WO2023241391A1

WO2023241391A1 - Tcr molecule binding to ssx2 antigen and application thereof

Info

Publication number: WO2023241391A1
Application number: PCT/CN2023/098275
Authority: WO
Inventors: 战凯; 杨东雪; 刘敏; 殷倩霞; 王定国; 唐先青; 马瑞娟
Original assignee: 香雪生命科学技术（广东）有限公司
Priority date: 2022-06-17
Filing date: 2023-06-05
Publication date: 2023-12-21
Also published as: CN117304301A

Abstract

The present invention provides a TCR molecule binding to an SSX2 antigen and an application thereof. The TCR molecule can specifically bind to an SSX2 antigen short peptide complex AQIPEIQK-HLA A1101. Meanwhile, effector cells transducing the TCR further have a very strong killing function.

Description

A TCR molecule binding to SSX2 antigen and its application

Related applications

This application claims priority to the Chinese patent application filed on June 17, 2022, with application number 202210691744.7 and titled "A TCR molecule binding SSX2 antigen and its application", the full text of which is hereby incorporated by reference.

Technical field

The invention belongs to the field of biotechnology and relates to a TCR molecule that binds SSX2 antigen and its application. Specifically, it relates to a T cell receptor (TCR) capable of recognizing polypeptides derived from SSX2 protein and its preparation method and application.

Background technique

There are only two types of molecules that can recognize antigens in a specific manner. One is an immunoglobulin or antibody; the other is a T cell receptor (TCR). TCR is a glycoprotein on the cell membrane surface that exists as a heterodimer of α chain/β chain or γ chain/δ chain. In humans, 95% of T cell receptors in TCR are composed of α chain and β chain, respectively. Encoded by TRA and TRB. The recognition of pMHC (antigenic peptide-major histocompatibility complex) by TCR involves two combinations: the binding of TCR to MHC molecules and the binding of TCR to polypeptide antigens.

The binding surface on the TCR comes from 6 regions: CDR1, CDR2 and CDR3 on the TCRα and β chains. CDR1 and CDR2 mainly bind to relatively conserved MHC molecules, while CDR3 mainly binds to variable antigen peptides. This binding mode can well ensure that TCR specifically binds MHC molecules and can recognize variable antigens. Among them, CDR3 has the greatest variation, which directly determines the antigen-binding specificity of TCR.

In the immune system, the binding of the antigen-specific TCR to the pMHC complex triggers direct physical contact between T cells and antigen-presenting cells (APCs), and then other cell membrane surface molecules of the T cells and APCs interact, which This triggers a series of subsequent cell signaling and other physiological responses, allowing T cells with different antigen specificities to exert immune effects on their target cells.

SSX2 is synovial sarcoma X breakpoint, also known as HOM-MEL-40. SSX2 is one of ten highly homologous nucleic acid proteins of the SSX family. SSX protein is a tumor testis antigen that is only expressed 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, and prostate cancer. After SSX2 is produced in cells, it is degraded into small peptides, combines with MHC (major histocompatibility complex) molecules to form a complex, and is presented to the cell surface. AQIPEKIQK is a short peptide derived from the SSX2 antigen.

This application is dedicated to the development of TCRs that can bind the AQIPEKIQK-HLA A1101 complex and have high application value in the treatment of tumors. For example, a TCR capable of targeting this tumor cell marker could be used to deliver a cytotoxic or immunostimulatory agent to the target cell, or be transformed into a T cell such that the T cell expressing the TCR is able to destroy the tumor cell in order to achieve the desired outcome in what is known as Adoptive immunotherapy is administered to patients during treatment.

Contents of the invention

In view of the shortcomings of the existing technology, the purpose of this application is to provide a TCR molecule that binds to SSX2 antigen and its application. This application provides a TCR molecule that specifically recognizes and binds to the AQIPEKIQK-HLA A1101 complex. This application also provides a preparation method for the TCR molecule and the use of the TCR molecule.

In order to achieve the purpose of this application, this application adopts the following technical solutions:

A first aspect of the present application provides a TCR molecule, the TCR molecule comprising a TCRα chain variable domain and a TCRβ chain variable domain, the TCR molecule has the activity of binding the AQIPEKIQK-HLA A1101 complex, and the TCRα The amino acid sequence of the chain variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO: 1 (for example, it can be 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or 100%); and the amino acid sequences of the three CDR regions of the TCRβ chain variable domain are:
βCDR1-SGHVS (SEQ ID NO:16)
βCDR2-FQNEAQ (SEQ ID NO:17)
βCDR3-ASSLVGYEQY (SEQ ID NO:18).

In another preferred embodiment, the amino acid sequence of the TCRα 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 TCRβ chain variable domain has at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:1. The amino acid sequence shown in SEQ ID NO:7 has at least 90% sequence homology (for example, it can be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%).

In this application, relative to the sequence shown in SEQ ID NO: 1, the alpha chain variable domain of the TCR molecule has 1, 2, 3, 4, 5, 6, Insertion, deletion, substitution or combinations of 7, 8, 9, 10 or 11 amino acid residues.

In this application, the β chain variable domain of the TCR molecule has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 relative to the sequence shown in SEQ ID NO:7 Amino acid residue insertion, deletion, substitution or combinations thereof.

In another preferred embodiment, the TCR molecule is αβ heterodimeric TCR. Preferably, the TCR molecule has an alpha chain constant region sequence TRAC*01 and a beta chain constant region sequence TRBC1*01 or TRBC2*01.

In another preferred embodiment, the TCR molecule includes 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 embodiment, the constant regions of the α and β chains of the TCR molecule are the constant regions of the murine α and β chains respectively.

In another preferred embodiment, the TCRα chain variable domain includes 3 CDR regions, and the TCRβ chain variable domain includes 3 CDR regions, wherein the amino acid sequences of the 3 CDR regions of the TCRβ chain variable domain are for:
βCDR1-SGHVS (SEQ ID NO:16)
βCDR2-FQNEAQ (SEQ ID NO:17)
βCDR3-ASSLVGYEQY (SEQ ID NO:18).

In another preferred embodiment, the α chain variable domain of the TCR molecule is an amino acid sequence that has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO: 1, and the β chain of the TCR molecule The variable domain is an amino acid sequence that has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO:7.

In another preferred example, the amino acid sequence of the β chain variable domain of the TCR molecule is SEQ ID NO: 7.

In another preferred embodiment, the TCRα chain variable domain includes 3 CDR regions, and the TCRβ chain variable domain includes 3 CDR regions, wherein the amino acid sequences of the 3 CDR regions of the TCRα chain variable domain for:
αCDR1-SSYSPS (SEQ ID NO:13)
αCDR2-YTSAATLV (SEQ ID NO:14)
αCDR3-VVSSGNTPLV (SEQ ID NO:15).

In another preferred embodiment, the α chain variable domain of the TCR molecule is an amino acid sequence that has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO: 1; and the TCR β chain variable domain The amino acid sequences of the three CDR regions are:
βCDR1-SGHVS (SEQ ID NO:16)
βCDR2-FQNEAQ (SEQ ID NO:17)
βCDR3-ASSLVGYEQY (SEQ ID NO:18).

In another preferred embodiment, the α chain variable domain of the TCR molecule is an amino acid sequence that has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO: 1; and the β chain of the TCR molecule The variable domain amino acid sequence is SEQ ID NO:7.

In another preferred example, the TCR has a CDR selected from the following group, as shown in Table 1:

Table 1

As a preferred technical solution of the present application, the amino acid sequences of the three CDR regions of the α chain variable domain of the TCR molecule are:
αCDR1-SSYSPS (SEQ ID NO:13)
αCDR2-YTSAATLV (SEQ ID NO:14)
αCDR3-VVSSGNTPLV (SEQ ID NO:15);

The amino acid sequences of the three CDR regions of the β chain variable domain of the TCR molecule are:
βCDR1-SGHVS (SEQ ID NO:16)
βCDR2-FQNEAQ (SEQ ID NO:17)
βCDR3-ASSLVGYEQY (SEQ ID NO:18).

As a preferred technical solution of the present application, the amino acid sequences of the three CDR regions of the α chain variable domain of the TCR molecule are:
αCDR1-SSYSPY (SEQ ID NO:24)
αCDR2-YTSAATLV (SEQ ID NO:14)
αCDR3-VISLGNTPLV (SEQ ID NO:25);

In another preferred example, the amino acid sequence of the α chain variable domain of the TCR molecule is: one of SEQ ID NO: 1 and SEQ ID NO: 21; and/or the amino acid sequence of the β chain variable domain of the TCR molecule Selected from: SEQ ID NO:7.

In another preferred embodiment, the TCR molecule is selected from the following group:

(1) The alpha chain variable domain sequence is SEQ ID NO:1, and the beta chain variable domain sequence is SEQ ID NO:7;

(2) The alpha chain variable domain sequence is SEQ ID NO:21, and the beta chain variable domain sequence is SEQ ID NO:7.

In another preferred embodiment, the TCR molecule includes (i) all or part of the TCRα chain except its transmembrane domain, and (ⅱ) all or part of the TCRβ chain except its transmembrane domain, wherein (i) ) and (ii) both include the variable domain and at least part of the constant domain of the TCR chain.

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

Preferably, the cysteine residue forming an artificial interchain disulfide bond is substituted for one or more groups of sites selected from the following:

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

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

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

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

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

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

Pro89 in exon 1 of TRAC*01 and Ala19 in exon 1 of TRBC1*01 or TRBC2*01; and,

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

In another preferred embodiment, the TCR molecules described in this application are of human origin.

In another preferred embodiment, the TCR molecule described in this application is humanized.

In another preferred embodiment, the TCR molecule is soluble.

In another preferred embodiment, the TCR molecule is a single-chain TCR. Preferably, the single-chain TCR is composed of an α-chain variable domain and a β-chain variable domain connected by a flexible short peptide sequence (linker).

In another preferred embodiment, a conjugate is bound to the C- or N-terminus of the α chain and/or β chain of the TCR molecule. Preferably, the conjugate includes any one or a combination of at least two of a detectable label, a therapeutic agent or a PK modifying moiety. Most preferably, the therapeutic agent that binds to the TCR molecule is an anti-CD3 antibody linked to the C- or N-terminus of the alpha or beta chain of the TCR molecule.

A second aspect of the present application provides a multivalent TCR complex. The multivalent TCR complex contains at least two TCR molecules, and at least one of the TCR molecules is the TCR molecule described in the first aspect of the present application.

The third aspect of the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the TCR molecule described in the first aspect of the present application, or a nucleic acid encoding the TCR molecule described in the first aspect of the present application. The complementary sequence of the nucleotide sequence.

The fourth aspect of the present application provides a vector containing the nucleic acid molecule described in the third aspect of the present application. Preferably, the vector is a viral vector. More preferably, the vector is a lentiviral vector.

The fifth aspect of the present application provides a host cell, the host cell contains the vector described in the fourth aspect of the present application, or the genome of the host cell integrates the exogenous vector described in the third aspect of the present application. nucleic acid molecules.

A sixth aspect of the present application provides an isolated cell expressing the TCR molecule described in the first aspect of the present application. Preferably, the isolated cells include T cells, NK cells or NKT cells.

In another preferred embodiment, the isolated cells express the TCR molecule described in the first aspect of the application, and also express exogenous CD8 receptors. Preferably, the CD8 receptor is CD8α.

The seventh aspect of this application provides a pharmaceutical composition, which contains the TCR molecule described in the first aspect of this application, the multivalent TCR complex described in the second aspect of this application or the sixth aspect of this application. Any one or a combination of at least two of the isolated cells described in the aspect. Preferably, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

The eighth aspect of this application provides a method of treating a disease. The method of treating a disease includes administering to a subject in need of treatment an appropriate amount of the TCR molecule described in the first aspect of this application, or the TCR molecule described in the second aspect of this application. Any one or a combination of at least two of the multivalent TCR complex, the isolated cells described in the sixth aspect of the application, or the pharmaceutical composition described in the seventh aspect of the application. Preferably, the disease includes SSX2 positive tumors.

The ninth aspect of the application provides any one of the TCR molecules described in the first aspect of the application, the multivalent TCR complex described in the second aspect of the application, the isolated cells described in the sixth aspect of the application, or Use of a combination of at least two in the preparation of medicaments for treating tumors or autoimmune diseases. Preferably, the tumor includes an SSX2 positive tumor.

The tenth aspect of the application provides any one of the TCR molecules described in the first aspect of the application, the multivalent TCR complex described in the second aspect of the application, or the isolated cells described in the sixth aspect of the application, or A combination of at least two is used as a drug for treating tumors or autoimmune diseases. Preferably, the tumor includes an SSX2 positive tumor.

An eleventh aspect of the present application provides a cell transduced with the nucleic acid molecule described in the third aspect of the present application or the vector described in the fourth aspect of the present application. Preferably, the cells include T cells, NK cells or NKT cells.

A twelfth aspect of the present application provides a method for preparing the TCR molecule described in the first aspect of the present application. The preparation method includes the following steps:

(i) Cultivate the host cell described in the fifth aspect of the application to express the TCR molecule described in the first aspect of the application; and,

(ii) Isolate or purify the TCR molecule.

It should be understood that within the scope of the present application, the above-mentioned technical features of the present application and the technical features specifically described below (such as embodiments) can be combined with each other to constitute a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

The numerical range described in this application not only includes the above-mentioned point values, but also includes any point value between the above-mentioned numerical ranges that are not exemplified. Due to space limitations and for the sake of simplicity, this application will not exhaustively list all the points. The specific point values included in the stated range.

Compared with the existing technology, this application has the following beneficial effects:

(1) The TCR molecule of the present application can specifically bind to the SSX2 antigen short peptide complex AQIPEKIQK-HLA A1101.

(2) For positive target cells, the effector cells transduced with the TCR molecule of the present application can be specifically activated. At the same time, the effector cells transduced with the TCR molecule of the present application also have a strong killing function.

Description of the drawings

Figure 1 shows the CD8-APC and tetramer-PE double positive staining results of monoclonal cells in Example 1;

Figure 2 is the ELISPOT activation function verification result of the T cell clone in Example 1;

Figure 3a is an SDS-PAGE electrophoresis gel image of soluble TCR1 in Example 3. The right lane is a non-reducing gel, and the left lane is a molecular weight marker (marker);

Figure 3b is an SDS-PAGE electrophoresis gel image of soluble TCR1 in Example 3. The right lane is reducing gel, and the left lane is molecular weight marker (marker);

Figure 3c is an SDS-PAGE electrophoresis gel image of soluble TCR2 in Example 3. The left lane is a non-reducing gel, and the right lane is a molecular weight marker (marker);

Figure 3d is an SDS-PAGE electrophoresis gel image of soluble TCR2 in Example 3. The left lane is reducing gel, and the right lane is molecular weight marker (marker);

Figure 4a is a BIAcore kinetic diagram of the binding of TCR1 to the AQIPEKIQK-HLA A1101 complex in Example 4;

Figure 4b is a BIAcore kinetic diagram of the binding of TCR2 to the AQIPEKIQK-HLA A1101 complex in Example 4;

Figure 5 shows the experimental results of the activation function of effector cells transfected with TCR2 for T2 cells loaded with short peptides in Example 5;

Figure 6a shows the experimental results of the activation function of effector cells transfected with TCR1 for tumor cell lines in Example 6;

Figure 6b shows the experimental results of the activation function of effector cells transfected with TCR2 for tumor cell lines in Example 6;

Figure 7 is the ELISA experimental results of the activation function of effector cells transfected with TCR2 for tumor cell lines in Example 7;

Figure 8 shows the results of the killing function LDH experiment of effector cells transfected with TCR2 on tumor cell lines in Example 8.

Detailed ways

Before the present application is described, it is to be understood that this application is not limited to the specific 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, and that the scope of the application will be limited only by the appended claims.

Unless otherwise defined, 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 application belongs.

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

the term

T cell receptor (TCR)

The International Immunogenetics Information System (IMGT) can be used to describe TCRs. Natural αβ heterodimeric TCRs have α and β chains. Broadly speaking, each chain contains a variable region (V region), a connecting region (J region) and a constant region (C region). The β chain usually also contains a short variable region (D) between the variable region and the connecting region. area), but the variable area is often considered part of the connecting area. 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 TRAC and TRBC of IMGT.

In the IMGT nomenclature, different numbers for TRAV and TRBV refer to different Vα types and Vβ types, respectively. In the IMGT system, the alpha chain constant domain has the following symbols: TRAC*01, where "TR" represents the T cell receptor gene; "A" represents the alpha chain gene; C represents the constant region; "*01" represents the allele Gene 1. The beta chain constant domain has the following symbols: TRBC1*01 or TRBC2*01, where "TR" represents the T cell receptor gene; "B" represents the beta chain gene; C represents the constant region; "*01" represents the allele 1. The constant region of the alpha chain is uniquely determined, 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 public IMGT database.

The α and β chains of TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain. The variable domain consists of linked variable regions and linker regions. Therefore, in the description and claims of this application, "TCR alpha chain variable domain" refers to the linked TRAV and TRAJ regions, and similarly, "TCR beta chain variable domain" refers to the linked TRBV and TRBD/TRBJ regions.

Each variable region contains three CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, embedded in the framework sequence. The three CDRs in the variable domain of the TCRα chain are CDR1α, CDR2α, and CDR3α; the three CDRs in the variable domain of the TCRβ chain are CDR1β, CDR2β, and CDR3β. The framework sequence of the TCR variable domain of the present application can be of murine or human origin, preferably of human origin. The constant domain of a TCR contains an intracellular part, a transmembrane region, and an extracellular part.

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 are shown in SEQ ID NO: 22 and SEQ ID NO: 23 respectively.

The TCR sequences used in this application are of human origin. In the present application, the terms "polypeptide of the present application", "TCR of the present application" and "T cell receptor of the present application" are used interchangeably. T cell receptors may be referred to as "TCR molecules" or "TCR" for short.

Natural interchain disulfide bonds and artificial interchain disulfide bonds

There is a set of disulfide bonds between the Cα and Cβ chains in the membrane-proximal region of natural TCR, which are called “natural interchain disulfide bonds” in this application. In this application, 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 this application are numbered in order from the N end to the C end. For example, in TRBC1*01 or TRBC2*01, the position numbers are from N The 60th amino acid in sequence from end to C end is P (proline), then in this application it can be described as Pro60 of TRBC1*01 or TRBC2*01 exon 1, or it can also be expressed as TRBC1* 01 or the 60th amino acid of exon 1 of TRBC2*01, and in TRBC1*01 or TRBC2*01, the 61st amino acid in order from the N terminus to the C terminus is Q (glutamine), then this In the application, it can be described as Gln61 in exon 1 of TRBC1*01 or TRBC2*01, or as the 61st amino acid in exon 1 of TRBC1*01 or TRBC2*01, and so on. In this application, the position numbering of the amino acid sequences of variable regions TRAV and TRBV follows the position numbering listed in IMGT. For example, for a certain amino acid in TRAV, the position number listed in IMGT is 46, then this application will describe it as the 46th amino acid of TRAV, and so on. In this application, if there are special instructions for the sequence position numbers of other amino acids, the special instructions will apply.

tumor

The term "tumor" is meant to include all types of cancer cell growth or carcinogenic processes, metastatic tissue or malignantly transformed cells, tissues or organs, regardless of pathological type or stage of infection. Examples of tumors include, without limitation: solid tumors, soft tissue tumors, and metastatic lesions. T cells transduced with the TCR of the present application can be used to treat diseases related to target cells presenting the SSX2 antigen short peptide AQIPEKIQK-HLA A1101 complex, including but not limited to tumors, such as colon cancer, breast cancer, etc.

Application details

During antigen processing, the antigen is degraded within the cell and then carried to the cell surface by MHC molecules. T cell receptors can Recognizes peptide-MHC complexes on the surface of antigen-presenting cells. Therefore, the first aspect of the present application provides a TCR, the TCR has the activity of binding the AQIPEKIQK-HLA A1101 complex, and the TCR alpha chain variable domain of the TCR has at least 90% with SEQ ID NO: 1 Amino acid sequences with sequence homology (for example, may be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, etc.), preferably having at least 95% An amino acid sequence with sequence homology; and/or the TCRβ chain variable domain of the present application is an amino acid sequence with at least 90% sequence homology with SEQ ID NO:7 (for example, it can be at least 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or 100%, etc.), preferably an amino acid sequence with at least 95% sequence homology.

In another preferred embodiment, the TCR described in this application is of human origin.

In a preferred example of the present application, the amino acid sequence of the alpha chain variable domain included in the TCR of the present application is one of SEQ ID NO: 1 and SEQ ID NO: 21; and the amino acid sequence of the beta chain variable domain of the TCR is SEQ ID NO:7.

The recognition of pMHC (antigenic peptide-major histocompatibility complex) by TCR involves two combinations: the binding of TCR to MHC molecules and the binding of TCR to polypeptide antigens. The binding surface on the TCR comes from 6 regions: CDR1, CDR2 and CDR3 on the TCRα and β chains. CDR1 and CDR2 mainly bind to relatively conserved MHC molecules, while CDR3 mainly binds to variable antigen peptides. This binding mode can well ensure that TCR specifically binds MHC molecules and can recognize variable antigens. Among them, CDR3 has the greatest variation, which directly determines the antigen-binding specificity of TCR. The CDR regions of the TCR of this application determined by sequencing are shown in Table 1.

The above-mentioned amino acid sequence of the CDR region of the present application can be embedded into any suitable framework structure to prepare chimeric TCR. In the art, substitutions with amino acids with similar or similar properties generally do not alter the function of the protein. Adding one or more amino acids to the C-terminus and/or N-terminus usually does not change the structure and function of the protein. Therefore, the TCR of the present application also includes at most 5, preferably at most 3, more preferably at most 2, and optimally 1 amino acid (especially the amino acid located outside the CDR region) of the TCR of the present application, with similar properties. Or a TCR that is replaced with a similar amino acid and still maintains its functionality.

In a preferred example of the present application, the constant domain of the TCR molecule of the present application is a human constant domain. Those skilled in the art know or can obtain the human constant domain amino acid sequence by consulting relevant books or the public database of IMGT (International Immunogenetic Information System). For example, the constant domain sequence of the α chain of the TCR molecule in this application can be "TRAC*01", and the constant domain sequence of the β chain of the TCR molecule can be "TRBC1*01" or "TRBC2*01". The 53rd position of the amino acid sequence given in TRAC*01 of IMGT is Arg, which is represented here as: Arg53 of exon 1 of TRAC*01, and so on.

In another preferred example, the TCR is an αβ heterodimeric TCR; preferably, the TCR has an α chain constant region sequence TRAC*01 and a β chain constant region sequence TRBC1*01 or TRBC2*01.

In a preferred example of the present application, the TCR molecule of the present application is a single-chain TCR molecule composed of part or all of the α chain and/or part or all of the β chain. For a description of single-chain TCR molecules, please refer to Chung et al (1994) Proc.Natl.Acad.Sci.USA 91,12654-12658. According to what is described in the literature, those skilled in the art can easily construct single-chain TCR molecules containing the CDRs region of the present application. Specifically, the single-chain TCR molecule contains Vα, Vβ and Cβ, preferably connected in order from the N-terminus to the C-terminus.

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

The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane region. Just like immunoglobulins (antibodies) as antigen recognition molecules, TCR can also be developed for diagnosis and treatment, in which case soluble TCR molecules need to be obtained. Soluble TCR molecules do not include their transmembrane regions. Soluble TCR has a wide range of uses. It can not only be used to study the interaction between TCR and pMHC, but can also be used as a diagnostic tool to detect infections or as a marker for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic compounds or immunostimulatory compounds) to cells presenting specific antigens, and soluble TCRs can also be combined with other molecules (e.g., anti-CD3 antibodies). to redirect T cells to target cells presenting specific antigens.

In order to obtain a soluble TCR, on the one hand, the TCR of the present application can be a TCR in which artificial disulfide bonds are introduced between residues in the constant domains of its α and β chains. Cysteine residues form artificial interchain disulfide bonds between the α and β chain constant domains of the TCR. Cysteine residues can replace other amino acid residues at appropriate positions in the native TCR to form artificial interchain disulfide bonds. For example, the cysteine residues replacing Thr48 in exon 1 of TRAC*01 and Ser57 in exon 1 of TRBC1*01 or TRBC2*01 form a disulfide bond. Other sites where cysteine residues are introduced to form disulfide bonds can also be:

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

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

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

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

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

Pro89 in exon 1 of TRAC*01 and Ala19 in 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, the cysteine residue replaces any set of positions in the constant domains of the above-mentioned α and β chains. Up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or less amino acids can be truncated at one or more C termini of the TCR constant domain of the present application so that it does not include Cysteine residues can be used to achieve the purpose of deleting natural disulfide bonds, or the above purpose can be achieved by mutating the cysteine residue that forms natural disulfide bonds into another amino acid. This application also obtained a soluble TCR specific for SSX2 antigen short peptide. As for the soluble TCR constructed in Example 3 of the present application, its alpha chain variable domain amino acid sequence is SEQ ID NO: 19, and its beta chain variable domain amino acid sequence is SEQ ID NO: 20.

As mentioned above, the TCR of the present application may contain artificial disulfide bonds introduced between residues in the constant domains of its α and β chains. It should be noted that the TCR of the present application may contain the TRAC constant domain sequence, and the TRBC1 or TRBC2 constant domain sequence, whether the constant domains contain or do not contain the artificial disulfide bonds introduced above. The TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence can be linked by natural disulfide bonds present in the TCR.

In addition, regarding stability, Patent Document 201680003540.2 also discloses that the introduction of artificial interchain disulfide bonds between the α chain variable region and β chain constant region of TCR can significantly improve the stability of TCR. Therefore, the TCR of the present application may also contain artificial inter-chain disulfide bonds between the α-chain variable region and the β-chain constant region.

Specifically, the cysteine residues that form an artificial interchain disulfide bond between the alpha chain variable region and the beta chain constant region of the TCR are replaced with:

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

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

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

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

Preferably, such a TCR may comprise (i) all or part of the TCRα chain except its transmembrane domain, and (ii) all or part of the TCRβ chain except its transmembrane domain, wherein (i) and (ii) ) both contain the variable domain of the TCR chain and at least part of the constant domain, and the α chain and β chain form a heterodimer.

More preferably, such a TCR may include an α chain variable domain and a β chain variable domain and all or part of the β chain constant domain except the transmembrane domain, but it does not include an α chain constant domain, and the α chain of the TCR The chain variable domain forms a heterodimer with the β chain.

Mutations may be performed using any suitable method, including, but not limited to, polymerase chain reaction (PCR)-based, restriction enzyme-based cloning, or ligation-independent cloning (LIC) methods. These methods are detailed in many standard molecular biology texts. Further 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 (3rd edition) CSHL Press. More information on the LIC method can be found (Rashtchian, (1995) Curr Opin Biotechnol 6(1):30-6).

The method of generating mutations can be, but is not limited to, screening out TCRs that specifically bind to the AQIPEKIQK-HLA A1101 complex from a diversity library of phage particles displaying such TCRs, as described in the literature (Li, et al (2005) Nature Biotech 23 (3):349-354).

The TCR of the present application can also be provided in the form of a multivalent complex. The multivalent TCR complex of the present application includes a multimer formed by combining two, three, four or more TCRs of the present application. For example, the tetramerization domain of p53 can be used to generate a tetramer, or multiple A complex formed by combining the TCR of this application with another molecule. The TCR complex of the present application can be used to track or target cells presenting specific antigens in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes with such applications.

The TCR of the present application can be used alone, or can be combined with a conjugate in a covalent or other manner, preferably in a covalent manner. The conjugate includes a detectable label (for diagnostic purposes, where the TCR is used to detect the presence of cells presenting the AQIPEKIQK-HLA A1101 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or any of the above. A combination of combinations or couplings.

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

Therapeutic agents that can be combined or coupled to the TCR of the present application include, but are not limited to:

1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539);

2. Biological toxicity (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565);

3. Cytokines such as IL-2 (Gillies et al., 1992, Proceedings of the National Academy of Sciences (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 174, 4381);

5. Antibody scFv fragment (Zhu et al., 1995, International Journal of Cancer (International Journal of Cancer) 62,319);

6. Gold nanoparticles/nanorods (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115);

7. Virus particles (Peng et al., 2004, Gene therapy 11, 1234);

8. Liposomes (Mamot et al., 2005, Cancer research 65, 11631);

9. Nanomagnetic particles;

10. Prodrug-activating enzyme (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL));

11. Chemotherapeutic agents (eg, cisplatin) or any form of nanoparticles, etc.

Antibodies or fragments thereof that bind to the TCR of the present application include anti-T cell or NK-cell determining antibodies, such as anti-CD3, anti-CD28 or anti-CD16 antibodies. The combination of the above antibodies or fragments thereof with the TCR can affect effector cells. Orientation to better target target cells. A preferred embodiment is that the TCR of the present application is combined with an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody. Specifically, the fusion molecule of TCR and anti-CD3 single chain antibody of the present application includes: (1) TCR α chain variable domain amino acid sequence is one of SEQ ID NO: 1 and SEQ ID NO: 21, and the TCR The amino acid sequence of the β chain variable domain is SEQ ID NO:7.

The present application also relates to nucleic acid molecules encoding the TCRs of the present application. The nucleic acid molecules of the present application may be in the form of DNA or RNA. DNA can be a coding strand or a non-coding strand. For example, the nucleic acid sequence encoding the TCR of the present application may be the same as or a degenerate variant of the nucleic acid sequence shown in the sequence listing of the present application. To illustrate the meaning of "degenerate variant", as used herein, "degenerate variant" in this application refers to a protein sequence that encodes SEQ ID NO:1, but is different from the sequence of SEQ ID NO:2 Different nucleic acid sequences.

The full-length sequence of the nucleic acid molecule of the present application 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 application (or its fragments, or its derivatives) can be obtained entirely through chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art.

The present application also relates to vectors containing the nucleic acid molecules of the present application, as well as host cells genetically engineered using the vectors or coding sequences of the present application.

The vectors containing the nucleic acid molecules of the present application include expression vectors, that is, constructs capable of expression in vivo or in vitro. Commonly used vectors include bacterial plasmids, bacteriophages, and animal and plant viruses. Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpesviral vectors, retroviral vectors, lentiviral vectors, or baculoviral vectors. Preferably, the vector can transfer the nucleotide of the present application into cells, such as T cells, so that the cells express HPV16E6 antigen-specific TCR. Ideally, the vector should be capable of sustained high-level expression in T cells.

The host cell contains the vector of the present application or the nucleic acid molecule of the present application is integrated into the chromosome of the host cell. Host cells are selected from: prokaryotic cells and eukaryotic cells, such as Escherichia coli, yeast cells or CHO cells.

In addition, the present application also includes isolated cells expressing the TCR of the present application, which can be T cells, NK cells, NKT cells, etc., and are preferably T cells. The T cells may be derived from T cells isolated from the subject, or may be part of a mixed cell population isolated from the subject, such as a peripheral blood lymphocyte (PBL) population. For example, the cells can be isolated from peripheral blood mononuclear cells (PBMC) and can be CD4 ⁺ helper T cells or CD8 ⁺ cytotoxic T cells. The cells may be in a mixed population of CD4 ⁺ helper T cells/CD8 ⁺ cytotoxic T cells. Generally, the cells can be activated with antibodies (e.g., anti-CD3 or anti-CD28 antibodies) to render them more receptive to transfection, for example, with a vector containing a nucleotide sequence encoding a TCR molecule of the present application. Transfection.

Alternatively, the cells of the present application may also be or be derived from stem cells, such as hematopoietic stem cells (HSC). Gene transfer to HSCs does not result in TCR expression on the cell surface because stem cells do not express CD3 molecules on their surfaces. However, when stem cells differentiate into lymphoid cells that migrate to the thymus The expression of CD3 molecules will initiate the expression of introduced TCR molecules on the surface of thymocytes when they are Lymphoid precursors.

There are many methods suitable for T cell transfection with DNA or RNA encoding the TCR of the present application (eg, Robbins et al., (2008) J. Immunol. 180:6116-6131). T cells expressing the TCR of the present application can be used for adoptive immunotherapy. Those skilled in the art will be aware of many suitable methods for conducting adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).

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

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

The TCR of the present application, the TCR complex or the T cells transfected with the TCR of the present application can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The TCR, multivalent TCR complex or cell of the present application is 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 is available in unit dosage form, usually in sealed containers, and may be provided as part of a kit. Such kits (but not required) include instructions for use. It may include a plurality of such unit dosage forms.

In addition, the TCR of the present application can be used alone, or can be used in combination or coupling 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 used for the administration of a therapeutic agent. The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies that are harmful to the individual receiving the pharmaceutical composition and do not exhibit undue toxicity 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, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.

Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerin, and ethanol. In addition, the carrier may also contain auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc.

Generally, the therapeutic compositions may be prepared as injectables, such as liquid solutions or suspensions; they may also be prepared in solid forms suitable for solution or suspension in liquid vehicles prior to injection.

Once the compositions of the present application are formulated, they may be administered by conventional routes, including but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal or topical administration, preferably parenterally. including 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 application is used for actual treatment, pharmaceutical compositions in various dosage forms can be used according to the usage conditions. Preferably, injections, oral agents, etc. can be exemplified.

The pharmaceutical composition 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, etc. Isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the preparation process can be carried out in conventional ways according to the dosage form.

The pharmaceutical composition of the present application can also be administered in the form of a sustained-release preparation. For example, the TCR of the present application can be incorporated into a pill or microcapsule using a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. Examples of sustained-release polymers include ethylene-vinyl acetate copolymer, polyhydromethaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, or Lactic acid-glycolic acid copolymer, etc. Preferably, biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer can be exemplified.

The present application provides a method for treating SSX2-related diseases, which method includes infusing isolated T cells expressing the TCR of the present application into a patient; preferably, the T cells are derived from the patient himself. Generally, it includes (1) isolating the patient's T cells; (2) transducing the T cells in vitro with the nucleic acid molecule of the present application or a nucleic acid molecule capable of encoding the TCR molecule of the present application; (3) injecting the genetically engineered T cells into the patient in vivo. When the pharmaceutical composition of the present application is used for actual treatment, the TCR or TCR complex of the present application as the active ingredient or the cells presenting the TCR of the present application can be determined according to the weight, age, gender, and symptom severity of each patient to be treated. It is determined rationally, and ultimately it is up to the physician to determine the reasonable dosage.

In addition, the TCR of the present application may also be a hybrid TCR comprising sequences derived from more than one species. For example, studies have shown that murine TCRs are expressed more efficiently in human T cells than human TCRs. Therefore, the TCR of the present application may comprise a human variable domain and a murine constant domain. The drawback of this approach is the potential for triggering an immune response. Therefore, when it is used for adoptive T-cell therapy, there should be an adjustment protocol for immunization. immune suppression to allow engraftment of murine-expressing T cells.

It should be understood that the amino acid names in this article are represented by an internationally accepted single English letter or three English letters. The corresponding relationship between the single English letter and the three English letters of the amino acid name is as follows: 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).

The following specific examples further illustrate this application. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. In addition, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only some, not all, of the embodiments of the present application.

Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as (Sambrook and Russell et al., Molecular Cloning: Laboratory Manual (Molecular Cloning-A Laboratory Manual) (Third Edition) (2001) published by CSHL company) or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise stated. Among them, E.coli DH5α was purchased from Tiangen, E.coli BL21 (DE3) was purchased from Tiangen, E.coli Tuner (DE3) was purchased from Novagen, and plasmid pET28a was purchased from Novagen.

The main advantages of this application are:

(1) The TCR of this application can specifically bind to the SSX2 antigen short peptide complex AQIPEKIQK-HLA A1101.

(2) For positive target cells, the effector cells transduced with the TCR of the present application can be specifically activated. At the same time, the effector cells transduced with the TCR of the present application also have a strong killing function.

The sequences involved in this application are as follows:

Example 1 Cloning SSX2 antigen short peptide-specific T cells

The synthetic short peptide AQIPEKIQK (synthesized by Jiangsu GenScript Biotechnology Co., Ltd.) was used to stimulate peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A1101. The AQIPEKIQK short peptide was renatured with biotin-labeled HLA-A1101 to prepare pMHC haploid. These haplotypes were combined with PE-labeled streptavidin (BD) to form PE-labeled tetramers, and the tetramers and anti-CD8-APC double-positive cells were sorted. The sorted cells were expanded and secondary sorted as described above, followed by monocloning using the limiting dilution method. Monoclonal cells were stained with tetramer, and the double-positive clones were screened. The results of CD8-APC and tetramer-PE double-positive staining of the monoclonal cells are shown in Figure 1. The double-positive clones obtained after layers of screening still need to meet further functional testing.

IFN-γ is a powerful immunoregulatory factor produced by activated T lymphocytes. Therefore, in this example, the number of IFN-γ is detected through the ELISPOT experiment, which is well known to those skilled in the art, to verify the activation function of cells transfected with the TCR of the present application and Antigen specificity. The function and specificity of the T cell clones were further tested through ELISPOT experiments. The effector cells used in the IFN-γ ELISPOT experiment in this example are the T cell clones obtained in this example, and the target cells are T2-A11 (referring to T2 cells transfected with HLA-A1101) and K562-A11 loaded with the AQIPEKIQK short peptide. -SSX2 (referring to K562 cells transfected with HLA-A1101 and SSX2), and the control group was T2-A11 and SW1088 (human brain astroglioma cells) loaded with other short antigen peptides. Among them, T2 cells and K562 cells were purchased from ATCC, and SW1088 was purchased from Guangzhou Saiku Biotechnology Co., Ltd.

First prepare the ELISPOT plate. The ELISPOT experiment steps are as follows: Add each component of the test to the ELISPOT plate in the following order: 20,000 target cells/well, 2,000 effector cells/well, then add 20 μL of the corresponding short peptide to the experimental group and control group. , the final concentration of the short peptide loaded with T2-A11 was 10 ^-5 M. Add 20 μL culture medium (test culture medium) to the blank group, and set up 2 duplicate wells. Then incubate overnight (37°C, 5% _CO2 ). The plate was then washed and subjected to secondary detection and color development. The plate was dried for 1 hour, and the spots formed on the membrane were counted using an immunospot plate reader (ELISPOT READER system; AID Company). The results of ELISPOT activation function verification of T cell clones are shown in Figure 2. The obtained T cell clones highly released IFN-γ for T2-A11 and K562-A11-SSX2 loaded with AQIPEKIQK short peptides, while for T2-A11 and K562-A11-SSX2 loaded with other antigen short peptides, The T2-A11 and SW1088 are basically unresponsive.

Example 2 Obtaining SSX2 antigen short peptide-specific TCR

Quick-RNA ^TM MiniPrep (ZYMO research) was used to extract the total RNA of the antigen short peptide AQIPEKIQK-specific and HLA-A1101-restricted T cell clones (double-positive clones) screened in Example 1. cDNA was synthesized using clontech's SMART RACE cDNA amplification kit, and the primers used were designed in the C-terminal conserved region of the human TCR gene. The sequence was cloned into T vector (TAKARA) and sequenced. It should be noted that the sequences are complementary and do not contain introns.

After sequencing, the α chain and β chain sequences of the TCR expressed by the double-positive clone are as follows:

For convenience of description, it is named TCR1. It was identified that the α chain of TCR1 contains a CDR with the following amino acid sequence:

αCDR1-SSYSPS (SEQ ID NO:13)

αCDR2-YTSAATLV (SEQ ID NO:14)

αCDR3-VVSSGNTPLV (SEQ ID NO:15)

The beta chain of TCR1 contains CDRs with the following amino acid sequence:

βCDR1-SGHVS (SEQ ID NO:16)

βCDR2-FQNEAQ (SEQ ID NO:17)

βCDR3-ASSLVGYEQY (SEQ ID NO:18).

This example further uses the method of site-directed mutagenesis, which is well known to those skilled in the art, to apply the TCR phage display and screening method described by Li et al. ((2005) Nature Biotech 23 (3): 349-354) to TCR1, and obtain TCR2 after mutation. Binding characterization using BIAcore shows that the above-mentioned TCR2 can also specifically bind to the complex AQIPEKIQK-HLA A1101. In addition, the present application unexpectedly discovered that for SSX2-positive target cell lines, effector cells transduced with TCR2 can also be specifically activated and have strong killing functions. The alpha chain of TCR2 has at least 95% sequence homology with the alpha chain of TCR1.

Specifically, compared to TCR1, TCR2 has three mutations in the CDR region of the α chain. The mutations are marked with double underlines. The specific CDRs are shown in Table 2:

Table 2

More specifically, the variable domain sequences of the α and β chains of the TCR of this embodiment are as follows:

(1) TCR1: The α chain variable domain sequence is SEQ ID NO: 1, the β variable domain sequence is SEQ ID NO: 7, and the dissociation equilibrium constant K _D is 2.41E-03M;

(2) TCR2: The alpha chain variable domain sequence is SEQ ID NO: 21 (the mutated residues are underlined), the beta variable domain sequence is SEQ ID NO: 7, and the dissociation equilibrium constant K _D is 1.94E -05M.

Example 3 Expression, refolding and purification of SSX2 antigen short peptide-specific soluble TCR

In this embodiment, a cysteine residue is introduced into the constant domains of the α and β chains of TCR to form artificial inter-chain disulfide bonds, thereby obtaining stable soluble TCR molecules (including soluble TCR1 molecules and soluble TCR2 molecules) in order to evaluate the interaction between TCR and the complex AQIPEKIQK-HLA A1101. The amino acid sequence of the α chain of the soluble TCR1 molecule is SEQ ID NO:19, in which the mutated cysteine residues are represented by bold letters; the amino acid sequence of the β chain is SEQ ID NO:20, in which the mutated cysteine residues are represented by bold letters. The following cysteine residues are indicated in bold letters. The amino acid sequence of the α chain of the soluble TCR2 molecule is SEQ ID NO:24, in which the mutated cysteine residues are represented by bold letters; the amino acid sequence of the β chain is SEQ ID NO:25, in which the mutated cysteine residues are represented by bold letters. The following cysteine residues are indicated in bold letters.

The target gene sequences of the above TCRα and β chains were synthesized and inserted into the expression vector pET28a+ (Novagene ), the upstream and downstream cloning sites are Nco I and Not I respectively. The insert was confirmed to be correct by sequencing.

The expression vectors of TCRα chain and TCRβ chain were transformed into expression bacteria BL21 (DE3) through chemical transformation. The bacteria were cultured in LB medium and induced with a final concentration of 0.5mM IPTG at OD ₆₀₀ = 0.6. The α and β chains of TCR The inclusion bodies formed after expression were extracted with BugBuster Mix (Novagene) and washed repeatedly with BugBuster solution. The inclusion bodies were finally dissolved in buffer (6M guanidine hydrochloride, 10mM dithiothreitol (DTT), 10mM ethylene glycol). aminetetraacetic acid (EDTA), 20mM Tris, pH 8.1).

The dissolved TCRα chain and TCRβ chain were quickly mixed in the buffer (5M urea, 0.4M arginine, 20mM Tris (pH8.1), 3.7mM cystamine, 6.6mMβ-mercapoethylamine, 4°C) at a mass ratio of 1:1. ), the final concentration is 60mg/mL. After mixing, the solution was dialyzed in 10 times the volume of deionized water (4°C). After 12 hours, the deionized water was replaced with buffer (20mM Tris, pH 8.0) and continued to be dialyzed at 4°C for 12 hours. After dialysis, the solution was filtered through a 0.45 μm filter membrane and purified through an anion exchange column (HiTrap Q HP, 5 mL, GE Healthcare). The elution peak containing TCR of successfully renatured α and β dimers was confirmed by SDS-PAGE gel. TCR was then further purified by gel filtration chromatography (HiPrep 16/60, Sephacryl S-100HR, GE Healthcare). The purity of the purified TCR was greater than 90% as measured by SDS-PAGE, and the concentration was determined by the BCA method.

The SDS-PAGE electrophoresis gel pattern of soluble TCR1 obtained in this example is shown in Figure 2a and Figure 2b, wherein the right lane of Figure 2a is a non-reducing gel, and the right lane of Figure 2b is a reducing gel, Figure 2a and Figure The left lanes of 2b are molecular weight markers; the SDS-PAGE electrophoresis gel images of soluble TCR2 are shown in Figure 2c and Figure 2d. The left lane of Figure 2c is a non-reducing gel, and the left lane of Figure 2d is a reducing gel. , the right lanes in Figure 2c and Figure 2d are molecular weight markers.

Example 4 Combination Characterization

This example uses the BIAcore T200 real-time analysis system to detect the binding activity of the soluble TCR molecules obtained according to Example 3 and the AQIPEKIQK-HLA A1101 complex. AQIPEKIQK-HLA A1101 complex is prepared using methods well known to those skilled in the art. The main processes include purification, renaturation, re-purification and biotinylation.

Add the anti-streptavidin antibody (GenScript) to the coupling buffer (10mM sodium acetate buffer, pH 4.77), and then flow the antibody through the CM5 chip that has been previously activated with EDC and NHS to immobilize the antibody on the chip. surface, and finally use ethanolamine hydrochloric acid solution to seal the unreacted activated surface to complete the coupling process. The coupling level is approximately 15,000RU. Flow low-concentration streptavidin through the antibody-coated chip surface, then flow the AQIPEKIQK-HLA A1101 complex through the detection channel, and use the other channel as the reference channel, and then add 0.05mM biotin at 10μL/min. The flow rate flows through the chip for 2 minutes to block the remaining binding sites of streptavidin.

BIAcore Evaluation software was used to calculate the kinetic parameters, and the kinetic spectrum of the combination of the soluble TCR molecule of the present application and the AQIPEKIQK-HLA A1101 complex was obtained. The maps are shown in Figure 3a and Figure 3b respectively. Figure 3a shows the complex between TCR1 and AQIPEKIQK-HLA A1101. Figure 3b shows the BIAcore kinetic map of TCR2 binding to the AQIPEKIQK-HLA A1101 complex. The spectrum shows that the obtained soluble TCR molecules are able to bind to the AQIPEKIQK-HLA A0101 complex.

Example 5 Activation function experiment of effector cells transfected with TCR against target cells loaded with short peptides

IFN-γ is a powerful immunoregulatory factor produced by activated T lymphocytes. Therefore, in this example, the number of IFN-γ is detected through the ELISPOT experiment, which is well known to those skilled in the art, to verify the activation function of cells transfected with the TCR of the present application. and antigen specificity. The TCR2 molecules obtained in Example 2 were transfected into CD3 ⁺ T cells isolated from the blood of healthy volunteers as effector cells, and the same volunteers were CD3 ⁺ T cells transfected with TCR (A6) targeting other antigens were used as controls. The target cells used were T2 cells loaded with SSX2 antigen short peptide AQIPEKIQK, loaded with other peptides or empty. Experimental steps: First prepare the ELISPOT plate. The ELISPOT plate is activated and coated with ethanol and kept overnight at 4°C. On the first day of the experiment, remove the coating solution, wash and block, incubate at room temperature for two hours, remove the blocking solution, and add each component of the test to the ELISPOT plate: 1×10 ⁴ /well for target cells and 2× for effector cells. 10 ³ /well (calculated based on the positive rate of transfection), the final concentration of short peptide is 1×10 ^-6 M/well, and two duplicate wells are set up. Incubate overnight (37°C, 5% _CO2 ).

On the second day of the experiment, wash the plate and perform secondary detection and color development, dry the plate, and then use an immunospot plate reader (ELISPOT READER system; AID20 Company) to count the spots formed on the membrane.

For T2 cells loaded with short peptides, the experimental results of the activation function of effector cells transfected with TCR2 are shown in Figure 5. For the target cells loaded with the SSX2 antigen short peptide AQIPEKIQK, the effector cells transfected with the TCR2 had a significant activation effect, while the effector cells transfected with other TCRs were basically inactive; at the same time, the effector cells transfected with the TCR2 were Other peptides or empty target cells showed little response.

Example 6 Experiment on the activation function of effector cells transfected with TCR for tumor cell lines

In order to once again verify the activation function and specificity of the effector cells transfected with the TCR, this example uses tumor cell lines to conduct two rounds of ELISPOT experiments. TCRs (TCR1, TCR2) were transfected into CD3 ⁺ T cells isolated from the blood of healthy volunteers as effector cells, and CD3 ⁺ T cells from the same volunteer transfected with other TCR (A6) were used as negative controls.

The SSX2-positive tumor cell line used in the first round of experiments was SK-MEL-28 (SSX2 overexpression), the negative tumor cell line was SK-MEL-28, and the TCR was TCR1;

The SSX2-positive tumor cell lines used in the second round of experiments were SK-MEL-28 (SSX2 overexpression) and Huh-1-A11 (HLA-A11 overexpression), and the negative tumor cell lines were SNU423, HUCC-T1, and SK-MEL. -1. MOG-G-UVW contains only effector cells, and the TCR is TCR2. Among them, SK-MEL-28, SNU423, HUCC-T1, SK-MEL-1, and MOG-G-UVW are all from Guangzhou Saiku Biotechnology Co., Ltd., and Huh-1 is from Nanjing Kebai Biotechnology Co., Ltd.

The experimental procedures are as shown in Example 5, in which the components added to the ELISPOT plate are: 2×10 ⁴ /well for target cells and 2×10 ³ /well for effector cells (calculated based on the positive rate of transfection).

The results of the two rounds of experiments are shown in Figures 6a and 6b. Figure 6a is the experimental results of the activation function of effector cells transfected with TCR1 against tumor cell lines; Figure 6b is the effect of transfection with TCR2 against tumor cell lines. Cell activation function experimental results.

For SSX2-positive tumor cell lines, effector cells transfected with TCR have a significant activation effect, while effector cells transfected with other TCRs and/or null transduced are inactive; at the same time, effector cells transfected with the TCR of this application are negative for SSX2. Tumor cell lines are essentially inactive.

Example 7 Experiment on the activation function of effector cells transfected with TCR of the present application for tumor cell lines

In order to once again verify the activation function and specificity of the effector cells transfected with the TCR, this example uses tumor cell lines to conduct ELISA experiments. The TCR of the present application (TCR2) was transfected into CD3 ⁺ T cells isolated from the blood of healthy volunteers as effector cells, and CD3 ⁺ T cells of the same volunteer transfected with other TCR (A6) were used as negative controls. The SSX2-positive tumor cell lines used in the experiment were HuH-1-A1101-B2M (overexpression of HLA-A1101 and B2M), and the negative tumor cell lines were HepG2-A1101-B2M, HepG2, HK-2-A1101, HK-2, and Eca. -109-A1101, Eca-109, SNU-423; among them, HuH-1 was purchased from Nanjing Kebai Biotechnology Co., Ltd., SNU423 and HK-2 were purchased from Guangzhou Saiku Biotechnology Co., Ltd., and HepG2 was purchased from the Cell Bank of the Chinese Academy of Sciences. , Eca-109 was purchased from Guangzhou Weijia Biotechnology Co., Ltd.

Experimental steps: Inoculate cells on the first day of the experiment: Inoculate the cell suspension of the tumor cell line and the TCR of the present application on a U-shaped plate: 3×10 ⁴ /well for target cells and 3×10 ⁴ /well for effector cells ( Calculated based on the positive rate of transfection), set up three duplicate wells, and incubate overnight (37°C, 5% CO ₂ ). IL-2 was coated with antibody on the ELISA plate and placed in a refrigerator at 4°C overnight.

On the second day of the experiment, remove the coating solution, wash and block, incubate at room temperature for two hours, and remove the blocking solution. Take the co-supernatant of the tumor cell line and the TCR and add it to the ELISA plate. Take another standard protein, dilute it 10 times, add it to the ELISA plate, and shake at room temperature for two hours. Wash the plate, add biotin-labeled secondary antibody, shake at room temperature for one hour, wash and add SA-HRP, shake at room temperature for one hour, wash, add TMB for color development for 5 to 10 minutes, terminate the reaction, and perform detection. The detection wavelength is 450nm.

For tumor cell lines, the ELISA experimental results of the activation function of effector cells transfected with the TCR2 are shown in Figure 7. For positive tumor cell lines, the effector cells transfected with the TCR2 have an obvious activation effect, while those transfected with other TCRs and null-transduced effector cells were inactive; at the same time, the effector cells transfected with the TCR2 were basically inactive against SSX2-negative tumor cell lines.

Example 8 Aiming at tumor cell lines, the killing function LDH experiment of effector cells transfected with TCR of the present application

Lactate dehydrogenase (LDH) is abundant in the cytoplasm and cannot pass through the cell membrane normally. It can be released outside the cell when the cell is damaged or dead. At this time, the LDH activity in the cell culture medium is proportional to the number of cell death. This example uses non-radioactive cytotoxicity experiments well known to those skilled in the art to measure the release of LDH, thereby verifying the killing function of cells transfected with the TCR (TCR2) of the present application.

CD3 ⁺ T cells isolated from the blood of healthy volunteers were used to transfect the TCR of the application as effector cells, and CD3 ⁺ T cells of the same volunteer transfected with other TCR (A6) were used as negative controls. The SSX2-positive tumor cell lines used were SK-MEL-28-SXX2 (SXX2 overexpression) and Huh-1-A11 (HLA-A11 overexpression), and the negative tumor cell lines were SK-MEL-5, SNU23, and SK-MEL. -1, SKM-1.

Experimental steps: First prepare the LDH plate, first add 3×10 ⁴ cells/well for target cells and 3×10 ⁴ cells/well for effector cells (calculated according to the positive rate of transfection) into the corresponding wells, and set up three replicates. hole. At the same time, set the effector cell spontaneous release hole, the target cell spontaneous release hole, the target cell maximum hole, the volume correction control hole and the culture medium background control hole. Incubate overnight (37°C, 5% _CO2 ). On the second day of the experiment, the color development was detected, and after the reaction was terminated, the absorbance value was recorded at 490 nm with a microplate reader (Bioteck).

For tumor cell lines, the killing function LDH experimental results of the effector cells transfected with the TCR2 are shown in Figure 8. For the SSX2-positive tumor cell lines, the effector cells transfected with the TCR2 also showed strong killing efficacy, and the transfection T cells with other TCRs basically did not react; at the same time, T cells transfected with the TCR2 basically had no killing effect on negative tumor cell lines, further demonstrating the very good specific killing function of the cells transfected with the TCR2.

In summary, the TCR provided by this application can specifically bind to the SSX2 antigen short peptide complex AQIPEKIQK-HLA A1101. Targeting positive target cells, the effector cells that transduce the TCR can be specifically activated. At the same time, the TCR of this application can be transduced. The effector cells also have strong killing function. The TCR can be used to deliver cytotoxic agents or immunostimulatory agents to target cells, or be transformed into T cells so that T cells expressing the TCR can destroy tumor cells. The TCR has important applications in adoptive immunotherapy. value.

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

Claims

A T cell receptor (TCR), characterized in that it has the activity of binding the AQIPEKIQK-HLA A1101 complex, and the α chain variable domain of the TCR is at least the same as the amino acid sequence shown in SEQ ID NO:1 The amino acid sequence with 90% sequence homology; and the amino acid sequence of the three CDR regions of the TCRβ chain variable domain are:

βCDR1-SGHVS (SEQ ID NO:16)

βCDR2-FQNEAQ (SEQ ID NO:17)

βCDR3-ASSLVGYEQY (SEQ ID NO:18).
The TCR of claim 1, wherein the TCR is an αβ heterodimeric TCR.
The TCR according to claim 2, characterized in that the TCR has an alpha chain constant region sequence TRAC*01 and a beta chain constant region sequence TRBC1*01 or TRBC2*01.
The TCR according to claim 1, the amino acid sequence of the β chain variable domain of the TCR is SEQ ID NO: 7.
The TCR of claim 1, wherein the alpha chain variable domain of the TCR is an amino acid sequence that has at least 95% sequence homology with the amino acid sequence shown in SEQ ID NO:1 and the TCR The amino acid sequence of the β chain variable domain is SEQ ID NO:7.
The TCR according to claim 1, characterized in that the TCR has a CDR selected from the following group:
The TCR according to claim 1, characterized in that the TCR is selected from:

(1) The alpha chain variable domain sequence is SEQ ID NO: 1, and the beta variable domain sequence is SEQ ID NO: 7;

(2) The alpha chain variable domain sequence is SEQ ID NO: 21, and the beta variable domain sequence is SEQ ID NO: 7.
The TCR according to claim 1, characterized in that the TCR comprises (i) TCRα chain variable domain and all or part of the TCRα chain constant region except the transmembrane domain; and (ii) TCRβ chain variable domain and all or part of the TCRβ chain constant region except the transmembrane domain.
The TCR according to claim 1, characterized in that an artificial interchain disulfide bond is contained between the α chain constant region and the β chain constant region of the TCR; preferably, the half of the artificial interchain disulfide bond is formed. Cystine residues are substituted for one or more groups of positions selected from:

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

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

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

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

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

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

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

and Tyr10 of exon 1 of TRAC*01 and Glu20 of exon 1 of TRBC1*01 or TRBC2*01.
The TCR of claim 1, wherein the TCR is soluble.
The TCR of claim 1, wherein the TCR is a single-chain TCR; preferably, the single-chain TCR is an α chain variable domain and a β chain variable domain composed of a flexible short peptide sequence (linker). connected.
The TCR of claim 1, wherein a conjugate is bound to the C- or N-terminus of at least one of the α chain and β chain of the TCR; preferably, the conjugate is an anti- CD3 antibodies.
A multivalent TCR complex, characterized in that it contains at least two TCR molecules, and at least one of the TCR molecules is the TCR described in any one of claims 1-12.
A nucleic acid molecule, characterized in that the nucleic acid molecule comprises a nucleic acid sequence encoding the TCR of any one of claims 1-12 or its complementary sequence.
A vector, characterized in that the vector contains the nucleic acid molecule described in claim 14.
A host cell, characterized in that the host cell contains the vector or chromosome described in claim 15 The exogenous nucleic acid molecule described in claim 14 is integrated into it.
An isolated cell, characterized in that the cell expresses the TCR according to any one of claims 1-12; preferably, the cell is a T cell.
A pharmaceutical composition, characterized in that the composition contains a pharmaceutically acceptable carrier and the TCR described in any one of claims 1-12, or the TCR complex described in claim 13, or the The cell described in claim 17.
A method for treating diseases, characterized by comprising administering the TCR described in any one of claims 1 to 12, or the TCR complex described in claim 13, or the TCR complex described in claim 17 to a subject in need of treatment. The cells described in claim 18, or the pharmaceutical composition described in claim 18.
The use of the T cell receptor according to any one of claims 1 to 12, the TCR complex described in claim 13 or the cell described in claim 17, characterized in that it is used to prepare drugs for treating tumors; Preferably, the tumor is an SSX2 positive tumor.
The T cell receptor described in any one of claims 1 to 12, the TCR complex described in claim 13 or the cell described in claim 17 is used as a drug for treating tumors; preferably, the tumor is SSX2-positive tumors.
A method for preparing the T cell receptor according to any one of claims 1 to 12, characterized by comprising the steps:

(i) culturing the host cell described in claim 16 to express the T cell receptor described in any one of claims 1-12; and,

(ii) Isolate or purify the T cell receptor.