CN112390875B

CN112390875B - High-affinity T cell receptor for identifying AFP

Info

Publication number: CN112390875B
Application number: CN201910760352.XA
Authority: CN
Inventors: 张婷婷; 唐先青
Original assignee: Xiangxue Life Science Technology Guangdong Co ltd
Current assignee: Xiangxue Life Science Technology Guangdong Co ltd
Priority date: 2019-08-16
Filing date: 2019-08-16
Publication date: 2023-01-24
Anticipated expiration: 2039-08-16
Also published as: WO2021032020A1; CN112390875A

Abstract

The present invention provides a T Cell Receptor (TCR) having the property of binding to the FMNKFIYEI-HLA a0201 complex; and the binding affinity of the TCR to the FMNKFIYEI-HLA A0201 complex is at least 2-fold greater than the binding affinity of a wild-type TCR to the FMNKFIYEI-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 FMNKFIYEI-HLA a0201 complex presenting tumor cells.

Description

High-affinity T cell receptor for identifying AFP

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 AFP proteins. 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 generated 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 for 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 reactions, 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 thereby presenting different short peptides of a single protein antigen to the cell surface of the respective APC. Human MHC is commonly referred to as an HLA gene or HLA complex.

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

Thus, the FMNKFIYEI-HLA a0201 complex provides a marker for targeting of TCRs to tumor cells. The TCR capable of being combined with the FMNKFIYEI-HLA A0201 compound has high application value in 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 to be transformed into T cells, such that T cells expressing the TCR can destroy tumor cells for administration to a patient in a treatment 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. Thus, 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 invention aims to provide a TCR with higher affinity to FMNKFIYEI-HLA A0201 complex.

It is a further object of the invention to provide a method of making a TCR of the type described above and uses thereof.

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

In another preferred example, the T Cell Receptor (TCR) has an activity of binding to the FMNKFIYEI-HLA A0201 complex, and the TCR comprises a TCR alpha chain variable domain comprising 3 CDR regions and a TCR beta chain variable domain, the reference sequences of the 3 CDR regions of the TCR alpha chain variable domain are as follows,

CDR1α：VGISA

CDR2α：LSSGK

CDR3 α: avetserdkvi, and contains at least one of the following mutations:

residues before mutation	Post-mutation residues
			1 st position V of CDR1 alpha	A or P
Position 3I of CDR 1. Alpha	L
		4 th position S of CDR1 alpha	Q
Position 2S of CDR2 alpha	P
		3 rd position S of CDR2 alpha	F or Y
Position 4G of CDR2 α	Q
		Position 5K of CDR2 α	T
5 th position S of CDR3 alpha	T or F
		Position 6Y of CDR3 α	R or N

And/or, the variable domain of the TCR beta chain comprises 3 CDR regions, the reference sequence of the 3 CDR regions of the variable domain of the TCR beta chain is as follows,

CDR1β：SGHVS

CDR2β：FNYEAQ

CDR3. Beta.: ASSYGAGGPLDTYY, and contains at least one of the following mutations:

residues before mutation	Residues after mutation
		4 th position E of CDR2 beta	V
Position 5A of CDR2 β	S
		Q at position 6 of CDR2 β	I
3 rd position S of CDR3 beta	A
		Position 4Y of CDR3 β	L or P or R or K or Q or F
Position 5G of CDR3 β	F or M or Y or H or S or W or A
		6 th position A of CDR3 beta	S or P or G
11 th position D of CDR3 β	G or S or M or E or A or R
		Position 12T of CDR3 β	S or A or E or G or M
Position 14Y of CDR3 beta	A or V or I or W or K or M or Q or R.

In another preferred embodiment, the mutation occurs in one or more CDR regions of the alpha chain and/or beta chain variable domains.

In another preferred embodiment, the number of mutations in the 3 CDR regions of the variable domain of the TCR α chain is 1 to 9, and/or the number of mutations in the 3 CDR regions of the variable domain of the TCR β chain is 1 to 10.

In another preferred embodiment, the number of mutations in the CDR regions of the TCR α chain can be 2,3, 4, 5, 6, 7, 8 or 9.

In another preferred embodiment, the number of mutations in the CDR regions of the TCR β chain may be 3, 4, 5, 6, 7, 8, 9 or 10.

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

In a preferred embodiment of the invention, the TCR has at least 2-fold greater affinity for the FMNKFIYEI-HLA a0201 complex than for 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 FMNKFIYEI-HLA a0201 complex than a wild-type TCR; preferably, at least 100 times; more preferably, at least 500 times.

In another preferred embodiment, the affinity of the TCR for the FMNKFIYEI-HLA A0201 complex is at least 10 of the wild type TCR ³ Doubling; preferably, at least 5 x 10 ³ Doubling; more preferably, at least 10 ⁴ Doubling; more preferably, at least 5 x 10 ⁴ And (4) doubling.

Specifically, the dissociation equilibrium constant KD of the TCR to the FMNKFIYEI-HLA A0201 complex is less than or equal to 20 mu M;

in another preferred embodiment, the TCR has a dissociation equilibrium constant for the FMNKFIYEI-HLA A0201 complex of 5 μ M ≦ KD ≦ 10 μ M; preferably, 0.1. Mu.M.ltoreq.KD.ltoreq.1. Mu.M; more preferably, 1 nM. Ltoreq. KD. Ltoreq.100 nM.

In another preferred embodiment, 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.

In another preferred embodiment, the TCR α chain variable domain comprises CDR1 α, CDR2 α and CDR3 α, and the TCR β chain variable domain comprises CDR1 β, CDR2 β and CDR3 β, characterized in that the amino acid sequence of CDR1 β is SGHVS.

In another preferred embodiment, the TCR α chain variable domain comprises CDR1 α, CDR2 α and CDR3 α, and the TCR β chain variable domain comprises CDR1 β, CDR2 β and CDR3 β, wherein the amino acid sequence of CDR3 β is: AS [3 betaX 1] [3 betaX 2] [3 betaX 3] [3 betaX 4] GGPL [3 betaX 5] [3 betaX 6] Q [3 betaX 7], wherein [3 betaX 1] is A or S; and/or [3 β X2] is Y, L, P, R, K, Q or F; and/or [3 β X3] is G, F, M, Y, H, S, W or A; and/or [3 β X4] is A, S, P or G; and/or [3 β X5] is D, S, G, R, M or E; and/or [3 β X6] is T, G, E, S, M or A; and/or [3 β X7] is Y, I, V, M, Q, R, A, W or K.

In another preferred embodiment, the TCR α chain variable domain comprises CDR1 α, CDR2 α and CDR3 α, and the TCR β chain variable domain comprises CDR1 β, CDR2 β and CDR3 β, wherein the CDR3 β is selected from the group consisting of: ASALMSGGPLDTQY, ASARYPGGPLDTQY, ASARHAGGPLDTQY, ASSYGAGGPLDTQY, ASALFSGGPLDTQY, ASAPFSGGPLDTQY, ASAKMSGGPLDTQY, ASSYGAGGPLGEQW, ASSYGAGGPGGQAQGA, ASSQSGGGPLDTQY, ASSYGAGGPLGEQV, ASSYGAGGPLMACQA, ASALYSGGPLDTQY, ASSPFSGGPLDTQY, YGAGGPLGQK, YGAGGPLEGQASSQV, ASSYGAGGPLSLSLSI, ASSLFGGGPLDTQY, ASSYGAGGPLSGQI, ASSYGAGGPLSSQQY, ASSYGAGSQTQM, ASSYGAGGPSQSQSQQQ, ASSYGAGLGSQSQSQSQSQSQVV, ASSYGAGGPAGGPLDA, LFY, ASSYGAGTQTQY, ASSYGGGGPTQTQY, ASSGPGGGPTQTQY and GPLDQQQQR.

In another preferred embodiment, the TCR α chain variable domain comprises CDR1 α, CDR2 α and CDR3 α, and the TCR β chain variable domain comprises CDR1 β, CDR2 β and CDR3 β, wherein the amino acid sequence of CDR2 β is selected from FNYEAQ and FNYVSI.

In another preferred embodiment, the TCR α chain variable domain comprises CDR1 α, CDR2 α and CDR3 α, and the TCR β chain variable domain comprises CDR1 β, CDR2 β and CDR3 β, wherein the amino acid sequence of CDR1 α is selected from the group consisting of: VGISA, AGLQA, VGLQA and PGLQA.

In another preferred embodiment, the TCR α chain variable domain comprises CDR1 α, CDR2 α and CDR3 α, and the TCR β chain variable domain comprises CDR1 β, CDR2 β and CDR3 β, characterized in that the amino acid sequence of CDR2 α is selected from the group consisting of: LSSGK, LPFGK and LPYQT.

In another preferred embodiment, the TCR α chain variable domain comprises CDR1 α, CDR2 α and CDR3 α, and the TCR β chain variable domain comprises CDR1 β, CDR2 β and CDR3 β, wherein the amino acid sequence of CDR3 α is selected from the group consisting of: AVETSYDKVI, avetrdkvi and avetfrdkvvi.

In another preferred example, the TCR α chain variable domain comprises CDR1 α, CDR2 α and CDR3 α, and the TCR β chain variable domain comprises CDR1 β, CDR2 β and CDR3 β, characterized in that the amino acid sequence of CDR1 α is VGISA, the amino acid sequence of CDR2 α is LSSGK, and the amino acid sequence of CDR3 α is avetssydkvi.

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

In another preferred embodiment, the mutation occurs in one or more CDR regions of the alpha chain and/or beta chain variable domain.

In a preferred embodiment of the invention, the T Cell Receptor (TCR), which has the activity of binding to the FMNKFIYEI-HLA A2 complex and comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, is represented in SEQ ID NO:1, the mutated amino acid residue positions include one or more of 27V, 29I, 30S, 50S, 51S, 52G, 53K, 92S and 93Y, wherein the amino acid residue numbering adopts the beta chain variable domain numbering shown in SEQ ID No. 1; and/or the TCR is as set out in SEQ ID NO:2, and the mutated amino acid residue positions comprise one or more of 52E, 53A, 54Q, 95S, 96Y, 97G, 98A, 103D, 104T and 106Y, wherein the amino acid residue numbers adopt the numbers shown in SEQ ID NO. 2;

preferably, the TCR α chain variable domain after mutation comprises one or more amino acid residues selected from the group consisting of: 27A or 27P;29L;30Q;50P;51F or 51Y;52Q;53T;92T or 92F and 93R or 93N, wherein the numbering of the amino acid residues adopts the numbering shown in SEQ ID NO 1; and/or the mutated TCR β chain variable domain comprises one or more amino acid residues selected from the group consisting of: 52V, 53S, 54I, 95A, 96L or 96P or 96R or 96K or 96Q or 96F, 97F or 97M or 97Y or 97H or 97S or 97W or 97A, 98S or 98P or 98G, 103G or 103S or 103M or 103E or 103A or 103R, 104S or 104A or 104E or 104G or 104M, 106A or 106V or 106I or 106W or 106K or 106M or 106Q or 106R, wherein the numbering of the amino acid residues is that shown in SEQ ID NO. 2.

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

in another preferred embodiment, the TCR is soluble.

In another preferred embodiment, the TCR is an α β heterodimeric TCR or a single chain TCR.

In another preferred embodiment, the α chain variable domain amino acid sequence of the TCR is selected from the group consisting of: 9-13 and 45-51 of SEQ ID NO; and/or the beta chain variable domain amino acid sequence of the TCR is selected from: 14-40 and 52-79 SEQ ID NOs.

In another preferred embodiment, the TCR of the invention is an α β heterodimeric TCR, preferably the TCR has an α chain constant region sequence TRAC 01 and a β chain constant region sequence TRBC1 × 01 or TRBC2 × 01.

In another preferred embodiment, the TCR 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 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 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.

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

In another preferred embodiment, the cysteine residue that forms the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR replaces one or more groups of sites selected from:

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 TRBC1 x 01 or TRBC2 x 01.

In another preferred embodiment, a TCR comprising an artificial interchain disulfide bond between an α chain variable region and a β chain constant region comprises an α chain variable domain and a β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise an α chain constant domain, the α chain variable domain of said TCR forming a heterodimer with the β chain.

In another preferred embodiment, a TCR comprising an artificial interchain disulfide bond between the α chain variable region and the β chain constant region 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 a TCR chain.

In another preferred embodiment, the TCR is an α β heterodimeric TCR comprising (i) all or part of a TCR α chain, excluding the transmembrane domain thereof, and (ii) all or part of a TCR β chain, excluding the transmembrane domain thereof, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain, the α chain constant region and the β chain constant region comprising an artificial interchain disulfide bond therebetween.

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

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

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

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

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

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

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

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

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

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 hydrophobic core of the TCR α chain variable domain and/or β chain variable domain is mutated.

In another preferred embodiment, the TCR with the mutated hydrophobic core is a single chain TCR consisting of an alpha variable domain and a beta variable domain, the alpha and beta variable domains being linked by a flexible short peptide sequence (linker).

In another preferred embodiment, 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 set forth 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 of amino acid sequence; and/or the β chain variable domain of the TCR comprises at least 85%, preferably at least 90% of the amino acid sequence set forth as 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.

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, the invention provides 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, for the manufacture of a medicament for the treatment of a tumour, preferably hepatocellular carcinoma.

In a tenth aspect of the invention, the T cell receptor, the TCR complex of claim 29 or the cell of claim 33 for use as a medicament for the treatment of a tumour. Preferably, the tumor is an AFP-positive tumor; more preferably, the tumor is hepatocellular carcinoma.

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 chain capable of specifically binding to the FMNKFIYEI-HLA A0201 complex, respectively.

FIGS. 2a and 2b show the amino acid sequences of the α chain variable domain and β chain variable domain, respectively, of a single-chain template TCR constructed according to 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, respectively, of the single-stranded template TCR constructed in accordance with the invention.

FIGS. 5 (1) - (5) show the amino acid sequences of the alpha chain variable domain of single chain TCRs with high affinity for FMNKFIYEI-HLA A0201 complex, respectively, with mutated residues underlined.

Fig. 6 (1) - (27) show the amino acid sequences of the β chain variable domain of single-chain TCRs with high affinity for FMNKFIYEI-HLA a0201 complex, respectively, with mutated residues underlined.

FIGS. 7a and 7b show the amino acid sequence and DNA sequence, respectively, of a single-stranded template TCR constructed in accordance with the invention.

Fig. 8a and 8b show the amino acid sequences of soluble reference TCR α and β chains, respectively, of the invention.

Fig. 9 (1) - (7) show the α chain variable domain amino acid sequences of heterodimeric TCRs with high affinity for FMNKFIYEI-HLA a0201 complex, respectively, with mutated residues underlined.

Fig. 10 (1) - (28) show the β chain variable domain amino acid sequences of heterodimeric TCRs with high affinity for FMNKFIYEI-HLA a0201 complex, respectively, with mutated residues underlined.

FIGS. 11a and 11b show the extracellular amino acid sequences of wild-type TCR alpha and beta chains, respectively, capable of specifically binding to the FMNKFIYEI-HLA A0201 complex.

Fig. 12a and 12b show the amino acid sequences of wild-type TCR α and β chains, respectively, capable of specifically binding to FMNKFIYEI-HLA a0201 complex.

FIG. 13 is a graph of binding curves of soluble reference TCR, i.e., wild-type TCR, to FMNKFIYEI-HLA A0201 complex.

FIGS. 14a and 14b are experimental results of the activation function of effector cells transfected with the high affinity TCR of the invention.

FIG. 15 is a graph showing the results of an experiment on the killing function of LDH in effector cells transfected with the high affinity TCR of the invention.

Detailed Description

The present invention, through extensive and intensive studies, resulted in a high-affinity T Cell Receptor (TCR) that recognizes FMNKFIYEI short peptides (derived from AFP protein) presented in the form of peptide-HLA a0201 complex. The high affinity TCR has 3 CDR regions in its alpha chain variable domain:

CDR1α：VGISA

CDR2α：LSSGK

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

CDR1β：SGHVS

CDR2β：FNYEAQ

CDR3. Beta.: mutations in ASSYGAGGPLDTQY; and, the affinity and/or binding half-life of the inventive TCR after mutation to the FMNKFIYEI-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, which are chimeric in the framework sequence. In the IMGT nomenclature, the different numbers of TRAV and TRBV refer to different va and vb types, respectively. In the IMGT system, the α chain constant domain has the following symbols: TRAC 01, wherein "TR" represents a T cell receptor gene; "A" represents an alpha chain gene; c represents a constant region; "x01" indicates allele 1. The beta-strand constant domain has the following notation: 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 α and β 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 consists 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 FMNKFIYEI-HLA A2 complex, the inventive TCR 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:82 and SEQ ID NO:83 as shown in fig. 12a and 12 b. The alpha chain amino acid sequence and the beta chain amino acid sequence of the 'reference TCR' are respectively SEQ ID NO 43 and SEQ ID NO:44 as shown in fig. 8a and 8 b. The extracellular amino acid sequences of the alpha chain and the beta chain of the wild-type TCR are respectively SEQ ID NO:80 and SEQ ID NO:81 as shown in fig. 11a and 11 b. In the present invention, the amino acid sequences of the α and β chain variable domains of the wild-type TCR capable of binding to the FMNKFIYEI-HLA A0201 complex are SEQ ID NO:1 and SEQ ID NO:2 as shown in fig. 1a and 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 in the membrane proximal region of native TCRs. In the present invention, the artificially introduced interchain covalent disulfide bond whose position is different from that of the natural interchain disulfide bond is referred to as an "artificial interchain disulfide bond".

For convenience of description, the amino acid sequences of TRAC × 01 and TRBC1 × 01 or TRBC2 × 01 are numbered sequentially from N to C, for example, in TRBC1 × 01 or TRBC2 × 01, the 60 th amino acid in the sequence from N to C is P (proline), and thus, in the present invention, it may be described as Pro60 of TRBC1 × 01 or TRBC2 × 01 exon 1, and also as the 60 th amino acid of TRBC1 × 01 or TRBC2 × 01 exon 1, and also as the 61 th amino acid in the sequence from N to C is Q (glutamine), and thus, in the present invention, it may be described as TRBC1 × 01 or TRBC2 × 01, and also as the Gln of TRBC1 × 61 or TRBC2 × 01 exon 1, and also as the other TRBC1 or TRBC2 × 01, and so forth, the position numbers of TRBC1 × 01 or TRBC2 × 01 may be numbered sequentially from N to C. 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 a certain amino acid in TRAV, the position number listed in IMGT is 46, it is described in the present invention as TRAV amino acid 46, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described.

Tumor(s)

The term "tumor" is meant to include all types of cancerous cell growth or carcinogenic processes, metastatic tissue or malignantly transformed cells, tissues or organs, regardless of the type of pathology or stage of infestation. Examples of tumors include, but are not limited to: solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include: malignancies 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, lung, 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 beta chain variable domain of the wild-type TCR capable of binding the antigen short peptide FMNKFIYEI and HLA a0201 complex (i.e., FMNKFIYEI-HLA a0201 complex) are SEQ ID NO:1 and SEQ ID NO:2, the sequence is discovered by the inventor for the first time. It has the following CDR regions:

alpha chain variable domain CDR

CDR1α：VGISA

CDR2α：LSSGK

CDR3α：AVETSYDKVI

Beta chain variable domain CDR

CDR1β：SGHVS

CDR2β：FNYEAQ

CDR3β：ASSYGAGGPLDTQY

According to the invention, the high-affinity TCR with the affinity of the FMNKFIYEI-HLA A0201 compound at least 2 times that of the wild-type TCR and the FMNKFIYEI-HLA A0201 compound is obtained by mutation screening of the CDR region.

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

The T cell receptor comprises a TCR alpha chain variable domain comprising 3 CDR regions and a TCR beta chain variable domain, the reference sequence of the 3 CDR regions of the TCR alpha chain variable domain is as follows,

CDR1α：VGISA

CDR2α：LSSGK

CDR3 α: AVETSYDKVI, and contains at least one of the following mutations:

CDR1β：SGHVS

CDR2β：FNYEAQ

CDR3. Beta.: ASSYGAGGPLDTQY, and contains at least one of the following mutations:

residues before mutation	Post-mutation residues
		4 th position E of CDR2 beta	V
Position 5A of CDR2 beta	S
		Q at position 6 of CDR2 beta	I
3 rd position S of CDR3 beta	A
		Position 4Y of CDR3 beta	L or P or R or K or Q or F
Position 5G of CDR3 beta	F or M or Y or H or S or W or A
		Position 6A of CDR3 beta	S or P or G
11 th position D of CDR3 beta	G or S or M or E or A or R
		Position 12T of CDR3 beta	S or A or E or G or M
Position 14Y of CDR3 beta	A or V or I or W or K or M or Q or R.

The wild type TCR alpha chain variable domain SEQ ID NO:1, i.e. CDR1, CDR2 and CDR3 are located in SEQ ID NO:1 from bits 27-31, from bits 49-53 and from bits 88-97. Accordingly, the amino acid residue numbering is as shown in SEQ ID NO 1, with 27V being the 1 st position V of CDR1 α,29I being the 3 rd position I of CDR1 α,30S being the 4 th position S of CDR1 α,50S being the 2 nd position S of CDR2 α, 51S being the 3 rd position S of CDR2 α, 52G being the 4 th position G of CDR2 α, 53K being the 5 th position K of CDR2 α, 92S being the 5 th position S of CDR3 α, and 93Y being the 5 th position Y of CDR3 α.

Similarly, the wild-type TCR β chain variable domain of SEQ ID NO:2, i.e. CDR1, CDR2 and CDR3 are located in SEQ ID NO:2, bits 27-31, bits 49-54, and bits 93-106. Thus, the amino acid residue numbers are as shown in SEQ ID NO 2, with 52E being the 4 th E of CDR2 β, 53A being the 5 th A of CDR2 β, 54Q being the 6 th Q of CDR2 β, 95S being the 3 rd S of CDR3 β, 96Y being the 4 th Y of CDR3 β, 97G being the 5 th G of CDR3 β, 98A being the 6 th A of CDR3 β, 103D being the 3 rd D of CDR11 β, 104T being the 12 th T of CDR3 β, 106Y being the 14 th Y of CDR3 β.

More specifically, the specific form of the mutation in the alpha chain variable domain comprises one or more of V27A/P, I29L, S30Q, S50P, S51F/Y, G52Q, K53T, S92T/F, Y93R/N; specific forms of the mutations described in the variable domain of the beta strand include one or more of E52V, A53S, Q54I, S95A, Y96L/P/R/K/Q/F, G97F/M/Y/H/S/W/A, A98S/P/G, D103G/S/M/E/A/R, T104S/A/E/G/M, Y106A/V/I/W/K/M/Q/R.

In a preferred embodiment of the invention, a TCR according to the invention comprises a TCR α chain variable domain comprising CDR1 α, CDR2 α, and CDR3 α, and a TCR β chain variable domain comprising CDR1 β, CDR2 β, and CDR3 β.

In another preferred embodiment, the CDR1 α comprises the sequence: [ 1. Alpha. X1] G [ 1. Alpha. X2] [ 1. Alpha. X3] A, wherein [ 1. Alpha. X1], [ 1. Alpha. X2] and [ 1. Alpha. X3] are each independently selected from any natural amino acid residue.

In another preferred embodiment, said [1 α X1] is V or a or P.

In another preferred embodiment, said [1 α X2] is I or L.

In another preferred embodiment, said [1 α X3] is S or Q.

In another preferred embodiment, the [1 α X1] is V or a or P, [1 α X2] is I or L, and [1 α X3] is S or Q.

In another preferred embodiment, the CDR1 α comprises a sequence selected from the group consisting of: VGISA, AGLQA, VGLQA and PGLQA.

In another preferred embodiment, the CDR2 α comprises the sequence: l [2 alpha X1] [2 alpha X2] [2 alpha X3] [2 alpha X4], wherein [2 alpha X1], [2 alpha X2], [2 alpha X3], [2 alpha X4] are independently selected from any natural amino acid residue.

In another preferred embodiment, said [2 α X1] is S or P.

In another preferred embodiment, said [2 α X2] is S or F or Y.

In another preferred embodiment, the [2 α X3] is G or Q.

In another preferred embodiment, said [2 α X4] is K or T.

In another preferred embodiment, the [2 α X1] is S or P, the [2 α X2] is S or F or Y, and the [2 α X3] is G or Q, and the [2 α X4] is K or T.

In another preferred embodiment, the CDR2 α comprises a sequence selected from the group consisting of: LSSGK, LPFGK and LPYQT.

In another preferred embodiment, the CDR3 α comprises the sequence: AVET [ 3. Alpha.X 1] [ 3. Alpha.X 2] DKKVI, wherein [ 3. Alpha.X 1] and [ 3. Alpha.X 2] are each independently selected from any natural amino acid residue.

In another preferred embodiment, said [3 α X1] is T or F.

In another preferred embodiment, the [3 α X2] is R or N.

In another preferred embodiment, the [3 α X1] is T or F, and the [3 α X2] is R or N.

In another preferred embodiment, the CDR 3a comprises a sequence selected from the group consisting of: avetsidkvi, avetrdkvi and avefndkvi.

In a preferred embodiment of the invention, the TCR comprises a TCR α chain variable domain comprising CDR1 α, CDR2 α, and CDR3 α, and a TCR β chain variable domain comprising CDR1 β, CDR2 β and CDR3 β, wherein the CDR1 β comprises the sequence: SGHVS.

In another preferred embodiment, the CDR2 β comprises the sequence: FNY [2 betaX 1] [2 betaX 2] [2 betaX 3], wherein [2 betaX 1], [2 betaX 2], [2 betaX 3] are independently selected from any natural amino acid residue.

In another preferred embodiment, the [2 β X1] is Q or H.

In another preferred embodiment, the [2 β X2] is N or G.

In another preferred embodiment, the [2 β X3] is E or D.

In another preferred embodiment, the CDR2 β comprises a sequence selected from the group consisting of: FNYEAQ and FNYVSI.

In another preferred embodiment, the CDR3 β comprises the sequence:

AS [ 3. Beta. X1] [ 3. Beta. X2] [ 3. Beta. X3] [ 3. Beta. X4] GGPL [ 3. Beta. X5] [ 3. Beta. X6] Q [ 3. Beta. X7]. Wherein [ 3. Beta.X 1], [ 3. Beta.X 2], [ 3. Beta.X 3], [ 3. Beta.X 4], [ 3. Beta.X 5], [ 3. Beta.X 6] and [ 3. Beta.X 7] are each independently selected from any natural amino acid residue.

In another preferred embodiment, the [3 β X1] is a or S.

In another preferred embodiment, said [3 β X2] is Y or L or P or R or K or Q or F.

In another preferred embodiment, said [3 β X3] is G or F or M or Y or H or S or W or a.

In another preferred embodiment, said [3 β X4] is S or a or P or G.

In another preferred embodiment, the [3 β X5] is D, G, S, M, E, a or R.

In another preferred embodiment, said [3 β X6] is T, S, a, E, G or M.

In another preferred embodiment, said [3 β X7] is Y, A, V, I, W, K, M, Q or R.

In another preferred embodiment, the CDR3 β comprises a sequence selected from the group consisting of: ASALMSGGPLDQY, ASARYPGGPLDQY, ASARHAGGPLDQY, ASSYGAGGPLDTQY, ASALFSGGPLDQY, ASAPFSGGPLDQY, ASAKMSGGPLDQY, ASSYGAGGPLGEQW, ASSYGAGGPLGAK QA, ASSQSGGGPLDTQY, ASSYGAGGPLGEQV, ASSYGAGGPLMAQA, ASALYSGGPLDQDTY, PFSGGPTQLDY, ASSYGAGGPLGQK, ASSYGGPLEGQV, ASSAGGPLSLSLSLSI, ASSLFGGGPLDTQY, ASSYGAGGPLSGQI, ASSYGAGGPLASQY, ASSYGAGGPLRGPSQM, ASSYGAGGPSQSQQ, ASSYGAGLGSQSQSQV, ASSYGAGGPSQUA, LFLDTQY, ASSYGSGTQTQY, ASSYGGGGPTQTQY and ASYGGPGGGPGGGPLDQQQQY.

In another preferred example, the TCR α chain variable domain comprises CDR1 α, CDR2 α and CDR3 α, wherein the amino acid sequence of CDR1 α is VGISA, the amino acid sequence of CDR2 α is LSSGK and the amino acid sequence of CDR3 α is avetsidkvi.

In more detail, the number of mutations in the CDR regions of the TCR α chain can be 3, 4, 5, 6, 7, 8 or 9; and/or the number of mutations in the CDR regions of the TCR β chain may be 4, 5, 6, 7, 8, 9 or 10.

Further, the TCR of the invention is an α β heterodimeric TCR, the α chain variable domain of the TCR comprising 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 of amino acid sequence; and/or the β chain variable domain of the TCR comprises at least 85%, preferably at least 90% of the amino acid sequence set forth as 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 a variable domain and at least part of a constant domain of a TCR chain.

The amino acid sequences of the reference TCRs, which are shown in figures 8a and 8b, respectively, were 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 × 01 or TRBC2 × 01 to cysteine according to site-directed mutagenesis methods well known to those skilled in the art, and the mutated cysteine residues are shown in bold letters. The cysteine substitutions described above allow for 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 allowing for a more convenient assessment of the binding affinity and/or half-life of the TCR and FMNKFIYEI-HLA A2 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 affect 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 FMNKFIYEI-HLA a0201 complex is considered to be the binding affinity between the wild-type TCR and the FMNKFIYEI-HLA a0201 complex. Similarly, if the binding affinity between the TCR of the invention and the FMNKFIYEI-HLA a0201 complex is determined to be at least 10 times greater than the binding affinity between the reference TCR and the FMNKFIYEI-HLA a0201 complex, i.e. equivalent to the binding affinity between the TCR of the invention and the FMNKFIYEI-HLA a0201 complex being at least 10 times greater than the binding affinity between the wild-type TCR and the FMNKFIYEI-HLA a0201 complex.

Binding affinity (equilibrium constant K to dissociation) can be determined by any suitable method _D Inversely 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 _D The number is reduced by half. T is _1/2 Calculated as In2 divided by the dissociation rate (K) _off ). Thus, T _1/2 Doubling can result in K _off And (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 in 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 FMNKFIYEI-HLA A2 complex _D 9.89E-06M, i.e., 9.89. Mu.M, the dissociation equilibrium constant K of the wild-type TCR to the FMNKFIYEI-HLA A2 complex is considered to be _D Also 9.89. Mu.M. Doubling of the affinity of the TCR will result in K _D Halved, so if the dissociation equilibrium constant K of the high affinity TCR for the FMNKFIYEI-HLA A2 complex is detected _D 9.89E-07M, i.e., 9.89E-01. Mu.M, indicates that the affinity of the high affinity TCR for the FMNKFIYEI-HLA A2 complex is 10 times greater than the affinity of the wild-type TCR for the FMNKFIYEI-HLA A2 complex. K is well known to those skilled in the art _D Conversion between units of value, i.e. 1M =10 ⁶ μM,1μM＝1000nM，1nM＝1000pM。

In a preferred embodiment of the invention, the TCR has at least 2-fold greater affinity for the FMNKFIYEI-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 FMNKFIYEI-HLA a0201 complex than for a wild-type TCR; preferably, at least 100 times; more preferably, at least 500 times.

In another preferred embodiment, the TCR has a dissociation equilibrium constant of 5 μ M ≦ K for the FMNKFIYEI-HLA A0201 complex _D Less than or equal to 10 mu M; preferably, 0.1. Mu.M.ltoreq.K _D Less than or equal to 1 mu M; more preferably, 1nM ≦ K _D ≤100nM；

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 having high affinity for the FMNKFIYEI-HLA-A2 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.

The high affinity TCRs of the invention comprise one of the alpha chain variable domain amino acid sequences SEQ ID NO 45-51 and/or one of the beta chain variable domain amino acid sequences SEQ ID NO 52-79. Thus, a TCR α chain comprising the α chain variable domain amino acid sequence of a wild-type TCR (SEQ ID NO: 1) can be combined with a TCR β chain comprising one of SEQ ID NOS: 52-79 to form a heterodimeric TCR or single chain TCR molecule. Alternatively, a TCR β chain containing the β variable domain amino acid sequence of a wild-type TCR (SEQ ID NO: 2) can be combined with a TCR α chain comprising one of SEQ ID NOS: 45-51 to form a heterodimeric TCR or single chain TCR molecule. Still alternatively, a TCR α chain comprising one of the TCR α chain variable domain amino acid sequences SEQ ID NO:45-51 can be combined with a TCR β chain comprising one of the TCR β chain variable domain amino acid sequences SEQ ID NO:52-79 to form a heterodimeric TCR or single chain TCR molecule. In the present invention, the amino acid sequences of the α chain variable domain and the β chain variable domain that form the heterodimeric TCR molecule are preferably selected from table 1 below:

TABLE 1

For the purposes of the present invention, the inventive TCRs are moieties 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 full-length chain (comprising the cytoplasmic and transmembrane domains) of an α β heterodimeric TCR can be transfected. The inventive TCRs may be used 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 TCR is preferably in soluble form.

For stability, it is disclosed in the prior art that introduction of an artificial interchain disulfide bond between the α and β chain constant domains of the 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 constant domains of its alpha and beta chains. 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 disulfide bond is formed by replacing Thr48 of exon 1 of TRAC × 01 and replacing Ser57 of exon 1 of TRBC1 × 01 or TRBC2 × 01. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 of TRAC × 01 exon 1 and TRBC1 × 01 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 of TRAC × 01 exon 1 and TRBC1 × 01 or Asp59 of TRBC2 × 01 exon 1; ser15 of TRAC × 01 exon 1 and TRBC1 × 01 or TRBC2 × 01 exon 1 Glu15; arg53 of TRAC × 01 exon 1 and TRBC1 × 01 or Ser54 of TRBC2 × 01 exon 1; pro89 of exon 1 TRAC × 01 and TRBC1 × 01 or TRBC2 × 01 of Ala19 of exon 1; or Tyr10 of exon 1 of TRAC × 01 and TRBC1 × 01 or TRBC2 × 01, glu20 of 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 disulphide bond, or by mutating the cysteine residues forming the native interchain disulphide 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 the TRBC1 or TRBC2 constant domain sequence of the TCR may be linked by the native interchain disulfide bonds present in the TCR.

In addition, for stability, 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 residues that form the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR replace: 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 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 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 increase 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 positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or

positions

3,5,7 from the last amino acid position of the short peptide of the alpha chain J gene (TRAJ) and/or positions 2,4,6 from the last amino acid position of the short peptide of the beta chain J gene (TRBJ), wherein the position numbers of the amino acid sequences are according to the position numbers listed in the International Immunogenetic information System (IMGT). The skilled person is aware of the above international immunogenetic information system and can derive from this database the position numbering of the amino acid residues of different TCRs in IMGT.

More specifically, the TCR with the mutated hydrophobic core region of the present invention may be a high stability single chain TCR comprising a flexible peptide chain connecting the variable domains of the α and β chains of the TCR. The CDR regions of the variable region of the TCR determine the affinity with the short peptide-HLA complex, and the mutation of the hydrophobic core can stabilize the TCR without affecting the affinity 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 containing the hydrophobic core mutation. By adopting the TCR with higher stability, the affinity between the TCR and the FMNKFIYEI-HLA-A0201 complex can be more conveniently evaluated.

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, 3 CDRs of the α chain variable domain are CDR1 α: VGISA, CDR2 α: the LSSGK is used for carrying out the operation of the system,

CDR3 α: the 3 CDRs of AVETSYDKVI and β chain variable domains are CDR1 β: SGHVS, CDR2 β: FNYEAQ, CDR3 β: ASSYGAGGPLDTQY. The amino acid sequence (SEQ ID NO: 41) and the nucleotide sequence (SEQ ID NO: 42) of the single-chain template TCR are shown in FIGS. 7a and 7b, respectively. Thus, a single-chain TCR composed of an alpha chain variable domain and a beta chain variable domain having high affinity for the FMNKFIYEI-HLA A0201 complex was selected.

The single-chain template TCR alpha chain variable domain SEQ ID NO:3, CDR1, CDR2 and CDR3 are located in SEQ ID NO: bits 27-31, bits 49-55, and bits 90-102 of 3. Accordingly, the amino acid residue numbering is as shown in SEQ ID NO. 3, with 27V being the 1 st position V of CDR1 α,29I being the 3 rd position I of CDR1 α,30S being the 4 th position S of CDR1 α,50S being the 2 nd position S of CDR2 α, 51S being the 3 rd position S of CDR2 α, 52G being the 4 th position G of CDR2 α, 53K being the 5 th position K of CDR2 α, 92S being the 5 th position S of CDR3 α, and 93Y being the 5 th position Y of CDR3 α.

Similarly, the single-chain template TCR beta chain variable domain SEQ ID NO:4, CDR1, CDR2 and CDR3 are located in SEQ ID NO:2, bits 27-31, bits 49-54, and bits 93-102. Thus, the amino acid residue numbering is as shown in SEQ ID NO 4, 52E is the 4 th E of CDR2 β, 53A is the 5 th A of CDR2 β, 54Q is the 6 th Q of CDR2 β, 95S is the 3 rd S of CDR3 β, 96Y is the 4 th Y of CDR3 β, 97G is the 5 th G of CDR3 β, 98A is the 6 th A of CDR3 β, 103D is the 3 rd D of CDR11 β, 104T is the 12 th T of CDR3 β, 106Y is the 14 th Y of CDR3 β.

The α β heterodimer having high affinity for FMNKFIYEI-HLA-A0201 complex of the present invention 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. And a part of the CDR region is obtained by artificially combining the mutation sites of the CDR regions obtained by screening.

The high affinity TCR of the invention further comprises one of the alpha chain variable domain amino acid sequences SEQ ID NO 9-13 and/or one of the beta chain variable domain amino acid sequences SEQ ID NO 14-40. Thus, the highly stable single chain TCR α chain variable domain (SEQ ID NO: 3) described above as a template chain can be combined with a TCR β chain variable domain having an amino acid sequence of one of SEQ ID NO:14-40 to form the single chain TCR molecule. Alternatively, the high stability single chain TCR beta chain variable domain (SEQ ID NO: 4) as the template chain can be combined with a TCR alpha chain variable domain having an amino acid sequence of one of SEQ ID NO:9-13 to form the single chain TCR molecule. Still alternatively, the TCR α chain variable domain SEQ ID NO:9-13 in combination with one of the TCR β chain variable domains SEQ ID NO 14-40 to form the single chain TCR molecule. In the present invention, the amino acid sequences of the α chain variable domain and the β chain variable domain of the high affinity single chain TCR molecule are preferably selected from table 2 below:

TABLE 2

TCR numbering	Alpha chain variable domain sequence	Beta chain variable domain sequence
			s-1	SQ3O	SQ14O
s-2	3	15
			s-3	3	16
s-4	3	17
			s-5	3	18
s-6	3	19
			s-7	3	20
s-8	3	21
			s-9	3	22
s-10	3	23
			s-11	3	24
s-12	3	25
			s-13	9	4
s-14	3	26
			s-15	3	27
s-16	3	28
			s-17	3	29
s-18	3	30
			s-19	3	31
s-20	3	32
			s-21	10	4
s-22	3	33
			s-23	3	34
s-24	3	35
			s-25	3	36
s-26	3	37
			s-27	3	38
s-28	3	39
			s-29	3	40
s-30	11	22
			s-31	12	22
s-32	13	4。

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 binding two, three, four or more TCRs of the invention, such as might be formed by a tetrameric domain of p53 to create a tetramer, or a complex of multiple TCRs of the invention bound to another molecule. The TCR complexes of the invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and also 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 FMNKFIYEI-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: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme 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. biotoxics (Chaudhary et al, 1989, nature 339, 394, epel et al, 2002, cancer Immunology and Immunotherapy) 51, 565); 3. cytokines such as IL-2 etc (Gillies et al, 1992, national institute of sciences (PNAS) 89, 1428, card et al, 2004, cancer Immunology and Immunotherapy) 53, 345, haiin et al, 2003, cancer Research (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 communications (Cancer letters) 239, 36, huang et al, 2006, journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, gene therapy 11, 1234); 8. liposomes (Mamot et al, 2005, cancer research 65, 11631); 9. nano magnetic particles; 10. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, 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 the binding of a TCR of the invention to an anti-CD 3 antibody or a functional fragment or variant of said anti-CD 3 antibody. Specifically, the fusion molecule of the TCR and the anti-CD 3 single chain antibody comprises TCR alpha chain variable domain amino acid sequences SEQ ID NO 9-13, 45-51 selected from the group and/or TCR beta chain variable domain amino acid sequences SEQ ID NO 14-40, 52-79 selected from the group.

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 present invention may be identical to or a degenerate variant of a nucleic acid sequence as set out in the figures of the present 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:41, but differs from the sequence of SEQ ID NO: 42.

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: 6116-6131). 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 Cancer8 (4): 299-308).

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

The invention also provides a method of treating a disease comprising administering to a subject in need thereof an 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 abbreviation corresponding to the amino acid names are: ala (A), arg (R), asn (N), asp (D), cys (C), gln (Q), glu (E), gly (G), his (H), ile (I), leu (L), lys (K), met (M), phe (F), pro (P), ser (S), thr (T), trp (W), tyr (Y), 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 "V27A/P" represents that V at position 27 is substituted by A or by P, and similarly, "I29L" represents that I at position 29 is substituted by L. 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 will 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 up to 1 amino acid (especially amino acids 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 effects glycosylation, such as 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 TCR, multivalent TCR complex or cell of the invention is typically provided as part of a sterile pharmaceutical composition, which typically comprises a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such kits (but not necessarily) include instructions for use. It may comprise a plurality of said unit dosage forms.

In addition, the TCRs of the invention can be used alone, or in combination or conjugation 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. Co., n.j.1991). Such vectors include (but are not limited to): saline, buffer, dextrose, 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 a liquid carrier before injection can 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 actual 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 may be formulated by mixing, dilution or dissolution according to a conventional method, and occasionally, suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents are added, and the formulation process may 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 sustained 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 the FMNKFIYEI-HLA-A2 complex is at least 2-fold, preferably at least 10-fold that of a wild-type TCR.

(2) The affinity and/or binding half-life of the inventive TCR to the FMNKFIYEI-HLA-A2 complex is at least 100-fold, preferably at least 500-fold, more preferably up to 10-fold that of a wild-type TCR ³ -5*10 ⁴ And (4) doubling.

(3) Effector cells transduced with the high affinity TCRs of the invention have a strong killing effect on 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 from commercial sources, as for example, e.coli DH5 α, e.coli BL21 (DE 3), e.coli Tuner (DE 3), novagen, and plasmid pET28a, without specific reference.

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 description in patent document WO2014/206304, wherein the amino acid and DNA sequences of the stable single-chain TCR molecule are respectively SEQ ID NO. 41 and SEQ ID NO. 42, and the stable single-chain TCR molecule is shown in figure 7a and figure 7 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 FIGS. 3a and 3 b; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are shown in

SEQ ID NO

7 and 8, respectively, as shown in FIGS. 4a and 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, spread on LB plates containing kanamycin, cultured at 37 ℃ for overnight inversion, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the correct sequence was determined, recombinant plasmids were extracted and transformed into e.coli BL21 (DE 3) for expression.

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

The entire BL21 (DE 3) colony containing the recombinant plasmid pET28 a-template strand prepared in example 1 was inoculated into LB medium containing kanamycin and cultured at 37 ℃ to OD ₆₀₀ At 0.6-0.8, IPTG was added to a final concentration of 0.5mM and incubation 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, centrifuged at 6000rpm for 15min, and the inclusion bodies were collected. The inclusion bodies were dissolved in a buffer (20 mM Tris-HCl pH8.0,8M urea) and centrifuged at high speedRemoving insoluble substances, 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 performed at 37 ℃ for 30min. The treated single-chain TCR was added dropwise to 125mL of renaturation buffer (100 mM Tris-HCl pH 8.1,0.4M L-arginine, 5M urea, 2mM EDTA,6.5mM beta-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 dialysis 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 (20 mM Tris-HCl pH 8.0), dialysis was continued for 8h at 4 ℃ and then the dialysate was changed to the same fresh buffer and dialysis was continued overnight. 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 by a 0-1M NaCl linear gradient eluent formulated in 20mM Tris-HCl pH8.0, the fractions collected were subjected to SDS-PAGE analysis, the fractions containing single-chain TCR were concentrated and then further purified by a gel filtration column (Superdex 75/300, 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 chromatographic column Agilent Bio SEC-3 (300A, phi 7.8X 300 mM) and the mobile phase are 150mM phosphate buffer solution, the flow rate is 0.5mL/min, the column temperature is 25 ℃, and the ultraviolet detection wavelength is 214nm.

Example 3 binding characterisation

BIAcore analysis

Binding activity of the TCR molecules to the FMNKFIYEI-HLA-A0201 complex was measured using a BIAcore T200 real-time assay system. Anti-streptavidin antibody (GenScript) was added to coupling buffer (10 mM sodium acetate buffer, pH 4.77), and then the antibody was passed through CM5 chip previously activated with EDC and NHS to immobilize the antibody on the chip surface, and finally the unreacted activated surface was blocked with ethanolamine hydrochloric acid solution to complete the coupling process at a coupling 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, and then the FMNKFIYEI-HLA-A0201 complex was flowed over the detection channel, the other channel was used as a reference channel, and 0.05mM of biotin was flowed over the chip at a flow rate of 10. Mu.L/min for 2min to block the remaining binding sites of streptavidin. The affinity was determined by single cycle kinetic assay by diluting TCR with HEPES-EP buffer (10 mM HEPES,150mM NaCl,3mM EDTA,0.005% P20, pH 7.4) to several different concentrations, sequentially flowing over the chip surface at a flow rate of 30. Mu.L/min for a binding time of 120s for each sample, and dissociating 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 FMNKFIYEI-HLA-A0201 complex is prepared as follows:

a. purification of

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

Resuspending the inclusion bodies in 5ml of BugBuster Master Mix, and rotary incubating at room temperature for 5min; adding 30ml of 10-fold diluted BugBuster, uniformly mixing, and centrifuging at 4 ℃ at 6000g for 15min; discarding supernatant, adding 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies, mixing, centrifuging at 4 ℃ for 15min, repeating twice, adding 30ml of 20mM Tris-HCl pH8.0 to resuspend the inclusion bodies, mixing, centrifuging at 4 ℃ for 15min at 6000g, finally dissolving the inclusion bodies by using 20mM Tris-HCl 8M urea, detecting the purity of the inclusion bodies by SDS-PAGE, and detecting the concentration by using a BCA kit.

b. Renaturation

The synthesized short peptide FMNKFIYEI (Beijing Saibance Gene technology Co., ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized using 8M urea, 20mM Tris pH8.0, 10mM DTT, and further denatured by addition of 3M guanidine hydrochloride, 10mM sodium acetate, 10mM EDTA prior to renaturation. FMNKFIYEI 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 light chain and 90mg/L heavy chain in sequence (final concentration, heavy chain was added in three portions, 8 h/time), and renaturation was performed at 4 ℃ for at least 3 days until completion, and SDS-PAGE was 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 pH8.0, at least twice 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 (5 ml bed volume). The protein was eluted using a linear gradient of 0-400mM NaCl prepared using an Akta purifier (GE general electric) at 20mM Tris pH8.0, pMHC was eluted at about 250mM NaCl, and the peak fractions were collected and subjected to purity detection using SDS-PAGE.

d. Biotinylation of the peptide

The purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while replacing the buffer with 20mM Tris pH8.0, 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 overnight at room temperature, and SDS-PAGE to determine whether biotinylation was complete.

e. Purification of the biotinylated Complex

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.) ^TM A16/60S 200HR 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, concentrated using Millipore ultrafiltration tubes, protein concentration was determined by BCA (Thermo), and biotinylated pMHC molecules were stored in aliquots at-80 ℃ by addition of the protease inhibitor cocktail (Roche).

Example 4 Generation of high affinity Single chain TCR

Phage display technology is one means of generating libraries of TCR high affinity variants to screen for high affinity variants. The TCR phage display and screening method described by Li et al ((2005) Nature Biotech 23 (3): 349-354) was applied to the single chain TCR template in example 1. Libraries of high affinity TCRs were created by mutating the CDR regions of the template chains and panning was performed. Phage libraries after several rounds of panning have specific binding with corresponding antigens, and monoclonal antibodies are picked and sequence analyzed.

Analysis of the interaction of TCR molecules with the FMNKFIYEI-HLA-A0201 complex using the BIAcore method of example 3, high affinity TCRs with affinity and/or binding half-life at least 2-fold greater than wild type TCRs were selected, i.e., the dissociation equilibrium constant K for the selected high affinity TCRs to bind to the FMNKFIYEI-HLA-A0201 complex _D Less than or equal to the dissociation equilibrium constant K of wild type TCR binding FMNKFIYEI-HLA-A0201 complex _D One-half of (a), the results are shown in table 3 below. K for reference TCR to interact with FMNKFIYEI-HLA-A0201 complex detected by the method described above _D The value was 9.39. Mu.M, and the interaction curve is shown in FIG. 12, i.e., the KD value for the interaction of wild-type TCR with FMNKFIYEI-HLA-A0201 complex is also 9.39. Mu.M, i.e., 9.39E-06M.

Specifically, using the numbering shown in SEQ ID NO 1, the α chain variable domain of these high affinity TCR mutants is mutated at one or more of the following amino acids: 27V, 29I, 30S, 50S, 51S, 52G, 53K, 92S and 93Y and/or using the numbering shown in SEQ ID NO 2, the beta chain variable domains of these high affinity TCR mutants are mutated at one or more of the following positions 52E, 53A, 54Q, 95S, 96Y, 97G, 98A, 103D, 104T and 106Y.

More specifically, the α chain variable domain of these high affinity TCRs comprises one or more amino acid residues 27A or 27P selected from the group consisting of SEQ ID No. 1; 29L;30Q;50P;51F or 51Y;52Q;53T;92T or 92F and 93R or 93N; and/or the β chain variable domain of these high affinity TCRs comprises one or more amino acid residues selected from the group consisting of 52V, 53S, 54I, 95A, 96L or 96P or 96R or 96K or 96Q or 96F, 97F or 97M or 97Y or 97H or 97W or 97A, 98S or 98P or 98G, 103G or 103S or 103M or 103E or 103A or 103R, 104S or 104E or 104G or 104M, 106A or 106V or 106I or 106W or 106K or 106M or 106Q or 106R using the numbering indicated in SEQ ID No. 2.

Specific amino acid sequences of the alpha chain variable domain (SEQ ID NOS: 9-13) and the beta chain variable domain (SEQ ID NOS: 14-40) of the high affinity single chain TCR are shown in FIGS. 5 (1) - (5) and FIGS. 6 (1) - (27), respectively.

TABLE 3

Example 5 Generation of high affinity α β Heterodimeric TCRs

The CDR region mutation of the high affinity single chain TCR selected in example 4 was introduced into the corresponding site of the variable domain of α β heterodimeric TCR and its affinity to fmnkfiyi-HLA-a 0201 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 of the TCR to FMNKFIYEI-HLA A0201 complex, the α β heterodimeric TCR may be a TCR in which a cysteine residue is 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. 8a (SEQ ID NO: 43) and 8b (SEQ ID NO: 44), 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 the CDR regions are introduced by overlap PCR (overlap PCR) which is well known to those skilled in the art. The insert was confirmed by sequencing without error.

Example 6 expression, renaturation and purification of α β heterodimeric TCR

The expression vectors of TCR alpha and beta chains are respectively transformed into expression bacteria BL21 (DE 3) by a chemical transformation method, and the bacteria grow in LB culture solution and grow on OD ₆₀₀ Induction with final concentration 0.5mM IPTG at 0.6 inclusion bodies formed after α and β chain expression of TCR were extracted by BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution for several times, the inclusion bodies finally being 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 are separated by a 1:1 was rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine,6.6mM ss-mericapoethylamine (4 ℃ C.) to a final concentration of 60mg/mL. After mixing, the solution was dialyzed against 10 times the volume 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 α and β dimers that were successfully renatured 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 method.

Example 7BIAcore analysis results

The affinity of the α β heterodimeric TCR with the FMNKFIYEI-HLA-a0201 complex introduced into the high affinity CDR regions was tested using the method described in example 3.

Transferring the CDR areas screened from the high affinity single chain TCR alpha and beta chain to the corresponding positions of the variable domain SEQ ID NO 1 and the variable domain SEQ ID NO 2 of the wild type TCR alpha chain respectively to form the alpha beta heterodimeric TCR. In addition, α β heterodimeric TCRs were formed by artificial combinations based on the mutation sites of the CDR regions obtained by screening, and the amino acid sequences of α and β chain variable domains of the new TCRs were obtained as shown in fig. 9 (1) - (7) and fig. 10 (1) - (28), respectively. Since the CDR regions of the TCR molecule determine their affinity to the corresponding pMHC complex, one skilled in the art would be able to expect that α β heterodimeric TCRs incorporating high affinity mutations also have high affinity for the fmnkfiyi-HLA-a 0201 complex. Expression vectors were constructed using the method described in example 5, and the above α β heterodimeric TCRs introduced with high affinity mutations were expressed, renatured and purified using the method described in example 6, and then their affinity to FMNKFIYEI-HLA-a0201 complex was determined using BIAcore T200, as shown in table 4 below.

TABLE 4

As can be seen from Table 4 above, the α β heterodimeric TCR with the CDR region mutation points introduced maintains high affinity for the FMNKFIYEI-HLA-A0201 complex. The affinity of the heterodimeric TCR is at least 2-fold greater than the affinity of a wild-type TCR for the fmnkfiyi-HLA-a 0201 complex.

Example 8 expression, renaturation and purification of fusions of anti-CD 3 antibodies with high affinity Single chain TCR

The high affinity single chain TCR molecule of the invention is fused with a single chain molecule (scFv) of an anti-CD 3 antibody to construct a fusion molecule. Through the overlapping PCR method, primers are designed, the genes of the anti-CD 3 antibody and the high-affinity single-chain TCR molecule are connected, the middle connecting short peptide (linker) is designed to be GGGGS, and the gene segments of the fusion molecule are provided with restriction enzyme sites NcoI and Not I. The PCR amplification product 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 is transformed into E.coli DH5 alpha competent cells, an LB plate containing kanamycin is coated, inverted culture is carried out at 37 ℃ overnight, positive clones are selected for PCR screening, positive recombinants are sequenced, and after the sequence is determined to be correct, recombinant plasmids are extracted and transformed into E.coli BL21 (DE 3) competent cells for expression.

Expression of fusion proteins

The expression plasmid containing the gene of interest was transformed into E.coli strain BL21 (DE 3), and an LB plate (kanamycin 50. Mu.g/ml) was plated and incubated at 37 ℃ overnight. The next day, the selected clones were inoculated into 10ml of LB liquid medium (kanamycin 50. Mu.g/ml) for 2-3 hours, inoculated into 1L of LB medium (kanamycin 50. Mu.g/ml) at a volume ratio of 1 ₆₀₀ 0.5-0.8, and then IPTG was used at a final concentration of 0.5mM to induce the expression of the protein of interest. 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, corresponding to 200ml of bacterial culture, lysed with 5ml of BugBuster Master Mix (Novagen) and centrifuged at 6000g for 15min to collect inclusion bodies. 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 detergents and salts. Finally, the inclusion body is dissolved by Tris buffer solution containing 8M urea, the concentration of the inclusion body is measured, and the inclusion body is subpackaged and then is frozen and preserved at minus 80 ℃.

Refolding of fusion proteins

Approximately 10mg of inclusion bodies were removed from an ultra-low temperature freezer at-80 ℃ and thawed, dithiothreitol (DTT) was added to a final concentration of 10mM, and incubated at 37 ℃ for 30min to 1 hour to ensure complete disulfide bond opening. The inclusion body sample solutions were then dropped into 200ml of 4 ℃ pre-chilled refolding buffer (100 mM Tris pH 8.1, 400mM L-arginine, 2mM EDTA,5M urea, 6.5mM beta-mercaptoethylamine, 1.87mM Cystamine), respectively, and slowly stirred at 4 ℃ for about 30 minutes. Renaturation solution with 8 times volume of precooled H ₂ O dialysis for 16-20 hours. The mixture was dialyzed twice against 8 volumes of 10mM Tris pH8.0 for about 8 hours at 4 ℃ and then filtered to purify the sample as follows.

First step purification of fusion proteins

Dialyzed refolded material (10 mM Tris pH 8.0) was subjected to gradient elution with 0-600mM NaCl in an AKTA purifier (GE Healthcare) using POROS HQ/20 anion exchange chromatography pre-packed column (Applied Biosystems). The individual fractions were analyzed by Coomassie blue stained SDS-PAGE and then pooled.

Second step purification of fusion proteins

The combined sample solutions from the first purification step were concentrated for this purification step, the fusion protein was purified using Superdex 75/300 GL gel filtration chromatography pre-column (GE Healthcare) pre-equilibrated in PBS buffer, and the peak fractions were analyzed by Coomassie blue-stained SDS-PAGE and then combined.

Example 9 expression, renaturation and purification of fusions of anti-CD 3 antibodies with high affinity α β hetero-dimeric TCRs

Fusion molecules were prepared by fusing anti-CD 3 single chain antibodies (scFv) to α β heterodimeric TCR. The scFv of the 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 alpha beta heterodimeric TCR is subjected to double enzyme digestion by Nco I and Not I and is connected with a pET28a vector subjected to double enzyme digestion by 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 overnight inversion, positive clones were selected for PCR screening, positive recombinants were sequenced, and after the correct sequence was determined, recombinant plasmids were extracted and transformed into e.coli Tuner (DE 3) for expression.

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

By the overlap PCR method, primers are designed to connect the anti-CD 3scFv and the high-affinity heterodimeric TCR beta chain gene, the middle connecting short peptide (linker) is GGGGS, and the gene segment 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 to pET28a vector digested simultaneously with Nco I and Not I. The ligation product was transformed into E.coli DH 5. Alpha. 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 correct sequence was determined, recombinant plasmids were extracted and transformed into E.coli Tuner (DE 3) competent cells for expression.

Expression, renaturation and purification of fusion proteins

The expression plasmids were transformed into E.coli Tuner (DE 3) competent cells, respectively, and LB plates (kanamycin 50. Mu.g/mL) were plated and incubated at 37 ℃ overnight. The next day, the selected clones were inoculated into 10mL of LB liquid medium (kanamycin 50. Mu.g/mL) for 2-3 hours, inoculated into 1L of LB medium at a volume ratio of 1. After 4 hours of induction, the cells were harvested by centrifugation at 6000rpm for 10 min. The cells were washed once with PBS buffer and dispensed, and cells corresponding to 200mL of 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 detergents and salts. Finally, inclusion bodies were dissolved in a buffer solution containing 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 2mM Tris, pH 8.1, and the inclusion body concentration was measured, and they were stored in portions at-80 ℃ for cryopreservation.

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), 2mM Tris pH 8.1,3.7mM cystamine,6.6mM β -mer peptide (4 ℃ C.), to final concentrations of 0.1mg/mL and 0.25mg/mL for α chain and anti-CD 3 (scFv) - β chain, 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 HP5ml, GE healthcare). The eluted peaks contain TCR alpha chain and anti-CD 3 (scFv) -beta chain dimers of which the renaturation was successful TCR alpha chain was confirmed by SDS-PAGE gel. The TCR fusion molecules were subsequently further purified by size exclusion chromatography (S-100/60, GE healthcare) and re-purified on an anion exchange column (HiTrap Q HP5ml, 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 10 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 PBLs isolated from blood of healthy volunteers as effector cells.

The following two batches (I) and (II) were tested in sequence (in which the TCR and target cell lines were different):

(I) the TCR and its numbering is known from Table 4 as TCR13 (alpha chain variable domain SEQ ID NO:45, beta chain variable domain SEQ ID NO: 2), TCR21 (alpha chain variable domain SEQ ID NO:46, beta chain variable domain SEQ ID NO: 2), TCR9 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 60), TCR4 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 55), TCR1 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 52), TCR2 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 53), TCR12 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 63), TCR5 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 56), TCR3 (beta chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 54), TCR5 (alpha chain variable domain TCR 6) and TCR 59, as control for transfecting cells with the wild type cell variable domain SEQ ID NO:1, TCR8 and the variable domain TCR 6. The target cell lines were HepG2, HCCC9810-AFP (i.e., AFP-transfected HCCC 9810), SNU-398-AFP (i.e., AFP-transfected SNU-398), huh-7, SNU-398, and HCCC9810 cells. Wherein, target cell lines HepG2, HCCC9810-AFP and SNU-398-AFP are positive tumor cell lines; huh-7, SNU-398 and HCCC9810 were negative tumor cell lines used as controls.

(II) the TCR and its numbering is shown in Table 4 as TCR14 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 64), TCR26 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 75), TCR18 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 68), TCR19 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 69), TCR10 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 61), TCR22 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 71), TCR23 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 72), TCR24 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 73), TCR11 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 65), TCR17 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 15, TCR17, TCR15 chain variable domain TCR16, TCR17 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO:1, SEQ ID NO: 16, beta chain variable domain SEQ ID NO: 70), control effector cell designations wild-type TCR (cells transfected with wild-type TCR) and A6 (cells transfected with other TCR). The target cell lines are HepG2, HCCC9810-AFP (i.e., AFP transfected HCCC 9810), SK-HEP-1-AFP (i.e., AFP transfected SK-HEP-1), huh-7, HCCC9810 and SK-HEP-1 cells. Wherein, target cell lines HepG2, HCCC9810-AFP and SK-HEP-1-AFP are positive tumor cell lines; huh-7, HCCC9810 and SK-HEP-1 are negative tumor cell lines, used as controls.

The following experimental procedures were carried out for both batches: first, an ELISPOT plate is prepared. ELISPOT plate ethanol activation coating, 4 degrees C overnight. Day 1 of the experiment, coating was removed, washed and blocked, incubated at room temperature for two hours, blocking solution removed, and the components of the experiment were added to ELISPOT plates: target cell line 2 x 10 ⁴ One/well, effector cell 10 ³ One/well (calculated as positive rate of transfection) and two duplicate wells were set. Incubation overnight (37 ℃,5% CO) ₂ ). Experiment day 2, plates were washed and secondary detection and visualization was performedThe plates were dried, and spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID20 Co.).

The results of the two batches of experiments are shown in fig. 14a and 14b, respectively, and the effector cells transfected with the high affinity TCR of the invention generate good specific activation effect for the positive target cell line, and the function is far better than that of the effector cells transfected with the wild TCR. While cells transduced other TCRs produced essentially no activation.

Example 11 experiment of LDH killing function of Effector cells transfected with high affinity TCR of the invention

This example demonstrates the killing function of cells transduced with the TCR of the invention by measuring LDH release by non-radioactive cytotoxicity assays. The test is ⁵¹ Colorimetric substitution test for Cr release cytotoxicity test Lactate Dehydrogenase (LDH) released after cell lysis was quantitatively determined. LDH released in the medium was detected using a 30min coupled enzymatic reaction in which LDH converted a tetrazolium salt (INT) to red formazan (formazan). The amount of red product produced is proportional to the number of cells lysed. 490nm visible absorbance data can be collected using a standard 96-well plate reader. The formula is calculated as% cytotoxicity =100 × (experiment-effector cell spontaneous-target cell spontaneous)/(target cell maximal-target cell spontaneous)

Methods for detecting cell function using LDH release assays are well known to those skilled in the art. Random selection of TCR of the invention transfection of CD3 isolated from blood of healthy volunteers ⁺ T cells, as effector cells. The TCRs and their numbering are known from Table 4 as TCR4 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 55), TCR1 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 52), TCR2 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 53), TCR12 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 63) and TCR5 (alpha chain variable domain SEQ ID NO:1, beta chain variable domain SEQ ID NO: 56), and control effector cell numbers as wild type TCR (cells transfected with wild type TCR) and A6 (cells transfected with other TCRs). Target cell lines were HepG2, HCCC9810 and SNU-398 cells. Wherein, hepG2 is a positive tumor cell line;HCCC9810 and SNU-398 were negative tumor cell lines as controls.

LDH plates were first prepared. On experiment day 1, the individual components of the experiment were added to the plate: target cell line 3 x 10 ⁴ Individual cells/well, effector cells 3 x 10 ⁴ One cell per well and three duplicate wells were set. Meanwhile, an effector cell spontaneous hole, a target cell maximum hole, a volume correction control hole and a culture medium background control hole are arranged. Incubation overnight (37 ℃,5% CO) ₂ ). On the 2 nd day of the experiment, color development was detected, and after the reaction was terminated, the absorbance was recorded at 490nm using a microplate reader (Biotech).

The experimental results are shown in fig. 15, the cells transduced with the TCR of the present invention all had a strong killing effect on the positive target cells, and the killing effect was much higher than that of the cells transduced with the wild type TCR; while cells transduced with other TCRs had essentially no killing effect on positive target cells.

Sequence listing

<110> Guangdong Xiangxue accurate medical technology Limited

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Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala Leu

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His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met

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Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn

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Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro Arg

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

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Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Ile Pro

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Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

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Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

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Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

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

100 105 110

Leu Thr Val Leu

115

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Lys Asn Glu Val Glu Gln Ser Pro Gln Asn Leu Thr Val Gln Glu Gly

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Glu Asn Val Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala Leu

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His Trp Leu Arg Gln Asp Pro Gly Gly Gly Ile Val Ser Leu Phe Met

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Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Asn Ala Thr Ile Asn

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Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Asp Val His Pro Arg

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Asp Ser Ala Val Tyr Phe Cys Ala Val Glu Thr Ser Tyr Asp Lys Val

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Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Thr Pro

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<213> Artificial Sequence (Artificial Sequence)

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Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

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Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

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Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

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

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Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

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Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

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

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Leu Thr Val Asp

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aaaaatgaag ttgaacagag cccgcaaaac ctgaccgtgc aggaaggtga aaacgttacc 60

atcaactgca gctacagcgt gggtattagc gcgctgcatt ggctgcgtca agatccgggt 120

ggcggtatcg ttagcctgtt catgctgagc agcggcaaga aaaagcacgg tcgtctgaac 180

gcgaccatca acattcaaga gaaacacagc agcctgcaca ttaccgacgt tcacccgcgt 240

gatagcgcgg tgtacttttg cgcggttgaa accagctatg acaaagtgat ttttggtccg 300

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ggtgcgggtg tgagccagag cccgcgttac ctgagcgtga aacgtggcca agacgttacc 60

ctgcgttgcg atccgatcag cggtcacgtt agcctgtact ggtaccgtca agatccgggt 120

caaggtccgg agttcctgac ctactttaac tatgaagcgc agcaagacaa gagcggcctg 180

ccgaacgatc gtttcagcgc ggagcgtccg gaaggtagca tcagcaccct gaccattcag 240

cgtgtggagc cgcgtgatag cgcgatgtat ttttgcgcga gcagctacgg tgcgggcggt 300

ccgctggaca cccaatattt tggcccgggt 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

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<213> Artificial Sequence (Artificial Sequence)

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ggcggtggca gcgagggtgg cggtagcgaa ggcggtggca gcgagggtgg cggtagcgaa 60

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<213> Artificial Sequence (Artificial Sequence)

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Lys Asn Glu Val Glu Gln Ser Pro Gln Asn Leu Thr Val Gln Glu Gly

1 5 10 15

Glu Asn Val Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala Leu

20 25 30

His Trp Leu Arg Gln Asp Pro Gly Gly Gly Ile Val Ser Leu Phe Met

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Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Asn Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Asp Val His Pro Arg

65 70 75 80

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85 90 95

Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Thr Pro

100 105

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<213> Artificial Sequence (Artificial Sequence)

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Lys Asn Glu Val Glu Gln Ser Pro Gln Asn Leu Thr Val Gln Glu Gly

1 5 10 15

Glu Asn Val Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala Leu

20 25 30

His Trp Leu Arg Gln Asp Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Asn Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Asp Val His Pro Arg

65 70 75 80

Asp Ser Ala Val Tyr Phe Cys Ala Val Glu Thr Phe Asn Asp Lys Val

85 90 95

Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Thr Pro

100 105

<210> 11

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Lys Asn Glu Val Glu Gln Ser Pro Gln Asn Leu Thr Val Gln Glu Gly

1 5 10 15

Glu Asn Val Thr Ile Asn Cys Ser Tyr Ser Val Gly Leu Gln Ala Leu

20 25 30

His Trp Leu Arg Gln Asp Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Pro Phe Gly Lys Lys Lys His Gly Arg Leu Asn Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Asp Val His Pro Arg

65 70 75 80

Asp Ser Ala Val Tyr Phe Cys Ala Val Glu Thr Thr Arg Asp Lys Val

85 90 95

Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Thr Pro

100 105

<210> 12

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Lys Asn Glu Val Glu Gln Ser Pro Gln Asn Leu Thr Val Gln Glu Gly

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Glu Asn Val Thr Ile Asn Cys Ser Tyr Ser Ala Gly Leu Gln Ala Leu

20 25 30

His Trp Leu Arg Gln Asp Pro Gly Gly Gly Ile Val Ser Leu Phe Met

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Leu Pro Phe Gly Lys Lys Lys His Gly Arg Leu Asn Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Asp Val His Pro Arg

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Asp Ser Ala Val Tyr Phe Cys Ala Val Glu Thr Ser Tyr Asp Lys Val

85 90 95

Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Thr Pro

100 105

<210> 13

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Lys Asn Glu Val Glu Gln Ser Pro Gln Asn Leu Thr Val Gln Glu Gly

1 5 10 15

Glu Asn Val Thr Ile Asn Cys Ser Tyr Ser Pro Gly Leu Gln Ala Leu

20 25 30

His Trp Leu Arg Gln Asp Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Pro Phe Gly Lys Lys Lys His Gly Arg Leu Asn Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Asp Val His Pro Arg

65 70 75 80

Asp Ser Ala Val Tyr Phe Cys Ala Val Glu Thr Ser Tyr Asp Lys Val

85 90 95

Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Thr Pro

100 105

<210> 14

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ala Leu

85 90 95

Met Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 15

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ala Arg

85 90 95

Tyr Pro Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 16

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ala Arg

85 90 95

His Ala Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 17

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ala Pro

85 90 95

Phe Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 18

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ala Lys

85 90 95

Met Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 19

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

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

100 105 110

Leu Thr Val Asp

115

<210> 20

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

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

100 105 110

Leu Thr Val Asp

115

<210> 21

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

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

85 90 95

Ser Gly Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 22

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ala Leu

85 90 95

Phe Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 23

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

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

100 105 110

Leu Thr Val Asp

115

<210> 24

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Met Ala Gln Ala Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 25

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ala Leu

85 90 95

Tyr Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 26

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Pro

85 90 95

Phe Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 27

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Gly Ala Gln Lys Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 28

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

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

100 105 110

Leu Thr Val Asp

115

<210> 29

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Ser Ser Gln Ile Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 30

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Leu

85 90 95

Phe Gly Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 31

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Ser Gly Gln Ile Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 32

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Ala Ser Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 33

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Arg Thr Gln Met Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 34

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Gly Ser Gln Gln Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 35

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Gly Ser Gln Val Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 36

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Gly Ser Gln Ala Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 37

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Leu

85 90 95

Phe Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 38

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Leu

85 90 95

Trp Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 39

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 39

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Phe

85 90 95

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

100 105 110

Leu Thr Val Asp

115

<210> 40

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 40

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser Val Lys Arg Gly

1 5 10 15

Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Gly Met Gln Arg Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Asp

115

<210> 41

<211> 248

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 41

Lys Asn Glu Val Glu Gln Ser Pro Gln Asn Leu Thr Val Gln Glu Gly

1 5 10 15

Glu Asn Val Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala Leu

20 25 30

His Trp Leu Arg Gln Asp Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Asn Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Asp Val His Pro Arg

65 70 75 80

Asp Ser Ala Val Tyr Phe Cys Ala Val Glu Thr Ser Tyr Asp Lys Val

85 90 95

Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Thr Pro Gly Gly Gly Ser

100 105 110

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

115 120 125

Gly Gly Thr Gly Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Leu Ser

130 135 140

Val Lys Arg Gly Gln Asp Val Thr Leu Arg Cys Asp Pro Ile Ser Gly

145 150 155 160

His Val Ser Leu Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu

165 170 175

Phe Leu Thr Tyr Phe Asn Tyr Glu Ala Gln Gln Asp Lys Ser Gly Leu

180 185 190

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

195 200 205

Leu Thr Ile Gln Arg Val Glu Pro Arg Asp Ser Ala Met Tyr Phe Cys

210 215 220

Ala Ser Ser Tyr Gly Ala Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly

225 230 235 240

Pro Gly Thr Arg Leu Thr Val Asp

245

<210> 42

<211> 744

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

aaaaatgaag ttgaacagag cccgcaaaac ctgaccgtgc aggaaggtga aaacgttacc 60

atcaactgca gctacagcgt gggtattagc gcgctgcatt ggctgcgtca agatccgggt 120

ggcggtatcg ttagcctgtt catgctgagc agcggcaaga aaaagcacgg tcgtctgaac 180

gcgaccatca acattcaaga gaaacacagc agcctgcaca ttaccgacgt tcacccgcgt 240

gatagcgcgg tgtacttttg cgcggttgaa accagctatg acaaagtgat ttttggtccg 300

ggtaccagcc tgagcgttac cccgggcggt ggcagcgagg gtggcggtag cgaaggcggt 360

ggcagcgagg gtggcggtag cgaaggcggt accggcggtg cgggtgtgag ccagagcccg 420

cgttacctga gcgtgaaacg tggccaagac gttaccctgc gttgcgatcc gatcagcggt 480

cacgttagcc tgtactggta ccgtcaagat ccgggtcaag gtccggagtt cctgacctac 540

tttaactatg aagcgcagca agacaagagc ggcctgccga acgatcgttt cagcgcggag 600

cgtccggaag gtagcatcag caccctgacc attcagcgtg tggagccgcg tgatagcgcg 660

atgtattttt gcgcgagcag ctacggtgcg ggcggtccgc tggacaccca atattttggc 720

ccgggtaccc gtctgaccgt tgat 744

<210> 43

<211> 202

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 43

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

1 5 10 15

Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala Leu

20 25 30

His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro Arg

65 70 75 80

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

85 90 95

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

100 105 110

Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys

115 120 125

Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln

130 135 140

Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met

145 150 155 160

Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys

165 170 175

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

180 185 190

Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200

<210> 44

<211> 246

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 44

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Asp Thr Gln Tyr 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> 45

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 45

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

1 5 10 15

Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala Leu

20 25 30

His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro Arg

65 70 75 80

Asp Ser Ala Val Tyr Ile Cys Ala Val Glu Thr Thr Arg Asp Lys Val

85 90 95

Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Ile Pro

100 105

<210> 46

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 46

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

1 5 10 15

Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala Leu

20 25 30

His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro Arg

65 70 75 80

Asp Ser Ala Val Tyr Ile Cys Ala Val Glu Thr Phe Asn Asp Lys Val

85 90 95

Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Ile Pro

100 105

<210> 47

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 47

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

1 5 10 15

Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Val Gly Leu Gln Ala Leu

20 25 30

His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Pro Phe Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro Arg

65 70 75 80

Asp Ser Ala Val Tyr Ile Cys Ala Val Glu Thr Thr Arg Asp Lys Val

85 90 95

Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Ile Pro

100 105

<210> 48

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 48

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

1 5 10 15

Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Ala Gly Leu Gln Ala Leu

20 25 30

His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Pro Phe Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro Arg

65 70 75 80

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

85 90 95

Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Ile Pro

100 105

<210> 49

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 49

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

1 5 10 15

Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Pro Gly Leu Gln Ala Leu

20 25 30

His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Pro Phe Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro Arg

65 70 75 80

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

85 90 95

Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Ile Pro

100 105

<210> 50

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 50

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

1 5 10 15

Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Ala Gly Leu Gln Ala Leu

20 25 30

His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Pro Tyr Gln Thr Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro Arg

65 70 75 80

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

85 90 95

Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Ile Pro

100 105

<210> 51

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 51

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

1 5 10 15

Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Ala Gly Leu Gln Ala Leu

20 25 30

His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Pro Tyr Gln Thr Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro Arg

65 70 75 80

Asp Ser Ala Val Tyr Ile Cys Ala Val Glu Thr Thr Arg Asp Lys Val

85 90 95

Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Ile Pro

100 105

<210> 52

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 52

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ala Leu

85 90 95

Met Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 53

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 53

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ala Arg

85 90 95

Tyr Pro Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 54

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 54

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ala Arg

85 90 95

His Ala Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 55

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 55

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ala Pro

85 90 95

Phe Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 56

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 56

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ala Lys

85 90 95

Met Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 57

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 57

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

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

100 105 110

Leu Thr Val Leu

115

<210> 58

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 58

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

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

100 105 110

Leu Thr Val Leu

115

<210> 59

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 59

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Gln

85 90 95

Ser Gly Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 60

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 60

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ala Leu

85 90 95

Phe Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 61

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 61

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

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

100 105 110

Leu Thr Val Leu

115

<210> 62

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 62

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Met Ala Gln Ala Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 63

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 63

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ala Leu

85 90 95

Tyr Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 64

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 64

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Pro

85 90 95

Phe Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 65

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 65

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Gly Ala Gln Lys Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 66

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 66

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

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

100 105 110

Leu Thr Val Leu

115

<210> 67

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 67

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Ser Ser Gln Ile Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 68

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 68

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Leu

85 90 95

Phe Gly Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 69

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 69

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Ser Gly Gln Ile Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 70

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 70

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Ala Ser Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 71

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 71

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Arg Thr Gln Met Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 72

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 72

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Gly Ser Gln Gln Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 73

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 73

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Gly Ser Gln Val Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 74

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 74

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Gly Ser Gln Ala Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 75

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 75

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Leu

85 90 95

Phe Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 76

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 76

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Leu

85 90 95

Trp Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 77

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 77

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Phe

85 90 95

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

100 105 110

Leu Thr Val Leu

115

<210> 78

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 78

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Gly Met Gln Arg Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 79

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 79

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

Phe Asn Tyr Val Ser Ile Gln Asp Lys Ser Gly Leu Pro Asn Asp Arg

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ala Leu

85 90 95

Phe Ser Gly Gly Pro Leu Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg

100 105 110

Leu Thr Val Leu

115

<210> 80

<211> 202

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 80

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

1 5 10 15

Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala Leu

20 25 30

His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro Arg

65 70 75 80

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

85 90 95

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

100 105 110

Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys

115 120 125

Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln

130 135 140

Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met

145 150 155 160

Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys

165 170 175

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

180 185 190

Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200

<210> 81

<211> 246

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 81

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Asp Thr Gln Tyr 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> 82

<211> 249

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 82

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

1 5 10 15

Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile Ser Ala Leu

20 25 30

His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser Leu Phe Met

35 40 45

Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Ile Ala Thr Ile Asn

50 55 60

Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser His Pro Arg

65 70 75 80

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

85 90 95

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

100 105 110

Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys

115 120 125

Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln

130 135 140

Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met

145 150 155 160

Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys

165 170 175

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

180 185 190

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

195 200 205

Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser

210 215 220

Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu

225 230 235 240

Leu Met Thr Leu Arg Leu Trp Ser Ser

245

<210> 83

<211> 295

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 83

Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr Lys Arg Gly

1 5 10 15

Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His Val Ser Leu

20 25 30

Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

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

50 55 60

Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu Thr Ile Gln

65 70 75 80

Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala Ser Ser Tyr

85 90 95

Gly Ala Gly Gly Pro Leu Asp Thr Gln Tyr 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

Claims

1. A T Cell Receptor (TCR) having binding activity to the FMNKFIYEI-HLA a0201 complex, and comprising a TCR a chain variable domain comprising 3 CDR regions and a TCR β chain variable domain comprising 3 CDR regions; the TCR has a CDR combination corresponding to any one of the following CDR numbers:

2. a TCR as claimed in claim 1 which is soluble.

3. A TCR as claimed in claim 1 which is an α β heterodimeric TCR.

4. A TCR as claimed in claim 3 which has the α chain constant region sequence TRAC 01 and the β chain constant region sequence TRBC1 01 or TRBC2 01.

5. A TCR as claimed in claim 1 which 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.

6. A TCR as claimed in claim 1 which comprises an artificial interchain disulphide bond between the α chain constant region and the β chain constant region of the TCR.

7. A TCR as claimed in claim 6 wherein the cysteine residue which forms an artificial interchain disulphide bond between the constant regions of the TCR α and β chains is substituted at one or more groups selected from:

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

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

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

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

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

tyr10 of exon 1 TRAC × 01 and TRBC1 × 01 or TRBC2 × 01 Glu20 of exon 1.

8. A TCR as claimed in claim 1 which has α and β chain variable domain sequences corresponding to any one of the following TCR numbering:

9. a TCR as claimed in claim 1 which is a single chain TCR.

10. A TCR as claimed in claim 1 which is a single chain TCR consisting of an α chain variable domain and a β chain variable domain, the α chain variable domain and β chain variable domain being linked by a flexible short peptide sequence.

11. A TCR as claimed in claim 1 which has a combination of α and β chain variable domain sequences selected from any one of the following TCR numbering:

12. a TCR as claimed in any one of claims 1 to 11 which has a conjugate attached to the C-or N-terminus of the α chain and/or β chain of the TCR.

13. A TCR as claimed in claim 12 wherein the conjugate to which the TCR is bound is a detectable label, a therapeutic agent or a combination of any of these.

14. A TCR as claimed in claim 13 wherein the therapeutic agent to which the TCR binds is an anti-CD 3 antibody linked to the C-or N-terminus of the α or β chain of the TCR.

15. A multivalent TCR complex comprising at least two TCR molecules, at least one of which is a TCR as claimed in any one of the preceding claims.

16. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR as claimed in any one of claims 1 to 14.

17. A vector comprising the nucleic acid molecule of claim 16.

18. A host cell comprising the vector of claim 17 or a nucleic acid molecule of claim 16 integrated into the chromosome.

19. An isolated cell expressing a TCR as claimed in any one of claims 1 to 14.

20. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1 to 14, or a TCR complex according to claim 15, or a cell according to claim 19.

21. Use of a T cell receptor according to any one of claims 1 to 14, a TCR complex according to claim 15 or a cell according to claim 19 for the manufacture of a medicament for the treatment of a tumour;

the tumor is hepatocellular carcinoma.

22. A method of preparing a T cell receptor according to any one of claims 1 to 14, comprising the steps of:

(i) Culturing the host cell of claim 18 so as to express the T cell receptor of any one of claims 1-14;

(ii) Isolating or purifying said T cell receptor.