WO2022262835A1

WO2022262835A1 - Tcr for identifying afp antigen and coding sequence thereof

Info

Publication number: WO2022262835A1
Application number: PCT/CN2022/099352
Authority: WO
Inventors: 杨东雪; 钟时; 廖有平; 郑文静; 温桥生; 马瑞娟; 孙含丽
Original assignee: 香雪生命科学技术（广东）有限公司
Priority date: 2021-06-18
Filing date: 2022-06-17
Publication date: 2022-12-22
Also published as: CN115490767A

Abstract

The present application provides a T cell receptor (TCR) capable of specifically binding to a short peptide TSSELMAITR (SEQ ID NO: 9) derived from an AFP antigen, wherein the antigenic short peptide TSSELMAITR (SEQ ID NO: 9) may form a complex with HLA A1101 and be presented to the cell surface together with HLA A1101. The present application also provides a nucleic acid molecule encoding the TCR and a vector containing the nucleic acid molecule. In addition, the present application also provides a cell for transducing the TCR of the present application.

Description

A TCR for recognizing AFP antigen and its coding sequence

technical field

This application belongs to the field of biotechnology, and specifically relates to the TCR and its coding sequence that can recognize short peptides derived from the AFP antigen. The application also relates to the AFP-specific T cells obtained by transducing the above-mentioned TCR, and their role in the prevention and treatment of AFP. use in disease.

Background technique

AFP (αFetoprotein), also known as α-fetoprotein, is a protein expressed during embryonic development and is the main component of embryonic serum. During development, AFP has relatively high expression levels in the yolk sac and liver, and is subsequently repressed. In hepatocellular carcinoma, the expression of AFP is activated (Butterfield et al. J Immunol., 2001, Apr 15; 166(8): 5300-8). After AFP 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. TSSELMAITR (SEQ ID NO: 9) is a short peptide derived from the AFP antigen and is a target for the treatment of AFP-related diseases.

T cell adoptive immunotherapy is the transfer of reactive T cells specific to target cell antigens into the patient's body so that they can act against the target cells. T cell receptor (TCR) is a membrane protein on the surface of T cells, which can recognize short antigenic peptides on the surface of corresponding target cells. In the immune system, direct physical contact between T cells and antigen-presenting cells (APCs) is triggered by the combination of antigenic short peptide-specific TCR and short peptide-major histocompatibility complex (pMHC complex), and then T cells and The other cell membrane surface molecules of APC and APC interact, causing 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. Therefore, those skilled in the art are committed to isolating the TCR with specificity to the short peptide of AFP antigen, and transducing T cells with the TCR to obtain T cells with specificity to the short peptide of AFP antigen, so that they can be used in cellular immunotherapy. play a role in.

Contents of the invention

The present application provides a T cell receptor that recognizes short peptides of AFP antigens.

In the first aspect of the present application, a T cell receptor (TCR) is provided, and the TCR can bind to the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex.

In another preferred example, the TCR comprises a TCRα chain variable domain and a TCRβ chain variable domain, and the amino acid sequence of CDR3 of the TCRα chain variable domain is SGGSNYKLT (SEQ ID NO: 12); and/or the The amino acid sequence of CDR3 of the TCR β chain variable domain is ASSPGTGVGYT (SEQ ID NO: 15).

In another preferred example, the three complementarity determining regions (CDRs) of the variable domain of the TCRα chain are:

αCDR1-DSVNN (SEQ ID NO: 10);

αCDR2-IPSGT (SEQ ID NO: 11);

αCDR3-SGGSNYKLT (SEQ ID NO: 12); and/or

The three complementarity-determining regions of the TCRβ chain variable domain are:

βCDR1-SEHNR (SEQ ID NO: 13);

βCDR2-FQNEAQ (SEQ ID NO: 14);

βCDR3-ASSPGTGVGYT (SEQ ID NO: 15).

In another preferred example, the TCR comprises a TCRα chain variable domain and a TCRβ chain variable domain, and the TCRα chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1; and/ Or the TCR beta chain variable domain is an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5.

In another preferred example, the TCR comprises the amino acid sequence of an alpha chain variable domain of SEQ ID NO: 1.

In another preferred example, the TCR comprises the amino acid sequence of the β-chain variable domain of SEQ ID NO: 5.

In another preferred embodiment, the TCR is an αβ heterodimer, which comprises a TCR α chain constant region TRAC*01 and a TCR β chain constant region TRBC1*01 or TRBC2*01.

In another preferred example, the amino acid sequence of the α chain of the TCR is SEQ ID NO: 3 and/or the amino acid sequence of the β chain of the TCR is SEQ ID NO: 7.

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

In another preferred embodiment, the TCR is soluble.

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

In another preferred example, the TCR is formed by linking the α-chain variable domain and the β-chain variable domain through a peptide linker sequence.

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

In another preferred example, the TCR is at the 11th, 13th, 19th, 21st, 53rd, 76, 89, 91st, or 94th amino acid position in the α-chain variable region, and/or at the reciprocal amino acid position of the J gene short peptide of the α-chain There are one or more mutations in the 3rd, penultimate 5th or penultimate 7th position; and/or the TCR is in the 11th, 13th, 19th, 21st, 53rd, 76th, 89th, 91st amino acid of the β-chain variable region , or position 94, and/or one or more mutations in the second-to-last, fourth-to-last or sixth-to-last amino acid positions of the short peptide of the β-chain J gene, wherein the amino acid positions are numbered according to IMGT (International Immunogenetics Information system) at the location number listed.

In another preferred example, the amino acid sequence of the α-chain variable domain of the TCR comprises SEQ ID NO: 32 and/or the amino acid sequence of the β-chain variable domain of the TCR comprises SEQ ID NO: 34.

In another preferred example, the amino acid sequence of the TCR is SEQ ID NO: 30.

In another preferred example, 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 except the transmembrane domain All or part of the TCRβ chain constant region.

In another preferred embodiment, the cysteine residue forms an artificial disulfide bond between the α and β chain constant domains of the TCR.

In another preferred example, the cysteine residues forming artificial disulfide bonds in the TCR 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 of the TCR is SEQ ID NO: 26 and/or the amino acid sequence of the β chain of the TCR is SEQ ID NO: 28.

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

In another preferred example, it is characterized in that the cysteine residues forming artificial interchain disulfide bonds in the TCR are substituted for one or more groups of sites selected from the following:

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

The 47th amino acid of TRAV and the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1;

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

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

In another preferred example, the TCR comprises 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 contain an α-chain constant domain, and the TCR The α-chain variable domain forms a heterodimer with the β-chain.

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

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

Preferably, the therapeutic agent is an anti-CD3 antibody.

The second aspect of the present application 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 application.

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

In another preferred example, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 2 or SEQ ID NO: 33 encoding the variable domain of the TCRα chain.

In another preferred example, the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 6 or SEQ ID NO: 35 encoding the variable domain of the TCR β chain.

In another preferred example, the nucleic acid molecule comprises a nucleotide sequence encoding a TCRα chain of SEQ ID NO: 4 and/or comprises a nucleotide sequence encoding a TCR β chain of SEQ ID NO: 8.

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 lentivirus Viral vector.

The fifth aspect of the present application provides an isolated host cell containing the vector described in the fourth aspect of the present application or the exogenous nucleic acid molecule described in the third aspect of the application integrated in the genome .

The sixth 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 cell is a T cell, NK cells, NKT cells or stem cells.

The seventh aspect of the present application provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR described in the first aspect of the present application, the TCR complex described in the second aspect of the present application, the TCR complex described in the second aspect of the present application, The nucleic acid molecule described in the third aspect of the application, the vector described in the fourth aspect of the application, or the cell described in the sixth aspect of the application.

The eighth aspect of the present application provides the use of the T cell receptor described in the first aspect of the present application, or the TCR complex described in the second aspect of the present application, or the cells described in the sixth aspect of the present application, for Medicines for treating tumors or autoimmune diseases are prepared, preferably, the tumors are liver cancers.

The ninth aspect of the present application provides the T cell receptor described in the first aspect of the present application, or the TCR complex described in the second aspect of the present application, or the cells described in the sixth aspect of the present application for treating tumors or Drugs for autoimmune diseases; preferably, the tumor is liver cancer.

The tenth aspect of the present application provides a method for treating diseases, comprising administering an appropriate amount of the T cell receptor described in the first aspect of the present application or the TCR complex described in the second aspect of the present application to a subject in need of treatment , or the cell described in the sixth aspect of the present application, or the pharmaceutical composition described in the seventh aspect of the present application; preferably, the disease is a tumor, preferably, the tumor is liver cancer.

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 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, Figure 1b, Figure 1c, Figure 1d, Figure 1e and Figure 1f are the amino acid sequence of the TCRα chain variable domain, the nucleotide sequence of the TCRα chain variable domain, the amino acid sequence of the TCRα chain, the nucleotide sequence of the TCRα chain, and The amino acid sequence of the TCRα chain of the leader sequence and the nucleotide sequence of the TCRα chain having the leader sequence.

Figure 2a, Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the amino acid sequence of the TCRβ chain variable domain, the TCRβ chain variable domain nucleotide sequence, the TCRβ chain amino acid sequence, the TCRβ chain nucleotide sequence, and The amino acid sequence of the TCRβ chain of the leader sequence and the nucleotide sequence of the TCRβ chain with the leader sequence.

Figure 3 shows the double positive staining results of CD8 ⁺ -APC and tetramer-PE of monoclonal cells.

Figure 4a and Figure 4b are the amino acid sequence and nucleotide sequence of the soluble TCRα chain, respectively.

Figure 5a and Figure 5b are the amino acid sequence and nucleotide sequence of the soluble TCRβ chain, respectively.

Figures 6a and 6b are gel images of purified soluble TCRs. Wherein, the right swimming lanes of Figures 6a and 6b are reducing gels and non-reducing gels respectively, and the left swimming lanes are molecular weight markers.

Figure 7a and Figure 7b are the amino acid sequence and nucleotide sequence of the single-chain TCR, respectively, and the amino acid sequence and nucleotide sequence of the linker are underlined.

Figure 8a and Figure 8b are the amino acid sequence and nucleotide sequence of the variable domain of the single-chain TCRα chain, respectively.

Figure 9a and Figure 9b are the amino acid sequence and nucleotide sequence of the single-chain TCRβ chain variable domain, respectively.

Figures 10a and 10b are gel images of purified soluble single-chain TCRs. The right swimming lanes in Figures 10a and 10b are reducing gels and non-reducing gels respectively, and the left swimming lanes are molecular weight markers.

Figure 11 is a BIAcore kinetic profile of the combination of the soluble TCR of the present application and the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex.

Figure 12 is the BIAcore kinetic profile of the combination of the soluble single-chain TCR of the present application and the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex.

Figure 13 shows the results of the ELISPOT activation function verification of the obtained T cell clones.

Figure 14 is the result of ELISPOT activation function verification of T2 cells transfected with TCR effector cells of the present application.

Fig. 15 is the result of ELISPOT activation function verification of effector cells transfected with the TCR of the present application for tumor cell lines.

Figure 16 is the verification result of the killing function of the effector cells transfected with the TCR of the present application.

detailed description

After extensive and in-depth research, the inventors have found a TCR that can specifically bind to the AFP antigen short peptide TSSELMAITR (SEQ ID NO: 9), which can form with HLA A1101 The complex is presented to the cell surface together. The present application also provides a nucleic acid molecule encoding the TCR and a vector comprising the nucleic acid molecule. In addition, the present application also provides cells transduced with the TCR of the present application.

the term

MHC molecules are proteins of the immunoglobulin superfamily and can be class I or class II MHC molecules. Therefore, it is specific for the presentation of antigens, and different individuals have different MHCs, which can present different short peptides in a protein antigen to the surface of their respective APC cells. Human MHC is often referred to as HLA genes or HLA complexes.

T cell receptor (TCR), is the sole receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC). 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.

TCR 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. TCR heterodimers consist of alpha and beta chains in 95% of T cells, whereas 5% of T cells have TCRs consisting of gamma and delta chains. The native αβ heterodimeric TCR has an α chain and a β chain, which constitute the subunits of the αβ heterodimeric TCR. Broadly speaking, each chain of α and β contains a variable region, a connecting region, and a constant region, and the β chain usually also contains a short variable region between the variable region and the connecting region, but this variable region is often regarded as the connecting region a part of. Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are embedded in framework regions. The CDR region determines the combination of the TCR and the pMHC complex, in which CDR3 is recombined from the variable region and the linker region, known as the hypervariable region. The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain, the variable domains are composed of linked variable and linker regions. The sequence of the TCR constant domain can be found in the public database of the International Immunogenetics Information System (IMGT). For example, the constant domain sequence of the α chain of the TCR molecule is "TRAC*01", and the constant domain sequence of the β chain of the TCR molecule is "TRBC1* 01" or "TRBC2*01". In addition, the α and β chains of TCR also contain a transmembrane region and a cytoplasmic region, which is very short.

In the present application, the terms "polypeptide of the present application", "TCR of the present application", "T cell receptor of the present application" 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 this application. In the present application, artificially introduced interchain covalent disulfide bonds whose positions are different from those of natural interchain disulfide bonds are called "artificial interchain disulfide bonds".

In order to facilitate the description of the position of the disulfide bond, the position numbers of the amino acid sequences of TRAC*01 and TRBC1*01 or TRBC2*01 in this application are numbered in sequence from the N-terminal to the C-terminal, such as TRBC1*01 or TRBC2*01 , the 60th amino acid in the sequence from N-terminal to C-terminal is P (proline), then it can be described as Pro60 of exon 1 of TRBC1*01 or TRBC2*01 in this application, and can also be described as It is expressed as the 60th amino acid of exon 1 of TRBC1*01 or TRBC2*01, and for example, in TRBC1*01 or TRBC2*01, the 61st amino acid in the order from N-terminal to C-terminal is Q (glutamine amide), then it can be described as Gln61 of TRBC1*01 or TRBC2*01 exon 1 in this application, and it can also be expressed as the 61st amino acid of TRBC1*01 or TRBC2*01 exon 1, other and so on. In the present application, the position numbers of the amino acid sequences of the variable regions TRAV and TRBV are according to the position numbers 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 this application, and so on. In this application, if there are special instructions for the sequence position numbers of other amino acids, the special instructions shall be followed.

Detailed description of the invention

TCR molecules

During antigen processing, antigens are degraded inside the cell and then carried to the cell surface by MHC molecules. T cell receptors recognize peptide-MHC complexes on the surface of antigen-presenting cells. Therefore, the first aspect of the application provides a TCR molecule capable of binding TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex. Preferably, said TCR molecule is isolated or purified. The α and β chains of this TCR each have 3 complementarity determining regions (CDRs).

In a preferred embodiment of the present application, the α chain of the TCR comprises a CDR having the following amino acid sequence:

αCDR1-DSVNN (SEQ ID NO: 10);

αCDR2-IPSGT (SEQ ID NO: 11);

αCDR3-SGGSNYKLT (SEQ ID NO: 12); and/or

βCDR1-SEHNR (SEQ ID NO: 13);

βCDR2-FQNEAQ (SEQ ID NO: 14);

βCDR3-ASSPGTGVGYT (SEQ ID NO: 15).

A chimeric TCR can be prepared by embedding the amino acid sequences of the above CDR regions of the present application into any suitable framework structure. As long as the framework structure is compatible with the CDR region of the TCR of the present application, those skilled in the art can design or synthesize TCR molecules with corresponding functions based on the CDR region disclosed in the present application. Therefore, the TCR molecule in the present application refers to a TCR molecule comprising the above-mentioned α and/or β chain CDR region sequence and any suitable framework structure. The TCR α chain variable domain of the present application is an amino acid sequence having at least 90%, preferably 95%, and more preferably 98% sequence identity with SEQ ID NO: 1; and/or the TCR β chain variable domain of the present application is an amino acid sequence identical to SEQ ID NO: 5 amino acid sequences having at least 90%, preferably 95%, more preferably 98% sequence identity.

In a preferred example of the present application, the TCR molecule of the present application is a heterodimer composed of α and β chains. Specifically, on the one hand, the α chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the amino acid sequence of the variable domain of the α chain comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 10) of the above α chain. ID NO: 11) and CDR3 (SEQ ID NO: 12). Preferably, the TCR molecule comprises an alpha chain variable domain amino acid sequence SEQ ID NO: 1. More preferably, the amino acid sequence of the α-chain variable domain of the TCR molecule is SEQ ID NO:1. On the other hand, the β chain of the heterogeneous dimeric TCR molecule comprises a variable domain and a constant domain, and the amino acid sequence of the variable domain of the β chain comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 13) of the above β chain. NO: 14) and CDR3 (SEQ ID NO: 15). Preferably, the TCR molecule comprises the amino acid sequence of the β chain variable domain of SEQ ID NO: 5. More preferably, the amino acid sequence of the β chain variable domain of the TCR molecule is SEQ ID NO: 5.

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 the description of single-chain TCR molecules, reference can be made to Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658. According to the literature, those skilled in the art can easily construct single-chain TCR molecules comprising the CDRs region of the present application. Specifically, the single-chain TCR molecule comprises Vα, Vβ and Cβ, preferably connected in order from N-terminus to C-terminus.

The α-chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12) of the above-mentioned α-chain. Preferably, the single-chain TCR molecule comprises the amino acid sequence of an α-chain variable domain of SEQ ID NO: 1. More preferably, the amino acid sequence of the α-chain variable domain of the single-chain TCR molecule is SEQ ID NO:1. The β chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) of the above β chain. Preferably, the single-chain TCR molecule comprises the amino acid sequence of the β-chain variable domain of SEQ ID NO: 5. More preferably, the amino acid sequence of the β-chain variable domain of the single-chain TCR molecule is SEQ ID NO: 5.

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 Immunogenetics Information System). For example, the constant domain sequence of the α chain of the TCR molecule in the present application may be "TRAC*01", and the constant domain sequence of the β chain of the TCR molecule may be "TRBC1*01" or "TRBC2*01". The 53rd position of the amino acid sequence given in TRAC*01 of IMGT is Arg, which is expressed here as: Arg53 of exon 1 of TRAC*01, and so on. Preferably, the amino acid sequence of the α chain of the TCR molecule of the present application is SEQ ID NO: 3, and/or the amino acid sequence of the β chain is SEQ ID NO: 7.

The naturally occurring TCR is a membrane protein that is stabilized by its transmembrane region. Like immunoglobulin (antibody) as an antigen recognition molecule, TCR can also be developed for diagnosis and treatment, and soluble TCR molecules need to be obtained at this time. Soluble TCR molecules do not include their transmembrane domains. Soluble TCR has a wide range of applications. It can not only be used to study the interaction between TCR and pMHC, but also can be used as a diagnostic tool for detecting infection or as a marker for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents, such as cytotoxic or immunostimulatory compounds, to cells presenting specific antigens, and additionally, soluble TCRs can be conjugated to other molecules, such as anti-CD3 antibodies To redirect T cells so that they target cells presenting specific antigens. The present application also obtained a soluble TCR specific for the short peptide of the AFP antigen.

To obtain a soluble TCR, on the one hand, the TCR of the present application can be a TCR that introduces an artificial disulfide bond between the residues of the constant domains of its α and β chains. 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 TRAC*01 exon 1 and substitution of cysteine residues of Ser57 of TRBC1*01 or TRBC2*01 exon 1 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 50, or a maximum of 30, or a maximum of 15, or a maximum of 10, or a maximum of 8 or less amino acids may be truncated at the C-terminus of one or more TCR constant domains of the present application, so that it does not include Cysteine residues can be used to delete natural disulfide bonds, or by mutating a cysteine residue that forms a natural disulfide bond to another amino acid.

As noted above, the TCRs of the present application may contain artificial 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 application can contain both the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence, with or without the introduced artificial disulfide bond 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 disulfide bonds present in the TCR.

In order to obtain soluble TCR, on the other hand, the TCR of the present application 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 soluble TCR of the present application. 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 acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or Or the penultimate 3rd, 5th, and 7th amino acid positions of the α-chain J gene (TRAJ) short peptide, and/or the penultimate 2nd, 4th, and 6th amino acid positions of the β-chain J gene (TRBJ) short peptide amino acid position, 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.

In this application, the TCR with mutations in the hydrophobic core region may be a stable soluble single-chain TCR composed of a flexible peptide chain connecting the variable domains of the α and β chains of the TCR. It should be noted that the flexible peptide chain in this application can be any peptide chain suitable for linking the variable domains of TCRα and β chains. For the single-chain soluble TCR constructed in Example 4 of the present application, the amino acid sequence of the α-chain variable domain is SEQ ID NO: 32, and the encoded nucleotide sequence is SEQ ID NO: 33; the amino acid sequence of the β-chain variable domain It is SEQ ID NO: 34, and the encoded nucleotide sequence is SEQ ID NO: 35.

In addition, in terms of stability, patent document 201680003540.2 also discloses that the introduction of artificial interchain disulfide bonds between the α-chain variable region and the β-chain constant region of TCR can significantly improve the stability of TCR. Therefore, the TCR of the present application 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.

The TCRs of the present application may also be provided in the form of multivalent complexes. The multivalent TCR complex of the present application comprises two, three, four or more multimers formed by combining the TCRs of the present application, 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 application and another molecule. The TCR complexes 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, 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 TSSELMAITR (SEQ ID NO:9)-HLA A1101 complex), therapeutic agents, PK (protein kinase) A modifying moiety or any combination of the above is combined or coupled.

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

Therapeutic agents that can be combined or coupled with the TCR of the present application include but are not limited to: 1. Radionuclides (Koppe et al., 2005, Cancer metastasis reviews (Cancer metastasis reviews) 24, 539); 2. Biological toxicity (Chaudhary et al., 1989 , Nature (Nature) 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy (Cancer Immunology and Immunotherapy) 51, 565); 3. Cytokines such as IL-2 etc. (Gillies et al., 1992, 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 /Nanorods (Lapotko et al., 2005, Cancer letters (Cancer letters) 239, 36; Huang et al., 2006, Journal of the American Chemical Society (Journal of the American Chemical Society) 128, 2115); 7. Virus particles (Peng et al., 2004 , Gene therapy (Gene therapy) 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research (Cancer research) 65, 11631); 9. Nanomagnetic particles; 10. Prodrug activating enzymes (for example, DT - diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)); and 11. A chemotherapeutic agent (eg cisplatin) or any form of nanoparticle or the like.

In addition, the TCRs of the present application may also be hybrid TCRs comprising sequences derived from more than one species. For example, it has been shown that murine TCRs are more efficiently expressed in human T cells than human TCRs. Thus, the TCRs of the present application may comprise human variable domains and murine constant domains. A drawback of this approach is the potential for eliciting an immune response. Therefore, its use in adoptive T cell therapy should have regulatory protocols for immunosuppression to allow engraftment of murine-expressing T cells.

It should be understood that the names of amino acids in this article are represented by a single English letter or three English letters commonly used in the world, and the corresponding relationship between a single English letter and three English letters in an 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).

nucleic acid molecule

The second aspect of the present application provides a nucleic acid molecule encoding the TCR molecule of the first aspect of the present application or a part thereof, the part may be one or more CDRs, variable domains of α and/or β chains, and α chains and/or or beta strand.

The nucleotide sequence encoding the α-chain CDR region of the TCR molecule in the first aspect of the application is as follows:

CDR1α-gactctgtgaacaat (SEQ ID NO: 16);

CDR2α-attccctcagggaca (SEQ ID NO: 17); and

CDR3α-agtggaggtagcaactataaactgaca (SEQ ID NO: 18);

The nucleotide sequence encoding the CDR region of the β chain of the TCR molecule in the first aspect of the application is as follows:

CDR1β-tctgaacacaaccgc (SEQ ID NO: 19);

CDR2β-ttccagaatgaagctcaa (SEQ ID NO: 20); and

CDR3β-gccagcagccccgggacaggggttggctacacc (SEQ ID NO: 21).

Therefore, the nucleotide sequence of the nucleic acid molecule of the present application encoding the TCR alpha chain of the present application comprises SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and/or the nucleic acid molecule of the present application encoding the TCR beta chain of the present application Nucleotide sequences include SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.

The nucleotide sequence of the nucleic acid molecule of the present application may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not contain introns. Preferably, the nucleotide sequence of the nucleic acid molecule of the present application does not contain introns but can encode the polypeptide of the present application, for example, the nucleotide sequence of the nucleic acid molecule of the present application encoding the TCRα chain variable domain of the present application includes SEQ ID NO: 2 and /Or the nucleotide sequence of the nucleic acid molecule of the present application encoding the TCRβ chain variable domain of the present application comprises SEQ ID NO:6. Alternatively, the nucleotide sequence of the nucleic acid molecule encoding the variable domain of the TCR α chain of the application comprises SEQ ID NO: 33 and/or the nucleotide sequence of the nucleic acid molecule of the application encoding the variable domain of the TCR β chain of the application comprises SEQ ID NO: 35. More preferably, the nucleotide sequence of the nucleic acid molecule of the present application comprises SEQ ID NO: 4 and/or SEQ ID NO: 8. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present application is SEQ ID NO: 31.

It is understood that due to the degeneracy of the genetic code, different nucleotide sequences may encode the same polypeptide. Therefore, the nucleic acid sequence encoding the TCR of the present application may be the same as the nucleic acid sequence shown in the drawings of the present application or a degenerate variant. Taking one of the examples in this application as an illustration, "degenerate variant" refers to a nucleic acid sequence that encodes a protein sequence with SEQ ID NO: 1, but differs from the sequence of SEQ ID NO: 2.

Nucleotide sequences may be codon optimized. Different cells use different codons, and the codons in the sequence can be changed to increase the expression according to the cell type. Codon usage tables for mammalian cells, as well as for a variety of other organisms, are well known to those skilled in the art.

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 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. DNA can be either the coding strand or the non-coding strand.

carrier

The present application also relates to vectors comprising the nucleic acid molecules of the present application, including expression vectors, ie 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 viral (AAV) vectors, herpesviral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.

Preferably, the vector can transfer the nucleotide of the present application into cells, such as T cells, so that the cells express AFP antigen-specific TCR. Ideally, the vector should be consistently expressed at high levels in T cells.

cell

The present application also relates to host cells produced by genetic engineering using the vectors or coding sequences of the present application. The host cell contains the vector of the present application or the nucleic acid molecule of the present application is integrated in the chromosome. The host cell is selected from: prokaryotic cells and eukaryotic cells, such as Escherichia coli, yeast cells, CHO cells and the like.

In addition, the present application also includes isolated cells expressing the TCR of the present application, which can be but not limited to T cells, NK cells, NKT cells, stem cells, especially 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 (such as anti-CD3 or anti-CD28 antibodies) so that they can be more easily transfected, for example, transfected with a vector comprising a nucleotide sequence encoding a TCR molecule of the present application dye.

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 did not result in TCR expression on the cell surface because stem cells do not express the CD3 molecule on the surface. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of the thymocyte.

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. Many suitable methods of performing adoptive therapy are known to those skilled in the art (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).

AFP antigen-related diseases

The present application also relates to a method for treating and/or preventing AFP-related diseases in a subject, which includes the step of adoptively transferring AFP-specific T cells to the subject. The AFP-specific T cells recognize the TSSELMAITR (SEQ ID NO:9)-HLA A1101 complex.

The AFP-specific T cells of the present application can be used to treat any AFP-related diseases that present the AFP antigen short peptide TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex, including but not limited to tumors such as liver cancer.

treatment method

Treatment can be carried out by isolating T cells from patients or volunteers suffering from diseases related to AFP antigens, introducing the TCR of the present application into the above T cells, and then returning these genetically modified cells to the patients. Therefore, the present application provides a method for treating AFP-related diseases, comprising injecting isolated T cells expressing the TCR of the present application, preferably, the T cells are derived from the patient itself, into the patient. Generally, it includes (1) isolating T cells from patients; (2) transducing T cells in vitro with nucleic acid molecules of the present application or nucleic acid molecules capable of encoding TCR molecules of the present application; and (3) importing T cells modified by genetic engineering into inside the patient. Wherein, the number of isolated, transfected and reinfused cells can be determined by the physician.

The main advantages of this application are:

(1) The TCR of the present application can specifically bind to the AFP antigen short peptide complex TSSELMAITR (SEQ ID NO: 9)-HLA A1101, and at the same time, the effector cells transduced with the TCR of the present application can be specifically activated.

(2) The effector cells transduced with the TCR of the present application can specifically kill AFP-positive target cells.

The following specific examples further illustrate the present 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. The experimental method that does not indicate specific condition in the following examples, generally according to conventional conditions, for example (Sambrook and Russell et al., Molecular Cloning: Laboratory Manual (Molecular Cloning-A Laboratory Manual) (Third Edition) (2001) CSHL publishes Conditions stated in the company), or in accordance with the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples can be obtained from commercially available channels unless otherwise specified.

Example 1 Cloning of AFP Antigen Short Peptide-Specific T Cells

Peripheral blood lymphocytes (PBL) from healthy volunteers with genotype HLA-A1101 were stimulated with the synthetic short peptide TSSELMAITR (SEQ ID NO: 9; Jiangsu GenScript Biotechnology Co., Ltd.). Refold TSSELMAITR (SEQ ID NO: 9) short peptide with biotin-labeled HLA-A1101 to prepare pMHC haploids. These haploids were combined with PE-labeled streptavidin (BD Company) to form PE-labeled tetramers, and the tetramers and anti-CD8-APC double-positive cells were sorted. Sorted cells were expanded and subjected to secondary sorting as described above, followed by monoclonalization by limiting dilution. Monoclonal cells were stained with tetramers, and the screened double-positive clones are shown in Figure 3. The double-positive clones obtained through layers of screening still need to meet further functional tests.

IFN-γ is a powerful immunoregulatory factor produced by activated T lymphocytes. Therefore, in this example, the number of IFN-γ is detected by the ELISPOT experiment well known to those skilled in the art to verify the activation function of cells transfected with the TCR of this application and Antigen specificity. The function and specificity of the T cell clone were further detected by ELISPOT experiment. The effector cells used in the IFN-γELISPOT experiment of this embodiment are the T cell clones obtained in this application, and the target cells are T2-A11 (referring to T2 cells transfected with HLA-A1101) loaded with TSSELMAITR (SEQ ID NO: 9) short peptide cells), SK-MEL-28-AFP (AFP overexpression), and the control group were T2 cells loaded with other antigen short peptides and SK-MEL-28.

First prepare the ELISPOT plate, and the ELISPOT experiment steps are as follows: Add the components of the test to the ELISPOT plate in the following order: 20,000 target cells/well and 2,000 effector cells/well, then add 20 μL of the corresponding short peptide to the experimental group and the control group , so that the final concentration of the short peptide was 10 ^-5 M, add 20 μL medium (test medium) to the blank group, and set up 2 duplicate wells. It was then incubated overnight (37°C, 5% CO ₂ ). Then the plate was washed for secondary detection and color development, and the plate was dried for 1 hour, and the spots formed on the membrane were counted by an immunospot plate reader (ELISPOT READER system; AID company). The experimental results are shown in Figure 13. The obtained T cell clones released high IFN-γ to T2-A11 and SK-MEL-28-AFP loaded with TSSELMAITR (SEQ ID NO: 9) short peptide, but to T2-A11 and SK-MEL-28-AFP loaded with other antigens The short peptides T2-A11 and SK-MEL-28 basically had no reaction.

Example 2 Obtaining the TCR gene and carrier construction of AFP antigen short peptide-specific T cell clone

The total RNA of the HLA-A1101-restricted T cell clone specific to the short antigen peptide TSSELMAITR (SEQ ID NO: 9) screened in Example 1 was extracted with Quick-RNA ^™ MiniPrep (ZYMO research). The cDNA was synthesized using clontech's SMART RACE cDNA amplification kit, and the primers used were designed at the C-terminal conserved region of the human TCR gene. The sequence was cloned into T vector (TAKARA) for sequencing. It should be noted that this sequence is complementary and does not contain introns. After sequencing, the sequence structures of the TCR α chain and β chain expressed by the double-positive clone are shown in Figure 1 and Figure 2, respectively. Variable domain amino acid sequence, TCRα chain variable domain nucleotide sequence, TCRα chain amino acid sequence, TCRα chain nucleotide sequence, TCRα chain amino acid sequence with leader sequence and TCRα chain nucleotide sequence with leader sequence; Figure 2a, Figure 2b, Figure 2c, Figure 2d, Figure 2e and Figure 2f are the amino acid sequence of the TCRβ chain variable domain, the nucleotide sequence of the TCRβ chain variable domain, the amino acid sequence of the TCRβ chain, the nucleotide sequence of the TCRβ chain, and the TCRβ chain amino acid sequence and TCRβ chain nucleotide sequence with leader sequence.

The alpha chain was identified to contain CDRs with the following amino acid sequence:

αCDR1-DSVNN (SEQ ID NO: 10);

αCDR2-IPSGT (SEQ ID NO: 11); and

αCDR3-SGGSNYKLT (SEQ ID NO: 12);

The beta strand contains CDRs with the following amino acid sequence:

βCDR1-SEHNR (SEQ ID NO: 13);

βCDR2-FQNEAQ (SEQ ID NO: 14); and

βCDR3-ASSPGTGVGYT (SEQ ID NO: 15).

The full-length genes of TCRα chain and β chain were respectively cloned into the lentiviral expression vector pLenti(addgene) by overlapping PCR. Specifically: use overlap PCR to connect the full-length genes of the TCRα chain and the TCRβ chain to obtain the TCRα-2A-TCRβ fragment. The lentiviral expression vector and TCRα-2A-TCRβ were digested and ligated to obtain the pLenti-TRA-2A-TRB-IRES-NGFR plasmid. As a control, a lentiviral vector pLenti-eGFP expressing eGFP was also constructed. Then use 293T/17 to package the fake virus.

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

In order to obtain a soluble TCR molecule, the α and β chains of the TCR molecule of the present application may only contain their variable domains and part of the constant domains, respectively, and a cysteine residue is introduced into the constant domains of the α and β chains respectively To form an artificial interchain disulfide bond, the amino acid sequence and nucleotide sequence of its α chain are shown in Figure 4a and Figure 4b, respectively, and the amino acid sequence and nucleotide sequence of its β chain are shown in Figure 5a and Figure 5b, respectively . 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 NcoI and NotI, respectively. The insert was confirmed by sequencing.

Transform the expression vectors of TCR α and β chains into the expressing bacteria BL21(DE3) by chemical transformation method respectively, grow the bacteria in LB culture medium, induce them with a final concentration of 0.5mM IPTG at OD ₆₀₀ =0.6, and express the α and β chains of TCR The inclusion bodies formed after that 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 (pH 8.1).

The dissolved TCRα and β chains were quickly mixed in 5M urea, 0.4M arginine, 20mM Tris (pH 8.1), 3.7mM cystamine, 6.6mMβ-mercapoethylamine (4°C) at a mass ratio of 1:1, and the final concentration was 60mg/mL. 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 solution (20mM 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, 5ml, 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. The SDS-PAGE gel images of the soluble TCR obtained in this application are shown in Figures 6a and 6b.

Example 4 Production of AFP antigen short peptide-specific soluble single-chain TCR

According to the patent document WO2014/206304, the variable domains of the TCRα and β chains in Example 2 were constructed by site-directed mutagenesis into a stable soluble single-chain TCR molecule linked by a flexible short peptide (linker). The amino acid sequence and nucleotide sequence of the single-chain TCR molecule are shown in Figure 7a and Figure 7b respectively, and the amino acid sequence and nucleotide sequence of the linker are underlined. The amino acid sequence and nucleotide sequence of its alpha chain variable domain are shown in Figure 8a and Figure 8b, respectively; the amino acid sequence and nucleotide sequence of its beta chain variable domain are shown in Figure 9a and Figure 9b, respectively.

The target gene was digested with Nco I and Not I, and connected to the pET28a vector that was digested with Nco I and Not I. 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 plasmids were extracted and transformed after confirming the sequence was correct to E.coli BL21(DE3) for expression.

Example 5 Expression, refolding and purification of soluble single-chain TCR specific for short peptides of AFP antigen

All the BL21 (DE 3) colonies containing the recombinant plasmid pET28a-template strand prepared in Example 4 were inoculated in LB medium containing kanamycin, cultivated at 37°C until the OD600 was 0.6-0.8, and added IPTG to the final concentration 0.5mM, 37 ℃ continue to culture 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 the inclusion body, then wash with Bugbuster (Merck) to remove cell debris and membrane components, centrifuge at 6000rpm for 15min, and collect the inclusion body body. The inclusion bodies were dissolved in the buffer solution (20mM Tris-HCl pH 8.0, 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 later use.

Add 2.5mL buffer solution (6M Gua-HCl, 50mM Tris-HCl pH 8.1, 100mM NaCl, 10mM EDTA) to 5mg 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-treated single-chain TCR dropwise into 125mL refolding buffer (100mM Tris-HCl pH 8.1, 0.4M L-arginine, 5M urea, 2mM EDTA, 6.5mM β-mercapthoethylamine, 1.87mM Cystamine) with a syringe , stirred at 4°C for 10 min, and then put the refolding solution into a cellulose membrane dialysis bag with a cutoff of 4kDa, placed the dialysis bag in 1L of pre-cooled water, and stirred slowly overnight at 4°C. After 17 hours, the dialysate was replaced with 1 L of pre-cooled buffer solution (20mM Tris-HCl pH 8.0), and dialysis was continued at 4°C for 8 hours, 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), and the protein was purified with a 0-1M NaCl linear gradient eluent prepared with 20mM Tris-HCl pH 8.0, The collected eluted fractions were analyzed by SDS-PAGE, and the fractions containing single-chain TCR were concentrated and further purified by gel filtration column (Superdex 75 10/300, GE Healthcare), and the target fractions were also analyzed by SDS-PAGE.

The eluted fractions for BIAcore analysis were further tested for purity by gel filtration. The conditions are: chromatographic column Agilent Bio SEC-3 (300A,

), the mobile phase was 150mM phosphate buffer, the flow rate was 0.5mL/min, the column temperature was 25°C, and the ultraviolet detection wavelength was 214nm.

The SDS-PAGE gel images of the soluble single-chain TCR obtained in this application are shown in Figures 10a and 10b.

Example 6 Combination Characterization

This example proves that the soluble TCR molecule of the present application can specifically bind to the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex through BIAcore analysis.

BIAcore T200 real-time analysis system was used to detect the binding activity of the TCR molecule obtained in Example 3 and Example 5 to the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex. Add the anti-streptavidin antibody (GenScript) to the coupling buffer (10mM sodium acetate buffer, pH 4.77), and then flow the antibody over the CM5 chip activated with EDC and NHS to immobilize the antibody on the chip surface , and finally the unreacted activated surface was blocked with ethanolamine hydrochloric acid solution to complete the coupling process, and the coupling level was about 15,000RU.

Let a low concentration of streptavidin flow over the surface of the antibody-coated chip, then flow the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex through the detection channel, and the other channel as a reference channel, and then 0.05mM Biotin flowed through the chip at a flow rate of 10 μL/min for 2 min to block the remaining binding sites of streptavidin.

The preparation process of the above-mentioned TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex is as follows:

a.Purification

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

Suspend the above inclusion body in 5mL 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 body , 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 TSSELMAITR (SEQ ID NO: 9) 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, and 10mM DTT. Before refolding, 3M guanidine hydrochloride, 10mM sodium acetate, and 10mM EDTA were added for further denaturation. TSSELMAITR (SEQ ID NO: 9) peptide was added 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 At 4°C for at least 3 days to complete, SDS-PAGE test whether the renaturation is successful.

c. Purification after renaturation

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

d. Biotinylation

Concentrate the purified pMHC molecules with Millipore ultrafiltration tubes, and 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, 100μg/mL BirA Enzyme (GST-BirA), the mixture was incubated overnight at room temperature, and SDS-PAGE was used to detect whether the biotinylation was complete.

e. Purification of biotinylated complexes

The biotinylated pMHC molecules were concentrated to 1 mL with Millipore ultrafiltration tubes, and the biotinylated pMHC was purified by gel filtration chromatography, and the HiPrep was pre-equilibrated with filtered PBS using an Akta purification instrument (GE General Electric Company). ^TM 16/60 S200 HR column (GE General Electric Company), loaded with 1 mL of concentrated biotinylated pMHC molecules, and then eluted with PBS at a flow rate of 1 mL/min. Biotinylated pMHC molecules elute as a single peak at approximately 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).

Kinetic parameters were calculated using BIAcore Evaluation software to obtain the kinetic profiles of the soluble TCR molecule of the application and the combination of the soluble single-chain TCR molecule constructed by the application and the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex, respectively as shown in Figure 11 and Figure 12 shows. The map shows that both the soluble TCR molecule and the soluble single-chain TCR molecule obtained in the present application can bind to the TSSELMAITR (SEQ ID NO: 9)-HLA A1101 complex. At the same time, the above method was also used to detect the binding activity of the soluble TCR molecules of the present application to the complexes of several other irrelevant antigen short peptides and HLA, and the results showed that the TCR molecules of the present application did not bind to other irrelevant antigens.

Example 7 For T2 cells loaded with short peptides, the effector cell activation experiment of transfection TCR of the present application

The effector cells used in this experiment were CD3 ⁺ T cells expressing the TCR of the present application, and CD3 ⁺ T cells transfected with other TCR (A6) from the same volunteer were used as the control group. The target cells used were T2-A11 loaded with AFP antigen short peptide TSSELMAITR (SEQ ID NO: 9), and empty T2-A11 loaded with other antigen short peptides was used as a control. Add each component of the test to the ELISPOT well plate: target cells 1×10 ⁴ target cells/well, effector cells 2×10 ³ /well (calculated according to the positive rate of transfection), and set up two duplicate wells. Then TSSELMAITR (SEQ ID NO: 9) short peptide was added to the corresponding wells, so that the final concentration of the short peptide in the ELISPOT well plate was 10 ⁻⁶ M.

Following the manufacturer's instructions, prepare well plates as follows: Dilute anti-human IFN-γ capture antibody 1:200 in 10 mL sterile PBS per plate, then aliquot 100 µl of the diluted capture antibody into each well . Incubate the plate overnight at 4°C. After incubation, wash the plate to remove excess capture antibody. Add 100 μl/well of RPMI 1640 medium containing 10% FBS and incubate the well plate at room temperature for 2 hours to seal the well plate. The medium was then washed from the well plate and any residual wash buffer was removed by flicking and patting the ELISPOT plate on the paper.

Then incubate the well plate overnight (37°C/5% CO ₂ ). The next day, discard the medium, wash the well plate twice with double distilled water, then wash three times with washing buffer, and pat on a paper towel to remove residual of wash buffer. The detection antibody was then diluted 1:200 with PBS containing 10% FBS, and added to each well at 100 microliters/well. Plates were incubated at room temperature for 2 hours, then washed 3 times with wash buffer, and excess wash buffer was removed by tapping the plate on a paper towel. Dilute streptavidin-alkaline phosphatase 1:100 with PBS containing 10% FBS, add 100 μl of diluted streptavidin-alkaline phosphatase to each well and incubate the plate at room temperature 1 hour. Then wash 4 times with wash buffer and 2 times with PBS, tapping the well plate on paper towels to remove excess wash buffer and PBS. After washing, 100 microliters/well of BCIP/NBT solution provided in the kit was added for development. Cover the well plate with tin foil to avoid light during the development period, and let it stand for 5-15 minutes. During this period, the spots on the developed well plate were routinely detected to determine the optimal time to terminate the reaction. Remove the BCIP/NBT solution and rinse the orifice plate with double distilled water to stop the development reaction, shake dry, then remove the bottom of the orifice plate, dry the orifice plate at room temperature until each well is completely dry, and then use the immunospot plate counter (CTL, Cellular Technology Limited (Cellular Technology Limited) counted the spots formed on the bottom membrane of the well plate. The number of ELSPOT spots observed in each well was plotted using GraphPad Prism6.

The experimental results are shown in Figure 14. For the T2 cells loaded with TSSELMAITR (SEQ ID NO: 9) short peptide, the target cells of the T cells transfected with the TCR of this application have an obvious activation effect, while the T cells transfected with other TCRs basically No response; at the same time, the T cells transfected with the TCR of the present application are inactive to T2 cells loaded with other antigen short peptides or empty.

Example 8. For tumor cell lines, the activation function experiment of the effector cells transfected with the TCR of the present application

In this embodiment, the function and specificity of the TCR of the present application in cells are also detected by ELISPOT experiment. The effector cells used were CD3 ⁺ T cells expressing the specific TCR of the AFP antigen short peptide of the application, and the same volunteers transfected with other TCR (A6) and empty transfected (NC) CD3 ⁺ T cells were used as the control group. The target cells are tumor cell lines, and the positive tumor cell lines used are HepG2-A11-B2M (overexpression of HLA A1101 and β2M), SK-MEL-28-AFP; the negative tumor cell lines used are HepG2, SK-MEL-28, SNU423 and HUCCT1, as a control group.

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% 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 15. For the positive tumor cell lines, the effector cells transfected with the TCR of the present application were specifically activated, and the empty transfected T cells transfected with other TCRs were basically not activated; while for the negative tumor cell lines, Effector cells transfected with the TCR of the present application are inactive.

Example 9 Killing function experiment of effector cells transfected with TCR of the present application

In this embodiment, the release of LDH was measured by non-radioactive cytotoxicity experiments 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 application. In the LDH experiment of this example, CD3+ T cells isolated from the blood of healthy volunteers were used to transfect the TCR of this application as effector cells, and the same volunteers were used to transfect other TCR (A6) or empty transfection (NC) CD3 ⁺ T cells served as negative controls. The target cells are tumor cell lines, and the positive tumor cell lines used are HepG2-A11-B2M (overexpression of HLA A1101 and β2M), SK-MEL-28-AFP; the negative tumor cell lines used are HepG2, SK-MEL-28, SNU423 and HUCCT1, as a control group. First prepare the LDH plate, and add the components of the test into the plate in the following order: target cells ³ ×104 cells/well, effector cells ³ ×104 cells/well (calculated according to the positive rate of transfection) were added to the corresponding wells , and set up three replicate holes. 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 Figure 16. For AFP-positive tumor cell lines, the effector cells transfected with the TCR of the present application showed a strong killing effect, while the effector cells transfected with other TCRs or empty transfection had no killing effect; at the same time, the effector cells transfected with the present application TCR T cells have no killing activity against negative tumor cell lines.

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

Claims

A kind of T cell receptor (TCR), it can combine with TSSELMAITR (SEQ ID NO:9)-HLA A1101 complex; And, described TCR comprises TCR α chain variable domain and TCR β chain variable domain, and described TCR α chain The three complementarity determining regions (CDRs) of the variable domains are:

αCDR1-DSVNN (SEQ ID NO: 10);

αCDR2-IPSGT (SEQ ID NO: 11);

αCDR3-SGGSNYKLT (SEQ ID NO: 12); and/or

The three complementarity-determining regions of the TCRβ chain variable domain are:

βCDR1-SEHNR (SEQ ID NO: 13);

βCDR2-FQNEAQ (SEQ ID NO: 14);

βCDR3-ASSPGTGVGYT (SEQ ID NO: 15).
The TCR of claim 1, wherein the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, and the TCR α chain variable domain is an amino acid having at least 90% sequence identity to SEQ ID NO: 1 sequence; and/or the TCR β chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO:5.
The TCR according to claim 1, wherein the TCR comprises the amino acid sequence of the α-chain variable domain of SEQ ID NO: 1; and/or the TCR comprises the amino acid sequence of the β-chain variable domain of SEQ ID NO: 5.
The TCR according to claim 1, wherein the TCR is an αβ heterodimer comprising a TCR α chain constant region TRAC*01 and a TCR β chain constant region TRBC1*01 or TRBC2*01; preferably, the TCR The amino acid sequence of the alpha chain of the TCR is SEQ ID NO: 3 and the amino acid sequence of the beta chain of the TCR is SEQ ID NO: 7.
The TCR of claim 1, wherein the TCR is soluble.
The TCR according to claim 5, wherein the TCR is a single chain; preferably, the TCR is formed by linking the variable domain of the α chain and the variable domain of the β chain through a peptide linker sequence.
The TCR according to claim 6, wherein the TCR is at the 11th, 13, 19, 21, 53, 76, 89, 91, or 94th amino acid position of the α-chain variable region, and/or the α-chain J gene There are one or more mutations in the penultimate 3rd, 5th or 7th amino acid position of the short peptide; and/or the TCR is in the 11th, 13th, 19th, 21st, 53rd, 76th amino acid of the β-chain variable region , 89, 91, or 94th, and/or one or more mutations in the penultimate 2nd, penultimate 4th or penultimate 6th amino acid position of the β chain J gene short peptide, wherein the numbering of amino acid positions is according to IMGT (International The position number listed in Immunogenetics Information System); Preferably, the α chain variable domain amino acid sequence of the TCR comprises SEQ ID NO: 32 and/or the β chain variable domain amino acid sequence of the TCR comprises SEQ ID NO: 34; more preferably, the amino acid sequence of the TCR is SEQ ID NO: 30.
The TCR of claim 1, wherein the TCR comprises (i) TCR α chain variable domain and all or part of the TCR α chain constant region except the transmembrane domain; and (ii) TCR β chain variable domain and All or part of the TCRβ chain constant region outside the transmembrane domain.
The TCR of claim 8, wherein a cysteine residue forms an artificial disulfide bond between the alpha and beta chain constant domains of the TCR; preferably, the cysteine residue that forms the artificial disulfide bond in the TCR Cysteine residues are substituted for one or more groups of positions selected from:

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.
The TCR according to claim 9, wherein the amino acid sequence of the alpha chain of the TCR is SEQ ID NO: 26 and/or the amino acid sequence of the beta chain of the TCR is SEQ ID NO: 28.
The TCR according to claim 8, wherein an artificial interchain disulfide bond is contained between the alpha chain variable region and the beta chain constant region of the TCR; preferably, an artificial interchain disulfide bond is formed in the TCR Cysteine residues are substituted for one or more groups of positions selected from the following:

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

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

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

or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1*01 or TRBC2*01.
The TCR according to claim 1, wherein the TCR comprises an α-chain constant region and a β-chain constant region, the α-chain constant region is of murine origin and/or the β-chain constant region is of murine origin.
The TCR according to claim 1, wherein the C- or N-terminus of the α-chain and/or β-chain of the TCR is bound with a conjugate; preferably, the conjugate that binds to the T cell receptor is a detectable marker or a therapeutic agent; more preferably, the therapeutic agent is an anti-CD3 antibody.
The TCR of claim 1, wherein the TCR is isolated or purified.
A multivalent TCR complex comprising at least two TCR molecules, wherein at least one TCR molecule is the TCR according to any one of the preceding claims.
A nucleic acid molecule comprising the nucleic acid sequence encoding the TCR molecule according to any one of the above claims or its complementary sequence.
The nucleic acid molecule of claim 16, wherein said nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 2 or SEQ ID NO: 33 encoding the variable domain of the TCR α chain.
The nucleic acid molecule according to claim 16 or 17, wherein said nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 6 or SEQ ID NO: 35 encoding the TCR β chain variable domain.
The nucleic acid molecule according to claim 16, wherein said nucleic acid molecule comprises the nucleotide sequence SEQ ID NO: 4 encoding a TCR α chain and/or comprises the nucleotide sequence SEQ ID NO: 8 encoding a TCR β chain.
A vector, which contains the nucleic acid molecule according to any one of claims 16-19; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.
An isolated host cell containing the vector of claim 20 or the nucleic acid molecule of any one of claims 16-19 integrated in the chromosome.
A cell transduced with the nucleic acid molecule of any one of claims 16-19 or the vector of claim 20; preferably, the cell is a T cell or a stem cell.
A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR according to any one of claims 1-14, the TCR complex according to claim 15, or the cell according to claim 22.
The use of the T cell receptor according to any one of claims 1-14, or the TCR complex described in claim 15, or the use of the cells described in claim 22, wherein it is used to prepare a drug for the treatment of tumors or autoimmunity A drug for a disease; preferably, the tumor is liver cancer.
The T cell receptor described in any one of claims 1-14, or the TCR complex described in claim 15 or the cell described in claim 22, used as a drug for treating tumors or autoimmune diseases; preferably Preferably, the tumor is liver cancer.
A method for treating a disease, comprising administering an appropriate amount of the TCR of any one of claims 1-14, the TCR complex of claim 15, the cell of claim 22, or the the pharmaceutical composition described in claim 23;

Preferably, the disease is a tumor, more preferably, the tumor is liver cancer.