TCR molecule for identifying tumor-associated antigen and use thereof

Abstract

Provided is a TCR capable of specifically binding to a tumor antigen PRAME-derived polypeptide SLLQHLIGL, the polypeptide being capable of being delivered to the cell surface by means of HLA-A02 molecules. An HLA-A02 complex incorporating the PRAME polypeptide can be used as a target of the TCR to develop TCR-related therapies. Also provided are a nucleic acid molecule encoding the TCR, a vector comprising the nucleic acid molecule, and a cell transducing the TCR.

Description

TCR molecules recognizing tumor-associated antigens and uses thereof Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to TCRs capable of recognizing short peptides derived from PRAME antigens and their coding sequences, T cells transduced with the TCRs, and their use in preventing and treating PRAME-related diseases. Background Art

[0002] TCRs (T cell receptors, TCRs) are typically expressed specifically on the surface of T cells as heterodimers. TCR heterodimers non-covalently bind to multiple different CD3 subunits in specific ratios to form a TCR-CD3 complex. T cells can be divided into two types: αβT cells and γδT cells. The corresponding TCRs can also be divided into two types: αβTCRs and γδTCRs. αβTCRs are composed of two chains, α and β, while γδTCRs are composed of two chains, γ and δ. The difference between αβTCRs and γδTCRs lies in their diversity. αβTCRs possess greater diversity and play a major role in cellular immunity. In peripheral blood, 90%-95% of T cells express αβTCRs. Each peptide chain of an αβTCR is divided into a variable region (V region), a constant region (C region), a transmembrane region, and an intracellular region. TCR molecules belong to the immunoglobulin superfamily, and their antigen specificity is determined by the V region sequence; the V region (Vα, Vβ) each has three hypervariable regions CDR1, CDR2, and CDR3, among which CDR3 has the greatest variation and directly determines the antigen binding specificity of TCR (Xu, X., Li, H. & Xu, C. Structural understanding of T cell receptor triggering. Cell Mol Immunol 17, 193-202 (2020).).

[0003] TCR mediates T cell recognition of target cells by recognizing a complex formed by a peptide and a major histocompatibility antigen (MHC). MHC is mainly divided into two types: MHC I and MHC II, which differ greatly in structure and tissue cell expression distribution. MHC I is composed of an α chain and a β2-microglobulin (B2M) chain, while MHC II is composed of an α chain and a β chain. During intracellular secretion and transport to the cell membrane, MHC molecules carry specific peptide molecules to form a peptide-MHC complex (R. A. Mariuzza, P. Agnihotri and J. Orban. The structural basis of T-cell receptor (TCR) activation: An enduring enigma. J Biol Chem 295(4); 914-925(2020)). When TCR recognizes the peptide-MHC complex, the TCR variable region CDR3 directly binds to the antigen peptide, determining the specificity of the TCR. TCR can be developed into TCR-T cell therapy or bispecific drugs.

[0004] T cell activation plays an important role in resisting the invasion of pathogenic microorganisms, foreign proteins, harmful environmental molecules, and in the prevention and treatment of diseases such as tumors and hyperplasia as part of the human immune protection mechanism. T cell surface expression is used to recognize receptor molecules TCR for short peptide antigens. These short peptide antigens are combined with major histocompatibility complex (MHC) molecules (also known as human leukocyte antigens (HLA)) and presented by antigen presenting cells (APC) (Davis et al.Ligand recognition by alpha beta T cell receptors.Annu Rev Immunol 16,523-44 (1998)). CD8+ T cells are also known as cytotoxic T cells and have TCRs that specifically recognize peptides bound to MHC class 1 molecules. CD8+ T cells are generally responsible for discovering and mediating the destruction of diseased cells, including cancerous cells and virus-infected cells.

[0005] TCR-T is an adoptive cell therapy that screens and identifies TCR sequences that specifically bind to target antigens. These sequences are then genetically engineered into peripheral blood-derived T cells, creating T cells capable of recognizing specific targets. These cells are called TCR-T cells. TCR bispecific antibodies combine TCRs and anti-CD3 antibodies through molecular design to form bispecific antibodies that simultaneously target tumor antigens and T cells. Currently, TCR-based therapies primarily include TCR-T cell therapy and TCR bispecific drugs. Due to the specific nature of their recognition and action mechanisms, TCRs can recognize intracellular targets, expanding the range of target options. However, the available targets remain limited. Selecting the appropriate target is crucial to the success of TCR therapy.

[0006] PRAME (Protein Preferentially Expressed Antigen in Melanoma) is a protein encoded by the PRAME gene and highly expressed in human melanoma tumors. As a member of the cancer-testis antigen family, PRAME is only expressed in low and selective amounts in normal human tissues, but is expressed at high levels in a variety of tumors, such as melanoma, non-small cell lung cancer, ovarian cancer, and breast cancer. These properties make PRAME an ideal target for tumor-targeted therapy. After processing, the PRAME protein contains a unique peptide sequence: SLLQHLIGL, which can be presented on the cell surface by HLA-A02 molecules. The HLA-A02 complex bound to the PRAME peptide can serve as a TCR target for the development of TCR-related therapies.

[0007] Therefore, there is an urgent need in the art for TCRs that can recognize PRAME polypeptides.

[0008] Summary of the Invention

[0009] The object of the present invention is to provide a T cell receptor that recognizes tumor-associated antigens, particularly PRAME polypeptide.

[0010] In a first aspect, the present invention provides a T cell receptor (TCR) capable of binding to a SLLQHLIGL polypeptide-HLA-A*02 complex.

[0011] In a preferred embodiment, the TCR comprises a TCR α chain variable domain and a TCR β chain variable domain,

[0012] The amino acid sequence of CDR1 of the TCR α chain variable domain is selected from the group consisting of SEQ ID NO: 12, 22, 32;

[0013] The amino acid sequence of CDR2 of the TCR α chain variable domain is selected from the group consisting of SEQ ID NO: 13, 23, 33;

[0014] The amino acid sequence of the CDR3 of the TCR α chain variable domain is selected from the group consisting of SEQ ID NO: 14, 24, 34, 42;

[0015] The amino acid sequence of CDR1 of the TCRβ chain variable domain is selected from the group consisting of SEQ ID NO: 17, 27, 37, 45;

[0016] The amino acid sequence of CDR2 of the TCRβ chain variable domain is selected from the group consisting of SEQ ID NO: 18, 28, 38, 46; and / or

[0017] The amino acid sequence of CDR3 of the TCRβ chain variable domain is selected from the following group: SEQ ID NO: 19, 29, 39, 47.

[0018] In a preferred embodiment, the amino acid sequences of CDR1, CDR2 and CDR3 of the TCR alpha chain variable domain are selected from the following combinations: SEQ ID NOs: 12, 13 and 14; SEQ ID NOs: 22, 23 and 24; SEQ ID NOs: 32, 33 and 34; SEQ ID NOs: 22, 23 and 42;

[0019] and / or

[0020] The amino acid sequences of CDR1, CDR2 and CDR3 of the TCRβ chain variable domain are selected from the following combinations: SEQ ID NOs: 17, 18 and 19; SEQ ID NOs: 27, 28 and 29; SEQ ID NOs: 37, 38 and 39; SEQ ID NOs: 45, 46 and 47.

[0021] In a preferred embodiment, the amino acid sequences of CDR1, CDR2 and CDR3 of the TCRα chain variable domain and the amino acid sequences of CDR1, CDR2 and CDR3 of the TCRβ chain variable domain are selected from the following combinations:

[0022] SEQ ID NOs: 12, 13, and 14 and SEQ ID NOs: 17, 18, and 19;

[0023] SEQ ID NOs: 22, 23, and 24 and SEQ ID NOs: 27, 28, and 29;

[0024] SEQ ID NOs: 32, 33, and 34 and SEQ ID NOs: 37, 38, and 39;

[0025] SEQ ID NOs: 22, 23 and 42 and SEQ ID NOs: 45, 46 and 47.

[0026] In a preferred embodiment, the TCR comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the TCR alpha chain variable domain is an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO: 11, 21, 31 or 41; and / or the TCR beta chain variable domain is an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO: 16, 26, 36 or 44.

[0027] In a preferred embodiment, the amino acid sequence of the TCRα chain variable domain of the TCR is shown in SEQ ID NO:11, 21, 31 or 41; and / or the amino acid sequence of the TCRβ chain variable domain is shown in SEQ ID NO:16, 26, 36 or 44.

[0028] In a preferred embodiment, the amino acid sequence of the TCRα chain variable domain and the amino acid sequence of the TCRβ chain variable domain of the TCR are selected from the following combinations:

[0029] SEQ ID NOs: 11 and 16; SEQ ID NOs: 21 and 26; SEQ ID NOs: 31 and 36; SEQ ID NOs: 41 and 44.

[0030] In a preferred embodiment, the TCR comprises a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain is an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO: 10, 20, 30 or 40; and / or the TCR beta chain variable domain is an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to SEQ ID NO: 15, 25, 35 or 43.

[0031] In a preferred embodiment, the amino acid sequence of the α chain of the TCR is selected from the group consisting of SEQ ID NO: 10, 20, 30 and 40; and / or

[0032] The amino acid sequence of the β chain of the TCR is selected from the group consisting of SEQ ID NO: 15, 25, 35 and 43.

[0033] In a preferred embodiment, the α chain amino acid sequence and the β chain amino acid sequence of the TCR are selected from the following combinations: SEQ ID NOs: 10 and 15; SEQ ID NOs: 20 and 25; SEQ ID NOs: 30 and 35; SEQ ID NOs: 40 and 43.

[0034] In a preferred embodiment, the TCR is soluble.

[0035] In a preferred embodiment, the TCR is a single-chain TCR.

[0036] In a preferred embodiment, the TCR is composed of an α chain variable domain and a β chain variable domain connected by a peptide linker sequence.

[0037] In a preferred embodiment, the TCR comprises (i) a TCR alpha chain variable domain and all or part of the TCR alpha chain constant region excluding the transmembrane domain; and (ii) a TCR beta chain variable domain and all or part of the TCR beta chain constant region excluding the transmembrane domain.

[0038] In a preferred embodiment, the TCR comprises an α chain variable domain and a β chain variable domain and all or part of the β chain constant domain excluding the transmembrane domain, but does not comprise an α chain constant domain, and the α chain variable domain of the TCR forms a heterodimer with the β chain.

[0039] In a preferred embodiment, a conjugate is bound to the C- or N-terminus of the α chain and / or β chain of the TCR.

[0040] In a preferred embodiment, the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modifying moiety, or a combination of any of these substances; preferably, the therapeutic agent is an anti-CD3 antibody.

[0041] In a second aspect, the present invention provides a multivalent TCR complex comprising at least two TCR molecules, wherein at least one TCR molecule is the TCR described in the first aspect.

[0042] In a third aspect, the present invention provides an isolated nucleic acid molecule encoding the TCR of the first aspect.

[0043] In a fourth aspect, the present invention provides a vector comprising the nucleic acid molecule described in the third aspect; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.

[0044] In a fifth aspect, the present invention provides an isolated host cell, wherein the host cell contains the vector described in the fourth aspect or the exogenous nucleic acid molecule described in the third aspect integrated into its genome.

[0045] In a sixth aspect, the present invention provides a cell, wherein the cell expresses the TCR described in the first aspect on its surface.

[0046] In a preferred embodiment, the cell is transduced with the nucleic acid molecule of the fourth aspect or the vector of the fifth aspect.

[0047] In a preferred embodiment, the cell is a T cell, a NK cell, a NKT cell or a stem cell.

[0048] In a preferred embodiment, the cells co-express the CD8 molecule.

[0049] In a preferred embodiment, the CD8 molecule is a human CD8 molecule.

[0050] In a preferred embodiment, the human CD8 molecule is a human CD8a molecule.

[0051] In a preferred embodiment, the human CD8a molecule has the sequence shown in SEQ ID NO:53.

[0052] In the seventh aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR described in the first aspect, the TCR complex described in the second aspect, the nucleic acid molecule described in the third aspect, the vector described in the fourth aspect, or the cell described in the sixth aspect.

[0053] In an eighth aspect, the present invention provides a use of the T cell receptor described in the first aspect, the TCR complex described in the second aspect, or the cell described in the sixth aspect for preparing a drug for treating tumors.

[0054] In a preferred embodiment, the tumor is a PRAME-associated tumor.

[0055] In a preferred embodiment, the tumor is a tumor that specifically expresses the complete or partial tumor antigen PRAME.

[0056] In a preferred embodiment, the tumor is a tumor that specifically expresses the SLLQHLIGL peptide segment.

[0057] In a preferred embodiment, the tumor is melanoma, non-small cell lung cancer, ovarian cancer, or breast cancer.

[0058] In a preferred embodiment, the tumor is non-small cell lung cancer.

[0059] In a ninth aspect, the present invention provides the T cell receptor of the first aspect, the TCR complex of the second aspect, or the cell of the sixth aspect for use as a drug for treating tumors.

[0060] In a preferred embodiment, the tumor is a PRAME-associated tumor.

[0061] In a preferred embodiment, the tumor is a tumor that specifically expresses the complete or partial tumor antigen PRAME.

[0062] In a preferred embodiment, the tumor is a tumor that specifically expresses the SLLQHLIGL peptide segment.

[0063] In a preferred embodiment, the tumor is melanoma, non-small cell lung cancer, ovarian cancer, or breast cancer.

[0064] In a preferred embodiment, the tumor is non-small cell lung cancer.

[0065] In the tenth aspect, the present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of the T cell receptor described in the first aspect, the TCR complex described in the second aspect, or the cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect to a subject in need of treatment.

[0066] In a preferred embodiment, the tumor is a PRAME-associated tumor.

[0067] In a preferred embodiment, the tumor is a tumor that specifically expresses the complete or partial tumor antigen PRAME.

[0068] In a preferred embodiment, the tumor is a tumor that specifically expresses the SLLQHLIGL peptide segment.

[0069] In a preferred embodiment, the tumor is melanoma, non-small cell lung cancer, ovarian cancer, or breast cancer.

[0070] In a preferred embodiment, the tumor is non-small cell lung cancer.

[0071] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] FIG1 shows the affinity level of the TCR molecules screened by the present invention to the target on the surface of the reporter gene cells;

[0073] FIG2 shows the activation effect of T2 cells loaded with target polypeptide on cells expressing the TCR molecule reporter gene of the present invention;

[0074] FIG3 shows the activation effect of T2 cells loaded with different concentrations of target polypeptide on cells expressing the TCR molecule reporter gene of the present invention;

[0075] FIG4 shows the specificity of the TCR molecules of the present invention in binding to targets;

[0076] FIG5 shows the expression efficiency of the TCR molecules of the present invention on primary T cells;

[0077] Figure 6 shows the killing effect of candidate TCR-T on target cells under different effector-target ratios;

[0078] FIG7 shows that target cells specifically activate TCR-T cells carrying the TCR sequences of the present invention and release cytokines;

[0079] FIG8 shows the results of in vivo animal efficacy evaluation of candidate TCR-T cells;

[0080] Figure 9 shows the results of the test of the killing ability of T cells in different groups against tumor cells, where E: effector; T: target;

[0081] FIG10 shows the results of the cytokine release ability test of T cells in different groups. DETAILED DESCRIPTION

[0082] After extensive and in-depth research, the inventors unexpectedly discovered a TCR that specifically binds to the PRAME-derived peptide SLLQHLIGL (SEQ ID NO: 1). This peptide is presented on the cell surface by HLA-A02 molecules. The present invention also provides nucleic acid molecules encoding this TCR and vectors containing this nucleic acid molecule. Furthermore, the present invention provides cells transduced with the TCR of the present invention. This is based on the present invention.

[0083] Definition of terms

[0084] The terms used herein have the meanings commonly understood by those skilled in the art. For a clear understanding of the present invention, some terms are defined as follows.

[0085] MHC molecules

[0086] MHC molecules are proteins from the immunoglobulin superfamily and can be either class I or class II. Therefore, they are specific for antigen presentation. Different individuals have different MHCs, capable of presenting different short peptides from a protein antigen on their APC cell surfaces. The human MHC is often referred to as the HLA gene or HLA complex.

[0087] SLLQHLIGL peptide

[0088] The SLLQHLIGL peptide of the present invention is derived from a polypeptide sequence in the tumor PRAME. The peptide is presented on the cell surface by the HLA-A02 molecule. Therefore, the SLLQHLIGL peptide-HLA-A*02 complex provides an ideal target for TCR-T immunotherapy of PRAME-associated tumors. The tumor is a PRAME-associated tumor.

[0089] In a preferred embodiment, the tumor is a tumor that specifically expresses the complete or partial tumor antigen PRAME.

[0090] T cell receptor

[0091] As used herein, T cell receptor has the meaning generally understood by those skilled in the art. It is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC). In the immune system, the binding of the antigen-specific TCR to the pMHC complex triggers direct physical contact between T cells and antigen-presenting cells (APCs). Then, other cell membrane surface molecules of both the T cell and the APC interact, which triggers a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells with different antigen specificities to exert immune effects on their target cells.

[0092] TCRs are glycoproteins found on the cell membrane, consisting of heterodimers of either α / β or γ / δ chains. In 95% of T cells, TCR heterodimers are composed of α and β chains, while 5% have TCRs composed of γ and δ chains. Natural αβ heterodimeric TCRs have α and β chains, which together constitute the subunits of the αβ heterodimeric TCR. Broadly speaking, each α and β chain contains a variable region, a joining region, and a constant region. The β chain typically also contains a short multivariable region between the variable and joining regions, but this multivariable region is often considered part of the joining region. Each variable region consists of three complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3, embedded within a framework structure. The CDR regions determine TCR binding to the pMHC complex. CDR3, formed by the recombination of the variable and joining regions, is known as the hypervariable region. The α and β chains of TCRs are generally considered to each have two "domains": a variable domain and a constant domain. The variable domain consists of a variable region and a connecting region. Sequences of TCR constant domains can be found in the public database of the International Immunogenetics Information System (IMGT). For example, the constant domain sequence of the TCR α chain is "TRAC*01," and the constant domain sequence of the TCR β chain is "TRBC1*01" or "TRBC2*01." In addition, the TCR α and β chains also contain transmembrane regions and cytoplasmic regions, the latter of which are very short.

[0093] In the present invention, the terms "polypeptide of the present invention", "TCR of the present invention" and "T cell receptor of the present invention" are used interchangeably.

[0094] During antigen processing, antigens are degraded within the cell and then carried to the cell surface by MHC molecules. T cell receptors are capable of recognizing peptide-MHC complexes on the surface of antigen-presenting cells. Therefore, the present invention provides a TCR molecule capable of binding to the SLLQHLIGL polypeptide-HLA-A*02 complex. Preferably, the TCR molecule is isolated or purified. The α and β chains of the TCR each have three complementarity determining regions (CDRs).

[0095] In a preferred embodiment of the present invention, the amino acid sequence of CDR1 of the TCRα chain variable domain is selected from the group consisting of SEQ ID NO: 12, 22, 32; the amino acid sequence of CDR2 of the TCRα chain variable domain is selected from the group consisting of SEQ ID NO: 13, 23, 33; the amino acid sequence of CDR3 of the TCRα chain variable domain is selected from the group consisting of SEQ ID NO: 14, 24, 34, 42; the amino acid sequence of CDR1 of the TCRβ chain variable domain is selected from the group consisting of SEQ ID NO: 17, 27, 37, 45; the amino acid sequence of CDR2 of the TCRβ chain variable domain is selected from the group consisting of SEQ ID NO: 18, 28, 38, 46; and / or the amino acid sequence of CDR3 of the TCRβ chain variable domain is selected from the group consisting of SEQ ID NO: 19, 29, 39, 47.

[0096] The CDR region amino acid sequence of the present invention described above 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, those skilled in the art can design or synthesize a TCR molecule with corresponding function 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 domain of the present invention is an amino acid sequence having at least 90%, preferably 95%, and more preferably 98% sequence identity with SEQ ID NO: 11, 21, 31 or 41; and / or the TCR β chain variable domain of the present invention is an amino acid sequence having at least 90%, preferably 95%, and more preferably 98% sequence identity with SEQ ID NO: 16, 26, 36 or 44.

[0097] In a preferred embodiment of the present invention, the TCR molecule of the present invention is a heterodimer consisting of an α and a β chain. Specifically, on the one hand, the α chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the α chain variable domain amino acid sequence comprises the CDR1, CDR2 and CDR3 of the above-mentioned α chain. Preferably, the TCR molecule comprises an α chain variable domain amino acid sequence of SEQ ID NO: 11, 21, 31 or 41. On the other hand, the β chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the β chain variable domain amino acid sequence comprises the CDR1, CDR2 and CDR3 of the above-mentioned β chain. Preferably, the TCR molecule comprises a β chain variable domain amino acid sequence of SEQ ID NO: 16, 26, 36 or 44.

[0098] 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 a description of single-chain TCR molecules, reference can be made to the document Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658. 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 single-chain TCR molecule comprises Vα, Vβ and Cβ, preferably connected in the order from N-terminus to C-terminus.

[0099] The amino acid sequence of the α chain variable domain of the single-chain TCR molecule comprises the CDR1, CDR2 and CDR3 of the above-mentioned α chain. Preferably, the single-chain TCR molecule comprises the amino acid sequence of the α chain variable domain of SEQ ID NO: 11, 21, 31 or 41. The amino acid sequence of the β chain variable domain of the single-chain TCR molecule comprises the CDR1, CDR2 and CDR3 of the above-mentioned β chain. Preferably, the single-chain TCR molecule comprises the amino acid sequence of the β chain variable domain of SEQ ID NO: 16, 26, 36 or 44.

[0100] In a preferred embodiment of the present invention, the constant domain of the TCR molecule of the present invention 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 of the present invention can be "TRAC*01", and the constant domain sequence of the β chain of the TCR molecule can be "TRBC1*01" or "TRBC2*01". The 53rd position of the amino acid sequence given in TRAC*01 of IMGT is Arg, which is expressed herein as: Arg53 of TRAC*01 exon 1, and the rest are deduced by analogy. Preferably, the amino acid sequence of the α chain of the TCR molecule of the present invention is SEQ ID NO: 10, 20, 30 and 40, and / or the amino acid sequence of the β chain is SEQ ID NO: 15, 25, 35 and 43.

[0101] Naturally occurring TCR is a membrane protein that is stabilized by its transmembrane region. Just like immunoglobulins (antibodies) as antigen recognition molecules, TCR can also be developed for diagnosis and treatment, in which case it is necessary to obtain soluble TCR molecules. Soluble TCR molecules do not include their transmembrane region. Soluble TCR has a wide range of uses. It can not only be used to study the interaction between TCR and pMHC, but also be used as a diagnostic tool for detecting infection or as a marker for autoimmune disease. Similarly, soluble TCR can be used to deliver therapeutic agents (such as cytotoxic compounds or immunostimulatory compounds) to cells that present specific antigens. In addition, soluble TCR can also be combined with other molecules (such as anti-CD3 antibodies) to redirect T cells, thereby targeting cells that present specific antigens. The TCR molecules of the present invention are all soluble, thereby providing more selectivity for the development of TCR-T cell therapy or TCR protein drugs.

[0102] To obtain a soluble TCR, the transmembrane region of the TCR can be removed. Preferably, such a TCR may comprise (i) all or part of the TCR α chain except its transmembrane domain, and (ii) all or part of the TCR β chain except its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least a portion of the constant domain of the TCR chain, 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 the β chain constant domain except the transmembrane domain, but it does not comprise an α chain constant domain, and the α chain variable domain of the TCR forms a heterodimer with the β chain.

[0103] The TCR of the present invention can 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 the present invention can be any peptide chain suitable for connecting the variable domains of the TCR α and β chains.

[0104] The TCRs of the present invention can also be provided as multivalent complexes. The multivalent TCR complexes of the present invention comprise multimers formed by the association of two, three, four, or more TCRs of the present invention, such as tetramers produced using the tetramerization domain of p53, or complexes formed by the association of multiple TCRs of the present invention with another molecule. The TCR complexes of the present invention 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 similar applications.

[0105] The TCRs of the present invention can be used alone or covalently or otherwise conjugated, preferably covalently, to a conjugate comprising a detectable label (for diagnostic purposes, wherein the TCR is used to detect the presence of cells presenting the GLSPTVWLSV polypeptide-HLA-A*02 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination thereof.

[0106] 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 enzymes capable of producing a detectable product.

[0107] Therapeutic agents that can be combined or coupled to the TCR of the present invention include, but are not limited to: 1. radionuclides (Koppe et al., 2005, Cancer metastasis reviews 24, 539); 2. biotoxins (Chaudhary et al., 1989, Nature 339, 394; Epel et al., 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines, such as IL-2 (Gillies et al., 1992, PNAS 89, 1428; Card et al., 2004, Cancer Immunology and Immunotherapy 53, 345; Halin et al., 2003, Cancer Research 63, 3202); 4. antibody Fc fragments (Mosquera et al., 2005, The Journal of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al., 1995, International Journal of Cancer 62,319); 6. Gold nanoparticles / nanorods (Lapotko et al., 2005, Cancer letters 239, 36; Huang et al., 2006, Journal of the American Chemical Society 128, 2115); 7. Viral particles (Peng et al., 2004, Gene therapy 11, 1234); 8. Liposomes (Mamot et al., 2005, Cancer research 65, 11631); 9. Nanomagnetic particles; 10. Prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. Chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0108] In addition, the TCR of the present invention can also be a hybrid TCR containing sequences derived from more than one species. For example, studies have shown that murine TCRs can be expressed more effectively in human T cells than human TCRs. Therefore, the TCR of the present invention can contain human variable domains and murine constant domains. The disadvantage of this method is that it may trigger an immune response. Therefore, when it is used in adoptive T cell therapy, a regulatory program should be included to perform immunosuppression to allow the implantation of murine T cells expressing murine T cells.

[0109] It should be understood that the amino acid names herein are represented by single letters or three letters in the internationally accepted English alphabet, 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).

[0110] Immunoconjugates

[0111] The term "immunoconjugate" as used herein has the meaning generally understood by those skilled in the art. The term refers to an artificially synthesized molecule comprising different parts (usually proteins) coupled by chemical means and having an immunological effect.

[0112] Nucleic acid molecules

[0113] The present invention provides nucleic acid molecules encoding a TCR of the present invention or a portion thereof, which may be one or more CDRs, the variable domains of the α and / or β chains, and the α and / or β chains.

[0114] The nucleotide sequence of the nucleic acid molecule of the present invention can be single-stranded or double-stranded, and the nucleic acid molecule can be RNA or DNA, and can contain or 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 present invention.

[0115] 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 identical to the nucleic acid sequence shown in the present invention or a degenerate variant.

[0116] The nucleotide sequence can be codon-optimized. Different cells utilize different codons, and the codons in the sequence can be altered to increase expression depending on the cell type. Codon usage tables for mammalian cells and various other organisms are well known to those skilled in the art.

[0117] The full-length sequence of the nucleic acid molecule of the present invention or its fragments can generally be obtained by, but not limited to, PCR amplification, recombinant methods, or artificial synthesis. Currently, DNA sequences encoding the TCR of the present invention (or its fragments, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. The DNA can be either a coding strand or a non-coding strand.

[0118] carrier

[0119] The present invention also relates to vectors comprising the nucleic acid molecules of the present invention, including expression vectors, i.e. constructs capable of in vivo or in vitro expression. Commonly used vectors include bacterial plasmids, bacteriophages, and animal and plant viruses.

[0120] Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.

[0121] Preferably, the vector can transfer the nucleotide of the present invention into cells, such as T cells, so that the cells express HBV antigen-specific TCR. Ideally, the vector should be able to be expressed at a high level in T cells.

[0122] cell

[0123] The present invention also relates to host cells produced by genetic engineering using the vector or coding sequence of the present invention. The host cells contain the vector of the present invention or have the nucleic acid molecule of the present invention integrated into their chromosomes. The host cells are selected from prokaryotic and eukaryotic cells, such as Escherichia coli, yeast cells, and CHO cells.

[0124] In addition, the present invention also includes cells expressing the separation of the TCR of the present invention, which can be, but are not limited to, T cells, NK cells, NKT cells, stem cells, particularly T cells. The T cell can be derived from the T cell separated from the subject, or can be a mixed cell population separated from the subject, such as a part for a peripheral blood lymphocyte (PBL) group. As, the cell can be separated from peripheral blood mononuclear cells (PBMC), can be CD4+ helper T cells or CD8+ cytotoxic T cells. The cell can be in a mixed group of CD4+ helper T cells / CD8+ cytotoxic T cells. Generally, the cell can be activated with an antibody (such as, anti-CD3 or anti-CD28 antibody) to enable them to be more easily transfected, for example, transfected with a vector comprising a nucleotide sequence encoding the TCR molecule of the present invention.

[0125] Alternatively, the cells of the present invention may also be or be derived from stem cells, such as hematopoietic stem cells (HSCs). Gene transfer to HSCs will not result in TCR expression on the cell surface because CD3 molecules are not expressed on the stem cell surface. However, when the stem cells differentiate into lymphoid precursors that migrate to the thymus, the expression of CD3 molecules will initiate expression of the introduced TCR molecules on the surface of the thymocytes.

[0126] There are many methods suitable for transfecting T cells with DNA or RNA encoding the TCR of the present invention (e.g., Robbins et al., (2008) J. Immunol. 180:6116-6131). T cells expressing the TCR of the present invention can be used for adoptive immunotherapy. Those skilled in the art will be aware of many suitable methods for adoptive therapy (e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).

[0127] To enhance the biological activity of the TCR molecule of the present invention in CD4-positive T cells, cells expressing the TCR of the present invention may further co-express a CD8 molecule, such as a human CD8 molecule, preferably a human CD8a molecule.

[0128] Treatment

[0129] Treatment can be performed by isolating T cells from patients or volunteers infected with HBV-induced HCC or chronic hepatitis, introducing the TCR of the present invention into the above-mentioned T cells, and then returning these genetically engineered cells to the patient. Therefore, the present invention provides a method for treating HBV-related diseases, comprising: infusing isolated T cells expressing the TCR of the present invention, preferably, the T cells are derived from the patient himself, into the patient. Generally, it comprises (1) isolating the patient's T cells, (2) transducing the T cells in vitro with the nucleic acid molecules of the present invention or nucleic acid molecules capable of encoding the TCR molecules of the present invention, and (3) infusing the genetically engineered T cells into the patient. The number of cells to be isolated, transfected, and returned can be determined by the physician.

[0130] Advantages of the present invention:

[0131] (1) The TCR of the present invention can specifically bind to a complex composed of the GLSPTVWLSV peptide segment and HLA-A*02, and effector cells transduced with the TCR of the present invention can be specifically activated;

[0132] (2) Effector cells transduced with the TCR of the present invention can specifically kill GLSPTVWLSV peptide-positive target cells;

[0133] (3) After stimulation by target cells, the effector cells transduced with the TCR of the present invention are able to secrete a large amount of cytokines; and

[0134] (4) The present invention provides TCR molecules with different levels of affinity activity with target pMHC complexes, all of which are soluble, which can provide more selectivity for the development of TCR-T cell therapy or TCR protein drugs.

[0135] The following specific examples further illustrate the present invention. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook and Russell et al., (Molecular Cloning: A Laboratory Manual (Molecular Cloning-A Laboratory Manual) (3rd Edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0136] Example:

[0137] Example 1: Obtaining candidate TCR molecules

[0138] 1.1 Antigen-specific T cell activation

[0139] Peripheral blood T cells were activated by in vitro activation of healthy donor PBMCs (Peripheral blood mononuclear cells, Miaoshun (Shanghai) Biotechnology Co., Ltd.) or by using a peptide immunization method or a peptide-loaded DCs immunization method. The T cells were then stained using fluorescently labeled polymers (Tetramer, etc.), and flow cytometry was used to sort T cells that could be stained by the polymers. This group of T cells may be T cells that are specifically activated by the antigen. The polypeptide sequence used in this study is: SLLQHLIGL (SEQ ID NO: 1). The polypeptide sequence used in this example is derived from the PRAME protein.

[0140] 1.2 TCR sequence acquisition

[0141] TCR screening and cloning were performed with reference to published literature (Paria BC, Levin N, Lowery FJ, et al. Rapid Identification and Evaluation of Neoantigen-reactive T-Cell Receptors From Single Cells. Journal of Immunotherapy (Hagerstown, Md.: 1997). 2021 Jan; 44(1): 1-8. Pai, JA, Satpathy, AT High-throughput and single-cell T cell receptor sequencing technologies. Nat Methods 18, 881-892(2021). De Simone M, Rossetti G and Pagani M(2018) Single Cell T Cell Receptor Sequencing: Techniques and Future Challenges. Front. Immunol. 9: 1638. doi: 10.3389 / fimmu.2018.01638). The resulting TCR clones were named TCR-01, TCR-02, TCR-03, and TCR-04. The full-length amino acid sequences of the TCR variable regions are shown in Table 1. TCR variable region subtypes and CDR region sequences were analyzed using the IMGT website to obtain the sequences of the CDR1, CDR2, and CDR3 regions of different TCRs (amino acid sequences are shown in Table 1).

[0142] Table 1. Amino acid sequence listing

[0143] Example 2: Expression and biological function evaluation of candidate TCR molecules

[0144] 2.1 TCR gene vector construction

[0145] The variable region sequences of the TCR alpha and TCR beta genes cloned in Example 1 were connected to the constant regions of mouse TCR alpha and TCR beta, respectively, to construct the full-length sequences of TCR alpha and TCR beta.

[0146] The paired full-length TCR alpha and TCR beta sequences were then linked via the 2A fragment and constructed into a lentiviral vector for lentiviral packaging. The lentivirus was concentrated by high-speed centrifugation and stored in a -80°C freezer. The basic experimental steps are as follows:

[0147] 1) On the first day, HEK-293T cells (purchased from the Chinese Academy of Sciences Cell Bank) were seeded at a density of approximately 1.0 × 10^7 cells / T175 flask (cultured in 30 mL of culture medium). Transfection was performed when the cell density reached 80-90% on the second day.

[0148] 2) The next day, plasmid transfection: Before transfection, the culture medium should be replaced with DMEM supplemented with 10% FBS but without the anti-antibody. First, prepare the plasmid complexes separately: add the following plasmids to 1.5 ml of Opti-MEM (Thermo Fisher Scientific; 31985-070) and mix thoroughly: 18 μg of psPAX2 plasmid (Addgene; Catalog No. 12260), 9 μg of pMD2.G plasmid (Addgene; Catalog No. 12259), and 18 μg of viral vector plasmid. The lentiviral vector plasmids carrying the TCR-01, TCR-02, TCR-03, and TCR-04 genes are named lenti-TCR-01, lenti-TCR-02, lenti-TCR-03, and lenti-TCR-04, respectively. Prepare the transfection reagent complex: Add 45 μL (3 mg / mL) PEI (Polysciences: 24765) to 1.5 mL of Opti-MEM at a plasmid to PEI mass ratio of 1:3. Mix thoroughly and let stand at room temperature for 5 minutes. Then, add the transfection reagent complex dropwise to the plasmid complex, mix thoroughly, and let stand for 20 minutes. Finally, slowly drip the transfection complex into the 293T cell culture flask, gently mix, and incubate in a 37°C cell culture incubator with 5% CO2.

[0149] 3) On the fifth day, virus collection: 72 hours after transfection, the culture supernatant was collected and centrifuged at 2000 rpm for 10 minutes to remove cell debris. The supernatant was filtered through a 0.45 μM filter membrane and the filtrate was transferred to a dedicated centrifuge tube for balancing. Ultracentrifuge at 25000 rpm for 2 hours. After discarding the supernatant, the lentivirus was resuspended in X-VIVO-15 medium and the lentivirus was aliquoted and stored in an ultra-low temperature freezer at -80°C. According to this process, lentivirus containing TCR-01, TCR-02, TCR-03, and TCR-04 genes were prepared respectively.

[0150] 2.2 Preparation of TCR-reporter cell lines.

[0151] The reporter gene cell line used in this study is a Jurkat cell line that knocks out the endogenous TCRα and β genes, carries the NFAT promoter and luciferase gene, and expresses the CD8a and CD8b genes. It is named Jurkat-TCRKO-031 (Smith-Garvin JE et al., 2009. T Cell Activation. Ann. Rev. Immunol. 27: 591-619). Lentivirus carrying the candidate TCR gene is added to the reporter gene cells respectively. After 2-3 days, TCR expression is detected by polymer staining and flow cytometry. The basic steps are as follows:

[0152] 1) Synthesize the HLA-A2-restricted PRAME target peptide (Nanjing GenScript Biotechnology Co., Ltd., SEQ ID No.: 1) and prepare a PE-fluorescein-coupled MHC I-peptide complex multimer Dextramer according to the kit instructions (Immudex, Catalog No.: U-LX02) for detecting TCRs that specifically recognize the target peptide.

[0153] 2) Take Jurkat-TCRKO-031 cells grown to the logarithmic phase and add 5×10 5 The density of 100 μg / well was inoculated into 6-well plates, and the volume of each well was supplemented with culture medium to 2 mL;

[0154] 3) Take 100 μL of packaged lentiviral solution and add it dropwise to the well plate. Gently shake to thoroughly mix the virus solution and culture medium.

[0155] 4) The plate was placed in a 37°C, 5% CO2 incubator for 48 hours. The tool cells transfected with the TCR-01, TCR-02, TCR-03, and TCR-04 genes were named TCR-P1, TCR-P2, TCR-P3, and TCR-P4, respectively. The NTD group consisted of untransfected cells, serving as a negative control.

[0156] 5) After 48 h, remove 200 μL of cell suspension, collect cells by centrifugation, and wash cells once with FACS buffer (PBS containing 2% FBS);

[0157] 6) Resuspend the cells in 100 μL FACS buffer, add 1 μL APC anti-huam CD3 and 2 μL Dextramer prepared above to each tube, mix well, and incubate at 4°C for 30 minutes;

[0158] 7) After incubation, wash the cells twice with FACS buffer and collect the cells by centrifugation;

[0159] 8) Resuspend the cells in 100 μL FACS buffer and use flow cytometry to detect the proportion of CD3 and Dextramer-positive cell populations. The flow cytometry results are shown in Figure 1.

[0160] The results showed that the four TCR sequences screened by the present invention can be integrated into the cell genome and expressed on the cell surface through lentiviral transfection, and the transfection positive rate is high; Dextramer staining results show that all four TCR molecules can bind to Dextramer, among which TCR-P1 and TCR-P2 have higher binding levels (>50%); while TCR-P3 and TCR-P4 have only weak binding.

[0161] 2.3 Evaluation of T cell activation effect

[0162] Using T2 cells as target cells, loading different concentrations of peptides, activating reporter cells, and calculating the EC50 value. The basic steps are as follows:

[0163] 1) Take T2 cells grown to the logarithmic phase and resuspend them in complete culture medium to 2×10 6 / mL;

[0164] 2) Dilute the target peptide to 60 μM using complete culture medium, mix the diluted peptide solution with the cell suspension at a volume ratio of 1:1, and incubate at 37°C for 1-2 hours to allow the peptide to load onto the T2 cell surface;

[0165] 3) After incubation, the excess peptide was washed away with culture medium, the cells were collected by centrifugation, and the cells were resuspended in culture medium to 2×10 5 / mL, T2 cells loaded with different concentrations of peptides were seeded into 96-well white opaque cell culture plates at 50 μL / well;

[0166] 4) Take the TCR-P1, TCR-P2, TCR-P3 and TCR-P4 cells expressing the TCR molecules of the present invention prepared in Example 2, as well as the negative control cell NTD group, and adjust the cell density to 1×10 6 / mL, add 50 μL / well to a 96-well plate, place on a microplate shaker to mix evenly, and then transfer the cells to a 37°C incubator and incubate for 4-6 hours;

[0167] 5) After the incubation, D-luciferin reaction substrate (purchased from Thermo Fisher Scientific, Catalog No. L2912) was added to the culture plate at 100 μL / well. The reaction was allowed to proceed at room temperature in the dark for 10 minutes. The fluorescence signal value (RLU) was then measured using a microplate reader (Thermo Fisher Scientific, Varioskan.LUX). The results are shown in Figure 2.

[0168] The results showed that Jurkat-TCRKO-031 cells expressing the four TCR molecules shown in the present invention could all be activated by T2 cells loaded with target polypeptides and express fluorescent signals. Among them, the activation effects of TCR-P1 and TCR-P2 were higher, comparable to the positive control level, showing good biological activity; the activation levels of TCR-P3 and TCR-P4 molecules were lower, expressing only weak fluorescent signals.

[0169] 2.4 Evaluation of the biological activity of candidate TCR molecules

[0170] To more comprehensively evaluate the biological activity of candidate TCR molecules, this example used T2 cells loaded with different concentrations of peptides as target cells, activated reporter cells, fitted dose curves, and calculated EC50 values. The basic steps are as follows:

[0171] 1) Take T2 cells grown to the logarithmic phase and resuspend them in complete culture medium to 2×10 6 / mL, the cell suspension was inoculated into a 96-well U-bottom deep-well plate at 100 μL / well;

[0172] 2) Dilute the target peptide in complete culture medium, starting at 50 μM, and adjust the concentration by 4-fold gradient to 10 concentration points. Add 100 μL / well of the diluted peptide solution to the cells in step 1, mix well, and incubate at 37°C for 1-2 hours to allow the peptide to load onto the T2 cell surface.

[0173] 3) After incubation, excess peptide was washed off with culture medium, cells were collected by centrifugation, 0.9 mL of culture medium was added to each well to resuspend the cells, and T2 cells loaded with different concentrations of peptide were seeded into 96-well white opaque cell culture plates at a rate of 50 μL / well;

[0174] 4) Take the TCR-P1, TCR-P2, TCR-P3 and TCR-P4 cells expressing the TCR molecules of the present invention prepared in Example 2 and adjust the cell density to 1×10 6 / mL, add 50 μL / well to a 96-well plate (untransfected Jurkat-TCRKO-031 cells were used as a negative control), place on a microplate shaker to mix evenly, and then transfer the cells to a 37°C incubator and incubate for 4-6 hours;

[0175] 5) After the incubation, D-luciferin substrate (purchased from Thermo Fisher Scientific, Cat. No. L2912) was added to the culture plate at 100 μL / well. The reaction was allowed to proceed at room temperature in the dark for 10 minutes. The fluorescence signal (RLU) was then measured using a microplate reader (Thermo Fisher Scientific, Varioskan.LUX).

[0176] 6) Graphpad Prism software was used for data analysis, dose-dependent curves were fitted, and EC50 values ​​were calculated. The results are shown in FIG3 .

[0177] The results showed that the reporter gene cells expressing the four TCR molecules shown in the present invention could all be activated by T2 cells loaded with target polypeptides and express fluorescent signals. Among them, the activation signals of TCR-P1 and TCR-P2 were higher, with EC50 values ​​of 0.02409 and 0.02163 μM, respectively, and showed a clear dose-dependent effect, indicating good biological activity; the activation signals of TCR-P3 and TCR-P4 were weaker and had no dose-dependence, which is presumably caused by their low molecular affinity.

[0178] Example 3: Analysis of off-target effects of candidate TCR molecules

[0179] Referring to the experimental steps of Example 2.4, peptides with similar sequences (Nanjing GenScript Biotechnology Co., Ltd., SEQ ID No.: 2-9) were used as potential off-target peptides, and T2 cells loaded with different off-target peptides were used as target cells. The peptide sequences are shown in Table 2. Jurkat-TCRKO-031 cells expressing the TCR molecules described in this patent were used as effector cells, and luciferase activity was detected after co-culture. The basic steps are as follows:

[0180] 1) Dissolve the off-target peptides in DMSO to a 10 mM stock solution. Add 3 μL of the peptide stock solution to 1 mL of complete culture medium (RPMI 1640 medium containing 10% FBS) to a working concentration of 30 μM.

[0181] 2) Take T2 cells with good growth status, collect the cells by centrifugation, and resuspend the cells to 1×10 6 / mL density, and the cell suspension was placed in a cell culture incubator and incubated for 1 hour;

[0182] 3) Take the Jurkat-TCRKO-031 cells expressing the candidate TCR prepared in Example 1 and resuspend them in culture medium to 1×10 6 / mL, add the cell suspension to a 96-well white opaque cell culture plate (purchased from Thermo, product number 136101) at 50 μL / well;

[0183] 4) The T2 cells loaded with peptides were washed twice with culture medium and the cells were washed with 2×10 5 Resuspend the cells at a density of 100 μL / mL, and add T2 cells loaded with different peptides to the corresponding wells at 50 μL / well. Use T2 cells without peptide loading as a negative control, and T2 cells loaded with PRAME target peptide as a positive control;

[0184] 5) Place the plate on a microplate shaker for 10 minutes to mix the cells, remove the plate, and incubate in an incubator for 4-6 hours.

[0185] 6) After the incubation, D-luciferin substrate (purchased from Thermo Fisher Scientific, Cat. No. L2912) was added to the culture plate at 100 μL / well. The reaction was allowed to proceed at room temperature in the dark for 10 minutes. The fluorescence signal (RLU) was then measured using a microplate reader (Thermo Fisher Scientific, Varioskan.LUX).

[0186] 7) Data analysis was performed using Graphpad Prism software, and the results are shown in Figure 4.

[0187] The results showed that all four candidate TCR molecules showed good specificity and safety, could only be specifically activated by T2 cells presenting the target peptide, and could not recognize the eight off-target peptides, and were worthy of further development.

[0188] Table 2. List of off-target analysis peptide sequences used in the present invention:

[0189] Example 4: Functional evaluation of candidate TCR molecules in primary human T cells

[0190] 4.1 TCR-T cell preparation

[0191] The lentiviral solution carrying the candidate TCR gene prepared in Example 2 was used to infect primary human T cells to prepare TCR-T cells carrying different TCR genes. The TCR-T cells carrying the four TCR clonal T genes were named: TCR-T-01, TCR-T-02, TCR-T-03, and TCR-T-04. The expression of the TCR gene was detected using an antibody against the mouse constant region. The specific steps are as follows:

[0192] 1) CD3+ T cells from healthy human peripheral blood (Miaoshun (Shanghai) Biotechnology Co., Ltd.) were resuspended in T cell culture medium containing 300 IU / mL IL-2 to a density of 1×10 6 / mL, T cell activator CD3 / CD28 magnetic beads (purchased from ACROBiosystems, catalog number: MBS-C001) were added at a ratio of 1:1 between cells and magnetic beads, and after thorough mixing, the cells were seeded in 6-well plates for culture;

[0193] 2) After 48 hours of culture, the cells were aspirated, the beads were removed by magnetic adsorption, and the cells were collected by centrifugation;

[0194] 3) Resuspend the cells in fresh culture medium and inoculate them into new 6-well plates, with 5×10 cells per well.5 Cells were cultured with 100 μL of lentiviral solution carrying different TCR genes. T cells without virus solution were used as negative control NTD. The cells were placed in an incubator and cultured for 5 days.

[0195] 4) Take 100 μL of cell suspension, collect cells by centrifugation, resuspend cells in 100 μL FACS buffer, add 1 μL APC anti-mouse TCRβ antibody (Biolegend, Cat. No. 109212) to each tube of cells, mix well, and incubate at 4°C for 30 minutes;

[0196] 5) Wash the cells twice, resuspend the cells in 100 μL FACS buffer, and detect the expression efficiency of TCR on T cells by flow cytometry.

[0197] The results showed that the four TCR molecules of the present invention were all successfully expressed on T cells, with an expression positive rate of 40-80% (Figure 5). The TCR-T cells prepared by the present invention can be used for subsequent biological function evaluation.

[0198] 4.2 Cell killing assay

[0199] The TCR-T cells prepared by the present invention can specifically recognize and kill target cells. This experiment used four HLA-A2 and PRAME target expression double-positive cell lines as target cells, namely non-small cell lung cancer NCI-H1755, ovarian cancer OVCAR-3, melanoma HS695T and osteosarcoma U2OS cell lines; PRAME-negative, HLA-A2-positive MCF-7 cells, and PRAME and HLA-A2 expression-negative A549 cells were used as negative cells, and the TCR-T cells prepared in the above examples were co-cultured with target cells to detect the killing effect and evaluate the biological function of TCR-T. The specific steps are as follows:

[0200] 1) GFP-luciferase-expressing lentiviral vectors were prepared according to the method of Example 2.1, and the lentiviral vectors were used to transfect different target cells to obtain luciferase-labeled cell lines: NCI-H1755-GFP, OVCAR-3-GFP, HS695T-GFP, U2OS-GFP, MCF-7-GFP, and A549-GFP;

[0201] 2) Resuspend different tumor cell lines in culture medium to 2×10 5 / mL, and tumor cells were inoculated into 96-well flat-bottom opaque cell culture plates at 50 μL / well and temporarily incubated at 37°C;

[0202] 3) Use NTD cells to adjust the positive ratio of the four TCR-T cells to 40%, and resuspend the four TCR-T cells to 3×10 6 / mL, 1×10 6 / mL and 2×10 5 / mL three different cell densities (effector-target ratios were 15:1, 5:1, and 1:1, respectively), with NTD cells as negative control, and the effector cells of different densities were seeded into 96-well culture plates at 50 μL / well, mixed thoroughly, and incubated in a 37°C incubator for 24 hours;

[0203] 4) Remove the opaque 96-well flat-bottom plate after co-culture and add 100 μL of an equal volume of D-luciferin substrate (Thermo Fisher Scientific: 88293) to the wells. Mix well and incubate in the dark for 10 minutes. Fluorescence intensity is measured using a microplate reader in chemiluminescence mode. Since luciferase is expressed only in target cells, the remaining luciferase activity in the well is directly related to the number of viable target cells. Culture medium is added to target cells to obtain maximum luciferase activity as a control. The target cell apoptosis ratio is calculated by subtracting the viable cell fluorescence signal, which represents the TCR-T cell-mediated killing effect on target cells. The results are shown in Figure 6 (Figure 6A: NCI-H1755-GFP; Figure 6B: OVCAR-3-GFP; Figure 6C: HS695T-GFP; Figure 6D: U2OS-GFP; Figure 6E: MCF-7-GFP; Figure 6F: A549-GFP).

[0204] The results showed that the four TCR molecules described in the present invention can all mediate T cell killing of three target-positive cells, among which the killing effect on NCI-H1755 cells is the strongest.

[0205] 4.3 Cytokine secretion detection

[0206] To further evaluate the specific activation function of target cells, in this example, candidate TCR-T cells were co-cultured with target cells expressing positive or negative targets at a 1:1 effector-target ratio for 24 hours, and the level of IFN-γ cytokine secreted by T cells in the culture supernatant was measured. The specific steps are as follows:

[0207] 1) Take the tumor cells used in Example 4.2, HepG2 cells (target expression negative), and T2 cells, and resuspend them in complete culture medium to 2×10 6 / mL, the cell suspension was inoculated into a 96-well U-bottom deep-well plate at 50 μL / well;

[0208] 2) Take the four types of TCR-T cells prepared in Example 4.1 (with untransfected NTD cells as negative control), adjust the transfection positive rate to about 40% with NTD cells, and adjust the cell density to 2×10 6 / mL, add 50 μL / well to a 96-well plate, place on a microplate shaker to mix evenly, and then transfer the cells to a 37°C incubator and incubate for 24 hours;

[0209] 3) After incubation, 50 μL of culture supernatant was collected and transferred to a new U-bottom 96-well plate. IFN-γ cytokine secretion in T cells was detected using an ELISA kit (Thermo Fisher Scientific; Cat. No. 88-7316). Plate preparation and supernatant cytokine detection were performed according to the instructions provided by the kit.

[0210] The results showed (Figure 7) that the candidate TCR-T cells could effectively recognize tumor cells expressing positive targets, activate TCR-T cells and transmit immune signals, and secrete IFN-γ cytokines, among which the TCR-T-01 molecule had relatively better biological activity.

[0211] Based on the above results, the TCR sequences described in the present invention have good affinity activity and biological function. Among them, the antigen binding activity, biological function and specificity of the TCR-01 and TCR-02 molecules show higher potential, suggesting that they are worthy of further development and application.

[0212] Example 5: In vivo efficacy evaluation in animals

[0213] In this example, the TCR molecules TCR-P1 and TCR-P2 were further evaluated for their efficacy in mice. Two TCR-T cells were prepared with reference to Example 4: TCR-T-01 and TCR-T-02. T cells that were not transfected with lentivirus were used as control T cells (NTD, no transduction). The human melanoma A375 tumor cell line (ATCC; Product No. CRL-1619) was amplified and cultured under conventional culture conditions, and cells in the logarithmic growth phase were harvested after at least three passages. After centrifugation to collect the tumor cells, they were resuspended in 1x PBS to 2.5×10 7 10 cells / mL, and Matrigel was added at a ratio of 1:1. 30 female fully immunodeficient NOG mice (Wei Tong Li Hua Co., Ltd.) were taken. Under sterile conditions, all animals were subcutaneously inoculated with 0.2 mL of A375 tumor cell suspension (5 x 10 6 Tumor cells). Seven days after inoculation of tumor cells, the tumor size was measured using a ruler. When the tumor reached 100 mm 3 The mice were randomly divided into groups.

[0214] In this experiment, mice were randomly divided into 4 groups: buffer group, non-transfected control T cell group (NTD), TCR-T-01 group, and TCR-T-02 group, with 5 mice in each group. The prepared T cells of different groups were centrifuged at 1800 rpm for 5 minutes. After resuspending the cells in 1x PBS buffer, the cell density of the test sample was adjusted. Each group of animals was given 1×10 7 Total T cells / mouse (about 5×10 6 The mice were injected with the IL-2 cytokine solution (200,000 IU / mouse) after the T cell transfusion. The tumor diameter was measured with a vernier caliper twice a week to measure and evaluate the tumor size of the mice. The mice were observed for 30 days, and their diet, activity, and other health indicators were also monitored. After the experiment, the tumor volume was used as the vertical axis and the number of days as the horizontal axis for plotting.

[0215] The study found that both the TCR-T-01 and TCR-T-02 groups were able to eliminate tumors and inhibit their growth. Starting on day 20, tumors in the TCR-T-02 group began to grow again. However, the TCR-T-01 group effectively controlled tumor growth over a long period of time (see Figure 8), demonstrating a strong anti-tumor effect. Therefore, both TCR-T-01 and TCR-T-02 cells demonstrated excellent in vivo animal efficacy. TCR-T-01 cells completely eliminated tumor cells in mice and effectively controlled tumor recurrence, making the TCR-P1 molecule a promising candidate for further development.

[0216] Example 6: Optimization of TCR molecular structure

[0217] The TCR molecules discovered in this study are all CD8-dependent and require the assistance of CD8 molecules to better recognize HLA-A molecules. Therefore, in CD4-positive T cells, the TCR molecules discovered in this study cannot recognize HLA-A molecules very well. CD4-positive T cells can release a large amount of cytokines after activation, thereby increasing the activity of CD8-positive T cells. In order to improve the biological activity of the TCR molecules of the present invention in CD4-positive T cells, this study connected the example TCR molecule (TCR01, with its signal peptide replaced by a human signal peptide, shown in bold) and the human CD8a molecule (Uniprot: P01732) and co-expressed them. The TCR after connecting the CD8a molecule is named: TCR-P1-CD8, and the amino acid sequence is shown in Table 3.

[0218] Table 3. TCR-P1-CD8 molecule amino acid sequence

[0219] With reference to Example 4, T cells co-expressing CD8 were prepared and named TCR-T-01CD8. With reference to Example 4, experimental groups were designed: three groups of T cells, non-transfected control group (NTD), TCR-T-01, and TCR-T-01CD8. With reference to Example 4, this study evaluated the cell killing ability and cytokine IFN-gamma release ability of the three T cells against target cells OVCAR3 (cell bank of the Chinese Academy of Sciences). The results of the cell killing ability test are shown in Figure 9. The results of the cytokine release ability test are shown in Figure 10.

[0220] The results showed that TCR-T cells co-expressing CD8 (TCR-T-01CD8) exhibited superior cytotoxicity compared to TCR-T-01 cells lacking CD8 expression (Figure 9). Furthermore, TCR-T cells co-expressing CD8a released more IFN-gamma cytokines (Figure 10). These results suggest that TCR-T cells co-expressing CD8 have enhanced biological activity. TCRs co-expressing CD8 may serve as candidate molecules for further development.

[0221] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A T cell receptor (TCR), wherein the TCR is capable of binding to a SLLQHLIGL polypeptide-HLA-A*02 complex.

2. The T cell receptor (TCR) according to claim 1, characterized in that The TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, The amino acid sequence of CDR1 of the TCR α chain variable domain is selected from the group consisting of SEQ ID NO: 12, 22, 32; The amino acid sequence of CDR2 of the TCR α chain variable domain is selected from the group consisting of SEQ ID NO: 13, 23, 33; The amino acid sequence of CDR3 of the TCR α chain variable domain is selected from the group consisting of SEQ ID NO: 14, 24, 34, 42; The amino acid sequence of CDR1 of the TCRβ chain variable domain is selected from the group consisting of SEQ ID NO: 17, 27, 37, 45; The amino acid sequence of CDR2 of the TCRβ chain variable domain is selected from the group consisting of SEQ ID NO: 18, 28, 38, 46; and / or The amino acid sequence of CDR3 of the TCRβ chain variable domain is selected from the following group: SEQ ID NO: 19, 29, 39, 47.

3. The T cell receptor (TCR) according to claim 2, characterized in that The amino acid sequences of CDR1, CDR2 and CDR3 of the TCR alpha chain variable domain are selected from the following combinations: SEQ ID NOs: 12, 13 and 14; SEQ ID NOs: 22, 23 and 24; SEQ ID NOs: 32, 33 and 34; SEQ ID NOs: 22, 23 and 42; and / or The amino acid sequences of CDR1, CDR2 and CDR3 of the TCR β chain variable domain are selected from the following combinations: SEQ ID NOs: 17, 18 and 19; SEQ ID NOs: 27, 28 and 29; SEQ ID NOs: 37, 38 and 39; SEQ ID NOs: 45, 46 and 47.

4. The T cell receptor (TCR) according to claim 3, characterized in that The amino acid sequences of CDR1, CDR2 and CDR3 of the TCR α chain variable domain and the amino acid sequences of CDR1, CDR2 and CDR3 of the TCR β chain variable domain are selected from the following combinations: SEQ ID NOs: 12, 13 and 14 and SEQ ID NOs: 17, 18 and 19; SEQ ID NOs: 22, 23 and 24 and SEQ ID NOs: 27, 28 and 29; SEQ ID NOs: 32, 33 and 34 and SEQ ID NOs: 37, 38 and 39; SEQ ID NOs: 22, 23 and 42 and SEQ ID NOs: 45, 46 and 47.

5. The T cell receptor (TCR) according to claim 1, characterized in that The TCR comprises a TCR α chain variable domain and a TCR β chain variable domain, wherein the TCR α chain variable domain is an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 11, 21, 31 or 41; and / or the TCR β chain variable domain is an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 16, 26, 36 or 44.

6. The T cell receptor (TCR) according to claim 5, characterized in that The amino acid sequence of the TCR α chain variable domain of the TCR is shown in SEQ ID NO: 11, 21, 31 or 41; and / or the amino acid sequence of the TCR β chain variable domain is shown in SEQ ID NO: 16, 26, 36 or 44.

7. The T cell receptor (TCR) according to claim 6, characterized in that The amino acid sequence of the TCR α chain variable domain of the TCR and the amino acid sequence of the TCR β chain variable domain of the TCR are selected from the following combinations: SEQ ID NOs: 11 and 16; SEQ ID NOs: 21 and 26; SEQ ID NOs: 31 and 36; SEQ ID NOs: 41 and 44.

8. The T cell receptor (TCR) according to claim 1, characterized in that The TCR comprises a TCR α chain and a TCR β chain, wherein the TCR α chain is an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 10, 20, 30 or 40; and / or the TCR β chain variable domain is an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with SEQ ID NO: 15, 25, 35 or 43.

9. The T cell receptor (TCR) according to claim 8, characterized in that The α chain amino acid sequence of the TCR is selected from the group consisting of SEQ ID NO: 10, 20, 30 and 40; and / or The β chain amino acid sequence of the TCR is selected from the following group: SEQ ID NO:15, 25, 35 and 43.

10. The T cell receptor (TCR) according to claim 9, characterized in that The α chain amino acid sequence and β chain amino acid sequence of the TCR are selected from the following combinations: SEQ ID NOs: 10 and 15; SEQ ID NOs: 20 and 25; SEQ ID NOs: 30 and 35; SEQ ID NOs: 40 and 43.

11. The T cell receptor (TCR) according to claim 1, characterized in that The TCR is soluble.

12. The T cell receptor (TCR) according to claim 1, characterized in that The TCR is a single-chain TCR.

13. The T cell receptor (TCR) according to claim 1, characterized in that The TCR is formed by connecting the α chain variable domain and the β chain variable domain via a peptide connecting sequence.

14. The T cell receptor (TCR) according to claim 1, characterized in that The TCR comprises (i) a TCR α chain variable domain and all or part of the TCR α chain constant region excluding the transmembrane domain; and (ii) a TCR β chain variable domain and all or part of the TCR β chain constant region excluding the transmembrane domain.

15. The T cell receptor (TCR) according to claim 1, characterized in that The TCR comprises an α chain variable domain and a β chain variable domain and all or part of the β chain constant domain excluding the transmembrane domain, but does not comprise an α chain constant domain, and the α chain variable domain of the TCR forms a heterodimer with the β chain.

16. The T cell receptor (TCR) according to claim 1, characterized in that A conjugate is bound to the C- or N-terminus of the α chain and / or β chain of the TCR.

17. The T cell receptor (TCR) according to claim 16, characterized in that The conjugate bound to the T cell receptor is a detectable marker, a therapeutic agent, a PK modifying moiety, or a combination of any of these substances; preferably, the therapeutic agent is an anti-CD3 antibody.

18. A multivalent TCR complex comprising at least two TCR molecules, wherein at least one TCR molecule is the TCR described in any one of claims 1-17.

19. An isolated nucleic acid molecule encoding the TCR of any one of claims 1-17.

20. A vector comprising the nucleic acid molecule of claim 19; preferably, the vector is a viral vector; more preferably, the vector is a lentiviral vector.

21. An isolated host cell, wherein the host cell contains the vector according to claim 20 or an exogenous nucleic acid molecule according to claim 19 integrated into its genome.

22. A cell expressing the TCR of any one of claims 1-19 on its surface.

23. A cell according to claim 22, characterized in that The cell is transduced with the nucleic acid molecule of claim 19 or the vector of claim 20.

24. The cell according to claim 23, wherein The cells are T cells, NK cells, NKT cells or stem cells.

25. The cell according to claim 24, wherein The cells co-express CD8 molecules; preferably human CD8 molecules; more preferably human CD8a molecules, such as human CD8a molecules having the sequence shown in SEQ ID NO:

53.

26. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the TCR of any one of claims 1-17, the TCR complex of claim 18, the nucleic acid molecule of claim 19, the vector of claim 20, or the cell of any one of claims 23-25.

27. Use of the T cell receptor according to any one of claims 1 to 17, the TCR complex according to claim 18, and the cell according to any one of claims 23 to 25 for preparing a drug for treating tumors.

28. The use according to claim 27, characterized in that The tumor is a PRAME-associated tumor.

29. The use according to claim 28, characterized in that The tumor is a tumor that specifically expresses the complete or partial tumor antigen PRAME.

30. The use according to claim 29, characterized in that The tumor is a tumor that specifically expresses the SLLQHLIGL peptide segment.

31. The use according to claim 30, characterized in that The tumor is melanoma, non-small cell lung cancer, ovarian cancer, and breast cancer.

32. The use according to claim 31, characterized in that The tumor is non-small cell lung cancer.

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