T-cell receptors

Abstract

The present invention provides novel tumour-antigen specific T-cell receptors (TCRs).

Description

[0001] T-CELL RECEPTORS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to tumour-antigen specific T-cell receptors (TCRs).

[0004] BACKGROUND TO THE INVENTION

[0005] The fifth pillar of cancer care is represented by immunotherapy, which exploits the innate ability of the immune system to recognize cancer cells. Indeed, the presence of tumourinfiltrating lymphocytes is correlated with a better prognosis (see e.g. Zhang, L., et al. New England journal of medicine, 348(3), pp.203-213).

[0006] The ability of T lymphocytes to recognise an antigen is determined by the expression of a T cell receptor (TCR). TCR gene therapy is based on the genetic transfer of high-avidity tumourspecific TCR genes into T lymphocytes, thus enabling the specific targeting of the desired tumour-associated antigens and leading to a less toxic and more specific and effective therapy. This approach has shown promise in clinical trials.

[0007] One of the main barriers limiting the exploitation of TCR gene therapy for clinical treatment of cancers is the lack of tumour-specific T-cells and corresponding TCRs. Tumour-specific T cells against tumour-associated antigens (TAAs), self-proteins overexpressed on cancer cells, are interesting targets in the context of adoptive T cell therapy. However, the number of isolated TAA-specific T cells is still limited, particularly in the context of blood malignancies.

[0008] Thus, the low availability of tumour-specific TCRs still remains an open issue limiting the broad exploitation of TCR-based immunotherapeutic approaches.

[0009] SUMMARY OF THE INVENTION

[0010] The present inventors have identified novel tumour-antigen specific TCRs. The present inventors have determined the amino acid sequences of the TCRs, including the amino acid sequences of their CDR regions, which are responsible for binding specificity.

[0011] The inventors’ studies facilitate the identification of tumor-relevant immunogenic proteins within the patients' proteome, and the expansion of tumor-specific T cells and TCRs that target these identified molecules. In particular, the inventors have further identified the targets of the tumour-antigen specific TCRs of the invention: assays including killing and cytokine release assays identified a target derived from the fatty acid-binding protein 5 (FABP5) presented by HLA-B*1801 as the target of the TCR from Pt#14, and a target derived from histone H2B (HIST1 H2B) presented by HLA-B*0801 as the target of the TCR from Pt#15. In one aspect, the present invention provides a T cell receptor (TCR) comprising a CDR3a comprising the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3P comprising the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11) or a variant thereof having up to three amino acid substitutions, additions or deletions. In some embodiments, the TCR comprises the following CDR sequences: CDR1a - NSASDY (SEQ ID NO: 4), CDR2a - IRSNMDK (SEQ ID NO: 5), CDR3a - CAENPPGTGNQFYF (SEQ ID NO: 6), CDR1 P - SEHNR (SEQ ID NO: 9), CDR2P - FQNEAQ (SEQ ID NO: 10), and CDR3P - CASSLQGPGELFF (SEQ ID NO: 11), or variants thereof each having up to three amino acid substitutions, additions or deletions. In some embodiments, the TCR comprises an a chain variable domain comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a p chain variable domain comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the TCR comprises an a chain comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a p chain comprising the amino acid sequence of SEQ ID NO: 13 or 14, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the TCR binds to a fatty acid-binding protein 5 (FABP5) peptide when presented by a major histocompatibility complex (MHC). In some embodiments, the TCR is restricted to HLA-B*1801.

[0012] In some embodiments, the TCR comprises an a chain comprising the amino acid sequence of SEQ ID NO: 70 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a p chain comprising the amino acid sequence of SEQ ID NO: 71 , or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0013] In one aspect, the present invention provides a T cell receptor (TCR) comprising a CDR3a comprising the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3P comprising the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22) or a variant thereof having up to three amino acid substitutions, additions or deletions. In some embodiments, the TCR comprises the following CDR sequences: CDR1a - TSENNYY (SEQ ID NO: 15), CDR2a - QEAYKQQN (SEQ ID NO: 16), CDR3a - CAFMKTGGTSYGKLTF (SEQ ID NO: 17), CDR1P - KGHSH (SEQ ID NO: 20), CDR2 - LQKENI (SEQ ID NO: 21), and CDR3P - CASSPVAGGFYEQYF (SEQ ID NO: 22), or variants thereof each having up to three amino acid substitutions, additions or deletions. In some embodiments, the TCR comprises an a chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a p chain variable domain comprising the amino acid sequence of SEQ ID NO: 23 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the TCR comprises an a chain comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a p chain comprising the amino acid sequence of SEQ ID NO: 24 or 25, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the TCR binds to a histone H2B (HIST1 H2B) peptide when presented by a major histocompatibility complex (MHC). In some embodiments, the TCR is restricted to HLA-B*0801+.

[0014] In some embodiments, the TCR comprises an a chain comprising the amino acid sequence of SEQ ID NO: 72 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a p chain comprising the amino acid sequence of SEQ ID NO: 73, or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0015] In some embodiments, the TCR comprises one or more mutations at the a chain / p chain interface, such that when the a chain and the p chain are expressed in a T-cell, the frequency of mispairing between said chains and endogenous TCR a and p chains is reduced. In some embodiments, the TCR comprises one or more mutations at the a chain / p chain interface, such that when the a chain and the p chain are expressed in a T-cell, the level of expression of the TCR a and p chains is increased. In some embodiments, the one or more mutations introduce a cysteine residue into the constant region domain of each of the a chain and the p chain, wherein the cysteine residues are capable of forming a disulphide bond between the a chain and the p chain. In some embodiments, the one or more mutations are at amino acid positions selected from those disclosed in Table 1 of Boulter, J.M et al. (2003) Protein Engineering 16: 707-711.

[0016] In some embodiments, the TCR comprises one or more mutations to remove one or more N- glycosylation sites (see, for example, Kuball, J et al. (2009) J Exp Med 206: 463-75). Suitably, the N-glycosylation sites are in the TCR constant domains. In some embodiments, the mutation is a substitution of the amino acid N in an N-X-S / T motif with the amino acid Q. For example, the substitution may occur at one or more of the positions: TCR alpha constant gene position 36, 90 or 109; and / or TCR beta constant gene position 85.6. In some embodiments, the substitution is at position 36 of the TCR alpha constant gene.

[0017] In some embodiments, the TCR comprises a murinised constant region.

[0018] In some embodiments, the TCR is a soluble TCR.

[0019] In another aspect, the invention provides a polynucleotide encoding the a chain of a T-cell receptor (TCR) according to the invention, and / or the p chain of a TCR according to the invention. In some embodiments, the polynucleotide encodes the a chain linked to the chain. In some embodiments, the polynucleotide further encodes one or more short interfering RNA (siRNA) or other agents capable of reducing or preventing expression of one or more endogenous TCR genes.

[0020] In another aspect, the invention provides a vector comprising a polynucleotide according to the invention. In some embodiments, the vector is a plasmid or a viral vector. In some embodiments, the vector comprises a polynucleotide, which encodes one or more CD3 chains, CD8, a suicide gene and / or a selectable marker.

[0021] In another aspect, the invention provides a cell comprising a TCR of the invention, a polynucleotide of the invention or a vector of the invention. In some embodiments, the cell further comprises a vector which encodes one or more CD3 chains, CD8, a suicide gene and / or a selectable marker.

[0022] The cell of the invention may be any suitable cell. Suitably, the cell is a T-cell, a lymphocyte, or a stem cell. In some embodiment, the cell is selected from the group consisting of CD4+ cells, CD8+ cells, naive T-cells, memory stem T-cells, central memory T-cells, double negative T-cells, effector memory T-cells, effector T-cells, ThO cells, TcO cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, gamma / delta T-cells, natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, hematopoietic stem cells and pluripotent stem cells. In some embodiments, the cell is a T-cell. In some embodiments, the cell is a T- cell which has been isolated from a subject. In some embodiments, an endogenous gene encoding a TCR a chain and / or an endogenous gene encoding a TCR p chain in the cell is disrupted, suitably such that the endogenous gene encoding a TCR a chain and / or the endogenous gene encoding a TCR chain is not expressed. In some embodiments, the endogenous gene encoding a TCR a chain and / or the endogenous gene encoding a TCR p chain is disrupted by insertion of an expression cassette comprising a polynucleotide sequence encoding the TCR of the invention. In some embodiments, one or more endogenous genes encoding an MHC is disrupted, suitably wherein the cell is a non-alloreactive universal T-cell. In some embodiments, an endogenous gene involved in persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capacity, or other T-cell functions is disrupted, suitably wherein the endogenous gene involved in persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capacity, or other T-cell functions is selected from the group consisting of PD1, TIM3, LAG3, 2B4, KLRG1, TGFbR, CD160, TIGIT, CTLA4 and CD39. In some embodiments, the endogenous gene involved in persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capacity, or other T-cell functions is disrupted by integration of an expression cassette, wherein the expression cassette comprises a polynucleotide sequence encoding a TCR of the invention.

[0023] In another aspect, the invention provides a T-cell genetically engineered (e.g. genetically edited) to modify the persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capacity or other T cell functions, wherein the T-cell expresses a TCR a chain of the invention and / or a TCR p chain of the invention.

[0024] In another aspect, the invention provides a T cell genetically engineered (e.g. genetically edited) by a protocol which comprises the step of targeted integration of an expression cassette into an endogenous gene involved in persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capacity or other T-cell functions disrupted by an artificial nuclease, wherein the expression cassette comprises a polynucleotide sequence encoding TCR a chain of the invention and / or a TCR chain of the invention.

[0025] In another aspect, the invention provides a method of preparing a cell, which comprises the step of introducing the polynucleotide or vector of the invention into a cell in vitro, ex vivo or in vivo, for example by transfection or transduction.

[0026] In another aspect, the invention provides a method of preparing a cell, which comprises the step of transducing a cell in vitro, ex vivo or in vivo with one or more vectors of the invention. In some embodiments, the cell to be transduced or transfected is selected from the group consisting of T-cells, lymphocytes or stem cells, such as hematopoietic stem cells or induced pluripotent stem cells (iPS). In some embodiments, the cell to be transduced or transfected is selected from the group consisting of CD4+ cells, CD8+ cells, ThO cells, TcO cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, gamma / delta T-cells, natural killer (NK) cells, natural killer T (NKT) cells, double negative T-cells, naive T-cells, memory stem T-cells, central memory T-cells, effector memory T-cells, effector T cells, cytokine-induced killer (CIK) cells, hematopoeitic stem cells and pluripotent stem cells.

[0027] In some embodiments, the method of preparing a cell comprises the step of T-cell editing, which comprises disrupting an endogenous gene encoding a TCR a chain and / or an endogenous gene encoding a TCR p chain with an artificial nuclease, optionally wherein the artificial nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR / Cas systems.

[0028] In some embodiments, the method of preparing a cell comprises a step of T-cell editing, which comprises disrupting an endogenous gene encoding a TCR a chain and / or an endogenous gene encoding a TCR chain by base editing. Base editing may comprise contacting a cell with a catalytically inactive Cas operably linked to a deaminase (e.g. a cytosine deaminase or an adenine deaminase). Reagents for T-cell editing (e.g. the artificial nuclease and / or guide RNA; or the base editing enzyme and / or guide RNA) may be delivered to a T-cell by any suitable means, such as electroporation or using lipid nanoparticles (LNPs).

[0029] In some embodiments, the method of preparing a cell comprises the step of targeted integration of an expression cassette into the endogenous gene encoding the TCR a chain and / or the endogenous gene encoding the TCR p chain disrupted by the artificial nuclease, wherein the expression cassette comprises a polynucleotide sequence encoding the TCR of the invention or a polynucleotide sequence of the invention.

[0030] In some embodiments, the method of preparing a cell comprises the step of disrupting one or more endogenous genes encoding an MHC, suitably wherein the cell prepared by the method is a non-alloreactive universal T-cell.

[0031] In some embodiments, the method of preparing a cell comprises the step of disrupting one or more endogenous genes to modify the persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capacity, or other T-cell functions, optionally wherein the method comprises the step of targeted integration of an expression cassette into an endogenous gene involved in persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capacity, or other T-cell functions disrupted by an artificial nuclease, wherein the expression cassette comprises a polynucleotide sequence encoding the TCR of the invention, suitably wherein the endogenous gene is selected from the group consisting of PD1, TIM3, LAG3, 2B4, KLRG1, TGFbR, CD160, TIGIT, CTLA4 and CD39.

[0032] In another aspect, the invention provides a chimeric molecule comprising the TCR of the invention, or a portion thereof, conjugated to a non-cellular substrate, a toxin and / or an antibody. In some embodiments, the non-cellular substrate is selected from the group consisting of nanoparticles, exosomes and other non-cellular substrates.

[0033] In another aspect, the invention provides a pharmaceutical composition comprising the TCR of the invention, the polynucleotide of the invention, the vector of the invention, the cell of the invention, a cell prepared by the method of the invention, or the chimeric molecule of the invention.

[0034] In another aspect, the invention provides the cell of the invention or a cell prepared by the method of the invention for use in adoptive cell transfer, suitably adoptive T-cell transfer, optionally wherein the adoptive T-cell transfer is allogenic adoptive T-cell transfer, autologous adoptive T-cell transfer, or universal non-alloreactive adoptive T-cell transfer.

[0035] In another aspect, the invention provides the TCR of the invention, the polynucleotide of the invention, the vector of the invention, the cell of the invention, a cell prepared by the method of the invention, the chimeric molecule of the invention, or the pharmaceutical composition of the invention for use in therapy.

[0036] In another aspect, the invention provides the TCR of the invention, the polynucleotide of the invention, the vector of the invention, the cell of the invention, a cell prepared by the method of the invention, a chimeric molecule of the invention, or a pharmaceutical composition of the invention for use in treating and / or preventing a proliferative disorder.

[0037] In another aspect, the invention provides a method for treating and / or preventing a proliferative disorder, which comprises the step of administering the TCR of the invention, the polynucleotide of the invention, the vector of the invention, the cell of the invention, a cell prepared by the method of the invention, a chimeric molecule of the invention, or a pharmaceutical composition of the invention to a subject in need thereof.

[0038] The proliferative disorder may be a hematological malignancy or a solid tumour. Suitably, the hematological malignancy is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, acute lymphocytic leukemia (ALL), myelodisplastic syndromes, lymphoma, multiple myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma. Suitably, the solid tumour is selected from the group consisting of lung cancer, breast cancer, oesophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancers, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, oral cancer, hepatocellular carcinoma, bladder cancer, endometrial cancer, neuroblastoma, rabdomyosarcoma, liver cancer, melanoma, prostate cancer, renal cancer, soft tissue sarcoma, urothelial cancer, biliary cancer, glioblastoma, cervical cancer, mesothelioma and colorectal cancer. In preferred embodiments, the proliferative disorder is acute myeloid leukemia (AML). In other embodiments, the proliferative disorder is renal cancer.

[0039] DESCRIPTION OF DRAWINGS

[0040] Figure 1 - Schematic of the in vitro expansion protocol

[0041] Bone-marrow infiltrating T cells from Pt#14 and Pt#15 at the moment of relapse were sorted into a fraction not expressing (IR_) and a fraction expressing (IR+) multiple Inhibitory Receptors and then expanded in vitro by means of Leukemic-Antigen Presenting Cells (L-APCs).

[0042] Figure 2 - The IR+ fraction enrich for a dominant TCR clone upon L-APCs stimulation

[0043] (A) CDR3 a-chain and (B) CDR3 p-chain clonotype frequency was assessed in both the IR+and IR' fractions, before and after sequential L-APCs stimulation, for Pt#14 and Pt#15. The expanding dominant IR+clone is highlighted by the shadow. The IR+fraction preferentially enrich with a dominant clone upon L-APC stimulation.

[0044] Figure 3 - The IR+-derived TCRs lyse autologous leukemic cells while sparing healthy bone marrow cells

[0045] In each patient, the dominant end-of-stimulation IR+TCR was reconstructed and engineered into T cells derived from N=3 different healthy donors. Edited T cells were then challenged with either autologous leukemic blasts (black lines) or autologous healthy bone marrow (grey lines). The elimination index is reported. The elimination index was calculated using the formula [1- (number of alive target cells co-cultured with engineered T cells / number of alive target cells co-cultured alone)]. Two-way ANOVA was used to compare curves. The mean and the SEM are shown * P-value <0.05. These results indicate that TCRs retrieved from the IR+fraction are specific toward a leukemic epitope, not shared by the healthy tissue counterpart (bone marrow).

[0046] Figure 4 - The IR+- derived TCRs degranulate and activate when co-cultured with leukemic cells In each patient, the dominant end-of-stimulation IR+TCR was reconstructed and engineered into T cells derived from N=4 different healthy donors. Edited T cells were then challenged with either autologous leukemic blasts (dark grey bars) or autologous healthy hematopoietic cells (light grey bars). The cytokine production (A-B), degranulation (C) and activation (D) of engineered T cells are reported. Paired multiple student T tests were used to compare groups. The mean and the SEM are shown. * P-value <0.05.

[0047] Figure 5 - T cells engineered with the IR+can transfer their CD3 receptor to target leukemia

[0048] The trogocytosis effect was quantified in T cells engineered with the IR+-derived orphan TCRs. Representative gates (A) and quantification (B) of the trogocytosis effect, defined as the percentage of blasts acquiring the CD3 T cell marker, in blast cultured with T cells engineered with Pt#14-15 TCRs, at different effector target ratios. These results show that the trogocytosis effect is quantifiable in the represented experimental context.

[0049] DETAILED DESCRIPTION

[0050] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0051] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0052] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes", "containing", or "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms "comprising", "comprises" and "comprised of" also include the term "consisting of".

[0053] Numeric ranges are inclusive of the numbers defining the range. As used herein the term “about” means approximately, in the region of, roughly, or around. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0054] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. All publications mentioned in the specification are herein incorporated by reference.

[0055] T-cell receptors (TCRs)

[0056] In one aspect, the present invention provides a T-cell receptor (TCR). The present invention also provides an a chain or a p chain from such a T cell receptor.

[0057] As used herein the term "T-cell receptor” (TCR) may refer to molecule capable of recognising a peptide (e.g. derived from a tumour-associated antigen) when presented by an MHC molecule. TCRs may be found on the surface of T-cells and are responsible for recognising an antigen peptide bound to an MHC molecule. During antigen processing, antigens are degraded inside cells and then antigen peptides carried to the cell surface by MHC molecules. Binding of the TCR to the peptide:MHC complex results in activation of the T lymphocyte on which the TCR is expressed through a series of biochemical events mediated by associated enzymes, co-receptors, and specialized accessory molecules.

[0058] The TCR of the invention may be a tumour-specific or tumour-antigen specific TCR. TCR affinity for the peptide may be determined by the association (kon) and dissociation rates (kOff) and represented as the equilibrium dissociation constant (KD). The TCR affinity may be determined by any suitable method, for example by titration calorimetry, surface plasmon resonance (SPR) or fluorescence microscopy-based methods (see e.g. Piepenbrink, K.H., et al., 2009. Methods Enzymol, 466, pp:359-381). The TCR may bind to a peptide when presented by an MHC with an affinity of about 100 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less, about 20 pM or less, or about 10 pM or less. The TCR may bind to a peptide when presented by an MHC with an affinity of about 0.1 pM to about 100 pM, about 1 pM to about 100 pM, or about 5 pM to about 100 pM. The TCR may bind to a peptide when presented by an MHC with an affinity of about 0.1 pM to about 10 pM, about 1 pM to about 10 pM, or about 5 pM to about 10 pM.

[0059] The TCR of the invention may be capable of re-directing T-cells to tumour cells (e.g. leukemic blasts). To redirect T-cells against tumour cells, T-cells can be engineered ex vivo to express tumour-antigen specific T cell receptors (TCRs), generating products known as TCR- engineered T cells. Engineered T-cells expressing the TCR of the invention may be capable of recognising tumour cells (e.g. leukemic blasts). Engineered T-cells expressing the TCR of the invention may be capable of specifically lysing tumour cells (e.g. leukemic blasts). The TCR of the invention may not recognise non-tumour cells (e.g. healthy hematopoietic cells). Engineered T-cells expressing the TCR of the invention may not recognise non-tumour cells (e.g. healthy hematopoietic cells). The naturally-occurring TCR heterodimer consists of an alpha (a) and beta (P) chain in around 95% of T-cells, whereas around 5% of T-cells have TCRs consisting of gamma (y) and delta (5) chains. Each chain of a natural TCR is a member of the immunoglobulin superfamily and possesses an N-terminal immunoglobulin (Ig)-variable (V) region, a Ig-constant (C) region, a transmembrane / cell membrane-spanning region, and a short cytoplasmic tail at the C-terminal end. The TCR of the present invention may be a heterodimer of two chains a and (or optionally y and 5) or it may be a single chain TCR construct.

[0060] A variable domain may determine the specificity of the TCR. The variable domain of both the TCR a chain and p chain have three hypervariable or complementarity determining regions (CDRs). A TCR a chain or p chain, for example, comprises a CDR1 , a CDR2, and a CDR3 in amino to carboxy terminal order. In general, CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the beta chain interacts with the C-terminal part of the peptide. CDR2 is thought to recognize the MHC molecule. Suitable variable domains are described herein.

[0061] A TCR of the invention may comprise a constant domain. A constant domain may allow the TCR to associate with other molecules like CD3 which possess three distinct chains (y, 5, and E) in mammals and the ^-chain. These accessory molecules have negatively charged transmembrane regions and are vital to propagating the signal from the TCR into the cell. The CD3- and ^-chains, together with the TCR, form what is known as the T cell receptor complex.

[0062] The constant domain may consist of short connecting sequences in which a cysteine residue forms a disulfide bond, making a link between the two chains. The constant domain may comprise an Ig-constant (C) region, a transmembrane / cell membrane-spanning region and a cytoplasmic tail.

[0063] An a chain of a TCR of the present invention may comprise a constant domain encoded by a TRAC gene. An a chain constant domain may have the amino acid sequence set out in UniProt entry P01848. An example a chain constant domain has the amino acid sequence set out below in SEQ ID NO: 1.

[0064] A TCR of the invention may comprise an a chain constant domain comprising or consisting of the amino acid sequence of SEQ ID NO: 1 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK SNSAVAWSNKSDFACANAFNNS I IPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNL SVIGFRILLLKVAGFNLLMTLRLWSS

[0065] Example a chain constant domain ( SEQ ID NO : 1 )

[0066] A p chain of a TCR of the present invention may comprise a constant domain encoded by a TRBC1 or a TRBC2 gene. A chain constant domain may have the amino acid sequence set out in UniProt entry P01850 or A0A5B9. An example p chain constant domain encoded by a TRBC1 gene has the amino acid sequence set out below in SEQ ID NO: 2. An example p chain constant domain encoded by a TRBC2 gene has the amino acid sequence set out below in SEQ ID NO: 3.

[0067] A TCR of the invention may comprise a p chain constant domain comprising the amino acid sequence of SEQ ID NO: 2 or 3 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0068] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVST DPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAK PVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV KRKDF

[0069] Example p chain constant domain ( SEQ ID NO : 2 )

[0070] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVST DPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAK PVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV KRKDSRG

[0071] Example p chain constant domain ( SEQ ID NO : 3 )

[0072] The TCR of the invention may have one or more additional cysteine residues in each of the a and p chains such that the TCR may comprise two or more disulphide bonds in the constant domains.

[0073] The signal from the T cell complex may be enhanced by simultaneous binding of the MHC molecules by a specific co-receptor. For helper T-cells, this co-receptor may be CD4 (specific for class II MHC); whereas for cytotoxic T-cells, this co-receptor may be CD8 (specific for class I MHC). The co-receptor can allow prolonged engagement between the antigen presenting cell and the T cell and can recruit essential molecules (e.g., LCK) inside the cell involved in the signalling of the activated T lymphocyte.

[0074] The TCR of the invention may be a hybrid TCR comprising sequences derived from more than one species. For example, it has been found that murine TCRs are more efficiently expressed in human T-cells than human TCRs. The TCR may therefore comprise a human variable domain and murine sequences within a constant domain. A disadvantage of this approach is that the murine constant sequences may trigger an immune response, leading to rejection of the transferred T-cells. However, the conditioning regimens used to prepare patients for adoptive T-cell therapy may result in sufficient immunosuppression to allow the engraftment of T-cells expressing murine sequences.

[0075] A TCR of the invention may be a soluble TCR, e.g. omitting or altering one or more constant domains. Other suitable methods for engineering soluble TCRs are known in the art (see e.g. Robinson, R.A., et al., 2021. The FEBS Journal, 288(21), pp.6159-6173).

[0076] In some embodiments, the TCR comprises one or more mutations to remove one or more N- glycosylation sites. Suitably, the N-glycosylation sites are in the TCR constant domains. Deletion of N-glycosylation sites in TCR constant domains is described in Kuball, J et al. (2009) J Exp Med 206: 463-75. In some embodiments, the one or more mutations are substitutions of the amino acid N in an N-X-S / T motif with the amino acid Q. For example, the substitution may at one or more of the positions: TCR alpha constant gene position 36, 90 or 109; and / or TCR beta constant gene position 85.6. Suitably, the substitution is at position 36 of the TCR alpha constant gene. Mutations of TCR constant domains disclosed herein may be described based on a numbering convention in which the first amino acid of each of SEQ ID NOs: 1-3 is assigned to be position 2.

[0077] Complementarity determining regions (CDRs)

[0078] T-cell receptor diversity is focused on CDR3 and this region is primarily responsible for antigen recognition. The sequences of the CDR3 regions of the TCR of the invention may be selected from those described herein. A TCR of the present invention may comprise CDRs that comprise or consist of a CDR3a and a CDR3P pair described herein.

[0079] The portion of the TCR that establishes the majority of the contacts with the antigenic peptide bound to the major histocompatibility complex (MHC) is the complementarity determining region 3 (CDR3), which is unique for each T cell clone. The CDR3 region is generated upon somatic rearrangement events occurring in the thymus and involving non-contiguous genes belonging to the variable (V), diversity (D, for p and 5 chains) and joining (J) genes. Furthermore, random nucleotides inserted / deleted at the rearranging loci of each TCR chain gene greatly increase diversity of the highly variable CDR3 sequence. Thus, the frequency of a specific CDR3 sequence in a biological sample indicates the abundance of a specific T cell population. The great diversity of the TCR repertoire in healthy human beings provides a wide range protection towards a variety of foreign antigens presented by MHC molecules on the surface of antigen presenting cells. In this regard, it is of note that theoretically up to 1015different TCRs can be generated in the thymus.

[0080] The sequences of the CDR1 and CDR2 regions of the TCR of the invention may also be selected from those described herein. A TCR of the present invention may comprise CDRs that comprise of consist of CDR1a, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P sets described herein.

[0081] The CDRs may, for example, comprise one, two, or three substitutions, additions or deletions from the given sequence, provided that the TCR retains the capacity to bind the corresponding immunogenic peptide when presented by an MHC molecule. In some embodiments, each CDR3 comprises up to three substitutions, additions or deletions, up to two substitutions, additions or deletions, up to one substitution, addition or deletion, or any combination thereof. In some embodiments, each CDR3 comprises up to three substitutions, up to two substitutions, up to one substitution, or any combination thereof. In some embodiments, each CDR comprises up to three substitutions, additions or deletions, up to two substitutions, additions or deletions, up to one substitution, addition or deletion, or any combination thereof. In some embodiments, each CDR comprises up to three substitutions, up to two substitutions, up to one substitution, or any combination thereof.

[0082] Major histocompatability complex (MHC) molecules

[0083] Typically, TCRs bind to peptides as part of peptide: MHC complex. The TCR of the present invention may bind to an MHC I and / or MHC II peptide complex. The MHC peptide complex may be on the surface of an antigen presenting cell, such as a dendritic cell or a B cell, or any other cell, including cancer cells, or it may be immobilised by, for example, coating on to a bead or plate.

[0084] The human leukocyte antigen system (HLA) is the name of the gene complex which encodes MHC in humans and includes HLA class I antigens (A, B & C) and HLA class II antigens (DP, DQ, & DR). HLA alleles A, B and C present peptides derived mainly from intracellular proteins, e.g. proteins expressed within the cell. The TCR may be restricted to a human leukocyte antigen (HLA) allele.

[0085] During T-cell development in vivo, T-cells undergo a positive selection step to ensure recognition of self MHCs followed by a negative step to remove T-cells that bind too strongly to MHC which present self-antigens. As a consequence, certain T-cells and the TCRs they express will only recognise peptides presented by certain types of MHC molecules, i.e. those encoded by particular HLA alleles. This is known as HLA restriction. The preferred MHC binding motifs of different HLA alleles are disclosed in The Immune Epitope Database (IEDB) (Vita, R., et al., 2019. Nucleic acids research, 47(D1), pp.D339-D343).

[0086] Immunogenic peptides

[0087] The TCRs of the present invention may bind to an immunogenic peptide when presented by an MHC.

[0088] As used herein, the term “immunogenic peptide” may refer to a peptide which is capable of being presented by an MHC molecule and subsequently recognised by a TCR. As used herein, the term “peptide” may refer to a plurality of amino acid residues linked by peptide bonds. A peptide may consist of less than about 30, less than about 25, less than about 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13, less than 12, less than 11 , less than 10, less than 9, less than 8, less than 7, less than 6, or less than 5 amino acid residues in length. Suitably, a peptide is about 5 to 20 amino acids in length or about 8 to 15 amino acid residues in length.

[0089] As used herein, the term “protein” may include single-chain polypeptide molecules as well as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means. As used herein, the term “polypeptide” may refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulphide bonds.

[0090] The immunogenic peptide may be derived from e.g. a tumour-associated antigen. As used herein, a “tumour-associated antigen” may refer to an antigenic protein produced in tumour cells. Tumour-associated antigens may act as useful tumour markers and as potential candidates for use in immunotherapy. The immunogenic peptide may be a fragment of a tumour-associated antigen.

[0091] The immunogenic peptide may be derived from e.g. an AML-associated antigen. As used herein, an “AML-associated antigen” may refer to an antigenic protein produced in acute myeloid leukaemia cells. AML-associated antigens may act as useful tumour markers and as potential candidates for use in immunotherapy.

[0092] Example TCR #1

[0093] Example TCR amino acid sequences are provided in the table below. The inventors have identified the target of Example TCR #1 as a target derived from the fatty acid-binding protein 5 (FABP5) presented by HLA-B*1801 (e.g. Manfredi et al. (2023) Sci. Adv. 9: eadg8014). FABP5 is highly expressed in AML patients at diagnosis and at relapse.

[0094] In one aspect, the present invention provides a TCR comprising a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to three amino acid substitutions, additions or deletions, and / or a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11) or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0095] In some embodiments, the TCR comprises a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to two amino acid substitutions, additions or deletions, and / or a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11) or a variant thereof having up to two amino acid substitutions, additions or deletions. In some embodiments, the TCR comprises a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to one amino acid substitution, addition or deletion, and / or a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11) or a variant thereof having up to one amino acid substitution, addition or deletion. In some embodiments, the TCR comprises a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to three amino acid substitutions, and / or a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11) or a variant thereof having up to three amino acid substitutions. In some embodiments, the TOR comprises a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to two amino acid substitutions, and / or a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11) or a variant thereof having up to two amino acid substitutions. In some embodiments, the TCR comprises a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to one amino acid substitution, and / or a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11) or a variant thereof having up to one amino acid substitution. In some embodiments, the TCR comprises a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6), and / or a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11).

[0096] In some embodiments, the TCR further comprises (a) a CDR1a comprising or consisting of the amino acid sequence of NSASDY (SEQ ID NO: 4), or a variant thereof having up to three amino acid substitutions, additions or deletions; and (b) a CDR2a comprising or consisting of the amino acid sequence of IRSNMDK (SEQ ID NO: 5), or a variant thereof having up to three amino acid substitutions, additions or deletions; and / or (a) a CDRi p comprising or consisting of the amino acid sequence of SEHNR (SEQ ID NO: 9), or a variant thereof having up to three amino acid substitutions, additions or deletions; and (b) a CDR2P comprising or consisting of the amino acid sequence of FQNEAQ (SEQ ID NO: 10), or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0097] In some embodiments, the TCR comprises an a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and / or a p chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0098] In some embodiments, the TCR comprises an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and / or a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or 14, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0099] In one aspect, the present invention provides a TOR comprising (a) a CDR1a comprising or consisting of the amino acid sequence of NSASDY (SEQ ID NO: 4), or a variant thereof having up to three amino acid substitutions, additions or deletions; (b) a CDR2a comprising or consisting of the amino acid sequence of IRSNMDK (SEQ ID NO: 5), or a variant thereof having up to three amino acid substitutions, additions or deletions; (c) a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to three amino acid substitutions, additions or deletions; and / or (a) a CDR1 p comprising or consisting of the amino acid sequence of SEHNR (SEQ ID NO: 9), or a variant thereof having up to three amino acid substitutions, additions or deletions; (b) a CDR2 comprising or consisting of the amino acid sequence of FQNEAQ (SEQ ID NO: 10), or a variant thereof having up to three amino acid substitutions, additions or deletions; (c) a CDR3 comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11), or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0100] In some embodiments, the TCR comprises (a) a CDR1 a comprising or consisting of the amino acid sequence of NSASDY (SEQ ID NO: 4), or a variant thereof having up to two amino acid substitutions, additions or deletions; (b) a CDR2a comprising or consisting of the amino acid sequence of IRSNMDK (SEQ ID NO: 5), or a variant thereof having up to two amino acid substitutions, additions or deletions; (c) a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to two amino acid substitutions, additions or deletions; and / or (a) a CDRi comprising or consisting of the amino acid sequence of SEHNR (SEQ ID NO: 9), or a variant thereof having up to two amino acid substitutions, additions or deletions; (b) a CDR2P comprising or consisting of the amino acid sequence of FQNEAQ (SEQ ID NO: 10), or a variant thereof having up to two amino acid substitutions, additions or deletions; (c) a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11), or a variant thereof having up to two amino acid substitutions, additions or deletions.

[0101] In some embodiments, the TCR comprises (a) a CDR1 a comprising or consisting of the amino acid sequence of NSASDY (SEQ ID NO: 4), or a variant thereof having up to one amino acid substitution, addition or deletion; (b) a CDR2a comprising or consisting of the amino acid sequence of IRSNMDK (SEQ ID NO: 5), or a variant thereof having up to one amino acid substitution, addition or deletion; (c) a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to one amino acid substitution, addition or deletion; and / or (a) a CDRi p comprising or consisting of the amino acid sequence of SEHNR (SEQ ID NO: 9), or a variant thereof having up to one amino acid substitution, addition or deletion; (b) a CDR2P comprising or consisting of the amino acid sequence of FQNEAQ (SEQ ID NO: 10), or a variant thereof having up to one amino acid substitution, addition or deletion; (c) a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11), or a variant thereof having up to up to one amino acid substitution, addition or deletion.

[0102] In some embodiments, the TCR comprises (a) a CDR1 a comprising or consisting of the amino acid sequence of NSASDY (SEQ ID NO: 4); (b) a CDR2a comprising or consisting of the amino acid sequence of IRSNMDK (SEQ ID NO: 5); (c) a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6); and / or (a) a CDRi p comprising or consisting of the amino acid sequence of SEHNR (SEQ ID NO: 9); (b) a CDR2P comprising or consisting of the amino acid sequence of FQNEAQ (SEQ ID NO: 10); (c) a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11).

[0103] In one aspect, the present invention provides a TCR comprising an a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and / or a p chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0104] In one aspect, the present invention provides a TCR comprising an a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain variable domain comprises a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to three amino acid substitutions, additions or deletions; and / or a p chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the p chain variable domain comprises a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11), or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0105] In one aspect, the present invention provides a TOR comprising an a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain variable domain comprises a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6); and / or a p chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the chain variable domain comprises a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11).

[0106] In one aspect, the present invention provides a TCR comprising an a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain variable domain comprises (a) a CDR1a comprising or consisting of the amino acid sequence of NSASDY (SEQ ID NO: 4), or a variant thereof having up to three amino acid substitutions, additions or deletions; (b) a CDR2a comprising or consisting of the amino acid sequence of IRSNMDK (SEQ ID NO: 5), or a variant thereof having up to three amino acid substitutions, additions or deletions; (c) a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to three amino acid substitutions, additions or deletions; and / or a p chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the p chain variable domain comprises (a) a CDRi p comprising or consisting of the amino acid sequence of SEHNR (SEQ ID NO: 9), or a variant thereof having up to three amino acid substitutions, additions or deletions; (b) a CDR2P comprising or consisting of the amino acid sequence of FQNEAQ (SEQ ID NO: 10), or a variant thereof having up to three amino acid substitutions, additions or deletions; (c) a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11), or a variant thereof having up to three amino acid substitutions, additions or deletions. In one aspect, the present invention provides a TCR comprising an a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain variable domain comprises (a) a CDR1a comprising or consisting of the amino acid sequence of NSASDY (SEQ ID NO: 4); (b) a CDR2a comprising or consisting of the amino acid sequence of IRSNMDK (SEQ ID NO: 5); (c) a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6); and / or a p chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the chain variable domain comprises (a) a CDRi p comprising or consisting of the amino acid sequence of SEHNR (SEQ ID NO: 9); (b) a CDR2P comprising or consisting of the amino acid sequence of FQNEAQ (SEQ ID NO: 10); (c) a CDR3P comprising or consisting of the amino acid sequence of CASS LOG PG ELF F (SEQ ID NO: 11).

[0107] In one aspect, the present invention provides a TCR comprising an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and / or a p chain comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or 14, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0108] In one aspect, the present invention provides a TCR comprising an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain comprises a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to three amino acid substitutions, additions or deletions; and / or a p chain comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or 14, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the p chain comprises a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11), or a variant thereof having up to three amino acid substitutions, additions or deletions. In one aspect, the present invention provides a TCR comprising an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain comprises a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6); and / or a p chain comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or 14, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the chain comprises a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11).

[0109] In one aspect, the present invention provides a TCR comprising an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain comprises (a) a CDR1a comprising or consisting of the amino acid sequence of NSASDY (SEQ ID NO: 4), or a variant thereof having up to three amino acid substitutions, additions or deletions; (b) a CDR2a comprising or consisting of the amino acid sequence of IRSNMDK (SEQ ID NO: 5), or a variant thereof having up to three amino acid substitutions, additions or deletions; (c) a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to three amino acid substitutions, additions or deletions; and / or a p chain comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or 14, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the p chain comprises (a) a CDRi p comprising or consisting of the amino acid sequence of SEHNR (SEQ ID NO: 9), or a variant thereof having up to three amino acid substitutions, additions or deletions; (b) a CDR2P comprising or consisting of the amino acid sequence of FQNEAQ (SEQ ID NO: 10), or a variant thereof having up to three amino acid substitutions, additions or deletions; (c) a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11), or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0110] In one aspect, the present invention provides a TCR comprising an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain comprises (a) a CDR1a comprising or consisting of the amino acid sequence of NSASDY (SEQ ID NO: 4); (b) a CDR2a comprising or consisting of the amino acid sequence of IRSNMDK (SEQ ID NO: 5); (c) a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6); and / or a p chain comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or 14, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the chain comprises (a) a CDRi p comprising or consisting of the amino acid sequence of SEHNR (SEQ ID NO: 9); (b) a CDR2P comprising or consisting of the amino acid sequence of FQNEAQ (SEQ ID NO: 10); (c) a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11).

[0111] In one aspect, the present invention provides a TCR comprising an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain comprises (a) a CDR1a comprising or consisting of the amino acid sequence of NSASDY (SEQ ID NO: 4); (b) a CDR2a comprising or consisting of the amino acid sequence of IRSNMDK (SEQ ID NO: 5); (c) a CDR3a comprising or consisting of the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6); and a p chain comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or 14, or variants thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the p chain comprises (a) a CDRi p comprising or consisting of the amino acid sequence of SEHNR (SEQ ID NO: 9); (b) a CDR2P comprising or consisting of the amino acid sequence of FQNEAQ (SEQ ID NO: 10); (c) a CDR3P comprising or consisting of the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11).

[0112] In preferred embodiments, the TCR binds to a fatty acid-binding protein 5 (FABP5) peptide when presented by a major histocompatibility complex (MHC). In some embodiments, the TCR is restricted to HLA-B*1801.

[0113] FABP5 is an intracellular carrier of long-chain fatty acids and related lipids that regulate the metabolism and actions of the ligands they bind. It may selectively deliver specific fatty acids from the cytosol to the nucleus.

[0114] An example FABP5 protein has the amino acid sequence set out in UniProt entry Q01469.

[0115] An example FABP5 protein has the amino acid sequence set out below:

[0116] MATVQQLEGRWRLVDSKGFDEYMKELGVGIALRKMGAMAKPDCI ITCDGKNLTIKTESTLKTTQFSCT LGEKFEETTADGRKTQTVCNFTDGALVQHQEWDGKESTITRKLKDGKLVVECVMNNVTCTRIYEKVE ( SEQ ID NO : 68 )

[0117] For example, a FABP5 peptide may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 25 contiguous amino acid residues of a FABP5 protein amino acid sequence.

[0118] Example TCR #2

[0119] Example TCR amino acid sequences are provided in the table below.

[0120] The inventors have identified the target of Example TCR #2 as a target derived from histone

[0121] H2B (HIST1 H2B) presented by HLA-B*0801+(e.g. Manfredi et al. (2023) Sci. Adv. 9: eadg8014). HIST1 H2B isoforms are highly expressed in AML patients at diagnosis and at relapse.

[0122] In one aspect, the present invention provides a TCR comprising a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to three amino acid substitutions, additions or deletions, and / or a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22) or a variant thereof having up to three amino acid substitutions, additions or deletions. In some embodiments, the TCR comprises a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to two amino acid substitutions, additions or deletions, and / or a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22) or a variant thereof having up to two amino acid substitutions, additions or deletions. In some embodiments, the TCR comprises a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to one amino acid substitution, addition or deletion, and / or a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22) or a variant thereof having up to one amino acid substitution, addition or deletion. In some embodiments, the TCR comprises a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to three amino acid substitutions, and / or a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22) or a variant thereof having up to three amino acid substitutions. In some embodiments, the TCR comprises a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to two amino acid substitutions, and / or a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22) or a variant thereof having up to two amino acid substitutions. In some embodiments, the TCR comprises a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to one amino acid substitution, and / or a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22) or a variant thereof having up to one amino acid substitution. In some embodiments, the TCR comprises a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17), and / or a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22).

[0123] In some embodiments, the TCR further comprises (a) a CDR1a comprising or consisting of the amino acid sequence of TSENNYY (SEQ ID NO: 15), or a variant thereof having up to three amino acid substitutions, additions or deletions; and (b) a CDR2a comprising or consisting of the amino acid sequence of QEAYKQQN (SEQ ID NO: 16), or a variant thereof having up to three amino acid substitutions, additions or deletions; and / or (a) a CDRi p comprising or consisting of the amino acid sequence of KGHSH (SEQ ID NO: 20), or a variant thereof having up to three amino acid substitutions, additions or deletions; and (b) a CDR2P comprising or consisting of the amino acid sequence of LQKENI (SEQ ID NO: 21), or a variant thereof having up to three amino acid substitutions, additions or deletions. In some embodiments, the TCR comprises an a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and / or a p chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0124] In some embodiments, the TCR comprises an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and / or a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 24 or 25, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0125] In one aspect, the present invention provides a TCR comprising (a) a CDR1a comprising or consisting of the amino acid sequence of TSENNYY (SEQ ID NO: 15), or a variant thereof having up to three amino acid substitutions, additions or deletions; (b) a CDR2a comprising or consisting of the amino acid sequence of QEAYKQQN (SEQ ID NO: 16), or a variant thereof having up to three amino acid substitutions, additions or deletions; (c) a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to three amino acid substitutions, additions or deletions; and / or (a) a CDRi p comprising or consisting of the amino acid sequence of KGHSH (SEQ ID NO: 20), or a variant thereof having up to three amino acid substitutions, additions or deletions; (b) a CDR2P comprising or consisting of the amino acid sequence of LQKENI (SEQ ID NO: 21), or a variant thereof having up to three amino acid substitutions, additions or deletions; (c) a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22), or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0126] In some embodiments, the TCR comprises (a) a CDR1 a comprising or consisting of the amino acid sequence of TSENNYY (SEQ ID NO: 15), or a variant thereof having up to two amino acid substitutions, additions or deletions; (b) a CDR2a comprising or consisting of the amino acid sequence of QEAYKQQN (SEQ ID NO: 16), or a variant thereof having up to two amino acid substitutions, additions or deletions; (c) a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to two amino acid substitutions, additions or deletions; and / or (a) a CDRi p comprising or consisting of the amino acid sequence of KGHSH (SEQ ID NO: 20), or a variant thereof having up to two amino acid substitutions, additions or deletions; (b) a CDR2P comprising or consisting of the amino acid sequence of LQKENI (SEQ ID NO: 21), ora variant thereof having up to two amino acid substitutions, additions or deletions; (c) a CDR3 comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22), or a variant thereof having up to two amino acid substitutions, additions or deletions.

[0127] In some embodiments, the TCR comprises (a) a CDR1 a comprising or consisting of the amino acid sequence of TSENNYY (SEQ ID NO: 15), or a variant thereof having up to one amino acid substitution, addition or deletion; (b) a CDR2a comprising or consisting of the amino acid sequence of QEAYKQQN (SEQ ID NO: 16), or a variant thereof having up to one amino acid substitution, addition or deletion; (c) a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to one amino acid substitution, addition or deletion; and / or (a) a CDRi p comprising or consisting of the amino acid sequence of KGHSH (SEQ ID NO: 20), or a variant thereof having up to one amino acid substitution, addition or deletion; (b) a CDR2P comprising or consisting of the amino acid sequence of LQKENI (SEQ ID NO: 21), or a variant thereof having up to one amino acid substitution, addition or deletion; (c) a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22), or a variant thereof having up to up to one amino acid substitution, addition or deletion.

[0128] In some embodiments, the TCR comprises (a) a CDR1 a comprising or consisting of the amino acid sequence of TSENNYY (SEQ ID NO: 15); (b) a CDR2a comprising or consisting of the amino acid sequence of QEAYKQQN (SEQ ID NO: 16); (c) a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17); and / or (a) a CDRi p comprising or consisting of the amino acid sequence of KGHSH (SEQ ID NO: 20); (b) a CDR2P comprising or consisting of the amino acid sequence of LQKENI (SEQ ID NO: 21); (c) a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22).

[0129] In one aspect, the present invention provides a TCR comprising an a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and / or a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In one aspect, the present invention provides a TCR comprising an a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain variable domain comprises a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to three amino acid substitutions, additions or deletions; and / or a p chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the chain variable domain comprises a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22), or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0130] In one aspect, the present invention provides a TCR comprising an a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain variable domain comprises a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17); and / or a p chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the P chain variable domain comprises a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22).

[0131] In one aspect, the present invention provides a TCR comprising an a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain variable domain comprises (a) a CDR1a comprising or consisting of the amino acid sequence of TSENNYY (SEQ ID NO: 15), or a variant thereof having up to three amino acid substitutions, additions or deletions; (b) a CDR2a comprising or consisting of the amino acid sequence of QEAYKQQN (SEQ ID NO: 16), or a variant thereof having up to three amino acid substitutions, additions or deletions; (c) a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to three amino acid substitutions, additions or deletions; and / or a p chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the p chain variable domain comprises (a) a CDRi p comprising or consisting of the amino acid sequence of KGHSH (SEQ ID NO: 20), or a variant thereof having up to three amino acid substitutions, additions or deletions; (b) a CDR2P comprising or consisting of the amino acid sequence of LQKENI (SEQ ID NO: 21), or a variant thereof having up to three amino acid substitutions, additions or deletions; (c) a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22), or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0132] In one aspect, the present invention provides a TCR comprising an a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain variable domain comprises (a) a CDR1a comprising or consisting of the amino acid sequence of TSENNYY (SEQ ID NO: 15); (b) a CDR2a comprising or consisting of the amino acid sequence of QEAYKQQN (SEQ ID NO: 16); (c) a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17); and / or a chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the p chain variable domain comprises (a) a CDRi p comprising or consisting of the amino acid sequence of KGHSH (SEQ ID NO: 20); (b) a CDR2P comprising or consisting of the amino acid sequence of LQKENI (SEQ ID NO: 21); (c) a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22).

[0133] In one aspect, the present invention provides a TCR comprising an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto; and / or a p chain comprising or consisting of the amino acid sequence of SEQ ID NO: 24 or 25, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0134] In one aspect, the present invention provides a TCR comprising an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain comprises a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to three amino acid substitutions, additions or deletions; and / or a p chain comprising or consisting of the amino acid sequence of SEQ ID NO: 24 or 25, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the chain comprises a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22), or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0135] In one aspect, the present invention provides a TCR comprising an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain comprises a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17); and / or a p chain comprising or consisting of the amino acid sequence of SEQ ID NO: 24 or 25, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the p chain comprises a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22).

[0136] In one aspect, the present invention provides a TCR comprising an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain comprises (a) a CDR1a comprising or consisting of the amino acid sequence of TSENNYY (SEQ ID NO: 15), or a variant thereof having up to three amino acid substitutions, additions or deletions; (b) a CDR2a comprising or consisting of the amino acid sequence of QEAYKQQN (SEQ ID NO: 16), or a variant thereof having up to three amino acid substitutions, additions or deletions; (c) a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to three amino acid substitutions, additions or deletions; and / or a p chain comprising or consisting of the amino acid sequence of SEQ ID NO: 24 or 25, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the p chain comprises (a) a CDRi p comprising or consisting of the amino acid sequence of KGHSH (SEQ ID NO: 20), or a variant thereof having up to three amino acid substitutions, additions or deletions; (b) a CDR2P comprising or consisting of the amino acid sequence of LQKENI (SEQ ID NO: 21), or a variant thereof having up to three amino acid substitutions, additions or deletions; (c) a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22), or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0137] In one aspect, the present invention provides a TCR comprising an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain comprises (a) a CDR1a comprising or consisting of the amino acid sequence of TSENNYY (SEQ ID NO: 15); (b) a CDR2a comprising or consisting of the amino acid sequence of QEAYKQQN (SEQ ID NO: 16); (c) a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17); and / or a p chain comprising or consisting of the amino acid sequence of SEQ ID NO: 24 or 25, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the p chain comprises (a) a CDR1 p comprising or consisting of the amino acid sequence of KGHSH (SEQ ID NO: 20); (b) a CDR2P comprising or consisting of the amino acid sequence of LQKENI (SEQ ID NO: 21); (c) a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22).

[0138] In one aspect, the present invention provides a TCR comprising an a chain comprising or consisting of the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the a chain comprises (a) a CDR1a comprising or consisting of the amino acid sequence of TSENNYY (SEQ ID NO: 15); (b) a CDR2a comprising or consisting of the amino acid sequence of QEAYKQQN (SEQ ID NO: 16); (c) a CDR3a comprising or consisting of the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17); and a p chain comprising or consisting of the amino acid sequence of SEQ ID NO: 24 or 25, or variants thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the p chain comprises (a) a CDRi p comprising or consisting of the amino acid sequence of KGHSH (SEQ ID NO: 20); (b) a CDR2P comprising or consisting of the amino acid sequence of LQKENI (SEQ ID NO: 21); (c) a CDR3P comprising or consisting of the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22). In preferred embodiments, the TCR binds to a histone H2B (HIST1 H2B) peptide when presented by a major histocompatibility complex (MHC). In some embodiments, the TCR is restricted to HLA-B*0801+.

[0139] Histone H2B is one of the histone proteins involved in the structure of chromatin. Histone H2B has a main globular domain and N- and C-terminal tails.

[0140] An example Histone H2B protein has the amino acid sequence set out in UniProt entry P62807. An example Histone H2B protein has the amino acid sequence set out below:

[0141] MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQVHPDTGI SSKAMGIMNS FVN DI FERIAGEASRLAHYNKRST ITSRE IQTAVRLLLPGELAKHAVSEGTKAVTKYTSSK

[0142] ( SEQ ID NO : 69 )

[0143] For example, a Histone H2B peptide may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 25 contiguous amino acid residues of a Histone H2B protein amino acid sequence.

[0144] Reduced mispairing and improved TCR expression

[0145] The TCR of the invention may be expressed in a T-cell to alter the antigen specificity of the T- cell. TCR-transduced T-cells may express at least two TCR alpha and two TCR beta chains. While the endogenous TCR alpha / beta chains form a receptor that is self-tolerant, the introduced TCR alpha / beta chains form a receptor with defined specificity for the given target antigen.

[0146] However, TCR gene therapy requires sufficient expression of transferred TCRs. Transferred TCR might be diluted by the presence of the endogeneous TCR, resulting in suboptimal expression of the tumour specific TCR. Furthermore, mispairing between endogenous and introduced chains may occur to form novel receptors, which might display unexpected specificities for self-antigens and cause autoimmune damage when transferred into patients.

[0147] Hence, several strategies have been explored to reduce the risk of mispairing between endogenous and introduced TCR chains. Mutations of the TCR alpha / beta interface is one strategy currently employed to reduce unwanted mispairing. For example, the introduction of a cysteine in the constant domains of the alpha and beta chain can allow the formation of a disulfide bond and enhance the pairing of the introduced chains while reducing mispairing with wild type chains. Accordingly, the TCRs of the invention may comprise one or more mutations at the a chain / p chain interface, such that when the a chain and the chain are expressed in a T-cell, the frequency of mispairing between said chains and endogenous TCR a and p chains is reduced. In some embodiments, the one or more mutations introduce a cysteine residue into the constant region domain of each of the a chain and the p chain, wherein the cysteine residues are capable of forming a disulphide bond between the a chain and the p chain. Such modification of TCRs is described in e.g. Boulter, J.M et al. (2003) Protein Engineering 16: 707-711 and Kuball, L. et al. (2007) Blood 109: 2331-8.

[0148] In some embodiments, the one or more mutations are at amino acid positions selected from those disclosed in Table 1 of Boulter, J.M et al. (2003) Protein Engineering 16: 707-711. As described above, mutations of TCR constant domains disclosed herein may be described based on a numbering convention in which the first amino acid of each of SEQ ID NOs: 1-3 is assigned to be position 2. In some embodiments, the one or more mutations are a substitution of one or more of the following amino acids with cysteine:

[0149] In some embodiments, the TCR comprises one or more of the following groups of mutations:

[0150] (a) a substitution of threonine at position 48 of the TCR alpha constant domain with cysteine; and / or a substitution of serine at position 57 of the TCR beta constant domain with cysteine;

[0151] (b) a substitution of threonine at position 45 of the TCR alpha constant domain with cysteine; and / or a substitution of serine at position 77 of the TCR beta constant domain with cysteine; (c) a substitution of serine at position 61 of the TCR alpha constant domain with cysteine; and / or a substitution of serine at position 57 of the TCR beta constant domain with cysteine;

[0152] (d) a substitution of leucine at position 50 of the TCR alpha constant domain with cysteine; and / or a substitution of serine at position 57 of the TCR beta constant domain with cysteine;

[0153] (e) a substitution of tyrosine at position 10 of the TCR alpha constant domain with cysteine; and / or a substitution of serine at position 17 of the TCR beta constant domain with cysteine;

[0154] (f) a substitution of serine at position 15 of the TCR alpha constant domain with cysteine; and / or a substitution of valine at position 13 of the TCR beta constant domain with cysteine;

[0155] (g) a substitution of serine at position 15 of the TCR alpha constant domain with cysteine; and / or a substitution of glutamate at position 15 of the TCR beta constant domain with cysteine;

[0156] (h) a substitution of threonine at position 45 of the TCR alpha constant domain with cysteine; and / or a substitution of aspartate at position 59 of the TCR beta constant domain with cysteine;

[0157] (i) a substitution of leucine 12 at position 48 of the TCR alpha constant domain with cysteine; and / or a substitution of serine at position 17 of the TCR beta constant domain with cysteine;

[0158] (j) a substitution of serine at position 61 of the TCR alpha constant domain with cysteine; and / or a substitution of arginine at position 79 of the TCR beta constant domain with cysteine;

[0159] (k) a substitution of leucine at position 12 of the TCR alpha constant domain with cysteine; and / or a substitution of phenylalanine at position 14 of the TCR beta constant domain with cysteine;

[0160] (l) a substitution of valine at position 22 of the TCR alpha constant domain with cysteine; and / or a substitution of phenylalanine at position 14 of the TCR beta constant domain with cysteine; and / or (m) a substitution of tyrosine at position 43 of the TCR alpha constant domain with cysteine; and / or a substitution of leucine at position 63 of the TCR beta constant domain with cysteine.

[0161] In some embodiments, the TCR comprises a substitution of threonine at position 48 of the TCR alpha constant domain with cysteine; and / or a substitution of serine at position 57 of the TCR beta constant domain with cysteine.

[0162] Another strategy to reduce mispairing relies on the introduction of polynucleotide sequences encoding siRNA, added to the genes encoding for the tumour specific TCR a and or p chains, and designed to limit the expression of the endogenous TCR genes (see e.g. Okamoto S. Cancer research 69, 9003-9011 , 2009). Suitably, the vector or polynucleotide encoding the TCRs of the invention may comprise one or more siRNA or other agents aimed at limiting or abrogating the expression of the endogenous TCR genes.

[0163] It is also possible to combine artificial nucleases, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) or CRISPR / Cas systems, designed to target the constant regions of the endogenous genes, e.g. TCR genes (TRAC and, or TRBC), to obtain the permanent disruption of the endogenous TCR alpha and / or beta chain genes, thus allowing full expression of the tumour specific TCR and thus reducing or abrogating the risk of TCR mispairing. This process, known as TCR gene editing, proved superior to TCR gene transfer in vitro and in vivo (see e.g. Provasi E., Genovese P., Nature Medicine May; 18(5):807-15; 2012; Mastaglio S. et al. (2017) Blood 130: 606-618). Suitably, the TCRs of the invention may be used to edit T cell specificity by TCR disruption and genetic addition of the tumour specific TCR.

[0164] In addition, the genome editing technology allows targeted integration of an expression cassette, comprising a polynucleotide encoding a TCR of the invention, and optionally one or more promoter regions and / or other expression control sequences, into an endogenous gene disrupted by the artificial nucleases (see e.g. Lombardo A., Nature biotechnology 25, 1298- 1306; 2007). Suitably, the TCRs of the invention may be used to edit T-cell specificity by targeted integration of a polynucleotide encoding a TCR of the invention at a genomic region. The integration may be targeted by an artificial nuclease.

[0165] A cell, such as a T cell, may therefore be genetically engineered to comprise a TCR of the invention. In addition, a cell, such as a T cell, may be genetically edited by gene disruption, for example TRAC and / or TRBC disruption obtained by, for example, CRISPR / Cas9, or by targeted integration, for example of an expression cassette into an endogenous gene (such as an endogenous gene involved in antigen specificity, persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capacity or other T-cell functions).

[0166] Any suitable TCR gene-editing agents may be used to genetically engineer a cell, such as a T cell. For example, a RNA-guided gene editing system comprising a guide RNA and a RNA- guided nuclease may be used to specifically introduce a double strand break (DSB) into an endogenous gene (e.g. TRAC and / or TRBC or a gene encoding an inhibitory receptor such as CD39, PD-1 , LAG-3, Tim-3, or 2B4). Subsequently, a nucleotide sequence insert encoding a TCR of the invention can be introduced at a DSB site by homology-directed repair (HDR). A CRISPR / Cas9 system is an example of a commonly used RNA-guided gene editing system, but other RNA-guided gene editing systems may also be used.

[0167] Any suitable guide RNA may be used. A “guide RNA” (gRNA) confers target sequence specificity to a RNA-guided nuclease. Guide RNAs are non-coding short RNA sequences which bind to the complementary target DNA sequences. For example, in the CRISPR / Cas9 system, gRNA first binds to the Cas9 enzyme and the gRNA sequence guides the resulting complex via base-pairing to a specific location on the DNA, where Cas9 performs its nuclease activity by cutting the target DNA strand.

[0168] The guide RNA may comprise a trans-activating CRISPR RNA (tracrRNA) that provides the stem loop structure and a target-specific CRISPR RNA (crRNA) designed to cleave the gene target site of interest. The tracrRNA and crRNA may be annealed, for example by heating them at 95°C for 5 minutes and letting them slowly cool down to room temperature for 10 minutes. Alternatively, the guide RNA may be a single guide RNA (sgRNA) that consists of both the crRNA and tracrRNA as a single construct.

[0169] The guide RNA may comprise of a 3’-end, which forms a scaffold for nuclease binding, and a 5'-end which is programmable to target different DNA sites. For example, the targeting specificity of CRISPR-Cas9 may be determined by the 15-25 bp sequence at the 5' end of the guide RNA. The desired target sequence typically precedes a protospacer adjacent motif (PAM) which is a short DNA sequence usually 2-6 bp in length that follows the DNA region targeted for cleavage by the CRISPR system, such as CRISPR-Cas9. The PAM is required for a Cas nuclease to cut and is typically found 3-4 bp downstream from the cut site. After base pairing of the guide RNA to the target, Cas9 mediates a double strand break about 3-nt upstream of PAM.

[0170] Numerous tools exist for designing guide RNAs (e.g. Cui, Y., et al., 2018. Interdisciplinary Sciences: Computational Life Sciences, 10(2), pp.455-465). For example, COSMID is a web- based tool for identifying and validating guide RNAs (Cradick TJ, et al. Mol Ther - Nucleic Acids. 2014;3(12):e214).

[0171] A list of exemplary guide sequences for use in the present invention is provided in the table below. The term "guide sequence" may refer to a sequence within a gRNA that recognizes, e.g., is complementary to, a target sequence, e.g. in a gene, that directs the gRNA to the target sequence for cleavage by a nuclease. A “guide sequence” may also be indicated as a “targeting sequence,” or a “spacer sequence. In some embodiments, the present invention provides a cell, such as a T cell, that has been genetically engineered to insert a polynucleotide encoding a TCR of the present invention, using one or more guide RNA comprising a guide sequence selected from any of SEQ ID NOs: 26-57, or variants thereof having at least 90% sequence identity or at least 95% sequence identity thereto. A cell of the present invention may comprise a polynucleotide encoding a TCR of the present invention at any of the genomic coordinates listed in the table above.

[0172] In some embodiments, the present invention provides a cell, such as a T cell, that has been genetically engineered to insert a polynucleotide encoding a TCR of the present invention, using: (i) one or more guide RNA comprising a guide sequence selected from any of SEQ ID NOs: 26-35, or variants thereof having at least 90% sequence identity or at least 95% sequence identity thereto; (ii) one or more guide RNA comprising a guide sequence selected from any of SEQ ID NOs: 36-48, or variants thereof having at least 90% sequence identity or at least 95% sequence identity thereto; and optionally (iii) one or more guide RNA comprising a guide sequence selected from any of SEQ ID NOs: 49-52, or variants thereof having at least 90% sequence identity or at least 95% sequence identity thereto.

[0173] In some embodiments, the present invention provides a cell, such as a T cell, that has been genetically engineered to insert a polynucleotide encoding a TCR of the present invention, using: (i) one or more guide RNA comprising a guide sequence selected from any of SEQ ID NOs: 26-35, or variants thereof having at least 90% sequence identity or at least 95% sequence identity thereto; (ii) one or more guide RNA comprising a guide sequence selected from any of SEQ ID NOs: 38-42, or variants thereof having at least 90% sequence identity or at least 95% sequence identity thereto.

[0174] In some embodiments, the present invention provides a cell, such as a T cell, that has been genetically engineered to insert a polynucleotide encoding a TCR of the present invention, using: (i) a guide RNA comprising the guide sequence of SEQ ID NO: 26, or a variant thereof having at least 90% sequence identity or at least 95% sequence identity thereto; (ii) a guide RNA comprising the guide sequence of SEQ ID NO: 40, or a variant thereof having at least 90% sequence identity or at least 95% sequence identity thereto; and optionally (iii) a guide RNA comprising the guide sequence of SEQ ID NO: 51 , or a variant thereof having at least 90% sequence identity or at least 95% sequence identity thereto.

[0175] Any suitable RNA-guided nuclease may be used. An “RNA-guided nuclease” is a nuclease which can be directed to a specific site by a guide RNA. RNA-guided nucleases include, but are not limited to, Type II CRISPR nucleases such as Cas9, and Type V CRISPR nucleases such as Cas12a and Cas12b, as well as other nucleases derived therefrom. Suitably, the RNA-guided nuclease is a Type II CRISPR nuclease, for example a Cas9 nuclease. The RNA- guided nuclease may be in a complex with the guide RNA, i.e. the guide RNA and the RNA- guided nuclease may together form a ribonucleoprotein (RNP). Suitably, the RNP is a Cas9 RNP. A RNP may be formed by any method known in the art, for example by incubating a RNA-guided nuclease with a guide RNA for 5-30 minutes at room temperature.

[0176] Another strategy developed to increase expression of the transferred TCR and to reduce TCR mispairing is murinization, which replaces the human TCR a and TCR p constant regions (e.g. the TRAC, TRBC1 and TRBC2 regions) by their murine counterparts. Murizination of TCR constant regions is described in, for example, Sommermeyer and llckert J Immunol; 2010 (184:6223-6231). Suitably, the TCRs of the invention may be murinized.

[0177] Polynucleotides

[0178] In one aspect, the present invention provides a polynucleotide encoding a TCR of the invention or a part thereof, such as the a chain and / or the chain, a variable domain or a portion thereof.

[0179] The polynucleotide may be double or single stranded, and may be RNA or DNA. The polynucleotide may be an isolated polynucleotide.

[0180] It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that the skilled person may, using routine techniques, make nucleotide substitutions, additions or deletions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.

[0181] Polynucleotides such as DNA polynucleotides may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. Longer polynucleotides will generally be produced using recombinant means, for example using polymerase chain reaction (PCR) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking the target sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture with an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0182] The polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of the polynucleotides of the invention. Example TCR #1 sequences

[0183] Examples TCR nucleotide sequences are provided in the tables below.

[0184] The present invention also encompasses the TCR amino acid sequences encoded by the TCR nucleotide sequences provided in the tables below, and variants thereof.

[0185] In one aspect, the present invention provides a polynucleotide comprising or consisting of one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 58 to 62, or variants thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0186] In one aspect, the present invention provides a polynucleotide encoding an a chain, comprising of consisting of SEQ ID NO: 58, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In one aspect, the present invention provides a polynucleotide encoding a p chain, comprising of consisting of SEQ ID NO: 59, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0187] In one aspect, the present invention provides a polynucleotide encoding a chain, comprising of consisting of SEQ ID NO: 60, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0188] In one aspect, the present invention provides a polynucleotide encoding an a chain, comprising of consisting of SEQ ID NO: 61 , or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0189] In one aspect, the present invention provides a polynucleotide encoding a p chain, comprising of consisting of SEQ ID NO: 62, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0190] In one aspect, the present invention provides a TCR comprising an a chain encoded by the nucleotide sequence of SEQ ID NO: 58, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0191] In one aspect, the present invention provides a TCR comprising a p chain encoded by the nucleotide sequence of SEQ ID NO: 59, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0192] In one aspect, the present invention provides a TCR comprising a p chain encoded by the nucleotide sequence of SEQ ID NO: 60, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0193] In one aspect, the present invention provides a TCR comprising an a chain encoded by the nucleotide sequence of SEQ ID NO: 61 , or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In one aspect, the present invention provides a TCR comprising a p chain encoded by the nucleotide sequence of SEQ ID NO: 62, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0194] Further provided by the invention are polynucleotide sequences derived from the sequences present in the table above. For example, the present invention provides a polynucleotide encoding a variable region of a TCR according to the invention, wherein the polynucleotide comprises a stretch of nucleotides of any one of SEQ ID NOs: 58 to 62.

[0195] The variant sequences may have additions, deletions or substitutions, of one or more bases. If the variation involves addition(s) or deletion(s) they may either occur in threes or be balanced (i.e. an addition for each deletion) so that the variation does not cause a frame-shift for translation of the remainder of the sequence. Some or all of the variations may be “silent” in the sense that they do not affect the sequence of the encoded protein due to the degeneracy of the genetic code.

[0196] Some or all of the variations may produce conservative amino acid substitutions, additions or deletions as explained above. The variation may be concentrated in one or more regions, such as the regions encoding the constant regions, the linker, or the framework regions of the a or P chains, or they may be spread throughout the molecule. The variant sequence should retain the capacity to encode all or part of a TCR amino acid sequence which binds to a peptide.

[0197] Example TCR #2 sequences

[0198] Examples of TCR nucleotide sequences are provided in the tables below.

[0199] The present invention also encompasses the TCR amino acid sequences encoded by the TCR nucleotide sequences provided in the tables below, and variants thereof.

[0200] In one aspect, the present invention provides a polynucleotide comprising or consisting of one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 63 to 67, or variants thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0201] In one aspect, the present invention provides a polynucleotide encoding an a chain, comprising of consisting of SEQ ID NO: 63, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0202] In one aspect, the present invention provides a polynucleotide encoding a p chain, comprising of consisting of SEQ ID NO: 64, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0203] In one aspect, the present invention provides a polynucleotide encoding a chain, comprising of consisting of SEQ ID NO: 65, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0204] In one aspect, the present invention provides a polynucleotide encoding an a chain, comprising of consisting of SEQ ID NO: 66, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In one aspect, the present invention provides a polynucleotide encoding a p chain, comprising of consisting of SEQ ID NO: 67, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0205] In one aspect, the present invention provides a TOR comprising an a chain encoded by the nucleotide sequence of SEQ ID NO: 63, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0206] In one aspect, the present invention provides a TCR comprising a chain encoded by the nucleotide sequence of SEQ ID NO: 64, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0207] In one aspect, the present invention provides a TCR comprising a p chain encoded by the nucleotide sequence of SEQ ID NO: 65, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0208] In one aspect, the present invention provides a TCR comprising an a chain encoded by the nucleotide sequence of SEQ ID NO: 66, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0209] In one aspect, the present invention provides a TCR comprising a p chain encoded by the nucleotide sequence of SEQ ID NO: 67, or a variant thereof having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0210] Further provided by the invention are polynucleotide sequences derived from the sequences present in the table above. For example, the present invention provides a polynucleotide encoding a variable region of a TCR according to the invention, wherein the polynucleotide comprises a stretch of nucleotides of any one of SEQ ID NOs: 63 to 67.

[0211] The variant sequences may have additions, deletions or substitutions, of one or more bases. If the variation involves addition(s) or deletion(s) they may either occur in threes or be balanced (i.e. an addition for each deletion) so that the variation does not cause a frame-shift for translation of the remainder of the sequence. Some or all of the variations may be “silent” in the sense that they do not affect the sequence of the encoded protein due to the degeneracy of the genetic code.

[0212] Some or all of the variations may produce conservative amino acid substitutions, additions or deletions as explained above. The variation may be concentrated in one or more regions, such as the regions encoding the constant regions, the linker, or the framework regions of the a or P chains, or they may be spread throughout the molecule. The variant sequence should retain the capacity to encode all or part of a TCR amino acid sequence which binds to a peptide.

[0213] Codon optimisation

[0214] The polynucleotides of the present invention may be codon-optimised.

[0215] Codon optimisation has previously been described in WO 1999 / 41397 and WO 2001 / 79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available.

[0216] Many viruses, including HIV and other lentiviruses, use a large number of rare codons and by changing these to correspond to commonly used mammalian codons, increased expression of the packaging components in mammalian producer cells can be achieved. Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms.

[0217] Codon optimisation may also involve the removal of mRNA instability motifs and cryptic splice sites.

[0218] Variants, derivatives, analogues, and fragments

[0219] In addition to the specific polypeptides and polynucleotides mentioned herein, the invention also encompasses variants, derivatives, analogues, and fragments thereof.

[0220] In the context of the present invention, a “variant” of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question substantially retains at least one of its functions. Suitably, for example, any amino acid changes in the TCR sequences should maintain the capacity of the TCR to bind the peptide presented by MHC molecules.

[0221] A variant amino acid or nucleic acid sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the naturally-occurring polypeptide or polynucleotide. A variant amino acid sequence of the invention referred to as having up to three amino acid substitutions, additions or deletions may have, for example, one, two or three amino acid substitutions, additions or deletions. Suitably, for example, the variation may be concentrated in one or more regions, such as the constant regions, the linker, or the framework regions of the a or p chains, or they may be spread throughout the TCR.

[0222] The term “derivative” as used herein, in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and / or addition of one (or more) amino acid residues from or to the sequence providing that the resultant protein or polypeptide substantially retains at least one of its functions.

[0223] The term “analogue” as used herein, in relation to polypeptides or polynucleotides includes any mimetic, that is, a chemical compound that possesses at least one of the functions of the polypeptides or polynucleotides which it mimics.

[0224] Polypeptides of the present invention may have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues as long as the function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

[0225] Typically, amino acid substitutions may be made, for example from 1 , 2 or 3 to 10 or 20 substitutions provided that the modified sequence substantially retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues. A substitution may involve replacement of an amino acid for a similar amino acid (a conservative substitution). A similar amino acid is one which has a side chain moiety with related properties as grouped together, for example as shown below:

[0226] (i) basic side chains: lysine (K), arginine (R), histidine (H);

[0227] (ii) acidic side chains: aspartic acid (D) and glutamic acid (E); (iii) uncharged polar side chains: asparagine (N), glutamine (Q), serine (S), threonine (T) and tyrosine (Y); or

[0228] (iv) non-polar side chains: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W) and cysteine (C).

[0229] Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

[0230] The effect of additions, deletions, substitutions, modifications, replacements and / or variations may be predicted using any suitable prediction tool e.g. SIFT (Vaser, R., et al., 2016. Nature protocols, 11(1), pp.1-9), PolyPhen-2 (Adzhubei, I., et al., 2013. Current protocols in human genetics, 76(1), pp.7-20), CADD (Rentzsch, P., et al., 2021. Genome medicine, 13(1), pp.1- 12), REVEL (loannidis, N.M., et al., 2016. The American Journal of Human Genetics, 99(4), pp.877-885), MetaLR (Dong, C., et al., 2015. Human molecular genetics, 24(8), pp.2125- 2137), and / or MutationAssessor (Reva, B., et al., 2011. Nucleic acids research, 39(17), pp.e118-e118).

[0231] Typically, a variant may have a certain identity with the subject amino acid sequence or the subject nucleotide sequence. Suitably, reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.

[0232] In the present context, a variant amino acid sequence is taken to include an amino acid sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although a variant can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to express in terms of sequence identity. In the present context, a variant nucleotide sequence is taken to include a nucleotide sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although a variant can also be considered in terms of similarity, in the context of the present invention it is preferred to express it in terms of sequence identity.

[0233] Sequence identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent identity between two or more sequences.

[0234] Percent identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0235] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent identity when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall identity score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local identity.

[0236] However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum percent identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (see e.g. Devereux, J., et al., 1984. Nucleic acids research, 12(1), pp.387-395). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see e.g. Altschul, S.F., et al., 1990. Journal of molecular biology, 215(3), pp.403-410), BLAST 2 (see e.g. Tatusova, T.A. and Madden, T.L., 1999. FEMS microbiology letters, 174(2), pp.247-250), FASTA (see e.g. Pearson, W.R. and Lipman, D.J., 1988. PNAS, 85(8), pp.2444-2448.), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47(W1), pp.W636-W641) and the GENEWORKS suite of comparison tools. For some applications, it is preferred to use EMBOSS Needle.

[0237] Although the final percent identity can be measured, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix.

[0238] Once the software has produced an optimal alignment, it is possible to calculate percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to.

[0239] “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- length polypeptide or polynucleotide.

[0240] Such variants, derivatives, analogues and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded polypeptide. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used. Vectors

[0241] In one aspect, the present invention provides a vector comprising a polynucleotide of the present invention.

[0242] A “vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the invention, and by way of example, some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. The vector may serve the purpose of maintaining the heterologous nucleic acid (DNA or RNA) within the cell, facilitating the replication of the vector comprising a segment of nucleic acid, or facilitating the expression of the protein encoded by a segment of nucleic acid.

[0243] Examples of vectors used in recombinant nucleic acid techniques include, but are not limited to, plasmids, chromosomes, artificial chromosomes and viruses. The vector may be single stranded or double stranded. The vector may be linear or circular. The vector may also be, for example, a naked nucleic acid (e.g. DNA). In its simplest form, the vector may itself be a nucleotide of interest.

[0244] The term “vector" includes an expression vector i.e. a construct capable of in vivo or in vitro / ex vivo expression. Expression may be controlled by a vector sequence, or, for example in the case of insertion at a target site, expression may be controlled by a target sequence. A vector may be integrated or tethered to the cell’s DNA.

[0245] The vectors used in the invention may be, for example, plasmid or viral vectors and may include a promoter for the expression of a polynucleotide and optionally a regulator of the promoter.

[0246] Viral vectors include but are not limited to adenovirus vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a retroviral vector, a lentiviral vector, and a baculoviral vector.

[0247] In some embodiments, the vector is a retroviral vector. Retroviruses are RNA viruses with a life cycle different to that of lytic viruses. In this regard, a retrovirus is an infectious entity that replicates through a DNA intermediate. When a retrovirus infects a cell, its genome is converted to a DNA form by a reverse transcriptase enzyme. The DNA copy serves as a template for the production of new RNA genomes and virally encoded proteins necessary for the assembly of infectious viral particles.

[0248] There are many retroviruses, for example murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV) and all other retroviridiae including lentiviruses. A detailed list of retroviruses may be found in Coffin et al (“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763). Lentiviruses also belong to the retrovirus family, but they can infect both dividing and non-dividing cells (Lewis et al (1992) EMBO J. 3053-3058).

[0249] The vector may be capable of transferring a nucleotide sequence encoding a TCR described herein to a cell, such as a T-cell, such that the cell expresses the TCR. Suitably, the vector will be capable of sustained high-level expression in T-cells, so that the introduced TCR may compete successfully with the endogenous TCR for a limited pool of CD3 molecules.

[0250] Increasing the supply of CD3 molecules may increase TCR expression, for example, in a cell that has been modified to express the TCRs of the invention. Accordingly, the vector of the invention may further comprise one or more genes encoding CD3-gamma, CD3-delta, CD3- epsilon and / or CD3-zeta. In some embodiments, the vector of the invention comprises a gene encoding CD3-zeta. The vector may comprise a gene encoding CD8. The vector may encode a selectable marker or a suicide gene, to increase the safety profile of the genetically engineered cell, e.g. a cell of the invention, or a cell that has been modified to express the TCRs of the invention (Bonini, C., et al., 1997. Science, 276(5319), pp.1719-1724; Ciceri, F., et al., 2009. The lancet oncology, 10(5), pp.489-500; Oliveira, G., et al., 2015. Science translational medicine, 7(317), p.317ra198). The genes comprised in the vector of the invention may be linked by self-cleaving sequences, such as the 2A self-cleaving sequence.

[0251] Alternatively one or more separate vectors encoding a CD3 gene may be provided for cotransfer to a cell simultaneously, sequentially or separately with one or more vectors of the invention, e.g. one or more vectors encoding TCRs of the invention.

[0252] Cells

[0253] In one aspect, the present invention provides a cell or a population of cells comprising a TCR, a polynucleotide or a vector according to the present invention.

[0254] The cell is not particularly limited and any suitable cell may be used. For example, the cell may be a T-cell, a lymphocyte, or a stem cell. The T-cell, the lymphocyte, or the stem cell may be selected from the group consisting of CD4+ cells, CD8+ cells, naive T-cells, memory stem T-cells, central memory T-cells, double negative T-cells, effector memory T-cells, effector T- cells, ThO cells, TcO cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, gamma / delta T-cells, natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, hematopoietic stem cells and pluripotent stem cells.

[0255] The type of cell may be selected in order to provide desirable and advantageous in vivo persistence and to provide desirable and advantageous functions and characteristics to the cells of invention.

[0256] The cell may have been isolated from a subject.

[0257] The cell of the invention may be provided for use in adoptive cell transfer. As used herein, the term “adoptive cell transfer” may refer to the administration of a cell population to a patient. Typically, the cells are T-cells isolated from a subject and then genetically modified and cultured in vitro in order to express a TCR of the invention before being administered to the patient. Adoptive cell transfer may be allogenic or autologous.

[0258] By “autologous cell transfer” it is to be understood that the starting population of cells (which are then transduced according to a method of the invention, or are transduced with a vector according to the invention) is obtained from the same subject as that to which the transduced cell population is administered. Autologous transfer is advantageous as it avoids problems associated with immunological incompatibility and is available to subjects irrespective of the availability of a genetically matched donor.

[0259] By “allogeneic cell transfer” it is to be understood that the starting population of cells (which are then transduced according to a method of the invention, or are transduced with a vector according to the invention) is obtained from a different subject as that to which the transduced cell population is administered. Suitably, the donor will be genetically matched to the subject to which the cells are administered to minimise the risk of immunological incompatibility. Alternatively, the donor may be mismatched and unrelated to the patient.

[0260] Suitable doses of transduced cell populations are such as to be therapeutically and / or prophylactically effective. The dose to be administered may, for example, depend on the subject and condition to be treated, and may be readily determined by a skilled person.

[0261] In some embodiments, the cell is a T-cell. The cell may be derived from a T-cell isolated from a subject. The T-cell may be part of a mixed cell population isolated from the subject, such as a population of peripheral blood lymphocytes (PBL). T-cells within the PBL population may be activated by methods known in the art, such as using anti-CD3 and / or anti-CD28 antibodies or cell sized beads conjugated with anti-CD3 and / or anti-CD28 antibodies. The T-cell may be a CD4+helper T cell or a CD8+cytotoxic T cell. The cell may be in a mixed population of CD4+helper T cell / CD8+cytotoxic T-cells. Polyclonal activation, for example using anti-CD3 antibodies optionally in combination with anti-CD28 antibodies will trigger the proliferation of CD4+and CD8+T-cells.

[0262] The cell may be isolated from the subject to which the genetically modified cell is to be adoptively transferred. In this respect, the cell may be made by isolating a T-cell from a subject, optionally activating the T-cell, transferring the TCR gene to the cell ex vivo. Subsequent immunotherapy of the subject may then be carried out by adoptive transfer of the TCR- transduced cells. As used herein this process refers to autologous T-cell transfer i.e. the TCR- transduced cells are administered to the same subject from which the T-cells were originally derived.

[0263] Alternatively the T-cell may be isolated from a different subject, such that it is allogeneic. The T-cell may be isolated from a donor subject. For example, if the subject is undergoing allogeneic hematopoietic stem cell transplantation (Allo-HSCT) or solid organ transplantation or cell transplantation or stem cell therapy, the cell may be derived from the donor, from which the organs, tissues or cells are derived. The donor and the subject undergoing treatment may be siblings.

[0264] Alternatively the cell may be, or may be derived from, a stem cell, such as a hematopoietic stem cell (HSC). Gene transfer into HSCs does not lead to TCR expression at the cell surface as stem cells do not express CD3 molecules. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, the initiation of CD3 expression leads to the surface expression of the introduced TCR in thymocytes.

[0265] An advantage of this approach is that the mature T-cells, once produced, express only the introduced TCR and little or no endogenous TCR chains, because the expression of the introduced TCR chains suppresses rearrangement of endogenous TCR gene segments to form functional TCR alpha and beta genes. A further benefit is that the gene-modified stem cells are a continuous source of mature T-cells with the desired antigen specificity. The cell may therefore be a gene-modified stem cell, preferably a gene-modified hematopoietic stem cell, which, upon differentiation, produces a T-cell expressing a TCR of the invention.

[0266] Other approaches known in the art may be used to reduce, limit, prevent, silence, or abrogate expression of endogenous genes in the cells of the invention or cells prepared by the methods of the invention. As used herein the term “disrupting” refers to reducing, limiting, preventing, silencing, or abrogating expression of a gene. The person skilled in the art is able to use any method known in the art to disrupt an endogenous gene, e.g., any suitable method for genome editing, gene silencing, gene knock-down or gene knock-out.

[0267] For example, an endogenous gene may be disrupted with an artificial nuclease. An artificial nuclease is, e.g., an artificial restriction enzyme engineered to selectively target a specific polynucleotide sequence (e.g. encoding a gene of interest) and induce a double strand break in said polynucleotide sequence. Typically, the double strand break (DSB) will be repaired by error-prone non-homologous end joining (NHEJ) thereby resulting in the formation of a nonfunctional polynucleotide sequence, which may be unable to express an endogenous gene. In some embodiments, the artificial nuclease is selected from the group consisting of zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and CRISPR / Cas (e.g. CRISPR / Cas9).

[0268] The methods of preparing a cell (e.g. a T-cell) of the invention may comprise the step of targeted integration of an expression cassette into an endogenous gene (e.g. an endogenous TCR a chain gene and / or an endogenous TCR p chain gene). As used herein the term expression cassette refers to a polynucleotide sequence (e.g. a DNA polynucleotide sequence) comprising one or more polynucleotide sequences encoding one or more genes of interest such that said genes of interest are capable of expression. Endogenous sequences may facilitate expression from the expression cassette, and / or transcription control sequences within the expression cassette may facilitate expression. For example, the expression cassette may comprise a polynucleotide sequence of the invention, or a polynucleotide sequence encoding a TCR of the invention, operably linked to an expression control sequence, e.g. a promoter or an enhancer sequence. The one or more genes of interest may be located between one or more sets of restriction sites. Suitably, the restriction sites may facilitate the integration of the expression cassette into, e.g., a vector, a plasmid, or genomic DNA (e.g. host cell genomic DNA).

[0269] For example, an expression cassette of the invention may be transferred from a first polynucleotide sequence, e.g. on a vector, to another by 'cutting', e.g. excising, the expression cassette using one or more suitable restriction enzymes and 'pasting', e.g. integrating, the expression cassette into a second polynucleotide sequence.

[0270] The expression cassette may comprise a polynucleotide of the invention. The expression cassette may comprise a polynucleotide encoding one or more TCRs of the invention. The expression cassette may further comprise an antibiotic resistance gene or other selectable marker gene that allows cells that have successfully integrated the expression cassette into their DNA to be identified. The polynucleotide sequences comprised in the expression cassette may be operably linked to expression control sequences, e.g. a suitable promoter or enhancer sequence. The person skilled in the art will be able to select suitable expression control sequences.

[0271] The invention also contemplates a cell expressing a TCR of the invention, which has been engineered to disrupt one or more endogenous MHC genes. Disruption of an endogenous MHC gene can reduce or prevent expression of MHC on the engineered cell surface. Accordingly, such an engineered cell with reduced or no MHC expression will have limited or no capacity to present antigens on its cell surface. Such a cell is particularly advantageous for adoptive cell transfer since the cell will be non-alloreactive, e.g., the cell will not present antigens which could be recognized by the immune system of a subject receiving the adoptively transferred cell. As a result, the transferred cell will not be recognized as ‘non-self’ and an adverse immune reaction to the cell can be avoided. Such a cell is termed a ‘universal cell’ since it is suitable for adoptive transfer to a variety of different hosts regardless of HLA type. The invention provides a method of preparing a non-alloreactive universal T-cell, which expresses a TCR of the invention. Further provided by the invention is a non-alloreactive universal T-cell, which expresses a TCR of the invention.

[0272] The invention further contemplates cells which have been engineered to disrupt one or more endogenous genes to modify the cell to enhance advantageous properties, characteristics or functions of the cell and / or reduce undesirable properties, characteristics or functions. For example, by disrupting an endogenous cell the persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capacity, or other cell functions may be modified. As used in this context, the term “modify” may refer to a change in one or more characteristics relative to an equivalent unmodified cell, e.g. a cell in which an endogenous gene has not been disrupted. For example, the change may be an increase, an enhancement or an introduction of a characteristic or function of the cell relative to an equivalent unmodified cell. Alternatively, the change may be a decrease, suppression or abrogation of a characteristic or function of the cell relative to an equivalent unmodified cell.

[0273] The polynucleotides and vectors of the invention may be transferred into specific T-cell subsets, including CD4 and / or CD8, naive, memory stem T cells, central memory, effector memory or effector cells, or in other cellular subsets such as to promote different in vivo length of persistence and function in the cells of the invention. The polynucleotides and vectors of the invention may also be transferred into T-cell subsets with different polarizations, such as ThO / TcO, Th1 / Tc1 , Th2 / Tc2, Th17, Th22 or others, depending on the cytokine background most appropriate to target a particular tumour type.

[0274] Furthermore, the polynucleotides and vectors of the invention encoding the antigen-specific regions of the TCRs of the present invention may be transferred into other cellular subsets, including gamma / delta T-cells, NK cells, NKT cells, cytokine-induced killer (CIK) cells, hematopoietic stem cells or other cells, in order to obtain the therapeutic effect.

[0275] Methods of preparing a cell

[0276] In one aspect, the present invention provides a method of preparing a cell or a population of cells comprising a TCR, a polynucleotide or a vector according to the present invention.

[0277] The polynucleotide or vector of the present invention may be introduced into the cell or population of cells in vitro, ex vivo or in vivo. Suitably, the polynucleotide or vector of the present invention is introduced in vitro or ex vivo.

[0278] The polynucleotide or vector of the present invention may be introduced into cells using a variety of techniques known in the art, such as transformation, transfection and transduction. Several techniques are known in the art, for example transduction with recombinant viral vectors, such as retroviral, lentiviral, adenoviral, adeno-associated viral, baculoviral and herpes simplex viral vectors, Sleeping Beauty vectors; direct injection of nucleic acids and biolistic transformation.

[0279] Non-viral delivery systems include but are not limited to transfection methods. Here, transfection includes a process using a non-viral vector to deliver a gene to a target cell. Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs), and combinations thereof.

[0280] In addition, the invention may employ gene targeting protocols, for example the delivery of DNA-modifying agents.

[0281] In one aspect, the present invention provides a method of preparing a cell, which comprises the step of transducing a cell in vitro or ex vivo with a vector of the invention. In one aspect, the present invention provides a method of preparing a T-cell expressing a TCR of the invention by inducing the differentiation of a stem cell which comprises a polynucleotide or a vector of the invention.

[0282] A population of cells may be purified selectively for cells that exhibit a specific phenotype or characteristic, and from other cells which do not exhibit that phenotype or characteristic, or exhibit it to a lesser degree. For example, a population of cells that expresses a specific marker (e.g. CD3, CD4, CD8, CD25, CD127, CD152, CXCR3, or CCR4) may be purified from a starting population of cells. Alternatively, or in addition, a population of cells that does not express another marker may be purified.

[0283] By “enriching” a population of cells for a certain type of cells it is to be understood that the concentration of that type of cells is increased within the population. The concentration of other types of cells may be concomitantly reduced. Purification or enrichment may result in the population of cells being substantially pure of other types of cell.

[0284] Purifying or enriching for a population of cells expressing a specific marker (e.g. CD3, CD4, CD8, CD25, CD127, CD152, CXCR3, or CCR4) may be achieved by using an agent that binds to that marker, preferably substantially specifically to that marker. An agent that binds to a cellular marker may be an antibody, for example antibody which binds to CD3, CD4, CD8, CD25, CD127, CD152, CXCR3, or CCR4.

[0285] The term “antibody” may refer to complete antibodies or antibody fragments capable of binding to a selected target, including Fv, ScFv, F(ab’) and F(ab’)2, monoclonal and polyclonal antibodies, engineered antibodies including chimeric, CDR-grafted and humanised antibodies, and artificially selected antibodies produced using phage display or alternative techniques. In addition, alternatives to classical antibodies may also be used in the invention, for example “avibodies”, “avimers”, “anticalins”, “nanobodies” and “DARPins”.

[0286] The agents that bind to specific markers may be labelled so as to be identifiable using any of a number of techniques known in the art. The agent may be inherently labelled, or may be modified by conjugating a label thereto. By “conjugating” it is to be understood that the agent and label are operably linked. This means that the agent and label are linked together in a manner which enables both to carry out their function (e.g. binding to a marker, allowing fluorescent identification, or allowing separation when placed in a magnetic field) substantially unhindered. Suitable methods of conjugation are well known in the art and would be readily identifiable by the skilled person. A label may allow, for example, the labelled agent and any cell to which it is bound to be purified from its environment (e.g. the agent may be labelled with a magnetic bead or an affinity tag, such as avidin), detected or both. Detectable markers suitable for use as a label include fluorophores (e.g. green, cherry, cyan and orange fluorescent proteins) and peptide tags (e.g. His tags, Myc tags, FLAG tags and HA tags).

[0287] A number of techniques for separating a population of cells expressing a specific marker are known in the art. These include magnetic bead-based separation technologies (e.g. closed- circuit magnetic bead-based separation), flow cytometry, fluorescence-activated cell sorting (FACS), affinity tag purification (e.g. using affinity columns or beads, such as biotin columns to separate avidin-labelled agents) and microscopy-based techniques.

[0288] It may also be possible to perform the separation using a combination of different techniques, such as a magnetic bead-based separation step followed by sorting of the resulting population of cells for one or more additional (positive or negative) markers by flow cytometry. Clinical grade separation may be performed, for example, using the CliniMACS® system (Miltenyi). This is an example of a closed-circuit magnetic bead-based separation technology.

[0289] It is also envisaged that dye exclusion properties (e.g. side population or rhodamine labelling) or enzymatic activity (e.g. ALDH activity) may be used to enrich for HSCs.

[0290] Chimeric molecules

[0291] In one aspect, the present invention provides a chimeric molecule comprising a TCR of the invention, a TCR encoded by a polynucleotide of the invention, ora portion thereof, conjugated to a non-cellular substrate.

[0292] The conjugation may be covalent or non-covalent. The non-cellular substrate may be a nanoparticle, an exosome, or any non-cellular substrate known in the art. The chimeric molecule of the invention may be soluble.

[0293] In one aspect, the present invention provides a chimeric molecule comprising a TCR of the invention, a TCR encoded by a polynucleotide of the invention, ora portion thereof, conjugated to a toxin or an antibody.

[0294] The toxin or antibody may be cytotoxic. The toxin may be a cytotoxic molecule or compound, e.g. a radioactive molecule or compound. The TCR portion of the chimeric molecule may confer the ability to recognize tumour cells. Thus, the chimeric molecule may specifically recognize and / or bind to tumour cells. Accordingly, the chimeric molecules of the invention may provide tumour-targeted delivery of cytotoxic toxins, antibodies and / or compounds. Pharmaceutical compositions

[0295] In one aspect, the present invention provides a pharmaceutical composition comprising a TCR according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, a cell or a population of cells according to the present invention, a cell or a population of cells prepared by a method according to the present invention, or a chimeric molecule according to the present invention.

[0296] A “pharmaceutical composition” is a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent. A pharmaceutical composition preferably includes a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).

[0297] By “pharmaceutically acceptable” is included that the formulation is sterile and pyrogen free. The carrier, diluent, and / or excipient must be “acceptable” in the sense of being compatible with the pharmaceutically active agent and not deleterious to the recipients thereof.

[0298] Typically, the carriers, diluents, and excipients will be saline or infusion media which will be sterile and pyrogen free, however, other acceptable carriers, diluents, and excipients may be used. Acceptable carriers, diluents, and excipients for therapeutic use are well known in the pharmaceutical art. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s).

[0299] The TCR, polynucleotide, vector, cell, population of cells, chimeric molecule, or pharmaceutical composition of the present invention may be administered in a manner appropriate for treating and / or preventing the diseases described herein. Suitable administration routes will be known to the skilled person. The quantity and frequency of administration may be determined by the skilled person, for example depending by such factors as the condition of the subject, and the type and severity of the subject's disease. The pharmaceutical composition may be formulated accordingly.

[0300] In one aspect, the present invention provides a pharmaceutical composition comprising the cell or population of cells according to the present invention. The cells of the invention may be formulated for administration to subjects with a pharmaceutically acceptable carrier, diluent or excipient. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline, and potentially contain human serum albumin. Handling of the cell therapy products is preferably performed in compliance with FACT-JACIE International Standards for cellular therapy.

[0301] The pharmaceutical composition may further comprise one or more other therapeutic agents

[0302] The invention further includes kits comprising the TCR, polynucleotide, vector, cell, population of cells, chimeric molecule, or pharmaceutical composition of the present invention. Preferably said kits are for use in the methods and used as described herein, e.g., the therapeutic methods as described herein. Preferably said kits comprise instructions for use of the kit components.

[0303] Methods of treatment

[0304] In one aspect, the present invention provides the TCR, polynucleotide, vector, cell, population of cells, chimeric molecule, or pharmaceutical composition according to the present invention for use as a medicament.

[0305] In one aspect, the present invention provides use of the TCR, polynucleotide, vector, cell, population of cells, chimeric molecule, or pharmaceutical composition according to the present invention in the manufacture of a medicament.

[0306] In one aspect, the present invention provides a method of administering a therapeutically effective amount of the TCR, polynucleotide, vector, cell, population of cells, chimeric molecule, or pharmaceutical composition according to the present invention to a subject in need thereof.

[0307] The TCR, polynucleotide, vector, cell, population of cells, chimeric molecule, or pharmaceutical composition may be administered to any subject in need thereof. The subject may be a mammal. The subject may be a human subject. The human subject may be a child. For example, the child may be less than 10 years in age, less than 9 years in age, less than 8 years in age, less than 7 years in age, less than 6 years in age, less than 5 years in age, less than 4 years in age, less than 3 years in age, or less than 2 years in age. The human subject may be an infant. The subject may be a non-human animal subject.

[0308] The subject may have a proliferative disorder. The subject may be at risk of developing a proliferative disorder. The subject may have been previously determined to be at risk of developing a proliferative disorder. The subject may have an increased risk of developing a proliferative disorder. The increased risk may have been determined by genetic screening and / or by reviewing the subject’s family history. The subject may express genetic markers indicative of increased risk of developing a proliferative disorder. Suitably, a person skilled in the art will be aware of genetic risk factors (e.g. genetic markers) associated with increased risk of developing a proliferative disorder. The skilled person may be able to use any suitable method or technique known in the art to determine whether the subject has an increased risk of developing a proliferative disorder.

[0309] The subject may have previously received treatment for a proliferative disorder. The subject may be in remission. The subject may be resistant to chemotherapy.

[0310] Proliferative disorders

[0311] The TCR, polynucleotide, vector, cell, population of cells, chimeric molecule, or pharmaceutical composition according to the present invention may be used to prevent and / or treat a proliferative disorder.

[0312] In one aspect, the present invention provides the TCR, polynucleotide, vector, cell, population of cells, chimeric molecule, or pharmaceutical composition according to the present invention for use in preventing and / or treating a proliferative disorder.

[0313] In one aspect, the present invention provides use of the TCR, polynucleotide, vector, cell, population of cells, chimeric molecule, or pharmaceutical composition according to the present invention in the manufacture of a medicament for preventing and / or treating a proliferative disorder.

[0314] In one aspect, the present invention provides a method of preventing and / or treating a proliferative disorder, the method comprising administering a therapeutically effective amount of the TCR, polynucleotide, vector, cell, population of cells, chimeric molecule, or pharmaceutical composition according to the present invention to a subject in need thereof.

[0315] As used herein, the term “preventing” may refer to averting, delaying, impeding or hindering the contraction of the disease. The treatment may, for example, prevent or reduce the likelihood of developing or contracting a proliferative disorder.

[0316] As used herein, the term “treating” may refer to caring for a diseased subject, in order to ameliorate, cure or reduce the symptoms of the disease, or in order to reduce, halt or delay the progression of the disease.

[0317] The proliferative disorder may be a hematological malignancy or a solid tumour.

[0318] As used herein, a “hematological malignancy” may refer to a cancer that affects the blood, bone marrow, and lymph nodes. The hematological malignancy may be selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, acute lymphocytic leukemia (ALL), myelodisplastic syndromes, lymphoma, multiple myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma.

[0319] As used herein, a “solid tumour” may refer to an abnormal mass of tissue that usually does not contain cysts or liquid areas. The solid tumour may be selected from the group consisting of lung cancer, breast cancer, oesophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancers, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, oral cancer, hepatocellular carcinoma, bladder cancer, endometrial cancer, neuroblastoma, rabdomyosarcoma, liver cancer, melanoma, prostate cancer, renal cancer, soft tissue sarcoma, urothelial cancer, biliary cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.

[0320] The proliferative disorder may be selected from a group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, acute lymphocytic leukemia (ALL), myelodisplastic syndromes, lymphoma, multiple myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma, lung cancer, breast cancer, oesophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancers, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, oral cancer, hepatocellular carcinoma, bladder cancer, endometrial cancer, neuroblastoma, rabdomyosarcoma, liver cancer, melanoma, prostate cancer, renal cancer, soft tissue sarcoma, urothelial cancer, biliary cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.

[0321] In some embodiments, the proliferative disorder is acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). In some embodiments, the proliferative disorder is acute myeloid leukemia (AML).

[0322] In some embodiments, the proliferative disorder is renal cancer.

[0323] In some embodiments, the method for treating and / or preventing a proliferative disorder comprises the step of administering a chemotherapy to the subject. The chemotherapy may be administered to the subject simultaneously, sequentially or separately with the TCR, polynucleotide, vector, cell, population of cells, chimeric molecule, or pharmaceutical composition of the invention.

[0324] The practice of the invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology, histology, immunology, oncology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example: Skoog, D.A., et al. (2013) Fundamentals of Analytical Chemistry, 9th edition, Cengage learning; Walker J. M. (2009) The Protein Protocols Handbook, 3rd edition, Springer Nature; Green, M.R. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M., et al. (2003) Current Protocols in Molecular Biology, John Wiley & Sons; Hill, A. J. (2013) DNA Sequencing Protocols, Humana Press; Nielsen, B.S. and Jones, J. (2021) In Situ Hybridization Protocols, Springer US; Herdewijn, P. (2010) Oligonucleotide Synthesis: Methods and Applications, Humana Press; and Luo, Y. (2019) CRISPR Gene Editing: Methods and Protocols, Springer New York. Each of these general texts is herein incorporated by reference.

[0325] EXAMPLES

[0326] The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0327] Example 1 - Isolation of tumour-specific TCRs

[0328] The inventors investigated whether the T cell fraction co-expressing multiple inhibitory receptors (I Rs) could represent a target population to isolate tumour-specific TCRs.

[0329] For this purpose, T cells from two acute myeloid leukaemia patients (Pt#14-15) were harvested from the bone marrow at the moment of relapse, when IR expression was the highest. T cells were sorted into a fraction positive for multiple I Rs (IR+) and into a control group negative for all I Rs (IR-) and serially stimulated with autologous blasts forced to mature into Leukemic antigen-presenting cells (L-APCs) expressing co-stimulatory molecules and the blast ligandome (Figure 1).

[0330] The IR' and IR+TCR clonality was then assessed for the two patients. Despite a comparable baseline, at the end of L-APCs stimulations the IR+fraction was enriched for a dominant TCR clone, present at low frequency ex vivo (Figure 2).

[0331] The dominant IR+TCRs were then reconstructed and engineered into previously TCR-KO T cells. Engineered T cells efficiently lysed autologous leukemic blasts (Figure 3) while releasing cytokines, degranulating and activating, but failed to recognize autologous healthy hematopoietic cells harvested at remission before allo-HSCT (Figure 3 and 4).

[0332] Example 2 - Identification of target cells expressing the antigens recognized by the orphan TCRs The inventors investigated whether trogocytosis phenomenon could be adapted to de- orphanize TCRs expressed by engineered T cells and specific for patients’ blasts. Trogocytosis is a phenomenon of intracellular cell membrane exchange where T cells can translocate part of their immunological synapse, such as the TCR / CD3 complex, on the proper target cells. The inventors co-incubated T cells engineered to express the two orphan IR+- derived Pt#14 and Pt#15 TCRs with autologous blasts and observed that a limited but detectable fraction of blasts acquired CD3 on cell surface, in a target: effector-dependent manner (Figure 5). These data suggest that the fraction of blasts that had “eaten up” pieces of engineered T cell membranes were the ones that specifically express or express at higher levels the antigens recognized by the orphan TCRs #14 and #15.

[0333] Example 3 - Materials and Methods

[0334] Inhibitory receptor-based T cell sorting and expansion

[0335] Lymphocyte samples from frozen bone marrow samples from two patients who underwent allo-HSCT were typed for the surface expression of Inhibitory receptors. T cells were sorted via Fluorescence-Activated Cell Sorting into two fractions, one expressing no IRs (IR-) and the other co-expressing multiple IRs (IR+), according to the IR asset specific of each patient. Cells were then resuspended in complete X-Vivo and subsequently stimulated using autologous Leukemic antigen-presenting cells (L-APC), derived from autologous Leukemic blasts forced to express an array of co-stimulatory molecules while maintain the blast ligandome (Casucci, M., et al. Molecular Therapy, 21(2), pp.466-475), in order to convey a leukemia-specific activatory signal. Stimulation was performed every two weeks, until expansion halted. Autologous blasts were forced to differentiated into L-APC via a two-days culture in complete X-Vivo supplemented with IL-4 (250 lU / ml) and Calcimycin (375 ng / ml, Sigma).

[0336] TCR repertoire sequencing

[0337] RNA samples were collected at each round of stimulation to perform TCR sequencing. RNA was extracted by using Arcturus Pico Pure RNA extraction kit. Complementarity determining region (CDR) 3 sequences of all different antigen-specific T cells were amplified by using a modified RACE approach (Ruggiero, E., et al. Nature communications, 6(1), pp.1-7). Samples were sequenced by using an Illumina MiSeq sequencer and CDR3 clonotypes identified using the MiXCR software (Bolotin, D.A., et al., 2015. Nature methods, 12(5), pp.380-381).

[0338] Co-culture assays against primary cells T cells engineered with either Pt#14 or Pt#15 TCRs were co-cultured with autologous AML blasts (targets) or autologous Bone Marrow cells (controls) in X-VIVO supplemented with 5% human AB serum and supplemented with the CD28 monoclonal antibody (1 pg / ml). To assess cytokine release, Golgi Stop (BD) and CD107a-FITC antibody was added to the culture, and after 6 hours cells were fixed (FoxP3 Fix / Perm buffer (Biolegend), 20 mins), permeabilized (FoxP3 Perm buffer Biolegend, 20 mins) and then treated to perform an intracellular staining. T cell specific response was assessed by measuring CD107a, I FNy, and TNFa expression. For killing assays, the number of alive target or control cells remaining in culture after 24 hours was assessed by flow cytometry-based cell count. HLA-DR expression on engineered T cells was quantified by flow cytometry.

[0339] Troqocytosis-based sorting of blasts targeted by Pt#14 and Pt#15 TCRs

[0340] In order to characterize the fraction of target cells recognized by the previously isolated orphan IR+ TCRs, we harnessed the mechanism of trogocytosis, a phenomenon of intracellular cell membrane exchange, where T cells can translocate part of their immunological synapse on target cells (e.g. tumour cells), so that target cells can acquire the T cell specific TCR or its co-receptors such as CD3.

[0341] Edited T cells expressing Pt#14 or Pt#15 orphan TCRs were co-cultured with autologous blasts. Then, blasts were sorted into fractions experiencing (i.e., CD3+ blasts) or not experiencing (CD3- blasts) trogocytosis. Sorted blasts can be used to identify the antigen recognized by the newly identified orphan TCRs.

[0342] EMBODIMENTS

[0343] Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paras).

[0344] 1. A T-cell receptor (TCR) comprising a CDR3a comprising the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3P comprising the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11) or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0345] 2. The TCR according to para 1 , wherein the TCR comprises the following CDR sequences:

[0346] CDR1a - NSASDY (SEQ ID NO: 4),

[0347] CDR2a - IRSNMDK (SEQ ID NO: 5),

[0348] CDR3a - CAENPPGTGNQFYF (SEQ ID NO: 6),

[0349] CDR1 P - SEHNR (SEQ ID NO: 9),

[0350] CDR2P - FQNEAQ (SEQ ID NO: 10), and

[0351] CDR3P - CASSLQGPGELFF (SEQ ID NO: 11), or variants thereof each having up to three amino acid substitutions, additions or deletions.

[0352] 3. The TCR according to para 1 or 2, wherein the TCR comprises an a chain variable domain comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a p chain variable domain comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0353] 4. The TCR according to any of paras 1 to 3, wherein the TCR comprises an a chain comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a p chain comprising the amino acid sequence of SEQ ID NO: 13 or 14, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. 5. A T-cell receptor (TCR) comprising a CDR3a comprising the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3P comprising the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22) or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0354] 6. The TCR according to para 5, wherein the TCR comprises the following CDR sequences:

[0355] CDR1a - TSENNYY (SEQ ID NO: 15),

[0356] CDR2a - QEAYKQQN (SEQ ID NO: 16),

[0357] CDR3a - CAFMKTGGTSYGKLTF (SEQ ID NO: 17),

[0358] CDR1 P - KGHSH (SEQ ID NO: 20),

[0359] CDR2P - LQKENI (SEQ ID NO: 21), and

[0360] CDR3P - CASSPVAGGFYEQYF (SEQ ID NO: 22), or variants thereof each having up to three amino acid substitutions, additions or deletions.

[0361] 7. The TCR according to para 5 or 6, wherein the TCR comprises an a chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a p chain variable domain comprising the amino acid sequence of SEQ ID NO: 23 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0362] 8. The TCR according to any of paras 5 to 7, wherein the TCR comprises an a chain comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; and a p chain comprising the amino acid sequence of SEQ ID NO: 24 or 25, or variants thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0363] 9. The TCR according to any preceding para, wherein the TCR comprises one or more mutations at the a chain / p chain interface, such that when the a chain and the p chain are expressed in a T-cell, the frequency of mispairing between said chains and endogenous TCR a and p chains is reduced, optionally wherein the one or more mutations introduce a cysteine residue into the constant region domain of each of the a chain and the p chain, wherein the cysteine residues are capable of forming a disulphide bond between the a chain and the chain.

[0364] 10. The TCR according to any preceding para, wherein the TCR comprises a murinised constant region.

[0365] 11 . The TCR according to any preceding para, wherein the TCR is a soluble TCR.

[0366] 12. The TCR according to any preceding para, wherein the TCR is a tumour-antigen specific TCR.

[0367] 13. A polynucleotide encoding the a chain and / or the p chain of a TCR according to any preceding para.

[0368] 14. The polynucleotide according to para 13, wherein the polynucleotide encodes the a chain linked to the p chain.

[0369] 15. The polynucleotide according to para 13 or 14, wherein the polynucleotide further encodes one or more short interfering RNA (siRNA) or other agents capable of reducing or preventing expression of one or more endogenous TCR genes.

[0370] 16. A vector comprising a polynucleotide according to any of paras 13-15.

[0371] 17. The vector according to para 16, wherein the vector is a plasmid or a viral vector.

[0372] 18. The vector according to para 16 or 17, wherein the vector comprises a polynucleotide which encodes one or more CD3 chains, CD8, a suicide gene and / or a selectable marker.

[0373] 19. A cell comprising a TCR according to any of paras 1-12, a polynucleotide according to any of paras 13-15 or a vector according to any of paras 16-18.

[0374] 20. The cell according to para 19, wherein the cell further comprises a vector which encodes one or more CD3 chains, CD8, a suicide gene and / or a selectable marker.

[0375] 21. The cell according to para 19 or 20, wherein the cell is a T-cell, a lymphocyte, or a stem cell.

[0376] 22. The cell according to para 21 , wherein the T-cell, the lymphocyte, or the stem cell is selected from the group consisting of CD4+ cells, CD8+ cells, naive T-cells, memory stem T- cells, central memory T-cells, double negative T-cells, effector memory T-cells, effector T- cells, ThO cells, TcO cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, gamma / delta T-cells, natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, hematopoietic stem cells and pluripotent stem cells.

[0377] 23. The cell according to any of paras 19 to 22, wherein the cell is a T-cell, optionally wherein the cell is a T-cell which has been isolated from a subject.

[0378] 24. The cell according to any of paras 19 to 23, wherein an endogenous gene encoding a TCR a chain and / or an endogenous gene encoding a TCR p chain is disrupted, preferably such that the endogenous gene encoding a TCR a chain and / or the endogenous gene encoding a TCR chain is not expressed, optionally wherein the endogenous gene encoding a TCR a chain and / or the endogenous gene encoding a TCR p chain is disrupted by insertion of an expression cassette comprising a polynucleotide sequence encoding the TCR of any of paras 1-12.

[0379] 25. The cell according to any of paras 19 to 24, wherein one or more endogenous genes encoding an MHC is disrupted.

[0380] 26. The cell according to any of paras 19 to 25, wherein an endogenous gene involved in persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capacity, or other T-cell functions is disrupted, optionally wherein the endogenous gene involved in persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capacity, or other T-cell functions is selected from the group consisting of PD1, TIM3, LAG3, 2B4, KLRG1, TGFbR, CD160, TIGIT, CTLA4 and CD39.

[0381] 27. A method of preparing a cell, which comprises the step of introducing a polynucleotide according to any of paras 13-15 or a vector according any of paras 16-18 into a cell in vitro, ex vivo or in vivo, for example by transfection or transduction.

[0382] 28. The method according to para 27, wherein the method comprises the step of T-cell editing, which comprises disrupting an endogenous gene encoding a TCR a chain and / or an endogenous gene encoding a TCR p chain with an artificial nuclease, preferably wherein the artificial nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR / Cas systems.

[0383] 29. The method according to para 28, wherein the method comprises the step of targeted integration of an expression cassette into the endogenous gene encoding the TCR a chain and / or the endogenous gene encoding the TCR chain disrupted by the artificial nuclease, wherein the expression cassette comprises a polynucleotide sequence encoding the TCR of any of paras 1-12. 30. The method according to any of paras 27 to 29, wherein the method comprises the step of disrupting one or more endogenous genes encoding an MHC, optionally wherein the cell prepared by the method is a non-alloreactive universal T-cell.

[0384] 31. The method according to any of paras 27 to 30, wherein the method comprises the step of disrupting one or more endogenous genes to modify the persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capacity, or other T-cell functions, optionally wherein the method comprises the step of targeted integration of an expression cassette into an endogenous gene involved in persistence, expansion, activity, resistance to exhaustion / senescence / inhibitory signals, homing capacity, or other T-cell functions disrupted by an artificial nuclease, wherein the expression cassette comprises a polynucleotide sequence encoding the TCR of any of paras 1-12, optionally wherein the endogenous gene is selected from the group consisting of PD1, TIM3, LAG3, 2B4, KLRG1, TGFbR, CD160, TIGIT, CTLA4 and CD39.

[0385] 32. A chimeric molecule comprising the TCR of any of paras 1-12, or a portion thereof, conjugated to a non-cellular substrate, a toxin and / or an antibody, optionally wherein the non- cellular substrate is selected from the group consisting of nanoparticles, exosomes and other non-cellular substrates.

[0386] 33. A pharmaceutical composition comprising a TCR according to any of paras 1-12, a polynucleotide according to any of paras 13-15, a vector according to any of paras 16-18, a cell according to any of paras 17-26, a cell prepared by a method according to any of paras 27-31 , or a chimeric molecule according to para 32.

[0387] 34. A cell according to any of paras 17-26 or a cell prepared by the method of any of paras 27-31 for use in adoptive cell transfer, preferably adoptive T-cell transfer, optionally wherein the adoptive T-cell transfer is allogenic adoptive T-cell transfer, autologous adoptive T-cell transfer, or universal non-alloreactive adoptive T-cell transfer.

[0388] 35. A TCR according to any of paras 1-12, a polynucleotide according to any of paras 13-15, a vector according to any of paras 16-18, a cell according to any of paras 19-26, a cell prepared by a method according to any of paras 27-31 , a chimeric molecule according to para 32, or a pharmaceutical composition according to para 33 for use in therapy.

[0389] 36. A TCR according to any of paras 1-12, a polynucleotide according to any of paras 13-15, a vector according to any of paras 16-18, a cell according to any of paras 19-26, a cell prepared by a method according to any of paras 27-31 , a chimeric molecule according to para 32, or a pharmaceutical composition according to para 33 for use in treating and / or preventing a proliferative disorder.

[0390] 37. The TCR, polynucleotide, vector, cell, chimeric molecule, or pharmaceutical composition for use according to para 43, wherein the proliferative disorder is a hematological malignancy or a solid tumour, optionally wherein the hematological malignancy is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, acute lymphocytic leukemia (ALL), myelodisplastic syndromes, lymphoma, multiple myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma, optionally wherein the solid tumour is selected from the group consisting of lung cancer, breast cancer, oesophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancers, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, oral cancer, hepatocellular carcinoma, bladder cancer, endometrial cancer, neuroblastoma, rabdomyosarcoma, liver cancer, melanoma, prostate cancer, renal cancer, soft tissue sarcoma, urothelial cancer, biliary cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.

[0391] 38. The TCR, polynucleotide, vector, cell, chimeric molecule, chimeric molecule, or pharmaceutical composition for use according to para 36, wherein the proliferative disorder is acute myeloid leukemia (AML).

[0392] 39. A method for treating and / or preventing a proliferative disorder, which comprises the step of administering a therapeutically effective amount of the TCR of any of paras 1-12, the polynucleotide of any of paras 13-15, the vector of any of paras 16-18, the cell of any of paras 19-26, a cell prepared by the method of any of paras 27-31 , the chimeric molecule of para 32, or the pharmaceutical composition of para 33 to a subject in need thereof.

[0393] 40. The method according to para 39, wherein the proliferative disorder is a hematological malignancy or a solid tumour, optionally wherein the hematological malignancy is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, acute lymphocytic leukemia (ALL), myelodisplastic syndromes, lymphoma, multiple myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma, optionally wherein the solid tumour is selected from the group consisting of lung cancer, breast cancer, oesophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancers, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, oral cancer, hepatocellular carcinoma, bladder cancer, endometrial cancer, neuroblastoma, rabdomyosarcoma, liver cancer, melanoma, prostate cancer, renal cancer, soft tissue sarcoma, urothelial cancer, biliary cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.

[0394] 41. The method according to para 39, wherein the proliferative disorder is acute myeloid leukemia (AML).

[0395] 42. Use of the TCR of any of paras 1-12, the polynucleotide of any of paras 13-15, the vector of any of paras 16-18, the cell of any of paras 19-26, a cell prepared by the method of any of paras 27-31 , the chimeric molecule of para 32, or the pharmaceutical composition of para 33 in the manufacture of a medicament for the treatment and / or prevention of a proliferative disorder.

[0396] 43. The use according to para 42, wherein the proliferative disorder is a hematological malignancy or a solid tumour, optionally wherein the hematological malignancy is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, acute lymphocytic leukemia (ALL), myelodisplastic syndromes, lymphoma, multiple myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma, optionally wherein the solid tumour is selected from the group consisting of lung cancer, breast cancer, oesophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancers, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, oral cancer, hepatocellular carcinoma, bladder cancer, endometrial cancer, neuroblastoma, rabdomyosarcoma, liver cancer, melanoma, prostate cancer, renal cancer, soft tissue sarcoma, urothelial cancer, biliary cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer.

[0397] 44. The use according to para 42, wherein the proliferative disorder is acute myeloid leukemia (AML).

[0398] Various further preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paras).

[0399] 1. A T-cell receptor (TCR) comprising:

[0400] (a) a CDR3a comprising the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3P comprising the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11) or a variant thereof having up to three amino acid substitutions, additions or deletions; or (b) a CDR3a comprising the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3P comprising the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22) or a variant thereof having up to three amino acid substitutions, additions or deletions.

[0401] 2. The TOR according to para 1 , wherein the TOR comprises a CDR3a comprising the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3P comprising the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11) or a variant thereof having up to three amino acid substitutions, additions or deletions, optionally wherein the TCR comprises the following CDR sequences: CDR1a - NSASDY (SEQ ID NO: 4), CDR2a - IRSNMDK (SEQ ID NO: 5), CDR3a - CAENPPGTGNQFYF (SEQ ID NO: 6), CDRip - SEHNR (SEQ ID NO: 9), CDR2P - FQNEAQ (SEQ ID NO: 10), and CDR3P - CASSLQGPGELFF (SEQ ID NO: 11), or variants thereof each having up to three amino acid substitutions, additions or deletions.

[0402] 3. The TCR according to para 2, wherein the TCR comprises an a chain variable domain comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least 70% sequence identity thereto; and a p chain variable domain comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 70% sequence identity thereto, optionally wherein the TCR comprises an a chain comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 70% sequence identity thereto; and a p chain comprising the amino acid sequence of SEQ ID NO: 13 or 14, or variants thereof having at least 70% sequence identity thereto.

[0403] 4. The TCR according to para 1 , wherein the TCR comprises a CDR3a comprising the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3P comprising the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22) or a variant thereof having up to three amino acid substitutions, additions or deletions, optionally wherein the TCR comprises the following CDR sequences: CDR1 a - TSENNYY (SEQ ID NO: 15), CDR2a - QEAYKQQN (SEQ ID NO: 16), CDR3a - CAFMKTGGTSYGKLTF (SEQ ID NO: 17), CDRi p - KGHSH (SEQ ID NO: 20), CDR2P - LQKENI (SEQ ID NO: 21), and CDR3P - CASSPVAGGFYEQYF (SEQ ID NO: 22), or variants thereof each having up to three amino acid substitutions, additions or deletions.

[0404] 5. The TCR according to para 4, wherein the TCR comprises an a chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 70% sequence identity thereto; and a p chain variable domain comprising the amino acid sequence of SEQ ID NO: 23 ora variant thereof having at least 70% sequence identity thereto, optionally wherein the TOR comprises an a chain comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 70% sequence identity thereto; and a chain comprising the amino acid sequence of SEQ ID NO: 24 or 25, or variants thereof having at least 70% sequence identity thereto.

[0405] 6. A polynucleotide encoding the a chain and / or the p chain of a TCR according to any preceding para.

[0406] 7. A vector comprising a polynucleotide according to para 6.

[0407] 8. A cell comprising a TCR according to any of paras 1-5, a polynucleotide according to para 6, or a vector according to para 7.

[0408] 9. The cell according to para 8, wherein the cell is a T-cell, a lymphocyte, or a stem cell, optionally wherein the T-cell, the lymphocyte, or the stem cell is selected from the group consisting of CD4+ cells, CD8+ cells, naive T-cells, memory stem T-cells, central memory T- cells, double negative T-cells, effector memory T-cells, effector T-cells, ThO cells, TcO cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, gamma / delta T-cells, natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, hematopoietic stem cells and pluripotent stem cells, preferably wherein the cell is a T-cell which has been isolated from a subject.

[0409] 10. A chimeric molecule comprising the TCR of any of paras 1-5, or a portion thereof, conjugated to a non-cellular substrate, a toxin and / or an antibody, optionally wherein the non- cellular substrate is selected from the group consisting of nanoparticles, exosomes and other non-cellular substrates.

[0410] 11. A pharmaceutical composition comprising a TCR according to any of paras 1-5, a polynucleotide according to para 6, a vector according to para 7, a cell according to para 8 or 9, or a chimeric molecule according to para 10.

[0411] 12. A cell according to para 8 or 9 for use in adoptive cell transfer, preferably adoptive T-cell transfer, optionally wherein the adoptive T-cell transfer is allogenic adoptive T-cell transfer, autologous adoptive T-cell transfer, or universal non-alloreactive adoptive T-cell transfer.

[0412] 13. A TCR according to any of paras 1-5, a polynucleotide according to para 6, a vector according to para 7, a cell according to para 8 or 9, a chimeric molecule according to para 10 or a pharmaceutical composition according to para 11 for use in therapy. 14. A TCR according to any of paras 1-5, a polynucleotide according to para 6, a vector according to para 7, a cell according to para 8 or 9, a chimeric molecule according to para 10, or a pharmaceutical composition according to para 11 for use in treating and / or preventing a proliferative disorder.

[0413] 15. The TCR, polynucleotide, vector, cell, chimeric molecule, or pharmaceutical composition for use according to para 14, wherein the proliferative disorder is a hematological malignancy or a solid tumour, optionally wherein the hematological malignancy is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, acute lymphocytic leukemia (ALL), myelodisplastic syndromes, lymphoma, multiple myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma, optionally wherein the solid tumour is selected from the group consisting of lung cancer, breast cancer, oesophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancers, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, endometrial cancer, neuroblastoma, rabdomyosarcoma, liver cancer, melanoma, prostate cancer, renal cancer, soft tissue sarcoma, urothelial cancer, biliary cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer, preferably wherein the proliferative disorder is acute myeloid leukemia (AML).

Claims

CLAIMS1. A T-cell receptor (TCR) comprising:(a) a CDR3a comprising the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3P comprising the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11) or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the TCR binds to a fatty acid-binding protein 5 (FABP5) peptide when presented by a major histocompatibility complex (MHC); or(b) a CDR3a comprising the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3P comprising the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22) or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the TCR binds to a histone H2B (HIST1 H2B) peptide when presented by a major histocompatibility complex (MHC).

2. The TCR according to claim 1 , wherein the TCR comprises a CDR3a comprising the amino acid sequence of CAENPPGTGNQFYF (SEQ ID NO: 6) or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3P comprising the amino acid sequence of CASSLQGPGELFF (SEQ ID NO: 11) or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the TCR binds to a fatty acid-binding protein 5 (FABP5) peptide when presented by a major histocompatibility complex (MHC), optionally wherein the TCR comprises the following CDR sequences: CDR1 a - NSASDY (SEQ ID NO: 4), CDR2a - IRSNMDK (SEQ ID NO: 5), CDR3a - CAENPPGTGNQFYF (SEQ ID NO: 6), CDR1 P - SEHNR (SEQ ID NO: 9), CDR2P - FQNEAQ (SEQ ID NO: 10), and CDR3P - CASSLQGPGELFF (SEQ ID NO: 11), or variants thereof each having up to three amino acid substitutions, additions or deletions.

3. The TCR according to claim 2, wherein the TCR comprises an a chain variable domain comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least 70% sequence identity thereto; and a p chain variable domain comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 70% sequence identity thereto, optionally wherein the TCR comprises an a chain comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least 70% sequence identity thereto; and a chain comprising the amino acid sequence of SEQ ID NO: 13 or 14, or variants thereof having at least 70% sequence identity thereto.

4. The TCR according to claim 1 , wherein the TCR comprises a CDR3a comprising the amino acid sequence of CAFMKTGGTSYGKLTF (SEQ ID NO: 17) or a variant thereof having up to three amino acid substitutions, additions or deletions, and a CDR3P comprising the amino acid sequence of CASSPVAGGFYEQYF (SEQ ID NO: 22) or a variant thereof having up to three amino acid substitutions, additions or deletions, wherein the TCR binds to a histone H2B (HIST1 H2B) peptide when presented by a major histocompatibility complex (MHC), optionally wherein the TCR comprises the following CDR sequences: CDR1a - TSENNYY (SEQ ID NO: 15), CDR2a - QEAYKQQN (SEQ ID NO: 16), CDR3a - CAFMKTGGTSYGKLTF (SEQ ID NO: 17), CDR1 P - KGHSH (SEQ ID NO: 20), CDR2P - LQKENI (SEQ ID NO: 21), and CDR3P - CASSPVAGGFYEQYF (SEQ ID NO: 22), or variants thereof each having up to three amino acid substitutions, additions or deletions.

5. The TCR according to claim 4, wherein the TCR comprises an a chain variable domain comprising the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 70% sequence identity thereto; and a p chain variable domain comprising the amino acid sequence of SEQ ID NO: 23 ora variant thereof having at least 70% sequence identity thereto, optionally wherein the TCR comprises an a chain comprising the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 70% sequence identity thereto; and a p chain comprising the amino acid sequence of SEQ ID NO: 24 or 25, or variants thereof having at least 70% sequence identity thereto.

6. A polynucleotide encoding the a chain and / or the p chain of a TCR according to any preceding claim.

7. A vector comprising a polynucleotide according to claim 6.

8. A cell comprising a TCR according to any of claims 1-5, a polynucleotide according to claim 6, or a vector according to claim 7.

9. The cell according to claim 8, wherein the cell is a T-cell, a lymphocyte, or a stem cell, optionally wherein the T-cell, the lymphocyte, or the stem cell is selected from the group consisting of CD4+ cells, CD8+ cells, naive T-cells, memory stem T-cells, central memory T- cells, double negative T-cells, effector memory T-cells, effector T-cells, ThO cells, TcO cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, gamma / delta T-cells, natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, hematopoietic stem cells and pluripotent stem cells, preferably wherein the cell is a T-cell which has been isolated from a subject.

10. A chimeric molecule comprising the TCR of any of claims 1-5, or a portion thereof, conjugated to a non-cellular substrate, a toxin and / or an antibody, optionally wherein the non- cellular substrate is selected from the group consisting of nanoparticles, exosomes and other non-cellular substrates.

11. A pharmaceutical composition comprising a TCR according to any of claims 1-5, a polynucleotide according to claim 6, a vector according to claim 7, a cell according to claim 8 or 9, or a chimeric molecule according to claim 10.

12. A cell according to claim 8 or 9 for use in adoptive cell transfer, preferably adoptive T-cell transfer, optionally wherein the adoptive T-cell transfer is allogenic adoptive T-cell transfer, autologous adoptive T-cell transfer, or universal non-alloreactive adoptive T-cell transfer.

13. A TCR according to any of claims 1-5, a polynucleotide according to claim 6, a vector according to claim 7, a cell according to claim 8 or 9, a chimeric molecule according to claim 10 or a pharmaceutical composition according to claim 11 for use in therapy.

14. A TCR according to any of claims 1-5, a polynucleotide according to claim 6, a vector according to claim 7, a cell according to claim 8 or 9, a chimeric molecule according to claim 10, or a pharmaceutical composition according to claim 11 for use in treating and / or preventing a proliferative disorder.

15. The TCR, polynucleotide, vector, cell, chimeric molecule, or pharmaceutical composition for use according to claim 14, wherein the proliferative disorder is a hematological malignancy or a solid tumour, optionally wherein the hematological malignancy is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), lymphoblastic leukemia, acute lymphocytic leukemia (ALL), myelodisplastic syndromes, lymphoma, multiple myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma, optionally wherein the solid tumour is selected from the group consisting of lung cancer, breast cancer, oesophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancers, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, endometrial cancer, neuroblastoma, rabdomyosarcoma, liver cancer, melanoma, prostate cancer, renal cancer, soft tissue sarcoma, urothelial cancer, biliary cancer, glioblastoma, mesothelioma, cervical cancer, and colorectal cancer, preferably wherein the proliferative disorder is acute myeloid leukemia (AML).

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