CN106459176B

CN106459176B - High-affinity HCV T cell receptors

Info

Publication number: CN106459176B
Application number: CN201580024400.9A
Authority: CN
Inventors: 李懿; 陈磊; 张剑冰
Original assignee: Guangdong Xiangxue Precision Medical Technology Co Ltd
Current assignee: Xiangxue Life Science Technology Guangdong Co ltd
Priority date: 2014-06-18
Filing date: 2015-06-15
Publication date: 2018-07-06
Anticipated expiration: 2035-06-15
Also published as: WO2015192757A1; CN106459176A

Abstract

A kind of T cell receptor (TCR) is provided, with reference to KLVALGINAV HLA A₂Compound and include TCR α variable domains and/or TCR β variable domains.Additionally provide the fusion of the TCR and therapeutic agent.The TCR can be used alone, and can be also combined with therapeutic agent, and KLVALGINAV HLA A are presented with targeting₂The HCV infection cell of compound.

Description

High affinity HCV T cell receptors

Technical Field

The present invention relates to the field of biotechnology, and more specifically to a T Cell Receptor (TCR) capable of specifically recognizing a polypeptide derived from HCV NS3 protein. The invention also relates to the preparation and use of said receptors.

Background

the general TCR repertoire of the immune system results from recombination by V (D) J in the thymus, followed by positive and negative selection.

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.

On the T cell membrane, the TCR binds to the constant protein CD3 involved in signal transduction to form a complex. TCRs exist in a variety of forms and are structurally similar, however T cells expressing these TCRs can exist in different anatomical locations and may have different functions. The extracellular portion of the TCR consists of two membrane-proximal constant domains and two membrane-distal variable domains with variable loops similar to the Complementarity Determining Regions (CDRs) of the antibody. It is these loops that form the antigen (pMHC) binding site of the T cell receptor molecule and that determine the specificity of the TCR. 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.

Hepatitis C Virus (HCV) is a major cause of acute hepatitis and long-term liver diseases including cirrhosis and hepatocellular carcinoma. HCV-infected hepatocytes degrade viral proteins into short peptides, KLVALGINAV peptide derived from the non-structural protein NS3 of HCV, which is loaded by HLA-a2 and presented on the surface of HCV-infected cells to form an epitope of HCV.

Thus, the KLVALGINAV-HLA A2 complex provides a marker for targeting of the TCR to HCV. For example, TCRs capable of targeting HCV markers can be used to deliver cytotoxic or immunostimulatory agents to infected cells, or transformed into T cells, enabling them to destroy HCV-infected cells for administration to patients in a therapeutic process known as adoptive immunotherapy. For the former purpose, the ideal TCR is of higher affinity, so that it can reside on the targeted cell for a long period of time. For the latter purpose, it is preferred to use a medium affinity TCR, since a sharp increase in affinity is associated with a loss of antigen specificity in TCR gene-modified CD8T cells, leading to non-specific activation of these TCR-transfected CD8T cells (see Zhao et al, (2007) J immunol.179: 5845-54; Robbins et al, (2008) J immunol.180: 6116-31; see also WO 2008/038002). The skilled artisan is therefore working to develop TCRs that target HCV markers that can be used to meet different objectives.

Disclosure of Invention

The invention aims to provide a new medicine for treating HLA-A2/HCV NS 3: the 1406-1415(KLVALGINAV, SEQ IDNO: 1) complex has a higher affinity TCR.

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

in a first aspect of the invention, there is provided a T Cell Receptor (TCR) having the property of binding KLVALGINAV (SEQ id no: 1) the HLA-a2 complex and comprising a TCR β chain variable domain and/or a TCR beta chain variable domain, wherein:

(i) the TCR having a mutation in the β chain variable domain as shown in SEQ ID NO. 2 and/or in the beta chain variable domain amino acid residue as shown in SEQ ID NO. 3, and

(ii) the TCR has at least twice the affinity and/or binding half-life of the KLVALGINAV-HLA-a2 complex as compared to a wild-type HCV NS3 TCR; or

The TCR binds to the dissociation equilibrium constant K of the KLVALGINAV-HLA-A2 complex_DLess than or equal to the dissociation equilibrium constant K for wild-type HCV NS3TCR to bind the KLVALGINAV-HLA-A2 complex_DOne half of (a).

in another preferred embodiment, the amino acid sequence of the TCR before mutation of the α chain variable domain is as shown in SEQ ID No. 2.

in another preferred embodiment, the amino acid sequence of the TCR before mutation of the β chain variable domain is as shown in SEQ ID No. 3.

in another preferred embodiment, the α chain variable domain of the TCR has at least 80% sequence identity to the amino acid sequence set forth in SEQ ID No. 2 and/or the β chain variable domain of the TCR has at least 80% sequence identity to the amino acid sequence set forth in SEQ ID No. 3;

in another preferred embodiment, the TCR has a dissociation equilibrium constant K for the KLVALGINAV-HLA-A2 complex_D≤3.3μM。

In another preferred embodiment, the TCR has a dissociation equilibrium constant of 0.5 μ M ≦ K for the KLVALGINAV-HLA-A2 complex_DLess than or equal to 3.3 mu M; preferably, 1 μ M ≦ K_DLess than or equal to 3.3 mu M; more preferably, 1. mu.M.ltoreq.K_D≤2μM。

In another preferred embodiment, the TCR has a dissociation equilibrium constant K for the KLVALGINAV-HLA-A2 complex_DLess than or equal to 500 nM; preferably, 10 pM. ltoreq.K_D≤500nM。

in another preferred embodiment, the mutation occurs in the CDR regions of the α chain and/or the β chain.

in another preferred embodiment, the mutation occurs in CDR1 and/or CDR2 and/or CDR3 of the β chain and/or CDR1 and/or CDR2 and/or CDR3 of the beta chain, preferably the mutation occurs in CDR1 and/or CDR2 and/or CDR3 of the β chain and/or CDR3 of the beta chain, more preferably the mutation occurs in CDR1 of the β chain and/or CDR3 of the beta chain.

in another preferred embodiment, the mutation occurs at one or more amino acid residue positions selected from the group consisting of 30S, 31D, 33Y, 53A, 54Y, 55K, 56Q, 57Q, 58N, 96E, 98D, 99K, 100I and 101I in the α chain variable domain shown in SEQ ID No. 2, wherein the amino acid residue numbering is as shown in SEQ ID No. 2.

in another preferred embodiment, the mutation occurs at one or more amino acid residue positions selected from the group consisting of 29H, 50Y, 53N, 54S, 97P, 98Y, 99E, 100Q and 101Y in the variable domain of the beta strand as set forth in SEQ ID No. 3, wherein the numbering of the amino acid residues is as set forth in SEQ ID No. 3.

in another preferred embodiment, the mutated TCR α chain variable domain comprises one or more amino acid residues selected from the group consisting of 30N, 31E, 33I, 53S, 54D, 54W or 54A, 55N, 55S or 55Y, 56T, 56K, 56M, 56P or 56N, 57H, 57E, 57D, 57I or 57N, 58S or 58I, 96D, 98N, 98T, 98Q or 98V, 99S, 99Y or 99P, 100L and 101T, wherein the numbering of the amino acid residues is as shown in SEQ ID NO. 2.

in another preferred embodiment, the mutated TCR β chain variable domain comprises one or more amino acid residues selected from the group consisting of 29Y, 50L, 53G, 54H, 97S, 98A, 98L, 98E, 98S or 98G, 99A or 99P, 100L, 100M or 100V, 101I, 101F or 101S, wherein the numbering of the amino acid residues is as shown in SEQ ID NO. 3.

in another preferred embodiment, the amino acid sequence of the α chain variable domain of the TCR is selected from the group consisting of SEQ ID No. 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71 and 72.

in another preferred embodiment, the amino acid sequence of the β chain variable domain of the TCR is selected from the group consisting of SEQ ID No. 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87 and 88.

in another preferred embodiment, the TCR comprises a combination of α and β chain variable domains as shown in the following table:

in another preferred embodiment, the TCR is an α β heterodimeric TCR having α and β chain constant domain sequences with cysteine residues forming disulfide bonds between the α and β chain constant domains of the TCR.

in another preferred embodiment, in the TCR the cysteine residues form an artificial disulfide bond between the α and β chain constant domains of the TCR.

In another preferred embodiment, in the TCR the cysteine residues forming the artificial disulfide bond replace one or more groups of sites selected from the group consisting of:

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

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

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

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

ser15 and TRBC1 × 01 or Glu15 of TRBC2 × 01 exon 1 substituted for TRAC × 01 exon 1.

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 is a single chain TCR consisting of an α variable domain and a β variable domain linked by a flexible short peptide sequence (linker).

in another preferred embodiment, the hydrophobic core mutation occurs at one or more amino acid residue positions selected from the group consisting of 11M, 21L, 48V and 110I in the α chain variable domain as set forth in SEQ ID No. 2, wherein the numbering of the amino acid residues is as set forth in SEQ ID No. 2.

in another preferred embodiment, the hydrophobic core mutation occurs at one or more amino acid residue positions 78L and 81A of the variable domain of its beta strand as set forth in SEQ ID No. 3, wherein the numbering of the amino acid residues is as set forth in SEQ ID No. 3.

in another preferred embodiment, the α chain variable domain of the TCR after hydrophobic core mutation comprises one or more amino acid residues selected from the group consisting of 11L, 21I, 48L and 110V, wherein the amino acid residue numbering is as shown in SEQ ID No. 2.

in another preferred embodiment, the variable domain of the beta chain of the TCR after the hydrophobic core has been mutated comprises one or more amino acid residues selected from the group consisting of 78I and 81L, wherein the numbering of the amino acid residues is as shown in SEQ ID No. 3.

in another preferred embodiment, the amino acid sequence of the α chain variable domain of the TCR is selected from the group consisting of SEQ ID No. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30 and 31.

in another preferred embodiment, the amino acid sequence of the β chain variable domain of the TCR is selected from the group consisting of SEQ ID No. 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46 and 47.

in another preferred embodiment, the α chain and/or β chain of the TCR is conjugated to a conjugate.

In another preferred embodiment, the conjugate is selected from one or more of the group consisting of:

(1) a detectable label;

(2) a therapeutic agent; and/or

(3) A PK modifying moiety.

Preferably, the detectable label comprises: 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.

Preferably, the therapeutic agent comprises: radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles, and the like.

In another preferred embodiment, the therapeutic agent is an anti-CD 3 antibody.

in another preferred embodiment, the conjugate is attached to the C-or N-terminus of the α and/or β chains of the TCR.

in another preferred embodiment, the amino acid sequence of the β chain variable domain of the TCR binding to the conjugate is selected from the group consisting of SEQ ID NO. 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71 and 72 and/or the amino acid sequence of the beta chain variable domain of the TCR binding to the conjugate is selected from the group consisting of SEQ ID NO. 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87 and 88.

in another preferred embodiment, the amino acid sequence of the TCR β chain fused to the anti-CD 3 antibody is selected from the group consisting of SEQ ID Nos. 95, 96 and 97.

in another preferred embodiment, the amino acid sequence of the β chain variable domain of the TCR binding to the conjugate is selected from the group consisting of SEQ ID NO. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30 and 31 and/or the amino acid sequence of the beta chain variable domain of the TCR binding to the conjugate is selected from the group consisting of SEQ ID NO. 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46 and 47.

In another preferred embodiment, the amino acid sequence of the TCR fused to the anti-CD 3 antibody is selected from the group consisting of: SEQ id No.: 89. 90, 91, 92, 93 and 94.

In a second aspect of the invention there is provided a multivalent TCR complex comprising at least two TCR molecules, and at least one of which is a TCR according to any one of the first aspects 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 the complement thereof;

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

In a fifth aspect of the invention, there is provided a host cell comprising a vector 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, 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, a TCR complex according to the second aspect of the invention, a cell according to the sixth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention.

In a ninth aspect of the invention, there is provided a use of a T cell receptor according to the first aspect of the invention for the manufacture of a medicament for the treatment of a tumour or an HCV viral infection.

In a tenth 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

FIG. 1a and FIG. 1b show the wt-NS3TCR α chain variable domain amino acid sequence (SEQ ID NO: 2) and β chain variable domain amino acid sequence (SEQ ID NO: 3), respectively.

FIGS. 2a and 2b show the DNA sequences encoding the wt-NS3TCR α chain variable domain (SEQ ID NO: 4) and β chain variable domain (SEQ ID NO: 5), respectively.

FIG. 3a shows the amino acid sequence of wt-NS3 sTv (SEQ ID NO: 6), with its hydrophobic core mutation site indicated in bold letters; FIG. 3b shows the DNA sequence (SEQ ID NO: 7) encoding wt-NS3 sTv.

FIGS. 4a and 4b show the α chain variable domain amino acid sequence (SEQ ID NO: 8) and β chain variable domain amino acid sequence (SEQ ID NO: 9), respectively, of wt-NS3 sTv.

FIG. 5 shows the linker sequence (SEQ ID NO: 10) for wt-NS3 sTv.

FIG. 6 shows the ELISA results of HCV NS3 sTv monoclonal phage after 3 rounds of screening.

FIGS. 7a-s show the HCV NS3 KLVALGINAV-HLA-A2 complex high affinity HCV NS3 sTv α chain variable domain amino acid sequences (SEQ ID NOS: 11-22 and 25-31), respectively, with the mutated residues shown in bold and underlined.

FIGS. 8a-o show the HCV NS3 KLVALGINAV-HLA-A2 complex having high affinity for the HCV NS3 sTv β chain variable domain amino acid sequence (SEQ ID NOS: 32-41 and 43-47), respectively, with the mutated residues shown in bold and underlined.

FIGS. 9a and 9b show the α chain extracellular amino acid sequence (SEQ ID NO: 48) and β chain extracellular amino acid sequence (SEQ ID NO: 49), respectively, of the wt-NS3TCR, with cysteine residues introduced into the constant regions shown in italics and underlined.

FIGS. 10a and 10b show the α chain extracellular DNA sequence (SEQ ID NO: 50) (constant region containing the introduced cysteine) and β chain extracellular DNA sequence (SEQ ID NO: 51) (constant region containing the introduced cysteine) of wt-NS3TCR, respectively.

FIGS. 11a-s show the HCV NS3 KLVALGINAV-HLA-A2 complex high affinity HCV NS3TCR α chain variable domain amino acid sequence (SEQ ID NOS: 52-61, 63, 64, 66-72), respectively, with mutated residues shown in bold and underlined.

FIGS. 12a-o show the HCV NS3 KLVALGINAV-HLA-A2 complex high affinity HCV NS3TCR β chain variable domain amino acid sequence (SEQ ID NOS: 73-82 and 84-88), respectively, with mutated residues shown in bold and underlined.

FIG. 13 shows the BIAcore profile of an affinity assay for the KLVALGINAV-HLA-A2 complex of wt-NS3 TCR.

FIGS. 14a and 14b show the amino acid sequences of the fusion of anti-CD 3scFv at the N and C termini of molecule sTv (α chain variable domain SEQ ID NO: 8 and β chain variable domain SEQ ID NO: 32), respectively.

FIGS. 15a and 15b show the amino acid sequences of the fusion of anti-CD 3scFv at the N and C termini of molecule sTv (α chain variable domain SEQ ID NO: 17 and β chain variable domain SEQ ID NO: 33), respectively.

FIGS. 16a and 16b show the amino acid sequences of the fusion of anti-CD 3scFv at the N and C termini of molecule sTv (α chain variable domain SEQ ID NO: 14 and β chain variable domain SEQ ID NO: 32), respectively.

FIGS. 17a and 17b show BIAcore maps of affinity assays for the KLVALGINAV-HLA-A2 complex of fusion molecules of sTv molecules (α chain variable domain SEQ ID NO: 8 and β chain variable domain SEQ ID NO: 32) fused to anti-CD 3scFv at the N-and C-termini, respectively.

FIGS. 18a and 18b show BIAcore profiles of affinity assays for the KLVALGINAV-HLA-A2 complex of fusion molecules of sTv molecules (α chain variable domain SEQ ID NO: 17 and β chain variable domain SEQ ID NO: 33) fused to anti-CD 3scFv at the N-and C-termini, respectively.

FIGS. 19a and 19b show BIAcore profiles of affinity assays for the KLVALGINAV-HLA-A2 complex of fusion molecules of sTv molecules (α chain variable domain SEQ ID NO: 14 and β chain variable domain SEQ ID NO: 32) fused to anti-CD 3scFv at the N-and C-termini, respectively.

FIG. 20 shows the amino acid sequence of a beta chain fusion molecule of an scFv of anti-CD 3 with a TCR.

FIGS. 21a and 21b show experimental results of staining T2 cells with the high affinity sTv molecule of the invention.

FIG. 22 shows the results of Elisa experiments on T cell activation by the fusion molecule of high affinity HCV NS3 sTv and anti-CD 3 antibody.

FIG. 23 shows the results of an Elispot experiment in which the fusion molecule of high affinity HCV NS3TCR and anti-CD 3 antibody activates T cells.

FIG. 24 shows the results of cell killing experiments with the high affinity HCV NS3 sTv fused to anti-CD 3 antibody.

FIG. 25 shows the results of cell killing experiments with the high affinity HCV NS3TCR fusion molecules of the present invention with anti-CD 3 antibody.

Detailed Description

the present inventors have conducted extensive and intensive studies to obtain a high affinity T Cell Receptor (TCR) recognizing KLVALGINAV peptide (derived from HCV NS3 protein), said KLVALGINAV peptide being presented in the form of peptide-HLA-A2 complex, the TCR of the invention being mutated in its α chain variable domain and/or β chain variable domain relative to the wild-type HCV NS3TCR, and the TCR of the invention having at least twice the affinity and/or binding half-life to the KLVALGINAV-HLA A2 complex as compared to the wild-type HCV NS3 TCR.

Term(s) for

T Cell Receptor (TCR)

the international immunogenetic information system (IMGT) may be used to describe TCRs. native α - β heterodimeric TCRs have β 1 and β 0 chains. broadly, each chain comprises a variable region, a connecting region and a constant region, the β 3 chain usually also contains a short variable region between the variable and connecting regions, but the variable region normally being considered part of the variable region connecting region, the connecting region of a TCR is defined by unique IMGT TRAJ and TRBJ nomenclature, the constant region is defined by IMGT TRAC and TRBC nomenclature, each variable region comprises 3 CDRs (complementarity determining regions) chimeric in a framework sequence, one of which is CDR3, which is recombined from the variable and connecting regions and is referred to as hypervariable region, the sequences of CDRs 1 and 2, and the partially defined CDR3 sequences, the β 2 (va) variable regions may be divided into several classes, the β 4 variable regions (va 5) may be divided into several classes, the variable regions of the β 4 variable regions (va 5) may be designated by IMGT and TRBV types, respectively, the imv α - β 1-C-.

thus, in the specification and claims of this application, "TCR α variable domain" refers to the linked TRAV and TRAJ regions and TCR α constant domain refers to the extracellular TRAC region or C-terminally truncated TRAC sequence.

Callender, et al 2006 reported TCRs specific for HCV NS 3: 1406-1415(KLVALGINAV)/HLA-A2 (Callender, et al., (2006), Hepatology43 (5): 981-973), whose α and beta chains are AV38s2/AJ30/AC and BV11s1/BD2s1/BJ2s7/BC2, respectively, for convenience of description, this clone was designated as a wt-NS3TCR, and the amino acid sequences of the variable domains of the α and beta chains of this wt-NS3TCR were shown as SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

Detailed Description

the present invention provides a T Cell Receptor (TCR) comprising a TCR β variable domain and/or a TCR beta variable domain having the property of binding KLVALGINAV (SEQ ID NO: 1) the HLA-A2 complex, wherein (i) the TCR is mutated in its β chain variable domain amino acid as shown in SEQ ID NO: 2 and/or its beta chain variable domain amino acid as shown in SEQ ID NO: 3 and (ii) the TCR has at least twice the affinity for the KLVALGINAV (SEQ ID NO: 1) HLA-A2 complex as compared to the wild type HCV NS3 TCR.

wherein, in (i) above, mutations in amino acid residues of the α and/or β variable domains occur in one or more Complementarity Determining Regions (CDRs) of the α and/or beta variable domains, which mutations are capable of providing a higher interaction force and/or a slower dissociation rate between the inventive TCR and KLVALGINAV (SEQ ID NO: 1) HLA-A2 complex.

Binding affinity (equilibrium constant K to dissociation) can be determined by any suitable method_DInversely proportional) and binding half-life (denoted T)_1/2). It will be appreciated that doubling the affinity of the TCR will result in K_DAnd (4) halving. T is_1/2Calculated as In2 divided by dissociation rate (K)_off). Thus, T_1/2Doubling can result in K_offin a preferred embodiment, these assays are performed using the surface plasmon resonance (BIAcore) method of the examples herein, this method detects wt-NS3TCR (artificial disulfide bond introduced between the constant domains, with the extracellular α and β chain amino acid sequences being SEQ ID NO: 48 and SEQ ID NO: 49, respectively) versus K for the KLVALGINAV (SEQ ID NO: 1) HLA-A2 complex_DIs 6.7. mu.M, K_offIs 5.1X 10^-1s^-1(i.e. T)_1/2About 1.4 s). It is understood that the introduction of this artificial disulfide bond does not affect the affinity of the TCR. Thus, the affinity of the wild-type HCV NS3TCR for the KLVALGINAV-HLA-A2 complex is the same as the affinity of the wt-NS3TCR for the KLVALGINAV-HLA-A2 complex.

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).

Methods for generating TCRs of the invention can be, but are not limited to, screening libraries of diverse phage particles displaying such TCRs for TCRs with high affinity for the KLVALGINAV-HLA-a2 complex, such pooling and screening methods being well known to those skilled in the art.

it will be appreciated that both the use of genes comprising similar amino acids to the alpha and β chain variable domains of the wt-NS3TCR and the slightly modified genes comprising the alpha and β chain variable domain amino acids of the wt-NS3TCR may be used to prepare the template TCR.

the inventive TCRs comprise TCRs having at least 80%, preferably at least 85%, or more preferably at least 90% sequence identity with the amino acid sequence set forth in SEQ ID No. 2 (i.e., less than 20%, preferably less than 15%, or more preferably less than 10% mutation of the amino acid residues of the TCR β chain variable domain set forth in SEQ ID No. 2) and/or the inventive TCRs comprise TCRs having at least 80%, preferably at least 85%, or more preferably at least 90% sequence identity with the amino acid sequence set forth in SEQ ID No. 3 (i.e., less than 20%, preferably less than 15%, or more preferably less than 10% mutation of the amino acid residues of the TCR beta chain variable domain set forth in SEQ ID No. 3) sequence identity can be determined, for example, by sequence alignment, either manually or using a computer program.

in some preferred embodiments of the invention, a TCR of the invention comprises the β chain variable domain as set forth in SEQ ID NO: 2 but has one or more mutations in amino acid residues 30S, 31D, 33Y, 53A, 54Y, 55K, 56Q, 57Q, 58N, 96E, 98D, 99K, 100I and 101I thereof (using the numbering as set forth in SEQ ID NO: 2) and/or comprises a beta chain variable domain as set forth in SEQ ID NO: 3 but has one or more mutations in amino acid residues 29H, 50Y, 53N, 54S, 97P, 98Y, 99E, 100Q, 101Y thereof (using the numbering as set forth in SEQ ID NO: 3). for example, a TCR of the invention may have α chain variable domain amino acid residues 30N, 31E, 33I, 53S, 54D, 54W or 54A, 55N, 55S or 55Y, 56T, 56K, 56M, 56P or 56N, 57H, 57E, 33I, 53S, 54W or 54A, 55N, 55S or 55S, 55Y, 56T, 56K, 56M, 57N, 57H, or 99L, or 98G, or 98G, 98 or the group of the amino acid residues 29H, 98 or 98G, 98 or 98, 98 or 98, or groups of the groups of amino acid residues as set forth in SEQ ID NO, 98 or 98, or.

the high affinity TCRs of the invention comprise one of the amino acid sequences of the alpha chain variable domains SEQ ID NOs 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71, 72 and/or one of the amino acid sequences of the α chain variable domains SEQ ID NOs 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87, 88 therefore a TCR alpha chain comprising the amino acid sequence of the alpha chain variable domain of the wt-NS3TCR β (SEQ ID NO: 2) may be combined with the TCR alpha chain comprising one of the amino acid sequences of the TCR IDs SEQ ID NOs 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87, 88 or the TCR α 1 variable domain amino acid sequence of the wt-NS3TCR α chain comprising one of the amino acid sequences of the TCR IDs of the chains SEQ ID NOs 52, 53, 54, 55, 56, 57, 58, 70, 71, 72, 71, 75, 76, 77, 78, 79, 80, 82, 84, 86, 87, 88, or 71, 72, 71, 72, 75, 76, 77, 76, 78, 79, 80, 82, 84, 72, or 72.

for the purposes of the present invention, the inventive TCR is a portion having at least one TCR alpha and/or TCR beta chain variable domain, which typically comprises both a TCR alpha chain variable domain and a TCR beta chain variable domain, which can be in either an α β heterodimer or single chain form or any other form that is capable of stable existence.

for stability, in one aspect, the inventive TCR may be a TCR in which an artificial disulfide bond is introduced between residues of its α and β chain constant domains, as described in the literature (Boulter et al (2003) Protein Engineering) 16: 707-711. cysteine residues form an artificial disulfide bond between its α and β chain constant domains of the TCR.for example, the substitution of Thr48 of exon TRAC 01 and the substitution of Ser57 of exon TRBC 101 or TRBC2 to form a disulfide bond.other sites where cysteine residues are introduced to form a disulfide bond may also be Thr45 and TRBC1 of exon 1 of TRAC 01 or TRBC2 exon 1, the substitution of more Ser77 of the Ser57 of exon TRAC 6301 or the deletion of more Ser 638 or Ser 5848 residues of the most native Ser 638 or Ser 638 of exon 1 of the same, or more than 5 residues of the same, the more than just mentioned above, or less than just the original Ser 588, Ser 638, Ser 59, Ser 638 or Ser 8, Ser 59 of exon 1 of the TrBC 638, TrBC 59, or TrBC 59, and Trbc 588, or Trbc 588, which may be deleted for the purpose.

it is noted that the constant domains may or may not contain the artificial disulfide bonds introduced above, and that the TCRs of the invention may contain TRAC constant domain sequences and TRBC1 or TRBC2 constant domain sequences.

for stability, in another aspect, the inventive TCRs further include TCRs having mutations in the hydrophobic core region, preferably mutations that improve the stability of the inventive TCRs, as described in WO2014/206304, such TCRs may have mutations in the hydrophobic core positions of the following variable domains (α and/or β) variable region amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or the reciprocal positions 3, 5, 7 of the amino acid positions of the short peptide of the α chain J gene (TRAJ), and/or the reciprocal positions 2, 4, 6 of the amino acid positions of the short peptide of the beta chain J gene (TRBJ), wherein the amino acid sequences are numbered according to the position numbers listed in the International Immunogenetic information System (IMGT).

the TCR with the mutated hydrophobic core region of the invention may be a hyperstable TCR variable domain formed by a flexible peptide chain connecting the variable domains of the α and β chains of the TCR (sTv). it is noted that the flexible peptide chain of the invention may be any peptide chain suitable for connecting the TCR α and β variable domains.

To more conveniently assess the binding affinity and binding half-life of the TCRs of the invention, the template strand for screening high affinity HCV NS3 sTv constructed in example 1 of the invention contains a mutation in the hydrophobic core region. It should be noted that mutations in the hydrophobic core region do not affect the binding affinity and binding half-life of sTv.

in some embodiments of the invention, one or more of the α variable domain hydrophobic core amino acid residue 11M (i.e. position 11 of the α variable region listed in IMGT), 21L (i.e. position 21 of the α variable region listed in IMGT), 48V (i.e. position 53 of the α variable region listed in IMGT) or 110I (i.e. position 3 of the amino acid reciprocal of the short peptide of the α chain J gene listed in IMGT) of the TCR β chain variable domain hydrophobic core amino acid residue 78L (i.e. position 91 of the β variable region listed in IMGT) or 81A (i.e. position 94 of the β variable region listed in IMGT) is mutated using the numbering shown in SEQ ID No. 2.

in some preferred embodiments of the invention, the α variable domain hydrophobic core of the invention comprises one or more of amino acid residues 11L, 21I, 48L or 110V using the numbering shown in SEQ ID No. 2 and/or the TCR β variable domain comprises one or more of amino acid residues 78I or 81L using the numbering shown in SEQ ID No. 3.

the high affinity TCR of the invention further comprises one of the α chain variable domain amino acid sequences SEQ ID NO 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30, 31 and/or β chain variable domain amino acid sequences SEQ ID NO 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47 thus a TCR α chain comprising the α chain variable domain amino acid sequence of wt-NS3 sTv (SEQ ID NO 8) can be combined with a TCR α chain comprising one of the TCR α chain variable domain amino acid sequences SEQ ID NO 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47 or a TCR beta chain comprising the beta 1 chain variable domain amino acid sequence of wt-NS3 sTv (SEQ ID NO 9) can be combined with a TCR beta chain comprising one of the TCR α chain variable domain amino acid sequences of SEQ ID NO 11, 12, 13, 14, 16, 22, 25, 16, 26, 19, 31, 19, 31, 19, 31, 19, 41, 19, 41, 40, 41, 19, 40, 19, 40, 19, 40, 19, 32, or 19.

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

The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the KLVALGINAV HLA-a2 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. biological toxins (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and immunotherapy (Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research (Cancer Research)63, 3202); 4. antibody Fc fragment (Mosquera et al, 2005, Journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62, 319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, Gene therapy 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 nanoparticles in any form, and the like.

antibodies or fragments thereof that bind to the inventive TCR 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 is capable of targeting effector cells better.A preferred embodiment is the inventive TCR to bind to an anti-CD 3 antibody or a functional fragment or variant of said anti-CD 3 antibody.specifically, the inventive TCR-anti-CD 3 single chain antibody fusion includes a TCR α chain variable domain amino acid sequence selected from the group consisting of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30, 31, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 66, 67, 68, 69, 70, 71, 72 and a TCR β chain variable domain amino acid sequence selected from the group consisting of SEQ ID NO: 32, 33, 78, 77, 78, 76, 77, 89, 78, 76, 77, 95, or more specifically to the inventive TCR.

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. As used herein, "degenerate variant" means in the present invention a variant that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2, but has a sequence identical to SEQ ID NO: 4, or a nucleic acid sequence having a sequence difference.

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 genetically engineered host cells 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. One skilled in the art will be able to know many suitable methods for adoptive therapy (e.g., Rosenberg et al, (2008) NatRev 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 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 1 amino acid (especially outside the CDR regions) of the TCR of the invention has been replaced by amino acids of similar or analogous nature, and still retain its functionality. The substitution of these amino acids with similar or analogous properties is preferably carried out according to Table A.

TABLE A

Initial residue(s)	Representative substitutions	Preferred substitutions
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

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

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

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Such vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack pub. co., n.j.1991). Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.

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

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

Once formulated, the compositions of the present invention may be administered by conventional routes including, but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably parenteral including subcutaneous, intramuscular, or intravenous. The subject to be prevented or treated may be an animal; especially a human.

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

These pharmaceutical compositions 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 slow release polymer, which pellet or microcapsule is then surgically implanted into the tissue to be treated. As examples of the sustained-release polymer, ethylene-vinyl acetate copolymer, polyhydroxymethacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer and the like can be exemplified, and biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer can be preferably exemplified.

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

The main advantages of the invention are:

(1) the invention screens out the TCR with high affinity to the KLVALGINAV-HLA-A2 complex by taking high-stability sTv molecules with hydrophobic core mutation as templates, and has antigen specificity and cell killing effect.

(2) The TCR of the invention has at least twice the affinity and/or binding half-life to the KLVALGINAV-HLA-a2 complex as compared to the wild-type HCV NS3 TCR.

(3) The affinity and/or binding half-life of the high affinity TCRs of the invention to the KLVALGINAV-HLA-a2 complex can be up to 10 for the wild-type HCV NS3TCR³-10⁵More than twice.

(4) The high affinity fusion molecule of HCV NS3TCR and anti-CD 3 antibody of the invention can well activate T cells, while the fusion molecule of wild type TCR and anti-CD 3 antibody has no activation effect on T cells.

(5) Fusion molecules comprising the high affinity TCRs of the invention and anti-CD 3 antibodies are capable of redirecting T cells and thereby killing target cells.

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

Materials and methods

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

Example 1 stability of hydrophobic core mutations production of HCV NS3 sTv

the document (Callender, et al., (2006), Hepatology43 (5): 981-973) discloses a T cell receptor (abbreviated herein as wt-NS3 TCR) specific for the HCV NS3KLVALGINAV-HLA A2 complex, whose α chain and beta chain variable domain amino acid sequences (SEQ ID NO: 2 and 3) are shown in FIG. 1a and FIG. 1b, respectively, the corresponding DNA sequences (SEQ ID NO: 4 and 5) are shown in FIG. 2a and FIG. 2 b. patent document WO2014/206304 discloses a platform technique for constructing high stability sTv by mutating the hydrophobic core.the core site in this high sTv platform technique is introduced into the beta 0 and beta β chains of wt-NS3TCR, and the variable domains of the α chain and beta 1 chains are linked with a flexible short peptide (linker) to construct high affinity HCVNS3 sTv, which the high affinity TCR3 sTv is screened by the PCR using the amino acid sequences shown in SEQ ID NO: 26 a and the amino acid sequence shown in SEQ ID NO: 11b, the amino acid sequence of the amino acid sequences shown in SEQ ID NO: 11b, the amino acid sequences shown in SEQ ID NO: 11 and SEQ ID NO: 11b, respectively, and the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 14b, which the amino acid sequences shown in SEQ ID NO: 5, which overlap by the PCR primer (SEQ ID NO: 14, and SEQ ID NO: 26 b) are shown in the sequence of the hydrophobic core sequence of the aforementioned SEQ ID NO: 26 b, and the sequence of the polypeptide of SEQ ID NO: 26 b, and the sequence of the sequence shown in SEQ ID NO: 26 b, respectively, and the sequence of SEQ ID NO: 26 b, shown.

carrying the wt-NS3 sTv target gene through Nco I and Not I double digestion, with Nco I and Not I double digestion pET28a carrier connection, the ligation product transformation to E.coli DH5 α, coating LB plate containing kanamycin, 37 ℃ inversion culture overnight, picking positive clone for PCR screening, positive recombinant sequencing, after the sequence is determined to be correct, extracting recombinant plasmid transformation to E.coli BL21(DE3), for expression.

Example 2 stability of hydrophobic core mutations wt-NS3 sTv expression, renaturation and purification

The BL21(DE3) colonies containing the recombinant plasmid pET28a-sTv prepared in example 1 were all inoculated into LB medium containing kanamycin and cultured at 37 ℃ to OD₆₀₀0.6-0.8, IPTG was added to a final concentration of 0.5mM and incubation was continued for 4h at 37 ℃. The cell pellet was harvested by centrifugation at 5000rpm for 15min, lysed with a Bugbuster Master Mix (Merck), and centrifuged at 6000rpm for 1The inclusion bodies were recovered at 5min, 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 buffer (20mM Tris-HCl pH8.0, 8M urea), the insoluble material was removed by high speed centrifugation, the supernatant was quantified by BCA method and split charged, and stored at-80 ℃ for further use.

to 5mg of solubilized wt-NS3 sTv inclusion body protein, 2.5mL of buffer (6M Gua-HCl, 50mM Tris-HCl pH8.1, 100mM NaCl, 10mM EDTA) was added, DTT was added to a final concentration of 10mM, treated at 37 ℃ for 30min, 125mL of renaturation buffer (100mM Tris-HCl pH8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM β -mercapthylamine, 1.87mM Cystamine) was added dropwise with a syringe, the treated wt-NS3 sTv was stirred at 4 ℃ for 10min, the renaturation was then filled into a dialysis bag with a cut-off of 4kDa, the dialysis bag was placed in 1L of water, slowly stirred overnight at 4 ℃ on the morning, the dialysate was changed to 1L of buffer (20mM Tris-HCl pH 8.0), the dialysate was further changed to the same fresh dialysate after dialysis solution, purified on a Sepharose gel column (Sepharose gel electrophoresis) containing 20. mu. SDS-PAGE, purified fraction of target protein by filtration on a Sepharose gel column, purified by SDS-PAGE, and further purification by a gradient PAGE under vacuum filtration, whereby fraction of 20. mu.20 mM Tris-HCl, pH8.

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 300mM) 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 214 nm.

Example 3 binding characterisation

BIAcore analysis

The BIAcore T200 real-time assay system was used to detect the binding activity of HCV NS3 sTv to the HCV NS3 KLVALGINAV-HLA-A2 complex. Anti-streptavidin antibody (GenScript) was added to coupling buffer (10mM 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.

A low concentration of streptavidin was flowed over the antibody-coated chip surface, followed by HCV NS 3: 1406-1415(KLVALGINAV)/HLA A2 complex was flowed through the detection channel, the other channel served as a reference channel, and 0.05mM 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 sTv with HEPES-EP buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% P20, pH 7.4) to 5 different concentrations, sequentially flowing over the chip surface at a flow rate of 30. mu.L/min, with a binding time of 120s for each injection, and dissociating for 600s after the end of the last injection. 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 HLA-A2/HCV NS 3: the 1406-1415(KLVALGINAV) complex is prepared as follows:

a. purification of

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

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

b. Renaturation

The synthesized short peptide (NS 3: 1408-1415 KLVALGINAV) was dissolved in DMSO to a concentration of 20 mg/ml. Inclusion of light and heavy chains was solubilized with 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. And (3) connecting NS 3: 1408-1415KLVALGINAV peptide was added to 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 carried out at 4 ℃ for at least 3 days until completion, and SDS-PAGE checked for success or failure.

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 (5ml bed volume). Using Akta purifiers (GE general electric company), 20mM Tris pH8.0 prepared 0-400mM NaCl linear gradient elution protein, pMHC approximately 250mM NaCl elution, collecting the peak components, SDS-PAGE detection purity.

d. Biotinylation of the compound

The purified pMHC molecules were concentrated using Millipore ultrafiltration tubes while displacing the buffer to 20mM Tris 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 the completion of biotinylation.

e. Purification of biotinylated complexes

The biotinylated pMHC molecules were concentrated to 1ml using Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using Akta purifier (GE general electric Co., Ltd.)^TM16/60S200HR column (GE general electric) was loaded with 1ml of concentrated biotinylated pMHC molecules and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules appeared as a unimodal 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 HCV NS3 sTv variant

Phage display technology is one means of generating libraries of TCR high affinity variants to identify the 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 wt-NS3 sTv in example 1. A library of high affinity sTv was created by mutating the CDR regions in this wt-NS3 sTv and panning was performed. Those skilled in the art are familiar with the above described library construction and screening methods. I.e., by using a primer having the desired codon change or changes and a plasmid containing the relevant sTv strand DNA as a template. The phage libraries after 3 rounds of panning all have specific binding with corresponding antigens, and the ELISA results are shown in FIG. 6. From these, single clones were picked and subjected to sequence analysis.

Analysis of the interaction of HCV NS3 sTv with the peptide KLVALGINAV-HLA-A2 complex using the BIAcore method of example 3, a high affinity sTv mutant with an affinity and/or binding half-life at least twice that of wild-type HCV NS3TCR, i.e., a high affinity sTv mutant selected to bind the dissociation equilibrium constant K of the peptide KLVALGINAV-HLA-A2 complex_DLess than or equal to dissociation equilibrium constant K of wild type HCV NS3TCR binding peptide KLVALGINAV-HLA-A2 complex_DOne-half of (a), the results are shown in table 1 below. No detection of wt-NS3 sTv with the peptide KLV was obtained using the above methodalgnav-HLA-a 2 complex interaction.

specifically, the β chain variable domain of these high affinity sTv mutants was mutated at one or more of the following amino acids 30S, 31D, 33Y, 53A, 54Y, 55K, 56Q, 57Q, 58N, 96E, 98D, 99K, 100I, 101I using the numbering shown in SEQ ID NO. 2 and/or the beta chain variable domain of these high affinity sTv mutants was mutated at one or more of the following amino acids 29H, 50Y, 53N, 54S, 97P, 98Y, 99E, 100Q, 101Y using the numbering shown in SEQ ID NO. 3.

more specifically, these high affinity sTv mutants have one or more of the following β chain variable domain amino acid residues 30N, 31E, 33I, 53S, 54D, 54W, 54A, 55N, 55S, 55Y, 56T, 56K, 56M, 56P, 56N, 57H, 57E, 57D, 57I, 57N, 58S, 58I, 96D, 98N, 98T, 98Q, 98V, 99S, 99Y, 99P, 100L, 101T and/or the numbering shown in SEQ ID NO. 3 using the numbering shown in SEQ ID NO. 2, these high affinity sTv mutants have one or more of the following beta chain variable domain amino acids 29Y, 50L, 53G, 54H, 97S, 98A, 98L, 98E, 98S, 98G, 99A, 99P, 100L, 100M, 100V, 101I, 101F, 101S.

specific examples of the amino acid sequences of the α chain variable domain (SEQ ID NOS: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30, 31) and β chain variable domain (SEQ ID NOS: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46, 47) of high affinity HCV NS3 sTv are shown in FIGS. 7a-u and FIGS. 8a-p, respectively.

TABLE 1

Example 5 Generation of high affinity HCV NS3TCR variants

the high affinity CDR region mutations selected in example 4 were introduced into the corresponding sites of the variable domain of wt-NS3TCR and their affinity to the HCV NS3 KLVALGINAV-HLA-A2 complex was detected by BIAcore the method for introducing high affinity mutations described above employed site-directed mutagenesis methods well known to those skilled in the art, the α and β chain extracellular amino acid sequences of the above wt-NS3TCR are shown in FIGS. 9a (SEQ ID NO: 48) and 9b (SEQ ID NO: 49), respectively, without the leader methionine introduced for efficient initiation of expression in bacteria, the corresponding DNA sequences are shown in FIGS. 10a (SEQ ID NO: 50) and 10b (SEQ ID NO: 51), respectively, it should be noted that cysteine residues were introduced into the constant regions of the above α and β chains to form artificial interchain during refolding, the TCR variable domain of wt- β chain and the TCR chain variable domain of FIGS. 1 and 3b, respectively, with underlined italic letters 3 in the amino acid sequences.

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), and mutations in the cdr regions at upstream and downstream Cloning sites were NcoI and noti, respectively, were introduced by overlap PCR (overlap PCR) well known to those skilled in the art.

Example 6 expression, renaturation and purification of HCV NS3TCR

the expression vectors of TCR α and beta chains are transformed into expression bacteria BL21(DE3) by chemical transformation method, and the bacteria are grown in LB culture solution and OD₆₀₀Inclusion bodies formed after alpha and β chain expression of the TCR were extracted by BugBuster Mix (Novagene) and back-extracted with BugBuster solution at 0.6 with a final concentration of 0.5mM IPTGAfter multiple washes, the inclusion bodies were finally dissolved in 6M guanidinium hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 20mM Tris (pH 8.1).

the solubilized TCR α and β chains were rapidly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH8.1), 3.7mM cystamine, 6.6mM β -mecapothylamine (4 ℃) in a mass ratio of 1: 1 to a final concentration of 60mg/mL, after mixing the solution was dialyzed (4 ℃) against 10 volumes of deionized water and 12 hours later deionized water was changed to a buffer (20mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours, the dialyzed solution 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 with the eluted peak containing the α and β dimers with success was confirmed by SDS-PAGE gel, the TCR after purification was further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare).

Example 7 BIAcore analysis results

The method described in example 3 was used to detect HCV NS3TCR and HLA-A2/HCV NS3 with the high affinity mutation points introduced: 1406-1415(KLVALGINAV) affinity of the complex.

the CDR region mutations in the high affinity HCV NS3 sTv are introduced into the corresponding positions of SEQ ID NO. 2 and/or SEQ ID NO. 3 to obtain new TCR alpha and β chain variable domain amino acid sequences, as shown in FIGS. 11 and 12, respectively, the expression vector is constructed by the method described in example 5, the HCV NS3TCR introduced with the high affinity mutation is expressed, renatured and purified by the method described in example 6, and then the affinity of the complex with HLA-A2/HCV NS 3: 1406-1415(KLVALGINAV) is determined by BIAcore T200. the affinity of the wt-NS3TCR for the HLA-A2/HCV NS 3: 1406-1415(KLVALGINAV) complex, K-NS 3TCR, K-X-Y and K-Y are determined by the method described in example 3 and multi-cycle kinetics_DThe value was 6.668. mu.M, and the BIAcore spectrum is shown in FIG. 13. HCV NS3TCR with high affinity mutations introduced and HLA-a2/HCV NS 3: 1406-1415(KLVALGINAV) complex, as shown in Table 2, has a much higher affinity than the wt-NS3TCR for HLA-A2/HCV NS 3: 1406-1415(KLVALGINAV) affinity of the complex.

TABLE 2

From the data in Table 18 above, it can be seen that the HCV NS3TCR heterodimer with the same CDR mutations as the high affinity HCV NS3 sTv is specific for HLA-A2/HCV NS 3: the affinity of the 1406-1415(KLVALGINAV) complex is higher than that of the high affinity HCV NS3 sTv for HLA-A2/HCV NS 3: 1406-1415(KLVALGINAV) complex, and therefore, it is surmised that after all high affinity mutant sites screened in HCV NS3 sTv were introduced into the HCV NS3TCR heterodimer, the heterodimeric TCR was also able to satisfy the following conditions for HLA-a2/HCV NS 3: the requirement that the affinity and/or binding half-life of the 1406-1415(KLVALGINAV) complex is at least two-fold greater than that of wild-type HCV NS3TCR, i.e., the dissociation equilibrium constant K of the high affinity heterodimeric TCR mutant binding peptide KLVALGINAV-HLA-A2 complex_DLess than or equal to the dissociation equilibrium constant K of wild-type HCV NS3 TCR-binding peptide KLVALGINAV-HLA-A2 complex_DOne half of (a).

We also tested the high affinity heterodimeric TCR as described above for its lack of binding activity to 10 other non-specific antigens, as shown in table 3 below. The high affinity heterodimeric TCR did not bind to any of the other 10 non-specific antigens, indicating that it has strong specificity.

TABLE 3

Example 8 expression, renaturation and purification of anti-CD 3 antibody with high affinity HCV NS3 sTv fusion

Against HLA-A2/HCV NS 3: the high affinity sTv of 1406-1415(KLVALGINAV) and the single chain antibody (scFv) of the anti-CD 3 antibody were fused at the gene level in two ways: one was to fuse the anti-CD 3scFv to the N-terminus of sTv, and the other was to fuse the anti-CD 3scFv to the C-terminus of sTv. In both fusion modes, the two molecules are linked by a flexible short peptide (linker) GGGGS, the choice of polypeptide linker is not exclusive, and any suitable linker sequence may be used in the fusion molecule. In such molecules, the TCR may be a full-length heterodimeric TCR or may be a single-chain sTv.

the selected high affinity sTv molecules include (1) a sTv molecule consisting of α chain variable domain of SEQ ID NO: 8 and the β chain variable domain of SEQ ID NO: 32 fused to the scFv of anti-CD 3 at the N and C termini of the sTv molecule, respectively, (2) a sTv molecule consisting of α chain variable domain of SEQ ID NO: 17 and the β chain variable domain of SEQ ID NO: 33 fused to the scFv of anti-CD 3 at the N and C termini of the sTv molecule, respectively, as shown in FIGS. 15a and 15b, (3) a methionine sTv molecule consisting of α chain variable domain of SEQ ID NO: 14 and the β chain variable domain of SEQ ID NO: 32 fused to the scFv of anti-CD 3 at the N and C termini of the sTv molecule, respectively, (3) a methionine sTv molecule consisting of the fusion of α chain variable domain of SEQ ID NO: 14 and the β chain variable domain of SEQ ID NO: 32 fused to the scFv of anti-CD 3 at the N and C termini of the sTv molecule, respectively, as shown in the fusion process of the leader 16 molecule:

expression of fusion proteins

Transformation of expression plasmids into the Large intestineIn Bacillus strain BL21(DE3), LB plates (kanamycin 50. mu.g/ml) were plated and incubated overnight at 37 ℃. The next day, the selected clones were inoculated into 10ml LB liquid medium (kanamycin 50. mu.g/ml) for 2-3h, inoculated into 1L LB medium (kanamycin 50. mu.g/ml) at a volume ratio of 1: 100, and cultured until OD₆₀₀0.5-0.8, 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 the detergent and salts. Finally, the inclusion bodies were dissolved in Tris buffer containing 8M urea, and the inclusion body concentration was measured, and after subpackaging, the inclusion bodies were stored at-80 ℃ for freezing.

Refolding of fusion proteins

approximately 10mg of inclusion bodies were thawed from-80 ℃ ultra-low temperature freezer, Dithiothreitol (DTT) was added to a final concentration of 10mM, incubated at 37 ℃ for 30min to 1 hour to ensure complete disulfide bond opening, then the inclusion body sample solutions were each dropped into 200ml of 4 ℃ pre-chilled refolding buffer (100mM Tris pH8.1, 400mM L-arginine, 2mM EDTA, 5M Urea, 6.5mM β -mercaptoethylamine, 1.87mM Cystamine), slowly stirred at 4 ℃ for approximately 30 minutes, the refolding solution was thawed with 8 volumes of 8 times the volume of H₂O dialysis for 16-20 hours. The dialysis was performed twice with 8 volumes of 10mM Tris pH8.0, and the dialysis was continued at 4 ℃ for about 8 hours, after which the samples were filtered and then subjected to the following purification.

First step purification of fusion proteins

Dialyzed refolded material (10mM Tris pH 8.0) was subjected to gradient elution with 0-600mM NaCl using POROS HQ/20 anion exchange chromatography pre-packed column (Applied Biosystems) in AKTA purifier (GE Healthcare). 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 7510/300 GL gel filtration chromatography pre-packed 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 affinity assay for 9 sTv fusion molecules

the affinity of the group 3 fusion molecules of example 8 for the HLA-A2/HCV NS 3: 1406-1415(KLVALGINAV) complex was determined as described in example 3 to evaluate the effect of fusion of sTv and anti-CD 3 molecules on the affinity of sTv, wherein the group (1) fusion molecules, i.e., sTv molecules composed of the α chain variable domain SEQ ID NO: 8 and β chain variable domain SEQ ID NO: 32, have BIAcore maps of fusion molecules fused to the scFv of anti-CD 3 at the N and C termini of sTv molecules as shown in FIGS. 17a and 17b, respectively, and their K is K_Dvalues of 9.6nM and 4.0nM, respectively, and K of sTv molecule consisting of α chain variable domain SEQ ID NO 8 and β chain variable domain SEQ ID NO 32_Dthe BIAcore maps of the fusion molecules of group (2) with a value of 7.0 nM., i.e., sTv molecules consisting of α chain variable domain SEQ ID NO: 17 and β chain variable domain SEQ ID NO: 33, fused to the scFv of anti-CD 3 at the N-and C-termini of sTv molecules are shown in FIGS. 18a and 18b, respectively, and their Ks are shown_Dthe values are 227.6pM and 75.4pM respectively, while the K of sTv molecule consisting of α chain variable domain SEQ ID NO: 17 and β chain variable domain SEQ ID NO: 33_Dthe BIAcore maps of the fusion molecules of group (3) with a value of 0.6 nM., i.e., sTv molecules consisting of α chain variable domain SEQ ID NO: 14 and β chain variable domain SEQ ID NO: 32 fused to the scFv of anti-CD 3 at the N-and C-termini of sTv molecules are shown in FIGS. 19a and 19b, respectively, and their Ks are shown_Dthe values are 881.2pM and 536.4pM respectively, while the K of sTv molecule consisting of α chain variable domain SEQ ID NO. 14 and β chain variable domain SEQ ID NO. 32_DThe value was 1.8 nM. It can be seen that the fusion of sTv molecule and anti-CD 3 molecule has no influence on the affinity of sTv molecule, and can ensure that sTv molecule has no influence on the affinity of HLA-A2/HCV NS 3: 14Binding potency of 06-1415(KLVALGINAV) complex.

Example 10 expression, renaturation and purification of anti-CD 3 antibody and high affinity HCV NS3TCR fusions

the scFv of anti-CD 3 is fused to the β chain of the TCR, which may comprise the β chain variable domain of any of the above-described high affinity HCV NS3 TCRs, and the TCR β chain variable domains shown in SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 87 are used in this example the TCR α chain of the fusion molecule may comprise the α chain variable domain of any of the above-described high affinity HCV NS3 TCRs, and the α chain variable domains shown in SEQ ID NO: 2 and SEQ ID NO: 58 are used in this example the amino acid sequences of the anti-CD 3 fusion molecule with the β chain of the scFv are shown as 20a, 20b, and 20c, respectively, and the amino acid sequences contain the leader methionine introduced for efficient expression in bacteria.

Construction of fusion molecule expression vectors

construction of alpha chain expression vectors

the vector carrying HCV NS3TCR α chain gene is double digested by Nco I and Not I and is connected with pET28a vector which is double digested by Nco I and Not I, the ligation product is transformed into E.coli DH5 α, the E.coli DH5 α is coated on an LB plate containing kanamycin and is inversely cultured overnight at 37 ℃, 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 Tuner (DE3) for expression.

2. construction of anti-CD 3(scFv) - β chain expression vector

the primers are designed to connect anti-CD 3scFv and high affinity HCV NS3TCR β chain gene by overlapping (overlap) PCR method, the middle connecting short peptide (linker) is GGGGS, and the gene fragment of the fusion protein of the anti-CD 3scFv and the high affinity HCV NS3TCR β chain is provided with restriction endonuclease sites Nco I (CCATGG) and Not I (GCGGCCGC). the PCR amplification product is double-digested by Nco I and Not I and is connected with pET28a vector double-digested by Nco I and Not I.the ligation product is transformed into E.coli DH5 α competent cells, coated with LB plate containing kanamycin, inverted cultured overnight at 37 ℃, the positive clone is picked up for PCR screening, the positive recombinant is sequenced, and after the sequence is determined to be correct, the recombinant plasmid is extracted and transformed into E.coli Tuner (DE3) competent cells for expression.

Expression, renaturation and purification of fusion proteins

The expression plasmids were transformed into E.coli Tuner (DE3) competent cells, respectively, and LB plates (kanamycin 50. mu.g/mL) were plated and incubated at 37 ℃ overnight. The next day, the selected clones were inoculated into 10mL LB liquid medium (kanamycin 50. mu.g/mL) for 2-3h of culture, inoculated into 1L of LB medium at a volume ratio of 1: 100, cultured until OD600 became 0.5-0.8, and added with 1mM IPTG to induce the expression of the target protein. After 4 hours of induction, cells were harvested by centrifugation at 6000rpm for 10 min. The cells were washed once with PBS buffer and aliquoted, and 200mL of the cells from the bacterial culture were lysed with 5mL of BugBuster Master Mix (Merck) and the inclusion bodies were collected by centrifugation at 6000g for 15 min. 4 detergent washes were then performed to remove cell debris and membrane components. The inclusion bodies are then washed with a buffer such as PBS to remove the detergent and salts. Finally, inclusion bodies were dissolved in a buffer solution containing 6M guanidine hydrochloride, 10mM Dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 20mM Tris, pH8.1, and the inclusion body concentration was measured, and they were stored frozen at-80 ℃ after being dispensed.

the solubilized TCR α chain and anti-CD 3(scFv) - β chain were rapidly mixed in 5M Urea (urea), 0.4M L-arginine (L-arginine), 20mM Tris pH8.1, 3.7mM cystamine, 6.6mM β -mercaptamine (4 ℃) at a mass ratio of 2: 5 to give final concentrations of α chain and anti-CD 3(scFv) - β chain of 0.1mg/mL and 0.25mg/mL, respectively.

after mixing, the solution was dialyzed against 10 volumes of deionized water (4 ℃) for 12 hours, then deionized water was exchanged for buffer (10mM Tris, pH 8.0) and dialysis was continued at 4 ℃ for 12 hours, the dialyzed solution was filtered through a 0.45 μ M filter and purified by anion exchange column (HiTrap Q HP5ml, GE healthcare), the eluted peaks contained TCR alpha chain and anti-CD 3(scFv) - β chain dimers, which were confirmed by SDS-PAGE gel, the TCR fusion molecules were then further purified by size exclusion chromatography (S-10016/60, GE healthcare), and anion exchange column (HiTrap Q HP5ml, GE healthcare), the purified TCR fusion molecules were repurified by SDS-PAGE at a purity of greater than 90%, as determined by BCA method.

The fusion molecules were tested for HLA-A2/HCV NS 3: 1406-1415(KLVALGINAV, SEQ ID No.: 1) the affinity of the complex. The results showed that the fusion molecule was specific for HLA-A2/HCV NS 3: the affinity of the 1406-1415(KLVALGINAV) complex with its corresponding native TCR molecule for HLA-A2/HCV NS 3: the affinity of the 1406-1415(KLVALGINAV) complexes did not differ significantly.

Example 11 staining of T2 cells with the high affinity HCV NS3 sTv variant

First, the high affinity HCV NS3 sTv variant of the invention was biotinylated according to the method described in example 3.

Staining of T2 cells with biotinylated high affinity sTv molecules was subsequently determined using a microbeam staining method. The reagents used included: test medium: 10% FBS (heat-inactivated, Gibco, Cat. No. 10108-165); 1640 medium without phenol red: (Life, catalog number: 11835-030); 1% penicillin/streptomycin: (invitrogen, catalog number 15070063); washing liquid: PBS pH 7.4w/o Ca Mg (Invitrogen, cat #10010015) and 5% FBS; culture plate: nunc 8 orifice plate (Fisher, Cat # TKT-210-051T); culture plate coating solution: poly L-lysine solution (SIGMA, Cat # P8920-100 ML).

The method comprises the following specific steps: diluting poly L-lysine solution with PBS containing Ca and Mg at a ratio of 1: 100, adding into 8-well plate at 200 μ L/well, incubating at 37 deg.C for 30min, removing poly L-lysine solution, washing with PBS buffer solution for 3 times, and air drying at room temperature. Taking and incubating with related short peptide for 1 hour1x10 of⁶T2 cells were suspended in 1ml of washing solution, divided into 4 groups, and the cells were washed by centrifugation 1 time at 500g for 5 minutes using a 75004250 centrifuge (Thermo Co.) and the supernatant was removed. Biotinylated high affinity sTv molecules were diluted with washing solution to final concentrations of 10-7, 10-8, 10-9, 10-10M, respectively. The centrifuged T2 cells were suspended in group 4, incubated on ice for 20min, and the cells were washed by centrifugation 2 times at 500g for 5min using a 75004250 centrifuge (Thermo Co.). Streptavidin-PE (BD Co.) was diluted 1: 100 with a washing solution, centrifuged cells were suspended, incubated on ice for 20min, and centrifuged 2 times at 500g for 5min using an 75004250 centrifuge (Thermo Co.) to wash the cells. The centrifuged cells were suspended in 500. mu.l of washing solution, and 200. mu.l of the cell suspension was added to a dry 8-well plate.

And observing the cells under a 63-fold microscope by using a Zeiss microscope Observer Z.01PE channel, scanning and photographing the cells along a Z axis, and superposing and calculating the surface fluorescence quantity of the cells by using the pictures along the Z axis. Three-dimensional images of the cells were obtained by Z-stack acquisition (21 planes, 1um intervals).

The results of the experiment are shown in fig. 21a and 21b, which show that the high affinity sTv molecule of the invention was able to successfully bind to T2 cells loaded with the relevant short peptide.

Example 12 high affinity HCV NS3 sTv fusion molecules with anti-CD 3 antibodies

This example demonstrates that the high affinity HCV NS3 sTv fusion molecule with anti-CD 3 antibody of the present invention activates T cells well by Elisa assay. The activation effect was verified by the release amount of IL-2. Several fusion proteins of the present invention, high affinity HCV NS3 sTv and anti-CD 3 antibody, were selected to perform Elisa experiments, including fusion protein 1(SEQ ID NO: 91), fusion protein 2(SEQ ID NO: 92), fusion protein 3(SEQ ID NO: 89), and fusion protein 4(SEQ ID NO: 90).

The main materials used in the Elisa experiment included: u-bottom 96-well plates (Nick, Cat. No. 163320), bovine serum (heat-inactivated, Gibbo, Cat. No. 10108-165), RPMI 1640 contains no phenol red (Invitrogen, Cat. No. 32404014), L-glutamine (Invitrogen, Cat. No. 25030024), penicillin/streptomycin (Invitrogen, Cat. No. 15070063) PBS pH 7.4 (Invitrogen, Cat. No. 10010015), anti-human interleukin-2 (IL-2) (e-Bioscience Cat. No. 14-7029-85), anti-human IL-2-biotin (e-Bioscience Cat. No. 13-7028-85), IL-2 standard (eBioscience Cat. No. 39-8021), streptavidin-HRP (Invitrogen, Cat. No. 43-4323), chromogenic substrate (Sigma, Cat. No. 54827-17-7), Flat-bottomed 96-well plates (nikk, cat # 163320), target cells, effector cells, fusion molecules of high affinity HCV NS3 sTv with anti-CD 3 antibody (prepared as described in example 8, which can be diluted with assay medium), and 1M H2SO 4.

The solvents used include: culture solution: RPMI 1640 contains no phenol red, 10% fetal bovine serum and 1% L-glutamine and 1% penicillin/streptomycin; washing liquid: PBS containing 0.5% tween; and a sealing liquid: PBS containing 5% milk powder.

The target cells used in this example were K562 cells transfected with HLA-A2 and NS 3. Sufficient target cells (100,000 cells/well) were washed by centrifugation 1 time at 500g for 5 minutes using an 75004250 centrifuge (Thermo). The cells were then resuspended at 2X106 cells/ml in the test medium.

The effector cells (T cells) used in this example were CD8 positive T cells (obtained from PBL by negative selection (using CD8 negative isolation kit, MACS, catalog No. 130-094-156)). Effector cells were thawed, placed in test medium, and then washed by centrifugation at 500g for 5 minutes using an 75004250 centrifuge (Thermo corporation). The cells were then resuspended in the test medium at 4-fold the desired final concentration.

The specific experimental steps are as follows: anti-human IL-2 coated antibody was diluted 1: 250 with PBS. 100ul of diluted antibody per well was added to a 96-well flat-bottom plate. The plate was placed on a shaker and shaken overnight at 4 degrees. Spin off the coated antibody from the plate, add 200ul of wash solution per well, spin dry, repeat this step 6 times. 100ul of blocking solution was added to each well and shaken at room temperature for 2 h. The blocking solution was spun off the plate, 200ul of wash solution was added to each well, spun dry, and this step was repeated 6 times.

Add culture supernatant to the sealed plates at 100ul per well, dilute the human IL-2 standard to 10, 5, 2.5, 1.25, 0.625, 0.31, 0.16, 0.08ng/ml for the standard curve, place the plates in a shaker for 2h at room temperature. The supernatant was spun off the plate, 200ul of wash solution was added to each well, spun dry, and this step was repeated 6 times. The anti-human gamma interferon-biotin was diluted with blocking solution at a ratio of 1: 500, and 100ul was added per well. Spin off the plate, add 200ul of wash per well, spin dry, repeat this step 6 times. streptavidin-HRP was diluted 1: 2500 with blocking solution, 100ul was added per well. Removing the antibody from the plate, adding 200ul of washing solution into each well, spin-drying, repeating the step for 6 times, adding chromogenic substrate into each well, and developing for 5-10 min. The reaction was stopped by adding 1M H2SO4 to each well and absorbance was read at 45 nm. All experimental groups were assigned 3 parallel controls and the final volume was 200 ul.

The results of the experiments are shown in FIG. 22, and the fusion molecule of the high affinity HCV NS3 sTv and the anti-CD 3 antibody of the present invention can activate T cells well.

Example 13 high affinity HCV NS3TCR fusion molecules with anti-CD 3 antibodies

This example compares the ability of several of the high affinity HCV NS3TCR fusion molecules with anti-CD 3 antibody and wild-type TCR fusion molecules with anti-CD 3 antibody to activate Cytotoxic T Lymphocytes (CTLs) in tumor cell lines presenting the HCV short peptide-HLA-a 2 complex. The production of IFN-. gamma.was measured as a readout of T cell activation using the ELISPOT assay.

the ELISPOT experiment described above was performed by selecting high affinity HCV NS3TCR molecules of the invention including high affinity TCR1(V α SEQ ID NO: 2; V β SEQ ID NO: 73), high affinity TCR2(V β 0 SEQ ID NO: 2; V β 1 SEQ ID NO: 75), high affinity TCR3(V α SEQ ID NO: 2; V β SEQ ID NO: 74), high affinity TCR4(V α SEQ ID NO: 58; V β SEQ ID NO: 74), high affinity TCR5(V α SEQ ID NO: 58; V β SEQ ID NO: 87) fused to the anti-CD 3 antibody, and wild type TCR (V α SEQ ID NO: 2; V β SEQ ID NO: 3) fused to the anti-CD 3 antibody.

Reagents used in the ELISPOT experiment were as follows: assay medium, wash buffer, PBS and human IFN- γ elispot tpvdf-enzymatic kit, target cells, effector cells and fusion molecules of high affinity HCV NS3TCR with anti-CD 3 antibody (prepared as described in example 10, which can be diluted with assay medium).

The target cells were prepared as follows: the target cells used in this example were K562 cells transfected with HLA-A2 and NS 3. Sufficient target cells (20,000 cells/well) were washed by centrifugation 1 time at 500g for 5 minutes using an 75004250 centrifuge (Thermo). The cells were then resuspended at 4X 105 cells/ml in the test medium.

The effector cells were prepared as follows: the effector cells (T cells) used in this example were CD8 positive T cells (obtained from PBL by negative selection (using CD8 negative isolation kit, MACS, catalog No. 130-094-156)). Effector cells were thawed, placed in test medium, and then washed by centrifugation at 500g for 5 minutes using an 75004250 centrifuge (Thermo corporation). The cells were then resuspended in the test medium at 4-fold the desired final concentration.

The ELISPOT plate preparation process is as follows: add 50. mu.l of 35% alcohol per well to pre-wet the plate bottom and dilute 100. mu.l of anti-IFN γ capture antibody with 10ml of sterile PBS per plate. 100 microliters of the diluted capture antibody was then aliquoted into each well. The plates were incubated at 4 ℃ overnight. After incubation, the plates were washed (procedure 1, plate type 2, 96 well plate washer; BioTech) to remove the capture antibody. 1640 medium containing 10% serum was then added to each well at 100. mu.l/well and the plates were incubated at room temperature for 2 hours to block the plates. The media was then washed from the plates (procedure 1, plate type 2, 96-well plate washer; BioTech) by flicking and tapping the ELISPOT plates on a paper towel to remove any remaining wash buffer.

The ELISPOT experiment procedure was as follows: the components of the assay were added to the ELISPOT plate in the following order: 50 μ l of target cells 4X 105 cells/ml (giving a total of 20,000 target cells/well), 50 μ l of reagent (high affinity TCR-anti-CD 3 fusion molecule; different concentrations), 50 μ l of medium (test medium) and 50 μ l of effector cells (1000 CD8+ cells/well) and then incubate the plates overnight (37 ℃, 5% CO 2). The plates were then washed and subjected to secondary detection and development, dried for 1 hour, and the spots formed on the membrane were counted using an immuno-spot plate READER (ELISPOT READER system; AID Co.).

The experimental results are shown in FIG. 23, which shows that the fusion molecule of wild-type TCR and anti-CD 3 antibody has little effect on T cell activation, while the fusion molecule of high affinity HCV NS3TCR and anti-CD 3 antibody of the present invention can well activate T cells.

Example 14 redirection of T cells by fusion molecules of high affinity T cell receptor and anti-CD 3 antibodies

This example demonstrates that the high affinity HCV NS3 sTv and anti-CD 3 antibody fusion molecules of the present invention, as well as the high affinity HCV NS3TCR and anti-CD 3 antibody fusion molecules, are able to redirect T cells, thereby killing the target cells.

This example uses a non-radioactive cytotoxicity assay to verify killing. The test is a colorimetric substitution test for a 51Cr release cytotoxicity test, and quantitatively determines milk released after cell lysis

Acid dehydrogenases (LDHs). The released LDH in the culture supernatant was detected using a 30-minute coupled enzyme assay, which converted the tetrazolium salt (INT) to the red formazan product. The amount of color formed is proportional to the number of lysed cells. Absorbance data at 490nm was collected using a standard 96 well plate reader.

The materials used were as follows: CytoTox96 nonradioactive cytotoxicity assay (promegag) (G1780) contained a substrate mixture, assay buffer, lysis solution, and stop solution; test medium: 10% FBS (heat-inactivated, Gibbo Corp., Cat. No. 10108-165), 1640 medium without phenol red (Life), Cat. No.: 11835-030); 1% penicillin/streptomycin (invitrogen, cat # 15070063); nunc microwell round bottom 96 well tissue culture plates (Nunc, Cat. No. 163320); Nunc-Immunoplate Maxisorb (New York company, Cat. No. 442404).

The target cells used in this example were derived from K562 cells transfected with HLA-A2 and NS3 proteins from a tumor cell line. Preparing target cells in a test medium; the target cell concentration was adjusted to 2x106 cells/ml, resulting in 1x105 cells/well, 50 μ l.

The effector cells (T cells) used in this example were CD8 positive T cells (obtained from PBL by negative selection (using CD8 negative isolation kit, MACS, catalog No. 130-094-156)). Effector cells were thawed, placed in test medium, and then washed by centrifugation at 500g for 5 minutes using an 75004250 centrifuge (Thermo corporation). The cells were then resuspended at 2X 106/ml in the test medium.

Fusion molecules of affinity HCV NS3 sTv and anti-CD 3 antibody and high affinity HCV NS3TCR and anti-CD 3 antibody were prepared as described in example 8 and example 10, respectively, and diluted to different concentrations (10nM to 0.001pM) by assay medium.

The components of the assay were added to the plate in the following order: 50 μ l of target cells (prepared as described above) were added to each well; 50 μ l of effector cells (prepared as described above) were added to each well; mu.l of fusion protein was added to each well.

Several controls were prepared as follows: target cells release spontaneously: only 200. mu.l of target cells; maximum release of target cells: there were only 200. mu.l of target cells. All wells were made in triplicate and the final volume was 200. mu.l. The plates were centrifuged at 250x g for 4 minutes and then incubated at 37 ℃ for 24 hours. Mu.l of lysis solution was added to the target cell maximum release control wells and the supernatant was collected after 45 minutes. The plate was centrifuged at 250Xg for 4 minutes. 50 μ l of each well of the assay plate was transferred to the corresponding well of a flat-bottomed 96-well Nunc Vlnxisorb plate. The substrate mixture was reconstituted with assay buffer (12 ml). Then 50. mu.l of reconstituted substrate mixture was added to each well of the plate. The plates were covered with aluminum foil and incubated at room temperature for 30 minutes. 50 μ l of stop solution was added to each well of the plate to stop the reaction. Absorbance at 490nm was recorded using an Elisa plate reader within 1 hour after addition of the stop solution.

And (4) calculating a result: the average absorbance value of the background is subtracted from the absorbance values obtained from the experimental and target cell spontaneous release controls, and the average absorbance value of the volume correction controls is subtracted from the absorbance values obtained from the target cell maximal release controls. The corrected values obtained in the first two steps were used to calculate the percent cytotoxicity as follows: % cytotoxicity is 100 × (experiment-target cell spontaneous/(target cell maximal-target cell spontaneous).

FIGS. 24 and 25 are the results of cell killing experiments with the high affinity HCV NS3 sTv and anti-CD 3 antibody fusion molecules and the high affinity HCV NS3TCR and anti-CD 3 antibody fusion molecules, respectively, of the present invention, showing that both fusion molecules of the present invention are capable of redirecting T cells and thereby killing target cells.

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

Sequence listing

<110> Guangzhou Xiangxue pharmaceutical products Co., Ltd

<120> high affinity HCV T cell receptor

<130>P2016-1665

<150>201410274355.X

<151>2014-06-18

<160>101

<170>PatentIn version 3.5

<210>1

<211>10

<212>PRT

<213> hepatitis C Virus

<400>1

Lys Leu Val Ala Leu Gly Ile Asn Ala Val

1 5 10

<210>2

<211>112

<212>PRT

<213> human

<400>2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>3

<211>111

<212>PRT

<213> human

<400>3

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>4

<211>336

<212>DNA

<213> human

<400>4

gctcagaccg ttacccagtc gcaaccggaa atgtcagttc aagaagcaga aaccgttacc 60

ctgtcctgta cctacgatac cagtgaaagt gattactacc tgttttggta caagcagccg 120

ccgtcccgtc aaatgattct ggttatccgc caggaagcat acaagcagca aaacgctacc 180

gaaaatcgtt ttagtgtcaa cttccagaaa gcggccaaga gtttctccct gaaaatttca 240

gattcgcagc tgggcgacgc agctatgtat ttttgcgcat acggtgaaga tgacaaaatt 300

atcttcggca agggtacgcg tctgcatatt ctgccg 336

<210>5

<211>333

<212>DNA

<213> human

<400>5

gaagccgaca tctatcagac gccgcgctac ctggttattg gcacgggcaa gaaaatcacg 60

ctggaatgct ctcagacgat gggtcatgat aaaatgtatt ggtaccagca agacccgggc 120

atggaactgc atctgattca ctatagctac ggcgtgaaca gcaccgaaaa gggtgatctg 180

agctctgaaa gtaccgtttc ccgtatccgc acggaacact tcccgctgac cctggaatcc 240

gcccgtccgt ctcatacctc gcaatatctg tgcgcctctc gtcgcggtcc gtatgaacag 300

tactttggtc cgggtacccg tctgaccgtc acc 333

<210>6

<211>247

<212>PRT

<213> Artificial sequence

<400>6

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

1 5 10 15

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

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Thr Leu Leu

35 40 45

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

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

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

115 120 125

Gly Gly Ser Glu Gly Gly Ser Gly Glu Ala Asp Ile Tyr Gln Thr Pro

130 135 140

Arg Tyr Leu Ser Ile Gly Thr Gly Lys Lys Ile Thr Leu Glu Cys Ser

145 150 155 160

Gln Thr Met Gly His Asp Lys Met Tyr Trp Tyr Gln Gln Asp Pro Gly

165 170 175

Gln Glu Leu His Leu Ile His Tyr Ser Tyr Gly Val Asn Ser Thr Glu

180 185 190

Lys Gly Asp Leu Ser Ser Glu Ser Thr Val Ser Arg Ile Arg Thr Glu

195 200 205

His Phe Pro Leu Thr Ile Glu Ser Leu Arg Pro Ser Asp Ser Ala Val

210 215 220

Tyr Leu Cys Ala Ser Arg Arg Gly Pro Tyr Glu Gln Tyr Phe Gly Pro

225 230 235 240

Gly Thr Arg Leu Thr Val Thr

245

<210>7

<211>741

<212>DNA

<213> Artificial sequence

<400>7

gctcaaacgg tgacccaatc ccagccggaa ctgtctgtcc aagaagcgga aacggttacc 60

atctcctgta cctacgatac cagcgaatcg gattattacc tgttttggta taaacagccg 120

ccgagccgtc aaatgacgct gctgattcgc caggaagcgt acaagcagca aaacgccacc 180

gaaaatcgtt tttctgtcaa cttccagaaa gcggccaaga gtttctccct gaaaatctca 240

gattcgcaac cgggcgacag cgcaacctat ttttgcgctt acggtgaaga tgacaaaatt 300

atcttcggca aaggtacccg cctgcacgtt acgccgggcg gtggcagcga aggtggcggt 360

tctgaaggcg gtggcagtga aggtggcggt tcagaaggcg gttcgggcga agcagatatt 420

tatcagacgc cgcgctacct gtctattggc accggtaaaa agatcacgct ggaatgtagt 480

cagacgatgg gccatgataa aatgtattgg taccagcaag acccgggtca agaactgcat 540

ctgatccact attcctacgg cgtgaattca accgaaaaag gtgatctgag ctctgaaagt 600

accgtttccc gtattcgcac ggaacacttt ccgctgacca tcgaatccct gcgcccgagc 660

gactctgccg tttatctgtg cgcctctcgc cgtggtccgt atgaacagta ttttggtccg 720

ggcacccgcc tgacggttac c 741

<210>8

<211>112

<212>PRT

<213> Artificial sequence

<400>8

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

1 5 10 15

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

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Thr Leu Leu

35 40 45

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

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>9

<211>111

<212>PRT

<213> Artificial sequence

<400>9

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>10

<211>24

<212>PRT

<213> Artificial sequence

<400>10

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 Ser Gly

20

<210>11

<211>112

<212>PRT

<213> Artificial sequence

<400>11

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

1 5 10 15

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

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Thr Leu Leu

35 40 45

Ile Arg Gln Glu His Asp Lys Thr His Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>12

<211>112

<212>PRT

<213> Artificial sequence

<400>12

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

1 5 10 15

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

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Thr Leu Leu

35 40 45

Ile Arg Gln Glu Ala Trp Asn Asn Ile Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

8590 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>13

<211>112

<212>PRT

<213> Artificial sequence

<400>13

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

1 5 10 15

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

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Thr Leu Leu

35 40 45

Ile Arg Gln Glu Ala Trp Asn Lys Glu Ser Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>14

<211>112

<212>PRT

<213> Artificial sequence

<400>14

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

1 5 10 15

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

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Thr Leu Leu

35 40 45

Ile Arg Gln Glu Ser Tyr Asn Met Asp Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>15

<211>112

<212>PRT

<213> Artificial sequence

<400>15

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

1 5 10 15

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

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Thr Leu Leu

35 40 45

Ile Arg Gln Glu Ser Trp Ser Pro Gln Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Lys Ile Ile PheGly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>16

<211>112

<212>PRT

<213> Artificial sequence

<400>16

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>17

<211>112

<212>PRT

<213> Artificial sequence

<400>17

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>18

<211>112

<212>PRT

<213> Artificial sequence

<400>18

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asn Pro Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100105 110

<210>19

<211>112

<212>PRT

<213> Artificial sequence

<400>19

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Arg Gln Glu Ser Trp Ser Pro Gln Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>20

<211>112

<212>PRT

<213> Artificial sequence

<400>20

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>21

<211>112

<212>PRT

<213> Artificial sequence

<400>21

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

1 5 10 15

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

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Thr Leu Leu

35 40 45

Ile Arg Gln Glu Ala Trp Asn Asn Ile Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>22

<211>112

<212>PRT

<213> Artificial sequence

<400>22

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>23

<211>9

<212>PRT

<213> Artificial sequence

<400>23

Gly Ile Leu Gly Phe Val Phe Thr Leu

1 5

<210>24

<211>9

<212>PRT

<213> Artificial sequence

<400>24

Gly Ala Phe Glu His Leu Pro Ser Leu

1 5

<210>25

<211>112

<212>PRT

<213> Artificial sequence

<400>25

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

1 5 10 15

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

20 25 30

Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Thr Leu Leu

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asn Pro Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>26

<211>112

<212>PRT

<213> Artificial sequence

<400>26

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Arg Gln Glu Ser Tyr Asn Met Asp Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>27

<211>112

<212>PRT

<213> Artificial sequence

<400>27

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Thr Ser Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>28

<211>112

<212>PRT

<213> Artificial sequence

<400>28

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Gln Pro Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>29

<211>112

<212>PRT

<213> Artificial sequence

<400>29

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Asp Ser Gln Pro Gly Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu

85 90 95

Asp Asp Tyr Leu Thr Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>30

<211>112

<212>PRT

<213> Artificial sequence

<400>30

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 5560

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

65 70 75 80

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

85 90 95

Asp Val Pro Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>31

<211>112

<212>PRT

<213> Artificial sequence

<400>31

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Arg Gln Glu Ser Tyr Asn Met Asp Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asn Pro Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr Pro

100 105 110

<210>32

<211>111

<212>PRT

<213> Artificial sequence

<400>32

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>33

<211>111

<212>PRT

<213> Artificial sequence

<400>33

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>34

<211>111

<212>PRT

<213> Artificial sequence

<400>34

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>35

<211>111

<212>PRT

<213> Artificial sequence

<400>35

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

6570 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

Ser Ser Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr

100 105 110

<210>36

<211>111

<212>PRT

<213> Artificial sequence

<400>36

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 7580

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>37

<211>111

<212>PRT

<213> Artificial sequence

<400>37

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>38

<211>111

<212>PRT

<213> Artificial sequence

<400>38

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser AspSer Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>39

<211>111

<212>PRT

<213> Artificial sequence

<400>39

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>40

<211>111

<212>PRT

<213> Artificial sequence

<400>40

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

Ser Ser Glu Met Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Thr

100 105 110

<210>41

<211>111

<212>PRT

<213> Artificial sequence

<400>41

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

8590 95

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

100 105 110

<210>42

<211>9

<212>PRT

<213> Artificial sequence

<400>42

Arg Leu Leu Val Pro Thr Gln Phe Val

1 5

<210>43

<211>111

<212>PRT

<213> Artificial sequence

<400>43

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>44

<211>111

<212>PRT

<213> Artificial sequence

<400>44

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>45

<211>111

<212>PRT

<213> Artificial sequence

<400>45

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>46

<211>111

<212>PRT

<213> Artificial sequence

<400>46

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly Tyr Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>47

<211>111

<212>PRT

<213> Artificial sequence

<400>47

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu Ile His Tyr

35 40 45

Ser Leu Gly Val Gly His Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Ile Glu Ser

65 70 75 80

Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>48

<211>198

<212>PRT

<213> Artificial sequence

<400>48

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

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

115 120 125

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

130 135 140

Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys

145 150 155 160

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

165 170 175

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

180 185 190

Ile Ile Pro Glu Asp Thr

195

<210>49

<211>241

<212>PRT

<213> Artificial sequence

<400>49

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

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

115 120 125

Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala

130 135 140

Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly

145 150 155 160

Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu

165 170 175

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

180 185 190

Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe Arg Cys Gln

195 200 205

Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg

210 215 220

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

225 230 235 240

Asp

<210>50

<211>594

<212>DNA

<213> Artificial sequence

<400>50

gctcagaccg ttacccagtc gcaaccggaa atgtcagttc aagaagcaga aaccgttacc 60

ctgtcctgta cctacgatac cagtgaaagt gattactacc tgttttggta caagcagccg 120

ccgtcccgtc aaatgattct ggttatccgc caggaagcat acaagcagca aaacgctacc 180

gaaaatcgtt ttagtgtcaa cttccagaaa gcggccaaga gtttctccct gaaaatttca 240

gattcgcagc tgggcgacgc agctatgtat ttttgcgcat acggtgaaga tgacaaaatt 300

atcttcggca agggtacgcg tctgcatatt ctgccgaaca tccagaatcc ggatccggcc 360

gtttatcagc tgcgtgatag caaaagcagc gataaaagcg tgtgcctgtt caccgatttt 420

gatagccaga ccaacgtgag ccagagcaaa gatagcgatg tgtacatcac cgataaatgc 480

gtgctggata tgcgcagcat ggatttcaaa agcaatagcg cggttgcgtg gagcaacaaa 540

agcgattttg cgtgcgcgaa cgcgtttaac aacagcatca tcccggaaga tacg 594

<210>51

<211>723

<212>DNA

<213> Artificial sequence

<400>51

gaagccgaca tctatcagac gccgcgctac ctggttattg gcacgggcaa gaaaatcacg 60

ctggaatgct ctcagacgat gggtcatgat aaaatgtatt ggtaccagca agacccgggc 120

atggaactgc atctgattca ctatagctac ggcgtgaaca gcaccgaaaa gggtgatctg 180

agctctgaaa gtaccgtttc ccgtatccgc acggaacact tcccgctgac cctggaatcc 240

gcccgtccgt ctcatacctc gcaatatctg tgcgcctctc gtcgcggtcc gtatgaacag 300

tactttggtc cgggtacccg tctgaccgtc accgaagacc tgaaaaacgt ctttccgcca 360

gaggttgcgg tcttcgaacc ttcagaagca gagatctctc atactcagaa agcgaccttg 420

gtttgtttgg caaccggatt ttaccctgac catgttgagt tgagctggtg ggtaaatgga 480

aaagaggtac acagcggggt gtgtaccgac cctcaaccac tgaaagaaca acccgcgttg 540

aatgattccc gctatgcttt aagctcccgt ctgcgtgtta gcgcgacttt ctggcaagat 600

ccgcgcaacc atttccgttg ccaagtccaa ttttacggat taagcgaaaa cgacgagtgg 660

actcaggacc gcgcgaaacc tgtcacacag atcgtgagtg ccgaagcctg gggccgcgca 720

gat 723

<210>52

<211>112

<212>PRT

<213> Artificial sequence

<400>52

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Arg Gln Glu His Asp Lys Thr His Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>53

<211>112

<212>PRT

<213> Artificial sequence

<400>53

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Arg Gln Glu Ala Trp Asn Asn Ile Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>54

<211>112

<212>PRT

<213> Artificial sequence

<400>54

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Arg Gln Glu Ala Trp Asn Lys Glu Ser Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>55

<211>112

<212>PRT

<213> Artificial sequence

<400>55

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Arg Gln Glu Ser Tyr Asn Met Asp Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>56

<211>112

<212>PRT

<213> Artificial sequence

<400>56

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Arg Gln Glu Ser Trp Ser Pro Gln Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>57

<211>112

<212>PRT

<213> Artificial sequence

<400>57

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

1 5 10 15

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

20 25 30

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

3540 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>58

<211>112

<212>PRT

<213> Artificial sequence

<400>58

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>59

<211>112

<212>PRT

<213> Artificial sequence

<400>59

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210>60

<211>112

<212>PRT

<213> Artificial sequence

<400>60

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Arg Gln Glu Ser Trp Ser Pro Gln Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>61

<211>112

<212>PRT

<213> Artificial sequence

<400>61

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>62

<211>9

<212>PRT

<213> Artificial sequence

<400>62

Ile Leu Ser Leu Glu Leu Met Lys Leu

1 5

<210>63

<211>112

<212>PRT

<213> Artificial sequence

<400>63

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>64

<211>112

<212>PRT

<213> Artificial sequence

<400>64

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Arg Gln Glu Ser Trp Ser Pro Gln Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>65

<211>9

<212>PRT

<213> Artificial sequence

<400>65

Gly Leu Met Lys Tyr Ile Gly Glu Val

1 5

<210>66

<211>112

<212>PRT

<213> Artificial sequence

<400>66

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210>67

<211>112

<212>PRT

<213> Artificial sequence

<400>67

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Arg Gln Glu Ser Tyr Asn Met Asp Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>68

<211>112

<212>PRT

<213> Artificial sequence

<400>68

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Thr Ser Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>69

<211>112

<212>PRT

<213> Artificial sequence

<400>69

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Gln Pro Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>70

<211>112

<212>PRT

<213> Artificial sequence

<400>70

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Asp Tyr Leu Thr Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>71

<211>112

<212>PRT

<213> Artificial sequence

<400>71

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Val Pro Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu Pro

100 105 110

<210>72

<211>112

<212>PRT

<213> Artificial sequence

<400>72

Ala Gln Thr Val Thr Gln Ser Gln Pro Glu MetSer Val Gln Glu Ala

1 5 10 15

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

20 25 30

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

35 40 45

Ile Arg Gln Glu Ser Tyr Asn Met Asp Asn Ala Thr Glu Asn Arg Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210>73

<211>111

<212>PRT

<213> Artificial sequence

<400>73

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>74

<211>111

<212>PRT

<213> Artificial sequence

<400>74

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

15 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>75

<211>111

<212>PRT

<213> Artificial sequence

<400>75

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 1015

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>76

<211>111

<212>PRT

<213> Artificial sequence

<400>76

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

Ser Ser Glu Leu Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr

100 105 110

<210>77

<211>111

<212>PRT

<213> Artificial sequence

<400>77

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu GluCys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>78

<211>111

<212>PRT

<213> Artificial sequence

<400>78

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>79

<211>111

<212>PRT

<213> Artificial sequence

<400>79

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>80

<211>111

<212>PRT

<213> Artificial sequence

<400>80

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

2025 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>81

<211>111

<212>PRT

<213> Artificial sequence

<400>81

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

Ser Ser Glu Met Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Thr

100 105 110

<210>82

<211>111

<212>PRT

<213> Artificial sequence

<400>82

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>83

<211>9

<212>PRT

<213> Artificial sequence

<400>83

Ser Leu Leu Met Trp Ile Thr Gln Val

1 5

<210>84

<211>111

<212>PRT

<213> Artificial sequence

<400>84

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>85

<211>111

<212>PRT

<213> Artificial sequence

<400>85

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>86

<211>111

<212>PRT

<213> Artificial sequence

<400>86

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>87

<211>111

<212>PRT

<213> Artificial sequence

<400>87

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 5 10 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly Tyr Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>88

<211>111

<212>PRT

<213> Artificial sequence

<400>88

Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly

1 510 15

Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met

20 25 30

Tyr Trp Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr

35 40 45

Ser Leu Gly Val Gly His Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser

50 55 60

Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser

65 70 75 80

Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly

85 90 95

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

100 105 110

<210>89

<211>498

<212>PRT

<213> Artificial sequence

<400>89

Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly

20 25 30

Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys

50 55 60

Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala Ile Gln Met

130 135 140

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

145 150155 160

Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser

180 185 190

Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gln Thr Val Thr

245 250 255

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

260 265 270

Ser Cys Thr Tyr Asp Thr Ser Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr

275 280 285

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

290 295 300

Tyr Lys Gln Gln Asn Ala Thr Glu Asn Arg Phe Ser Val Asn Phe Gln

305 310 315 320

Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile Ser Asp Ser Gln Pro Gly

325 330 335

Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu Asp Asp Lys Ile Ile

340 345 350

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

355 360 365

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

370 375 380

Gly Ser Gly Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile

385 390 395 400

Gly Thr Gly Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His

405 410 415

Asp Lys Met Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu

420 425 430

Ile His Tyr Ser Tyr GlyVal Asn Ser Thr Glu Lys Gly Asp Leu Ser

435 440 445

Ser Glu Ser Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr

450 455 460

Ile Glu Ser Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser

465 470 475 480

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

485 490 495

Val Thr

<210>90

<211>499

<212>PRT

<213> Artificial sequence

<400>90

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

1 5 10 15

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

20 25 30

Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Thr Leu

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Glu Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr

100 105 110

Pro Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu

115 120 125

Gly Gly Gly Ser Glu Gly Gly Ser Gly Glu Ala Asp Ile Tyr Gln Thr

130 135 140

Pro Arg Tyr Leu Ser Ile Gly Thr Gly Lys Lys Ile Thr Leu Glu Cys

145 150 155 160

Ser Gln Thr Met Gly His Asp Lys Met Tyr Trp Tyr Gln Gln Asp Pro

165 170 175

Gly Gln Glu Leu His Leu Ile His Tyr Ser Tyr Gly Val Asn Ser Thr

180 185 190

Glu Lys Gly Asp Leu Ser Ser Glu Ser Thr Val Ser Arg Ile Arg Thr

195 200 205

Glu His Phe Pro Leu Thr Ile Glu Ser Leu Arg Pro Ser Asp Ser Ala

210 215 220

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

225 230 235 240

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

245 250 255

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

260 265 270

Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn

275 280 285

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile

290 295 300

Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg

305 310 315 320

Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met

325 330 335

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser

340 345 350

Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly

355 360 365

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Gly Gly Ala Ile Gln Met Thr Gln Ser Pro

385 390 395 400

Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg

405 410 415

Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro

420 425 430

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser

435 440 445

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr

450 455 460

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

465 470 475 480

Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val

485 490 495

Glu Ile Lys

<210>91

<211>498

<212>PRT

<213> Artificial sequence

<400>91

Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly

20 25 30

Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys

50 55 60

Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala

65 70 75 80

Tyr Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala Ile Gln Met

130 135 140

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

145 150 155 160

Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser

180 185 190

Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gln Thr Val Thr

245 250 255

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

260 265 270

Ser Cys Thr Tyr Asp Thr Ser Glu Asn Asp Tyr Ile Leu Phe Trp Tyr

275 280 285

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

290 295 300

Tyr Lys Gln Gln Asn Ala Thr Glu Asn Arg Phe Ser Val Asn Phe Gln

305 310 315 320

Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile Ser Asp Ser Gln Pro Gly

325 330 335

Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu Asp Asp Lys Ile Ile

340 345 350

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

355360 365

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

370 375 380

Gly Ser Gly Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile

385 390 395 400

Gly Thr Gly Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His

405 410 415

Asp Lys Met Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu

420 425 430

Ile His Tyr Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser

435 440 445

Ser Glu Ser Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr

450 455 460

Ile Glu Ser Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser

465 470 475 480

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

485 490 495

Val Thr

<210>92

<211>499

<212>PRT

<213> Artificial sequence

<400>92

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Glu Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr

100 105 110

Pro Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly GlySer Glu

115 120 125

Gly Gly Gly Ser Glu Gly Gly Ser Gly Glu Ala Asp Ile Tyr Gln Thr

130 135 140

Pro Arg Tyr Leu Ser Ile Gly Thr Gly Lys Lys Ile Thr Leu Glu Cys

145 150 155 160

Ser Gln Thr Met Gly His Asp Lys Met Tyr Trp Tyr Gln Gln Asp Pro

165 170 175

Gly Gln Glu Leu His Leu Ile His Tyr Ser Tyr Gly Val Asn Ser Thr

180 185 190

Glu Lys Gly Asp Leu Ser Ser Glu Ser Thr Val Ser Arg Ile Arg Thr

195 200 205

Glu His Phe Pro Leu Thr Ile Glu Ser Leu Arg Pro Ser Asp Ser Ala

210 215 220

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

225 230 235 240

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

245 250 255

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

260 265 270

Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn

275 280 285

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile

290 295 300

Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg

305 310 315 320

Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met

325 330 335

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser

340 345 350

Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly

355 360 365

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Gly Gly Ala Ile Gln Met Thr Gln Ser Pro

385 390 395 400

Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg

405 410 415

Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro

420 425 430

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser

435 440 445

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr

450 455 460

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

465 470 475 480

Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val

485 490 495

Glu Ile Lys

<210>93

<211>498

<212>PRT

<213> Artificial sequence

<400>93

Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 510 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly

20 25 30

Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys

50 55 60

Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala Ile Gln Met

130 135 140

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

145 150 155 160

Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser

180 185 190

Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gln Thr Val Thr

245 250 255

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

260 265 270

Ser Cys Thr Tyr Asp Thr Ser Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr

275 280 285

Lys Gln Pro Pro Ser Arg Gln Met Thr Leu Leu Ile Arg Gln Glu Ser

290 295 300

Tyr Asn Met Asp Asn Ala Thr Glu Asn Arg Phe Ser Val Asn Phe Gln

305 310 315 320

Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile Ser Asp Ser Gln Pro Gly

325 330 335

Asp Ser Ala Thr Tyr Phe Cys Ala Tyr Gly Glu Asp Asp Lys Ile Ile

340 345 350

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

355 360 365

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

370 375 380

Gly Ser Gly Glu Ala Asp Ile Tyr Gln Thr Pro Arg Tyr Leu Ser Ile

385 390 395 400

Gly Thr Gly Lys Lys Ile Thr Leu Glu Cys Ser Gln Thr Met Gly His

405 410 415

Asp Lys Met Tyr Trp Tyr Gln Gln Asp Pro Gly Gln Glu Leu His Leu

420 425 430

Ile His Tyr Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser

435 440 445

Ser Glu Ser Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr

450 455 460

Ile Glu Ser Leu Arg Pro Ser Asp Ser Ala Val Tyr Leu Cys Ala Ser

465 470 475 480

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

485 490 495

Val Thr

<210>94

<211>499

<212>PRT

<213> Artificial sequence

<400>94

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

1 5 10 15

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

20 25 30

Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Thr Leu

35 40 45

Leu Ile Arg Gln Glu Ser Tyr Asn Met Asp Asn Ala Thr Glu Asn Arg

50 55 60

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

65 70 75 80

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

85 90 95

Glu Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Val Thr

100 105 110

Pro Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu

115 120 125

Gly Gly Gly Ser Glu Gly Gly Ser Gly Glu Ala Asp Ile Tyr Gln Thr

130 135 140

Pro Arg Tyr Leu Ser Ile Gly Thr Gly Lys Lys Ile Thr Leu Glu Cys

145 150 155 160

Ser Gln Thr Met Gly His Asp Lys Met Tyr Trp Tyr Gln Gln Asp Pro

165 170 175

Gly Gln Glu Leu His Leu Ile His Tyr Ser Tyr Gly Val Asn Ser Thr

180 185 190

Glu Lys Gly Asp Leu Ser Ser Glu Ser Thr Val Ser Arg Ile Arg Thr

195 200 205

Glu His Phe Pro Leu Thr Ile Glu Ser Leu Arg Pro Ser Asp Ser Ala

210 215 220

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

225 230 235 240

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

245 250 255

Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

260 265 270

Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn

275 280 285

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile

290 295 300

Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg

305 310 315 320

Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met

325 330 335

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser

340 345 350

Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly

355 360 365

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

370 375 380

Ser Gly Gly Gly Gly Ser Gly Gly Ala Ile Gln Met Thr Gln Ser Pro

385 390 395 400

Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg

405 410 415

Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro

420 425 430

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser

435 440 445

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr

450 455 460

Leu Thr IleSer Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

465 470 475 480

Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val

485 490 495

Glu Ile Lys

<210>95

<211>493

<212>PRT

<213> Artificial sequence

<400>95

Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly

20 25 30

Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys

50 55 60

Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala Ile Gln Met

130 135 140

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

145 150 155 160

Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser

180 185 190

Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210215 220

Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Glu Ala Asp Ile

245 250 255

Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly Lys Lys Ile Thr

260 265 270

Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met Tyr Trp Tyr Gln

275 280 285

Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr Ser Tyr Gly Val

290 295 300

Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser Thr Val Ser Arg

305 310 315 320

Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser Ala Arg Pro Ser

325 330 335

His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly Ser Ala Glu Leu

340 345 350

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

355 360 365

Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile

370 375 380

Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr

385 390 395 400

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

405 410 415

Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu

420 425 430

Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr

435 440 445

Phe Trp Gln Asp Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr

450 455 460

Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val

465 470 475 480

Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp

485 490

<210>96

<211>493

<212>PRT

<213> Artificial sequence

<400>96

Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly

20 25 30

Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys

50 55 60

Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala Ile Gln Met

130 135 140

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

145 150 155 160

Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser

180 185 190

Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Glu Ala Asp Ile

245 250 255

Tyr Gln Thr Pro Arg Tyr LeuVal Ile Gly Thr Gly Lys Lys Ile Thr

260 265 270

Leu Glu Cys Ser Gln Thr Met Gly His Asp Lys Met Tyr Trp Tyr Gln

275 280 285

Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr Ser Tyr Gly Val

290 295 300

Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser Thr Val Ser Arg

305 310 315 320

Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser Ala Arg Pro Ser

325 330 335

His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly Ser Leu Glu Leu

340 345 350

Val Phe Gly Pro Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn

355 360 365

Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile

370 375 380

Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr

385 390 395 400

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

405 410 415

Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu

420 425 430

Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr

435 440 445

Phe Trp Gln Asp Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr

450 455 460

Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val

465 470 475 480

Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp

485 490

<210>97

<211>493

<212>PRT

<213> Artificial sequence

<400>97

Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser CysAla Ala Ser Gly Tyr Ser Phe Thr Gly

20 25 30

Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys

50 55 60

Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ala Ile Gln Met

130 135 140

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

145 150 155 160

Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser

180 185 190

Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Glu Ala Asp Ile

245 250 255

Tyr Gln Thr Pro Arg Tyr Leu Val Ile Gly Thr Gly Lys Lys Ile Thr

260 265 270

Leu Glu Cys Ser Gln Thr Met Gly Tyr Asp Lys Met Tyr Trp Tyr Gln

275 280 285

Gln Asp Pro Gly Met Glu Leu His Leu Ile His Tyr Ser Tyr Gly Val

290 295300

Asn Ser Thr Glu Lys Gly Asp Leu Ser Ser Glu Ser Thr Val Ser Arg

305 310 315 320

Ile Arg Thr Glu His Phe Pro Leu Thr Leu Glu Ser Ala Arg Pro Ser

325 330 335

His Thr Ser Gln Tyr Leu Cys Ala Ser Arg Arg Gly Ser Leu Glu Leu

340 345 350

Val Phe Gly Pro Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn

355 360 365

Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile

370 375 380

Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr

385 390 395 400

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

405 410 415

Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu

420 425 430

Asn Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr

435 440 445

Phe Trp Gln Asp Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr

450 455 460

Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val

465 470 475 480

Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp

485 490

<210>98

<211>9

<212>PRT

<213> Artificial sequence

<400>98

Phe Ile Leu Pro Val Leu Gly Ala Val

1 5

<210>99

<211>10

<212>PRT

<213> Artificial sequence

<400>99

Ala Leu Gly Gly His Pro Leu Leu Gly Val

1 5 10

<210>100

<211>9

<212>PRT

<213> Artificial sequence

<400>100

Glu Val Asp Pro Ile Gly His Leu Tyr

1 5

<210>101

<211>9

<212>PRT

<213> Artificial sequence

<400>101

Lys Tyr Lys Asp Tyr Phe Pro Val Ile

1 5

Claims

1. A T Cell Receptor (TCR) having the property of binding the KLVALGINAV HLA-A2 complex and comprising a TCR β chain variable domain and/or a TCR beta chain variable domain,

wherein the TCR comprises a combination of α and β chain variable domains as shown in the following table:

or,

the TCR comprises a combination of α and β chain variable domains as shown in the following table:

2. the T cell receptor of claim 1, wherein the TCR is an α β heterodimeric TCR having α and β chain constant domain sequences with cysteine residues forming a disulfide bond between the α and β chain constant domains of the TCR.

3. a T cell receptor according to claim 2 wherein the cysteine residues form an artificial disulphide bond between the α and β chain constant domains of the TCR.

4. A T cell receptor according to claim 3 wherein in the TCR the cysteine residues which form the artificial disulphide bond are substituted at one or more groups selected from,

5. the T cell receptor of any one of claims 1 to 4, wherein the hydrophobic core of the α chain variable domain and/or the β chain variable domain of the TCR is mutated.

6. the T cell receptor of claim 1, wherein the TCR is a single chain TCR consisting of an α variable domain and a β variable domain, the α and β variable domains being linked by a flexible short peptide sequence.

7. the T cell receptor of claim 5, wherein the hydrophobic core mutation occurs at one or more amino acid residue positions 11M, 21L, 48V and 110I of the α chain variable domain of SEQ ID No. 2, wherein the amino acid residue numbering is as shown in SEQ ID No. 2 and/or

the hydrophobic core mutation occurs at one or more amino acid residue positions 78L and 81A of a variable domain of a beta strand as shown in SEQ ID No. 3, wherein the numbering of the amino acid residues is as shown in SEQ ID No. 3.

8. the T cell receptor of claim 7, wherein the α chain variable domain of the TCR after hydrophobic core mutation comprises one or more amino acid residues selected from the group consisting of 11L, 21I, 48L and 110V, wherein the amino acid residue numbering adopts the numbering shown in SEQ ID No. 2, and/or

the β chain variable domain of the TCR after hydrophobic core mutation comprises one or more amino acid residues selected from the group consisting of 78I and 81L, wherein the numbering of the amino acid residues is as shown in SEQ ID No. 3.

9. the T cell receptor of claim 1, wherein the TCR comprises a combination of α and β chain variable domains as set forth in the following table:

10. the T cell receptor of claim 1, wherein the C-or N-terminus of the α chain and/or β chain of the TCR is conjugated to a conjugate.

11. The T cell receptor of claim 10, wherein the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modifying moiety or a combination thereof.

12. the T cell receptor of claim 11, wherein the therapeutic agent that binds to the T cell receptor is an anti-CD 3 antibody linked to the C-or N-terminus of the α or β chain of the TCR.

13. the T cell receptor of claim 12, wherein the TCR β chain has an amino acid sequence selected from the group consisting of SEQ ID nos 95, 96, and 97, upon binding to an anti-CD 3 antibody.

14. The T cell receptor of claim 12, wherein the amino acid sequence of the TCR fused to an anti-CD 3 antibody is selected from the group consisting of: 89, 90, 91, 92, 93 and 94.

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 the T cell receptor of any one of claims 1-14 or a complement thereof.

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 as claimed in claim 15 or a cell according to claim 19 for the manufacture of a medicament for the treatment of a tumour or HCV viral infection.

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

(i) culturing the host cell of claim 18, thereby expressing the T cell receptor of claim 1;

(ii) isolating or purifying said T cell receptor.