CN110156889B - High affinity HBs T cell receptor - Google Patents

Abstract

The present invention provides a T Cell Receptor (TCR) having the property of binding to the ilspflplll-HLA A2 complex; and the binding affinity of the TCR to the ILSPFLLL-HLA A2 complex is at least 1.3 times greater than the binding affinity of a wild-type TCR to the ILSPFLLL-HLA A2 complex. Such TCRs can be used alone or in combination with therapeutic agents to target ilspflplll-HLA A2 complex-presenting tumor cells.

Description

High affinity HBs T cell receptor

technical field

The invention relates to the field of biotechnology, and more specifically relates to a T cell receptor (T cell receptor, TCR) capable of recognizing polypeptides derived from HBV surface antigen (HBsAg) protein. The invention also relates to the preparation and use of said receptors.

Background technique

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

TCR is the sole receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC), and such exogenous or endogenous peptides may be the only sign of abnormalities in cells. In the immune system, the combination of antigen-specific TCR and pMHC complexes triggers direct physical contact between T cells and antigen-presenting cells (APCs), and then other cell membrane surface molecules of T cells and APCs interact. It causes a series of subsequent cell signal transmission and other physiological responses, so that T cells with different antigen specificities exert immune effects on their target cells.

The ligands of MHC class I and class II molecules corresponding to TCR are also proteins of the immunoglobulin superfamily, but they are specific for the presentation of antigens. Different individuals have different MHCs, so they can present different short sequences in a protein antigen. Peptides to the surface of the respective APC cells. Human MHC is often referred to as HLA genes or HLA complexes.

The short peptide ILSPFLPLL (SEQ ID NO:53) is derived from HBsAg expressed by HBV-infected hepatocytes or HBV-induced liver cancer cells (Roh, S., et al. (2001). Virus Research 73(1): 17 -26.). The type Ⅰ HLA molecules of tumor cells present short peptides derived from HBV surface antigen (HBV surface antigen), including ILSPFLPLL. Thus, the ILSPFLPLL-HLA A2 complex provides a TCR-targetable marker for tumor cells. The TCR that can bind to the ILSPFLPLL-HLA A2 complex has high application value for the treatment of tumors. For example, a TCR capable of targeting this tumor cell marker can be used to deliver a cytotoxic or immunostimulatory agent to the target cell, or be transformed into a T cell, enabling the T cell expressing the TCR to destroy the tumor cell, so that in what is known as Administered to patients during the course of treatment with adoptive immunotherapy. For the former purpose, the ideal TCR is one with high affinity, allowing the TCR to reside on the targeted cell for a long period of time. For the latter purpose, the use of intermediate affinity TCRs is preferred. Therefore, those skilled in the art are devoting themselves to developing TCRs targeting tumor cell markers that can be used for different purposes.

Contents of the invention

The purpose of the present invention is to provide a TCR with higher affinity to ILSPFLPLL-HLA A2 complex.

Another object of the present invention is to provide a preparation method of the above-mentioned type of TCR and an application of the above-mentioned type of TCR.

The first aspect of the present invention provides a T cell receptor (TCR), which has the activity of binding ILSPFLPLL-HLA A2 complex.

In another preferred example, the T cell receptor (TCR) has the activity of binding ILSPFLPLL-HLA A2 complex, and the T cell receptor comprises a TCRα chain variable domain and a TCRβ chain variable domain, and the TCRα The chain variable domain contains 3 CDR regions, and the reference sequence of the 3 CDR regions of the TCRα chain variable domain is as follows,

CDR1α:DRGSQS

CDR2α: IYSNGD

CDR3α: AVNLYAGNMLT with at least one of the following mutations:

Residue before mutation Mutated residues N at position 3 of CDR3α Q or A Position 4 L of CDR3α D, E, G, S or H Y at position 5 of CDR3α P, Q, S or G Position 6A of CDR3α S, T, W or D G at position 7 of CDR3α R, K, M, Q, N, T, S or H

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

CDR1β:SGHVS

CDR2β: FQNEAQ

CDR3β:ASSSDFGNQPQH.

In another preferred example, the number of mutations in the CDR region of the TCRα chain can be 1, 2, 3, 4, or 5.

In another preferred embodiment, the T cell receptor (TCR) according to the present invention comprises a TCRα chain variable domain and a TCRβ chain variable domain, and the TCRα chain variable domain includes CDR1α, CDR2α, and CDR3α.

In another preferred example, the CDR1α comprises the sequence: DRGSQS.

In another preferred example, the CDR2α comprises the sequence: IYSNGD.

In another preferred example, the CDR3α comprises the sequence:

AV[3αX1][3αX2][3αX3][3αX4][3αX5]NMLT, wherein [3αX1], [3αX2], [3αX3], [3αX4], [3αX5] are independently selected from any natural amino acid residues.

In another preferred example, the [3αX1] is N, Q or A.

In another preferred example, the [3αX2] is L, D, E, G, S or H.

In another preferred example, the [3αX3] is Y, P, Q, S or G.

In another preferred example, the [3αX4] is A, S, T, W or D.

In another preferred example, the [3αX5] is G, R, K, M, Q, N, T, S or H.

In another preferred example, the CDR3α comprises a sequence selected from the following group:

AVNLYAGNMLT、AVQDPSRNMLT、AVADQSRNMLT、AVQDPSKNMLT、AVQDPSMNMLT、AVQDPSQNMLT、AVQDPTNNMLT、AVQDSSRNMLT、AVQDPAKNMLT、AVQEPSRNMLT、AVQDPTKNMLT、AVAGGWRNMLT、AVQSPDRNMLT、AVQHPATNMLT、AVADPSKNMLT、AVAHPSKNMLT、AVQSPDQNMLT、AVQDPASNMLT、AVQDPSHNMLT、AVQDPSTNMLT。

In another preferred embodiment, the T cell receptor (TCR) according to the present invention comprises a TCRα chain variable domain and a TCRβ chain variable domain, and the TCRβ chain variable domain comprises CDR1β, CDR2β and CDR3β.

In another preferred example, the CDR1β comprises the sequence: SGHVS.

In another preferred example, the CDR2β comprises the sequence: FQNEAQ.

In another preferred example, the CDR3β comprises the sequence: ASSSDFGNQPQH.

In another preferred example, the TCRα chain variable domain of the TCR does not contain the following CDRs at the same time:

CDR1α: DRGSQS; CDR2α: IYSNGD; and CDR3α: AVNLYAGNMLT.

In another preferred example, the TCRβ chain variable domain of the TCR does not contain the following CDRs at the same time:

CDR1β: SGHVS; CDR2β: FQNEAQ; and CDR3β: ASSSDFGNQPQH.

In another preferred example, the mutation occurs in one or more CDR regions of the α-chain and/or β-chain variable domains.

In another preferred example, the mutation occurs in CDR3 of the α chain.

In another preferred example, the amino acid sequence of the α-chain variable domain of the TCR is selected from the group consisting of: SEQ ID NO:28-46.

In another preferred example, the affinity of the TCR to the ILSPFLPLL-HLA A2 complex is at least 1.3 times that of the wild-type TCR; preferably, at least 2 times; more preferably, at least 5 times.

In another preferred example, the affinity of the TCR to the ILSPFLPLL-HLA A2 complex is at least 10 times that of the wild-type TCR; preferably, at least 20 times.

In another preferred example, the dissociation equilibrium constant K _D of the TCR to the ILSPFLPLL-HLA A2 complex is ≤3.4 μM.

In another preferred example, the TCR dissociation equilibrium constant for the ILSPFLPLL-HLA A2 complex is 10nM≤K _D≤2.58 μM; preferably, 100nM≤K _D≤700nM .

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

TCR number CDR1α CDR2α CDR3α CDR1β CDR2β CDR3β s-1 DRGSQS IYSNGD AVQDPSRNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-2 DRGSQS IYSNGD AVADQSRNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-3 DRGSQS IYSNGD AVQDPSKNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-4 DRGSQS IYSNGD AVQDPSMNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-5 DRGSQS IYSNGD AVQDPSQNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-6 DRGSQS IYSNGD AVQDPTNNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-7 DRGSQS IYSNGD AVQDSSRNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-8 DRGSQS IYSNGD AVQDPAKNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-9 DRGSQS IYSNGD AVQEPSRNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-10 DRGSQS IYSNGD AVQDPTKNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-11 DRGSQS IYSNGD AVAGGWRNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-12 DRGSQS IYSNGD AVQSPDRNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-13 DRGSQS IYSNGD AVQHPATNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-14 DRGSQS IYSNGD AVADPSKNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-15 DRGSQS IYSNGD AVAHPSKNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-16 DRGSQS IYSNGD AVQSPDQNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-17 DRGSQS IYSNGD AVQDPASNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-18 DRGSQS IYSNGD AVQDPSHNMLT SGHVS FQNEAQ ASSSDFGNQPQH s-19 DRGSQS IYSNGD AVQDPSTNMLT SGHVS FQNEAQ ASSSDFGNQPQH

In another preferred embodiment, the TCR is soluble.

In another preferred example, the TCR is an αβ heterodimeric TCR or a single-chain TCR.

In another preferred embodiment, the TCR of the present invention is an αβ heterodimeric TCR, and the α chain variable domain of the TCR contains at least 85% of the amino acid sequence shown in SEQ ID NO: 24, preferably, at least 90%; more preferably, at least 92%; most preferably, at least 94% (e.g., can be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% sequence homology) amino acid sequence of sequence homology; and/or the β chain variable domain of the TCR comprises at least the amino acid sequence shown in SEQ ID NO:25 90%, preferably at least 92%; more preferably, at least 94%; most preferably, at least 97%; (e.g., may be at least 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences with sequence homology of 97%, 98%, 99% sequence homology).

In another preferred example, the TCR comprises (i) all or part of the TCRα chain except its transmembrane domain, and (ii) all or part of the TCRβ chain except its transmembrane domain, wherein (i) and (ii) each comprise a variable domain and at least a portion of a constant domain of a TCR chain.

In another preferred example, the amino acid sequence of the α-chain variable domain of the TCR is selected from the group consisting of: SEQ ID NO:28-46.

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

In another preferred example, the cysteine residues forming an artificial interchain disulfide bond between the α-chain variable region and the β-chain constant region of the TCR are substituted for one or more groups selected from the following point:

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

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

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

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

Wherein, the amino acid sequence position number is according to the position number listed in IMGT (International Immunogenetics Information System).

In another preferred example, the TCR containing an artificial interchain disulfide bond between the variable region of the α chain and the constant region of the β chain comprises the variable domain of the α chain and the variable domain of the β chain and all or Part of the beta chain constant domain, but it does not contain the alpha chain constant domain, the alpha chain variable domain of the TCR forms a heterodimer with the beta chain.

In another preferred example, the TCR containing an artificial interchain disulfide bond between the variable region of the α chain and the constant region of the β chain comprises (i) all or part of the TCR α chain except its transmembrane domain, and (ii) All or part of a TCR beta chain other than its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least a portion of the constant domain of the TCR chain.

In another preferred example, the TCR is an αβ heterodimeric TCR, which includes (i) all or part of the TCRα chain except its transmembrane domain, and (ii) all but its transmembrane domain Or part of the TCRβ chain, wherein (i) and (ii) both comprise the variable domain of the TCR chain and at least a part of the constant domain, and the constant region of the α chain and the constant region of the β chain contain an artificial interchain disulfide bond.

In another preferred example, the cysteine residues forming an artificial interchain disulfide bond between the constant regions of the TCRα and β chains are substituted for one or more groups of sites selected from the following:

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

Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1;

Tyr10 of TRAC*01 exon 1 and Ser17 of TRBC1*01 or TRBC2*01 exon 1;

Thr45 of TRAC*01 exon 1 and Asp59 of TRBC1*01 or TRBC2*01 exon 1;

Ser15 of TRAC*01 exon 1 and Glu15 of TRBC1*01 or TRBC2*01 exon 1;

Arg53 of TRAC*01 exon 1 and Ser54 of TRBC1*01 or TRBC2*01 exon 1; Pro89 of TRAC*01 exon 1 and Ala19 of TRBC1*01 or TRBC2*01 exon 1; and

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

Wherein, the amino acid sequence position number is according to the position number listed in IMGT (International Immunogenetics Information System).

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

In another preferred example, the TCR is a single-chain TCR composed of an α-chain variable domain and a β-chain variable domain, and the α-chain variable domain and the β-chain variable domain are composed of a flexible short peptide sequence (linker )connect.

In another preferred embodiment, the hydrophobic core of the TCR is mutated.

In another preferred example, the hydrophobic core of the TCR α chain variable domain and/or β chain variable domain is mutated.

In another preferred example, the TCR in which the hydrophobic core is mutated is a single-chain TCR composed of an α variable domain and a β variable domain, and the α variable domain and β variable domain consist of a flexible short peptide sequence ( linker) connection.

In another preferred embodiment, the TCR of the present invention is a single-chain TCR, and the α-chain variable domain of the TCR comprises at least 85%, preferably at least 90%, of the amino acid sequence shown in SEQ ID NO: 2; more Preferably, at least 92%; most preferably, at least 94% (e.g., may be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology) amino acid sequence of sequence homology; and/or the β chain variable domain of the TCR comprises at least 90% of the amino acid sequence shown in SEQ ID NO:3, preferably , at least 92%; more preferably, at least 94%; most preferably, at least 97%; (e.g., may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% sequence homology) amino acid sequence of sequence homology.

In another preferred example, the amino acid sequence of the α-chain variable domain of the TCR is selected from the group consisting of: SEQ ID NO:5-23.

In another preferred example, the hydrophobic core mutation of the TCR occurs at one or more amino acid residue positions selected from the group consisting of 19A, 21L, 39Y, 40S, 79S and 88L; wherein, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:24; and/or

The hydrophobic core mutation occurs at one or more amino acid residue positions selected from the group consisting of 11K, 13A, 36Q, 38A, 39L, 43P, 82T, 84Q of the beta chain variable domain shown in SEQ ID NO:25 , 91L and 113I, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:25.

In another preferred example, the α-chain variable domain of the TCR after the hydrophobic core mutation includes one or more amino acid residues selected from the group consisting of 19V, 21I, 39D, 40P, 79V and 88F; and/or The beta chain variable domain of the TCR after hydrophobic core mutation comprises one or more amino acid residues selected from the group consisting of 11L, 13V, 36R, 38D, 39P, 43L, 82V, 84P, 91F and 113V.

In a preferred embodiment of the present invention, the T cell receptor (TCR), which has the activity of binding ILSPFLPLL-HLA A2 complex, and comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, the TCR A mutation occurs in the α-chain variable domain shown in SEQ ID NO: 24, and the mutated amino acid residue position includes one or more of 92N, 93L, 94Y, 95A or 96G, wherein the amino acid residue numbering adopts The number shown in SEQ ID NO:24.

In another preferred example, the mutated TCRα chain variable domain includes one or more amino acid residues selected from the group consisting of: 92Q or 92A; 93D, 93E, 93G, 93S or 93H; 94P, 94Q, 94S or 94G; 95S, 95T, 95W or 95D; 96R, 96K, 96M, 96Q, 96N, 96T, 96S or 96H; wherein, the amino acid residue numbering adopts the numbering shown in SEQ ID NO:24.

In another preference, the TCR is selected from the following group:

In another preference, the TCR is selected from the following group:

In another preferred example, a conjugate is bound to the C- or N-terminus of the α chain and/or β chain of the TCR.

In another preferred embodiment, the conjugate that binds to the TCR is a detectable marker, a therapeutic agent, a PK modification moiety, or any combination of these substances.

In another preferred example, the therapeutic agent that binds to the TCR is an anti-CD3 antibody linked to the C- or N-terminus of the α or β chain of the TCR.

The second aspect of the present invention provides a multivalent TCR complex, comprising at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect of the present invention.

A third aspect of the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the TCR molecule described in the first aspect of the present invention or the multivalent TCR complex described in the second aspect of the present invention or its complement sequence;

The fourth aspect of the present invention provides a vector containing the nucleic acid molecule described in the third aspect of the present invention.

The fifth aspect of the present invention provides a host cell containing the vector of the fourth aspect of the present invention or the exogenous nucleic acid molecule of the third aspect of the present invention integrated in the chromosome.

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

The seventh aspect of the present invention provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, Or the cell described in the sixth aspect of the present invention.

The eighth aspect of the present invention provides a method for treating diseases, comprising administering an appropriate amount of the TCR described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the The cell according to the sixth aspect of the invention, or the pharmaceutical composition according to the seventh aspect of the invention.

The ninth aspect of the present invention provides the use of the TCR described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, or the use of the cells described in the sixth aspect of the present invention, for the preparation and treatment of tumors Drug.

The tenth aspect of the present invention provides a method for preparing the T cell receptor described in the first aspect of the present invention, comprising the steps of:

(i) cultivating the host cell described in the fifth aspect of the present invention, thereby expressing the T cell receptor described in the first aspect of the present invention;

(ii) isolating or purifying the T cell receptor.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1a and Figure 1b are the amino acid sequence and DNA sequence of the single-stranded template TCR constructed in the present invention, respectively.

Figure 2a and Figure 2b are the amino acid sequence of the alpha variable domain and the amino acid sequence of the beta chain variable domain of the single-chain template TCR constructed in the present invention, respectively.

Figure 3a and Figure 3b are the DNA sequence of the alpha variable domain and the DNA sequence of the beta chain variable domain of the single-chain template TCR constructed in the present invention, respectively.

Figure 4a and Figure 4b are the amino acid sequence and nucleotide sequence of the short linker peptide (linker) of the single-chain template TCR constructed in the present invention, respectively.

Figures 5a-s show the amino acid sequence of the α-chain variable domain of a single-chain TCR with high affinity for the ILSPFLPLL-HLA A2 complex, respectively, with mutated residues being underlined.

Figure 6a and Figure 6b respectively show the amino acid sequences of wild-type TCRα and β chain variable domains that can specifically bind to ILSPFLPLL-HLA A2 complex.

Figure 7a and Figure 7b respectively show the amino acid sequences of the reference TCR α and β chains in the present invention.

Figures 8a-s show the amino acid sequence of the α-chain variable domain of a heterodimeric TCR with high affinity for the ILSPFLPLL-HLA A2 complex, respectively, with mutated residues being underlined.

Fig. 9 is the affinity curve of wild-type TCR (ie reference TCR) and ILSPFLPLL-HLA A2 complex.

Figure 10a and Figure 10b respectively show the amino acid sequences of wild-type TCRα and β chains in the present invention.

Figures 11a-h respectively show the results of Elispot experiments of effector cells transfected with part of the high-affinity TCR of the present invention against T2 cells loaded with short peptides.

Figures 12a-h respectively show the results of Elispot experiments of effector cells transfected with part of the high-affinity TCR of the present invention against empty T2 cells and T2 cells loaded with a specific short peptide at a concentration of 10 ^-6 M.

Detailed ways

Through extensive and in-depth research, the present invention obtains a high-affinity T cell receptor (TCR) that recognizes ILSPFLPLL short peptide (derived from HBVSurface Antigen), and the ILSPFLPLL short peptide uses the peptide (peptide)-HLAA2 complex The form is presented. The high-affinity TCR has three CDR regions in its α-chain variable domain

CDR1α:DRGSQS

CDR2α: IYSNGD

CDR3α: Mutations in AVNLYAGNMLT; and/or in the 3 CDR regions of its β-chain variable domain

CDR1β:SGHVS

CDR2β: FQNEAQ

CDR3β: mutated in ASSSDFGNQPQH; and, after mutation, the affinity and/or binding half-life of the TCR of the present invention to the above-mentioned ILSPFLPLL-HLAA2 complex is at least 1.3 times that of the wild-type TCR.

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

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

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

the term

T cell receptor (T cell receptor, TCR)

TCRs can be described using the International Immunogenetics Information System (IMGT). Natural αβ heterodimeric TCRs have an α chain and a β chain. Broadly speaking, each chain comprises a variable region, a connecting region, and a constant region, and the beta strands usually also contain a short variable region between the variable and connecting regions, but this variable region is often considered part of the connecting region. The junction region of the TCR is determined by the unique TRAJ and TRBJ of IMGT, and the constant region of the TCR is determined by the TRAC and TRBC of IMGT.

Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, chimeric in the framework sequence. In the IMGT nomenclature, different numbers of TRAV and TRBV refer to different Vα types and Vβ types, respectively. In the IMGT system, the α-chain constant domain has the following symbols: TRAC*01, where "TR" indicates the T cell receptor gene; "A" indicates the α-chain gene; C indicates the constant region; "*01" indicates allele Gene 1. The beta chain constant domain has the following symbols: TRBC1*01 or TRBC2*01, where "TR" indicates the T cell receptor gene; "B" indicates the beta chain gene; C indicates the constant region; "*01" indicates the allele 1. The constant region of the alpha chain is uniquely defined, and in the form of the beta chain, there are two possible constant region genes "C1" and "C2". Those skilled in the art can obtain the constant region gene sequences of TCRα and β chains through the published IMGT database.

The alpha and beta chains of a TCR are generally considered to have two "domains" each, a variable domain and a constant domain. A variable domain consists of linked variable and linker regions. Therefore, in the specification and claims of the present application, "TCRα chain variable domain" refers to the connected TRAV and TRAJ regions, and similarly, "TCRβ chain variable domain" refers to the connected TRBV and TRBD/TRBJ regions. The three CDRs of the variable domain of the TCRα chain are CDR1α, CDR2α and CDR3α; the three CDRs of the variable domain of the TCRβ chain are CDR1β, CDR2β and CDR3β. The framework sequence of the TCR variable domain of the present invention can be of murine or human origin, preferably of human origin. The constant domain of a TCR comprises an intracellular portion, a transmembrane region and an extracellular portion. In order to obtain a soluble TCR in order to determine the affinity between the TCR and the ILSPFLPLL-HLA A2 complex, the TCR of the invention preferably does not comprise a transmembrane region. More preferably, the amino acid sequence of TCR in the present invention refers to the extracellular amino acid sequence of TCR.

The α-chain amino acid sequence and β-chain amino acid sequence of the "wild-type TCR" in the present invention are SEQ ID NO: 51 and SEQ ID NO: 52, respectively, as shown in Figures 10a and 10b. The α-chain amino acid sequence and β-chain amino acid sequence of the "reference TCR" in the present invention are SEQ ID NO: 26 and SEQ ID NO: 27, respectively, as shown in Figures 7a and 7b. In the present invention, the amino acid sequences of the α and β chain variable domains of the wild-type TCR capable of binding to the ILSPFLPLL-HLA A2 complex are SEQ ID NO: 24 and SEQ ID NO: 25, respectively, as shown in Figures 6a and 6b. In the present invention, the terms "polypeptide of the present invention", "TCR of the present invention", and "T cell receptor of the present invention" are used interchangeably.

In another preferred example, the TCR comprises a TCRα chain variable domain and a TCRβ chain variable domain, and the TCRβ chain variable domain comprises CDR1β, CDR2β and CDR3β, wherein the CDR1β comprises the sequence: SGHVS.

In another preferred embodiment, CDR2β comprises the sequence: FQNEAQ.

In another preferred example, CDR3β comprises the sequence: ASSSDFGNQPQH.

In a preferred embodiment of the present invention, the T cell receptor (TCR) according to the present invention comprises a TCRα chain variable domain and a TCRβ chain variable domain, and the TCRα chain variable domain includes CDR1α, CDR2α, and CDR3α.

In another preferred example, the CDR1α comprises the sequence: DRGSQS.

In another preferred example, the CDR2α comprises the sequence: IYSNGD.

In another preferred example, the CDR3α comprises the sequence:

AV[3αX1][3αX2][3αX3][3αX4][3αX5]NMLT, wherein [3αX1], [3αX2], [3αX3], [3αX4], [3αX5] are independently selected from any natural amino acid residues.

In another preferred example, the [3αX1] is N, Q or A.

In another preferred example, the [3αX2] is L, D, E, G, S or H.

In another preferred example, the [3αX3] is Y, P, Q, S or G.

In another preferred example, the [3αX4] is A, S, T, W or D.

In another preferred example, the [3αX5] is G, R, K, M, Q, N, T, S or H.

In another preferred example, the CDR3α comprises a sequence selected from the following group:

AVNLYAGNMLT、AVQDPSRNMLT、AVADQSRNMLT、AVQDPSKNMLT、AVQDPSMNMLT、AVQDPSQNMLT、AVQDPTNNMLT、AVQDSSRNMLT、AVQDPAKNMLT、AVQEPSRNMLT、AVQDPTKNMLT、AVAGGWRNMLT、AVQSPDRNMLT、AVQHPATNMLT、AVADPSKNMLT、AVAHPSKNMLT、AVQSPDQNMLT、AVQDPASNMLT、AVQDPSHNMLT、AVQDPSTNMLT。

In another preferred example, the TCRα chain variable domain of the TCR does not contain the following CDRs at the same time:

CDR1α: DRGSQS; CDR2α: IYSNGD; and CDR3α: AVNLYAGNMLT.

Natural interchain disulfide bonds vs. artificial interchain disulfide bonds

There is a group of disulfide bonds between the Cα and Cβ chains in the near-membrane region of the natural TCR, which is called "natural inter-chain disulfide bonds" in the present invention. In the present invention, artificially introduced interchain covalent disulfide bonds whose positions are different from those of natural interchain disulfide bonds are called "artificial interchain disulfide bonds".

For the convenience of description, the position numbers of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in the present invention are numbered in sequence from N-terminal to C-terminal, such as in TRBC1*01 or TRBC2*01, according to the sequence from N The 60th amino acid in the sequence from end to C end is P (proline), then it can be described as Pro60 of TRBC1*01 or TRBC2*01 exon 1 in the present invention, and it can also be expressed as TRBC1* 01 or the 60th amino acid of exon 1 of TRBC2*01, and for example in TRBC1*01 or TRBC2*01, the 61st amino acid is Q (glutamine) in the order from N-terminal to C-terminal, then this In the invention, it can be described as Gln61 of exon 1 of TRBC1*01 or TRBC2*01, or as amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01, and so on. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. For example, if a certain amino acid in TRAV is numbered 46 in IMGT, it will be described as the 46th amino acid in TRAV in the present invention, and so on. In the present invention, if there are special instructions for the sequence position numbers of other amino acids, follow the special instructions.

the tumor

The term "neoplastic" is meant to include all types of cancer cell growth or oncogenic process, metastatic tissue or malignantly transformed cells, tissues or organs, regardless of pathological type or stage of invasion. Examples of tumors include, but are not limited to: solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include: malignant tumors of different organ systems, such as sarcomas, squamous cell carcinomas of the lung, and carcinomas. Examples: Infected prostate, lung, breast, lymphatic, gastrointestinal (eg colon), and genitourinary (eg kidney, epithelial), pharynx. Squamous cell carcinoma of the lung includes malignancies such as most colon, rectal, renal cell, liver, non-small cell carcinomas of the lung, small intestine, and esophagus. Metastatic lesions of the above cancers can also be treated and prevented using the methods and compositions of the present invention.

Detailed description of the invention

It is well known that the α-chain variable domain and β-chain variable domain of TCR each contain 3 CDRs, which are similar to the complementarity determining regions of antibodies. CDR3 interacts with antigenic short peptides, and CDR1 and CDR2 interact with HLA. Therefore, the CDR of the TCR molecule determines its interaction with the antigen short peptide-HLA complex. According to the present inventors, the amino acid sequence of the α-chain variable domain and the β-chain variable domain amino acid sequence of the wild-type TCR that can bind to the antigenic short peptide ILSPFLPLL and HLA A2 complex (ie, ILSPFLPLL-HLA A2 complex) are SEQ ID NO :24 and SEQ ID NO:25. It has the following CDR regions:

Alpha chain variable domain CDR CDR1α:DRGSQS

CDR2α: IYSNGD

CDR3α: AVNLYAGNMLT

and β chain variable domain CDR CDR1β: SGHVS

CDR2β: FQNEAQ

CDR3β:ASSSDFGNQPQH

The present invention obtains a high-affinity TCR whose affinity with the ILSPFLPLL-HLA A2 complex is at least 1.3 times that of the wild-type TCR and the ILSPFLPLL-HLA A2 complex by performing mutation screening on the above CDR region.

The present invention provides a T cell receptor (TCR), which has the activity of binding ILSPFLPLL-HLA A2 complex.

The T cell receptor comprises a TCRα chain variable domain and a TCRβ chain variable domain, the TCRα chain variable domain comprises 3 CDR regions, and the reference sequence of the 3 CDR regions of the TCRα chain variable domain is as follows,

CDR1α:DRGSQS

CDR2α: IYSNGD

CDR3α: AVNLYAGNMLT with at least one of the following mutations:

Residue before mutation Mutated residues N at position 3 of CDR3α Q or A Position 4 L of CDR3α D, E, G, S or H Y at position 5 of CDR3α P, Q, S or G Position 6A of CDR3α S, T, W or D G at position 7 of CDR3α R, K, M, Q, N, T, S or H

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

CDR1β:SGHVS

CDR2β: FQNEAQ

CDR3β:ASSSDFGNQPQH.

More specifically, the number of mutations in the CDR region of the TCRα chain is 1, 2, 3, 4, or 5.

Further, the TCR of the present invention is an αβ heterodimeric TCR, and the α chain variable domain of the TCR comprises at least 85%, preferably at least 90%, of the amino acid sequence shown in SEQ ID NO: 24; more preferably , at least 92%; most preferably, at least 94% (e.g., may be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% sequence homology) amino acid sequence of sequence homology); and/or the β chain variable domain of the TCR comprises at least 90% of the amino acid sequence shown in SEQ ID NO:25, preferably, at least 92%; more preferably, at least 94%; most preferably, at least 97%; (e.g., can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% % sequence homology) amino acid sequence of sequence homology.

Further, the TCR of the present invention is a single-chain TCR, and the α-chain variable domain of the TCR comprises at least 85%, preferably at least 90%, of the amino acid sequence shown in SEQ ID NO: 2; more preferably, at least 92%; most preferably, at least 94% (e.g., may be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% % sequence homology) amino acid sequence of sequence homology); and/or the beta chain variable domain of the TCR comprises at least 90%, preferably at least 92% of the amino acid sequence shown in SEQ ID NO:3 %; more preferably, at least 94%; most preferably, at least 97%; (e.g., may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence of sequence homology).

Preferably, the TCR comprises (i) all or part of the TCR alpha chain except its transmembrane domain, and (ii) all or part of the TCR beta chain except its transmembrane domain, wherein (i) and (ii) Both comprise the variable domain of the TCR chain and at least a portion of the constant domain.

In the present invention, the three CDRs of the wild-type TCRα chain variable domain SEQ ID NO: 24, namely CDR1, CDR2 and CDR3 are respectively located at positions 27-32, 50-55 and 90-100 of SEQ ID NO: 24 . Accordingly, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 24, 51Y is the second Y of CDR2α, 52S is the third S of CDR2α, 53N is the fourth N of CDR2α, and 54G is The 5th G of CDR2α, 55D is the 6th D of CDR2α, 91V is the 2nd V of CDR3α, 96G is the 7th G of CDR3α, 97N is the 8th N of CDR3α, 98M is The 9th M of CDR3α, 99L is the 10th L of CDR3α, and 100T is the 11th T of CDR3α.

Similarly, the three CDRs of the wild-type TCRβ chain variable domain SEQ ID NO: 25 in the present invention, that is, CDR1, CDR2 and CDR3 are respectively located at positions 27-31, 49-54 and 93 of SEQ ID NO: 25 -104 bits. Therefore, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:25, 27S is the first S of CDR1β, 28G is the second G of CDR1β, 29H is the third H of CDR1β, and 30V is The fourth V of CDR1β, 31S is the fifth S of CDR1β, 96S is the fourth S of CDR3β, 97D is the fifth D of CDR3β, 98F is the sixth F of CDR3β, and 99G is The 7th G of CDR3β, 100N is the 8th N of CDR3β, and 101Q is the 9th Q of CDR3β.

The present invention provides a TCR having the property of binding to the ILSPFLPLL-HLA A2 complex and comprising a TCR α chain variable domain and a TCR β chain variable domain, the TCR having a mutation in the α chain variable domain shown in SEQ ID NO: 24 , the mutated amino acid residue positions include one or more of 92N, 93L, 94Y, 95A or 96G, wherein the amino acid residue numbering adopts the numbering shown in SEQID NO:24.

In another preferred example, the mutated TCRα chain variable domain includes one or more amino acid residues selected from the group consisting of: 92Q or 92A; 93D, 93E, 93G, 93S or 93H; 94P, 94Q, 94S or 94G; 95S, 95T, 95W or 95D; 96R, 96K, 96M, 96Q, 96N, 96T, 96S or 96H; wherein, the amino acid residue numbering adopts the numbering shown in SEQ ID NO:24.

According to the method of site-directed mutagenesis well known to those skilled in the art, the Thr48 of the exon 1 of the TRAC*01 exon of the wild-type TCR α chain constant region was mutated to cysteine, and the β chain constant region TRBC1*01 or TRBC2*01 exon 1 was mutated. Ser57 was mutated to cysteine to obtain a reference TCR, the amino acid sequences of which are shown in Figures 7a and 7b, respectively, and the mutated cysteine residues are indicated in bold letters. The cysteine substitutions described above allow the formation of artificial interchain disulfide bonds between the constant regions of the α and β chains of the reference TCR to form a more stable soluble TCR, allowing for easier assessment of TCR complexation with ILSPFLPLL-HLA A2 binding affinity and/or binding half-life. It should be understood that the CDR region of the TCR variable region determines its affinity with the pMHC complex, therefore, the above-mentioned cysteine substitution in the TCR constant region will not affect the binding affinity and/or binding half-life of the TCR. Therefore, in the present invention, the measured binding affinity between the reference TCR and the ILSPFLPLL-HLA A2 complex is considered to be the binding affinity between the wild-type TCR and the ILSPFLPLL-HLA A2 complex. Likewise, if the binding affinity between the TCR of the present invention and the ILSPFLPLL-HLA A2 complex is determined to be at least 1.3 times the binding affinity between the reference TCR and the ILSPFLPLL-HLA A2 complex, it is equivalent to the TCR of the present invention and the ILSPFLPLL - The binding affinity between HLA A2 complexes is at least 1.3 times greater than the binding affinity between wild-type TCR and ILSPFLPLL-HLA A2 complexes.

Binding affinity (inversely proportional to the dissociation equilibrium constant _KD ) and binding half-life (expressed as T _1/2 ) can be determined by any suitable method. It will be appreciated that doubling the affinity of a TCR will result in a halving of the _KD . T _1/2 was calculated as In2 divided by the off-rate (K _off ). Therefore, doubling T _1/2 causes K _off to be halved. Preferably, the same assay protocol is used to detect the binding affinity or binding half-life of a given TCR several times, eg 3 times or more, and the results are averaged. In a preferred embodiment, these assays are performed using the Surface Plasmon Resonance (BIAcore) method described in the Examples herein. This method detects that the dissociation equilibrium constant K _D of the reference TCR to the ILSPFLPLL-HLA A2 complex is 3.4 μ M. In the present invention, it is considered that the dissociation equilibrium constant K _D of the wild-type TCR to the ILSPFLPLL-HLA A2 complex is also 3.4 μM. Since the doubling of the affinity of TCR will lead to the halving of _KD , if it is detected that the dissociation equilibrium constant _KD of high-affinity TCR for ILSPFLPLL-HLA A2 complex is 0.34 μM, it means that the high-affinity TCR for ILSPFLPLL-HLA A2 complex The affinity of TCR was 10 times higher than that of wild-type TCR for ILSPFLPLL-HLAA2 complex. Those skilled in the art are familiar with the conversion relationship between K _D value units, that is, 1 μM=1000 nM, 1 nM=1000 pM.

In a preferred example of the present invention, the affinity of the TCR to the ILSPFLPLL-HLA A2 complex is at least 1.3 times that of the wild-type TCR; preferably, at least 2 times; more preferably, at least 5 times.

In another preferred example, the affinity of the TCR to the ILSPFLPLL-HLA A2 complex is at least 10 times that of the wild-type TCR; preferably, at least 20 times.

In another preferred example, the dissociation equilibrium constant K _D of the TCR for the ILSPFLPLL-HLA A2 complex is ≤3.4 μM;

In another preferred example, the TCR dissociation equilibrium constant for the ILSPFLPLL-HLA A2 complex is 10nM≤K _D≤2.58 μM; preferably, 100nM≤K _D≤700nM .

Mutations may be performed using any suitable method, including but not limited to those based on polymerase chain reaction (PCR), restriction enzyme based cloning or ligation independent cloning (LIC) methods. These methods are detailed in many standard molecular biology textbooks. More details on polymerase chain reaction (PCR) mutagenesis and restriction enzyme based cloning can be found in Sambrook and Russell, (2001) Molecular Cloning - A Laboratory Manual (Third Edition) CSHL publishing house. More information on the LIC method can be found in (Rashtchian, (1995) Curr Opin Biotechnol 6(1):30-6).

The method for producing the TCR of the present invention may be, but not limited to, screening a TCR with high affinity for the ILSPFLPLL-HLA-A2 complex from a diversity library of phage particles displaying such TCR, as described in literature (Li, et al (2005) Nature Biotech 23(3):349-354).

It will be appreciated that genes expressing the variable domain amino acids of the alpha and beta chains of wild-type TCRs or genes expressing the amino acids of the variable domains of the alpha and beta chains of wild-type TCRs with slight modifications can be used to make template TCRs. The changes required to generate the high affinity TCRs of the invention are then introduced into the DNA encoding the variable domains of this template TCR.

In some preferred embodiments of the present invention, TCR of the present invention has one or more mutations in amino acid residues 92N, 93L, 94Y, 95A or 96G of the alpha chain variable domain shown in SEQ ID NO:24 ( Use the numbering shown in SEQ ID NO: 24). For example, the mutated TCRα chain variable domain includes one or more amino acid residues selected from the group consisting of: 92Q or 92A; 93D, 93E, 93G, 93S or 93H; 94P, 94Q, 94S or 94G; 95S, 95T, 95W, or 95D; 96R, 96K, 96M, 96Q, 96N, 96T, 96S, or 96H. More specifically, specific forms of the mutations in the α chain variable domain include N92Q/A, L93D/E/G/S/H, Y94P/Q/S/G, A95S/T/W/D or G96R/K One or several groups in /M/Q/N/T/S/H.

The high-affinity TCR of the present invention comprises an alpha chain variable domain amino acid sequence SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, One of 43, 44, 45, 46. Thus, a TCR beta chain comprising the amino acid sequence of the beta chain variable domain of a wild-type TCR (SEQ ID NO: 25) can be compared with a TCR comprising SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 , 38, 39, 40, 41, 42, 43, 44, 45, 46 TCRα chains combine to form heterodimeric TCR or single chain TCR molecules. In the present invention, the amino acid sequences of the α chain variable domain and the β chain variable domain forming the heterodimeric TCR molecule are preferably selected from the following table 1:

For the purposes of the present invention, a TCR of the present invention is a portion having at least one variable domain of a TCR alpha and/or TCR beta chain. They usually contain both TCRα chain variable domains and TCRβ chain variable domains. They may be αβ heterodimers or single-chain forms or any other forms that can exist stably. In adoptive immunotherapy, the full-length chain of the αβ heterodimeric TCR (including the cytoplasmic and transmembrane domains) can be transfected. The TCR of the present invention can be used as a targeting agent to deliver therapeutic agents to antigen-presenting cells or be combined with other molecules to prepare bifunctional polypeptides to target effector cells. In this case, the TCR is preferably in a soluble form.

In terms of stability, it is disclosed in the prior art that introducing an artificial interchain disulfide bond between the α and β chain constant domains of TCR can obtain a soluble and stable TCR molecule, as disclosed in the patent document PCT/CN2015/093806 stated. Thus, a TCR of the invention may be a TCR that incorporates an artificial interchain disulfide bond between residues in its alpha and beta chain constant domains. Cysteine residues form artificial interchain disulfide bonds between the alpha and beta chain constant domains of the TCR. Cysteine residues can be substituted for other amino acid residues at appropriate sites in native TCRs to form artificial interchain disulfide bonds. For example, substitution of Thr48 of exon 1 of TRAC*01 and substitution of Ser57 of exon 1 of TRBC1*01 or TRBC2*01 to form disulfide bonds. Other sites for introducing cysteine residues to form disulfide bonds can also be: Thr45 of TRAC*01 exon 1 and Ser77 of TRBC1*01 or TRBC2*01 exon 1; TRAC*01 exon Tyr10 of 1 and Ser17 of exon 1 of TRBC1*01 or TRBC2*01; Thr45 of exon 1 of TRAC*01 and Asp59 of exon 1 of TRBC1*01 or TRBC2*01; of exon 1 of TRAC*01 Ser15 and Glu15 of exon 1 of TRBC1*01 or TRBC2*01; Arg53 of exon 1 of TRAC*01 and Ser54 of exon 1 of TRBC1*01 or TRBC2*01; Pro89 of exon 1 of TRAC*01 and Ala19 of exon 1 of TRBC1*01 or TRBC2*01; or Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01. That is, cysteine residues replace any one group of positions in the above-mentioned α and β chain constant domains. A maximum of 15, or a maximum of 10, or a maximum of 8 or fewer amino acids may be truncated at the C-terminus of one or more of the TCR constant domains of the invention so that they do not include cysteine residues to achieve deletion of native The purpose of interchain disulfide bonds can also be achieved by mutating the cysteine residue that forms the natural interchain disulfide bond to another amino acid.

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

In addition, in terms of stability, the patent document PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond between the α-chain variable region and the β-chain constant region of TCR can significantly improve the stability of TCR. Therefore, the high-affinity TCR of the present invention may also contain an artificial interchain disulfide bond between the variable region of the α chain and the constant region of the β chain. Specifically, the cysteine residue that forms an artificial interchain disulfide bond between the α-chain variable region and the β-chain constant region of the TCR is substituted for: amino acid 46 of TRAV and TRBC1*01 or TRBC2* Amino acid 60 of exon 1 of 01; amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1*01 or TRBC2*01; amino acid 46 of TRAV and exon of TRBC1*01 or TRBC2*01 Amino acid 61 of exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01. Preferably, such a TCR may comprise (i) all or part of a TCR alpha chain except for its transmembrane domain, and (ii) all or part of a TCR beta chain except for its transmembrane domain, wherein (i) and (ii ) all contain the variable domain of the TCR chain and at least a part of the constant domain, and the α chain and the β chain form a heterodimer. More preferably, such a TCR may comprise an α-chain variable domain and a β-chain variable domain and all or part of a β-chain constant domain except the transmembrane domain, but it does not contain an α-chain constant domain, and the α-chain of said TCR Chain variable domains form heterodimers with beta chains.

In terms of stability, on the other hand, the TCR of the present invention also includes TCRs with mutations in its hydrophobic core region, and these mutations in the hydrophobic core region are preferably mutations that can improve the stability of the TCR of the present invention, as disclosed in Publication No. described in the patent literature of WO2014/206304. Such TCRs may have mutations at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable domain amino acids 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or Or the 3rd, 5th, and 7th positions from the bottom of the amino acid position of the α-chain J gene (TRAJ) short peptide, and/or the 2nd, 4th, and 6th position from the bottom of the amino acid position of the β-chain J gene (TRBJ) short peptide, where the position number of the amino acid sequence By position number as listed in the International Immunogenetics Information System (IMGT). Those skilled in the art are aware of the above-mentioned international immunogenetics information system, and can obtain the position numbers of the amino acid residues of different TCRs in IMGT according to the database.

More specifically, the TCR with mutations in the hydrophobic core region of the present invention may be a highly stable single-chain TCR composed of a flexible peptide chain connecting the variable domains of the α and β chains of the TCR. The CDR region of the TCR variable region determines its affinity with the short peptide-HLA complex. The mutation of the hydrophobic core can make the TCR more stable, but it will not affect its affinity with the short peptide-HLA complex. It should be noted that the flexible peptide chain in the present invention can be any peptide chain suitable for linking the variable domains of TCRα and β chains. The template strand used for screening high-affinity TCR constructed in Example 1 of the present invention is the above-mentioned highly stable single-chain TCR containing a hydrophobic core mutation. Using TCR with higher stability can more conveniently evaluate the affinity between TCR and ILSPFLPLL-HLA-A2 complex.

The CDR regions of the α-chain variable domain and the β-chain variable domain of the single-chain template TCR are completely identical to the CDR regions of the wild-type TCR. That is, the three CDRs of the variable domain of the α chain are CDR1α: DRGSQS, CDR2α: IYSNGD, CDR3α: AVNLYAGNMLT, and the three CDRs of the variable domain of the β chain are CDR1β: SGHVS, CDR2β: FQNEAQ, and CDR3β: ASSSDFGNQPQH. The amino acid sequence (SEQ ID NO: 1) and nucleotide sequence (SEQ ID NO: 47) of the single-stranded template TCR are shown in Figures 1a and 1b, respectively. In this way, a single-chain TCR composed of α-chain variable domain and β-chain variable domain with high affinity to ILSPFLPLL-HLA A2 complex was screened out.

In the present invention, the three CDRs of the single-chain template TCRα chain variable domain SEQ ID NO: 2, that is, CDR1, CDR2 and CDR3 are respectively located at positions 27-32, 50-55 and 90-100 of SEQ ID NO: 2 bit. Accordingly, the numbering of amino acid residues adopts the numbering shown in SEQID NO: 2, 51Y is the second Y of CDR2α, 52S is the third S of CDR2α, 53N is the fourth N of CDR2α, and 54G is The 5th G of CDR2α, 55D is the 6th D of CDR2α, 91V is the 2nd V of CDR3α, 96G is the 7th G of CDR3α, 97N is the 8th N of CDR3α, 98M is The 9th M of CDR3α, 99L is the 10th L of CDR3α, and 100T is the 11th T of CDR3α.

Similarly, the three CDRs of the single-chain template TCRβ chain variable domain SEQ ID NO: 3 in the present invention, that is, CDR1, CDR2 and CDR3 are respectively located at positions 27-31, 49-54 and 3 of SEQ ID NO: 3 93-104 bits. Therefore, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:3, 27S is the first S of CDR1β, 28G is the second G of CDR1β, 29H is the third H of CDR1β, and 30V is The fourth V of CDR1β, 31S is the fifth S of CDR1β, 96S is the fourth S of CDR3β, 97D is the fifth D of CDR3β, 98F is the sixth F of CDR3β, and 99G is The 7th G of CDR3β, 100N is the 8th N of CDR3β, and 101Q is the 9th Q of CDR3β.

The αβ heterodimer with high affinity for the ILSPFLPLL-HLA-A2 complex of the present invention is obtained by transferring the CDR regions of the α and β chain variable domains of the screened high-affinity single-chain TCR to the corresponding positions of the wild-type TCR α chain variable domain (SEQ ID NO: 24) and β chain variable domain (SEQ ID NO: 25).

In some embodiments of the present invention, using the numbering shown in SEQ ID NO: 24, amino acid residue 19A of the hydrophobic core of the α-chain variable domain of the TCR of the present invention (that is, the 19th position of the α-chain variable region listed in IMGT ), 21L (that is, the 21st position in the α-chain variable region listed in IMGT), 39Y (that is, the 39th position in the α-chain variable region listed in IMGT), 40S (that is, the α-chain variable region listed in IMGT One or more mutations in 79S (i.e., position 79 of the alpha chain variable region listed in IMGT) and 88L (i.e., position 88 of the alpha chain variable region listed in IMGT) and /or adopt the numbering shown in SEQ ID NO:25, the TCRβ chain variable domain hydrophobic core amino acid residue 11K (i.e. the 11th position of the β chain variable region listed in IMGT), 13A (i.e. listed in IMGT The 13th position in the β-chain variable region), 36Q (ie the 36th position in the β-chain variable region listed in IMGT), 38A (ie the 38th position in the β-chain variable region listed in IMGT), 39L (ie 39th of the beta-chain variable region listed in IMGT), 43P (ie, 43rd of the beta-chain variable region listed in IMGT), 82T (ie, 82nd of the beta-chain variable region listed in IMGT) , 84Q (ie, the 84th position of the β-chain variable region listed in IMGT), 91L (ie, the 91st position of the β-chain variable region listed in IMGT), and 113I (ie, the β-chain variable region listed in IMGT One or more mutations in position 113).

More specifically, in some preferred embodiments of the present invention, using the numbering shown in SEQ ID NO: 24, the hydrophobic core of the alpha chain variable domain of the present invention comprises amino acid residues 19V, 21I, 39D, 40P, 79V or 88F One or more of and/or using the numbering shown in SEQ ID NO: 25, said TCR variable domain hydrophobic core comprising amino acid residues 11L, 13V, 36R, 38D, 39P, 43L, 82V, 84P, 91F or 113V one or more of the . More specifically, the mutant form of the hydrophobic core of the TCRα variable domain includes one or more groups of A19V, L21I, Y39D, S40P, S79V or L88F; the mutant form of the hydrophobic core of the TCRβ variable domain includes K11L, A13V , Q36R, A38D, L39P, P43L, T82V, Q84P, L91F or I113V in one or more groups.

The high-affinity TCR of the present invention also comprises the amino acid sequence of the alpha chain variable domain SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , one of 20, 21, 22 and 23. Therefore, the above-mentioned highly stable single-chain TCRβ chain variable domain (SEQ ID NO: 3) as a template chain can be compared with the amino acid sequence of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 TCR alpha chain variable domains combine to form said single chain TCR molecule. In the present invention, the amino acid sequences of the α-chain variable domain and the β-chain variable domain of the high-affinity single-chain TCR molecule are preferably selected from the following table 2:

Table 2

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

The TCR of the present invention can be used alone, and can also be combined with a conjugate in a covalent or other manner, preferably in a covalent manner. The conjugate includes a detectable marker (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the ILSPFLPLL-HLA-A2 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or any of the above Combination of substances bound or coupled.

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

Therapeutic agents that can be combined or coupled with the TCR of the present invention include, but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews (Cancer metastasis reviews) 24, 539); 2. Biological toxicity (Chaudhary et al., 1989 , Nature (Nature) 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. Cytokines such as IL-2 etc. (Gillies et al., 1992, Proceedings of the National Academy of Sciences of the United States of America (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 (The Journal Of Immunology) 174, 4381); 5. Antibody scFv fragment (Zhu et al., 1995, International Journal of Cancer (International Journal of Cancer) 62,319); 6. Gold nanoparticles/nanorods ( Lapotko et al., 2005, Cancer letters (Cancer letters) 239, 36; Huang et al., 2006, Journal of the American Chemical Society (Journal of the American Chemical Society) 128, 2115); 7. Virus particles (Peng et al., 2004, Gene therapy ( Gene therapy) 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 9. Nanomagnetic particles; 10. Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)); 11. Chemotherapeutic agents (eg, cisplatin) or nanoparticles in any form, etc.

Antibodies or fragments thereof that bind to the TCR of the present invention include anti-T cells or NK-cell determination antibodies, such as anti-CD3 or anti-CD28 or anti-CD16 antibodies, and the combination of the above-mentioned antibodies or fragments thereof and TCR can carry out effects on effector cells. Orientation to better target target cells. A preferred embodiment is that the TCR of the present invention binds to an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody. Specifically, the fusion molecule of the TCR of the present invention and the anti-CD3 single chain antibody comprises the amino acid sequence of the variable domain of the TCR α chain selected from the group consisting of SEQ ID NO: 2, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 and the TCR β chain variable domain amino acid sequence shown in SEQ ID NO: 3; and specifically, the TCR and anti-CD3 of the present invention The fusion molecule of the single-chain antibody comprises the amino acid sequence of the variable domain of the TCRα chain selected from the group consisting of SEQ ID NO: 24, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45 or 46 and the amino acid sequence of the TCR beta chain variable domain shown in SEQ ID NO:25.

The invention also relates to nucleic acid molecules encoding the TCRs of the invention. A nucleic acid molecule of the invention may be in the form of DNA or RNA. DNA can be either the coding strand or the non-coding strand. For example, the nucleic acid sequence encoding the TCR of the present invention may be the same as the nucleic acid sequence shown in the figures of the present invention or a degenerate variant. Illustrate the meaning of "degenerate variant", as used herein, "degenerate variant" in the present invention refers to a protein sequence encoding a protein sequence having SEQ ID NO: 1, but having a sequence similar to that of SEQ ID NO: 47 different nucleic acid sequences.

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

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

The invention also includes isolated cells, particularly T cells, expressing a TCR of the invention. There are many methods suitable for transfection of T cells with DNA or RNA encoding the high affinity TCRs of the invention (eg, Robbins et al., (2008) J. Immunol. 180:6116-6131). T cells expressing the high-affinity TCR of the present invention can be used for adoptive immunotherapy. Many suitable methods for performing adoptive therapy are known to those skilled in the art (eg, Rosenberg et al., (2008) NatRev Cancer 8(4):299-308).

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

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

It should be understood that the names of amino acids in this article are identified by international single English letters, and the corresponding three-letter abbreviations of amino acid names are: Ala(A), Arg(R), Asn(N), Asp(D), Cys (C), Gln(Q), Glu(E), Gly(G), His(H), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro (P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V); in the present invention, Pro60 or 60P all represent the 60th proline. In addition, the expression of the specific form of the mutation in the present invention is such as "K52A" means that the K at the 52nd position is replaced by A, and similarly, "K52A/R/M/T" means that the K at the 52nd position is replaced by A or Substituted by R or by M or by T. Others and so on.

In this field, substitutions with amino acids with similar or similar properties usually do not change the function of the protein. The addition of one or several amino acids at the C-terminus and/or N-terminus usually does not alter the structure and function of the protein. Therefore, the TCR of the present invention also includes at most 5, preferably at most 3, more preferably at most 2, and most preferably 1 amino acid (especially the amino acid located outside the CDR region) of the TCR of the present invention, which are similar in nature or similar amino acid replacement, and still be able to maintain its functional TCR.

The present invention also includes slightly modified TCRs of the present invention. Modified (generally without altering the primary structure) forms include: chemically derivatized forms of the TCRs of the invention such as acetylation or carboxylation. Modifications also include glycosylation, such as those TCRs produced by glycosylation modifications during the synthesis and processing of the TCRs of the invention or during further processing steps. This modification can be accomplished by exposing the TCR to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are TCRs that have been modified to increase their resistance to proteolysis or to optimize solubility.

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

In addition, the TCR of the present invention can be used alone, or combined or coupled with other therapeutic agents (such as formulated in the same pharmaceutical composition).

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. The term refers to pharmaceutical carriers which do not, by themselves, induce the production of antibodies deleterious to the individual receiving the composition and which are not unduly toxic upon administration. These 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 carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

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

Typically, therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution, or suspension, in liquid carriers prior to injection can also be prepared.

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

When the pharmaceutical composition of the present invention is used for actual treatment, various dosage forms of the pharmaceutical composition can be used according to the usage conditions. Preferably, injections, oral preparations and the like can be exemplified.

These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, etc. (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in a conventional manner depending on the dosage form.

The pharmaceutical compositions of the present invention can also be administered in the form of sustained release formulations. For example, the TCRs of the present invention can be incorporated into pellets or microcapsules supported by slow-release polymers, which are then surgically implanted into the tissue to be treated. Examples of sustained-release polymers include ethylene-vinyl acetate copolymers, polyhydroxymethacrylate (polyhydrometaacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymers, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.

When the pharmaceutical composition of the present invention is used for actual treatment, the TCR or TCR complex of the present invention or the cells presenting the TCR of the present invention as the active ingredient can be selected according to the body weight, age, sex, and degree of symptoms of each patient to be treated. However, it should be reasonably determined, and finally the doctor will determine the reasonable dosage.

The main advantages of the present invention are:

(1) In the present invention, the TCR with high affinity for the ILSPFLPLL-HLA-A2 complex is screened out using the single-chain TCR molecule with a hydrophobic core mutation as a template.

(2) The affinity and/or binding half-life of the TCR of the present invention to the ILSPFLPLL-HLA-A2 complex is at least 1.3 times that of the wild-type TCR.

(3) The affinity and/or binding half-life of the high-affinity TCR of the present invention to the ILSPFLPLL-HLA-A2 complex can reach 1.3-24 times that of the wild-type TCR.

(4) The high-affinity TCR-transduced effector cell TCR-T of the present invention has a lower EC50 of T2 cell-loaded short peptide (ILSPFLPLL) than that of wild-type TCR-T, at least an order of magnitude lower.

(5) The high-affinity TCR-transduced effector cell TCR-T of the present invention has a better killing effect on liver cancer tumor cells expressing a specific antigen (ILSPFLPLL) than the wild-type TCR-T.

The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples, usually according to conventional conditions, for example (Sambrook and Russell et al., Molecular Cloning: Laboratory Manual (Molecular Cloning-A Laboratory Manual) (Third Edition) (2001) CSHL Press ), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Materials and methods

The experimental materials used in the examples of the present invention can be obtained from commercially available channels unless otherwise specified. Among them, E.coli DH5α was purchased from Tiangen, E.coli BL21 (DE3) was purchased from Tiangen, E.coli Tuner (DE3) was purchased from From Novagen, plasmid pET28a was purchased from Novagen.

Example 1 Generation of Stable Single-Strand TCR Template Strands for Hydrophobic Core Mutations

The present invention uses the method of site-directed mutagenesis, according to the patent document WO2014/206304, to construct a stable single-chain TCR molecule composed of a flexible short peptide (linker) connecting the TCRα and β chain variable domains, its amino acid and DNA The sequences are respectively SEQ ID NO:1 and SEQ ID NO:47, as shown in Figure 1a and Figure 1b. And use the single-chain TCR molecule as a template to screen for high-affinity TCR molecules. The amino acid sequences of the α variable domain (SEQ ID NO:2) and the β variable domain (SEQ ID NO:3) of the template chain are shown in Figures 2a and 2b; the corresponding DNA sequences are respectively SEQ ID NO:48 and 49, as shown in Figures 3a and 3b; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are SEQ ID NO: 4 and 50, respectively, as shown in Figures 4a and 4b.

The target gene carrying the template strand was digested with NcoI and NotI, and then ligated with the pET28a vector that had been digested with NcoI and NotI. The ligation product was transformed into E.coli DH5α, coated with kanamycin-containing LB plates, cultured upside down at 37°C overnight, and positive clones were picked for PCR screening, and the positive recombinants were sequenced, and the recombinant plasmid was extracted and transformed after confirming the correct sequence to E.coli BL21(DE3) for expression.

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

All the BL21(DE 3) colonies containing the recombinant plasmid pET28a-template strand prepared in Example 1 were inoculated in LB medium containing kanamycin, cultured at 37°C until the _OD600 was 0.6-0.8, and added IPTG until the final The concentration was 0.5mM, and culture was continued for 4h at 37°C. Centrifuge at 5000rpm for 15min to harvest the cell pellet, use Bugbuster Master Mix (Merck) to lyse the cell pellet, centrifuge at 6000rpm for 15min to recover inclusion bodies, then wash with Bugbuster (Merck) to remove cell debris and membrane components, centrifuge at 6000rpm for 15min to collect inclusion bodies body. The inclusion bodies were dissolved in the buffer solution (20mM Tris-HCl pH8.0, 8M urea), and the insoluble matter was removed by high-speed centrifugation. The supernatant was quantified by the BCA method, then aliquoted, and stored at -80°C for future use.

Add 2.5mL of buffer (6M Gua-HCl, 50mM Tris-HCl pH 8.1, 100mM NaCl, 10mM EDTA) to 5mg of dissolved single-chain TCR inclusion body protein, then add DTT to a final concentration of 10mM, and treat at 37°C for 30min . Add the above-mentioned single-chain TCR dropwise to 125mL refolding buffer (100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mMβ-mercapthoethylamine, 1.87mM Cystamine) with a syringe, Stir at 4°C for 10 minutes, then put the refolding solution into a dialysis bag with a cellulose membrane with a cutoff of 4kDa, place the dialysis bag in 1L of pre-cooled water, and stir slowly overnight at 4°C. After 17 hours, the dialysate was replaced with 1L pre-cooled buffer solution (20mM Tris-HCl pH 8.0), and the dialysis was continued at 4°C for 8h, and then the dialysate was replaced with the same fresh buffer solution to continue dialysis overnight. After 17 hours, the sample was filtered through a 0.45 μm filter membrane, vacuum degassed, and then passed through an anion exchange column (HiTrap Q HP, GE Healthcare) to purify the protein with a 0-1M NaCl linear gradient eluent prepared with 20 mM Tris-HCl pH 8.0, and collected The eluted fractions were analyzed by SDS-PAGE, and the fractions containing single-chain TCR were concentrated and further purified by gel filtration column (Superdex 7510/300, GE Healthcare), and the target fractions were also analyzed by SDS-PAGE.

The eluted fractions for BIAcore analysis were further tested for purity by gel filtration. The conditions are: chromatographic column Agilent Bio SEC-3 (300A, φ7.8×300mm), the mobile phase is 150mM phosphate buffer, the flow rate is 0.5mL/min, the column temperature is 25°C, and the ultraviolet detection wavelength is 214nm.

Example 3 Combination Characterization

BIAcore analysis

BIAcore T200 real-time analysis system was used to detect the binding activity of TCR molecule and ILSPFLPLL-HLA-A2 complex. Add the anti-streptavidin antibody (GenScript) to the coupling buffer (10mM sodium acetate buffer, pH 4.77), and then flow the antibody through the CM5 chip 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, and the coupling level was about 15,000 RU.

Let a low concentration of streptavidin flow over the surface of the antibody-coated chip, then flow the ILSPFLPLL-HLA-A2 complex through the detection channel, and the other channel as a reference channel, and then add 0.05 mM biotin at 10 μL/ The flow rate of min flows through the chip for 2 min to block the remaining binding sites of streptavidin. The affinity was determined by single-cycle kinetic analysis, and the TCR was diluted into several different concentrations with HEPES-EP buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% P20, pH 7.4) at a flow rate of 30μL/min , flowing over the surface of the chip in turn, the binding time of each injection is 120s, and it is allowed to dissociate for 600s after the last injection. The chip was regenerated with 10 mM Gly-HCl, pH 1.75, after each run. Kinetic parameters were calculated using BIAcore Evaluation software.

The preparation process of the above-mentioned ILSPFLPLL-HLA-A2 complex is as follows:

a.Purification

Collect 100ml of E.coli bacteria liquid induced to express heavy chain or light chain, centrifuge at 8000g at 4°C for 10min, wash the bacteria once with 10ml PBS, then resuspend the bacteria with 5ml BugBuster Master Mix Extraction Reagents (Merck) vigorously shake, and place in Rotate and incubate at room temperature for 20 min, then centrifuge at 6000 g for 15 min at 4 °C, discard the supernatant, and collect inclusion bodies.

Suspend the above inclusion bodies in 5ml of BugBuster Master Mix, incubate with rotation at room temperature for 5min; add 30ml of 10-fold diluted BugBuster, mix well, and centrifuge at 6000g at 4°C for 15min; discard the supernatant, add 30ml of 10-fold diluted BugBuster to resuspend the inclusion bodies , mix well, centrifuge at 6000g at 4°C for 15min, repeat twice, add 30ml 20mM Tris-HCl pH 8.0 to resuspend inclusion bodies, mix well, centrifuge at 6000g at 4°C for 15min, finally dissolve inclusion bodies with 20mM Tris-HCl 8M urea, SDS- The purity of inclusion bodies was detected by PAGE, and the concentration was measured by BCA kit.

b. Refolding

The synthetic short peptide ILSPFLPLL (Nanjing GenScript Biotechnology Co., Ltd.) was dissolved in DMSO to a concentration of 20 mg/ml. The inclusion bodies of the light chain and heavy chain were dissolved with 8M urea, 20mM Tris pH 8.0, 10mM DTT, and further denatured by adding 3M guanidine hydrochloride, 10mM sodium acetate, and 10mM EDTA before renaturation. Add ILSPFLPLL peptide at 25mg/L (final concentration) to refolding buffer (0.4M L-arginine, 100mM Tris pH 8.3, 2mM EDTA, 0.5mM oxidized glutathione, 5mM reduced glutathione, 0.2mM PMSF, cooled to 4°C), then sequentially add 20mg/L light chain and 90mg/L heavy chain (final concentration, heavy chain is added in three times, 8h/time), renaturation is carried out at 4°C for at least 3 days To completion, SDS-PAGE test whether the renaturation is successful.

c. Purification after renaturation

Replace the refolding buffer by dialysis against 10 volumes of 20 mM Tris pH 8.0, at least twice to sufficiently reduce the ionic strength of the solution. After dialysis, the protein solution was filtered through a 0.45 μm cellulose acetate filter and loaded onto a HiTrap Q HP (GE General Electric Company) anion exchange column (5 ml bed volume). Using Akta Purifier (GE General Electric Company), 0-400mM NaCl linear gradient solution prepared by 20mM Tris pH 8.0 was used to elute protein, and pMHC was eluted at about 250mM NaCl, and the peak components were collected, and the purity was detected by SDS-PAGE.

d. Biotinylation

Concentrate the purified pMHC molecules with Millipore ultrafiltration tubes, and at the same time replace the buffer with 20mM Tris pH8.0, then add biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50μM D-Biotin, 100μg/ml BirA enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and SDS-PAGE was used to detect whether the biotinylation was complete.

e. Purification of biotinylated complexes

The biotinylated pMHC molecules were concentrated to 1ml with Millipore ultrafiltration tubes, the biotinylated pMHC was purified by gel filtration chromatography, and HiPrep was pre-equilibrated with filtered PBS using an Akta purification instrument (GE General Electric Company). ^TM 16/60S200HR column (GE General Electric Company), loaded with 1 ml of concentrated biotinylated pMHC molecules, and then eluted with PBS at a flow rate of 1 ml/min. Biotinylated pMHC molecules elute as a single peak at about 55 ml. The protein-containing fractions were pooled, concentrated with Millipore ultrafiltration tubes, and the protein concentration was determined by the BCA method (Thermo). The biotinylated pMHC molecules were subpackaged and stored at -80°C by adding protease inhibitor cocktail (Roche).

Example 4 Generation of high-affinity single-chain TCR

Phage display technology is a means to generate a library of TCR high-affinity variants to screen for high-affinity variants. The TCR phage display and screening method described by Li et al. ((2005) Nature Biotech 23(3):349-354) was applied to the single-chain TCR template in Example 1. A library of high-affinity TCRs is created and panned by mutating the CDR regions of the template strand. Those skilled in the art can obtain the library construction and screening methods by reading the above documents. That is, by using primers with the desired change of one or more codons and a plasmid containing the relevant DNA as a template. After several rounds of panning, the phage library specifically binds to the corresponding antigen, and a single clone is picked from it for sequence analysis.

Using the BIAcore method in Example 3 to analyze the interaction between the TCR molecule and the ILSPFLPLL-HLA-A2 complex, the high-affinity TCR whose affinity and/or binding half-life is at least 2 times that of the wild-type TCR was screened, that is, the screened The dissociation equilibrium constant K _D of the high-affinity TCR binding ILSPFLPLL-HLA-A2 complex is less than or equal to half of the dissociation equilibrium constant K _D of the wild-type TCR binding ILSPFLPLL-HLA-A2 complex, the results are shown in Table 3 shown. The _KD value of the interaction between the reference TCR and the ILSPFLPLL-HLA-A2 complex detected by the above method is 3.4 μM, and the interaction curve is shown in Figure 9, that is, the wild-type TCR interacts with the ILSPFLPLL-HLA-A2 complex The _KD value of is also 3.4 μM.

Specifically, using the numbering shown in SEQ ID NO: 24, the α-chain variable domains of these high-affinity TCR mutants are mutated at one or more of the following amino acid positions: 92N, 93L, 94Y, 95A or 96G.

More specifically, using the numbering shown in SEQ ID NO: 24, the α-chain variable domains of these high-affinity TCRs comprise one or more amino acid residues selected from the group consisting of 92Q or 92A; 93D, 93E, 93G, 93S or 93H; 94P, 94Q, 94S or 94G; 95S, 95T, 95W or 95D; 96R, 96K, 96M, 96Q, 96N, 96T, 96S or 96H.

The alpha chain variable domain (SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, The specific amino acid sequences of 21, 22, 23, 24, 25 and 26) are shown in Figure 5a-s, respectively.

table 3

After testing, the affinity and/or binding half-life of the TCRα chain variable domain and the TCRβ chain variable domain in the above table are at least 2 times that of the wild-type TCR.

Example 5 Generation of High Affinity αβ Heterodimeric TCR

The mutation of the CDR region of the high-affinity single-chain TCR screened in Example 4 was introduced into the corresponding site of the variable domain of the αβ heterodimeric TCR, and its complex with ILSPFLPLL-HLA-A2 was detected by BIAcore affinity. The introduction of the above-mentioned high-affinity mutation point in the CDR region adopts the method of site-directed mutation well known to those skilled in the art. The amino acid sequences of the α-chain and β-chain variable domains of the above wild-type TCR are shown in Figure 6a (SEQ ID NO: 24) and 6b (SEQ ID NO: 25), respectively.

It should be noted that in order to obtain a more stable soluble TCR for easier assessment of the binding affinity and/or binding half-life between the TCR and the ILSPFLPLL-HLAA2 complex, the αβ heterodimeric TCR can be in the constant region of the α and β chains A cysteine residue was introduced in the TCR to form an artificial interchain disulfide bond. In this embodiment, the amino acid sequences of the TCRα and β chains are shown in Figure 7a (SEQ ID NO: 26) and shown in Figure 7b (SEQ ID NO: 27), the introduced cysteine residues are indicated in bold letters.

The extracellular sequence genes of the TCR α and β chains to be expressed were synthesized and inserted into expression vectors, respectively, by standard methods described in Molecular Cloning a Laboratory Manual (Third Edition, Sambrook and Russell) For pET28a+ (Novagene), the upstream and downstream cloning sites are NcoI and NotI, respectively. Mutations in the CDR regions were introduced by overlapping PCR (overlap PCR) well known to those skilled in the art. The insert was confirmed by sequencing.

Example 6 Expression, refolding and purification of αβ heterodimeric TCR

The expression vectors of the TCRα and β chains were transformed into the expressing bacteria BL21(DE3) by chemical transformation method respectively, the bacteria were grown in LB culture medium, induced with a final concentration of 0.5mM IPTG at OD ₆₀₀ =0.6, and the α and β chains of TCR were expressed The inclusion bodies formed after were extracted by BugBuster Mix (Novagene), and repeatedly washed with BugBuster solution, and the inclusion bodies were finally dissolved in 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA ), 20mM Tris (pH8.1).

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

Embodiment 7BIAcore analysis result

The method described in Example 3 was used to detect the affinity between the αβ heterodimeric TCR introduced into the high-affinity CDR region and the ILSPFLPLL-HLA-A2 complex.

The CDR regions selected from the high-affinity single-chain TCRα and β chains were transferred to the corresponding positions of the wild-type TCRα chain variable domain SEQ ID NO:24 and the β chain variable domain SEQ ID NO:25, respectively, to form αβ heterogeneity Dimeric TCR. The obtained amino acid sequence of the new TCRα variable domain is shown in Fig. 8a-s. Since the CDR region of the TCR molecule determines its affinity with the corresponding pMHC complex, those skilled in the art can expect that the αβ heterodimeric TCR introduced with a high-affinity mutation point also has a high affinity for the ILSPFLPLL-HLA-A2 complex . The method described in Example 5 was used to construct an expression vector, and the method described in Example 6 was used to express, anneal and purify the αβ heterodimeric TCR introduced with a high-affinity mutation, and then use BIAcore T200 to determine its relationship with ILSPFLPLL- The affinity of the HLA-A2 complex is shown in Table 4 below.

Table 4

It can be seen from Table 4 above that the αβ heterodimeric TCR introduced with the mutation point in the CDR region maintains a high affinity for the ILSPFLPLL-HLA-A2 complex. The affinity of the heterodimeric TCR is at least 1.3 times greater than the affinity of the wild-type TCR for the ILSPFLPLL-HLA-A2 complex.

Example 8 Functional experiment of effector cells transfected with high-affinity TCR of the present invention

This example verifies that the effector cells transfected with the high-affinity TCR of the present invention can be specifically activated by T2 cells loaded with specific short peptides. It cannot be specifically activated by T2 cells loaded with non-specific short peptides or empty.

The function and specificity of the high-affinity TCR of the present invention in cells are detected by ELISPOT experiment. Those skilled in the art are familiar with the method of using ELISPOT assay to detect cell function. In the IFN-γELISPOT experiment of this example, PBL cells isolated from the blood of healthy volunteers were transfected with TCR with lentivirus as effector cells (respectively labeled TCR1, TCR2, TCR3, TCR4, TCR5, TCR6, TCR7, TCR8, TCR9, TCR10, TCR11, TCR12, TCR13, TCR14, TCR15, TCR16, TCR17, TCR18, TCR19), the control effector cells are marked as A6 (transfected with HTLV-1Tax TCR), and the wild-type effector cells are marked as TCRWT. The experimental group was T2-loaded antigen peptide pHBs (A0201HBs:ILSPFLPLL), in which the short peptide concentration gradient was 10 ^-13 M, 10 ^-12 M, 10 ^-11 M, 10 ^-10 M, 10 ^-9 M, 10 ^-8 M , 10 ^-7 M, 10 ^-6 M. The control group was T2 empty or loaded antigen peptide pNY-ESO-1 (A0201NY-ES0-1:SLLMWITQC), in which the short peptide concentration gradient was 10 ^-12 M, 10 ^-11 M, 10 ^-10 M, 10 ^-9 M, 10 ^-8 M, 10 ^-7 M, 10 ^-6 M.

First prepare the ELISPOT plate. ELISPOT plates were activated with ethanol and coated overnight at 4°C. On the first day of the experiment, remove the coating solution, wash and block, incubate at room temperature for two hours, remove the blocking solution, and add each component of the test to the ELISPOT plate in the following order: Adjust the effector cells to 2 X ¹⁰ cells/ ml, adjust the T2 cells to 4 X 10 ⁵ cells/ml in the medium, and adjust the concentration of short peptides in the medium to 4 X 10 ^-13 M, 4 X 10 ^-12 M, 4 X 10 ^-11 M, 4 X 10 ^{- 10M} , ^4X10-9M , ^4X10-8M , ^4X10-7M , ^4X10-6M . After mixing evenly, take 50 μL of short peptides (where T2 is empty, replace it with 50 μL of medium), 50 μL of T2 cells 4 X ¹⁰⁵ cells/ml (20,000 cells/well), 100 μL of effector cells 2 X10 ⁴ Add cells/ml (ie 2,000 cells/well) into corresponding wells, and set up two duplicate wells. Incubate overnight (37°C, 5% CO2). On the second day of the experiment, the plate was washed for secondary detection and color development, the plate was dried, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID20 company). The experimental results are shown in Figure 11a-h, which sequentially display the specific short peptide pHBs (A0201HBs: ILSPFLPLL) and IFN-γ release points of non-specific short peptide pNY-ESO-1 (A0201NY-ES0-1:SLLMWITQC) loaded with concentration gradient at T2. The experimental results are shown in Figure 12a-h, showing the number of IFN-γ release points and T2 loading of high-affinity effector cells TCR6, TCR10, TCR11, TCR12, TCR14, TCR15, TCR17, and TCR19 in turn when T2 is empty (T2 & non-peptide) The release points of IFN-γ at the concentration of 10 ^-6 M specific short peptide pHBs (A0201HBs:ILSPFLPLL) (T2&HBs-peptide or T2&pHBs). The effector cells transfected with the high-affinity TCR of the present invention have a good specific activation effect.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences

<120> High affinity HBs T cell receptor

<130> P2018-0072

<160> 53

<170> PatentIn version 3.5

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

1 5 10 15

Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Asn Leu Tyr Ala Gly

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro Gly

100 105 110

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

115 120 125

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

130 135 140

Tyr Leu Val Val Lys Arg Gly Gln Asp Val Ala Leu Arg Cys Asp Pro

145 150 155 160

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

165 170 175

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

180 185 190

Ser Gly Leu Pro Ser Asp Arg Phe Phe Ala Glu Arg Pro Glu Gly Ser

195 200 205

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

210 215 220

Tyr Phe Cys Ala Ser Ser Ser Asp Phe Gly Asn Gln Pro Gln His Phe

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Gly Asp Gly Thr Arg Leu Ser Val Leu

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

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Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

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

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Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

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

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Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Asn Leu Tyr Ala Gly

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Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

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

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

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

35 40 45

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

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

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

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Asp Phe Gly Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser

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Val Leu

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

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Gly Gly Ser Glu Gly Gly Thr Gly

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

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Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

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

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Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

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

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Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Gln Asp Pro Ser Arg

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Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

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

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Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Ala Asp Gln Ser Arg

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Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

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

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Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

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

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Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

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

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Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Gln Asp Pro Ser Lys

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Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

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

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Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

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Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Gln Asp Pro Ser Met

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Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

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

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Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

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Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

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

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Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

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

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Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

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

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Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Gln Asp Pro Thr Asn

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Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

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

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Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Gln Asp Ser Ser Arg

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

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

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Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Gln Asp Pro Ala Lys

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

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

1 5 10 15

Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

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

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 14

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

1 5 10 15

Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Gln Asp Pro Thr Lys

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

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

1 5 10 15

Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

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

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

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

1 5 10 15

Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Gln Ser Pro Asp Arg

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 17

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

1 5 10 15

Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Gln His Pro Ala Thr

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 18

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

1 5 10 15

Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

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

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 19

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

1 5 10 15

Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Ala His Pro Ser Lys

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 20

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

1 5 10 15

Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

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

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 21

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

1 5 10 15

Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

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

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 22

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

1 5 10 15

Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Phe Cys Ala Val Gln Asp Pro Ser His

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 23

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

1 5 10 15

Ala Ile Val Ser Ile Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser

20 25 30

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

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

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

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 24

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Leu Tyr Ala Gly

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 25

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Phe Gly Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser

100 105 110

Ile Leu

<210> 26

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Leu Tyr Ala Gly

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro His

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile

180 185 190

Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200 205

<210> 27

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<400> 27

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Phe Gly Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Arg Ala Asp

<210> 28

<211> 111

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<400> 28

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Asp Pro Ser Arg

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 29

<211> 111

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<400> 29

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Ala Asp Gln Ser Arg

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 30

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 30

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Asp Pro Ser Lys

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 31

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 31

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Asp Pro Ser Met

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 32

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 32

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Asp Pro Ser Gln

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 33

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 33

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Asp Pro Thr Asn

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 34

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 34

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Asp Ser Ser Arg

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 35

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 35

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Asp Pro Ala Lys

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 36

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 36

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

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

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 37

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 37

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Asp Pro Thr Lys

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 38

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 38

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

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

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 39

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 39

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Ser Pro Asp Arg

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 40

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 40

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln His Pro Ala Thr

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 41

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 41

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Ala Asp Pro Ser Lys

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 42

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 42

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Ala His Pro Ser Lys

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 43

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 43

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Ser Pro Asp Gln

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 44

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 44

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Asp Pro Ala Ser

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 45

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 45

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Asp Pro Ser His

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 46

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 46

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Asp Pro Ser Thr

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro

100 105 110

<210> 47

<211> 747

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 47

cagaaagagg tggaacaaaa cagcggtccg ctgagcgtgc cggagggtgc gatcgttagc 60

attaactgca cctacagcga ccgtggcagc cagagcttct tttggtatcg tcaagatccg 120

ggtaaaagcc cggagctgat catgtttat tacagcaacg gcgacaagga agatggtcgt 180

ttcaccgcgc agctgaacaa agcgagccaa tatgtgagcc tgctgatccg tgacgttcag 240

ccgagcgata gcgcgaccta cttttgcgcg gtgaacctgt atgcgggcaa catgctgacc 300

ttcggtggcg gtacccgtct gatggttaag ccgggcggtg gcagcgaggg tggcggtagc 360

gaaggcggtg gcagcgaggg tggcggtagc gaaggcggta ccggcggtgc gggtgttagc 420

cagagcccgc gttacctggt ggttaaacgt ggccaagacg tggcgctgcg ttgcgatccg 480

atcagcggtc acgttagcct gttttggtac cgtcaggacc cgggccaagg tctggagttc 540

ctgacctatt ttcagaacga agcgcaactg gacaagagcg gcctgccgag cgatcgtttc 600

tttgcggagc gtccggaagg tagcgtgagc accctgaaaa ttcagcgtgt gcaaccggaa 660

gatagcgcgg tttatttctg cgcgagcagc agcgactttg gtaaccagcc gcaacacttc 720

ggcgatggta cccgtctgag cgttctg 747

<210> 48

<211> 333

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 48

cagaaagagg tggaacaaaa cagcggtccg ctgagcgtgc cggagggtgc gatcgttagc 60

attaactgca cctacagcga ccgtggcagc cagagcttct tttggtatcg tcaagatccg 120

ggtaaaagcc cggagctgat catgtttat tacagcaacg gcgacaagga agatggtcgt 180

ttcaccgcgc agctgaacaa agcgagccaa tatgtgagcc tgctgatccg tgacgttcag 240

ccgagcgata gcgcgaccta cttttgcgcg gtgaacctgt atgcgggcaa catgctgacc 300

ttcggtggcg gtacccgtct gatggttaag ccg 333

<210> 49

<211> 342

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 49

ggtgcgggtg ttagccagag cccgcgttac ctggtggtta aacgtggcca agacgtggcg 60

ctgcgttgcg atccgatcag cggtcacgtt agcctgtttt ggtaccgtca ggacccgggc 120

caaggtctgg agttcctgac ctattttcag aacgaagcgc aactggacaa gagcggcctg 180

ccgagcgatc gtttctttgc ggagcgtccg gaaggtagcg tgagcaccct gaaaattcag 240

cgtgtgcaac cggaagatag cgcggtttat ttctgcgcga gcagcagcga ctttggtaac 300

cagccgcaac acttcggcga tggtacccgt ctgagcgttc tg 342

<210> 50

<211> 72

<212>DNA

<213> Artificial sequence (Artificial sequence)

<400> 50

ggcggtggca gcgaggtgg cggtagcgaa ggcggtggca gcgagggtgg cggtagcgaa 60

ggcggtaccg gc 72

<210> 51

<211> 205

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 51

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

1 5 10 15

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

20 25 30

Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met

35 40 45

Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

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

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Leu Tyr Ala Gly

85 90 95

Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro His

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile

180 185 190

Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200 205

<210> 52

<211> 244

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 52

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Phe Gly Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Arg Ala Asp

<210> 53

<211> 9

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 53

Ile Leu Ser Pro Phe Leu Pro Leu Leu

1 5

Claims (22)

1. A T cell receptor (TCR), characterized in that it has the activity of binding ILSPFLPLL-HLA A2 complex, and the T cell receptor comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, said The TCRα chain variable domain contains 3 CDR regions, and the reference sequence of the 3 CDR regions of the TCRα chain variable domain is as follows, CDR1α:DRGSQS CDR2α: IYSNGD CDR3α: AVNLYAGNMLT with at least one of the following mutations:

And/or, the TCRβ chain variable domain comprises 3 CDR regions, and the reference sequence of the 3 CDR regions of the TCRβ chain variable domain is as follows, CDR1β:SGHVS CDR2β: FQNEAQ CDR3β:ASSSDFGNQPQH; The TCR has a CDR selected from the group consisting of:

. 2. The T cell receptor (TCR) according to claim 1, wherein the affinity of the TCR to the ILSPFLPLL-HLA A2 complex is at least 1.3 times that of the wild-type TCR. 3. The T cell receptor (TCR) according to claim 2, wherein the affinity of the TCR to the ILSPFLPLL-HLA A2 complex is at least 2 times that of the wild-type TCR. 4. The T cell receptor (TCR) according to claim 3, wherein the affinity of the TCR to the ILSPFLPLL-HLA A2 complex is at least 5 times that of the wild-type TCR. 5. The T cell receptor (TCR) of claim 1, wherein said TCR is soluble. 6. The T cell receptor (TCR) according to claim 1, wherein the TCR is an αβ heterodimeric TCR or a single-chain TCR. 7. The T cell receptor (TCR) of claim 1, wherein said TCR comprises (i) all or part of the TCRα chain except for its transmembrane domain, and (ii) except for its transmembrane All or part of the TCRβ chain other than the structural domain, wherein (i) and (ii) both comprise the variable domain and at least a part of the constant domain of the TCR chain. 8. The T cell receptor (TCR) of claim 1, wherein the TCR is an αβ heterodimeric TCR comprising (i) all or part of the TCRα chain except its transmembrane domain , and (ii) all or part of the TCR beta chain except its transmembrane domain, where (i) and (ii) both comprise the variable domain and at least a portion of the constant domain of the TCR chain, the alpha chain constant region and the beta chain constant region contain artificial interchain disulfide bonds. 9. The T cell receptor (TCR) according to claim 1, wherein the TCR is a single-chain TCR composed of an α-chain variable domain and a β-chain variable domain, and the α-chain variable domain And the variable domain of the β chain is connected by a flexible short peptide sequence (linker). 10. The T cell receptor (TCR) according to claim 1, wherein the amino acid sequence of the α chain variable domain of the TCR is selected from the group consisting of: SEQ ID NO: 28-35, 37-38, 40-42 , 44-46, or SEQ ID NO: 5-12, 14-15, 17-19, 21-23. 11. The T cell receptor (TCR) according to claim 1, characterized in that the C- or N-terminus of the α-chain and/or β-chain of the TCR is bound with a conjugate. 12 . The T cell receptor (TCR) according to claim 11 , wherein the conjugate that binds to the TCR is a detectable marker, a therapeutic agent, a PK modifying moiety or any combination of these substances. 13. The T cell receptor (TCR) according to claim 12, wherein the therapeutic agent that binds to the TCR is an anti- CD3 antibody. 14. A T cell receptor (TCR), characterized in that the TCR is selected from the group consisting of:

Or, the TCR is selected from the group:

15. A multivalent TCR complex, characterized in that it comprises at least two TCR molecules, and at least one of the TCR molecules is the TCR according to any one of the preceding claims. 16. A nucleic acid molecule, characterized in that the nucleic acid molecule comprises a nucleic acid sequence encoding the TCR according to any one of claims 1 or 15 or a complementary sequence thereof. 17. A vector, characterized in that the vector contains the nucleic acid molecule as claimed in claim 16. 18. A host cell, characterized in that the host cell contains the vector according to claim 17 or the exogenous nucleic acid molecule according to claim 16 is integrated in the chromosome. 19. An isolated cell, characterized in that the cell expresses the TCR according to any one of claims 1 or 15. 20. A pharmaceutical composition, characterized in that the composition contains a pharmaceutically acceptable carrier and the TCR as claimed in claim 1, or the TCR complex as claimed in claim 15, or the TCR complex as claimed in claim 19. the aforementioned cells. 21. The use of the cell according to claim 19, characterized in that it is used to prepare a drug for treating tumor, the tumor is a tumor expressing ILSPFLPLL-HLA A2 complex, and the cell is a T cell. 22. A method for preparing the T cell receptor according to any one of claims 1 or 15, comprising the steps of: (i) culturing the host cell of claim 18 so as to express the T cell receptor of any one of claims 1 or 15; (ii) isolating or purifying the T cell receptor.

Images