CN118909085A - T Cell Receptor (TCR) recognizing WT1 antigen short peptide, complex of multivalent TCR, pharmaceutical composition and application - Google Patents

Abstract

The present invention provides complexes, pharmaceutical compositions and uses of T Cell Receptors (TCRs), multivalent TCRs, that recognize WT1 antigen short peptides, wherein the T cell receptors are capable of recognizing RMFPNAPYL-HLa-a 02:01 complexes with high affinity, the TCRs comprising a TCR alpha chain variable region comprising three Complementarity Determining Regions (CDRs) having the sequences of SEQ ID NOs: 2-4, 42-44, 78-80, 114-116 or 150-152; the TCR β chain variable region comprises three CDRs, the sequence of which is SEQ ID NO:5-7, 45-47, 81-83, 117-119 or 153-157; the TCR, the nucleic acid molecule and the like provided by the invention have the application of preparing medicines for treating tumors or other immune diseases.

Description

T cell receptor (TCR) recognizing WT1 antigen short peptide, multivalent TCR complex, drug composition and use

Technical Field

The present invention relates to a TCR capable of recognizing a short peptide derived from a WT1 antigen with high affinity, and also relates to WT1-specific T cells obtained by transducing the above TCR, and their use in preventing and treating WT1-related tumors.

Background Art

T cell immunotherapy is a very important method in tumor immunotherapy. By isolating tumor infiltrating lymphocytes (TIL) from tumor tissue, cloning and amplifying them in vitro and then returning them to the patient, patients with advanced cancer who are tolerant to radiotherapy and chemotherapy can achieve lasting remission and produce good clinical treatment effects (Zacharakis, N. et al. Nat Med. 2018, 24 (6): 724-730.). However, the isolation and cultivation of TILs are not only demanding, but also take a long time to reach the number of cells required for clinical treatment. More importantly, the tumor tissues from which TILs can be successfully isolated are still very limited, which limits the application of TILs in tumor clinics.

T cells recognize tumors mainly through T cell receptors (TCR) on their surfaces. TCR has the ability to recognize the human major histocompatibility complex (MHC)-antigen peptide complex on tumor cells. TCR is composed of two peptide chains, α and β, to form a heterodimer structure. Each peptide chain contains a variable region, a connecting region and a constant region. The β chain usually also contains a short variable region between the variable region and the connecting region, but the variable region is often regarded as part of the connecting region. Each variable region contains three CDRs (complementarity determining regions) embedded in the framework region, namely CDR1, CDR2 and CDR3. The CDR region determines the binding of TCR to the MHC complex, among which CDR3 is recombined by the variable region and the connecting region and is called the hypervariable region, which directly determines the antigen specificity of TCR. When TCR recognizes the MHC-antigen peptide complex, CDR3 can directly bind to the antigen peptide. CDR1 and CDR2 mainly contact the two α helices of the binding groove of the MHC molecule. These interactions ensure that TCR accurately recognizes the MHC-antigen peptide complex. The α and β chains of TCR are generally considered to have two "domains", namely the variable region and the constant region, in which the variable region contains the connecting region. The sequence of the TCR constant region can be found in the public database of the International Immunogenetics Information System (IMGT), such as the constant region sequence of the TCR molecule α chain is "TRAC", and the constant region sequence of the TCR molecule β chain is "TRBC1" or "TRBC2". In addition, the α and β chains of TCR also contain a transmembrane region and a short cytoplasmic region.

By using gene transduction to transfer the TCR that can recognize tumor cells into the patient's immune T cells, the patient's T cells can be transformed into tumor-specific cytotoxic T cells (TCR-T). When this genetically engineered TCR-T is delivered to the patient, these tumor-specific TCR-T will be activated by recognition when encountering the MHC-peptide complex on the tumor cell, thereby proliferating in the patient's body and achieving the effect of treating the tumor by killing the tumor cells.

WT1 is a tumor associated antigen (TAA). WT1 is a transcription factor in the process of embryonic development. In adults, WT1 is expressed in renal podocytes, testes, ovaries, breast myoepithelial cells and some CD34+ stem cells from bone marrow, but the expression of WT1 in normal tissues is very limited (Yang L. et al., Leukemia, 2007, 21(5): 868-876). In most leukemias, WT1 is significantly overexpressed (Yong AS. et al., Leukemia, 2008, 22(9): 1721-1727). In particular, it is significantly overexpressed in CD34+ stem cells of AML and CML (Saito Y. et al., Sci Transl Med, 2010, 2(17): 17ra9; Gerber JM. et al., Am J Hematol, 2011; 86(1): 31-37). Since the expression level of WT1 is negatively correlated with the prognosis, WT1 is currently used as a marker in clinical practice to monitor residual disease in leukemia patients (Cilloni D&Saglio G.Acta Haematol, 2004, 112(1-2): 79-84; Schroeder T. et al., Blood, 2014, 124(21): 4661-4662) and predict the recurrence of leukemia (Aude G.Chapuis. et al., Nat Med, 2019, 25(7): 1064-1072). In addition to being overexpressed in a variety of leukemias, WT1 is also overexpressed in a variety of solid tumors (Nakatsuka S. et al., Mod Pathol, 2006, 19 (6): 804-814; Keiko Naitoh. et al., Anticancer Res, 2016, 36 (7): 3715-3724.). Therefore, some studies have pointed out that WT1 is an excellent target antigen for tumor immunotherapy (Cheever MA. et al., Clin Cancer Res, 2009, 15 (17): 5323-5337; Haruo Sugiyama, Jpn J Clin Oncol, 2010, 40 (5): 377-387). After being produced in cells, WT1 is degraded into small molecular peptides, which are presented on the cell surface after binding to MHC molecules to form a complex. RMFPNAPYL is a short peptide derived from the WT1 antigen and is a target for the treatment of WT1-related tumors. For the above-mentioned WT1-related malignancies, traditional treatments include radiotherapy and chemotherapy, but both methods damage the patient's normal cells. The latest research shows that when using TCR-T that can recognize the RMFPNAPYL antigen peptide for tumor immunogene therapy, it can not only overcome the non-specificity and corresponding toxic side effects of traditional radiotherapy and chemotherapy, but also by injecting WT1-TCR-T that can recognize the RMFPNAPYL antigen peptide into the patient, 100% of the tumor patients will not relapse (Chapuis. et al., Nat Medicine, 2019, 25(7): 1064-1072), which is undoubtedly a very gratifying research result. The TCR reported in the literature is isolated from the T cells of human leukocyte antigen HLA-A2 positive donors, so according to immunological principles, it should not be a high-affinity TCR. This is because WT1 is a self-antigen, so in order to avoid autoimmune diseases, the cytotoxic T lymphocytes (CTLs) with high affinity for WT1 in HLA-A2 positive donors are deleted during the negative selection process of thymocytes. In this way, among the donor's own T cells, only WT1-CTLs with low affinity and tolerance to WT1, a self-antigen, can survive. Since low-affinity CTLs often have poor anti-tumor activity (Derby. et al., J. Immunol. 2001, 166: 1690-1697), a very important goal of T cell-based tumor immunotherapy is to be able to obtain CTLs with high affinity and specificity for tumor antigens (Zeh. et al., J. Immunol. 1999, 162: 989-994).

US Patent US8697854B2 describes a non-self-restricted or allo-restricted CTL scheme to obtain high-affinity CTL. By adopting the allo-restricted CTL scheme, they obtained CTLs and high-affinity TCRs that are specific to the tumor-related antigen tyrosinase. The principle of this technology is based on the use of one's own MHC-peptide complex to stimulate allo-CTLs. Since CTLs from allo-CTLs have not undergone the negative selection process of their own MHC, they contain CTLs that are highly sensitive to their own MHC and high-affinity TCRs. However, this scheme also has an obvious disadvantage that among the T cells that produce immune responses to their own MHC-peptide complexes, only a small part is both restricted by MHC and specific to the antigen peptide, while most of the cells often only recognize MHC and do not have specificity for the antigen peptide. In other words, most of the CTLs stimulated by this method are non-specific T cells. If such CTLs are used to treat tumors, they will cause serious non-specific immune reactions.

Therefore, there is still a need to find other CTLs that have both high affinity and specificity for the tumor antigen WT1, and then to isolate and obtain TCRs with high affinity and specificity for tumor antigens.

Summary of the invention

In order to obtain the small number of beneficial CTLs that are both restricted by MHC and specific to antigenic peptides, we performed single-cell cloning of mixed T cells that produced immune responses during stimulation. Since culturing T cells is a very difficult experiment in itself, it would be even more difficult if they were placed in an environment with only one T cell in each well, because it is difficult for a T cell to survive, so if you want it to survive and be expanded, the difficulty of the entire experiment will be greatly increased, or the possibility of success of the experiment will be greatly reduced. After many repeated experiments and a lot of screening work, we obtained a cytotoxic CTL with high affinity and specificity for WT1, and further separated and obtained its TCR with high affinity and specificity for WT1.

In the first aspect, the present invention provides a T cell receptor (TCR), which can recognize the RMFPNAPYL-HLA-A*02:01 complex with high affinity, and the TCR comprises a TCR α chain variable region and a TCR β chain variable region; wherein the TCR α chain variable region comprises three complementarity determining regions (CDRs), whose sequences are any one of SEQ ID NOs: 2-4, 42-44, 78-80, 114-116 or 150-152; the TCR β chain variable region comprises three complementarity determining regions (CDRs), whose sequences are any one of SEQ ID NOs: 5-7, 45-47, 81-83, 117-119 or 153-157.

Preferably, the TCR is an αβ heterodimer, which further comprises a TCR α chain constant region (TRAC) and a TCR β chain constant region (TRBC1 and/or TRBC2); preferably, the α chain amino acid sequence of the TCR is any one of the amino acid sequences listed in SEQ ID NO: 10, 50, 86, 122 or 158, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; the β chain amino acid sequence of the TCR is any one of the amino acid sequences listed in SEQ ID NO: 14, 54, 90, 126 or 162, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

Preferably, the TCR is a single chain; preferably, the amino acid sequence of the α chain variable region of the TCR is any one of SEQ ID NO: 26, 66, 102, 138 or 174, and the amino acid sequence of the β chain variable region of the TCR is any one of SEQ ID NO: 28, 68, 104, 140 or 176; more preferably, the TCR is composed of the α chain variable region and the β chain variable region connected by a connecting peptide chain SEQ ID NO: 32, and the amino acid sequence of the TCR is any one of SEQ ID NO: 30, 70, 106, 142 or 178.

Preferably, the TCR is a messenger ribonucleotide (mRNA); preferably, the α chain amino acid sequence of the TCR-mRNA is any one of SEQ ID NO: 34, 72, 108, 144 or 180, and the β chain amino acid sequence of the TCR-mRNA is any one of SEQ ID NO: 36, 74, 110, 146 or 182; more preferably, the TCR-mRNA is composed of an α chain and a β chain connected by a connecting peptide chain SEQ ID NO: 40, and the amino acid sequence of the TCR-mRNA is any one of SEQ ID NO: 38, 76, 112, 148 or 184.

In a second aspect, any one of the TCRs provided by the present invention is isolated or purified or recombinant, artificial, or monoclonal, wherein the antigen specificity is at least partially CD8-independent.

In a third aspect, the present invention provides a multivalent TCR complex comprising at least three TCR molecules, wherein at least one TCR molecule is the TCR described in the first and second aspects.

In a fourth aspect, the present invention provides a nucleic acid molecule, comprising a nucleotide sequence encoding the TCR described in the first or second aspect or its complementary sequence, or a codon-optimized nucleotide sequence corresponding to the amino acid sequence of the TCR described in the first or second aspect; preferably, the nucleic acid molecule comprises any one of the nucleotide sequences encoding the TCR α chain variable region of SEQ ID NO: 9, 27, 49, 67, 85, 103, 121, 139, 157 or 175, or a sequence having at least 90% sequence identity therewith; the nucleic acid molecule comprises any one of the nucleotide sequences encoding the TCR β chain variable region of SEQ ID NO: 13, 29, 53, 69, 89, 105, 125, 141, 161 or 177, or a sequence having at least 90% sequence identity therewith; preferably, the nucleic acid molecule comprises any one of the nucleotide sequences encoding the TCR α chain of SEQ ID NO: 11, 51, 87, 123 or 159, or a sequence having at least 90% sequence identity therewith, and the nucleic acid molecule comprises any one of the nucleotide sequences encoding the TCR β chain of SEQ ID NO: 123 or 159, or a sequence having at least 90% sequence identity therewith. NO: any one of 15, 55, 91, 127 or 163, or a sequence having at least 90% sequence identity therewith; preferably, the nucleic acid molecule comprises the messenger ribonucleotide sequence SEQ ID NO: any one of 35, 73, 109, 145 or 181 encoding the TCR alpha chain, or a sequence having at least 90% sequence identity therewith, and the nucleic acid molecule comprises the messenger ribonucleotide sequence SEQ ID NO: any one of 37, 75, 111, 147 or 183 encoding the TCR beta chain, or a sequence having at least 90% sequence identity therewith, wherein the messenger ribonucleotide sequence is replaced by Pseudo-UTP (Ψ) and/or 5-Methyl-CTP.

In a fifth aspect, the present invention provides a vector, wherein the vector contains the nucleic acid molecule described in the fourth aspect; preferably, the vector is a viral vector and/or a lipid nanoparticle vector; preferably, the vector is a lentiviral vector and/or a retroviral vector; more preferably, the vector is a lentiviral vector.

In a sixth aspect, the present invention provides an isolated host cell, wherein the host cell contains the vector described in the fifth aspect or the exogenous nucleic acid molecule described in the fourth aspect integrated into the chromosome.

In the seventh aspect, the present invention provides a cell, which is a cell transduced and transfected by the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect, wherein the TCR is heterologous to the cell; preferably, the cell is a T cell or a stem cell; more preferably, the cell is a primary T cell or stem cell from a subject.

In an eighth aspect, the present invention provides a pharmaceutical composition, comprising the TCR described in the first and second aspects, the TCR complex described in the third aspect, the nucleic acid molecule described in the fourth aspect, or the cell described in the seventh aspect, and a pharmaceutically acceptable carrier.

In the ninth aspect, the present invention provides the use of the T cell receptor described in the first and second aspects, or the TCR complex described in the third aspect, or the cell described in the seventh aspect for preparing a drug for treating tumors or other immune diseases.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions.

The present invention is described in detail below with reference to the accompanying drawings and embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the results of double positive staining of WT1-specific CTLs with CD8+ and HLA-A*02:01-WT1-tetramer-PE.

Figure 2 shows that after the WT1-TCR of the present invention is transferred into primary human T cells, the WT1-TCR can be expressed on the surface of the transduced T cells and can be stained with HLA-A*02:01-WT1-tetramer, thereby showing that they have WT1 specificity through TCR transduction; while the simulated transduced T cells cannot be stained with HLA-A*02:01-WT1-tetramer, indicating that they have no WT1 specificity.

Figures 3-7 show that the WT1-TCR-T cells of the present invention can produce IFN-γ after being stimulated by specific WT1 antigen peptides, but do not produce IFN-γ after being stimulated by control antigen peptides, indicating that they have their own antigen specificity and that the WT1-TCR-T cells of the present invention can specifically recognize WT1 antigen peptides at the nanomolar to picomolar level.

FIG. 8 shows A2-PE staining and GFP expression in the positive target cells K562-A2-GFP and the control target cells T2 used in the present invention.

Figures 9-13 show that the WT1-TCR-T cells of the present invention have a selective killing effect on leukemia target cells in the background of MHC of HLA-A*02:01.

DETAILED DESCRIPTION

In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined purpose, the specific implementation methods, structural features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and embodiments.

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and/or steps, can be combined in any manner.

Any feature disclosed in this specification (including any additional claims, abstract and drawings), unless otherwise stated, may be replaced by other equivalent or alternative features with similar purposes. That is, unless otherwise stated, each feature is only an example of a series of equivalent or similar features.

After repeated, in-depth and meticulous research, the inventors have found a TCR that can specifically bind to the WT1 antigen short peptide RMFPNAPYL (SEQ ID NO: 1), which can form a complex with HLA-A*02:01 and be presented to the cell surface together. The present invention also provides a nucleic acid molecule encoding the TCR and a vector containing the nucleic acid molecule, and a cell transduced with the TCR of the present invention. The present invention also provides the use of the TCR, nucleic acid molecule, vector and cell for preparing a drug for treating tumors or other immune diseases.

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

A CDR1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2, 42, 78, 114, or 150; a CDR2 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3, 43, 79, 115, or 151; a CDR3 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 4, 44, 80, 116, or 152;

The beta chain variable region of the TCR comprises a CDR having the following amino acid sequence:

A CDR1 comprising the amino acid sequence listed in any one of SEQ ID NOs: 5, 45, 81, 117 or 153; a CDR2 comprising the amino acid sequence listed in any one of SEQ ID NOs: 6, 46, 82, 118 or 154; and a CDR3 comprising the amino acid sequence listed in any one of SEQ ID NOs: 7, 47, 83, 119 or 155.

In one or more CDRs, up to three (preferably one or two) amino acid residues may be replaced by another amino acid residue. Usually, in these variants, some amino acids will be replaced by conservative amino acids. These conservative amino acids include the following groups: G, A; S, A, T; F, Y, W; D, E; N, Q and I, L, V.

The CDR region amino acid sequence of the present invention can be embedded into any suitable framework structure to prepare a chimeric TCR. As long as the framework structure is compatible with the CDR region of the TCR of the present invention, a person skilled in the art can design or synthesize a TCR molecule with corresponding functions based on the CDR region disclosed in the present invention. Therefore, the TCR molecule of the present invention 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 region of the present invention is an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 8, 48, 84, 120 or 156; the TCR β chain variable region of the present invention is an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 12, 52, 88, 124 or 160.

In a preferred embodiment of the present invention, the TCR molecule of the present invention 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 region and a constant region, and the amino acid sequence of the α chain variable region comprises CDR1 (SEQ ID NO: 2, 42, 78, 114 or 150 any one), CDR2 (SEQ ID NO: 3, 43, 79, 115 or 151 any one) and CDR3 (SEQ ID NO: 4, 44, 80, 116 or 152 any one) of the above α chain. Preferably, the TCR molecule comprises an α chain variable region amino acid sequence of SEQ ID NO: 8, 48, 84, 120 or 156 any one. More preferably, the amino acid sequence of the α chain variable region of the TCR molecule is SEQ ID NO: 8, 48, 84, 120 or 156 any one.

On the other hand, the β chain of the heterodimeric TCR molecule comprises a variable region and a constant region, and the amino acid sequence of the β chain variable region comprises CDR1 (SEQ ID NO: 5, 45, 81, 117 or 153), CDR2 (SEQ ID NO: 6, 46, 82, 118 or 154) and CDR3 (SEQ ID NO: 7, 47, 83, 119 or 155) of the above β chain. Preferably, the TCR molecule comprises the amino acid sequence of the β chain variable region of SEQ ID NO: 12, 52, 88, 124 or 160. More preferably, the amino acid sequence of the β chain variable region of the TCR molecule is SEQ ID NO: 12, 52, 88, 124 or 160.

In a preferred embodiment of the present invention, the TCR molecule of the present invention 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 descriptions of single-chain TCR molecules, reference may be made to S Chung. et al., Proc Natl Acad Sci USA, 1994, 91(26), 12654-12658; DH Aggen. et al., Gene Ther, 2012, 19(4): 365-374.

According to the literature, those skilled in the art can easily construct a single-chain TCR molecule comprising the CDRs region of the present invention. Specifically, the α chain and β chain of the single-chain TCR molecule are present in the same polypeptide chain, which comprises Vα, Vβ and Cβ, preferably connected in the order from N-terminus to C-terminus. In order to express a single-chain TCR, it is very useful to provide a construct encoding the constant region of the TCR α chain.

The amino acid sequence of the α chain variable region of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 2, 42, 78, 114 or 150), CDR2 (SEQ ID NO: 3, 43, 79, 115 or 151) and CDR3 (SEQ ID NO: 4, 44, 80, 116 or 152) of the above-mentioned α chain. Preferably, the single-chain TCR molecule comprises the amino acid sequence of the α variable region of SEQ ID NO: 8, 48, 84, 120 or 156. More preferably, the amino acid sequence of the α chain variable region of the single-chain TCR molecule is SEQ ID NO: 8, 48, 84, 120 or 156. The amino acid sequence of the variable region of the β chain of the single-chain TCR molecule comprises CDR1 (SEQ ID NO: 5, 45, 81, 117 or 153), CDR2 (SEQ ID NO: 6, 46, 82, 118 or 154) and CDR3 (SEQ ID NO: 7, 47, 83, 119 or 155) of the above-mentioned β chain. Preferably, the single-chain TCR molecule comprises the amino acid sequence of the variable region of the β chain of SEQ ID NO: 12, 52, 88, 124 or 160. More preferably, the amino acid sequence of the variable region of the β chain of the single-chain TCR molecule is SEQ ID NO: 12, 52, 88, 124 or 160.

In a preferred embodiment of the present invention, the TCR molecule of the present invention is a TCR-mRNA molecule composed of part or all of the α chain and/or part or all of the β chain. For a description of TCR-mRNA molecules, reference may be made to Parayath N.N. et al., Nature Communications, 2020, 11(1), 1-17.

As described in the references, those skilled in the art can easily construct a TCR-mRNA molecule comprising the CDRs region of the present invention. Specifically, the α chain and β chain of the TCR-mRNA molecule are connected together by a linker sequence, which comprises Vα and Vβ.

The α chain amino acid sequence of the TCR-mRNA molecule comprises the CDR1 (SEQ ID NO: 2, 42, 78, 114 or 150), CDR2 (SEQ ID NO: 3, 43, 79, 115 or 151) and CDR3 (SEQ ID NO: 4, 44, 80, 116 or 152) of the above α chain. Preferably, the TCR-mRNA molecule comprises the α variable region amino acid sequence SEQ ID NO: 8, 48, 84, 120 or 156. The β chain amino acid sequence of the TCR-mRNA molecule comprises the CDR1 (SEQ ID NO: 5, 45, 81, 117 or 153), CDR2 (SEQ ID NO: 6, 46, 82, 118 or 154) and CDR3 (SEQ ID NO: 7, 47, 83, 119 or 155) of the above β chain. Preferably, the TCR-mRNA molecule comprises any one of the amino acid sequences of the β chain variable region of SEQ ID NO: 12, 52, 88, 124 or 160.

In a preferred embodiment of the present invention, the constant region of the TCR molecule of the present invention is a human constant region. Those skilled in the art know or can obtain the constant region amino acid sequence of a human by consulting relevant books or the public database of IMGT (International Immunogenetics Information System). For example, the constant region sequence contained in the α chain of the TCR molecule of the present invention can be "TRAC", and the constant region sequence contained in the β chain of the TCR molecule can be "TRBC1" or "TRBC2". IMGT gives its 50th amino acid sequence as Leu in TRAC, which is expressed here as: Leu50 of TRAC, and the same applies to the others. Preferably, the amino acid sequence of the α chain of the TCR molecule of the present invention is any one of SEQ ID NO: 10, 50, 86, 122 or 158, and/or the amino acid sequence of the β chain is any one of SEQ ID NO: 14, 54, 90, 126 or 162.

In a specific embodiment, the TCR is a single chain; preferably, the amino acid sequence of the α chain variable region of the TCR is any one of SEQ ID NO: 26, 66, 102, 138 or 174, and the amino acid sequence of the β chain variable region of the TCR is any one of SEQ ID NO: 28, 68, 104, 140 or 176; more preferably, the TCR is composed of the α chain variable region and the β chain variable region connected by a connecting peptide chain SEQ ID NO: 32, and the amino acid sequence of the TCR is any one of SEQ ID NO: 30, 70, 106, 142 or 178.

In a specific embodiment, the TCR is TCR-mRNA; preferably, the α chain amino acid sequence of the TCR is any one of SEQ ID NO: 34, 72, 108, 144 or 180, and the β chain amino acid sequence of the TCR is any one of SEQ ID NO: 36, 74, 110, 146 or 182; more preferably, the TCR is composed of an α chain and a β chain connected by a connecting peptide chain SEQ ID NO: 40, and the amino acid sequence of the TCR is any one of SEQ ID NO: 38, 76, 112, 148 or 184.

In another preferred embodiment of the present invention, the amino acids on the Vα and Vβ domains outside the antigen binding CDR loop of the TCR molecule of the present invention are replaceable. Simple variable region modifications are performed at a distance away from the antigen binding loop (Thomas, S. et al., Nat Commun, 2019, 10, 4451). Therefore, the TCR of the present invention can be exposed to solvents, hydrophobic cores, Vα-Vβ interfaces, Vα-Cα or Vβ-Cβ interfaces. The residues in the variable regions of the α and β chains can be replaced with amino acids to increase the expression of TCR and improve effector function. Preferably, the amino acid residue replaces one or more sites selected from the following: the 5th, or 8th, or 19th, or 20th, or 24th, or 39th, or 50th, or 55th, or 66th, or 86th, or 96th amino acid of TRAV; the 9th, or 10th, or 43rd amino acid of TRBV.

In another preferred embodiment of the present invention, a new artificial disulfide bond can be introduced between Thr48 of the α chain constant region and Ser57 of the β chain constant region (by replacing these residues with cysteine. The original natural disulfide bond in the TCR connecting peptide can be retained in place or removed. Jonathan M Boulter. et al., Protein Eng, 2003, 16 (9): 707-711; Estelle Baulu. et al., Sci Adv, 2023, 15; 9 (7): eadf3700). Therefore, the TCR of the present invention may contain an artificial disulfide bond formed by cysteine introduced between the residues of its α and β chain constant regions. It should be noted that the constant region may or may not contain the artificial disulfide bond introduced as described above, and the TCR of the present invention may contain the TRAC constant region sequence and the TRBC1 or TRBC2 constant region sequence. There is a set of natural disulfide bonds between the Cα and Cβ chains in the near-membrane region of the natural TCR, which are referred to as "natural interchain disulfide bonds" in the present invention. We call the interchain covalent disulfide bonds artificially introduced in the present invention, whose positions are different from those of the natural interchain disulfide bonds, "artificial interchain disulfide bonds." Preferably, the cysteine residues of the artificial disulfide bond replace one or more groups of sites selected from the following: Thr48 of TRAC and Ser57 of TRBC1 or TRBC2; Tyr10 of TRAC and Ser17 of TRBC1 or TRBC2; Ser15 of TRAC and Val13 of TRBC1 or TRBC2; Thr45 of TRAC and Ser77 of TRBC1 or TRBC2; Thr45 of TRAC and Asp59 of TRBC1 or TRBC2; Leu50 of TRAC and Ser57 of TRBC1 or TRBC2; Arg53 of TRAC and Ser54 of TRBC1 or TRBC2; Ser61 of TRAC and Arg79 of TRBC1 or TRBC2; Pro89 of TRAC and Ala19 of TRBC1 or TRBC2.

According to the literature (Diana Knies. et al., Oncotarget, 2016, 7 (16): 21199-21221), the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of TCR can improve the stability of TCR. Therefore, the α chain variable region and the β chain constant region of the TCR of the present invention may also contain an artificial interchain disulfide bond. In the present invention, the position numbering of the amino acid sequence of the variable region TRAV and TRBV is in accordance with the position numbering listed in IMGT. 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 replaces: the 46th amino acid of TRAV and the 60th amino acid of TRBC1 or TRBC2; the 47th amino acid of TRAV and the 61st amino acid of TRBC1 or TRBC2; the 46th amino acid of TRAV and the 61st amino acid of TRBC1 or TRBC2; or the 47th amino acid of TRAV and the 60th amino acid of TRBC1 or TRBC2.

In another embodiment, the cysteine residue of the artificial disulfide bond also replaces one or more groups of sites selected from the following: the amino acid at position 48, or the 49th, or the 50th amino acid of TRAV, the amino acid at position 17, or the 18th, or the 19th amino acid of the connecting peptide chain between the α chain variable region and the β chain variable region.

In addition, the TCR of the present invention may also be a hybrid TCR comprising sequences derived from more than one species. For example, a study (Cyrille J Cohen. et al., Cancer Res, 2006, 66 (17): 8878-8886) showed that murine TCRs can be expressed more effectively in human T cells than human TCRs. Therefore, the TCR of the present invention may comprise a hybrid TCR consisting of a human variable region and a mouse constant region.

The double-chain TCR molecules of the present invention (e.g., α and β chain molecules containing the amino acid sequences given in SEQ ID NO: 10 and NO: 14) or chimeric TCR molecules containing the specific CDRs described above are introduced into human T cells to obtain antigen-specific CTLs; similarly, transduction of single-chain TCRs can also be used to obtain antigen-specific CTLs, and single-chain TCRs have the advantage of not pairing with endogenous TCRs. Single-chain TCRs can also be prepared into soluble TCRs in a manner similar to antibodies. In soluble TCRs, single-chain TCRs do not contain transmembrane regions (S Chung. et al., Proc Natl Acad Sci USA, 1994, 91 (26), 12654-12658; Jonathan M Boulter. et al., Protein Eng, 2003, 16 (9): 707-711).

It should be understood that the amino acid names herein are represented by single letters or three letters in the internationally accepted manner, and the correspondence between single letters and three letters of the amino acid names 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 fourth aspect of the present invention provides a nucleic acid molecule encoding the TCR molecule or part thereof of the first and second aspects of the present invention, wherein the part may be one or more CDRs, variable regions of α and/or β chains, and α chains and/or β chains.

The nucleotide sequence encoding the CDR region of the α chain of the TCR molecule of the first aspect of the present invention is as follows:

A CDR1 comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 16, 56, 92, 128, or 164; a CDR2 comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 17, 57, 93, 129, or 165; a CDR3 comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 18, 58, 94, 130, or 166;

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

A CDR1 comprising the nucleotide sequence listed in any one of SEQ ID NOs: 19, 59, 95, 131 or 167; a CDR2 comprising the nucleotide sequence listed in any one of SEQ ID NOs: 20, 60, 96, 132 or 168; and a CDR3 comprising the nucleotide sequence listed in any one of SEQ ID NOs: 21, 61, 97, 133 or 169.

Therefore, the nucleotide sequence of the nucleic acid molecule encoding the TCR α chain of the present invention includes any one of SEQ ID NO: 16, 56, 92, 128 or 164, any one of SEQ ID NO: 17, 57, 93, 129 or 165 and any one of SEQ ID NO: 18, 58, 94, 130 or 166, and/or the nucleotide sequence of the nucleic acid molecule encoding the TCR β chain of the present invention includes any one of SEQ ID NO: 19, 59, 95, 131 or 167, any one of SEQ ID NO: 20, 60, 96, 132 or 168 and any one of SEQ ID NO: 21, 61, 97, 133 or 169.

The nucleotide sequence of the nucleic acid molecule of the present invention may be single-stranded or double-stranded, the nucleic acid molecule may be DNA or RNA, and may or may not contain introns. Preferably, the nucleotide sequence of the nucleic acid molecule of the present invention does not contain introns but can encode the polypeptide of the TCR of the present invention, for example, the nucleotide sequence of the nucleic acid molecule encoding the variable region of the TCR α chain of the present invention comprises any one of SEQ ID NO: 9, 27, 49, 67, 85, 103, 121, 139, 157 or 175 and/or the nucleotide sequence of the nucleic acid molecule encoding the variable region of the TCR β chain of the present invention comprises any one of SEQ ID NO: 13, 29, 53, 69, 89, 105, 125, 141, 161 or 177. Alternatively, the nucleotide sequence of the nucleic acid molecule of the invention encoding the variable region of the TCR alpha chain of the invention comprises any one of SEQ ID NOs: 27, 67, 103, 139 or 175 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding the variable region of the TCR beta chain of the invention comprises any one of SEQ ID NOs: 29, 69, 105, 141 or 177. Alternatively, the messenger ribonucleotide sequence of the nucleic acid molecule of the invention encoding the TCR alpha chain of the invention comprises any one of SEQ ID NOs: 35, 73, 109, 145 or 181 and/or the messenger ribonucleotide sequence of the nucleic acid molecule of the invention encoding the TCR beta chain of the invention comprises any one of SEQ ID NOs: 37, 75, 111, 147 or 183. Alternatively, the nucleotide sequence of the nucleic acid molecule of the invention comprises any one of SEQ ID NOs: 11, 51, 87, 123 or 159 encoding the TCR alpha chain and/or comprises any one of SEQ ID NOs: 15, 55, 91, 127 or 163 encoding the TCR beta chain. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present invention is any one of SEQ ID NO: 31, 71, 107, 143 or 179. Alternatively, the messenger ribonucleotide sequence of the nucleic acid molecule of the present invention is any one of SEQ ID NO: 39, 77, 113, 149 or 185.

The sequence list of the present invention is as follows:

It should be understood that due to the degeneracy of the genetic code, different nucleotide sequences can encode the same polypeptide. Therefore, the nucleic acid sequence encoding the TCR of the present invention can be the same as the nucleic acid sequence shown in the present invention or a degenerate variant. Taking one of the examples in the present invention as an example, a "degenerate variant" refers to a nucleic acid sequence that encodes a protein sequence having SEQ ID NO: 8, but is different from the sequence of SEQ ID NO: 9.

The nucleotide sequence may be codon optimized. Different cells differ in the use of specific codons, and the codons in the sequence may be altered to increase expression depending on the cell type. Codon usage tables for mammalian cells and 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 invention or its fragment can usually be obtained by, but not limited to, PCR amplification, recombination or artificial synthesis. At present, the DNA sequence encoding the TCR of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. The DNA can be a coding strand or a non-coding strand.

The present invention also provides an expression vector comprising a polynucleotide of the present invention. When present in a suitable host cell, such an expression vector allows expression of a target polypeptide. Preferably, the expression vector is capable of expressing the polypeptide in a mammalian cell. More preferably, the expression vector is capable of expressing the polypeptide in a T cell (e.g., human CTL). Typically, the expression vector comprises a promoter active in a specific cell type, which promoter is likely to be controllable (e.g., inducible).

Preferably, the expression vector is a viral vector and/or a lipid nanoparticle vector; more preferably, the expression vector is suitably a lentiviral vector and/or a lipid nanoparticle vector, which can be transfected into a mammalian host cell such as a human T cell. Typically, the vector is a lentiviral vector.

Another aspect of the present invention provides a host cell comprising a polynucleotide of the present invention or a vector of the present invention. The host cell can be any cell, such as a bacterial cell, a yeast cell, an insect cell, a plant cell or a mammalian cell, and the method of transferring the polynucleotide into such a cell is well known in the art. Typically, bacterial cells, such as Escherichia coli cells, are used for the propagation and amplification of the polynucleotides and vectors of the present invention. Other host cells can be used to express the TCR molecules of the present invention. In order to express the TCR molecules of the present invention, the host cell needs to contain one or more nucleotides or vectors encoding both the α chain portion and the β chain portion. In particular, the cell can be a mammalian cell, such as a human cell. As described below for the treatment method using the TCR molecules of the present invention, it is particularly desirable that the host cell is a T cell and (preferably) is derived from a patient to be treated, typically a T cell of a patient with a malignant tumor expressing WT1.

Therefore, the present invention also includes cells expressing the TCR of the present invention, in particular T cells. Preferably, the T cell is a T cell from a tumor patient, and the T cell (taking the patient as an example) is usually derived from peripheral blood mononuclear cells (PBMC), which can be in a mixed cell population containing CD4+ helper T cells and/or CD8+ cytotoxic T cells. Generally, T cells can be activated with antibodies (such as anti-CD3 and/or anti-CD28 antibodies) and/or immunomagnetic beads (such as anti-CD3/CD28 coupled magnetic beads) so that they can more easily accept the transduction of the lentiviral vector encoding the TCR molecule of the present invention and promote the integration of the lentivirus and the stable expression of tumor-specific TCR, and also make them more easily accept the transfection of the lipid nanoparticle vector encoding the TCR molecule of the present invention and promote the short-term expression of tumor-specific TCR. The present invention also covers cells transduced and transfected by the nucleic acid or vector of the present invention; preferably, the cell is a T cell or a stem cell; more preferably, the cell is a primary T cell or stem cell from a subject.

Clinical treatment:

The TCR molecules of the present invention are transferred into the T cells of a patient with a malignant tumor expressing WT1 (or T cells from a donor), and then these TCR genetically engineered cells are transduced into the patient to achieve treatment of the patient. Therefore, another aspect of the present invention provides a method for treating a malignant tumor expressing WT1, the method comprising preferentially transducing T cells from the patient with the TCR molecules of the present invention, and then returning the T cells expressing the TCR molecules of the present invention to the patient. The treatment method generally comprises: (1) obtaining T cells from the patient, (2) transferring one or more polynucleotides encoding and capable of expressing the TCR molecules of the present invention into T cells in vitro, and (3) returning the TCR genetically engineered T cells to the patient. It is particularly preferred if the T cells are from the patient himself. The number of cells separated, transduced, and returned to the patient can be determined by the physician.

Alternatively, the cells of the present invention can also be or be derived from human stem cells, such as hematopoietic stem cells (HSC). Transferring the TCR gene into HSC will not result in the expression of TCR on its cell surface, because the CD3 molecule is not expressed on the stem cell surface. However, when the stem cells transduced with the TCR gene differentiate into lymphoid precursors that migrate to the thymus, the expression of the CD3 molecule will bring the transferred TCR molecule to the surface of the thymocytes to form tumor-specific T cells.

Typically, a lentiviral vector encoding the TCR molecule of the present invention can infect human CD4+ or CD8+ T lymphocytes and mediate the expression of the TCR gene: the lentiviral vector system is a preferred possibility. T cells expressing the TCR of the present invention can be used for adoptive immunotherapy of malignant tumors. Those skilled in the art will be aware of many suitable methods for adoptive immunotherapy.

The present invention also relates to a method for treating and/or preventing a disease associated with WT1 in a subject, comprising the step of adoptively transferring WT1-specific T cells into the subject. The WT1-specific T cells can recognize the HLA-A*02:01/RMFPNAPYL complex expressed on the surface of tumor cells.

The WT1-specific T cells of the present invention can be used to treat any WT1-related disease presenting the WT1 antigen short peptide RMFPNAPYL-HLA-A*02:01 complex, including but not limited to tumors, preferably the tumors include leukemia (such as AML, CML and MDS, etc.), lung cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, prostate cancer, thyroid cancer, head and neck cancer, glioblastoma and sarcoma, etc.

After clinical treatment, T cells can be removed from the patient and cryopreserved. If the patient relapses, the patient's T cells can be re-transduced with TCR and reinfused.

Whether a patient's tumor expresses WT1 can be determined using RT-PCR or intracellular staining techniques (using anti-WT1 antibodies).

Although animals can be used in research situations, the patient is preferably a human patient. Particularly preferably, the patient is HLA-A*02:01 positive. Whether a patient is HLA-A*02:01 positive can be determined by methods well known in the art.

Another aspect of the present invention provides a use of a T cell, preferably a T cell derived from a patient, which is modified to express the TCR molecule of the present invention so as to be made into a drug capable of resisting a tumor expressing WT1 in a patient.

The main advantage of the present invention is that the TCR of the present invention can bind to the WT1 antigen short peptide complex RMFPNAPYL-HLA-A*02:01 with high affinity, so the WT1-specific T cells of the present invention can be used to treat any tumor related to presenting the WT1 antigen short peptide RMFPNAPYL-HLA-A*02:01 complex. Since the T cells transduced with the TCR of the present invention can not only be specifically activated and amplified by the target cells presenting WT1, but also can produce antigen-specific immune factors (Figures 3-7), and can selectively kill tumor cells (Figures 9-13), it may have stronger tumor specificity and lower toxic side effects than traditional therapies. Especially when the tumor disease develops to the late stage and spreads, when traditional therapies are helpless, by injecting the WT1-specific T cells of the present invention into the patient's body, these T cells specific to WT1 can track any metastatic tumor cells through circulation in the body.

The present invention will be further described herein by the following specific examples. However, it should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

Example 1

Obtain WT1 antigen short peptide-specific T cell clones.

Peripheral blood mononuclear cells (PBMC) from healthy volunteers with negative HLA-A2 were stimulated with T2 cells (the cells themselves express HLA-A2, but because they lack the antigen processing-related factor TAP, they can effectively load exogenous peptides) loaded with the synthetic short peptide RMFPNAPYL (SEQ ID NO: 1; synthesized by Jiangsu GenScript Biotechnology Co., Ltd.), and T cells in PBMC that can recognize the HLA-A*02:01/RMFPNAPYL complex will be activated and expanded. Among them, T cells that are specific to the antigen short peptide RMFPNAPYL can be detected with PE-labeled HLA-A*02:01-RMFPNAPYL tetramer (MBL International). The amplified T cells are stained and sorted with tetramer-PE and anti-CD8-APC to obtain double-positive cells. In order to obtain true tumor-specific CTLs that are restricted by HLA-A*02:01 and can recognize the antigen peptide RMFPNAPYL, we used the limiting dilution method to culture the double-positive cells obtained by tetramer-PE and anti-CD8-APC staining sorting. After screening hundreds of monoclones, we obtained 5 tumor-specific T cell clones that are restricted by HLA-A*02:01 and can recognize the antigen peptide RMFPNAPYL. The FACS data of these monoclonal T cells after staining with CD8 and tetramer are shown in Figure 1.

Example 2

The TCR gene of WT1 antigen short peptide-specific T cell clone was obtained and the vector was constructed.

Methods for single-cell VDJ sequencing have been developed in recent years and are well known in the art and are described in detail in manuals such as 10×Genomics based on microfluidics and oil droplet encapsulation technology.

Specifically, gel beads with barcodes and primers are encapsulated in oil droplets with single cells of T cell clones specific to the antigenic short peptide RMFPNAPYL and restricted to HLA-A2 screened in Example 1. The gel beads are dissolved in each oil droplet, the cells are lysed to release mRNA, and barcoded cDNA for sequencing is produced by reverse transcription, followed by TCR library construction; wherein the V(D)J sequence of the TCR is enriched by nested PCR primers designed in the C region of the TCR. The library is then sequenced and tested using the Illumina sequencing platform to obtain the TCR data of the T cell clones in Example 1. After α and β pairing and verification, these double-positive clones express TCR α chains containing CDRs with the following amino acid sequences:

CDR1α - SEQ ID NO: 2, 42, 78, 114 or 150

CDR2α - SEQ ID NO: 3, 43, 79, 115 or 151

CDR3α - SEQ ID NO: 4, 44, 80, 116 or 152

The beta chain contains a CDR with the following amino acid sequence:

CDR1β - SEQ ID NO: 5, 45, 81, 117 or 153

CDR2β - SEQ ID NO: 6, 46, 82, 118 or 154

CDR3β - SEQ ID NO: 7, 47, 83, 119 or 155

By artificial gene synthesis, the partial or full-length genes of the TCR α chain and β chain are connected to the P2A sequence through the Furin GSG linker to obtain the TCR α-2A-TCR β fragment. By the standard method described in J Sambrook, David Russell. et al., Molecular Cloning: A Laboratory Manual, 2016, Third Edition, this fragment is double-digested with NotI+SalI and cloned into the lentiviral expression vector pLVX (addgene) to obtain the recombinant plasmid pLVX-TCRα-2A-TCRβ. The recombinant plasmid can express the amino acid sequence of the TCR α chain and β chain variable region shown in SEQ ID NO: 8, 48, 84, 120 or 156 and SEQ ID NO: 12, 52, 88, 124 or 160, and can also express the amino acid sequence of the single-chain TCR shown in SEQ ID NO: 30, 70, 106, 142 or 178. Or the TCR-mRNA molecule is cloned into the in vitro transcription vector pcDNA3.1(+), the recombinant plasmid can express the amino acid sequence of the TCR α chain and β chain shown in SEQ ID NO: 34, 72, 108, 144 or 180 and SEQ ID NO: 36, 74, 110, 146 or 182, and the mRNA expressing TCR is obtained by in vitro transcription, 5' Cap1 capping, Pseudo-UTP (Ψ) replacement, 5-Methyl-CTP replacement and 3' poly (A) tailing.

In order to enable the α and β chains of the TCR molecule of the present invention to form correct pairing more effectively during the transduction process, a cysteine residue was introduced into the constant region of the α and β chains of the TCR molecule of the present invention to form an artificial interchain disulfide bond, and the positions of the introduced cysteine residues were Thr48 of TRAC and Ser57 of TRBC, respectively.

Example 3

Preparation of TCR-lentivirus.

Preparation of recombinant plasmid: The recombinant plasmid pLVX-TCR obtained in Example 2 was transformed into Dh5a competent cells, evenly spread on LB solid culture medium plates containing ampicillin, and cultured at 37°C for 24 hours. Then, a single colony was picked up and transferred to LB liquid culture medium containing ampicillin, and cultured at 37°C and 220 rpm/min for 14-16 hours to extract the plasmid.

Packaging of recombinant plasmid: 293T cells in logarithmic growth phase were used as packaging cells and inoculated into a 10 cm dish containing culture medium (DMEM containing 10% FBS). When the cell density reached 80-85%, the recombinant plasmid pLVX-TCR described in Example 2 was transfected using the standard polyethyleneimine (PEI) method. After 6-8 hours of culture, the culture medium containing the transfection reagent was removed and replaced with fresh complete culture medium. After 24-48 hours, the culture fluid was collected and filtered with a 0.45 μm filter membrane to remove cell residues, and the TCR-lentiviral suspension was obtained and stored at -80°C.

Example 4

Preparation of WT1-specific TCR-T cells and analysis of their TCR expression.

Peripheral blood was obtained from healthy volunteers, and human peripheral blood mononuclear cells (PBMC) were isolated using a rapid mononuclear cell separation tube (Xiamen Sanyi Hematopoietic) and human peripheral blood lymphocyte separation fluid (Tianjin Haoyang Biological Products). The cell density was adjusted to 1×10 ⁶ cells/mL, and anti-CD3/CD28 coupled magnetic beads (Miltenyi Biotec) were added to the cell culture medium to activate the T cells. After 48 hours, the lentivirus was taken out from the -80℃ low-temperature refrigerator and quickly thawed in a 37℃ water bath. In a 24-well plate pre-coated with RetroNectin (Takara), 0.5×10 ⁶ PBMC was placed in each well, 1.5mL of viral supernatant was added, and IL-2 (600U/mL) was added at the same time, and the mixture was gently blown and mixed, and centrifuged at 931g at 32℃ for 90 minutes. Then it was placed in an incubator at 37℃ and 5% CO ₂ for continued culture. After 24 hours, the culture supernatant containing the virus was replaced with fresh culture medium and culture was continued. On the 4th day, FACS was used to detect the expression of WT1-TCR on T cells. As shown in Figure 2, the mock-transduced T cells could not be stained with WT1-tetramers, indicating that they had no WT1 specificity; while the WT1-TCR-transduced T cells could be stained with WT1-tetramers, indicating that they acquired WT1 specificity through TCR transduction. When the freshly transduced WT1-TCR-T shown in Figure 2 was stimulated with T2 cells loaded with the WT1 antigen peptide RMFPNAPYL, the WT1-specific T cells therein were significantly expanded.

Example 5

Intracellular immune factor staining was used to detect the function of WT1-specific TCR-T cells.

The artificially synthesized WT1 antigen peptide RMFPNAPYL and the control peptide KLPQLCTEL were incubated with T2 cells at 37°C and 5% CO2 for ₂ h (the concentration of the peptide was 10 μM, 1 μM, 100 nM, 10 nM, 1 nM, 100 pM, and the concentration of T2 cells was 4 × 10 ⁶ cells/mL). After irradiation for 70 gy, the unbound antigen peptide and control peptide were washed to remove the cells, and then the cells were collected to obtain T2 cells loaded with antigen peptides and control peptides.

The WT1-specific TCR-T cells obtained in Example 4 were co-cultured with T2 target cells loaded with the specific antigen peptide RMFPNAPYL or the control antigen peptide KLPQLCTEL in a 96-well plate at 37°C and 5% _CO2 , wherein the concentration of T cells and target cells was 0.4×10 ⁶ /well, and BFA (the role of BFA is to retain the immune factors in T cells in the cells without being released, so as to monitor staining by FACS) was added to make the final concentration 1.5 μg/mL. After 24 hours of co-culture, the cells were collected, and the cell surface was first stained with anti-CD3/CD8-APC, and then the intracellular immune factor staining was performed using the Fix and Perm kit (Invitrogen) according to the manufacturer's instructions. The stained cells were detected by FACS for the production of various immune factors. Figures 3-7 show that after WT1-TCR-T and T2 target cells are co-cultured, T2 cells loaded with WT1 antigen peptides can stimulate TCR-T to secrete IFN-γ, while T2 cells loaded with control antigen peptides cannot cause TCR-T to express IFN-γ; and the concentration of specific antigen peptides recognized by WT1-TCR-T can be as low as picomolar levels. These results show that the high-affinity TCR obtained by the present invention can specifically recognize the WT1 antigen peptide RMFPNAPYL at the nanomolar to picomolar level, and the T cells transduced with the TCR of the present invention can secrete IFN-γ after recognizing target cells, thereby playing a role in killing target cells, and when encountering control target cells, IFN-γ is not produced, thereby avoiding unnecessary side effects.

Example 6

Preparation of target cells K562-A2-GFP and T2.

HLA-A*02:01-GFP can be obtained by artificial gene synthesis, wherein A2 and GFP are connected by the P2A sequence. The recombinant plasmid pLVX-HLA-A*02:01-GFP can be obtained by the same method as in Example 2. HLA-A*02:01-GFP can be transferred into the leukemia cell line K562 expressing WT1 by the same method as in Examples 3 and 4 to obtain the positive target cell K562-A2-GFP required by the present invention. The HLA-A2 staining and GFP expression of the target cell are shown in Figure 8. As can be seen from Figure 8, the target cell expresses both HLA-A2 and GFP, wherein the expression of HLA-A*02:01 and WT1 can make it recognized by the WT1-TCR-T of the present invention, and the expression of GFP can be used to show whether the target cell is killed. In addition, T2 cells are prepared, which express HLA-A2 by themselves but do not express WT1, and are used as control target cells T2.

Example 7

Flow cytometry was used to detect the killing effect of WT1-TCR-T cells on target cells.

The K562-A2-GFP constructed in Example 6 was used as a positive target cell, and T2 was used as a control negative target cell. 0.1×10 ⁶ positive target cells were mixed with 0.1×10 ⁶ negative target cells in 200 μL RPMI1640 medium in a round-bottom 96-well plate, and then 1×10 ⁶ WT1-TCR-T cells were added for mixed culture. After 24 hours, all cells in each well were aspirated into a 1.5 mL centrifuge tube, washed with 1 mL FACS buffer, and first stained with HLA-A2 to separate the target cells from the mixed cell population, and then the change in the ratio between the positive target cells (GFP+) and the control target cells (GFP-) was observed in the HLA-A2+ target cells. Figures 9-13 show that in the absence of WT1-TCR-T cells, both the control target cells T2 and the positive target cells K562-A2-GFP survived well, but in the presence of WT1-TCR-T cells, the positive target cells K562-A2-GFP were significantly killed, while the control target cells T2 still survived well, indicating that the WT1-TCR-T cells of the present invention can selectively kill leukemia cells that express both HLA-A*02:01 and WT1.

The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims (11)

1. A T Cell Receptor (TCR), comprising: the T cell receptor is capable of recognizing RMFPNAPYL-HLA-A 02:01 complex with high affinity, the TCR comprising a TCR a chain variable region and a TCR β chain variable region, wherein:

the TCR a chain variable region comprises three Complementarity Determining Regions (CDRs) of sequence SEQ ID NO:2-4, 42-44, 78-80, 114-116 or 150-152;

The TCR β chain variable region comprises three Complementarity Determining Regions (CDRs) of sequence SEQ ID NO:5-7, 45-47, 81-83, 117-119 or 153-157.

2. The T Cell Receptor (TCR) of claim 1, wherein: the TCR is an αβ heterodimer, further comprising a TCR α chain constant region (TRAC) and a TCR β chain constant region (TRBC 1 and/or TRBC 2);

preferably, the alpha chain amino acid sequence of the TCR is SEQ ID NO: 10. 50, 86, 122, or 158, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto;

The beta chain amino acid sequence of the TCR is SEQ ID NO: 14. 54, 90, 126 or 162 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

3. The T Cell Receptor (TCR) of claim 1, wherein: the TCR is single chain;

Preferably, the alpha chain variable region amino acid sequence of the TCR is SEQ ID NO: 26. 66, 102, 138 or 174;

the amino acid sequence of the beta chain variable region of the TCR is SEQ ID NO: 28. 68, 104, 140 or 176;

More preferably, the TCR is formed by a linkage of the alpha chain variable region to the beta chain variable region by a peptide chain SEQ ID NO:32, wherein the amino acid sequence of the TCR is SEQ ID NO: 30. 70, 106, 142 or 178.

4. The T Cell Receptor (TCR) of claim 1, wherein: the TCR is a messenger ribonucleotide (mRNA);

Preferably, the alpha chain amino acid sequence of the TCR-mRNA is SEQ ID NO: 34. 72, 108, 144 or 180;

the beta chain amino acid sequence of the TCR-mRNA is SEQ ID NO: 36. 74, 110, 146 or 182;

More preferably, the TCR-mRNA is formed from an alpha chain and a beta chain linked by a peptide chain SEQ ID NO:40, wherein the amino acid sequence of the TCR-mRNA is SEQ ID NO: 38. 76, 112, 148 or 184.

5. A complex of multivalent T Cell Receptors (TCRs), comprising at least three TCR molecules, and wherein at least one TCR molecule is a TCR as claimed in any one of claims 1 to 4.

6. A nucleic acid molecule characterized in that: the nucleic acid molecule comprising a nucleotide sequence encoding the TCR of any one of claims 1-4, or a complement thereof, or a codon-optimized nucleotide sequence corresponding to the amino acid sequence of the TCR of any one of claims 1-4;

Preferably, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 9. 27, 49, 67, 85, 103, 121, 139, 157 or 175, or a sequence having at least 90% sequence identity thereto;

The nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 13. 29, 53, 69, 89, 105, 125, 141, 161, or 177, or a sequence having at least 90% sequence identity thereto;

Preferably, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 11. 51, 87, 123 or 159, or a sequence having at least 90% sequence identity thereto, said nucleic acid molecule comprising a nucleotide sequence encoding a TCR β chain of SEQ ID NO: 15. 55, 91, 127 or 163, or a sequence having at least 90% sequence identity thereto;

Preferably, the nucleic acid molecule comprises the messenger ribonucleic acid sequence SEQ ID NO: 35. 73, 109, 145 or 181, or a sequence having at least 90% sequence identity thereto, said nucleic acid molecule comprising a messenger ribonucleic acid sequence encoding a TCR β chain SEQ ID NO: 37. 75, 111, 147 or 183, or a sequence having at least 90% sequence identity thereto, wherein said messenger ribonucleotide sequence is substituted with a pseudoutp (ψ) substitution and/or a 5-Methyl-CTP substitution.

7. A carrier, characterized in that: said vector comprising the nucleic acid molecule of claim 6; preferably, the vector is a viral vector and/or a lipid nanoparticle vector; preferably, the vector is a lentiviral vector and/or a retroviral vector; more preferably, the vector is a lentiviral vector.

8. An isolated host cell, characterized in that: the host cell comprises the vector of claim 11 or the nucleic acid molecule of claim 6 integrated with an exogenous source.

9. A cell, characterized in that: the cell is a cell transduced and transfected with the nucleic acid molecule of claim 6 or the vector of claim 11, wherein the TCR is heterologous to the cell; preferably, the cell is a T cell or a stem cell; more preferably, the cell is a primary T cell or stem cell from a subject.

10. A pharmaceutical composition characterized by: the composition comprises a TCR as claimed in any one of claims 1 to 4, a TCR complex as claimed in claim 9, a nucleic acid molecule as claimed in claim 6, or a cell as claimed in claim 9, and a pharmaceutically acceptable carrier.

11. Use of a T cell receptor according to any one of claims 1 to 4, or a TCR complex as claimed in claim 5 or a cell as claimed in claim 9, for the manufacture of a medicament for the treatment of a tumour or other immune disorder.