T-cell receptors

Abstract

The present disclosure relates to T cell receptors (TCRs) capable of binding to HLA-A * 02 restrictive peptides derived from PRAME. The disclosure also relates to complexes comprising the TCRs, nucleic acids and vectors encoding the TCRs or portions thereof, cells comprising the TCRs, pharmaceutical compositions, and methods of using the TCRs to treat diseases, such as cancer.

Description

Technical Field

[0001] The present disclosure relates to T cell receptors (TCRs) capable of binding to HLA-A*02-restricted peptides derived from PRAME. The present disclosure also relates to complexes comprising these TCRs, nucleic acids and vectors encoding these TCRs or portions thereof, cells comprising these TCRs, pharmaceutical compositions, and methods of using these TCRs to treat diseases such as cancer. Background Art

[0002] T cell receptors (TCRs) are naturally expressed by CD4+ and CD8+ T cells. TCRs recognize short peptide antigens complexed with major histocompatibility complex (MHC) molecules and presented on the surface of antigen-presenting cells. In humans, MHC molecules are also known as human leukocyte antigens, or HLA (Davis et al., Annu Rev Immunol. 1998; 16:523-44). CD8+ T cells, also known as cytotoxic T cells, have TCRs that can specifically recognize peptide segments bound to MHC class I molecules. CD8+ T cells are typically responsible for seeking out and mediating the destruction of diseased cells such as cancer cells and virus-infected cells. Due to thymic selection, the affinity of cancer-specific TCRs in the natural repertoire for their corresponding antigens is usually low, meaning that cancer cells can often evade T cell detection and clearance. Novel immunotherapeutic approaches aimed at enhancing T cell recognition of cancer offer highly promising strategies for developing effective anti-cancer therapies.

[0003] PRAME, or preferentially expressed antigen in melanoma, was initially identified as an antigen overexpressed in melanoma (Ikeda et al., Immunity. 1997 Feb; 6(2): 199-208). Subsequently, it was found to be overexpressed in a variety of other cancers, such as leukemia, lymphoma, lung cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, bladder cancer, and head and neck cancer (Doolan et al., Breast Cancer Res Treat. 2008 May; 109(2): 359-65; Epping et al., Cancer Res. 2006 Nov 15; 66(22): 10639-42; Ercolak et al., Acta Haematol. 2015; 134(4): 199-207; Matsushita et al., Leuk Lymphoma. 2003 Mar; 44(3): 439-44; Mitsuhashi et al., Int. J Hematol. 2014; 100(1): 88-95; Proto-Siqueira et al., Leuk Res. 2006 Nov; 30(11): 1333-9; Szczepanski et al., Oral Oncol. 2013 Feb; 49(2): 144-51; Van Baren et al., Br J Haematol. 1998 Sep; 102(5): 1376-9).

[0004] PRAME has also been referred to in the literature as CT130, MAPE, and OIP-4. The polypeptide sequence of human PRAME can be found in the Uniprot database accession number P78395. PRAME functions as an inhibitor of the retinoic acid receptor signaling pathway (Epping et al., Cell. 2005 Sep 23; 122(6): 835-47) and belongs to the family of germline-encoded antigens known as cancer-testis antigens. Cancer-testis antigens are a class of tumor-associated antigens that are expressed in human tumors of various histological origins but not in normal human tissues outside the testis and placenta. Thus, cancer-testis antigens, due to their limited or no expression in normal adult tissues, are attractive targets for immunotherapeutic intervention. Therefore, there is a need for high-affinity TCRs against cancer-testis antigens. SUMMARY OF THE INVENTION

[0005] Provided herein are TCRs that are capable of binding to a complex comprising a PRAME-derived peptide and HLA-A*02. These TCRs have significant advantages, for example, in the treatment of cancers expressing PRAME.

[0006] More specifically, the present inventors have identified a dominant TCR that binds to a complex comprising SLLQHLIGL (SEQ ID NO:1) and HLA-A*02. The peptide SLLQHLIGL (SEQ ID NO:1) corresponds to amino acids 425-433 of the full-length PRAME protein (Uniprot accession number P78395), which complexes with HLA-A*02 and is co-presented on the cell surface (Kessler et al., J Exp Med. 2001 Jan 1;193(1):73-88). The TCRs of the present disclosure are engineered to bind the target antigen with higher affinity compared to any naturally occurring TCR. Thus, the TCRs of the present disclosure have significant advantages, for example, in therapeutic applications such as cancer treatment.

[0007] Accordingly, the present disclosure provides a TCR that binds to a complex comprising SEQ ID NO:1 and HLA-A*02, the TCR comprising: (a) a variable domain of the TCR α-chain comprising an amino acid sequence having at least 50% sequence identity with SEQ ID NO:17; and (b) a variable domain of the TCR β-chain comprising an amino acid sequence having at least 50% sequence identity with SEQ ID NO:29.

[0008] The present disclosure also provides a complex comprising (a) a TCR of the present disclosure, and (b) a detectable label, therapeutic agent, or pharmacokinetic modification moiety. The present disclosure also provides nucleic acids encoding: (a) a TCR of the present disclosure; (b) the variable domain of the TCR α-chain of the TCR of the present disclosure, and optionally the constant domain of the TCR α-chain; or (c) the variable domain of the TCR β-chain of the TCR of the present disclosure, and optionally the constant domain of the TCR β-chain. The present disclosure also provides a vector comprising a nucleic acid of the present disclosure.

[0009] The present disclosure also provides a viral particle comprising the nucleic acid or the vector of the present disclosure. The present disclosure also provides a cell that (a) expresses the TCR of the present disclosure; (b) comprises at least one nucleic acid of the present disclosure; optionally, wherein the cell comprises (i) a first nucleic acid encoding the variable domain of the TCRα chain of the TCR of the present disclosure and optionally the constant domain of the TCRα chain, and (ii) a second nucleic acid encoding the variable domain of the TCRβ chain of the TCR of the present disclosure and optionally the constant domain of the TCRβ chain; or (c) comprises at least one vector of the present disclosure; optionally, wherein the cell comprises (i) a first vector comprising a nucleic acid encoding the variable domain of the TCRα chain of the TCR of the present disclosure and optionally the constant domain of the TCRα chain, and (ii) a second vector comprising a nucleic acid encoding the variable domain of the TCRβ chain of the TCR of the present disclosure and optionally the constant domain of the TCRβ chain. The present disclosure also provides a pharmaceutical composition comprising (i) the TCR, the complex, the nucleic acid, or the cell of the present disclosure, and (ii) one or more pharmaceutically acceptable carriers or excipients.

[0010] The present disclosure also provides a TCR, a nucleic acid, a cell, or a pharmaceutical composition of the present disclosure for use in a method of treating a disease in an individual, the method comprising administering to the individual the TCR, the nucleic acid, the cell, or the pharmaceutical composition. Similarly, the present disclosure provides a method of treating a disease in an individual, comprising administering to the individual the TCR, the nucleic acid, the cell, or the pharmaceutical composition of the present disclosure.

[0011] The present disclosure also provides a method of preparing one or more cells of the present disclosure, comprising: (i) introducing into the cell at least one nucleic acid or at least one vector of the present disclosure; and optionally (ii) culturing the cell to proliferate the cell to form a population. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : Functional avidity increment of a potency-enhanced TCR specific for HLA-A*02:01PRAME_SLLQHLIGL. A set of TCRs transduced into Jurkat cells were co-cultured with T2 cells pulsed with 10-fold serial dilutions (concentrations from 1×10 -5 M to 1×10 -12 M) of the PRAME_SLLQHLIGL peptide at a 1:1 ratio for 16 hours. The enhanced CD69 response detected by flow cytometry is clearly visible in the titration curve (left panel), with EC50 values ranging from 14 to 0.1 nM, spanning 2 logarithmic orders of magnitude. The EC 50 value represents the concentration inducing half-maximal activation of Jurkat cells (CD69 +The peptide concentration required for (cell percentage). The titrated peptide concentrations are labeled logarithmically (x-axis of the graph), and the EC50 values are likewise listed, along with the corresponding values converted to nanomolar (nM) (see the table on the right).

[0013] Figure 2 : As shown by IFNγ ELISA assay, the TCR-transduced T cell groups were specific for PRAME. Six engineered TCR-transduced donor T cells were studied by IFNγ ELISpot assay in the presence of 4 PRAME-positive tumor cell lines (Malme-3M, SK-Mel-5, NCI-H1755, NCI-H1703) and 2 PRAME-negative (or negligible) tumor cell lines (NCI-H2228, TCC-SUP). Assays for each target cell line included untransduced T cells (NTD) and a "Targets only" group containing only the target cells (response values for both groups were generally too low to observe). Spot counts measured after IFNγ release are shown in the figure. Data show that all engineered TCR T cells (dark to medium gray bars) were target-specific, which was consistent with the RNA expression levels of the tumor cell lines (Malme-3M ≥ SK-Mel-5 > NCI-H1755 > NCI-H1703). Cell lines with higher PRAME expression (Malme-3M, SK-Mel-5, and NCI-H1755) elicited strong T cell responses in all PRAME TCR T cell groups, while responses to the cell line with lower PRAME expression (NCI-H1703) were variable and weak. Negligible or no T cell activation responses were detected in the PRAME-negative cell lines (NCI-H2228, TCC-SUP), and no activation responses or only negligible responses were observed in all NTD T cell groups or the Targets only groups (lightest gray areas). Note that the maximum response signal provides a semi-quantitative reading as the count may exceed the maximum countable signal (500 spots). Values are the mean of replicate experiments, and error bars show the signal range. The legend within the box (bottom) indicates the gray shading color scheme for each effector T cell type plotted or no T cells added (Targets only).

[0014] Figure 3: Cytotoxicity of selected TCR-transduced T cells against PRAME-positive tumor cell line (Malme-3M). When co-cultured with untransduced (ntd) T cells or T cells transduced with selected PRAME-specific TCRs (ADB02780_063, _070, _077), the two-dimensional killing response against the target Malme-3M tumor (melanoma) cell line is shown in the upper row (main figure, left). Potent target cell killing was observed within 24 hours of co-culture with each PRAME-specific TCR T cell; cytotoxicity lasted for at least 3 days. No killing was observed with ntd T cells lacking the PRAME TCR (upper row, leftmost figure). In the lower row (main figure), the corresponding NTD or transduced effector T cells were incubated alone, without co-incubation with Malme-3M target cells, and no killing was observed. Malme-3M target cells incubated alone (without addition of effector T cells) also did not show cell killing (upper right, inset). This experiment monitored the apoptotic activity over time (in hours) in real time using the IncuCyte ZOOM live cell imaging system. The response value (green object count) represents the number of apoptotic cells measured per mm 2 . Data for all co-cultured and single-cultured T cells (main figure, left) are presented as the mean ± standard error of 3 replicate experiments. Data for Malme-3M targets alone were from a single replicate experiment. Images were limited to 96 hours after T cell addition.

[0015] Figure 4 : Functional avidity of an enhanced-potency TCR specific for PRAME_SLLQHLIGL presented by HLA-A*02:01. After a panel of TCRs was transduced into primary T cells, they were co-cultured with T2 target cells (PRAME-negative, HLA-A*02-positive) pulsed with 10-fold dilutions of the PRAME_SLLQHLIGL peptide (from 1×10 -5 M to 1×10 -11 M) at a 1:1 ratio for 48 hours. Their potency was measured by IFNγ supernatant ELISA. The EC 50 value represents the peptide concentration required to induce T-cell activation to half-maximum, and the values for this panel of mutants ranged from 10 -8.8 M to 10 -7.8 M.

[0016] Figure 5 : When tested against a panel of PRAME-positive and -negative cell lines, T cells transduced to express the enhanced-potency TCR showed specificity for PRAME. Target cells were incubated alone with the indicated TCR-transduced T cells (solid circles) or in 10 - 5Incubate for approximately 42 hours in the presence of the exogenous PRAME peptide "SLLQHLIGL" of M (open circles, only ADB02780_070), and then measure IFNγ in the supernatant by ELISA. T cells from 4 donors (DVA_002, GPA_025, KXA_039, and XKA_003, presented in separate graphs) were tested. The bottom graph shows the respective target tumor cell lines, with their transcriptional levels (measured by qPCR and normalized to the reference gene level) arranged in ascending order from left to right. Untransduced T cells (ntd) and the target-only condition were set as negative controls. The top graph provides the results of the IFNγ ELISA assay.

[0017] Figure 6 : When tested against a panel of antigen-negative primary cell lines, T cells transduced to express a potency-enhanced TCR showed specificity for PRAME. Using IFNγ supernatant ELISA assays, the potency of 3 engineered TCRs against a panel of antigen-negative (HLA*02-positive) primary cell lines was studied. The target cells were incubated with TCR-transduced T cells (solid circles) or in the presence of 10 -5 Incubate for approximately 42 hours in the presence of the exogenous "PRAME_SLLQHLIGL" peptide of M (open circles, only ADB02780_070). T cells from 4 donors (DVA_002, GPA_025, KXA_039, and XKA_003, presented in separate graphs) were tested. The primary cell lines were arranged in ascending order of transcriptional level (measured by qPCR) from left to right. Untransduced T cells (ntd) and the target-only condition were set as negative controls. Detailed Description

[0018] It should be understood that various applications of the disclosed products and methods can be customized according to specific needs in the art. It should also be understood that the terms used herein are for the purpose of describing particular embodiments of the present disclosure only and are not intended to be limiting.

[0019] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0020] General Definition

[0021] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0022] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a TCR" includes a plurality of such TCRs, reference to "an antibody" includes two or more such antibodies, reference to "a cell" includes two or more such cells, and so forth.

[0023] In general, the term "comprising" is to be construed as an open-ended term (i.e., meaning "including, but not limited to") unless otherwise specified.

[0024] In some aspects of the present disclosure, the phrase "comprising" is replaced by the phrase "consisting of". The term "consisting of" is intended to be limiting.

[0025] The terms "cancer", "neoplasm", and "tumor" are used interchangeably and in both singular and plural forms to refer to cells that have undergone malignant transformation and are pathologically significant to the host organism.

[0026] As used herein, the terms "protein" and "polypeptide" are used interchangeably and refer to a polymeric chain of amino acids of any length.

[0027] For the purposes of the present disclosure, to determine the percent identity between two sequences (e.g., two polynucleotide or two polypeptide sequences), the sequences are aligned for optimal alignment (e.g., gaps may be introduced in the first sequence for optimal alignment with the second sequence). Subsequently, the nucleotide residues at the nucleotide positions are compared. When a position in the first sequence is occupied by the same nucleotide residue as the corresponding position in the second sequence, the nucleotides at that position are identical. The percent identity between the two sequences is a function of the number of positions at which the sequences have the same nucleotide (i.e., % identity = number of identical positions / total number of positions in the reference sequence × 100).

[0028] Typically, sequence alignments are performed over the entire length of the reference sequence. For example, if a user wishes to determine whether a given ("test") sequence has a particular percent identity with SEQ ID NO:X, SEQ ID NO:X will be used as the reference sequence. For example, to determine whether a sequence has at least 80% identity with SEQ ID NO:X (an example of a reference sequence), one of ordinary skill in the art would perform a sequence alignment over the entire length of SEQ ID NO:X and determine how many positions in the test sequence are identical to SEQ ID NO:X. If at least 80% of the positions are identical, then the test sequence has at least 80% identity with SEQ ID NO:X. If the sequence is shorter than SEQ ID NO:X, gaps or missing positions should be considered non-identical positions.

[0029] Those skilled in the art are aware that there are a variety of computer programs available for determining the homology or identity between two sequences. For example, sequence comparison and determination of the percentage of identity between two sequences can be achieved by mathematical algorithms.

[0030] The term "T cell receptor" or "TCR" refers to a heterodimeric molecule comprising an alpha polypeptide chain (alpha chain) and a beta polypeptide chain (beta chain), wherein the heterodimeric receptor is capable of binding a peptide antigen presented by an MHC molecule. In this article, the IMGT TCR nomenclature is used to describe TCRs and a link to the IMGT public TCR sequence database is provided. A native alpha-beta heterodimeric TCR has one alpha chain and one beta chain. Generally, each chain comprises a variable region, a joining region, and a constant region, and the beta chain usually also contains a short diversity region between the variable region and the joining region, but this diversity region is usually considered part of the joining region. Each variable region contains three complementarity-determining regions (CDRs) embedded within framework sequences, with one of them being a hypervariable region called CDR3. There are multiple types of alpha chain variable domains (Vα) and beta chain variable domains (Vβ), which differ in their framework regions, CDR1 and CDR2 sequences, and in part, the CDR3 sequence. In the IMGT nomenclature, Vα types are identified by unique TRAV numbers. Thus, "TRAV21" defines a TCR Vα region with unique framework, CDR1, and CDR2 sequences, and its CDR3 sequence is in part defined by an amino acid sequence conserved between TCRs, and also contains amino acid sequences that vary between TCRs. Similarly, "TRBV5-1" defines a TCR Vβ region with unique framework and CDR1 and CDR2 sequences, but its CDR3 sequence is only partially defined.

[0031] The joining regions of TCRs are also defined by the unique IMGT TRAJ and TRBJ nomenclatures, and the constant regions are defined by the IMGT TRAC and TRBC nomenclatures. In the IMGT nomenclature, the variable region of the β-chain is abbreviated as TRBD; as previously mentioned, the tandem TRBD / TRBJ regions are generally collectively referred to as the joining region. The α-chain and β-chain of the αβ TCR are generally considered to each have two "domains", namely the variable domain and the constant domain. The variable domain is composed of the variable domain and the joining region in series. During the process of joining the TRAV and TRAJ gene segments, a small number of DNA bases are randomly added or deleted. This helps to generate the diversity of the CDR3 sequence, enabling each T cell to generate a unique TCR α-chain gene. Similarly, during the joining process of the TRBV and TRBJ gene segments, several DNA bases are randomly added or deleted, thereby generating diversity in the CDR3 sequence and enabling each T cell to generate a unique TCR β-chain gene. Therefore, in this specification and the claims, the term "TCR α variable domain" refers to the tandem combination of the TRAV and TRAJ regions, and the term "TCR α constant domain" refers to the extracellular TRAC region or the C-terminally truncated TRAC sequence. Similarly, the term "TCR β variable domain" refers to the tandem combination of the TRBV and the TRBD / TRBJ regions, and the term "TCR β constant domain" refers to the extracellular TRBC region or the C-terminally truncated TRBC sequence.

[0032] The unique sequences defined by the IMGT nomenclature are well-known in the TCR field and are accessible to researchers in the TCR field. For example, they can be queried in the IMGT public database. The "T Cell Receptor FactsBook" (2001, by LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8) also includes sequences defined according to the IMGT nomenclature, but due to its early publication time and information lag, some content in the book needs to be confirmed by referring to the IMGT database.

[0033] T Cell Receptor (TCR)

[0034] The TCRs of the present disclosure are capable of binding to a complex comprising the PRAME peptide sequence SLLQHLIGL (SEQ ID NO:1) and HLA-A*02. The TCRs of the present disclosure comprise (a) a TCR α-chain variable domain comprising an amino acid sequence having at least 50% sequence identity with SEQ ID NO:17; and (b) a TCR β-chain variable domain comprising an amino acid sequence having at least 50% sequence identity with SEQ ID NO:29.

[0035] SEQ ID NO:17 is the variable domain sequence of the α-chain of the parental TCR (named TCR1), and SEQ ID NO:29 is the variable domain sequence of its β-chain. As shown in the examples of this application, eight affinity-matured TCRs (referred to as TCR2-9) were generated from TCR1, and these TCRs are particularly effective. Table 1 below summarizes the sequences of each of TCR 2-9.

[0036] Table 1 - SEQ ID numbers of TCRs. In the specification, TCR numbers (1 to 9) can be used interchangeably with ADB reference numbers (ADB02780_001, ADB02780_022, etc.).

[0037]

[0038]

[0039] Variable domain

[0040] As described above, the TCRs of the present disclosure include a variable domain of the TCR α-chain, which contains an amino acid sequence having at least 50% sequence identity with SEQ ID NO:17. For example, the variable domain of the TCR α-chain may contain an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO:17.

[0041] For example, the variable domain of the TCR α-chain may contain CDR1, which contains (1) the sequence shown in SEQ ID NO:8, or (2) an amino acid sequence containing one, two or three amino acid insertions, deletions or substitutions relative to the sequence shown in SEQ ID NO:8. An amino acid sequence containing one, two or three amino acid insertions, deletions or substitutions relative to the SEQ ID NO:8 sequence may, for example, contain SEQ ID NO:9, 10, 11, 12, 13 or 14. SEQ ID NO:9, 10, 11, 12, 13 and 14 are single-point substitution mutants of SEQ ID NO:8. For example, the variable domain of the TCR α-chain may contain CDR2, which contains (1) the sequence shown in SEQ ID NO:15, or (2) an amino acid sequence containing one, two or three amino acid insertions, deletions or substitutions relative to the sequence shown in SEQ ID NO:15. For example, the variable domain of the TCR α-chain may contain CDR3, which contains (1) the sequence shown in SEQ ID NO:16, or (2) an amino acid sequence containing one, two or three amino acid insertions, deletions or substitutions relative to the sequence shown in SEQ ID NO:16.

[0042] For example, the variable domain of the TCR α chain may comprise (i) CDR1, comprising (1) the sequence shown in SEQ ID NO:8 or (2) an amino acid sequence comprising one, two, or three amino acid insertions, deletions, or substitutions relative to the sequence shown in SEQ ID NO:8; (ii) CDR2, comprising (1) the sequence shown in SEQ ID NO:15 or (2) an amino acid sequence comprising one, two, or three amino acid insertions, deletions, or substitutions relative to the sequence of SEQ ID NO:15; and / or (iii) CDR3, comprising (1) the sequence shown in SEQ ID NO:16 or (2) an amino acid sequence comprising one, two, or three amino acid insertions, deletions, or substitutions relative to the sequence of SEQ ID NO:16. For example, the variable domain of the TCR α chain may comprise (i); (ii); (iii); (i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii), and (iii). In one aspect of the present disclosure, the variable domain of the TCR α chain comprises (i), (ii), and (iii).

[0043] The TCR of the present disclosure further comprises a variable domain of the TCR β chain, which comprises an amino acid sequence having at least 50% sequence identity with SEQ ID NO:29. For example, the variable domain of the TCR β chain may comprise an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO:29.

[0044] For example, the variable domain of the TCR β chain may comprise CDR1, which comprises (1) the sequence shown in SEQ ID NO:24, or (2) an amino acid sequence comprising one, two, or three amino acid insertions, deletions, or substitutions relative to the sequence of SEQ ID NO:24. For example, the variable domain of the TCR β chain may comprise CDR2, which comprises (1) the sequence shown in SEQ ID NO:25, or (2) an amino acid sequence comprising one, two, or three amino acid insertions, deletions, or substitutions relative to the sequence of SEQ ID NO:25. An amino acid sequence comprising one, two, or three amino acid insertions, deletions, or substitutions relative to the sequence of SEQ ID NO:25 may for example comprise SEQ ID NO:26 or 27. SEQ ID NO:26 and 27 are single point substitution mutants of SEQ ID NO:25. For example, the variable domain of the TCR β chain may comprise CDR3, which comprises (1) the sequence shown in SEQ ID NO:28, or (2) an amino acid sequence comprising one, two, or three amino acid insertions, deletions, or substitutions relative to the sequence of SEQ ID NO:28.

[0045] For example, the variable domain of the TCR β chain may comprise (iv) CDR1, comprising (1) the sequence shown in SEQ ID NO: 24 or (2) an amino acid sequence comprising one, two, or three amino acid insertions, deletions, or substitutions relative to the sequence shown in SEQ ID NO: 24; (v) CDR2, comprising (1) the sequence shown in SEQ ID NO: 25 or (2) an amino acid sequence comprising one, two, or three amino acid insertions, deletions, or substitutions relative to the sequence of SEQ ID NO: 25; and / or (vi) CDR3, comprising (1) the sequence shown in SEQ ID NO: 28 or (2) an amino acid sequence comprising one, two, or three amino acid insertions, deletions, or substitutions relative to the sequence of SEQ ID NO: 28. For example, the variable domain of the TCR β chain may comprise (iv); (v); (vi); (iv) and (v); (iv) and (vi); (v) and (vi); or (iv), (v), and (vi). In one aspect of the present disclosure, the variable domain of the TCR β chain comprises (iv), (v), and (vi).

[0046] The TCR may comprise: (a) a variable domain of the TCR α chain, the variable domain comprising: (i) CDR1, comprising (1) the sequence shown in SEQ ID NO: 8 or (2) an amino acid sequence comprising one, two or three amino acid insertions, deletions or substitutions relative to the SEQ ID NO: 8 sequence; (ii) CDR2, comprising (1) the sequence shown in SEQ ID NO: 15 or (2) an amino acid sequence comprising one, two or three amino acid insertions, deletions or substitutions relative to the SEQ ID NO: 15 sequence; and / or (iii) CDR3, comprising (1) the sequence shown in SEQ ID NO: 16 or (2) an amino acid sequence comprising one, two or three amino acid insertions, deletions or substitutions relative to the SEQ ID NO: 16 sequence; and (b) a variable domain of the TCR β chain, the variable domain comprising: (iv) CDR1, comprising (1) the sequence shown in SEQ ID NO: 24 or (2) an amino acid sequence comprising one, two or three amino acid insertions, deletions or substitutions relative to the SEQ ID NO: 24 sequence; (v) CDR2, comprising (1) the sequence shown in SEQ ID NO: 25 or (2) an amino acid sequence comprising one, two or three amino acid insertions, deletions or substitutions relative to the SEQ ID NO: 25 sequence; and / or (vi) CDR3, comprising (1) the sequence shown in SEQ ID NO: 28 or (2) an amino acid sequence comprising one, two or three amino acid insertions, deletions or substitutions relative to the SEQ ID NO: 28 sequence. For example, the TCR may comprise (i) and (iv); (i) and (v); (i) and (vi); (i), (iv) and (v); (i), (iv) and (vi); (i), (v) and (vi); (i), (iv), (v) and (vi); (ii) and (iv); (ii) and (v); (ii) and (vi); (ii), (iv) and (v); (ii), (iv) and (vi); (ii), (v) and (vi); (ii), (iv), (v) and (vi); (iii) and (iv); (iii) and (v); (iii) and (vi); (iii), (iv) and (v); (iii), (iv) and (vi); (iii), (v) and (vi); (iii), (iv), (v) and (vi); (i), (ii) and (iv); (i), (ii) and (v); (i), (ii) and (vi); (i), (ii), (iv) and (v); (i), (ii), (iv) and (vi); (i), (ii), (v) and (vi); (i), (ii), (iv), (v) and (vi); (i), (iii) and (iv); (i), (iii) and (v); (i), (iii) and (vi);

[0047] (i), (iii), (iv) and (v); (i), (iii), (iv) and (vi); (i), (iii), (v) and (vi); (i), (iii), (iv), (v) and (vi); (ii), (iii) and (iv); (ii), (iii) and (v); (ii), (iii) and (vi); (ii), (iii), (iv) and (v); (ii), (iii), (iv) and (vi);

[0048] (ii), (iii), (v) and (vi); (ii), (iii), (iv), (v) and (vi); (i), (ii), (iii) and (iv); (i), (ii), (iii) and (v); (i), (ii), (iii) and (vi); (i), (ii), (iii), (iv) and (v); (i), (ii), (iii), (iv) and (vi); (i), (ii), (iii), (v) and (vi); or (i), (ii), (iii), (iv), (v) and (vi). In one aspect of the present disclosure, the TCRα chain variable domain comprises (i), (ii) and (iii), and the TCRβ chain variable domain comprises (iv), (v) and (vi).

[0049] In some cases, (a) the TCRα chain variable domain comprises a CDR1 containing SEQ ID NO:8, 9, 10, 11, 12, 13 or 14; (b) the TCRα chain variable domain comprises a CDR2 containing SEQ ID NO:15; (c) the TCRα chain variable domain comprises a CDR3 containing SEQ ID NO:16; (d) the TCRβ chain variable domain comprises a CDR1 containing SEQ ID NO:24; (e) the TCRβ chain variable domain comprises a CDR2 containing SEQ ID NO:25, 26 or 27; and / or (f) the TCRβ chain variable domain comprises a CDR3 containing SEQ ID NO:28. For example, in some cases: (a); (b); (c); (d); (e); (f); (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (b) and (c); (b) and (d); (b) and (e); (b) and (f); (c) and (d);

[0050] (c) and (e); (c) and (f); (d) and (e); (d) and (f); (e) and (f); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and (f); (a), (e) and (f); (b), (c) and (d); (b), (c) and (e); (b), (c) and (f); (b), (d) and (e); (b), (d) and (f); (b), (e) and (f); (c), (d) and (e); (c), (d) and (f); (c), (e) and (f); (d), (e) and (f); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (c) and (f); (a), (b), (d) and (e); (a), (b), (d) and (f); (a), (b), (e) and (f); (a), (c), (d) and (e); (a), (c), (d) and (f); (a), (c), (e) and (f); (a), (d), (e) and (f); (b), (c), (d) and (e); (b), (c), (d) and (f); (b), (c), (e) and (f); (b), (d), (e) and (f); (c), (d), (e) and (f); (a), (b), (c), (d) and (e); (a), (b), (c), (d) and (f); (a), (b), (c), (e) and (f); (a), (b), (d), (e) and (f); (a), (c), (d), (e) and (f); (b), (c), (d), (e) and (f); (a), (b), (c), (d), (e) and (f); For example, in some cases, (a) the variable domain of the TCR α chain contains a CDR1 comprising SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14; (b) the variable domain of the TCR α chain contains a CDR2 comprising SEQ ID NO: 15; (c) the variable domain of the TCR α chain contains a CDR3 comprising SEQ ID NO: 16; (d) the variable domain of the TCR β chain contains a CDR1 comprising SEQ ID NO: 24; (e) the variable domain of the TCR β chain contains a CDR2 comprising SEQ ID NO: 25, 26 or 27; and (f) the variable domain of the TCR β chain contains a CDR3 comprising SEQ ID NO: 28.

[0051] For example, the variable domain of the TCR α chain may comprise a CDR1 containing SEQ ID NO:8, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16. For example, the variable domain of the TCR α chain may comprise SEQ ID NO:17. For example, the variable domain of the TCR α chain may comprise a CDR1 containing SEQ ID NO:9, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16. For example, the variable domain of the TCR α chain may comprise SEQ ID NO:18. For example, the variable domain of the TCR α chain may comprise a CDR1 containing SEQ ID NO:10, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16. For example, the variable domain of the TCR α chain may comprise SEQ ID NO:19. For example, the variable domain of the TCR α chain may comprise a CDR1 containing SEQ ID NO:11, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16. For example, the variable domain of the TCR α chain may comprise SEQ ID NO:20. For example, the variable domain of the TCR α chain may comprise a CDR1 containing SEQ ID NO:12, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16. For example, the variable domain of the TCR α chain may comprise SEQ ID NO:21. For example, the variable domain of the TCR α chain may comprise a CDR1 containing SEQ ID NO:13, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16. For example, the variable domain of the TCR α chain may comprise SEQ ID NO:22. For example, the variable domain of the TCR α chain may comprise a CDR1 containing SEQ ID NO:14, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16. For example, the variable domain of the TCR α chain may comprise SEQ ID NO:23.

[0052] Any variable domain of an alpha chain disclosed herein may include a signal peptide at its N-terminus. For example, the variable domain of an alpha chain may be expressed via an N-terminal signal peptide that is excised before expression on the T cell surface. For example, the signal peptide may comprise or consist of SEQ ID NO:38. For example, the variable domain of the alpha chain may comprise SEQ ID NO:38 and SEQ ID NO:17, wherein the C-terminal "S" of SEQ ID NO:38 is directly linked via a peptide bond to the N-terminal "G" of SEQ ID NO:17. For example, the variable domain of the alpha chain may comprise SEQ ID NO:38 and SEQ ID NO:18, wherein the C-terminal "S" of SEQ ID NO:38 is directly linked via a peptide bond to the N-terminal "G" of SEQ ID NO:18. For example, the variable domain of the alpha chain may comprise SEQ ID NO:38 and SEQ ID NO:19, wherein the C-terminal "S" of SEQ ID NO:38 is directly linked via a peptide bond to the N-terminal "G" of SEQ ID NO:19. For example, the variable domain of the alpha chain may comprise SEQ ID NO:38 and SEQ ID NO:20, wherein the C-terminal "S" of SEQ ID NO:38 is directly linked via a peptide bond to the N-terminal "G" of SEQ ID NO:20. For example, the variable domain of the alpha chain may comprise SEQ ID NO:38 and SEQ ID NO:21, wherein the C-terminal "S" of SEQ ID NO:38 is directly linked via a peptide bond to the N-terminal "G" of SEQ ID NO:21. For example, the variable domain of the alpha chain may comprise SEQ ID NO:38 and SEQ ID NO:22, wherein the C-terminal "S" of SEQ ID NO:38 is directly linked via a peptide bond to the N-terminal "G" of SEQ ID NO:22. For example, the variable domain of the alpha chain may comprise SEQ ID NO:38 and SEQ ID NO:23, wherein the C-terminal "S" of SEQ ID NO:38 is directly linked via a peptide bond to the N-terminal "G" of SEQ ID NO:23.

[0053] For example, the variable domain of the TCR β chain may comprise a CDR1 containing SEQ ID NO:24, a CDR2 containing SEQ ID NO:25, and a CDR3 containing SEQ ID NO:28. For example, the variable domain of the TCR β chain may comprise SEQ ID NO:29. For example, the variable domain of the TCR β chain may comprise a CDR1 containing SEQ ID NO:24, a CDR2 containing SEQ ID NO:26, and a CDR3 containing SEQ ID NO:28. For example, the variable domain of the TCR β chain may comprise SEQ ID NO:30. For example, the variable domain of the TCR β chain may comprise a CDR1 containing SEQ ID NO:24, a CDR2 containing SEQ ID NO:27, and a CDR3 containing SEQ ID NO:28. For example, the variable domain of the TCR β chain may comprise SEQ ID NO:31.

[0054] Any variable domain of the β chain disclosed herein may comprise a signal peptide at its N-terminus. For example, the variable domain of the β chain may be expressed via an N-terminal signal peptide that is excised prior to expression on the T cell surface. For example, the signal peptide may comprise or consist of SEQ ID NO:39. For example, the variable domain of the β chain may comprise SEQ ID NO:39 and SEQ ID NO:29, wherein the C-terminal "L" of SEQ ID NO:39 is directly linked via a peptide bond to the N-terminal "E" of SEQ ID NO:29. For example, the variable domain of the β chain may comprise SEQ ID NO:39 and SEQ ID NO:30, wherein the C-terminal "L" of SEQ ID NO:39 is directly linked via a peptide bond to the N-terminal "E" of SEQ ID NO:30. For example, the variable domain of the β chain may comprise SEQ ID NO:39 and SEQ ID NO:31, wherein the C-terminal "L" of SEQ ID NO:39 is directly linked via a peptide bond to the N-terminal "E" of SEQ ID NO:31.

[0055] For example, the TCR may comprise a variable domain of the TCR α chain, which variable domain comprises a CDR1 containing SEQ ID NO:8, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16; and a variable domain of the TCR β chain, which variable domain comprises a CDR1 containing SEQ ID NO:24, a CDR2 containing SEQ ID NO:25, and a CDR3 containing SEQ ID NO:28. For example, the TCR may comprise a variable domain of the TCR α chain containing SEQ ID NO:17, and a variable domain of the TCR β chain containing SEQ ID NO:29. For example, the TCR may comprise a variable domain of the TCR α chain, which variable domain comprises a CDR1 containing SEQ ID NO:8, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16; and a variable domain of the TCR β chain, which variable domain comprises a CDR1 containing SEQ ID NO:24, a CDR2 containing SEQ ID NO:26, and a CDR3 containing SEQ ID NO:28. For example, the TCR may comprise a variable domain of the TCR α chain containing SEQID NO:17, and a variable domain of the TCR β chain containing SEQ ID NO:30. For example, the TCR may comprise a variable domain of the TCR α chain, which variable domain comprises a CDR1 containing SEQ ID NO:8, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQID NO:16; and a variable domain of the TCR β chain, which variable domain comprises a CDR1 containing SEQ ID NO:24, a CDR2 containing SEQ IDNO:27, and a CDR3 containing SEQ ID NO:28. For example, the TCR may comprise a variable domain of the TCR α chain containing SEQ ID NO:17, and a variable domain of the TCR β chain containing SEQ ID NO:31. For example, the TCR may comprise a variable domain of the TCR α chain, which variable domain comprises a CDR1 containing SEQ ID NO:9, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16; and a variable domain of the TCR β chain, which variable domain comprises a CDR1 containing SEQ ID NO:24, a CDR2 containing SEQ ID NO:27, and a CDR3 containing SEQ ID NO:28. For example, the TCR may comprise a variable domain of the TCR α chain containing SEQ ID NO:18, and a variable domain of the TCR β chain containing SEQ ID NO:31.For example, the TCR may comprise a variable domain of the TCR α chain, which variable domain comprises a CDR1 containing SEQ ID NO:10, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16; and a variable domain of the TCR β chain, which variable domain comprises a CDR1 containing SEQ ID NO:24, a CDR2 containing SEQ ID NO:27, and a CDR3 containing SEQ ID NO:28. For example, the TCR may comprise a variable domain of the TCR α chain containing SEQ ID NO:19, and a variable domain of the TCR β chain containing SEQ ID NO:31. For example, the TCR may comprise a variable domain of the TCR α chain, which variable domain comprises a CDR1 containing SEQ ID NO:11, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16; and a variable domain of the TCR β chain, which variable domain comprises a CDR1 containing SEQ ID NO:24, a CDR2 containing SEQ ID NO:27, and a CDR3 containing SEQ ID NO:28. For example, the TCR may comprise a variable domain of the TCR α chain containing SEQ ID NO:20, and a variable domain of the TCR β chain containing SEQ ID NO:31. For example, the TCR may comprise a variable domain of the TCR α chain, which variable domain comprises a CDR1 containing SEQ ID NO:12, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16; and a variable domain of the TCR β chain, which variable domain comprises a CDR1 containing SEQ ID NO:24, a CDR2 containing SEQ ID NO:27, and a CDR3 containing SEQ ID NO:28. For example, the TCR may comprise a variable domain of the TCR α chain containing SEQ ID NO:21, and a variable domain of the TCR β chain containing SEQ ID NO:31. For example, the TCR may comprise a variable domain of the TCR α chain, which variable domain comprises a CDR1 containing SEQ ID NO:13, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16; and a variable domain of the TCR β chain, which variable domain comprises a CDR1 containing SEQ ID NO:24, a CDR2 containing SEQ ID NO:27, and a CDR3 containing SEQ ID NO:28. For example, the TCR may comprise a variable domain of the TCR α chain containing SEQ ID NO:22, and a variable domain of the TCR β chain containing SEQ ID NO:31.For example, the TCR may comprise a variable domain of the TCR α-chain, which variable domain comprises a CDR1 containing SEQ ID NO:14, a CDR2 containing SEQ ID NO:15, and a CDR3 containing SEQ ID NO:16; and a variable domain of the TCR β-chain, which variable domain comprises a CDR1 containing SEQ ID NO:24, a CDR2 containing SEQ ID NO:27, and a CDR3 containing SEQ ID NO:28. For example, the TCR may comprise a variable domain of the TCR α-chain containing SEQ ID NO:23, and a variable domain of the TCR β-chain containing SEQ ID NO:31. In any case, a signal peptide may be present at the N-terminus of the α-chain variable domain and / or the N-terminus of the β-chain variable domain. The α-chain signal peptide may comprise SEQ ID NO:38. The β-chain signal peptide may comprise SEQ ID NO:39.

[0056] The variable domain of the TCR α-chain and the variable domain of the TCR β-chain, each defined by its CDR sequence as described above, may further comprise one or more framework sequences. For example, the one or more framework sequences may be located (1) before CDR1, (2) between CDR1 and CDR2, (3) between CDR2 and CDR3, and / or (4) after CDR3. For example, the one or more framework sequences may be located at: (1); (2); (3); (4); (1) and (2); (1) and (3); (1) and (4); (2) and (3); (2) and (4); (3) and (4); (1), (2) and (3); (1), (2) and (4); (1), (3) and (4); (2), (3) and (4); or (1), (2), (3) and (4). In the variable domain of the TCR α-chain, the one or more framework sequences may, for example, have at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with one or more corresponding framework sequences in SEQ ID NO:17. In the variable domain of the TCR β-chain, the one or more framework sequences may, for example, have at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with one or more corresponding framework sequences in SEQ ID NO:29.

[0057] Constant domain

[0058] For example, the TCR may comprise a constant domain of the TCR α-chain. For example, the TCR may comprise a constant domain of the TCR β-chain. For example, the TCR may comprise a constant domain of the TCR α-chain and a constant domain of the TCR β-chain.

[0059] The TCR can include any TCR α-chain constant domain. For example, the TCR α-chain constant domain can include a TRAC constant domain sequence. For example, the TRAC constant domain sequence can include SEQ ID NO:2 or an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:2. SEQ ID NO:2 represents the native form of TRAC. For example, the TRAC constant domain sequence can include SEQ ID NO:32 or an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:32. SEQ ID NO:32 is a version of SEQ ID NO:2 without the N-terminal "N".

[0060] In another aspect of the present disclosure, the TRAC constant domain sequence can, for example, include a variant of SEQ ID NO:2 in which the threonine at position 48 is replaced by cysteine. That is, the TRAC constant domain sequence can include the T48C mutant of SEQ ID NO:2. For example, the TRAC constant domain sequence can include SEQ ID NO:3 or an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:3. The TRAC constant domain sequence can include SEQ ID NO:33 or an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:33. SEQ ID NO:33 is a version of SEQ ID NO:3 without the N-terminal "N".

[0061] In another aspect of the present disclosure, the TRAC constant domain sequence can encode a soluble version of TRAC. For example, the TRAC constant domain sequence can include SEQ ID NO:4 or an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:4. The TRAC constant domain sequence can include SEQ ID NO:34 or an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:34. SEQ ID NO:34 is a version of SEQ ID NO:4 without the N-terminal "N".

[0062] In one aspect of the present disclosure, the TRAC constant domain sequence can be modified, for example, by truncation or substitution to delete the native disulfide bond between Cys4 of TRAC exon 2 and Cys2 of TRBC1 or TRBC2 exon 2.

[0063] The various TCRα chain constant domains described above can be used interchangeably. That is, any TCRα chain constant domain can bind to any TCRα chain variable domain to form a TCRα chain.

[0064] The TCR can comprise any TCRβ chain constant domain structure. For example, the TCRβ chain constant domain can comprise a TRBC2 constant domain sequence. For example, the TRBC2 constant domain sequence can comprise SEQ ID NO:5 or an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:5. SEQ ID NO:5 represents the native form of TRBC2. For example, the TRBC2 constant domain sequence can comprise SEQ ID NO:35 or an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:35. SEQ ID NO:35 is a version of SEQ ID NO:5 without the N-terminal "E".

[0065] In another aspect of the present disclosure, the TRAC constant domain sequence can, for example, comprise a variant of SEQ ID NO:5 in which serine at position 57 is replaced by cysteine. That is, the TRBC2 constant domain sequence can comprise the S57C mutant of SEQ ID NO:5. For example, the TRBC2 constant domain sequence can comprise SEQ ID NO:6 or an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:6. The TRBC2 constant domain sequence can comprise SEQ ID NO:36 or an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:36. SEQ ID NO:36 is a version of SEQ ID NO:6 without the N-terminal "E".

[0066] In another aspect of the present disclosure, the TCR β-chain constant domain can be a soluble TCR β-chain constant domain. For example, the TCR β-chain constant domain can comprise a TRBC1 / 2 splice variant, such as SEQ ID NO:7 or an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:7. The TCR β-chain constant domain can comprise a TRBC1 / 2 splice variant, such as SEQ ID NO:37 or an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:37. SEQ ID NO:37 is a version of SEQ ID NO:7 without the N-terminal "E".

[0067] In one aspect of the present disclosure, the TCR β-chain constant domain (such as TCRB2 or TCRB1 / 2) sequence can be modified by truncation or substitution to remove the native disulfide bond between Cys4 of TRAC exon 2 and Cys2 of TRBC1 or TRBC2 exon 2.

[0068] In one aspect of the present disclosure, the TCR comprises a TCR α-chain constant domain with a T48C substitution and a TCR β-chain constant domain with an S57C substitution. The T48C substitution in the TCR α-chain and the S57C substitution in the β-chain are advantageous because it is these cysteines that form a disulfide bond between the α and β constant domains of the TCR. The addition of these cysteines enables the formation of a disulfide bond between the α and β constant domains of the TCR, thereby enhancing the preferential pairing of the α-chain and the β-chain (Blood. 2007 Mar 15;109(6):2331–2338).

[0069] The TCR according to the present disclosure can be, for example, an αβ heterodimer. An αβ heterodimer can be defined as a dimer comprising a TCR α-chain and a TCR β-chain. Generally, the TCR α-chain comprises a TCR α-chain variable domain and a TCR α-chain constant domain. Generally, the TCR β-chain comprises a TCR β-chain variable domain and a TCR β-chain constant domain. Thus, the TCR of the present disclosure can be an αβ heterodimer that comprises (a) a TCR α-chain comprising (i) a TCR α-chain variable domain as defined herein and (ii) a TCR α-chain constant domain; and (b) a TCR β-chain comprising (i) a TCR β-chain variable domain as defined herein and (ii) a TCR β-chain constant domain.

[0070] In another aspect of the present disclosure, the TCR can be a single-chain TCR having a Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, or Vα-L-Vβ-Cβ structure; wherein Vα is the variable domain of the TCR α-chain, Vβ is the variable domain of the TCR β-chain, Cα is the constant domain of the TCR α-chain, Cβ is the constant domain of the TCR β-chain, and L is a linker sequence.

[0071] In any case, the TCR of the present disclosure can exist in a form suitable for expression on the surface of T cells. Alternatively, the TCR of the present disclosure can be soluble. Using a stable, disulfide-linked soluble TCR as a reference TCR enables more convenient evaluation of binding affinity and binding half-life. For example, it is convenient to use a soluble TCR to provide measures of binding extent, binding affinity, specificity, and selectivity. As described in other parts, soluble TCRs can also be used for therapeutic purposes, such as targeting the delivery of cytotoxic agents conjugated to soluble TCRs to tumor sites.

[0072] The soluble property of the TCR can be conferred by the presence of a soluble TCR α-chain constant domain and / or a soluble TCR β-chain constant domain. Thus, a soluble TCR can comprise a soluble TCR α-chain constant domain. Soluble TCR α-chain constant domains are known in the art and are as described above. For example, a soluble TCR can comprise an α-chain constant domain that contains a sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:4. A soluble TCR can comprise a soluble TCR β-chain constant domain. Soluble TCR β-chain constant domains are known in the art and are as described above. For example, a soluble TCR can comprise a β-chain constant domain that contains a sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:7. A soluble TCR can comprise a soluble TCR α-chain constant domain and a soluble TCR β-chain constant domain. A soluble TCR can comprise an α-chain constant domain containing the sequence shown in SEQ ID NO:4 and a β-chain constant domain containing the sequence shown in SEQ ID NO:7.

[0073] Complex

[0074] The present disclosure also provides a complex comprising (a) the TCR of the present disclosure, and (b) a detectable label, therapeutic agent, or pharmacokinetic modification moiety.

[0075] For example, the detectable label can comprise a fluorescent label, radioactive label, enzyme, nucleic acid probe, or contrast agent.

[0076] For example, the therapeutic agent may comprise an immunomodulator, a radioactive compound, an enzyme (e.g., perforin), or a chemotherapeutic agent. Other suitable therapeutic agents include:

[0077] - Small molecule cytotoxic agents, i.e., compounds that are capable of killing mammalian cells and have a molecular weight of less than 700 daltons. These compounds may also contain toxic metals capable of producing a cytotoxic effect. In addition, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decompose or transform under physiological conditions to release a cytotoxic agent. Such agents include cisplatin, maytansine derivatives, calicheamicin, cactinomycin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, porfimer sodium photosensitizer II, temozolomide, topotecan, trimetrexate glucuronate, auristatin E, vincristine, and doxorubicin;

[0078] - Peptide cytotoxins, i.e., proteins or fragments thereof that are capable of killing mammalian cells. For example, ricin, diphtheria toxin, Pseudomonas exotoxin A, DNase, and RNase;

[0079] - Radionuclides, i.e., unstable isotopes of elements that emit one or more alpha particles, beta particles, or gamma rays upon decay. For example, iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and 213, actinium-225, and astatine-213; Chelating agents can be used to facilitate the binding of these radionuclides to high-affinity TCR or its multimer;

[0080] - Immunostimulants, i.e., immunoeffecter molecules that are capable of stimulating an immune response. For example, cytokines such as interleukin-2 (IL-2) and interferon gamma (IFN-γ),

[0081] - Superantigens and their mutants;

[0082] - TCR-HLA fusion proteins;

[0083] - Chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein;

[0084] - Antibodies or fragments thereof, including antibodies against T cell or natural killer (NK) cell determinants (e.g., anti-CD3,

[0085] anti-CD28, or anti-CD16 antibodies);

[0086] - Alternative protein scaffolds with antibody-like binding properties

[0087] - Complement activators;

[0088] - Xenoprotein domains, alloprotein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0089] For example, the pharmacokinetic modification moiety can confer pegylation characteristics. For example, the pharmacokinetic modification moiety can comprise polyethylene glycol (PEG).

[0090] In another aspect, the TCRs of the present disclosure can be included in a complex comprising multiple TCRs to form a multivalent TCR complex. A variety of human proteins contain multimerization domains that can be used to produce multivalent TCR complexes. For example, the tetramerization domain of p53 has been used to prepare tetramers of scFv antibody fragments, which exhibit longer serum residence times and significantly reduced dissociation rates compared to monomeric scFv fragments. (Willuda et al. (2001) J. Biol. Chem. 276(17) 14385 - 14392). Hemoglobin also has a tetramerization domain and may be suitable for such applications. The multivalent TCR complexes of the present disclosure can have enhanced binding ability to the HLA-A*02 complex SLLQHLIGL (SEQ ID NO:1) compared to the non-multimerized TCRs of the present disclosure. Thus, the TCR can form part of a multivalent TCR complex. Such multivalent TCR complexes are particularly suitable for tracking or targeting cells presenting a specific antigen in vitro or in vivo, and are also suitable as intermediates for producing other multivalent TCR complexes having such uses.

[0091] Post-translational modification

[0092] The TCRs of the present disclosure may undergo post-translational modification. Glycosylation is such a modification method, which involves covalently linking an oligosaccharide moiety to specific amino acids in the TCR chain. For example, asparagine residues or serine / threonine residues are known sites for oligosaccharide attachment. The glycosylation state of a particular protein depends on a variety of factors, including the protein sequence, protein conformation, and the availability of specific enzymes.

[0093] In addition, the glycosylation state (i.e., the type of oligosaccharide, the mode of covalent attachment, and the total number of attachment sites) can affect protein function. Therefore, it is generally desirable to control glycosylation when producing recombinant proteins. Controlled glycosylation has been used to improve antibody-based therapeutic drugs. (Jefferis R., Nat Rev Drug Discov. 2009 Mar; 8(3):226 - 34).

[0094] For the soluble TCRs of the present disclosure, glycosylation can be controlled in vivo by a specific cell line or in vitro by chemical modification. Such modifications are desirable because glycosylation can improve pharmacokinetic properties, reduce immunogenicity, and more closely mimic natural human proteins (Sinclair AM and Elliott S., Pharm Sci. 2005 Aug; 94(8):1626 - 35).

[0095] Binding characteristics

[0096] The TCR of the present disclosure is capable of binding to a complex comprising the PRAME peptide sequence SLLQHLIGL (SEQ ID NO:1) and HLA-A*02. This binding can be specific. Specificity refers to the binding strength between the TCR and its target antigen. Specificity can be described by the dissociation constant Kd, which represents the ratio of the bound state to the unbound state in the receptor-ligand system. Generally, the fewer different antigens that the TCR can bind to, other than SEQ ID NO:1, the stronger its binding specificity.

[0097] For example, the TCR can bind to a complex comprising SEQ ID NO:1 and HLA-A*02, and its dissociation constant (Kd) is between 0.01 μM and 100 μM, between 0.01 μM and 50 μM, between 0.01 μM and 20 μM, between 10 μM and 1000 μM, between 10 μM and 500 μM, or between 50 μM and 500 μM. For example, in a preferred aspect of the present disclosure, the TCR binds to a complex comprising SEQ ID NO:1 and HLA-A*02 with a Kd of 0.05 μM to 20.0 μM. For example, the Kd can be measured using surface plasmon resonance, optionally at 25°C, and optionally in a pH range of 6.5 to 6.9 or 7.0 to 7.5. The dissociation constant Kd or koff / kon can be determined by experimentally measuring the dissociation rate constant koff and the binding rate constant kon. The TCR dissociation constant can be measured using a soluble form of the TCR, wherein the TCR comprises the variable domain of the TCRα chain and the variable domain of the TCRβ chain.

[0098] The potency of the TCR can be quantified by measuring the degree of activation of T cells expressing the TCR upon contact with antigen-presenting cells presenting the target antigen (in the present disclosure, the target antigen is the complex comprising SEQ ID NO:1 and HLA-A*02). The TCR-expressing T cells disclosed herein can specifically bind to antigen-presenting cells (APCs) pulsed with a peptide segment comprising the sequence shown in SEQ ID NO:1 and expressing HLA-A*02. In certain aspects of the present disclosure, the TCR potency can be quantified by co-culturing T cells and APCs at an effector cell to target cell ratio of 1:1. In some aspects of the present disclosure, the half maximal effective concentration (EC50) can be determined by measuring the presence of CD69 on T cells. CD69 is an activation marker of T cells (Simms et al., Clinical and Diagnostic Laboratory Immunology, May 1996, p. 301–304; Maino et al., Cytometry 1995, 20:127-133; Abraham RT, Weiss A., Nat Rev Immunol., 2004, pp. 301-8). In some aspects of the present disclosure, the EC50 is determined by the CD4+ T cell population. Example 1 describes an exemplary CD69 T cell activation assay. The EC50 of the TCR can be, for example, less than 1 mM, less than 100 μM, less than 50 μM, less than 40 μM, less than 30 μM, less than 20 μM, less than 10 μM, less than 5 μM, less than 1 μM, less than 100 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 100 pM, less than 50 pM, less than 40 pM, less than 30 pM, less than 20 pM, less than 10 pM, less than 5 pM, or less than 1 pM. In certain cases, the EC50 of the TCR is less than 20 nM. In certain cases, the EC50 of the TCR is between 1 pM and 1 mM, such as 5 pM to 100 μM, 10 pM to 50 μM, 20 pM to 40 μM, 30 pM to 30 μM, 40 pM to 20 μM, 50 pM to 10 μM, 100 pM to 5 μM, 1 nM to 1 μM, 5 nM to 100 nM, 10 nM to 50 nM, or 20 nM to 40 nM. In certain cases, the EC50 of the TCR is between 50 pM and 20 nM.

[0099] Nucleic Acid

[0100] The present disclosure provides a nucleic acid encoding any TCR in the present disclosure. This nucleic acid can encode the TCR in a single open reading frame. That is, this nucleic acid can encode the variable domain of the α chain and the variable domain of the β chain in a single open reading frame (ORF). This nucleic acid can also encode (a) the constant domain of the α chain and / or (b) the constant domain of the β chain in this single open reading frame (ORF). In other words, this nucleic acid can encode the TCR α chain and the TCR β chain in this single open reading frame (ORF).

[0101] Alternatively, this nucleic acid can comprise (i) a first open reading frame encoding the TCR α chain comprising the variable domain of the TCR α chain and optionally the constant domain of the TCR α chain; and (ii) a second open reading frame encoding the TCR β chain comprising the variable domain of the TCR β chain and optionally the constant domain of the TCR β chain. That is, this nucleic acid can encode the variable domain of the α chain (and optionally the constant domain of the α chain) and the variable domain of the β chain (and optionally the constant domain of the β chain) in different open reading frames (ORFs). In other words, this nucleic acid can encode the TCR α chain and the TCR β chain in different open reading frames.

[0102] The present disclosure also provides a nucleic acid encoding the variable domain of the TCR α chain of any TCR in the present disclosure. This nucleic acid can also encode the constant domain of the TCR α chain. The constant domain of the TCR α chain can be any constant domain of the TCR α chain, such as the constant domain of the TCR α chain described herein.

[0103] The present disclosure also provides a nucleic acid encoding the variable domain of the TCR β chain of any TCR in the present disclosure. This nucleic acid can also encode the constant domain of the TCR β chain. The constant domain of the TCR β chain can be any constant domain of the TCR β chain, such as the constant domain of the TCR β chain described herein.

[0104] In any case, this nucleic acid can be isolated. This nucleic acid can be non-naturally occurring. This nucleic acid can be non-naturally occurring and purified. This nucleic acid can be non-naturally occurring and engineered. This nucleic acid can be non-naturally occurring and recombinant.

[0105] This nucleic acid can be DNA, such as cDNA. This nucleic acid can be RNA.

[0106] Vector

[0107] Also provided is a vector that can comprise the nucleic acid of the present disclosure.

[0108] This vector can be an expression vector and can also comprise regulatory elements for the expression of the product encoded by the nucleic acid of the present disclosure.

[0109] Viral Particle

[0110] The present disclosure also provides a viral particle comprising the nucleic acid or vector of the present disclosure. The viral particle may be suitable for transducing a sequence encoding a TCR, TCR α-chain, TCR β-chain, variable domain of TCR α-chain or variable domain of TCR β-chain described herein into a cell (e.g., a T cell).

[0111] For example, the viral particle may be a retroviral particle or a lentiviral particle.

[0112] The viral particle for transduction can be prepared according to known methods. For example, HEK293T cells can be transfected with a plasmid encoding viral packaging and envelope elements and a lentiviral vector containing the nucleic acid of the present disclosure. A VSVg pseudotyped viral vector containing the nucleic acid of the present disclosure can be produced in combination with the viral envelope glycoprotein G (VSVg) of vesicular stomatitis virus, thereby producing pseudotyped viral particles.

[0113] Cell

[0114] The present disclosure provides a cell expressing the TCR of the present disclosure.

[0115] The present disclosure also provides a cell comprising at least one nucleic acid of the present disclosure. The cell may comprise (i) a first nucleic acid of the present disclosure encoding the variable domain of TCR α-chain and optionally the constant domain of TCR α-chain of the TCR of the present disclosure, and (ii) a second nucleic acid of the present disclosure encoding the variable domain of TCR β-chain and optionally the constant domain of TCR β-chain of the TCR of the present disclosure. Thus, the cell can be a cell transduced with one or more nucleic acids that together encode the TCR of the present disclosure.

[0116] The present disclosure also provides a cell comprising at least one vector of the present disclosure. The cell may comprise (i) a first vector comprising a nucleic acid of the present disclosure encoding the variable domain of TCR α-chain of the TCR of the present disclosure and optionally the constant domain of TCR α-chain; and (ii) a second vector comprising a nucleic acid of the present disclosure encoding the variable domain of TCR β-chain of the TCR of the present disclosure and optionally the constant domain of TCR β-chain. Thus, the cell can be a cell transduced with one or more vectors that together encode the TCR of the present disclosure.

[0117] In any case, the cell can be a separated cell. The cell can be a non-naturally occurring cell. In other words, the cell can be an engineered cell.

[0118] The cell can be a T cell. The T cell can be a CD4+ T cell. That is, the T cell can be a T cell expressing an endogenous CD4 co-receptor. The T cell can be a CD8+ T cell. That is, the T cell can be a T cell expressing an endogenous CD8 co-receptor.

[0119] In any case, the T cell can express a heterologous CD8 co-receptor. In the context of the present disclosure, the term "heterologous" refers to a polypeptide or nucleic acid that is foreign to a particular biological system (such as a T cell), i.e., not naturally present within that system. A "heterologous" polypeptide or nucleic acid can be introduced into the system by artificial or recombinant means. Thus, heterologous expression of a TCR may alter the specificity of the T cell. Heterologous expression of the CD8 co-receptor can confer functions associated with that CD8 co-receptor to the T cell. For example, expression of a heterologous CD8 co-receptor can confer enhanced avidity and / or affinity of the T cell for PRAME, and / or an enhanced degree of activation upon binding to PRAME. Methods for measuring avidity, affinity, and T cell activation are well known in the art. Expression of a heterologous CD8 co-receptor can confer enhanced or increased CD40L expression, cytokine production, cytotoxic activity, induction of dendritic cell maturation, or induction of cytokine production by dendritic cells, e.g., in response to antigen (PRAME) binding. The improvement or enhancement may be relative to modified T cells that contain a heterologous TCR that binds to PRAME but lack the heterologous CD8 co-receptor.

[0120] The cells of the present disclosure have utility in adoptive therapy. For example, the cells of the present disclosure are suitable for cancer treatment based on adoptive therapy.

[0121] The cell can be allogeneic. For example, the cell can be allogeneic relative to the individual to whom the cell is to be administered. The cell can be autologous. For example, the cell can be autologous relative to the individual to whom the cell is to be administered. Autologous cells can be prepared by modifying endogenous cells obtained from an individual, such as peripheral blood mononuclear cells (PBMC). Methods of modification are known in the art.

[0122] The cells of the present disclosure can further comprise one or more exogenous or recombinant co-stimulatory ligands. For example, the cell can be transduced with one or more exogenous or recombinant co-stimulatory ligands.

[0123] The cell can comprise two or more, three or more, or four or more exogenous or recombinant co-stimulatory ligands.

[0124] The cell can express the one or more exogenous or recombinant co-stimulatory ligands. The cell can co-express (a) a TCR of the present disclosure, a product encoded by a nucleic acid of the present disclosure, or a product encoded by a vector of the present disclosure, and (b) one or more exogenous or recombinant co-stimulatory ligands. The combination of (a) and (b) can facilitate providing non-antigen-specific signals and cell antigen-specific activation.

[0125] Costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulating the acute-phase response. Its main function is to regulate immune cells. Members of the TNF superfamily have several common features. Most members of the TNF superfamily are synthesized as type II transmembrane proteins (extracellular C-terminus), which contain a short cytoplasmic segment and a relatively long extracellular region. Members of the TNF superfamily include, but are not limited to: nerve growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, TNF-α, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153), tumor necrosis factor β (TNFb) / lymphotoxin-α (LTa), lymphotoxin-β (LTβ), CD257 / B cell-activating factor (BAFF) / BLyS / THANK / Tall-1, glucocorticoid-induced TNF receptor ligand (GITRL), TNF-related apoptosis-inducing ligand (TRAIL), and LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins that participate in the processes of cell recognition, binding, or adhesion. These proteins have similar structural features to immunoglobulins - they possess an immunoglobulin domain (fold). Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both of which are ligands for CD28. In certain embodiments, the at least one costimulatory ligand is selected from the group consisting of 4-1BBL, CD275, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, and combinations thereof.

[0126] In one aspect, the exogenous or recombinant costimulatory ligand may comprise 4-1BBL or CD80. In one aspect, the exogenous or recombinant costimulatory ligand may be 4-1BBL. In one aspect, the exogenous or recombinant costimulatory ligand may be 4-1BBL and CD80.

[0127] In one aspect, the one or more exogenous or recombinant costimulatory ligands comprise cyclic adenosine monophosphate phosphodiesterase. In one aspect, the one or more exogenous or recombinant costimulatory ligands comprise a transforming growth factor β (TGFβ) receptor, such as a dominant-negative TGFβ receptor type II.

[0128] The cell can be designed to release cytokines that have a positive effect on its cytolytic activity. Such cytokines include, but are not limited to, interleukin-7, interleukin-15, and interleukin-21.

[0129] Pharmaceutical Composition

[0130] Also provided is a pharmaceutical composition comprising the TCR of the present disclosure, the nucleic acid of the present disclosure, or the cell of the present disclosure. The pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients. Suitable carriers and excipients include, for example, water, saline, glucose, glycerol, etc., and combinations thereof. Additionally, if desired, the pharmaceutical composition may contain small amounts of auxiliary substances such as wetting agents or emulsifying agents, and / or pH buffering agents.

[0131] The pharmaceutical composition may be provided in unit dosage forms. The pharmaceutical composition may be provided in a sealed container or as part of a kit. The kit may include instructions for use. The kit may include multiple said unit dosage forms.

[0132] Cancer Treatment

[0133] The present disclosure provides the TCR, nucleic acid, cell, or pharmaceutical composition of the present disclosure for use in a method of treating a disease in an individual, the method comprising administering to the individual the TCR, nucleic acid, cell, or pharmaceutical composition. The present disclosure also provides a method of treating a disease in an individual, comprising administering to the individual the TCR, nucleic acid, cell, or pharmaceutical composition of the present disclosure.

[0134] Since PRAME is a cancer antigen, the disease may for example be cancer. For example, the disease may be a solid tumor. For example, the disease may be selected from melanoma, leukemia, lymphoma, lung cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, bladder cancer, and head and neck cancer. For example, the lung cancer may be non-small cell lung cancer or small cell lung cancer. For example, the breast cancer may be triple-negative breast cancer.

[0135] The cancer may be a cancer expressing PRAME. "Cancer expressing PRAME" refers to any cancer in which cancer cells exhibit PRAME antigen expression. Such cancers include any of those listed above.

[0136] Preferably, the individual is a human. Non-human mammals, especially mammals commonly used as human therapeutic efficacy verification models (such as murine, primate, porcine, canine, or rabbit animals), may still be treated using the above method.

[0137] Treatment can be aimed at achieving a therapeutic effect. For example, the therapeutic effect can include inhibiting or delaying disease progression. For example, the therapeutic effect can include reducing the rate of disease progression; halting disease progression; improving the disease condition; curing or alleviating the disease (partially or completely); preventing, delaying, alleviating, or inhibiting one or more symptoms and / or signs of the disease, and / or extending the survival period of the subject or patient beyond what would be expected without treatment or compared to a control or control individual, for example. The therapeutic effect can be measured by, for example, TTP (time to progression), OS (overall survival), or PFS (progression-free survival).

[0138] For example, the therapeutic effect can be an anti-cancer effect. The anti-cancer effect can include reducing the tumor growth rate, shrinking the tumor volume, reducing the number of tumor cells, reducing the number of metastatic lesions, extending the life expectancy, or improving various physiological symptoms associated with the cancer condition. This can be measured in vivo or in vitro, optionally compared to a control or control individual treated with a vehicle or untreated.

[0139] Treatment can be aimed at achieving a preventive effect. For example, individuals susceptible or at risk of the occurrence or recurrence of a disease (such as cancer) can be treated as described herein. Such treatment can prevent or delay the occurrence or recurrence of the disease in the individual, optionally compared to a control group or control individual, for example.

[0140] Administration can be carried out by any convenient route. For example, administration can be parenteral, such as by infusion; intravenous; or subcutaneous. Infusion can involve the administration of a therapeutic agent through a needle or catheter.

[0141] Generally, cells such as T cells are infused intravenously or subcutaneously, although T cells can also be infused by other non-oral routes, such as intramuscular injection and the epidural route. Suitable infusion techniques are known in the art and commonly used in treatment (see, for example, Rosenberg et al., New Eng. J. of Med., 319:1676, 1988).

[0142] The therapeutic agent is administered in a manner compatible with its dosage form and in an amount that is therapeutically effective. The dosage administered depends on the subject being treated, the disease being treated, and the ability of the individual's immune system. The precise dosage of the therapeutic agent required may depend on the judgment of the physician and may vary from individual to individual. Determining the optimal dosage generally involves weighing the level of therapeutic benefit against any risks or harmful side effects of the treatment. The selected dosage level will depend on a variety of factors, including but not limited to: the activity of the particular therapeutic agent, the route of administration, the time of administration, the rate of loss or inactivation of the therapeutic agent, the duration of treatment, other drugs, compounds, and / or materials used in combination, and the age, sex, weight, physical condition, general health, and medical history of the patient.

[0143] When the therapeutic agent comprises T cells, for example, about 10 5 to about 10 10 T cells may be administered per kilogram of body weight, such as 2×10 5 to 2×10 10 cells per individual. For example, the administration time may last about 30 minutes and the treatment may be repeated as needed. The treatment may be repeated every few days to weeks. The appropriate dosage of T cells and compositions comprising T cells may vary among patients.

[0144] The method may comprise administering to the individual another therapeutic agent. The characteristics of the other therapeutic agent will depend on the disease being treated. Any known therapeutic agent for the disease may be used in the method. When the disease is cancer, the other therapeutic agent may be, for example, a chemotherapeutic agent or an immunomodulator. For example, the immunomodulator may comprise an antibody, an immune cell, or a checkpoint inhibitor.

[0145] Method for Preparing Cells

[0146] Also provided is a method of preparing the cells of the present disclosure. The method includes introducing into the cells at least one nucleic acid of the present disclosure or at least one vector of the present disclosure. The cells may be any suitable cell type. For example, the cells may be the cells as described above. Preferably, the cells are T cells. For example, the at least one nucleic acid or at least one vector may be contained in a viral particle. The viral particle is as described above.

[0147] The method may further comprise culturing the cells to proliferate the cells to form a population. Suitable culture conditions are well known in the art and are applicable to a variety of cell types, such as T cells. The method also includes separating or purifying the cells of the present disclosure from the resulting population. Methods for separating and purifying the desired cells from a population are well known in the art and include, for example, magnetic cell sorting and fluorescence-activated cell sorting.

[0148] Example

[0149] Example 1

[0150] Jurkat CD69 T cell activation assay

[0151] The Jurkat cell line and its derivative cell lines were widely used at the end of the 20th century to elucidate the major components of the T-cell signaling pathway. CD69 is a C-type lectin and is often used as an early activation marker for Jurkat cells and primary T cells - it is rapidly upregulated after TCR signaling and can be easily detected by flow cytometry. Transgenic TCRs can be expressed in Jurkat cells by transducing the TCR transgene using lentiviral particles as vectors. Antigen-presenting cells suitable for activating TCR-transduced Jurkat cells include cell lines expressing homologous HLA alleles and the target antigen, as well as HLA-A*02 pulsed with exogenous targets + , the TAP-deficient LCL-B / T cell hybrid line "T2".

[0152] TCR-transduced Jurkat cells were co-cultured with T2 cells pulsed with 10-fold serial dilutions of the PRAME_SLLQHLIGL peptide (concentration from 1×10 -5 M to 1×10 -12 M) at a 1:1 ratio for 16 hours. The co-cultures were stained with an antibody mixture to detect the TCR-transduced Jurkat cell population and any expression of the CD69 activation marker. Samples were acquired using a MACSQuant 10X flow cytometer (Miltenyi Biotec), and the data were analyzed using FlowJo v10.6.2 (BD). Peptide titration data were curve-fitted using a four-parameter logistic (agonist) vs. response non-linear regression model with GraphPad Prism v8.2.0 (GraphPad Software, Inc), and the best-fit value of EC 50 was obtained; this value represents the peptide concentration required to induce half-maximal activation of Jurkat cells (CD69 + cell percentage). The results are shown in Figure 1 .

[0153] References:

[0154] 1. Jurkat T cells and development of the T-cell receptor signalling paradigm. Abraham RT, Weiss A. 2004, Nat Rev Immunol., pp. 301-8.

[0155] Example 2

[0156] IFNγ ELISpot assay

[0157] The activation of a group of TCR-transduced donor T cells was studied by measuring the local IFNγ secretion captured by antibodies in the ELISpot assay. Briefly, the capture antibody was coated on a 96-well ELISpot plate and incubated at 4 °C for 3 days. On the day of assay setup, the unbound antibody was removed and the plate was blocked with R10 medium at room temperature for 2 hours. Next, 50,000 T cells were co-incubated with 50,000 target cells (or without target cells) at 37 °C in 5% CO2 by volume, with both concentrations at 1×10 6 / mL for 19 hours. Target cells included PRAME-positive cell lines: Malme-3M and SK-Mel-5 (melanoma); NCI-H1755 and NCI-H1703 (lung cancer); and 2 PRAME-negative (or negligible) tumor cell lines: NCI-H2228 (lung cancer), TCC-SUP (bladder cancer). As controls, target cells without added T cells and T cells only (without targets) were monitored. The plate was washed once in a plate washer to develop the color, and the detection antibody was added to each well. After incubation at room temperature for 2 hours, the plate was washed again, and streptavidin-HRP was added to each well. After continued incubation at room temperature for 1 hour, the plate was washed one last time. The AEC (3-amino-9-ethylcarbazole) chromogenic substrate was added to each well, and when the spots became detectable, the plate was washed with water to terminate the reaction. Subsequently, the plate was dried, the signal was read using a CTL Analyzer microplate reader (Series 6Ultra V), and analyzed using ImmunoCapture 6.5.8 software. The results are as Figure 2 shown.

[0158] Example 3

[0159] Two-dimensional killing experiment

[0160] The IncuCyte ZOOM live cell imaging system (Sartorius) was used to measure the number of target cells undergoing apoptosis over time, thereby evaluating the cytotoxicity of TCR T cells against target cells in co-cultures. One day prior to adding 50,000 T cells / well, Malme-3M (human melanoma) target cells were seeded at 20,000 cells / well in 96-well plates. IncuCyte Caspase-3 / 7 green dye (Sartorius) was used to measure the green fluorescence emitted after cleavage by caspase 3 / 7 released during apoptosis, and quantitatively determine the apoptosis of target cells induced by activated T cells. Untransduced T cells were used as a control for background cell death levels, and separate Malme-3M target cells (without T cells) were also monitored in the same way. Image acquisition and analysis were performed using IncuCyte ZOOM GUI software version 2018A (Sartorius). During image analysis, apoptotic (green fluorescent) T cells were excluded by size exclusion. After adding T cells, the plates were imaged every 3 hours for approximately 7 days. The data plots show the number of green fluorescent objects (apoptotic cells) per mm over time in hours 2 ( Figure 3 )。

[0161] Example 4

[0162] To further evaluate the functional avidity and specificity of the potency-enhanced engineered TCRs, we conducted studies. To assess the potency of the TCR against target cells expressing PRAME, HLA-A*02-positive, PRAME-negative T2 target cells were pulsed with 10-fold serial dilutions of the PRAME peptide SLLQHLIGL (SEQ ID NO:1) (from 1×10 -5 M to 1×10 -11 M). Primary T cells were transduced with one of the engineered TCRs provided herein and co-cultured with T2 target cells at a 1:1 ratio for 48 hours. Potency was measured by IFNγ supernatant ELISA. The results are provided in Figure 4 and Table 2. EC 50 values are shown in Table 2 and represent the peptide concentration required to induce T-cell activation to half-maximum, with values ranging from 10 -8.8 M to 10 -7.8 M。

[0163] Table 2: EC 50 values

[0164] TCR <![CDATA[EC 50 > ADB02780_25 -7.8 ADB02780_62 -8.7 ADB02780_63 -8.8 ADB02780_70 -8.8 ADB02780_77 -8.7 ADB02780_106 -7.8 ADB02780_107 -7.8

[0165] To evaluate the specificity for PRAME, the potency of engineered TCRs against cell lines with different PRAME expression levels was studied using IFNγ supernatant ELISA. The results are provided in Figure 5 T cells from 4 different donors were transduced with engineered TCRs and incubated with the tumor cell lines shown below the bottom panel, which are arranged in ascending order of PRAME transcript levels (measured by qPCR) from left to right.

[0166] Overall, as shown in Figure 5 regardless of which engineered TCR was transduced (although the potencies of different TCRs varied), the T cell responses were strongest against the tumor cell line with the highest PRAME expression level. Three cell lines with PRAME expression levels ranging from negligible to undetectable (<1000 transcript copies / 10 6 reference genes) were not recognized by any of the candidate TCRs in the absence of peptide. All cell lines triggered the activation of T cells transduced to express ADB02780_070 TCR in the presence of the PRAME_SLLQHLIGL peptide (positive control).

[0167] To further verify the specificity of the engineered TCRs, T cells from four different donors were transduced with engineered TCRs, and their responses against antigen-negative primary cell lines were detected using IFNγ supernatant ELISA. The results are provided in Figure 6 The transcript levels of different primary cell lines (measured by qPCR) are shown in the bottom panel. As shown in the top four panels, all target cells had the ability to present antigen, which was confirmed by the activation of ADB02780_070 TCR in the presence of exogenous PRAME_SLLQHLIGL peptide (open circles, positive control). However, in the absence of the PRAME_SLLQHLIGL peptide, no significant T cell responses were observed against any PRAME-negative primary cell lines, thus confirming the specificity of the engineered TCRs for the PRAME antigen.

[0168] Sequence Listing

[0169] SEQ ID NO:1

[0170] PRAME peptide

[0171] SLLQHLIGL

[0172] SEQ ID NO:2

[0173] Natural form of the alpha chain constant region (TRAC)

[0174] NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0175] SEQ ID NO:3

[0176] Alpha chain constant region (TRAC) with T48C mutation

[0177] NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0178] SEQ ID NO:4

[0179] Soluble form of alpha constant region (TRAC)

[0180] NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS

[0181] SEQ ID NO:5

[0182] Natural form of beta chain constant region (TRBC2)

[0183] EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0184] SEQ ID NO:6

[0185] Beta chain constant region (TRBC2) with S57C mutation

[0186] EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0187] SEQ ID NO:7

[0188] β constant domain soluble form (TRBC1 / 2 splice variant)

[0189] EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD

[0190] SEQ ID NO:8

[0191] α-chain CDR1 ADB02780_001, ADB02780_022, ADB02780_025

[0192] SIFNT

[0193] SEQ ID NO:9

[0194] α-chain CDR1 ADB02780_062

[0195] SIFLT

[0196] SEQ ID NO:10

[0197] α-chain CDR1 ADB02780_063

[0198] SIFET

[0199] SEQ ID NO:11

[0200] α-chain CDR1 ADB02780_070

[0201] SIFVT

[0202] SEQ ID NO:12

[0203] α-chain CDR1 ADB02780_077

[0204] SIFHT

[0205] SEQ ID NO:13

[0206] α-chain CDR1 ADB02780_0106

[0207] SIFAT

[0208] SEQ ID NO:14

[0209] α-chain CDR1 ADB02780_107

[0210] SIFMT

[0211] SEQ ID NO:15

[0212] α-chain CDR2

[0213] LYKAGEL

[0214] SEQ ID NO:16

[0215] α-chain CDR3

[0216] CAGQASGTYKYIF

[0217] SEQ ID NO:17

[0218] α-chain variable region ADB02780_001, ADB02780_022, ADB02780_025

[0219] GQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGQASGTYKYIFGTGTRLKVLA

[0220] SEQ ID NO:18

[0221] α-chain variable region ADB02780_062

[0222] GQQLNQSPQSMFIQEGEDVSMNCTSSSIFLTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGQASGTYKYIFGTGTRLKVLA

[0223] SEQ ID NO:19

[0224] Variable domain of α chain ADB02780_063

[0225] GQQLNQSPQSMFIQEGEDVSMNCTSSSIFETWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGQASGTYKYIFGTGTRLKVLA

[0226] SEQ ID NO:20

[0227] Variable domain of α chain ADB02780_070

[0228] GQQLNQSPQSMFIQEGEDVSMNCTSSSIFVTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGQASGTYKYIFGTGTRLKVLA

[0229] SEQ ID NO:21

[0230] Variable domain of α chain ADB02780_077

[0231] GQQLNQSPQSMFIQEGEDVSMNCTSSSIFHTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGQASGTYKYIFGTGTRLKVLA

[0232] SEQ ID NO:22

[0233] Variable domain of α chain ADB02780_0106

[0234] GQQLNQSPQSMFIQEGEDVSMNCTSSSIFATWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGQASGTYKYIFGTGTRLKVLA

[0235] SEQ ID NO:23

[0236] Variable domain of α chain ADB02780_107

[0237] GQQLNQSPQSMFIQEGEDVSMNCTSSSIFMTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGQASGTYKYIFGTGTRLKVLA

[0238] SEQ ID NO:24

[0239] β-chain CDR1

[0240] MNHEY

[0241] SEQ ID NO:25

[0242] β-chain CDR2 ADB02780_001

[0243] SMNVEV

[0244] SEQ ID NO:26

[0245] β-chain CDR2 ADB02780_022

[0246] SLNVEV

[0247] SEQ ID NO:27

[0248] β-chain CDR2 ADB02780_025, ADB02780_062, ADB02780_063, ADB02780_070, ADB02780_077, ADB02780_0106, ADB02780_107

[0249] SINVEV

[0250] SEQ ID NO:28

[0251] β-chain CDR3

[0252] CASSHGTSGEQYF

[0253] SEQ ID NO:29

[0254] β-chain variable region ADB02780_001

[0255] EAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSHGTSGEQYFGPGTRLTVT

[0256] SEQ ID NO:30

[0257] Variable domain of the β-chain, ADB02780_022

[0258] EAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSLNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSHGTSGEQYFGPGTRLTVT

[0259] SEQ ID NO:31

[0260] Variable domains of the β-chain, ADB02780_025, ADB02780_062, ADB02780_063, ADB02780_070, ADB02780_077, ADB02780_0106, ADB02780_107

[0261] EAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSINVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSHGTSGEQYFGPGTRLTVT

[0262] SEQ ID NO:32

[0263] Alternative α-chain constant domain, natural form (TRAC)

[0264] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0265] SEQ ID NO:33

[0266] Alternative α-chain constant domain (TRAC) with T48C mutation

[0267] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0268] SEQ ID NO:34

[0269] Alternative α constant domain soluble form (TRAC)

[0270] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS

[0271] SEQ ID NO:35

[0272] Alternative β-chain constant domain native form (TRBC2)

[0273] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0274] SEQ ID NO:36

[0275] Alternative β-chain constant domain with S57C mutation (TRBC2)

[0276] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0277] SEQ ID NO:37

[0278] Alternative β constant domain soluble form (TRBC1 / 2 splice variant)

[0279] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD

[0280] SEQ ID NO:38

[0281] Variable domain signal peptide of alpha chain

[0282] MLLEHLLIILWMQLTWVS

[0283] SEQ ID NO:39

[0284] Variable domain signal peptide of beta chain

[0285] MGPQLLGYVVLCLLGAGPL。

Claims

1. A T cell receptor (TCR) that binds to a complex comprising SEQ ID NO:1 and HLA-A*02 and comprises: (a) A variable domain of the TCR α chain comprising an amino acid sequence having at least 50% sequence identity with SEQ ID NO:17; and (b) A variable domain of the TCR β chain comprising an amino acid sequence having at least 50% sequence identity with SEQ ID NO:

29.

2. The TCR according to claim 1, wherein: (a) The variable domain of the TCR α chain comprises an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO:17; and / or (b) The variable domain of the TCR β chain comprises an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 90% or at least 95% sequence identity with SEQ ID NO:

29.

3. The TCR according to claim 1 or 2, wherein: (i) The variable domain of the TCR α chain comprises CDR1, and CDR1 comprises (1) the sequence shown in SEQ ID NO:8 or (2) an amino acid sequence comprising one, two or three amino acid insertions, deletions or substitutions relative to the sequence shown in SEQ ID NO:8; (ii) The variable domain of the TCR α chain comprises CDR2, and CDR2 comprises (1) the sequence shown in SEQ ID NO:15 or (2) an amino acid sequence comprising one, two or three amino acid insertions, deletions or substitutions relative to the sequence shown in SEQ ID NO:15; (iii) The variable domain of the TCR α chain comprises CDR3, and CDR3 comprises (1) the sequence shown in SEQ ID NO:16 or (2) an amino acid sequence comprising one, two or three amino acid insertions, deletions or substitutions relative to the sequence shown in SEQ ID NO:16; (iv) The variable domain of the TCR β chain comprises CDR1, and CDR1 comprises (1) the sequence shown in SEQ ID NO:24 or (2) an amino acid sequence comprising one, two or three amino acid insertions, deletions or substitutions relative to the sequence shown in SEQ ID NO:24; (v) The variable domain of the TCR β chain comprises CDR2, and CDR2 comprises (1) the sequence shown in SEQ ID NO:25 or (2) an amino acid sequence comprising one, two or three amino acid insertions, deletions or substitutions relative to the sequence shown in SEQ ID NO:25; and / or (vi) The variable domain of the TCR β chain comprises CDR3, and CDR3 comprises (1) the sequence shown in SEQ ID NO:28 or (2) an amino acid sequence comprising one, two or three amino acid insertions, deletions or substitutions relative to the sequence shown in SEQ ID NO:

28.

4. The TCR according to any one of the preceding claims, wherein: (i) The variable domain of the TCR α chain comprises a CDR1 containing SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14; (ii) The variable domain of the TCR α chain comprises a CDR2 containing SEQ ID NO: 15; (iii) The variable domain of the TCR α chain comprises a CDR3 containing SEQ ID NO: 16; (iv) The variable domain of the TCR β chain comprises a CDR1 containing SEQ ID NO: 24; (v) The variable domain of the TCR β chain comprises a CDR2 containing SEQ ID NO: 25, 26 or 27; and / or (vi) The variable domain of the TCR β chain comprises a CDR3 containing SEQ ID NO:

28.

5. The TCR according to any one of the preceding claims, wherein the variable domain of the TCR α chain comprises: (i) a CDR1 comprising SEQ ID NO: 8, a CDR2 comprising SEQ ID NO: 15, and a CDR3 comprising SEQ ID NO: 16; (ii) a CDR1 comprising SEQ ID NO: 9, a CDR2 comprising SEQ ID NO: 15, and a CDR3 comprising SEQ ID NO: 16; (iii) a CDR1 comprising SEQ ID NO: 10, a CDR2 comprising SEQ ID NO: 15, and a CDR3 comprising SEQ ID NO: 16; (iv) a CDR1 comprising SEQ ID NO: 11, a CDR2 comprising SEQ ID NO: 15, and a CDR3 comprising SEQ ID NO: 16; (v) a CDR1 comprising SEQ ID NO: 12, a CDR2 comprising SEQ ID NO: 15, and a CDR3 comprising SEQ ID NO: 16; (vi) a CDR1 comprising SEQ ID NO: 13, a CDR2 comprising SEQ ID NO: 15, and a CDR3 comprising SEQ ID NO: 16; or (vii) a CDR1 comprising SEQ ID NO: 14, a CDR2 comprising SEQ ID NO: 15, and a CDR3 comprising SEQ ID NO:

16.

6. The TCR according to any one of the preceding claims, wherein the variable domain of the TCR α chain comprises SEQ ID NO: 17, 18, 19, 20, 21, 22 or 23.

7. The TCR according to any one of the preceding claims, wherein the variable domain of the TCR β chain comprises: (i) a CDR1 comprising SEQ ID NO: 24, a CDR2 comprising SEQ ID NO: 25, and a CDR3 comprising SEQ ID NO: 28; (ii) a CDR1 comprising SEQ ID NO: 24, a CDR2 comprising SEQ ID NO: 26, and a CDR3 comprising SEQ ID NO: 28; or (iii) a CDR1 comprising SEQ ID NO:24, a CDR2 comprising SEQ ID NO:27, and a CDR3 comprising SEQ ID NO:

28.

8. The TCR according to any one of the preceding claims, wherein the variable domain of the TCR β chain comprises SEQ ID NO:29, 20 or 31.

9. The TCR according to any one of the preceding claims, wherein: (i) the variable domain of the TCR α chain comprises SEQ ID NO:17, and the variable domain of the TCR β chain comprises SEQ ID NO:29; (ii) the variable domain of the TCR α chain comprises SEQ ID NO:17, and the variable domain of the TCR β chain comprises SEQ ID NO:30; (iii) the variable domain of the TCR α chain comprises SEQ ID NO:17, and the variable domain of the TCR β chain comprises SEQ ID NO:31; (iv) the variable domain of the TCR α chain comprises SEQ ID NO:18, and the variable domain of the TCR β chain comprises SEQ ID NO:31; (v) the variable domain of the TCR α chain comprises SEQ ID NO:19, and the variable domain of the TCR β chain comprises SEQ ID NO:31; (vi) the variable domain of the TCR α chain comprises SEQ ID NO:20, and the variable domain of the TCR β chain comprises SEQ ID NO:31; (vii) the variable domain of the TCR α chain comprises SEQ ID NO:21, and the variable domain of the TCR β chain comprises SEQ ID NO:31; (viii) the variable domain of the TCR α chain comprises SEQ ID NO:22, and the variable domain of the TCR β chain comprises SEQ ID NO:31; or (ix) the variable domain of the TCR α chain comprises SEQ ID NO:23, and the variable domain of the TCR β chain comprises SEQ ID NO:

31.

10. The TCR according to any one of the preceding claims, wherein the TCR comprises a constant domain of the TCR α chain and / or a constant domain of the TCR β chain.

11. The TCR according to any one of the preceding claims, wherein the TCR is an αβ heterodimer having the following structure: (a) a TCR α chain, comprising (i) a variable domain of the TCR α chain and (ii) a constant domain of the TCR α chain; and (b) a TCR β chain, comprising (i) a variable domain of the TCR β chain and (ii) a constant domain of the TCR β chain.

12. The TCR according to any one of the preceding claims, wherein: The TCR is a single-chain TCR having a Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ or Vα-L-Vβ-Cβ structure; and wherein Vα is the variable domain of the TCR α chain, Vβ is the variable domain of the TCR β chain, Cα is the constant domain of the TCR α chain, Cβ is the constant domain of the TCR β chain, and L is a linker sequence.

13. The TCR according to claim 12, wherein the TCR is soluble.

14. The TCR according to any one of claims 10 to 13, wherein: (a) The constant domain of the TCR α-chain comprises a TRAC constant domain sequence, optionally wherein the TRAC constant domain sequence comprises SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4; and / or (b) The constant domain of the TCR β-chain comprises a TRBC2 constant domain sequence or a TRBC1 / 2 spliced constant domain sequence, optionally wherein the TCR β-chain constant domain sequence comprises SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:

7.

15. A complex comprising (a) a TCR as described in any one of the preceding claims, and (b) a detectable label, therapeutic agent or pharmacokinetic modification moiety.

16. A nucleic acid encoding: (a) a TCR as described in any one of claims 1 to 14; (b) a variable domain of a TCR α-chain as defined in any one of claims 1 to 14, and optionally a constant domain of the TCR α-chain; or (c) a variable domain of a TCR β-chain as defined in any one of claims 1 to 14, and optionally a constant domain of the TCR β-chain.

17. The nucleic acid according to claim 16, wherein the nucleic acid encodes a TCR as described in any one of claims 1 to 14, and: (a) the nucleic acid encodes the TCR in a single open reading frame; or (b) the nucleic acid comprises (i) a first open reading frame encoding a TCR α-chain comprising the variable domain of the TCR α-chain and optionally the constant domain of the TCR α-chain; and (ii) a second open reading frame encoding a TCR β-chain comprising the variable domain of the TCR β-chain and optionally the constant domain of the TCR β-chain.

18. A vector comprising the nucleic acid according to claim 16 or 17.

19. A viral particle comprising the nucleic acid according to claim 16 or 17, or the vector according to claim 18.

20. A cell, wherein the cell: (a) expresses a TCR as described in any one of claims 1 to 14; (b) comprises at least one nucleic acid according to claim 16 or claim 17; optionally, wherein the cell comprises (i) a first nucleic acid according to claim 16 encoding a variable domain of a TCR α-chain as defined in any one of claims 1 to 14 and optionally a constant domain of the TCR α-chain; and (ii) a second nucleic acid according to claim 16 encoding a variable domain of a TCR β-chain as defined in any one of claims 1 to 14 and optionally a constant domain of the TCR β-chain; or (c) comprising at least one vector as described in claim 18; optionally, wherein said cell comprises (i) a first vector, said first vector comprising a nucleic acid as described in claim 16, said nucleic acid encoding a TCRα chain variable domain as defined in any one of claims 1 to 14 and optionally a TCRα chain constant domain; and (ii) a second vector, said second vector comprising a nucleic acid as described in claim 16, said nucleic acid encoding a TCRβ chain variable domain as defined in any one of claims 1 - 14 and optionally a TCRβ chain constant domain.

21. The cell according to claim 20, wherein the cell is: (a) an isolated cell or a non - naturally occurring cell; and / or (b) a T cell.

22. The cell according to claim 20 or 21, wherein the cell is a T cell further comprising one or more exogenous or recombinant co - stimulatory ligands.

23. A pharmaceutical composition comprising (i) a TCR as described in any one of claims 1 to 14, a complex as described in claim 15, a nucleic acid as described in claim 16 or 17, or a cell as described in any one of claims 20 to 22, and (ii) one or more pharmaceutically acceptable carriers or excipients.

24. A method for using a TCR as described in any one of claims 1 to 14, a nucleic acid as described in claim 16 or 17, a cell as described in any one of claims 20 to 22, or a pharmaceutical composition as described in claim 23 to treat a disease in an individual, said method comprising administering the TCR, nucleic acid, cell or pharmaceutical composition to the individual.

25. A method for treating a disease in an individual, comprising administering a TCR as described in any one of claims 1 to 14, a nucleic acid as described in claim 16 or 17, a cell as described in any one of claims 20 to 22, or a pharmaceutical composition as described in claim 23 to the individual.

26. Use of the TCR, nucleic acid, cell or pharmaceutical composition as described in claim 24 or the method as described in claim 25, wherein the disease: (a) is cancer; (b) is a solid tumor; or (c) is selected from melanoma, leukemia, lymphoma, lung cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, bladder cancer and head and neck cancer, optionally wherein (i) the lung cancer is non - small cell lung cancer or small cell lung cancer, or (ii) the breast cancer is triple - negative breast cancer.

27. Use of the TCR, nucleic acid, cell or pharmaceutical composition as described in claim 24 or 26, or the method as described in claim 25 or 26, wherein the method comprises administering an additional therapeutic agent to the individual.

28. A method for preparing one or more cells as described in any one of claims 20 to 22, comprising: (i) introducing into a cell at least one nucleic acid as described in claim 16 or claim 17 or at least one vector as described in claim 18; and then optionally (ii) culturing the cell to proliferate the cell to form a population.

29. The method according to claim 28, wherein the at least one nucleic acid or the at least one vector is comprised within a viral particle.

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