CN115819555A - High-affinity TCR for identifying SSX2 - Google Patents

Abstract

The present invention provides a high affinity TCR that recognizes SSX2, which has the property of binding to the AQIPEKIQK-HLA a1101 complex. The invention also provides multivalent TCR complexes, nucleic acid molecules encoding such TCRs, vectors comprising these nucleic acids, cells expressing such TCRs, and pharmaceutical compositions comprising the foregoing, which are useful for diagnosing, treating, and preventing SSX 2-positive diseases. The invention also provides methods of making such TCRs.

Description

A high-affinity TCR that recognizes SSX2

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 a polypeptide derived from SSX2 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.

SSX2 is the synovial sarcoma X breakpoint, also known as HOM-MEL-40. SSX2 is one of ten highly homologous nucleic acid proteins of the SSX family. The SSX protein is a tumor-testis antigen expressed only in tumor cells and testicular germ cells without MHC expression. SSX2 is expressed in a variety of human cancer cells, including but not limited to, liver cancer, lung cancer, fibrosarcoma, breast cancer, colon cancer, prostate cancer. After SSX2 is produced in cells, it is degraded into small molecular polypeptides, and combines with MHC (major histocompatibility complex) molecules to form a complex, which is presented to the cell surface. AQIPEKIQK is a short peptide derived from SSX2 antigen.

Thus, the AQIPEKIQK-HLA A1101 complex provides a TCR-targetable marker for tumor cells. The TCR that can bind to the AQIPEKIQK-HLA A1101 complex has high application value in 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 high affinity for AQIPEKIQK-HLA A1101 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.

A first aspect of the present invention provides a T cell receptor (TCR) comprising an α chain variable domain and a β chain variable domain, which has the activity of binding to the AQIPEKIQK-HLA A1101 complex, and the TCR α chain The amino acid sequence of the variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:1 and the amino acid sequence of the TCR β chain variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:2 90% sequence identity.

In a preferred example, the amino acid sequence of the variable domain of the TCRα chain and the amino acid sequence of the variable domain of the TCRβ chain are different from the amino acid sequence of the variable domain of the wild-type TCRα chain and the amino acid sequence of the variable domain of the wild-type TCRβ chain sequence.

In a further preferred example, the amino acid sequence of the TCRα chain variable domain is not the amino acid sequence shown in SEQ ID NO:1, or the amino acid sequence of the TCRβ chain variable domain is not the amino acid sequence shown in SEQ ID NO:2 sequence.

In another preferred example, the α-chain variable domain of the TCR comprises at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98% or 99% sequence homology. Or relative to the sequence shown in SEQ ID NO: 1, there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid residues inserted, deleted, replaced or a combination thereof.

In another preferred example, the β chain variable domain of the TCR is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences with 98%, 99% or 100% sequence homology. Or relative to the sequence shown in SEQ ID NO: 1, there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid residues inserted, deleted, replaced or a combination thereof.

In another preferred example, the amino acid sequence of the variable domain of the TCRα chain has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence of the variable domain of the TCRβ chain is identical to that of SEQ ID NO:1. The amino acid sequence shown in ID NO:2 has at least 95% sequence homology.

In another preferred example, the TCR is mutated in CDR3α of the variable domain of the TCRα chain, and the number of mutations is 1-4, preferably 3 or 4.

In another preferred example, the three CDRs of the TCRβ chain variable domain are: CDR1β: SGHVS; CDR2β: FQNEAQ; and CDR3β: ASSLRAGGNTIY; preferably, the amino acid sequence of the TCRβ chain variable domain is SEQ ID NO :2.

In another preferred example, the CDR1α in the variable domain of the TCRα chain is SSYSPS, the CDR2α is YTSAATLV, and the CDR3α is selected from ALTLGNTPLV, GMTLGNTPLV and AITLGNTPLV.

In another preferred example, the amino acid mutation sites of the TCRα chain variable domain are positions 1, 2, 3 and 4 of CDR3α.

In another preferred example, the amino acid mutation sites of the TCRα chain variable domain are the 2nd, 3rd and 4th positions of CDR3α.

In another preferred example, the reference sequences of the three CDR regions (complementarity determining regions) of the variable domain of the TCRα chain are as follows,

CDR1α:SSYSPS

CDR2α: YTSAATLV

CDR3α: GGSIGNTPLV, and contains at least one of the following mutations:

Residue before mutation Mutated residues Position 4 I of CDR3α L or P or V or N S at position 3 of CDR3α T or H G at position 2 of CDR3α L or M or I or S G at position 1 of CDR3α A S at position 2 of CDR1α V or T or I S at position 4 of CDR1α T or D S at position 6 of CDR1α W or Y

In another preferred example, the amino acid mutation in CDR3α comprises:

Residue before mutation Mutated residues Position 4 I of CDR3α L

In another preferred example, the amino acid mutation in CDR3α comprises:

Residue before mutation Mutated residues S at position 3 of CDR3α T

Preferably, the amino acid mutation in the CDR3α comprises:

Residue before mutation Mutated residues G at position 1 of CDR3α A

In another preferred example, the amino acid mutation in CDR3α comprises:

Residue before mutation Mutated residues S at position 3 of CDR3α T Position 4 I of CDR3α L

In another preferred example, the reference sequences of the three CDR regions (complementarity determining regions) of the TCRβ chain variable domain are as follows,

CDR1β:SGHVS

CDR2β: FQNEAQ

CDR3β: ASSLRAGGNTIY, and CDR3β contains at least one of the following mutations:

In another preferred example, the affinity between the TCR and the AQIPEKIQK-HLA A1101 complex is at least 5 times that of the wild-type TCR.

In another preferred example, the TCR is mutated in the α chain variable domain shown in SEQ ID NO: 1, and the mutation is selected from I95L/P/V/N, S94T/H, G93L/M/I One or several groups of /S, G92A, S28V/T/I, S30T/D, S32W/Y, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:1.

In another preferred example, the TCR is mutated in the β chain variable domain shown in SEQ ID NO: 2, and the mutation is selected from S95Q, L96A/E/P, R97Y/F/H/W, A98P , G100S, N101S/T, T102D, I 103F, Y104T/H one or more groups, wherein the numbering of amino acid residues adopts the numbering shown in SEQ ID NO:2.

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

In another preferred embodiment, the TCR is soluble.

In another preferred example, the TCR is an αβ heterodimeric TCR, comprising a TRAC constant region sequence of the α chain and a TRBC1 or TRBC2 constant region sequence of the β chain.

In another preferred example, the TCR comprises (i) the TCRα chain variable domain and all or part of the TCRα chain constant region except its transmembrane domain; and (ii) the TCRβ chain variable domain and its transmembrane domain All or part of the constant region of the TCRβ chain outside the domain.

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

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.

In another preferred example, the amino acid sequence of the α-chain variable domain of the TCR is one of SEQ ID NO: 1, 13-30; and/or the amino acid sequence of the β-chain variable domain of the TCR is SEQ ID NO: 2. One of 31-42.

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

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

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

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 TCR comprises an α-chain constant region and a β-chain constant region, the α-chain constant region is a mouse constant region and/or the β-chain constant region is a mouse constant region.

In another preferred embodiment, the TCR comprises an α-chain constant region and a β-chain constant region, the α-chain constant region is a murine constant region and/or the β-chain constant region is a murine constant region.

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, preferably, the conjugate is a detectable marker or a therapeutic agent.

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, which comprises 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, preferably, the isolated cell is a T cell, NK cell or NKT cell, most preferably , the isolated cells are T cells.

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 In the cell according to the sixth aspect of the invention, or the pharmaceutical composition according to the seventh aspect of the invention, preferably, the disease is a SSX2-positive tumor.

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 Preferably, the disease is a SSX2 positive tumor.

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 respectively show the amino acid sequences of the variable domains of the wild-type TCR α chain and β chain that can specifically bind to the AQIPEKIQK-HLA A1101 complex.

Figure 2a and Figure 2b respectively show the amino acid sequence of the α-chain variable domain and the amino acid sequence of the β-chain variable domain of the single-chain template TCR constructed in the present invention.

Figure 3a and Figure 3b are the DNA sequences of the α-chain variable domain and the β-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 DNA sequence of the linker (linker) of the single-chain template TCR constructed in the present invention, respectively.

Figure 5a and Figure 5b are the amino acid sequence and DNA sequence of the single-stranded template TCR constructed in the present invention, respectively.

Figure 6a and Figure 6b are the amino acid sequences of the soluble reference TCR α chain and β chain in the present invention, respectively.

Figures 7(1)-(18) respectively show the amino acid sequences of the α-chain variable domains of heterodimeric TCRs with high affinity for the AQIPEKIQK-HLA A1101 complex, and the mutated residues are underlined.

Figures 8(1)-(12) respectively show the amino acid sequences of the β-chain variable domains of heterodimeric TCRs with high affinity for the AQIPEKIQK-HLA A1101 complex, and the mutated residues are underlined.

Figure 9a and Figure 9b respectively show the extracellular amino acid sequences of the wild-type TCR α chain and β chain that can specifically bind to the AQIPEKIQK-HLA A1101 complex.

Figure 10a and Figure 10b respectively show the amino acid sequences of the wild-type TCR α chain and β chain that can specifically bind to the AQIPEKIQK-HLA A1101 complex.

Fig. 11 is the binding curve of the soluble reference TCR, that is, the wild-type TCR, and the AQIPEKIQK-HLA A1101 complex.

Figure 12 is the binding curve of soluble single-chain TCR and AQIPEKIQK-HLA A1101 complex.

Fig. 13 is the experimental result of the activation function of the effector cells transfected with the high-affinity TCR of the present invention for T2 cells loaded with short peptides.

Fig. 14a, Fig. 14b and Fig. 14c are the experimental results of the activation function of the effector cells transfected with the high-affinity TCR of the present invention for tumor cell lines.

15a and 15b are the results of killing function LDH experiments on tumor cell lines and effector cells transfected with high-affinity TCR of the present invention.

Detailed ways

Through extensive and in-depth research, the present invention obtains a high-affinity T cell receptor (TCR) that recognizes AQIPEKIQK short peptide (derived from SSX2 protein), and the AQIPEKIQK short peptide is synthesized in the form of peptide-HLA A1101 complex present. The high-affinity TCR has three CDR regions in its α-chain variable domain:

CDR1α:SSYSPS

CDR2α: YTSAATLV

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

CDR1β:SGHVS

CDR2β: FQNEAQ

CDR3β: mutated in ASSLRAGGNTIY; and, after the mutation, the affinity and/or binding half-life of the TCR of the present invention to the above-mentioned AQIPEKIQK-HLAA1101 complex is at least 5 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 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 AQIPEKIQK-HLA A1101 complex are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, as shown in Figure 1a and Figure 1b. The α-chain amino acid sequence and β-chain amino acid sequence of the soluble "reference TCR" in the present invention are SEQ ID NO: 11 and SEQ ID NO: 12, respectively, as shown in Figure 6a and Figure 6b. The extracellular amino acid sequence of the α chain and the extracellular amino acid sequence of the β chain of the "wild-type TCR" described in the present invention are SEQ ID NO: 43 and SEQ ID NO: 44, respectively, as shown in Fig. 9a and Fig. 9b. The TCR sequences used in the present invention are of human origin. 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:45 and SEQ ID NO:46, respectively, as shown in Figures 10a and 10b. 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.

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. The amino acid sequence of the α-chain variable domain and the amino acid sequence of the β-chain variable domain of the wild-type TCR capable of binding the antigenic short peptide AQIPEKIQK and the HLA A1101 complex (that is, the AQIPEKIQK-HLA A1101 complex) are SEQ ID NO: 1 and SEQ ID NO: 1 and SEQ ID NO: 1, respectively. ID NO: 2, this sequence was discovered by the inventors for the first time. It has the following CDR regions:

Alpha chain variable domain CDR CDR1α: SSYSPS

CDR2α: YTSAATLV

CDR3α: GGSIGNTPLV

and β chain variable domain CDR CDR1β: SGHVS

CDR2β: FQNEAQ

CDR3β: ASSLRAGGNTIY

The present invention obtains a high-affinity TCR whose affinity with the AQIPEKIQK-HLA A1101 complex is at least 5 times that of the wild-type TCR with the AQIPEKIQK-HLA A1101 complex by performing mutation screening on the above CDR region.

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% of the amino acid sequence shown in SEQ ID NO: 1; preferably, at least 90%; more preferably , at least 92%; more preferably, at least 94% (e.g., may be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% sequence homology) 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: 2, preferably, at least 92%; more preferably, at least 94% (e.g., may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity ) amino acid sequence with 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:3; more preferably, at least 92%; most preferably, at least 94% (e.g., may be at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% % sequence homology) amino acid sequence of sequence homology); and/or the beta chain variable domain of the TCR comprises at least 85%, preferably at least 90% of the amino acid sequence shown in SEQ ID NO:4 %; more preferably, at least 92%; most preferably, at least 94%; (e.g., may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence of sequence homology).

In the present invention, the three CDRs of the wild-type TCRα chain variable domain SEQ ID NO: 1, namely CDR1, CDR2 and CDR3 are respectively located at positions 27-32, 50-57 and 92-101 of SEQ ID NO: 1 . Accordingly, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 1, 95I is the fourth I of CDR3α, 94S is the third S of CDR3α, 93G is the second G of CDR3α, and 92G is The first G and 28S of CDR3α are the second S of CDR1α, 30S is the fourth S of CDR1α, and 32S is the sixth S of CDR1α.

Specifically, the specific forms of the mutations in the variable domain of the α chain include I95L/P/V/N, S94T/H, G93L/M/I/S, G92A, S28V/T/I, S30T/D, S32W/ One or more groups in Y.

In the present invention, the three CDRs of the wild-type TCRβ chain variable domain SEQ ID NO: 2, namely CDR1, CDR2 and CDR3 are respectively located at positions 27-31, 49-54 and 93-104 of SEQ ID NO: 2 . Accordingly, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO: 1, 95S is the third S of CDR3β, 96L is the fourth L of CDR3β, 97R is the fifth R of CDR3β, and 98A is The 6th A of CDR3β, 100G is the 8th G of CDR3β, 101N is the 9th N of CDR3β, 102T is the 10th T of CDR3β, 103I is the 11th I of CDR3β, and 104Y is Y at position 12 of CDR3β.

Specifically, the specific form of the mutation in the β chain variable domain includes S95Q, L96A/E/P, R97Y/F/H/W, A98P, G100S, N101S/T, T102D, I 103F, Y104T/H one or several groups.

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 both represent the 60th proline. In addition, the expression of the specific form of the mutation in the present invention is such as "I95L/P/V/N" means that the I at the 95th position is replaced by L or by P or by V or by N, and so on .

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. The Ser57 of Ser57 is mutated to cysteine, that is, the reference TCR is obtained, and its amino acid sequences are respectively SEQ ID NO: 11 and SEQ ID NO: 12, as shown in Figure 6a and Figure 6b, the cysteine residue after mutation Indicated in bold letters. The cysteine substitutions described above enable the formation of artificial interchain disulfide bonds between the constant regions of the α and β chains of the reference TCR to form a more stable soluble TCR, allowing for easier assessment of TCR complexation with AQIPEKIQK-HLA A1101 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 AQIPEKIQK-HLA A1101 complex is considered to be the binding affinity between the wild-type TCR and the AQIPEKIQK-HLAA1101 complex. Similarly, if the binding affinity between the TCR of the present invention and the AQIPEKIQK-HLA A1101 complex is determined to be at least 5 times the binding affinity between the reference TCR and the AQIPEKIQK-HLA A1101 complex, it is equivalent to the binding affinity between the TCR of the present invention and the AQIPEKIQK - The binding affinity between HLA A1101 complexes is at least 5-fold higher than the binding affinity between wild-type TCR and AQIPEKIQK-HLA A1101 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, such as by surface plasmon resonance techniques. 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, the surface plasmon resonance (BIAcore) method in the examples herein is used to detect the affinity of soluble TCR, and the conditions are: temperature 25° C., pH value 7.1-7.5. This method detects that the dissociation equilibrium constant _K of the reference TCR to the AQIPEKIQK-HLAA1101 complex is 9.07E-04M, that is, 907 μM. In the present invention, it is considered that the dissociation equilibrium constant K of the wild-type TCR to the AQIPEKIQK-HLA A1101 complex _D is also 907 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 the high-affinity TCR to the AQIPEKIQK-HLA A1101 complex is 9.07E-05M, that is, 90.7μM, it means that the high-affinity TCR The affinity for the AQIPEKIQK-HLA A1101 complex was 10-fold higher than that of the wild-type TCR for the AQIPEKIQK-HLA A1101 complex. Those skilled in the art are familiar with the conversion relationship between K _D value units, that is, 1M=10 ⁶ μM, 1 μM=1000nM, 1nM=1000pM. In the present invention, the affinity between the TCR and the AQIPEKIQK-HLA A1101 complex is at least 5 times that of the wild-type TCR.

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 out a TCR with high affinity for the AQIPEKIQK-HLA A1101 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.

The high-affinity TCR of the present invention comprises the amino acid sequence of the α-chain variable domain as one of SEQ ID NO:1, 13-30; and/or the amino acid sequence of the β-chain variable domain of the TCR as SEQ ID NO:2, One of 31-42. The amino acid sequences of the α-chain variable domain and the β-chain variable domain of the heterodimeric TCR molecule of the present invention are preferably selected from the following table 1:

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 chain and β chain 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 AQIPEKIQK-HLA A1101 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 α-chain variable domain are CDR1α: SSYSPS; CDR2α: YTSAATLV; CDR3α: GGSIGNTPLV and the three CDRs of the β-chain variable domain are CDR1β: SGHVS; CDR2β: FQNEAQ; CDR3β: ASSLRAGGNTIY. The amino acid sequence (SEQ ID NO: 9) and nucleotide sequence (SEQ ID NO: 10) of the single-stranded template TCR are shown in Figures 5a and 5b, respectively. In this way, a single-chain TCR composed of an α-chain variable domain and a β-chain variable domain with high affinity to the AQIPEKIQK-HLA A1101 complex was screened out.

The αβ heterodimer with high affinity to the AQIPEKIQK-HLA A1101 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 wild-type TCR α chain variable domain (SEQ ID NO: 1) and β chain variable domain (SEQ ID NO: 2) were obtained.

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 conjugates include detectable markers (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the AQIPEKIQK-HLA A1101 complex), therapeutic agents, PK (protein kinase) modifying moieties, or any of the above combinations or couplings.

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. (Gill ies et al., 1992, National Academy of Sciences of the United States Journal (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, The 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/nanoparticles Rod (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 8. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 9. Nanomagnetic particles; 10. Prodrug activating enzymes (for example, DT-myocardial 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 an amino acid sequence selected from the variable domain of the TCR α chain as one of SEQ ID NO: 1, 13-30; and/or the variable β chain of the TCR The domain amino acid sequence is one of SEQ ID NO:2, 31-42.

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: 3, but with a sequence of SEQ ID NO: 5 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.

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) The affinity and/or binding half-life of the high-affinity TCR of the present invention to the AQIPEKIQK-HLA A1101 complex is at least 5 times that of the wild-type TCR.

(2) The high-affinity TCR of the present invention can specifically bind to the AQIPEKIQK-HLA A1101, and the cells transfected with the high-affinity TCR of the present invention can be specifically activated.

(3) The effector cells transfected with the high-affinity TCR of the present invention have a strong specific killing effect.

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 SEQ ID NO:9 and SEQ ID NO:10, respectively, as shown in Figure 5a and Figure 5b. And use the single-chain TCR molecule as a template to screen for high-affinity TCR molecules. The amino acid sequences of the α-chain variable domain (SEQ ID NO:3) and the β-chain variable domain (SEQ IDNO:4) of the template chain are shown in Figures 2a and 2b; the corresponding DNA sequences are respectively SEQ ID NO: 5 and SEQ ID NO:6, as shown in Figures 3a and 3b; the amino acid sequence and DNA sequence of the flexible short peptide (linker) are SEQ ID NO:7 and SEQ ID NO:8, 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 at 37°C for 4h. 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, The collected eluted fractions were subjected to SDS-PAGE analysis, and the fractions containing single-chain TCR were concentrated and further purified with a gel filtration column (Superdex 75 10/300, GE Healthcare), and the target fractions were also subjected to SDS-PAGE analysis.

The eluted fractions for BIAcore analysis were further tested for purity by gel filtration. The conditions are: 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

The binding activity of TCR molecule and AQIPEKIQK-HLA A1101 complex was detected by BIAcore T200 real-time analysis system. Add the anti-streptavidin antibody (GenScript) to the coupling buffer (10mM sodium acetate buffer, pH4.77), and then flow the antibody through the CM5 chip activated with EDC and NHS in advance 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 15,000RU. The conditions are: temperature 25°C, pH value 7.1-7.5.

Let a low concentration of streptavidin flow over the surface of the antibody-coated chip, then flow the AQIPEKIQK-HLAA1101 complex through the detection channel, and the other channel as a reference channel, and then 0.05mM biotin at 10μL/min The flow rate flowed through the chip for 2 minutes 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 AQIPEKIQK-HLA A1101 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, and resuspend with 5ml BugBuster Master Mix Extraction Reagents (Merck) vigorously Bacteria were incubated with rotation at room temperature for 20 minutes, and then centrifuged at 6000g at 4°C for 15 minutes, the supernatant was discarded, and the inclusion bodies were collected.

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 AQIPEKIQK (Jiangsu 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 AQIPEKIQK 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 Until the completion, SDS-PAGE test whether the renaturation is successful.

c. Purification after renaturation: use 10 volumes of 20mM Tris pH 8.0 for dialysis to replace the refolding buffer, and replace the buffer 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 the 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 a Millipore ultrafiltration tube, and at the same time replace the buffer with 20mM Tris pH 8.0, then add biotinylation reagent 0.05M Bicine pH 8.3, 10mM ATP, 10mM MgOAc, 50μM D -Biotin, 100 μg/ml BirA enzyme (GST-BirA), incubate the mixture overnight at room temperature, and check whether the biotinylation is complete by SDS-PAGE.

e. Purify the biotinylated complex: Concentrate the biotinylated pMHC molecules to 1ml with a Millipore ultrafiltration tube, purify the biotinylated pMHC using gel filtration chromatography, and use the Akta Purifier (GE general Electric Company), pre-equilibrated HiPrep ^TM 16/60S200 HR column (GE General Electric Company) with filtered PBS, loaded 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 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. After several rounds of panning, the phage library specifically binds to the corresponding antigen, and single clones are picked and analyzed.

The CDR region mutation of the screened high-affinity single-chain TCR was introduced into the corresponding site of the variable domain of the αβ heterodimeric TCR, and its affinity with the AQIPEKIQK-HLA A1101 complex was detected by BIAcore. 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 wild-type TCR are shown in Figure 1a (SEQ ID NO: 1) and 1b (SEQ ID NO: 2), 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 AQIPEKIQK-HLAA1101 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 6a (SEQ ID NO: 11) and 6b (SEQ ID NO: 12), 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 5 Expression, refolding and purification of high-affinity 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 6 BIAcore 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 AQIPEKIQK-HLA A1101 complex.

The present invention obtains the amino acid sequence of the variable domain of the TCRα chain and the amino acid sequence of the variable domain of the TCRβ chain, as shown in Figures 7 (1)-(18) and Figures 8 (1)-(12) respectively. 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 AQIPEKIQK-HLA A1101 complex. The method described in Example 4 was used to construct an expression vector, and the method described in Example 5 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 AQIPEKIQK- The affinity of the HLA A1101 complex is shown in Table 2 below.

Table 2

It can be seen from Table 2 above that the affinity of the heterodimeric TCR is at least 5 times that of the wild-type TCR to the AQIPEKIQK-HLA A1101 complex.

Example 7 Expression, refolding and purification of the fusion of anti-CD3 antibody and high-affinity αβ heterodimeric TCR

A fusion molecule was prepared by fusing an anti-CD3 single-chain antibody (scFv) to an αβ heterodimeric TCR. The scFv of anti-CD3 is fused with the β chain of TCR. The TCR β chain can contain any of the β chain variable domains of the above-mentioned high-affinity αβ heterodimeric TCR, and the TCR α chain of the fusion molecule can contain any of the above-mentioned high-affinity The α-chain variable domain of a sex αβ heterodimeric TCR.

Construction of Fusion Molecule Expression Vector

1. Construction of the α chain expression vector: the target gene carrying the α chain of αβ heterodimeric TCR 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, spread on LB plates containing kanamycin, cultured upside down at 37°C overnight, picked positive clones for PCR screening, sequenced positive recombinants, and extracted recombinant plasmids after confirming the sequence was correct Transformed into E.coli Tuner (DE3) for expression.

2. Construction of anti-CD3 (scFv)-beta chain expression vector: by overlapping (overlap) PCR, design primers to connect anti-CD3 scFv and high-affinity heterogeneous dimeric TCR beta chain gene, the middle connection The short peptide (linker) is GGGGS, and the gene fragment of the fusion protein of anti-CD3 scFv and high-affinity heterodimeric TCRβ chain is brought with restriction endonuclease sites NcoI (CCATGG) and NotI (GCGGCCGC ). The PCR amplified product 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 competent cells, coated with LB plates containing kanamycin, cultured upside down at 37°C overnight, and positive clones were picked for PCR screening, and the positive recombinants were sequenced, and extracted after confirming the correct sequence The recombinant plasmid was transformed into E.coli Tuner (DE3) competent cells for expression.

Expression, renaturation and purification of fusion protein

The expression plasmids were respectively transformed into E.coli Tuner (DE3) competent cells, spread on LB plates (kanamycin 50 μg/mL) and cultured at 37°C overnight. The next day, pick clones and inoculate them into 10 mL LB liquid medium (kanamycin 50 μg/mL) for 2-3 hours, inoculate them into 1L LB medium at a volume ratio of 1:100, and continue culturing until the OD600 is 0.5-0.8, adding The final concentration was 1mM IPTG to induce the expression of the target protein. After 4 hours of induction, cells were harvested by centrifugation at 6000 rpm for 10 min. The bacteria were washed once with PBS buffer, and the bacteria were subpackaged. The bacteria equivalent to 200 mL of bacterial culture were taken to lyse the bacteria with 5 mL of BugBusterMaster Mix (Merck), and the inclusion bodies were collected by centrifugation at 6000 g for 15 min. Four detergent washes were then performed to remove cell debris and membrane components. Then, the inclusion bodies are washed with a buffer such as PBS to remove detergents and salts. Finally, the inclusion bodies were dissolved with a buffer solution containing 6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylenediaminetetraacetic acid (EDTA), 20mM Tris, pH 8.1, and the concentration of the inclusion bodies was measured and divided into Store in -80°C freezer after loading.

The dissolved TCRα chain and anti-CD3 (scFv)-β chain were quickly mixed in 5M urea (urea), 0.4M L-arginine (L-arginine), 20mM Tris pH 8.1 at a mass ratio of 2:5, 3.7mM cystamine, 6.6mMβ-mercapoethylamine (4°C), the final concentration of α-chain and anti-CD3(scFv)-β-chain were 0.1mg/mL, 0.25mg/mL, respectively.

After mixing, the solution was dialyzed (4°C) in 10 times the volume of deionized water, and after 12 hours, the deionized water was replaced with buffer (10 mM Tris, pH 8.0) and continued to be dialyzed 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 peak contained the successfully refolded TCRα chain and anti-CD3(scFv)-β chain dimer TCR confirmed by SDS-PAGE gel. The TCR fusion molecule was then further purified by size exclusion chromatography (S-100 16/60, GE healthcare) and again by an anion exchange column (HiTrap Q HP5ml, GE healthcare). The purity of the purified TCR fusion molecule is determined by SDS-PAGE to be greater than 90%, and the concentration is determined by the BCA method.

Example 8 Activation function experiment of effector cells transfected with high-affinity TCR of the present invention for T2 target cells loaded with short peptides

IFN-γ is a powerful immunoregulatory factor produced by activated T lymphocytes. Therefore, this example detects the number of IFN-γ spots by ELISPOT experiments well known to those skilled in the art to verify the presence of cells transfected with the high-affinity TCR of the present invention. Activation function and antigen specificity. TCR was randomly selected for the experiment. The specific TCR sequence used can be learned from Table 2. TCR2, TCR4, TCR9, TCR21, TCR23, and TCR26 were transfected into CD3+ T cells isolated from the blood of healthy volunteers as effector cells. And the CD3+ T cells transfected with other TCR (A6) and CD3+ T cells transfected with wild-type TCR (WT-TCR) were used as controls in the same volunteer. The target cells used were T2-A11 loaded with SSX2 antigen short peptide AQIPEKIQK, T2-A11 loaded with other antigen short peptides, or empty T2-A11 (refers to T2 cells transfected with HLA-A1101, the same below).

First prepare the ELISPOT plate, first add 1× ¹⁰⁴ cells/well of target cells and 2× ¹⁰³ cells/well of effector cells (calculated according to the positive rate of transfection) into the corresponding wells, and then add SSX2 antigen short peptide to the experimental group AQIPEKIQK, and make the final concentration of 1×10 ^-6 M in the ELISPOT well plate; add other short antigen peptides in the control group, and make the final concentration of 1× ^{10 -6 M} , and set up two duplicate holes. 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 by an immunospot plate reader (ELISPOT READER system; AID20 company).

The experimental results are shown in Figure 13. For the target cells loaded with the SSX2 antigen short peptide AQIPEKIQK, the effector cells transfected with the high-affinity TCR of the present invention had a more obvious activation effect than the effector cells transfected with the wild-type TCR. The effector cells transfected with other TCRs or empty transfection are inactive; at the same time, the effector cells transfected with the TCR of the present invention are inactive for target cells loaded with other antigen short peptides or empty.

Example 9 Activation function experiment of effector cells transfected with high-affinity TCR of the present invention for cell lines

In this example, tumor cell lines were used to verify again the activation function and specificity of the effector cells transfected with the high-affinity TCR of the present invention. TCR molecules are randomly selected and detected by ELISPOT experiments well known to those skilled in the art. Transfect the high-affinity TCR of the present invention into CD3+ T cells isolated from the blood of healthy volunteers as effector cells, and use the same volunteer to transfect other TCR (A6) or transfect wild-type TCR (WT-TCR ) CD3+ T cells were used as control. The tumor cell lines used in the examples are SK-MEL-28, TT_THYROID, HUCC-T1, SNU423, SW620, Huh-1, LCLs-150909A, LCLs-151008A, respectively. Among them, SK-MEL-28, TT_THYROID, HUCC-T1, and SNU423 were purchased from Guangzhou Saiku Biotechnology Co., Ltd., Huh-1 was purchased from Nanjing Kebai Biotechnology Co., Ltd., and SW620 was purchased from ATCC. The following three batches (I), (II), (III) were successively tested:

(I) The high-affinity TCRs can be learned from Table 2, which are TCR2, TCR4, TCR5, and TCR23 respectively. The SSX2 positive tumor cell line used in this batch is SK-MEL-28-SSX2 (SSX2 overexpression), and the negative tumor cell line is SK-MEL-28, HUCC-T1, SNU423, LCLs-150909A, LCLs-151008A,, TT_THYROID.

(II) The high-affinity TCRs can be learned from Table 2, which are TCR1, TCR2, TCR20, TCR23, and TCR25, respectively. The SSX2 positive tumor cell lines used in this batch were SK-MEL-28-SSX2 (SSX2 overexpression), TT_THYROID-A11, TT_THYROID, and the negative tumor cell lines were HUCC-T1, SNU423, SW620-A11, SW620.

(III) The high-affinity TCR can be learned from Table 2, which is TCR3. The SSX2-positive tumor cell lines used in this batch were SK-MEL-28-SSX2 (SSX2 overexpression), HUCC-T1-SSX2 (SSX2 overexpression), Huh-1-A11, and the negative tumor cell lines were HUCC-T1, SNU423.

The above three batches all carried out the following steps: first prepare the ELISPOT plate. ELISPOT plates were activated with ethanol and coated at 4°C overnight. 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: 2* ¹⁰ cells/well for target cells, 2* for effector cells 10 ³ cells/well (calculated according to the positive rate of transfection), and set up two duplicate holes. Incubate overnight (37°C, 5% CO ₂ ). 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 by an immunospot plate reader (ELISPOT READER system; AID20 company).

The experimental results are shown in Figure 14a, Figure 14b, and Figure 14c. In three batches, for different SSX2-positive tumor cell lines, the effector cells transfected with the high-affinity TCR of the present invention were compared with the effector cells transfected with wild-type The effector cells transfected with other TCRs are basically inactive; at the same time, the effector cells transfected with the high-affinity TCR of the present invention are basically inactive against SSX2-negative cell lines.

Example 10 Killing function experiment of effector cells transfected with high-affinity TCR of the present invention for tumor cell lines

Lactate dehydrogenase (LDH) is abundant in the cytoplasm and cannot pass through the cell membrane under normal circumstances. When the cells are damaged or die, they can be released outside the cells. At this time, the activity of LDH in the cell culture medium is directly proportional to the number of dead cells. The TCR molecule of the present invention is randomly selected, and the release of LDH is measured through a non-radioactive cytotoxicity experiment well known to those skilled in the art, so as to verify the killing function of the cells transfected with the TCR of the present invention. In the LDH experiment of this example, CD3+ T cells isolated from the blood of healthy volunteers were used to transfect the high-affinity TCR of the present invention as effector cells, and CD3+ T cells transfected with other TCR (A6) from the same volunteer were used as negative control. The tumor cell lines used in the examples are SK-MEL-28, TT_THYROID, HUCC-T1, Huh-1, and SNU423, among which, SK-MEL-28, TT_THYROID, HUCC-T1, and SNU423 were all purchased from Guangzhou Saiku Biotechnology Co., Ltd. Co., Ltd., Huh-1 was purchased from Nanjing Kebai Biotechnology Co., Ltd. The following two batches (I), (II) were successively tested:

(I) The high-affinity TCRs can be learned from Table 2, which are TCR1, TCR2, TCR20, TCR23, and TCR25, respectively. The SSX2-positive tumor cell lines used in this batch were SK-MEL-28-SSX2 (SSX2 overexpression), TT_THYROID-A11, and the negative tumor cell lines were HUCC-T1, TT_THYROID.

(II) The high-affinity TCR can be learned from Table 2, which is TCR3. The SSX2-positive tumor cell lines used in this batch were SK-MEL-28-SSX2 (SSX2 overexpression), HUCC-T1-SSX2 (SSX2 overexpression), Huh-1-A11, and the negative tumor cell lines were HUCC-T1, SNU423.

The above two batches all carried out the following experimental steps: first prepare the LDH plate, and add the various components of the test to the plate in the following order: target cells 3× ¹⁰⁴ cells/well, effector cells 3× ¹⁰⁴ cells/well (according to Transfection positive rate calculation) were added to the corresponding wells, and three duplicate wells were set. Simultaneously set effector cell spontaneous wells, target cell spontaneous wells, target cell maximum wells, volume correction control wells and medium background control wells. Incubate overnight (37°C, 5% CO ₂ ). On the second day of the experiment, the color development was detected, and the absorbance value was recorded at 490 nm with a microplate reader (Bioteck) after the reaction was terminated.

The experimental results are shown in Figures 15a-15b. For SSX2-positive tumor cell lines, the effector cells transfected with the high-affinity TCR of the present invention still showed strong killing effect, while the T cells transfected with other TCRs basically did not respond; The T cells transfected with the high-affinity TCR of the present invention have basically no killing effect on negative tumor cell lines.

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> Xiangxue Life Science Technology (Guangdong) Co., Ltd.

<120> A high-affinity TCR that recognizes SSX2

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aacggcttcg aggcggagtt caaaaaatcc gaaaccagct tccatctgac caaaccgtct 240

gttcatccgt ctgacgcggc ggaatacttc tgcggcggta gtattggtaa taccccgtta 300

gtcttcggta aaggtacccg tctgagcgtt accccgggcg gtggttctga aggcggtggt 360

agtgaaggcg gcggtagcga aggcggcggt tctgaaggtg gtaccggcgg cgcaggcgtt 420

agtcagagtc cgcgctatct gagcgttaaa cgcggtcaag acgttgcatt acgttgcgat 480

ccgattagcg gtcacgtttc tctgttttgg tatcagcaag caccgggtca aggtccggaa 540

tttctgacct acttccagaa cgaagcgcag ctggataaat ctggtctgcc gtctgatcgt 600

ttctttgcag aacgtccgga aggtagcgtt agtaccctga aaattcagcg cgttcaaccg 660

gaagatagcg cggtttactt ctgcgcaagc agcttacgcg caggcggtaa taccatctac 720

ttcggcgaag gcagctggtt aaccgttgat 750

<210> 11

<211> 206

<212> PRT

<213> Artificial Sequence

<220>

<223> TCRAlpha amino acid sequence after introduction of cysteine residues

<400> 11

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Gly Gly Ser Ile Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

<210> 12

<211> 244

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of TCRBeta chain after introduction of cysteine residue

<400> 12

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 Leu

85 90 95

Arg Ala Gly Gly Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

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

115 120 125

Glu Pro Ser Glu Cys 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 Ser 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 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> 13

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 13

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Ala Leu Thr Leu Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 14

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 14

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Gly Met Thr Leu Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 15

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 15

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Ala Ile Thr Leu Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 16

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 16

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Ala Ile Ser Leu Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 17

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 17

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

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

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 18

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 18

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Ala Met Thr Leu Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 19

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 19

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Ala Met His Ile Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 20

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 20

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

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

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 21

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 21

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Ala Ile Thr Val Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 22

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 22

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Ala Ile Thr Pro Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 23

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 23

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Ala Ile Thr Asn Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 24

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 24

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Ala Ile His Ile Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 25

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 25

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Val Tyr Thr Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Gly Gly Ser Ile Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 26

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 26

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Thr Tyr Thr Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Gly Gly Ser Ile Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 27

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 27

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ile Tyr Thr Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Gly Gly Ser Ile Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 28

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 28

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Thr Pro Trp

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Gly Gly Ser Ile Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 29

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 29

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Ser Tyr Thr Pro Tyr

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Gly Gly Ser Ile Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 30

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain variable domain sequence

<400> 30

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Asp Pro Trp

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Gly Gly Ser Ile Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

<210> 31

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain variable domain sequence

<400> 31

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 Gln Ala

85 90 95

Tyr Pro Gly Gly Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val

<210> 32

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain variable domain sequence

<400> 32

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 Gln Glu

85 90 95

Tyr Pro Gly Gly Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val

<210> 33

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain variable domain sequence

<400> 33

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 Glu

85 90 95

Phe Ala Gly Gly Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val

<210> 34

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain variable domain sequence

<400> 34

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 Glu

85 90 95

His Ala Gly Gly Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val

<210> 35

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain variable domain sequence

<400> 35

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 Pro

85 90 95

His Ala Gly Gly Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val

<210> 36

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain variable domain sequence

<400> 36

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 Leu

85 90 95

Trp Ala Gly Ser Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val

<210> 37

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain variable domain sequence

<400> 37

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 Leu

85 90 95

Tyr Ala Gly Gly Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val

<210> 38

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain variable domain sequence

<400> 38

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 Leu

85 90 95

Tyr Ala Gly Ser Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val

<210> 39

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain variable domain sequence

<400> 39

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 Leu

85 90 95

Tyr Pro Gly Ser Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val

<210> 40

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain variable domain sequence

<400> 40

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 Leu

85 90 95

His Ala Gly Gly Ser Asp Phe Thr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val

<210> 41

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain variable domain sequence

<400> 41

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 Leu

85 90 95

His Ala Gly Gly Thr Asp Ile His Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val

<210> 42

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain variable domain sequence

<400> 42

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 Leu

85 90 95

His Ala Gly Gly Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val

<210> 43

<211> 206

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain extracellular amino acid sequence of "wild type TCR"

<400> 43

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Gly Gly Ser Ile Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

<210> 44

<211> 244

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain extracellular amino acid sequence of "wild-type TCR"

<400> 44

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 Leu

85 90 95

Arg Ala Gly Gly Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val 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> 45

<211> 253

<212> PRT

<213> Artificial Sequence

<220>

<223> Alpha chain amino acid sequence of "wild-type TCR"

<400> 45

Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val Ser Glu Gly

1 5 10 15

Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr Ser Pro Ser

20 25 30

Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln Leu Leu Leu

35 40 45

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

50 55 60

Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr Lys Pro Ser

65 70 75 80

Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Gly Gly Ser Ile Gly

85 90 95

Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

245 250

<210> 46

<211> 291

<212> PRT

<213> Artificial Sequence

<220>

<223> Beta chain amino acid sequence of "wild-type TCR"

<400> 46

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 Leu

85 90 95

Arg Ala Gly Gly Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr

100 105 110

Val Val 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 Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val

245 250 255

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

260 265 270

Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg

275 280 285

Lys Asp Phe

290

Claims (10)

1. A T cell receptor (TCR), said T cell receptor comprises TCR α chain variable domain and TCR β chain variable domain, it is characterized in that, it has the activity of binding AQIPEKIQK-HLA A1101 complex, and said The amino acid sequence of the TCR alpha chain variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:1 and the amino acid sequence of the TCR beta chain variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:2 There is at least 90% sequence identity. 2. 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 claim 1. 3. A nucleic acid molecule, characterized in that the nucleic acid molecule comprises a nucleic acid sequence encoding the TCR as claimed in claim 1 or a complementary sequence thereof. 4. A carrier, characterized in that said carrier contains the nucleic acid molecule as claimed in claim 3. 5. A host cell, characterized in that the host cell contains the vector of claim 4 or the nucleic acid molecule of claim 3 is integrated in the chromosome. 6. An isolated cell, characterized in that the cell expresses the TCR as claimed in claim 1. 7. A pharmaceutical composition, characterized in that the composition contains a pharmaceutically acceptable carrier and the TCR described in claim 1, or the TCR complex described in claim 2, or the TCR complex described in claim 6 the cells. 8. A method for treating diseases, comprising administering the TCR described in claim 1, or the TCR complex described in claim 2, or the cells described in claim 6, to a subject in need of treatment , or the pharmaceutical composition described in claim 7, preferably, the disease is a SSX2 positive tumor. 9. The use of the T cell receptor described in claim 1, the TCR complex described in claim 2, or the cells described in claim 6, characterized in that it is used to prepare a drug for treating tumors, preferably, The tumors are SSX2 positive tumors. 10. A method for preparing the T cell receptor described in claim 1, comprising the steps of: (i) cultivating the host cell described in claim 5, thereby expressing the T cell receptor described in claim 1; (ii) separating or purifying the T cells.

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