TCR α chain, except for its transmembrane domain, and (ii) all or part of a TCR β chain, except for its transmembrane domain, wherein both (i) and (ii) comprise the variable domain and at least part of the constant domain of the TCR chain.

In another preferred embodiment, the TCR comprises an artificial interchain disulfide bond 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 groups of sites selected from:

thr48 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser57 of TRBC2 × 01 exon 1;

thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1;

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

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

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

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

pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01;

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

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 alpha chain variable domain and a beta chain variable domain linked by a flexible short peptide sequence (linker).

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

In another preferred embodiment, the conjugate to which the TCR is bound is a detectable label, a therapeutic agent, a PK modifying moiety or a combination of any of these.

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

In a second aspect of the invention, there is provided a multivalent TCR complex comprising at least two TCR molecules, and wherein at least one TCR molecule is a TCR 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 TCR molecule according to the first aspect of the invention or a multivalent TCR complex according to the second aspect of the invention, or a complement thereof.

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

In a fifth aspect of the invention, there is provided a host cell comprising a vector or chromosome of the fourth aspect of the invention and, integrated therein, an exogenous nucleic acid molecule of the third aspect of the invention.

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

In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention.

In an eighth aspect of the invention, there is provided a method of treating a disease comprising administering to a subject in need thereof an amount of a TCR according to the first aspect of the invention, or a TCR complex according to the second aspect of the invention, or a cell according to the sixth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention.

In a ninth aspect of the invention there is provided the use of a TCR of the first aspect of the invention, or a TCR complex of the second aspect of the invention, or a cell of the sixth aspect of the invention, in the manufacture of a medicament for the treatment of a tumour, preferably a SSX2 positive tumour.

In a tenth aspect of the invention, the T cell receptor of the first aspect, the TCR complex of the second aspect or the cell of the sixth aspect, is for use as a medicament for the treatment of a tumour. Preferably, the tumor is an SSX2 positive tumor.

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

(i) culturing a host cell according to the fifth aspect of the invention, thereby expressing a T-cell receptor according to the first aspect of the invention;

(ii) isolating or purifying said T cell receptor.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIGS. 1a and 1b show the amino acid sequences of the variable domains of wild-type TCR alpha and beta chains, respectively, capable of specifically binding to the KASEKIFYV-HLA A0201 complex.

FIGS. 2a and 2b show the amino acid sequence of the alpha chain variable domain and the amino acid sequence of the beta chain variable domain, respectively, of a single-chain template TCR constructed in accordance with the invention.

FIGS. 3a and 3b are the DNA sequences of the alpha chain variable domain and beta chain variable domain, respectively, of a single-chain template TCR constructed in accordance with the invention.

FIGS. 4a and 4b show the amino acid sequence and nucleotide sequence of the linker of the single-chain template TCR constructed according to the present invention.

FIGS. 5a and 5b show the amino acid sequence and DNA sequence, respectively, of a single-stranded template TCR constructed in accordance with the invention.

Fig. 6a and 6b show the amino acid sequences of soluble reference TCR α and β chains, respectively, of the invention.

Fig. 7(1) -fig. 7(12) show the β chain variable domain amino acid sequences of hetero-dimeric TCRs with high affinity for KASEKIFYV-HLA a0201 complex, respectively, with mutated residues underlined.

FIGS. 8a and 8b show the extracellular amino acid sequences of wild-type TCR alpha and beta chains, respectively, capable of specific binding to the KASEKIFYV-HLA A0201 complex.

Fig. 9a and 9b show the amino acid sequences of wild type TCR α and β chains, respectively, capable of specific binding to the KASEKIFYV-HLA a0201 complex.

FIG. 10 is a binding curve of the soluble reference TCR, i.e., the wild-type TCR, to the KASEKIFYV-HLA A0201 complex.

FIG. 11 is a graph showing the results of an experiment on the activation function of effector cells transfected with the high affinity TCR of the invention.

Detailed Description

The present invention, through extensive and intensive studies, has resulted in obtaining a high affinity T Cell Receptor (TCR) that recognizes a KASEKIFYV short peptide (derived from an AFP protein) presented in the form of the KASEKIFYV-HLA A0201 complex. The high affinity TCR has 3 CDR regions in its alpha chain variable domain:

CDR1α：TSESDYY

CDR2α：QEAYKQQN

CDR3 α: AYRSGIIQGAQKLV; and/or in the 3 CDR regions of its beta chain variable domain:

CDR1β：PRHDT

CDR2β：FYEKMQ

CDR3 β: ASSSDRELLFYNEQF; and, the affinity and/or binding half-life of the inventive TCR after mutation to the KASEKIFYV-HLA a0201 complex described above is at least 2-fold that of the wild-type TCR.

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

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

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

Term(s) for

T Cell Receptor (TCR)

The TCR may be described using the international immunogenetics information system (IMGT). Native α β heterodimeric TCRs have an α chain and a β chain. In a broad sense, each chain comprises a variable region, a linker region and a constant region, and the beta chain also typically contains a short diversity region between the variable region and the linker region, but the diversity region is often considered part of the linker region. The TCR connecting region is defined by the unique TRAJ and TRBJ of IMGT, and the TCR constant region is defined by the TRAC and TRBC of IMGT.

Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2, and CDR3, chimeric in a framework sequence. In the IMGT nomenclature, the different numbers of TRAV and TRBV refer to different types of V α and V β, respectively. In the IMGT system, the α chain constant domain has the following symbols: TRAC 01, wherein "TR" denotes a T cell receptor gene; "A" represents an alpha chain gene; c represents a constant region; ". 01" indicates allele 1. The beta-strand constant domain has the following symbols: TRBC1 x 01 or TRBC2 x 01, wherein "TR" denotes 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 of the TCR alpha and beta chains can be obtained by those skilled in the art from published IMGT databases.

The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain. The variable domain is composed of linked variable regions and linked regions. Thus, in the description and claims of this application, the "TCR α chain variable domain" refers to the linked TRAV and TRAJ regions, and likewise the "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 murine or human, preferably human. The constant domain of the TCR comprises an intracellular portion, a transmembrane region, and an extracellular portion. To obtain a soluble TCR in order to determine the affinity between the TCR and the KASEKIFYV-HLA a0201 complex, the TCR of the invention preferably does not comprise a transmembrane region. More preferably, the amino acid sequence of the TCR of the invention refers to the extracellular amino acid sequence of the TCR.

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 respectively SEQ ID NO 27 and SEQ ID NO 28, as shown in FIGS. 9a and 9 b. The alpha chain amino acid sequence and the beta chain amino acid sequence of the reference TCR are respectively SEQ ID NO. 11 and SEQ ID NO. 12, as shown in FIG. 6a and FIG. 6 b. The extracellular amino acid sequence of the alpha chain and the extracellular amino acid sequence of the beta chain of the wild-type TCR are respectively SEQ ID NO 25 and SEQ ID NO 26, as shown in FIG. 8a and FIG. 8 b. In the present invention, the amino acid sequences of the alpha and beta chain variable domains of the wild type TCR capable of binding the KASEKIFYV-HLA A0201 complex are SEQ ID NO 1 and SEQ ID NO 2, respectively, as shown in FIG. 1a and FIG. 1 b. In the present invention, the terms "polypeptide of the invention", "TCR of the invention", "T cell receptor of the invention" are used interchangeably.

Natural interchain disulfide bond and artificial interchain disulfide bond

A set of disulfide bonds, referred to herein as "native interchain disulfide bonds," exist between the C α and C β chains of the membrane proximal region of native TCRs. In the present invention, the artificially introduced interchain covalent disulfide bond, which is located differently from the natural interchain disulfide bond, is referred to as an "artificial interchain disulfide bond".

For convenience of description, the amino acid sequences of TRAC 01 and TRBC1 × 01 or TRBC2 × 01 are position-numbered in the order from the N-terminus to the C-terminus, such as TRBC1 × 01 or TRBC2 × 01, and the 60 th amino acid is P (proline) in the order from the N-terminus to the C-terminus, and thus it may be described as TRBC1 × 01 or TRBC2 × 01 exon 1 Pro60 in the invention, or TRBC1 × 01 or TRBC2 × 01 exon 1, and as TRBC1 × 01 or TRBC2 × 01, and the 61 st amino acid is Q (glutamine) in the order from the N-terminus to the C-terminus, and thus it may be described as TRBC1 × 01 or TRBC2, and as glbc 8201 or TRBC 8536. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. If an amino acid in TRAV, the position listed in IMGT is numbered 46, it is described herein as the 46 th amino acid of TRAV, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described.

Tumor(s)

The term "tumor" is meant to include all types of cancer cell growth or carcinogenic processes, metastatic or malignant transformed cells, tissues or organs, regardless of the type of pathology or the stage of infection. Examples of tumors include, but are not limited to: solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include: malignant tumors of different organ systems, such as sarcomas, squamous carcinomas of the lung and cancers. For example: infected prostate, lung, breast, lymph, gastrointestinal (e.g., colon), and genitourinary tract (e.g., kidney, epithelial cells), pharynx. Squamous carcinoma of the lung includes malignant tumors, such as, for example, most cancers of the colon, rectum, renal cell, liver, non-small cell, small intestine and esophagus. Metastatic lesions of the above-mentioned cancers can likewise be treated and prevented using the methods and compositions of the present invention.

Detailed Description

It is well known that the α chain variable domain and β chain variable domain of a TCR each contain 3 CDRs, similar to the complementarity determining regions of an antibody. CDR3 interacts with antigen short peptides, CDR1 and CDR2 interact with HLA. Thus, the CDRs of the TCR molecule determine their interaction with the antigen short peptide-HLA complex. The amino acid sequences of the alpha chain variable domain and the amino acid sequence of the beta chain variable domain of the wild-type TCR capable of binding the antigen oligopeptide KASEKIFYV to the HLA A0201 complex (i.e., KASEKIFYV-HLA A0201 complex) are SEQ ID NO:1 and SEQ ID NO:2, respectively, which were the first discovery by the present inventors. It has the following CDR regions:

alpha chain variable domain CDR

CDR1α：TSESDYY

CDR2α：QEAYKQQN

CDR3α：AYRSGIIQGAQKLV

Beta chain variable domain CDR

CDR1β：PRHDT

CDR2β：FYEKMQ

CDR3β：ASSSDRELLFYNEQF

The invention obtains the high affinity TCR with the affinity of the KASEKIFYV-HLA A0201 complex being at least 2 times that of the wild type TCR and the KASEKIFYV-HLA A0201 complex by carrying out mutation screening on the CDR region.

The present invention provides a T Cell Receptor (TCR) having binding activity to the KASEKIFYV-HLA A0201 complex.

The 3 CDRs of the variable domain of the wild-type TCR alpha chain of SEQ ID NO. 1, i.e., CDR1, CDR2 and CDR3, are located at positions 27-33, 51-58 and 93-106 of SEQ ID NO. 1, respectively.

The 3 CDRs of the variable domain of wild-type TCR beta chain of the invention SEQ ID NO. 2, i.e., CDR1, CDR2 and CDR3, are located at positions 27-31, 49-54 and 92-106 of SEQ ID NO. 2, respectively. Thus, the amino acid residue numbering is as shown in SEQ ID NO 2, with 96D being the 5 th position D of CDR3 β, 98E being the 7 th position E of CDR3 β, 99L being the 8 th position L of CDR3 β, 100L being the 9 th position L of CDR3 β, 101F being the 10 th position F of CDR11 β, 102Y being the 11 th position Y of CDR3 β, 103N being the 12 th position N of CDR3 β, 104E being the 13 th position E of CDR3 β, 105Q being the 14 th position Q of CDR3 β, and 106F being the 15 th position F of CDR3 β.

Preferably, the TCR β chain variable domain after mutation comprises one or more amino acid residues selected from the group consisting of: 96V, 98I, 99P, 100E or 100I or 100N or 100P or 100V, 101D or 101H or 101N or 101Y, 102A or 102K or 102N or 102P or 102V, 103A or 103D or 103E or 103P, 104L or 104V, 105P or 105T, 106H, wherein the numbering of the amino acid residues is that shown in SEQ ID NO. 2.

More specifically, the specific forms of said mutations in the variable domain of the beta chain include one or several of D96V, E98I, L99P, L100E/I/N/P/V, F101D/H/N/Y, Y102A/K/N/P/V, N103A/D/E/P, E104L/V, Q105P/T, F106H.

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

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

Preferably, the TCR comprises (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain.

The reference TCR was obtained by mutating Thr48 of exon 1 of the α chain constant region TRAC 01 of the wild-type TCR to cysteine and Ser57 of exon 1 of the β chain constant region TRBC1 x 01 or TRBC2 x 01 to cysteine according to site-directed mutagenesis methods well known to those skilled in the art, the amino acid sequences of which are SEQ ID NO:11 and SEQ ID NO:12, respectively, as shown in fig. 6a and 6b, and the mutated cysteine residues are indicated in bold letters. The cysteine substitutions described above 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 a more convenient assessment of the binding affinity and/or binding half-life between the TCR and KASEKIFYV-HLA a0201 complex. It will be appreciated that the CDR regions of the TCR variable region determine their affinity for the pMHC complex and therefore cysteine substitutions in the TCR constant region as described above do not have an effect on the binding affinity and/or binding half-life of the TCR. Therefore, in the present invention, the measured binding affinity between the reference TCR and the KASEKIFYV-HLA A0201 complex is considered to be the binding affinity between the wild type TCR and the KASEKIFYV-HLA A0201 complex. Likewise, if the binding affinity between the inventive TCR and the KASEKIFYV-HLA a0201 complex is determined to be at least 10 times greater than the binding affinity between the reference TCR and the KASEKIFYV-HLA a0201 complex, i.e. equivalent to the binding affinity between the inventive TCR and the KASEKIFYV-HLA a0201 complex being at least 10 times greater than the binding affinity between the wild-type TCR and the KASEKIFYV-HLA a0201 complex.

Knots can be determined by any suitable methodSynthetic affinity (equilibrium constant K with dissociation)_DInversely proportional) and binding half-life (denoted as T)_1/2). Such as by surface plasmon resonance. It will be appreciated that doubling the affinity of the TCR will result in K_DAnd (4) halving. T is_1/2Calculated as In2 divided by dissociation rate (K)_off). Thus, T_1/2Doubling can result in K_offAnd (4) halving. Preferably, the binding affinity or binding half-life of a given TCR is measured several times, e.g. 3 times or more, using the same assay protocol, and the results are averaged. In a preferred embodiment, the surface plasmon resonance (BIAcore) method of the examples herein is used to detect the affinity of soluble TCRs, provided that: the temperature is 25 ℃, and the PH value is 7.1-7.5. The method detects the dissociation equilibrium constant K of the reference TCR to the KASEKIFYV-HLA A0201 complex_DAt 1.21E-04M, i.e., 121. mu.M, the dissociation equilibrium constant K of the wild-type TCR for the KASEKIFYV-HLA A0201 complex is considered in the present invention_DAlso 121. mu.M. Doubling of the affinity of the TCR will result in K_DHalving, so if the dissociation equilibrium constant K of the high affinity TCR for the KASEKIFYV-HLA A0201 complex is detected_DAt 1.21E-05M, i.e., 12.1. mu.M, this indicates that the high affinity TCR has 10-fold greater affinity for the KASEKIFYV-HLA A0201 complex than the wild type TCR. K is well known to those skilled in the art_DConversion between units of value, i.e. 1M 10⁶μM，1μM＝1000nM，1nM＝1000pM。

In a preferred embodiment of the invention, the TCR has at least 2-fold greater affinity for the KASEKIFYV-HLA a0201 complex than a wild-type TCR; preferably, at least 5 times; more preferably, at least 10 times.

In another preferred embodiment, the TCR has at least 50-fold greater affinity for the KASEKIFYV-HLA a0201 complex than a wild-type TCR; preferably, at least 100 times; more preferably, at least 300 times.

Specifically, the dissociation equilibrium constant KD of the TCR to the KASEKIFYV-HLA A0201 complex is less than or equal to 60 mu M;

in another preferred embodiment, the TCR has a dissociation equilibrium constant for the KASEKIFYV-HLA A0201 complex of 1 μ M ≦ KD ≦ 50 μ M.

The mutation may be performed 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. These methods are detailed in a number of standard molecular biology texts. For more details on Polymerase Chain Reaction (PCR) mutagenesis and Cloning by restriction enzymes, see Sambrook and Russell, (2001) Molecular Cloning-A Laboratory Manual (third edition) CSHL Press. More information on the LIC method can be found (Rashtchian, (1995) Curr Opin Biotechnol 6(1): 30-6).

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

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

In the present invention, the amino acid sequences of the α chain variable domain and the β chain variable domain that form the heterodimeric TCR molecule are preferably selected from table 1 below:

TABLE 1

For the purposes of the present invention, the inventive TCRs are parts having at least one TCR α and/or TCR β chain variable domain. They typically comprise both a TCR α chain variable domain and a TCR β chain variable domain. They may be α β heterodimers or single chain forms or any other form that is stable. In adoptive immunotherapy, the entire long chain (containing both cytoplasmic and transmembrane domains) of an α β heterodimeric TCR can be transfected. The TCRs of the invention are useful as targeting agents for delivering therapeutic agents to antigen presenting cells or in combination with other molecules to produce bifunctional polypeptides for targeting effector cells, where the TCRs are preferably in soluble form.

For stability, it is disclosed in the prior art that the introduction of an artificial interchain disulfide bond between the α and β chain constant domains of a TCR enables soluble and stable TCR molecules to be obtained, as described in patent document PCT/CN 2015/093806. Thus, the inventive TCR may be one in which an artificial interchain disulfide bond is introduced between residues of the α and β chain constant domains thereof. Cysteine residues form an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at appropriate positions in native TCRs to form artificial interchain disulfide bonds. For example, a substitution of Thr48 for exon 1 of TRAC × 01 and a substitution of Ser57 for exon 1 of TRBC1 × 01 or TRBC2 × 01 form disulfide bonds. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1; tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01; thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1; ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1; arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1; pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; or Tyr10 and TRBC1 and 01 of TRAC 01 exon 1 or Glu20 of TRBC2 and 01 exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. The TCR constant domains of the invention may be truncated at one or more of their C-termini by up to 15, or up to 10, or up to 8 or fewer amino acids, so as not to include cysteine residues for the purpose of deleting the native interchain disulfide bond, or by mutating the cysteine residues forming the native interchain disulfide bond to another amino acid.

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

In addition, for stability, patent document PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR can significantly improve the stability of the TCR. Thus, the high affinity TCRs of the invention may also contain an artificial interchain disulfide bond between the α chain variable region and the β chain constant region. Specifically, the cysteine residue that forms an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR is substituted for: 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 TRBC1 x 01 or TRBC2 x 01 exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain, the α chain forming a heterodimer with the β chain. More preferably, such a TCR may comprise the a chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the a chain constant domain, the a chain variable domain of the TCR forming a heterodimer with the β chain.

For stability, on the other hand, the inventive TCRs also include TCRs having mutations in their hydrophobic core region, preferably mutations that improve the stability of the inventive TCRs, as described in the patent publication WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable region amino acid 11, 13, 19, 21, 53, 76, 89, 91, 94 and/or alpha chain J gene (TRAJ) short peptide amino acid position reciprocal 3, 5, 7 and/or beta chain J gene (TRBJ) short peptide amino acid position reciprocal 2, 4, 6, wherein the position numbering of the amino acid sequence is according to the position numbering listed in the International Immunogenetic information System (IMGT). The above-mentioned international system of immunogenetics information is known to the skilled person and the position numbering of the amino acid residues of the different TCRs in IMGT can be derived from this database.

More specifically, the TCR with the mutated hydrophobic core region of the invention can be a high stability single chain TCR with a flexible peptide chain connecting the variable domains of the α and β chains of the TCR. The CDR regions of the TCR variable region determine the affinity of the TCR variable region with the short peptide-HLA complex, and the mutation of the hydrophobic core can stabilize the TCR without affecting the affinity of the TCR variable region with the short peptide-HLA complex. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the TCR α and β chain variable domains. The template chain for screening high affinity TCR constructed in example 1 of the present invention is the above-described high stability single chain TCR comprising the hydrophobic core mutation. Using a TCR with higher stability, the affinity between the TCR and the KASEKIFYV-HLA-A0201 complex can be more conveniently assessed.

The CDR regions of the alpha chain variable domain and the beta chain variable domain of the single-chain template TCR are completely identical to the CDR regions of the wild-type TCR. That is, the 3 CDRs of the α chain variable domain are CDR1 α: TSESDYY, CDR2 α: qeykqn, CDR3 α: AYRSGIIQGAQKLV and the 3 CDRs of the β chain variable domain are CDR1 β: PRHDT, CDR2 β: fYEKMQ, CDR3 β: ASSSDRELLFYNEQF are provided. The amino acid sequence (SEQ ID NO:9) and the nucleotide sequence (SEQ ID NO:10) of the single-chain template TCR are shown in FIG. 5a and FIG. 5b, respectively. Thus, a single-chain TCR composed of an alpha chain variable domain and a beta chain variable domain having high affinity for the KASEKIFYV-HLA A0201 complex is selected.

The 3 CDRs of the variable domain SEQ ID NO 3 of the single-chain template TCR alpha chain of the invention, namely CDR1, CDR2 and CDR3 are located at positions 27-33, 51-58 and 93-106 of SEQ ID NO 1, respectively.

The 3 CDRs of the variable domain SEQ ID NO. 4 of the single-chain template TCR beta chain of the invention, namely CDR1, CDR2 and CDR3 are located at positions 27-31, 49-54 and 92-106 of SEQ ID NO. 2, respectively. Thus, the amino acid residue numbering is as shown in SEQ ID NO 4, with 96D being the 5 th position D of CDR3 β, 98E being the 7 th position E of CDR3 β, 99L being the 8 th position L of CDR3 β, 100L being the 9 th position L of CDR3 β, 101F being the 10 th position F of CDR11 β, 102Y being the 11 th position Y of CDR3 β, 103N being the 12 th position N of CDR3 β, 104E being the 13 th position E of CDR3 β, 105Q being the 14 th position Q of CDR3 β, and 106F being the 15 th position F of CDR3 β.

The α β heterodimer of the present invention having high affinity for the KASEKIFYV-HLA-A0201 complex is obtained by transferring the CDR regions of the α and β chain variable domains of the selected high affinity single-chain TCR to the corresponding positions of the α chain variable domain (SEQ ID NO:1) and β chain variable domain (SEQ ID NO:2) of the wild-type TCR.

The TCRs of the invention may also be provided in the form of multivalent complexes. Multivalent TCR complexes of the invention comprise polymers formed by association of two, three, four or more TCRs of the invention, such as might be produced as a tetramer using the tetrameric domain of p53, or a complex formed by association of a plurality of TCRs of the invention with 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, and can also be used to generate intermediates for other multivalent TCR complexes having such applications.

The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the KASEKIFYV-HLA-a0201 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.

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

Therapeutic agents that may be associated or conjugated with the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. antibody Fc fragment (Mosquera et al, 2005, Journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62,319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, Gene therapy (Gene therapy)11, 1234); 8. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 9. nano magnetic particles; 10. a prodrug activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, 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, whose binding to the TCR directs effector cells to better target cells. A preferred embodiment is a TCR of the invention conjugated 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 of the present invention and the anti-CD 3 single chain antibody comprises the amino acid sequence of the TCR α chain variable domain: 1 and/or the variable domain amino acid sequence of the TCR beta chain: one of SEQ ID NOS 13-24.

The invention also relates to nucleic acid molecules encoding the inventive TCRs. The nucleic acid molecules of the invention may be in the form of DNA or in the form of RNA. The DNA may be the coding strand or the non-coding strand. For example, a nucleic acid sequence encoding a TCR of the invention may be identical to or a degenerate variant of a nucleic acid sequence as set out in the figures of the invention. By way of illustration of the meaning of "degenerate variant", as used herein, is meant a nucleic acid sequence which encodes a protein sequence having SEQ ID NO. 9, but differs from the sequence of SEQ ID NO. 10.

The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can be obtained by, but not limited to, PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the TCRs of the invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various 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, as well as to host cells genetically engineered with the vectors or coding sequences of the invention.

The invention also includes isolated cells, particularly T cells, expressing a TCR of the invention. There are many methods suitable for T cell transfection using DNA or RNA encoding the high affinity TCRs of the invention (e.g., Robbins et al, (2008) J.Immunol.180: 6116-. T cells expressing the high affinity TCRs of the invention may be used for adoptive immunotherapy. Those skilled in the art will be able to recognize many suitable methods for adoptive therapy (e.g., Rosenberg et al, (2008) Nat Rev Cancer 8 (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 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.

It should be understood that the amino acid names herein are given by the international single english letter designation, and the three english letters corresponding to the amino acid names are respectively: ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y) and Val (V); in the present invention, Pro60 or 60P both represent proline at position 60. In addition, the expression of a specific form of the mutation described in the present invention is such that "D96V" represents that D at position 96 is substituted by V, and "E104L/V" represents that E at position 104 is substituted by L or by V. Others may be analogized.

In the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the structure and function of the protein. Thus, the TCR of the invention also includes TCRs in which up to 5, preferably up to 3, more preferably up to 2, most preferably 1 amino acid (especially outside the CDR regions) of the TCR of the invention has been replaced by amino acids of similar or analogous nature, and still retain its functionality.

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

The TCR of the invention, the TCR complex or the TCR-transfected T cell of the invention may 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 dose form, typically in a sealed container, and may be provided as part of a kit. Such kits (but not necessarily) include instructions for use. It may comprise a plurality of said unit dosage forms.

In addition, the TCRs of the 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 further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Such vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences, Mack pub. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.

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

Generally, the therapeutic compositions can be prepared as injectables, e.g., as liquid solutions or suspensions; solid forms suitable for constitution with a solution or suspension, or liquid carrier, before injection, may also be prepared.

Once formulated, the compositions of the present invention 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 a human.

When the pharmaceutical composition of the present invention is used for practical treatment, various dosage forms of the pharmaceutical composition may be used depending on the use case. Preferably, injections, oral agents and the like are exemplified.

These pharmaceutical compositions can be formulated by mixing, dilution or dissolution according to a conventional method, and suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents are occasionally added, and the formulation process can be carried out in a conventional manner according to the dosage form.

The pharmaceutical compositions of the present invention may also be administered in the form of sustained release formulations. For example, the inventive TCR may be incorporated into a pellet or microcapsule carried by a slow release polymer, which pellet or microcapsule is then surgically implanted into the tissue to be treated. As examples of the sustained-release polymer, ethylene-vinyl acetate copolymer, polyhydroxymethacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer and the like can be exemplified, and biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer can be preferably exemplified.

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

The main advantages of the invention are:

(1) the affinity and/or binding half-life of the inventive TCR to said KASEKIFYV-HLA-a0201 complex is at least 2-fold, preferably at least 10-fold that of the wild-type TCR.

(2) The affinity and/or binding half-life of the inventive TCR to said KASEKIFYV-HLA-a0201 complex is at least 50-fold, preferably at least 100-fold, more preferably up to 300-fold that of the wild-type TCR.

(3) For target cells, effector cells that transduce the high affinity TCRs of the invention have a strong activation function on the target cells.

The following specific examples further illustrate the invention. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook and Russell et al, Molecular Cloning: A Laboratory Manual (third edition) (2001) CSHL Press, or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

Materials and methods

The experimental materials used in the examples of the present invention are commercially available as such, unless otherwise specified, wherein e.coli DH5 α is available from Tiangen, e.coli BL21(DE3) is available from Tiangen, e.coli Tuner (DE3) is available from Novagen, and plasmid pET28a is available from Novagen.

Example 1 Generation of Stable Single chain TCR template chains with hydrophobic core mutations

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 of the stable single-chain TCR molecule are SEQ ID NO. 9 and SEQ ID NO. 10 respectively, as shown in FIG. 5a and FIG. 5 b. And the single-chain TCR molecule is taken as a template to screen the high-affinity TCR molecule. The amino acid sequences of the alpha variable domain (SEQ ID NO:3) and the beta variable domain (SEQ ID NO:4) of the template strand are shown in FIGS. 2a and 2 b; the corresponding DNA sequences are SEQ ID NO 5 and 6, respectively, as shown in FIG. 3a and FIG. 3 b; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are SEQ ID NO 7 and 8, respectively, as shown in FIG. 4a and FIG. 4 b.

The target gene carrying the template strand was digested simultaneously with Nco I and Not I, and ligated to pET28a vector digested simultaneously with Nco I and Not I. The ligation product was transformed into e.coli DH5 α, coated with LB plates containing kanamycin, inverted cultured overnight at 37 ℃, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the sequence was determined to be correct, recombinant plasmids were extracted and transformed into e.coli BL21(DE3) for expression.

Example 2 expression, renaturation and purification of the Stable Single-chain TCR constructed in example 1

The BL21(DE3) 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 OD₆₀₀0.6-0.8, IPTG was added to a final concentration of 0.5mM and incubation was continued for 4h at 37 ℃. The cell pellet was harvested by centrifugation at 5000rpm for 15min, the 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 centrifuged at 6000rpm for 15min to collect the inclusion bodies. The inclusion bodies were dissolved in buffer (20mM Tris-HCl)pH 8.0, 8M urea), removing insoluble substances by high-speed centrifugation, quantifying the supernatant by using a BCA method, subpackaging, and storing at-80 ℃ for later use.

To 5mg of solubilized single-chain TCR inclusion body protein, 2.5mL of buffer (6M Gua-HCl, 50mM Tris-HCl pH 8.1, 100mM NaCl, 10mM EDTA) was added, DTT was added to a final concentration of 10mM, and treatment was carried out at 37 ℃ for 30 min. The treated single-chain TCR was added dropwise to 125mL of renaturation buffer (100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM beta-mercaptoethylamine, 1.87mM Cystamine) with a syringe, stirred at 4 ℃ for 10min, and then the renaturation solution was filled into a cellulose membrane dialysis bag with a cut-off of 4kDa, and the bag was placed in 1L of precooled water and stirred slowly at 4 ℃ overnight. After 17 hours, the dialysate was changed to 1L of pre-chilled buffer (20mM Tris-HCl pH 8.0), dialysis was continued at 4 ℃ for 8h, and then dialysis was continued overnight with the same fresh buffer. After 17 hours, the sample was filtered through a 0.45 μ M filter, vacuum degassed and then passed through an anion exchange column (HiTrap Q HP, GE Healthcare), the protein was purified using a 0-1M NaCl linear gradient eluent formulated in 20mM Tris-HCl pH 8.0, the collected fractions were subjected to SDS-PAGE analysis, the fractions containing single-stranded TCR were concentrated and then further purified using a gel filtration column (Superdex 7510/300, GE Healthcare), and the target fraction was also subjected to SDS-PAGE analysis.

The eluted fractions for BIAcore analysis were further tested for purity using gel filtration. The conditions are as follows: the chromatography 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 214 nm.

Example 3 binding characterisation

BIAcore analysis

The BIAcore T200 real-time assay system was used to detect the binding activity of TCR molecules to the KASEKIFYV-HLA-A0201 complex. The coupling process was completed by adding an anti-streptavidin antibody (GenScript) to a coupling buffer (10mM sodium acetate buffer, pH 4.77), then passing 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 ethanolamine hydrochloric acid solution to complete the coupling at a level of about 15,000 RU. 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, followed by flowing the KASEKIFYV-HLA-A0201 complex through the detection channel, the other channel as a reference channel, and then flowing 0.05 mM of biotin at a flow rate of 10. mu.L/min through the chip for 2min, blocking the remaining binding sites of streptavidin. The affinity of the TCR was determined by single cycle kinetic assay, in which the TCR was diluted with HEPES-EP buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% P20, pH 7.4) to several different concentrations, passed over the chip surface sequentially at a flow rate of 30. mu.L/min, with a binding time of 120s for each sample, and dissociated for 600s after the last sample. At the end of each assay run, the chip was regenerated with 10mM Gly-HCl pH 1.75. Kinetic parameters were calculated using BIAcore Evaluation software.

The above KASEKIFYV-HLA-A0201 complex is prepared as follows:

a. purification of

Collecting 100ml E.coli liquid for inducing expression of heavy chain or light chain, centrifuging at 4 ℃ for 10min at 8000g, washing the thalli once with 10ml PBS, then resuspending the thalli with 5ml BugBuster Master Mix Extraction Reagents (Merck) by vigorous shaking, rotatably incubating at room temperature for 20min, centrifuging at 4 ℃ for 15min at 6000g, discarding supernatant, and collecting inclusion body.

Resuspending the inclusion bodies in 5ml of BugBuster Master Mix, and rotary incubating at room temperature for 5 min; adding 30ml of 10-fold diluted BugBuster, uniformly mixing, and centrifuging at 4 ℃ at 6000g for 15 min; discarding the supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing, centrifuging at 4 ℃ for 15min at 6000g, repeating twice, adding 30ml of 20mM Tris-HCl pH 8.0 to resuspend the inclusion bodies, mixing, centrifuging at 4 ℃ for 15min at 6000g, 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 using a BCA reagent kit.

b. Renaturation

The synthesized short peptide KASEKIFYV (Beijing Saiban Gene technology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized with 8M Urea, 20mM Tris pH 8.0, 10mM DTT and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. The KASEKIFYV peptide was added to a renaturation buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidative glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4 ℃) at 25mg/L (final concentration), followed by the addition of 20mg/L of light chain and 90mg/L of heavy chain in sequence (final concentration, the heavy chain was added in three portions, 8 h/time), and the renaturation was carried out at 4 ℃ for at least 3 days until completion, and SDS-PAGE checked for success or failure of the renaturation.

c. Purification after renaturation

The renaturation buffer was replaced by dialysis against 10 volumes of 20mM Tris pH 8.0, at least two times to reduce the ionic strength of the solution sufficiently. 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) anion exchange column (5ml bed volume). Using Akta purifier (GE general electric company), 20mM Tris pH 8.0 prepared 0-400mM NaCl linear gradient elution protein, pMHC approximately 250mM NaCl elution, collecting the peak components, SDS-PAGE detection purity.

d. Biotinylation of the compound

The purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while displacing the buffer to 20mM Tris pH 8.0, followed by addition of biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50. mu. M D-Biotin, 100. mu.g/ml BirA enzyme (GST-BirA), incubation of the mixture at room temperature overnight, and SDS-PAGE to determine the completion of biotinylation.

e. Purification of biotinylated complexes

The biotinylated pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using an Akta purifier (GE general electric Co., Ltd.)^TM16/60S200HR column (GE general electric) was loaded with 1ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a single peak elution at approximately 55 ml. The fractions containing the protein were pooled and subjected to Millipore ultrafiltration tubesConcentration, BCA (Thermo) assay for protein concentration, addition of the protease inhibitor cocktail (Roche) and storage of the biotinylated pMHC molecules in aliquots at-80 ℃.

Example 4 Generation of high affinity TCR

Phage display technology is a means of generating libraries of TCR high affinity variants to screen for high affinity variants. The TCR phage display and screening methods described by Li et al ((2005) Nature Biotech 23(3):349-354) were applied to the single-chain TCR templates in example 1. Libraries of high affinity TCRs were created by mutating the CDR regions of the template strand and panning was performed. The phage library after several rounds of panning has specific binding with corresponding antigen, and single clone is picked out and sequence analysis is carried out.

The CDR region mutations of the selected high affinity single chain TCR were introduced into the corresponding sites of the variable domain of the α β heterodimeric TCR and their affinity to the KASEKIFYV-HLA-A0201 complex was examined by BIAcore. The introduction of the high affinity mutation points in the CDR regions described above is performed by site-directed mutagenesis methods well known to those skilled in the art. The amino acid sequences of the alpha chain and beta chain variable domains of the wild-type TCR are shown in FIG. 1a (SEQ ID NO:1) and FIG. 1b (SEQ ID NO:2), respectively.

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 KASEKIFYV-HLA A0201 complex, the α β heterodimeric TCR may be a TCR in which a cysteine residue has been introduced into the constant region of the α and β chains, respectively, to form an artificial interchain disulfide bond, in this example the amino acid sequences of the TCR α and β chains after introduction of the cysteine residue are shown in FIGS. 6a (SEQ ID NO:11) and 6b (SEQ ID NO:12), respectively, and the introduced cysteine residue is shown in bold letters.

Extracellular sequence genes of TCR α and β chains to be expressed were synthesized and inserted into expression vector pET28a + (Novagene) by standard methods described in Molecular Cloning a Laboratory Manual (third edition, Sambrook and Russell), with upstream and downstream Cloning sites being NcoI and NotI, respectively. Mutations in CDR regions are introduced by overlap pcr (overlap pcr), well known to those skilled in the art. The insert was confirmed by sequencing without error.

Example 5 expression, renaturation and purification of high affinity TCR

The expression vectors of TCR alpha and beta chains are transformed into expression bacteria BL21(DE3) by chemical transformation method, and the bacteria are grown in LB culture solution and OD₆₀₀Inclusion bodies formed after expression of the α and β chains of the TCR were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution several times at 0.6 final induction with final concentration of 0.5mM IPTG, and finally dissolved in 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 20mM Tris (pH 8.1).

The TCR α and β chains after lysis were separated by 1:1 was rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mM β -mercaptamine (4 ℃) to a final concentration of 60 mg/mL. After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ℃ C.) and after 12 hours, the deionized water was changed to a buffer (20mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours. The solution after completion of dialysis was filtered through a 0.45. mu.M filter and then purified by an anion exchange column (HiTrap Q HP, 5ml, GE Healthcare). The TCR eluted with peaks containing successfully renatured α and β dimers was confirmed by SDS-PAGE gel. The TCR was subsequently 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 6 BIAcore analysis results

The affinity of the α β heterodimeric TCR with the high affinity CDR regions introduced to the KASEKIFYV-HLA-A0201 complex was determined using the method described in example 3.

The invention obtains the amino acid sequence of the variable domain of the high affinity TCR beta chain, as shown in figure 7(1) -figure 7 (12). Since the CDR regions of the TCR molecule determine its affinity for the corresponding pMHC complex, one skilled in the art would be able to predict that α β heterodimeric TCRs incorporating high affinity mutation points also have high affinity for the KASEKIFYV-HLA-A0201 complex. Expression vectors were constructed using the method described in example 4, and the α β heterodimeric TCRs introduced with high affinity mutations described above were expressed, renatured and purified using the method described in example 5, and then their affinity to KASEKIFYV-HLA-a0201 complex was determined using BIAcore T200, as shown in table 2 below.

TABLE 2

As can be seen from Table 2, the affinity of the high affinity TCRs obtained in accordance with the invention is at least 2-fold greater than the affinity of the wild-type TCR for the KASEKIFYV-HLA-A0201 complex.

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

Fusion molecules were prepared by fusing an anti-CD 3 single chain antibody (scFv) to an α β heterodimeric TCR. The scFv of anti-CD 3 is fused to the β chain of the TCR, which β chain may comprise the β chain variable domain of any of the above-described high affinity α β heterodimeric TCRs, and the TCR α chain of the fused molecule may comprise the α chain variable domain of any of the above-described high affinity α β heterodimeric TCRs.

Construction of fusion molecule expression vectors

1. Construction of alpha chain expression vector

The target gene carrying the alpha chain of the α β heterodimeric TCR was double-digested with Nco i and Not i, and ligated to pET28a vector double-digested with Nco i and Not i. The ligation product was transformed into e.coli DH5 α, spread on LB plates containing kanamycin, cultured at 37 ℃ for inversion overnight, positive clones were picked for PCR screening, positive recombinants were sequenced, and after the sequence was determined to be correct, recombinant plasmids were extracted and transformed into e.coli Tuner (DE3) for expression.

2. Construction of anti-CD 3(scFv) -beta chain expression vector

By the overlap PCR method, primers were designed to link the anti-CD 3scFv and the high-affinity heterodimeric TCR beta chain gene, the middle linking short peptide (linker) is GGGGS, and the gene fragment of the fusion protein of the anti-CD 3scFv and the high-affinity heterodimeric TCR beta chain is provided with restriction enzyme sites Nco I (CCATGG) and Not I (GCGGCCGC). The PCR amplification product was digested simultaneously with Nco I and Not I, and ligated with pET28a vector digested simultaneously with Nco I and Not I. The ligation product was transformed into e.coli DH5 α competent cells, coated with LB plates containing kanamycin, inverted cultured overnight at 37 ℃, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the sequence was determined to be correct, recombinant plasmids were extracted and transformed into e.coli Tuner (DE3) competent cells for expression.

Expression, renaturation and purification of fusion proteins

The expression plasmids were transformed into E.coli Tuner (DE3) competent cells, respectively, and plated on LB plates (kanamycin 50. mu.g/mL) and incubated at 37 ℃ overnight. The next day, selecting clones, inoculating to 10mL LB liquid medium (50. mu.g/mL kanamycin) for culturing for 2-3h, inoculating to 1L LB medium according to the volume ratio of 1:100, continuing to culture until OD600 is 0.5-0.8, and adding 1mM IPTG to induce the expression of the target protein. After 4 hours of induction, cells were harvested by centrifugation at 6000rpm for 10 min. The cells were washed once with PBS buffer and aliquoted, and 200mL of the cells from the bacterial culture were lysed with 5mL of BugBuster Master Mix (Merck) and the inclusion bodies were collected by centrifugation at 6000g for 15 min. 4 detergent washes were then performed to remove cell debris and membrane components. The inclusion bodies are then washed with a buffer such as PBS to remove the detergent and salts. Finally, inclusion bodies were solubilized with a buffer solution containing 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 20mM Tris, pH 8.1, and the inclusion body concentration was determined, and they were stored frozen at-80 ℃ after being dispensed.

The TCR α chain and the anti-CD 3(scFv) - β chain after solubilization were separated by a 2: 5 in 5M Urea (urea), 0.4M L-arginine (L-arginine), 20mM Tris pH 8.1, 3.7mM cystamine, 6.6mM β -mer capoethylamine (4 ℃ C.), final concentrations of α chain and anti-CD 3(scFv) - β chain were 0.1mg/mL, 0.25 mg/mL, respectively.

After mixing, the solution was dialyzed against 10 volumes of deionized water (4 ℃ C.) and after 12 hours, the deionized water was changed to a buffer (10mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours. The solution after completion of dialysis was filtered through a 0.45. mu.M filter and then purified by an anion exchange column (HiTrap Q HP 5ml, GE healthcare). The eluted peaks contain TCR alpha chain and anti-CD 3(scFv) -beta chain dimers of which were successfully renatured TCR alpha chain and CD-PAGE gel confirmed. The TCR fusion molecules were then further purified by size exclusion chromatography (S-10016/60, GE healthcare) and re-purified on an anion exchange column (HiTrap Q HP 5ml, GE healthcare). The purity of the purified TCR fusion molecule was greater than 90% as determined by SDS-PAGE and the concentration was determined by BCA.

Example 8 activation function assay of Effector cells transfected with high affinity TCR of the invention

This example demonstrates that effector cells transfected with the high affinity TCRs of the invention have good specific activation of target cells. The function and specificity of the high affinity TCR of the invention in cells was examined by ELISPOT experiments.

Methods for detecting cell function using the ELISPOT assay are well known to those skilled in the art. The TCRs of the invention were randomly selected to transfect CD3+ T cells isolated from blood of healthy volunteers as effector cells. The TCRs and their numbering are known from Table 2 as TCR5 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO:17), TCR6 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO:18), TCR7 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO:19), TCR1 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO:13), TCR9 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 21), TCR10 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO:22), 2 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain TCR ID NO:14) and TCR8 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain TCR ID NO:20) and the wild type cell marker (wild type) transfected with the same marker as the control cell marker TCR6 (wild type) and the wild type transfected cell marker The cell of (a). The target cell lines are K562-A2(A2 overexpressed), SW620-SSX2 (SSX2 overexpressed), and K562-A11(A11 overexpressed) cells. Wherein the target cell lines K562-A2 and SW620-SSX2 are used as positive tumor cell lines; K562-A11 was a negative tumor cell line as a control.

First, an ELISPOT plate was prepared. ELISPOT plate ethanol activation coating, 4 degrees C overnight. On day 1 of the experiment, the coating solution was removed, washed and sealed, incubated at room temperature for two hours, the sealing solution was removed, and the test was runThe components tested were added to an ELISPOT plate: the target cell is 2X 10⁴One/well, effector cell 10³One/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 color development, dried, and spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID20 Co.).

The experimental results are shown in fig. 11, and for the positive target cell line, the effector cells transfected with the high affinity TCR of the invention generate a good specific activation effect, and the function is far better than that of effector cells transfected with wild TCR; while cells transduced other TCRs produced essentially no activation.

All documents referred to herein are incorporated by reference into this application as if each had been individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined in the appended claims.

Sequence listing

<110> Guangdong Xiangxue accurate medical technology Limited

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Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser Glu Ser Asp Tyr

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Ile Leu Val

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Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr Glu Asn Arg Phe

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Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile Ser

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Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys Ala Tyr Arg Ser

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Gly Ile Ile Gln Gly Ala Gln Lys Leu Val Phe Gly Gln Gly Thr Arg

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Leu Thr Ile Asn Pro

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Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

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Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Leu Phe Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

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Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Leu Ser Val Gln Glu Gly

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Glu Thr Val Thr Ile Ser Cys Thr Tyr Asp Thr Ser Glu Ser Asp Tyr

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Pro Ile Leu Val

35 40 45

Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr Glu Asn Arg Phe

50 55 60

Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile Ser

65 70 75 80

Asp Val Gln Pro Gly Asp Ala Ala Met Tyr Phe Cys Ala Tyr Arg Ser

85 90 95

Gly Ile Ile Gln Gly Ala Gln Lys Leu Val Phe Gly Gln Gly Thr Arg

100 105 110

Leu Thr Ile Asn Pro

115

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Ala Ala Gly Val Thr Gln Ser Pro Arg His Leu Ser Val Glu Lys Gly

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Glu Thr Val Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

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Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Leu Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Ile Ser Ser

65 70 75 80

Val Glu Pro Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Leu Phe Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

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gctcaaactg ttactcagag ccaaccggag ctgagcgtgc aagagggtga aaccgttacc 60

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ccgagccgtc aaccgatcct ggttattcgt caggaagcgt acaaacagca aaacgcgacc 180

gaaaaccgtt tcagcgtgaa ctttcagaag gcggcgaaaa gcttcagcct gaagatcagc 240

gacgttcaac cgggcgatgc ggcgatgtac ttttgcgcgt atcgtagcgg tatcattcag 300

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gcggcgggcg tgacccaaag cccgcgtcac ctgagcgtgg agaagggtga aaccgttacc 60

ctgaaatgct atccgatccc gcgtcacgac accgtttact ggtatcagca aggtccgggc 120

caggatctgc aattcctgat cagcttttac gagaagatgc agagcgacaa aggtagcatt 180

ccggatcgtt tcagcgcgca gcaatttagc gactatcaca gcgagctgaa cattagcagc 240

gtggaaccgg gtgacagcgc gctgtacttc tgcgcgagca gcagcgatcg tgagctgctg 300

ttttataacg aacagttctt tggtccgggc acccgtctga ccgttgat 348

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Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

1 5 10 15

Gly Gly Ser Glu Gly Gly Thr Gly

20

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<213> Artificial Sequence (Artificial Sequence)

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Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Leu Ser Val Gln Glu Gly

1 5 10 15

Glu Thr Val Thr Ile Ser Cys Thr Tyr Asp Thr Ser Glu Ser Asp Tyr

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Pro Ile Leu Val

35 40 45

Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr Glu Asn Arg Phe

50 55 60

Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile Ser

65 70 75 80

Asp Val Gln Pro Gly Asp Ala Ala Met Tyr Phe Cys Ala Tyr Arg Ser

85 90 95

Gly Ile Ile Gln Gly Ala Gln Lys Leu Val Phe Gly Gln Gly Thr Arg

100 105 110

Leu Thr Ile Asn Pro Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly

115 120 125

Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Thr Gly Ala Ala Gly

130 135 140

Val Thr Gln Ser Pro Arg His Leu Ser Val Glu Lys Gly Glu Thr Val

145 150 155 160

Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr Trp Tyr

165 170 175

Gln Gln Gly Pro Gly Gln Asp Leu Gln Phe Leu Ile Ser Phe Tyr Glu

180 185 190

Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser Ala Gln

195 200 205

Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Ile Ser Ser Val Glu Pro

210 215 220

Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp Arg Glu Leu

225 230 235 240

Leu Phe Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val

245 250 255

Asp

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gctcaaactg ttactcagag ccaaccggag ctgagcgtgc aagagggtga aaccgttacc 60

atcagctgca cctacgacac cagcgaaagc gattactacc tgttctggta taagcagccg 120

ccgagccgtc aaccgatcct ggttattcgt caggaagcgt acaaacagca aaacgcgacc 180

gaaaaccgtt tcagcgtgaa ctttcagaag gcggcgaaaa gcttcagcct gaagatcagc 240

gacgttcaac cgggcgatgc ggcgatgtac ttttgcgcgt atcgtagcgg tatcattcag 300

ggcgcgcaaa aactggtgtt cggtcagggc acccgtctga ccattaaccc gggtggcggt 360

agcgagggcg gtggcagcga aggtggcggt agcgagggcg gtggcagcga aggtggcacc 420

ggtgcggcgg gcgtgaccca aagcccgcgt cacctgagcg tggagaaggg tgaaaccgtt 480

accctgaaat gctatccgat cccgcgtcac gacaccgttt actggtatca gcaaggtccg 540

ggccaggatc tgcaattcct gatcagcttt tacgagaaga tgcagagcga caaaggtagc 600

attccggatc gtttcagcgc gcagcaattt agcgactatc acagcgagct gaacattagc 660

agcgtggaac cgggtgacag cgcgctgtac ttctgcgcga gcagcagcga tcgtgagctg 720

ctgttttata acgaacagtt ctttggtccg ggcacccgtc tgaccgttga t 771

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<213> Artificial Sequence (Artificial Sequence)

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Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser Val Gln Glu Ala

1 5 10 15

Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser Glu Ser Asp Tyr

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Ile Leu Val

35 40 45

Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr Glu Asn Arg Phe

50 55 60

Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile Ser

65 70 75 80

Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys Ala Tyr Arg Ser

85 90 95

Gly Ile Ile Gln Gly Ala Gln Lys Leu Val Phe Gly Gln Gly Thr Arg

100 105 110

Leu Thr Ile Asn Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln

115 120 125

Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp

130 135 140

Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr

145 150 155 160

Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser

165 170 175

Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn

180 185 190

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro

195 200 205

Glu Ser Ser

210

<210> 12

<211> 246

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Leu Phe Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala

115 120 125

Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr

130 135 140

Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser

145 150 155 160

Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro

165 170 175

Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu

180 185 190

Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn

195 200 205

His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu

210 215 220

Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu

225 230 235 240

Ala Trp Gly Arg Ala Asp

245

<210> 13

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Ile Pro Pro Tyr Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 14

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Val

85 90 95

Arg Glu Leu Asn Tyr Val Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 15

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Asn Asp Ala Pro Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 16

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Val Phe Asn Pro Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 17

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Leu Asp Ala Pro Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 18

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Leu Phe Pro Asp Leu Thr Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 19

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Ile His Pro Glu Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 20

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Leu Phe Tyr Ala Val Pro His Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 21

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Leu Asn Pro Glu Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 22

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Glu His Pro Glu Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 23

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Pro Phe Val Pro Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 24

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Val Phe Lys Pro Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 25

<211> 211

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser Val Gln Glu Ala

1 5 10 15

Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser Glu Ser Asp Tyr

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Ile Leu Val

35 40 45

Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr Glu Asn Arg Phe

50 55 60

Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile Ser

65 70 75 80

Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys Ala Tyr Arg Ser

85 90 95

Gly Ile Ile Gln Gly Ala Gln Lys Leu Val Phe Gly Gln Gly Thr Arg

100 105 110

Leu Thr Ile Asn Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln

115 120 125

Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp

130 135 140

Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr

145 150 155 160

Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser

165 170 175

Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn

180 185 190

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro

195 200 205

Glu Ser Ser

210

<210> 26

<211> 246

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Leu Phe Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala

115 120 125

Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr

130 135 140

Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser

145 150 155 160

Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro

165 170 175

Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu

180 185 190

Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn

195 200 205

His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu

210 215 220

Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu

225 230 235 240

Ala Trp Gly Arg Ala Asp

245

<210> 27

<211> 258

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser Val Gln Glu Ala

1 5 10 15

Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser Glu Ser Asp Tyr

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Ile Leu Val

35 40 45

Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr Glu Asn Arg Phe

50 55 60

Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile Ser

65 70 75 80

Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys Ala Tyr Arg Ser

85 90 95

Gly Ile Ile Gln Gly Ala Gln Lys Leu Val Phe Gly Gln Gly Thr Arg

100 105 110

Leu Thr Ile Asn Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln

115 120 125

Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp

130 135 140

Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr

145 150 155 160

Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser

165 170 175

Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn

180 185 190

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro

195 200 205

Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp

210 215 220

Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu

225 230 235 240

Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp

245 250 255

Ser Ser

<210> 28

<211> 295

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Ala Ala Gly Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg

1 5 10 15

Glu Thr Ala Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val

20 25 30

Tyr Trp Tyr Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser

35 40 45

Phe Tyr Glu Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe

50 55 60

Ser Ala Gln Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser

65 70 75 80

Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Ser Asp

85 90 95

Arg Glu Leu Leu Phe Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala

115 120 125

Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr

130 135 140

Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser

145 150 155 160

Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro

165 170 175

Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu

180 185 190

Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn

195 200 205

His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu

210 215 220

Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu

225 230 235 240

Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln

245 250 255

Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala

260 265 270

Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val

275 280 285

Lys Arg Lys Asp Ser Arg Gly

290 295