MAGEA1-specific T cell receptor and its use

Abstract

The present invention relates to isolated T cell receptors (TCRs) specific for MAGEA1-derived peptides and polypeptides comprising functional parts of TCRs. It further relates to multivalent TCR complexes, nucleic acids encoding TCRs, cells expressing TCRs and pharmaceutical compositions comprising TCRs. The present invention also relates to TCRs used as drugs, in particular TCRs for treating cancer.

Description

Technical Field

[0001] The present invention relates to isolated T cell receptors (TCRs) specific for MAGEA1-derived peptides and polypeptides comprising functional parts of TCRs. It further relates to multivalent TCR complexes, nucleic acids encoding TCRs, cells expressing TCRs and pharmaceutical compositions comprising TCRs. The present invention also relates to TCRs used as drugs, in particular TCRs for treating cancer. Background Art

[0002] MAGEA1, also known as melanoma-associated antigen 1 ( M MAGEA1 is a member of the MAGEA gene family that encodes many proteins with high homology to each other. MAGEA antigens belong to the cancer / testis antigen (CTA) family and are the first human tumor-associated antigens identified at the molecular level (Science 1991.254:1643-1647 / reprinted in J.Immunol.2007;178:2617-2621). The MAGEA gene family includes 12 highly homologous genes located on chromosome Xq28. Their expression is always detected in cancers of different histological origins, such as non-small cell lung cancer, bladder cancer, esophageal cancer, head and neck cancer and sarcoma, as well as myeloma, certain types of breast cancer, and also detected in germ cells (Front Med (Lausanne).2017;4:18.). MAGEA1 is a cytosolic / cytoplasmic protein, and peptides derived from MAGEA1 protein are presented in the context of MHC class I (i.e., on HLA molecules). More specifically, MAGEA1-derived epitopes are presented on HLA-A2 molecules, indicating antigen immunogenicity and suitability as a target for cancer immunotherapy. A large number of clinical trials have been conducted targeting MAGEA proteins in the field of immunotherapy (clinicaltrials.gov). The combination of the expression of MAGEA1 in a variety of tumor entities such as melanoma and lung cancer (Lancet Oncol 2003; 4: 104–09) and a large number of patients and the described antigen immunogenicity makes MAGEA1 a promising target for T cell-mediated immunotherapy. The principle of cancer immunotherapy using adoptive cell transfer (ACT) enables the use of the patient's own immune system for highly tumor-specific cancer treatment. ACT uses ex vivo expanded autologous (patient-derived) T cells that are genetically modified to express T cell receptors (TCRs) specific for defined epitopes derived from intracellular proteins (e.g., MAGEA1). Therefore, there is still a need for highly efficient TCRs that only target the tumor-specific / restricted antigen MAGEA1 and therefore have special potential for cancer immunotherapy. Therefore, specific TCRs targeting MAGEA1 are needed. Summary of the invention

[0003] One object of the present invention is to provide a T cell receptor (TCR) specific for MAGEA1.

[0004] In a specific embodiment, the isolated TCR comprises: a TCR alpha chain comprising a CDR1 having an amino acid sequence of SEQ ID NO:2, a CDR2 having an amino acid sequence of SEQ ID NO:3, and a CDR3 having an amino acid sequence of SEQ ID NO:4; a TCR beta chain comprising a CDR1 having an amino acid sequence of SEQ ID NO:5, a CDR2 having an amino acid sequence of SEQ ID NO:6, and a CDR3 having an amino acid sequence of SEQ ID NO:7.

[0005] TCR specifically recognizes the amino acid sequence SEQ ID NO: 1 or a fragment thereof.

[0006] In a specific embodiment, the TCR specifically recognizes the HLA-A2 and / or HLA-A26 binding form of the amino acid sequence of SEQ ID NO: 1, preferably the HLA-A2 binding form. More specifically, the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 1 presented by a molecule encoded by a gene selected from the group consisting of HLA-A*02:01, HLA-A*02:02, HLA-A*02:04, HLA-A*02:16, HLA-A*02:17 and HLA-A*26:01, more preferably the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 1 presented by a molecule encoded by a gene selected from the group consisting of HLA-A*02:01, HLA-A*02:04, HLA-A*02:16 and HLA-A*02:17, even more preferably the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 1 presented by a molecule encoded by HLA-A*02:01.

[0007] The TCR of the present invention is particularly useful because it exhibits high tumor cell recognition ability, high tumor cell killing ability and high functional affinity. In addition, the TCR does not exhibit a large amount of secretion of Th2 cytokines IL-4, IL-5 and IL-13, which is conducive to effective tumor regression.

[0008] Preferably, the TCR has no cross-reactivity with other MAGEA family members.

[0009] Another object of the present invention is to provide T cells expressing functional TCR specific for MAGEA1.

[0010] The TCR according to the invention is isolated and / or purified and may be soluble or membrane bound.

[0011] In some embodiments, the amino acid sequence of TCR may include one or more phenotypic silent replacements. In addition, the TCR of the present invention may be labeled. Useful labels are known in the art and can be coupled to TCR or TCR variants using conventional methods, optionally through various lengths of joints. The term "label" or "labeling group" refers to any detectable label. Additionally or alternatively, the amino acid sequence may be modified to include a therapeutic agent or a pharmacokinetic modification portion. The therapeutic agent may be selected from a group consisting of an immune effector molecule, a cytotoxic agent, and a radionuclide. The immune effector molecule may be, for example, a cytokine. The pharmacokinetic modification portion may be at least one polyethylene glycol repeating unit, at least one diol group, at least one sialic acid group, or a combination thereof.

[0012] TCRs according to the invention, particularly soluble forms of TCRs, can be modified by attaching additional functional parts, for example, to reduce immunogenicity, increase hydrodynamic size (size in solution), solubility and / or stability (e.g., by enhancing protection against proteolytic degradation) and / or extend serum half-life. Other useful functional parts and modifications include "suicide" or "safety switches" that can be used to turn off or on effector host cells carrying the TCR of the invention in a patient, or to turn off or on the transgenic TCR itself. TCRs with altered glycosylation patterns are also contemplated herein.

[0013] It is also conceivable to add drugs or therapeutic entities (eg small molecule compounds) to the TCR, in particular to a soluble form of the TCR of the invention.

[0014] TCRs, in particular soluble forms of the TCRs of the invention, may be additionally modified to introduce additional domains (tags) that facilitate identification, tracking, purification and / or isolation of the corresponding molecules.

[0015] In some embodiments, the TCR is a single-chain type in which the TCR alpha chain and the TCR beta chain are connected by a linker sequence.

[0016] Another aspect of the present invention relates to a polypeptide comprising a functional portion of a TCR as described herein, wherein the functional portion comprises one of the amino acid sequences of SEQ ID NO: 4 and 7, preferably wherein the functional portion comprises the amino acid sequences of SEQ ID NO: 2, 3, 4 and 5, 6, 7.

[0017] In a specific embodiment, the functional portion comprises a TCR alpha variable chain and / or a TCR beta variable chain.

[0018] Specific embodiments relate to multivalent TCR complexes comprising at least two TCRs as described herein. In more specific embodiments, at least one of the TCRs is associated with a therapeutic agent.

[0019] Some embodiments relate to a TCR of the present invention expressed as a functional polypeptide or a functional multivalent polypeptide on an effector cell, in particular an immune effector cell, wherein upon binding to the HLA-A2 binding form of the amino acid sequence SEQ ID NO: 2, IFN-γ secretion is induced in the aforementioned effector cell expressing the TCR.

[0020] Another aspect of the invention relates to a nucleic acid encoding a TCR as described herein or encoding a polypeptide as described above.

[0021] Another aspect of the present invention relates to a plasmid or vector comprising the nucleic acid of the present application as described above. Preferably, the vector is an expression vector, or a vector suitable for transduction or transfection of cells, especially eukaryotic cells. The vector can be, for example, a retroviral vector, such as a γ-retroviral or lentiviral vector.

[0022] Another aspect of the invention relates to a cell expressing a TCR as described herein. The cell may be an isolated primary cell or non-naturally occurring.

[0023] Another aspect of the present invention relates to a cell comprising a nucleic acid as described above or a plasmid or vector as described above. More specifically, the cell may comprise:

[0024] a) an expression vector comprising at least one nucleic acid as described above, or

[0025] b) a first expression vector comprising a nucleic acid encoding an alpha chain of a TCR as described herein, and a second expression vector comprising a nucleic acid encoding a beta chain of a TCR as described herein.

[0026] The cell can be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). Typically, the cell is an immune effector cell, particularly a T cell. Other suitable cell types include γ-δ T cells and NK-like T cells and NK cells, whether modified or unmodified.

[0027] Another aspect relates to an antibody or antigen-binding fragment thereof that specifically binds to a portion of a TCR that mediates specificity for MAGEA1 as described herein. In a specific embodiment, the portion of a TCR that mediates specificity for MAGEA1 comprises the α chain CDR3 of SEQ ID NO: 4 and / or the β chain CDR3 of SEQ ID NO: 7. In some embodiments, the portion of a TCR that mediates specificity for MAGEA1 comprises the amino acid sequences of SEQ ID NO: 2, 3, 4 and 5, 6, 7.

[0028] Another aspect of the invention relates to a pharmaceutical composition comprising a TCR as described herein, a polypeptide as described herein, a multivalent TCR complex as described herein, a nucleic acid as described herein, a vector as described herein, a cell as described herein, or an antibody as described herein.

[0029] Typically, pharmaceutical compositions comprise at least one pharmaceutically acceptable carrier.

[0030] Another aspect of the invention relates to a TCR as described herein, a polypeptide as described herein, a multivalent TCR complex as described herein, a nucleic acid as described herein, a vector as described herein, a cell as described herein, or an antibody as described herein, for use as a medicament, in particular for use in the treatment of cancer. The cancer may be a blood cancer or a solid tumor. The cancer may be selected from the group consisting of prostate cancer, uterine cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, cervical cancer, colorectal cancer, gastric adenocarcinoma, bile duct cancer, breast cancer, bladder cancer, myeloid leukemia and acute lymphoblastic leukemia, sarcoma or osteosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Effector pairs expressing TCR T15.8-4.3-83 loaded with MAGEA1 from different donors (donor A or B; grey or black columns) are shown. KVL (SEQ ID NO.1) peptide (KVL) specific recognition of T2 cells. As measured by IFN-γ secretion, TCR-mediated target recognition with a TCR version of the complete mouse C region (murC) is shown in the bottom figure, and TCR-mediated target recognition with a TCR version of the most mouse humanized human C region (mmC) is shown in the middle figure. Effector-mediated recognition transduced by the benchmark control MAGEA1-TCR is shown in the top figure. As a control, T2 cells loaded with an unrelated ASTN-1 peptide (SEQ ID NO.28) were incubated with T cells expressing the TCR of the present invention or a control TCR (Ctrl.MAGEA1-TCR).

[0032] Figure 2IFN-γ secretion after TCR T15.8-4.3-83 specific recognition of KVL peptide-loaded lymphoblastoid cell lines (LCL) expressed in three different effector cell preparations (shaded columns: white, light gray and dark gray) is shown. When unloaded LCL was co-cultured with corresponding effector cells (solid color columns: white, light gray, dark gray), no recognition was observed. The box represents the HLA allele responsible for presentation and recognition. In the case of HLA-A heterozygous LCL (as shown by the asterisk), recognition of peptides on other HLA-A can be excluded (data not shown).

[0033] Figure 3 Depict the recognition of various tumor cell lines of different sources mediated by the effector (upper figure) expressing TCR T15.8-4.3-83 or benchmark Ctrl.MAGEA1-TCR (lower figure).Effector cells are from three different donors (donor A, B or C; white, light grey or dark grey columns).Target recognition of effector cells (solid colour columns) expressing MAGEA1-TCR or untransduced control cells (shadow columns) is detected by measuring IFN-γ secretion.T2 cells loaded with KVL peptide or loaded with unrelated ASTN1 peptide are used as target controls and are shown on the right side of each figure (T2+KVL, T2+ASTN1).

[0034] Figure 4A Figures 1 to 3 show TCR-mediated lysis of tumor cell lines UACC-62 (A), NCI-H1703 (B), Saos-2 (C) and 647-V (D). Cell lysis was measured by the disappearance of fluorescently labeled target cells. Tumor cells that were not modified (black) or loaded with KVL peptide (grey) as a positive control were tested. The targets were co-cultured with effector cells from three different donors (donor A, B or C, solid circles) for up to 144 hours, and these cells expressed TCR T15.8-4.3-83 (upper figure), benchmark Ctrl.MAGEA1-TCR (middle figure) or were not transduced (lower figure). Target cells cultured alone are shown in each figure as a reference (open circles). IncuCyte was used every 2 hours. TM Images were captured and analyzed by the Zoom System and depict target cell counts / well over a 144-hour period.

[0035] Figure 5Shown is the recognition of T2 cells loaded with peptides for threonine replacement scanning after co-culture with effectors transduced (solid color columns) or untransduced (shaded columns) from three different donors (donor A, B or C; white, light gray or dark gray columns). The recognition of the original MAGEA1 derived epitope KVL of the TCR is shown on the left. The epitope sequence shown on the x-axis indicates the position where the original AA is exchanged to threonine. The recognition eliminated by the exchange of the original AA is highlighted with a black box. Target recognition is detected by measuring T cell-mediated IFN-γ secretion.

[0036] Figure 6 Shown is the recognition of T2 cells loaded with peptides for threonine replacement scanning after co-culture with TCR T15.8-4.3-83 transduced (solid color columns) or untransduced (shaded columns) effectors from three different donors (donor A, B or C; white, light gray or dark gray columns). The recognition of the original MAGEA1 derived epitope KVL of the TCR is shown on the left. The epitope sequence shown on the x-axis represents the position where the original AA is exchanged into threonine. The recognition eliminated by the exchange of the original AA is highlighted with a black box. Target recognition is detected by measuring T cell-mediated IFN-γ secretion.

[0037] Figure 7 The identification of HEK 293T cells (A), K562 cells (B) and LCL cells (C) mediated by TCR T15.8-4.3-83 is shown. The identification of target cells is detected by measuring the secretion of IFN-γ mediated by T cells. Cell lines and T2 cells are loaded with KVL peptides (+KVL) derived from MAGEA1 or KLY peptides (+ASTN1) derived from ASTN1 as controls, or ivtRNA transfections encoding MAGEA1, ASTN1 or 13 MAGE family members, as indicated on the x-axis. Targets are co-cultured with effector cells transduced (solid color columns) or not transduced (shadow columns) from two donors (light gray or dark gray). The background mediated by target cells (only target cells) cultured separately is given with white columns. Dashed line represents the independent background secretion of the target of IFN-γ.

[0038] Figure 8TCR T15.8-4.3-83 mediation is shown to be derived from healthy tissue or from the unmodified (unloaded) or loaded KVL target cell identification of the induced pluripotent stem cell (iPS) representing healthy human tissue. The identification of target cells is detected by measuring the IFN-γ secretion of the T cell from three different donors (donor A, B or C; white, light gray or dark gray columns) transduced with TCRT15.8-4.3-83. The background IFN-γ secretion of the independent target (only target) of each cell type is shown as a control, and the identification of the T2 cells (T2+KVL) loaded with epitope KVL identified by TCR is also shown on the right side of the figure, and the background (only effector) by the independent effector is shown.

[0039] Fig. 9 Representative images of normal cells co-cultured with effector cells taken using phase contrast microscopy are shown. While all KVL-loaded target cells had clear TCR-mediated lysis (complete disruption of the cell layer), the effector cells expressing the TCR did not lyse the unmodified normal cells.

[0040] Fig.10 The ability of a TCR transgenic effector T cell population to produce IFN-γ in response to a MAGEA1 and HLA-A*02:01 double-positive tumor cell line is shown. FH1, FH2, FH3 and FH4 refer to the TCR disclosed in WO 2018 / 170338, FH1: Vα=SEQ ID NO.:7 of WO'338, Vβ=SEQ ID NO.:5 of WO'338; FH2: Vα=SEQ ID NO.:11 of WO'338, Vβ=SEQ ID NO.:9 of WO'338, FH3: Vα=SEQ ID NO.:15 of WO'338, Vβ=SEQ ID NO:13 of WO'338; FH1: Vα=SEQ ID NO.:19 of WO'338, Vβ=SEQ ID NO:17 of WO'338; R37P1C9 refers to the MAGA1-TCR disclosed in WO2018 / 104438.

[0041] Fig.11 The ability of a TCR transgenic effector T cell population to lyse a MAGEA1 and HLA-A*02:01 double positive tumor cell line is shown. FH1, FH2, FH3 and FH4 refer to the TCR disclosed in WO 2018 / 170338 (see Fig.10 ). R37P1C9 refers to the MAGA1-TCR disclosed in WO2018 / 104438. UTD: untransduced.

[0042] Fig.12The functional avidity of different KVL peptide-specific TCRs is shown. Functional avidity refers to the cumulative strength of the multiple affinities of the individual non-covalent binding interactions between the transgenic TCR and the pMHC complex. FH1, FH2, FH3 and FH4 refer to the TCRs disclosed in WO 2018 / 170338 (see Fig.10 ). R37P1C9 refers to the MAGA1-TCR disclosed in WO2018 / 104438.

[0043] Fig.13 Serine residues were systematically replaced in MAGEA1 KVL Individual amino acids in the peptide (serine scanning) to determine the key amino acids in the epitope sequence.

[0044] Figure 14 shows the secretion pattern of TH2-specific cytokines IL-4, IL-5 and IL-13 by T cells transduced with different TCRs. UT: untransfected. T1367 represents the TCR disclosed in WO2014 / 118236. Target only: tumor cells were cultured without TCR. DETAILED DESCRIPTION

[0045] Before describing the present invention in detail with respect to some preferred embodiments of the invention, the following general definitions are provided.

[0046] The invention illustratively described below suitably may be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

[0047] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims.

[0048] When the term "comprising" is used in the present description and claims, other elements are not excluded. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising at least a certain number of embodiments, this is also to be understood as disclosing a group that preferably consists only of these embodiments.

[0049] For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable.” If in the following for example an antibody is defined as being obtainable from a particular source, this is also to be understood as disclosing the antibody obtained from this source.

[0050] Unless otherwise specifically stated, nouns not qualified by quantifiers include both the singular and plural forms of the noun. The term "about" or "approximately" in the context of the present invention indicates an accuracy interval that a person skilled in the art will understand still ensures the technical effect of the feature in question. The term generally indicates a deviation of ±10%, preferably ±5%, from the indicated value.

[0051] Technical terms are used according to their common sense or meaning to those skilled in the art. If specific meanings are conveyed to certain terms, the definitions of the terms will be given below in the context in which these terms are used.

[0052] TCR Background

[0053] TCR is composed of two different and independent protein chains, namely TCR alpha (α) and TCR beta (β) chains. The TCR α chain contains a variable region (V), a joining region (J), and a constant region (C). The TCR β chain contains a variable region (V), a diversity region (D), a joining region (J), and a constant region (C). The rearranged V(D)J region of both the TCR α and TCR β chains contains a hypervariable region (CDR, complementary determining region), of which the CDR3 region determines specific epitope recognition. In the C-terminal region, both the TCR α chain and the TCR β chain contain a hydrophobic transmembrane domain and end with a short cytoplasmic tail.

[0054] Typically, TCR is a heterodimer of one α chain and one β chain. This heterodimer can bind to MHC molecules presenting peptides.

[0055] The term "variable TCR alpha region" or "TCR alpha variable chain" or "variable domain" in the context of the present invention refers to the variable region of the TCR alpha chain. The term "variable TCR beta region" or "TCR beta variable chain" in the context of the present invention refers to the variable region of the TCR beta chain.

[0056] TCR loci and genes are described using the International Immunogenetics (IMGT) TCR nomenclature (IMGT database, www.IMGT.org; Giudicelli, V., et al. IMGT / LIGM-DB, the Comprehensive database of immunoglobulin and T cell receptor nucleotide sequences, Nucl. Acids Res., 34, D781-D784 (2006). PMID: 16381979; T cell Receptor Factsbook, LeFranc and LeFranc, Academic Press ISBN 0-12-441352-8) naming.

[0057] Target

[0058] The target of the TCR described herein is the peptide KVL (SEQ ID NO: 1) derived from MAGEA1 (NCBI Reference Sequence: NM_004988.4).

[0059] TCR-specific sequences

[0060] Some embodiments relate to an isolated TCR comprising a TCR alpha chain and a TCR beta chain, wherein

[0061] - the TCR alpha chain comprises a complementarity determining region 3 (CDR3) having the sequence of SEQ ID NO: 4,

[0062] - The TCR β chain comprises a CDR3 having the amino acid sequence of SEQ ID NO:7.

[0063] Particular embodiments relate to an isolated TCR comprising:

[0064] - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 2, a CDR 2 having the amino acid sequence of SEQ ID NO: 3, and a CDR 3 having the sequence of SEQ ID NO: 4;

[0065] - A TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:5, a CDR 2 having the amino acid sequence of SEQ ID NO:6, and a CDR 3 having the sequence of SEQ ID NO:7.

[0066] In some embodiments, the TCR comprises a variable TCR alpha region having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:8, and a variable TCR beta region having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:9.

[0067] A preferred embodiment relates to a TCR comprising a variable TCR alpha region having the amino acid sequence of SEQ ID NO:8 and a variable TCR beta region having the amino acid sequence of SEQ ID NO:9.

[0068] The TCR derived from T cell clone T15.8-4.3-83 (which is used in the examples in transgenic form) comprises: a TCR alpha chain comprising a complementarity determining region 3 (CDR3) having a sequence of SEQ ID NO: 4 and a TCR beta chain comprising a CDR3 having an amino acid sequence of SEQ ID NO: 7. In particular, the TCR of the present invention comprises a variable TCR alpha region having an amino acid sequence of SEQ ID NO: 8 and a variable TCR beta region having an amino acid sequence of SEQ ID NO: 9.

[0069] As can be seen from the examples, the TCR according to the present invention is specific for MAGEA1 and shows only very low cross-reactivity to other epitopes or antigens.

[0070] Other embodiments relate to an isolated TCR comprising a TCR alpha chain having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:10, and a TCR beta chain having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:11.

[0071] Specific embodiments relate to a TCR comprising a TCR alpha chain having an amino acid sequence of SEQ ID NO: 10 and a TCR beta chain having an amino acid sequence of SEQ ID NO: 11. Thus, the TCR described herein that is specific for a complex of HLA-A*02:01 and the MAGEA1 peptide of SEQ ID NO: 1 comprises a Valpha chain encoded by the TRAV19*01 gene and a Vbeta gene encoded by the TRAV30*01 gene.

[0072] Other embodiments relate to an isolated TCR comprising a TCR alpha chain and a TCR beta chain, wherein

[0073] - a variable TCR alpha region having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:8 and comprising the CDR3 region set forth in SEQ ID NO:3;

[0074] -The variable TCRβ region has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:9 and comprises the CDR3 region shown in SEQ ID NO:7.

[0075] Vector NTI Advance is preferred TM The percent identity between multiple sequences can be determined using the AlignX application of the 10 program (Invitrogen Corporation, Carlsbad CA, USA). This program uses a modified Clustal W algorithm (Thompson et al., 1994. Nucl Acids Res. 22: pp. 4673-4680; Invitrogen Corporation; Vector NTI AdvanceTM 10DNA and protein sequence analysis software. User's Manual, 2004, pp. 389-662). Percent identity was determined using standard parameters of the AlignX application.

[0076] The TCR according to the present invention is isolated or purified. "Isolated" in the context of the present invention means that the TCR is not present in the environment in which it originally occurs in nature. In the context of the present invention, "purified" means that, for example, the TCR is free of or substantially free of other proteins and non-protein parts of the cell from which it is derived.

[0077] In some embodiments, the amino acid sequence of a TCR may contain one or more phenotypically silent substitutions.

[0078] "Phenotypic silent substitutions" are also referred to as "conservative amino acid substitutions". The concept of "conservative amino acid substitutions" is understood by the skilled person, and preferably refers to codons encoding positively charged residues (H, K and R) replaced by codons encoding positively charged residues, codons encoding negatively charged residues (D and E) replaced by codons encoding negatively charged residues, codons encoding neutral polar residues (C, G, N, Q, S, T and Y) replaced by codons encoding neutral polar residues, and codons encoding neutral non-polar residues (A, F, I, L, M, P, V and W) replaced by codons encoding neutral non-polar residues. These variations can occur spontaneously, introduced by random mutagenesis, or can be introduced by directed mutagenesis. These changes can be made without destroying the basic characteristics of these polypeptides. Ordinary technicians can easily and routinely screen variant amino acids and / or nucleic acids encoding them by methods known in the art to determine whether these variations significantly reduce or destroy ligand binding ability.

[0079] The skilled person understands that the nucleic acid encoding the TCR can also be modified. Useful modifications in the entire nucleic acid sequence include codon optimization of the sequence. Conservative substitution changes in the amino acid sequence that result in expression can be made. These changes can be made in the complementary determining regions and non-complementary determining regions of the TCR chain amino acid sequence that do not affect function. Generally, additions and deletions should not be made in the CDR3 region.

[0080] According to some embodiments of the invention, the amino acid sequence of the TCR is modified to include a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety.

[0081] Non-limiting examples of detectable labels are radioactive labels, fluorescent labels, nucleic acid probes, enzymes and contrast agents. Therapeutic agents that can be associated with TCR include radioactive compounds, immunomodulators, enzymes or chemotherapeutic agents. The therapeutic agent can be encapsulated by liposomes connected to TCR so that the compound can be slowly released at the target site. This will avoid damage during transport in the body and ensure that the therapeutic agent (e.g., toxin) has maximum effect after the TCR binds to the relevant antigen presenting cell. Other examples of therapeutic agents are:

[0082] Peptide cytotoxins, i.e., proteins or peptides that have the ability to kill mammalian cells, such as ricin, diphtheria toxin, Pseudomonas exotoxin A, DNase and RNase. Small molecule cytotoxic agents, i.e., compounds with a molecular weight of less than 700 Daltons that have the ability to kill mammalian cells. Such compounds may contain toxic metals that can have cytotoxic effects. In addition, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decompose or transform under physiological conditions to release cytotoxic agents. Such agents may include, for example, docetaxel, gemcitabine, cisplatin, maytansine derivatives, rachelmycin, calicheamicin, etoposide, ifosfamide, irinotecan, porfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucoronate, mitoxantrone, auristatin E, vincristine, and doxorubicin; radionuclides, such as iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225, and astatine 213. The association of radionuclides with TCRs or their derivatives may be carried out, for example, via chelators; immunostimulants, also known as immunostimulants, are immune effector molecules that stimulate immune responses. Exemplary immunostimulants are cytokines, such as IL-2 and IFN-γ, antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (e.g., anti-CD3, anti-CD28 or anti-CD16); alternative protein scaffolds with antibody-like binding properties; superantigens, i.e., antigens and their mutants that cause non-specific activation of T cells, leading to polyclonal T cell activation and massive cytokine release; chemokines, such as IL-8, platelet factor 4, melanoma growth stimulating protein and other complement activators; heterologous protein domains, allogeneic protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0083] Antigen receptor molecules (T cell receptor molecules) on human T lymphocytes non-covalently associate with a CD3 (T3) molecule complex on the cell surface. Interference with this complex with anti-CD3 monoclonal antibodies induces T cell activation. Therefore, some embodiments relate to a TCR as described herein associated with an anti-CD3 antibody or a functional fragment or variant of the anti-CD3 antibody (usually by fusion to the N- or C-terminus of the α or β chain). Antibody fragments and variants / analogs suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab')2 fragments, dsFv and scFv fragments, Nanobodies TM (Ablynx (Belgium), molecules comprising synthetic single immunoglobulin variable heavy chain domains derived from camelid (e.g. camel or llama) antibodies) and domain antibodies (comprising affinity matured single immunoglobulin variable heavy chain domains or immunoglobulin variable light chain domains (Domantis (Belgium)) or alternative protein scaffolds exhibiting antibody-like binding properties, such as Affibodies (comprising the engineered protein A scaffold Affibody (Sweden)) or Anticalins (comprising the engineered anticalins Pieris (German)).

[0084] The therapeutic agent may preferably be selected from the group consisting of an immune effector molecule, a cytotoxic agent and a radionuclide. Preferably, the immune effector molecule is a cytokine.

[0085] The pharmacokinetic modifying moiety can be, for example, at least one polyethylene glycol repeating unit, at least one diol group, at least one sialic acid group or a combination thereof. The association of at least one polyethylene glycol repeating unit, at least one diol group, at least one sialic acid group can be caused in a variety of ways known to those skilled in the art. In a preferred embodiment, the unit is covalently linked to the TCR. The TCR according to the present invention can be modified by one or more pharmacokinetic modifying moieties. In particular, the soluble form of the TCR is modified by one or more pharmacokinetic modifying moieties. The pharmacokinetic modifying moiety can achieve beneficial changes to the pharmacokinetic characteristics of the therapeutic agent, such as improved plasma half-life, reduced or enhanced immunogenicity and improved solubility.

[0086] TCR according to the present invention can be soluble or membrane-bound.Term " soluble " refers to TCR in soluble form (i.e., without transmembrane or cytoplasmic domains), for example, used as a targeting agent for delivering therapeutic agents to antigen presenting cells.For stability, soluble α β heterodimer TCR preferably has an introduced disulfide bond between the residues of each constant domain, as described in, for example, WO 03 / 020763.One or two constant domains present in the α β heterodimer of the present invention can be truncated at one or two C-termini, for example, up to 15 or up to 10 or up to 8 or less amino acids.In order to use in adoptive therapy, α β heterodimer TCR can be, for example, transfected into full-length chains with both cytoplasm and transmembrane domains.TCR can include disulfide bonds corresponding to those found between corresponding α and β constant domains in nature, and in addition or alternatively, non-natural disulfide bonds can be present.

[0087] Therefore, TCRs according to the invention, in particular soluble forms of TCRs, can be modified by attaching additional functional parts, for example to reduce immunogenicity, increase hydrodynamic size (size in solution), solubility and / or stability (for example by enhancing protection from proteolytic degradation) and / or to extend serum half-life.

[0088] Other useful functional parts and modifications include "suicide" or "safety switches" that can be used to turn off effector host cells carrying the TCR of the present invention in patients. An example is the inducible caspase 9 (iCasp9) "safety switch" described by Gargett and Brown Front Pharmacol.2014; 5:235. In short, the effector host cells are modified to express the caspase 9 domain by a well-known method, and its dimerization depends on small molecule dimerization drugs such as AP1903 / CIP, and leads to rapid induction of apoptosis in modified effector cells. The system is described, for example, in EP2173869 (A2). Other examples of "suicide" and "safety switches" are known in the art, such as herpes simplex virus thymidine kinase (HSV-TK), expression of CD20 and subsequent consumption of anti-CD20 antibodies or myc tags (Kieback et al. Proc Natl Acad Sci US A. 2008 Jan 15; 105 (2): 623-8).

[0089] TCRs with altered glycosylation patterns are also contemplated herein. As known in the art, glycosylation patterns may depend on the amino acid sequence (e.g., the presence or absence of specific glycosylated amino acid residues discussed below) and / or the host cell or organism producing the protein. The glycosylation of a polypeptide is typically N-connected or O-connected. N-connection refers to the side chain of an asparagine residue to which a carbohydrate moiety is connected. By changing the amino acid sequence, it is convenient to add N-connected glycosylation sites to a binding molecule by including one or more tripeptide sequences selected from asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid except proline). O-connected glycosylation sites can be introduced by adding or replacing one or more serine or threonine residues to the starting sequence.

[0090] Another method of TCR glycosylation is by chemical or enzymatic coupling of glycosides to proteins. Depending on the coupling mode used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine or tryptophan, or (f) amide groups of glutamine. Similarly, deglycosylation (i.e., removal of carbohydrate moieties present on the binding molecule) can be accomplished chemically, for example by exposing the TCR to trifluoromethanesulfonic acid, or enzymatically by using endo- and exo-glycosidases.

[0091] It is also conceivable to add drugs such as small molecule compounds to TCRs, particularly soluble forms of the TCRs of the invention. Connection can be achieved by covalent bonds or non-covalent interactions (e.g., by electrostatic forces). Various linkers known in the art can be used to form drug conjugates.

[0092] TCR, in particular soluble forms of the TCR of the present invention, can be further modified to introduce additional domains (tags) that facilitate identification, tracking, purification and / or isolation of the corresponding molecule. Thus, in some embodiments, the TCR alpha chain or TCR beta chain can be modified to include an epitope tag.

[0093] Epitope tags are useful examples of tags that can be introduced into the TCR of the present invention. An epitope tag is a short stretch of amino acids that allows binding to specific antibodies, thereby enabling identification and tracking of the binding and movement of soluble TCRs or host cells or cultured (host) cells in patients. Detection of epitope tags can be achieved using a variety of different techniques, and therefore detection of tagged TCRs is achieved.

[0094] The tag can further be used to stimulate and expand host cells carrying the TCR of the invention by culturing the cells in the presence of a binding molecule (antibody) specific for the tag.

[0095] Typically, in some cases, a TCR can be modified with various mutations that change the affinity and off-rate of the TCR to the target antigen. In particular, mutations can increase affinity and / or reduce off-rate. Thus, a TCR can be mutated in at least one CDR and its variable domain framework region.

[0096] However, in a preferred embodiment, the CDR region of the TCR is not modified or affinity matured in vitro, such as for the TCR receptor in the examples. This means that the CDR region has a naturally occurring sequence. This may be advantageous because in vitro affinity maturation may lead to immunogenicity to the TCR molecule. This may lead to reduced or inactivated therapeutic efficacy and the generation of therapeutic anti-drug antibodies and / or cause side effects.

[0097] The mutation may be one or more substitutions, deletions or insertions. These mutations may be introduced by any suitable method known in the art, such as polymerase chain reaction, restriction enzyme-based cloning, ligation-independent cloning procedures, such as described in the examples of Sambrook, Molecular Cloning—4th edition (2012) Cold Spring Harbor Laboratory Press.

[0098] Theoretically, due to the mismatch between endogenous and exogenous TCR chains, unpredictable TCR specificity with a risk of cross-reaction may occur. To avoid the mismatch of TCR sequences, the recombinant TCR sequence can be modified to include the most mouse-specific Cα and Cβ regions, which has been proven to effectively enhance the correct pairing of TCR chains of various different transductions. The murinization of TCR (i.e., exchanging the human constant region in α and β chains by its mouse counterpart) is a technique commonly used to improve the cell surface expression of TCR in host cells. It is not desirable to be bound by a specific theory, and it is believed that murinized TCR is more effectively associated with CD3 co-receptors; and / or preferentially paired with each other and less likely to form mixed TCRs on human T cells, which are genetically modified in vitro to express the desired antigen-specific TCR, but still retain and express their "original" TCR.

[0099] Nine amino acids responsible for increasing murinized TCR expression have been identified (Sommermeyer and Uckert, J Immunol. 2010 Jun 1; 184 (11): 6223-31), and it is envisioned that one or all amino acid residues in the constant region of the TCR α and / or β chain are replaced with their mouse counterpart residues. This technology is also referred to as "ultimate mouse humanization", and has the advantage of enhancing cell surface expression while reducing the number of "foreign" amino acid residues in the amino acid sequence, thereby reducing the risk of immunogenicity.

[0100] Some embodiments relate to an isolated TCR as described herein, wherein the TCR is of a single chain type, wherein the TCR alpha chain and the TCR beta chain are connected by a linker sequence.

[0101] A suitable single-chain TCR format comprises a first segment consisting of an amino acid sequence corresponding to a variable TCR α region, a second segment consisting of an amino acid sequence corresponding to a variable TCR β region fused to the N-terminus of an amino acid sequence corresponding to an extracellular sequence of a constant region of a TCR β chain, and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment. Alternatively, the first segment may consist of an amino acid sequence corresponding to a variable region of a TCR β chain, and the second segment may consist of an amino acid sequence corresponding to a variable region sequence of a TCR α chain fused to the N-terminus of an amino acid sequence corresponding to an extracellular sequence of a constant region of a TCR α chain. The above-mentioned single-chain TCR may also comprise a disulfide bond between the first and second chains, and wherein the length of the linker sequence and the position of the disulfide bond are such that the variable domain sequences of the first and second segments are substantially oriented to each other as in a natural T cell receptor. More specifically, the first segment may be composed of an amino acid sequence corresponding to a TCR α chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to an extracellular sequence of a TCR α chain constant region, the second segment may be composed of an amino acid sequence corresponding to a TCR β chain variable region fused to the N-terminus of an amino acid sequence corresponding to an extracellular sequence of a TCR β chain constant region, and a disulfide bond may be provided between the first and second chains. The linker sequence may be any sequence that does not impair TCR function.

[0102] In the context of the present invention, a "functional" TCR alpha and / or beta chain fusion protein shall refer to a TCR or TCR variant that retains at least basic biological activity, for example modified by the addition, deletion or substitution of amino acids. In the case of the alpha and / or beta chains of the TCR, this would mean that the two chains are still able to form a T cell receptor (with unmodified alpha and / or beta chains or with another fusion protein alpha and / or beta chain of the present invention), which exerts its biological function, in particular binding to the specific peptide-MHC complex of the TCR, and / or functional signal transduction after specific peptide:MHC interaction.

[0103] In a specific embodiment, TCR can be modified to a functional T cell receptor (TCR) α and / or β chain fusion protein, wherein the epitope-tag is 6 to 15 amino acids in length, preferably 9 to 11 amino acids. In another embodiment, TCR can be modified to a functional T cell receptor (TCR) α and / or β chain fusion protein, wherein the T cell receptor (TCR) α and / or β chain fusion protein comprises two or more epitope-tags spaced apart or directly connected in series. Embodiments of the fusion protein may include 2, 3, 4, 5 or even more epitope-tags, as long as the fusion protein maintains its biological activity / multiple activities ("functionality").

[0104] Preferred is a functional T cell receptor (TCR) α and / or β chain fusion protein according to the present invention, wherein the epitope-tag is selected from but not limited to CD20 or Her2 / neu tags, or other conventional tags such as myc-tags, FLAG-tags, T7-tags, HA (hemagglutinin)-tags, His-tags, S-tags, GST-tags or GFP-tags. Myc, T7, GST, GFP tags are epitopes derived from existing molecules. In contrast, FLAG is a synthetic epitope tag designed for high antigenicity (see, e.g., U.S. Patent Nos. 4,703,004 and 4,851,341). The myc tag can be preferably used because high-quality reagents can be obtained for its detection. The epitope tag can certainly have one or more additional functions beyond antibody recognition. The sequences of these tags are described in the literature and are well known to those skilled in the art.

[0105] TCR variants

[0106] Another aspect of the present invention relates to a polypeptide comprising a functional portion of a TCR as described herein, wherein the functional portion comprises at least one of the amino acid sequences of SEQ ID NO: 4 and 7.

[0107] The functional portion can mediate the binding of TCR to antigen, particularly to antigen-MHC complex.

[0108] In one embodiment, the functional portion comprises a TCR alpha variable chain and / or a TCR beta variable chain as described herein.

[0109] TCR variant molecules may have the binding properties of a TCR receptor, but may be combined with a signaling domain of an effector cell (other than a T cell), in particular a signaling domain of an NK cell. Thus, some embodiments relate to proteins comprising a functional portion of a TCR as described herein combined with a signaling domain of an effector cell (e.g., a NK cell).

[0110] Another aspect of the present invention relates to a multivalent TCR complex comprising at least two TCRs as described herein. In one embodiment of this aspect, at least two TCR molecules are connected by a linker portion to form a multivalent complex. Preferably, the complex is water-soluble, so the linker portion should be selected accordingly. Preferably, the linker portion can be connected to a defined position on the TCR molecule, thereby minimizing the structural diversity of the complex formed. An embodiment of this aspect is provided by the TCR complex of the present invention, wherein the polymer chain or peptide linker sequence extends between the amino acid residues of each TCR that are not located in the TCR variable region sequence. Since the complex of the present invention can be used for drugs, their pharmaceutical suitability, such as their immunogenicity, should be appropriately considered to select the linker portion. Examples of linker portions that meet the above-mentioned required standards are known in the art, such as the field of connecting antibody fragments.

[0111] Examples of linkers are hydrophilic polymers and peptide linkers. Examples of hydrophilic polymers are polyalkylene glycols. The most commonly used of these are based on polyethylene glycol or PEG. However, others are based on other suitable, optionally substituted polyalkylene glycols, including polypropylene glycol, and copolymers of ethylene glycol and propylene glycol. Peptide linkers comprise amino acid chains, the function of which is to produce simple linkers or multimerization domains to which TCR molecules can be connected.

[0112] One embodiment relates to a multivalent TCR complex wherein at least one of the TCRs is associated with a therapeutic agent.

[0113] Cytokine and chemokine release

[0114] Some embodiments relate to an isolated TCR as described herein, a polypeptide as described herein, a multivalent TCR complex as described herein, wherein IFN-γ secretion is induced by binding of the TCR of the invention expressed on effector cells to an HLA-A*02 binding form of an amino acid sequence selected from the group consisting of SEQ ID NO: 1.

[0115] The IFN-γ secretion induced by the binding of the TCR of the invention expressed on effector cells to the HLA-A*02 binding form of the amino acid sequence selected from the group consisting of SEQ ID NO: 1 can exceed 500pg / ml, such as more than 1000pg / ml, more than 2000pg / ml, more preferably more than 4000pg / ml, most preferably even more than 6000pg / ml. The IFN-γ secretion can be at least 5 times higher when binding to the HLA-A*02 binding form of the amino acid sequence of SEQ ID NO: 1 compared to binding to the HLA-A*02 binding form of an irrelevant peptide (e.g., SEQ ID No: 28).

[0116] "Effector cells" may be peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC). Typically, effector cells are immune effector cells, particularly T cells. Other suitable cell types include γ-δ T cells and NK-like T cells.

[0117] The present invention also relates to a method for identifying a TCR or a fragment thereof that binds to a target amino acid sequence SEQ ID NO:1 or its HLA-A*02, preferably or HLA-A*02:01 bound form, wherein the method comprises contacting a candidate TCR or a fragment thereof with the amino acid sequence of SEQ ID NO:1 or its HLA-A*02, preferably or its HLA-A*02:01 bound form, and determining whether the candidate TCR or a fragment thereof binds to the target and / or mediates an immune response.

[0118] Whether a candidate TCR or fragment thereof mediates an immune response can be determined, for example, by measuring cytokine secretion, such as IFN-γ secretion. As described above, cytokine secretion can be measured by an in vitro assay, wherein K562 cells (or other APCs) transfected with ivtRNA encoding the amino acid sequence SEQ ID NO: 1 are incubated with CD8+-enriched PBMCs expressing a molecule of a TCR or a fragment of the TCR to be investigated.

[0119] In a specific embodiment, the TCR of the invention shows no significant secretion of the Th2 cytokines IL-4, IL-5 and IL-13. This may be advantageous because Th2 cytokine responses may increase tumor progression (Jager MJ, Desjardins L, T, Damato BE (eds): Current Concepts in Uveal Melanoma. Dev Ophthalmol. Basel, Karger, 2012, Vol. 49, pp. 137–149, J Immunother. 2018 Oct; 41(8): 369-378).

[0120] Nucleic acid, vector

[0121] Another aspect of the invention relates to a nucleic acid encoding a TCR as described herein or a nucleic acid encoding a polynucleotide encoding a TCR as described herein.

[0122]

[0123] "Nucleic acid molecule" generally refers to a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthetic or obtained from a natural source (e.g., isolated and / or purified), which can include natural, non-natural or altered nucleotides, and which can include natural, non-natural or altered internucleotide connections, such as phosphoramidate connections or phosphorothioate connections, rather than the phosphodiester found between the nucleotides of unmodified oligonucleotides. Preferably, nucleic acid as described herein is recombinant. As used herein, the term "recombinant" refers to (i) molecules constructed outside living cells by connecting natural or synthetic nucleic acid fragments to nucleic acid molecules that can be replicated in living cells, or (ii) molecules produced by the replication of those described in (i) above. For the purposes of this article, replication can be in vitro replication or in vivo replication. Nucleic acid can be constructed based on chemical synthesis and / or enzymatic ligation reactions using programs known in the art or commercially available (e.g., from companies such as Genscript, Thermo Fisher). For example, see Sambrook et al., for example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed after hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). The nucleic acid can comprise any nucleotide sequence encoding any recombinant TCR, polypeptide or protein, or a functional portion or functional variant thereof.

[0124] The present disclosure also provides variants of isolated or purified nucleic acids, wherein the variant nucleic acids comprise a nucleotide sequence at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence encoding the TCR described herein. Such variant nucleotide sequences encode functional TCRs that specifically recognize MAGEA1.

[0125] The present disclosure also provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any nucleic acid described herein.

[0126] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high stringency conditions. "High stringency conditions" refer to nucleotide sequences that specifically hybridize with target sequences (nucleotide sequences of any nucleic acid described herein) in a stronger amount than non-specific hybridization detectable. High stringency conditions include conditions that can distinguish polynucleotides with precise complementary sequences or polynucleotides containing only a few scattered mismatches from random sequences that happen to have several small regions (e.g., 3-10 bases) that match the nucleotide sequence. This small complementary region is easier to melt than the full-length complementation of 14-17 or more bases, and high stringency hybridization makes them easy to distinguish. Relatively high stringency conditions will include, for example, low salt and / or high temperature conditions, such as provided by about 0.02-0.1M NaCl or equivalents at a temperature of about 50-70°C. Such high stringency conditions can hardly tolerate (if any) mismatches between nucleotide sequences and templates or target chains, and are particularly suitable for detecting the expression of any TCR described herein. It is generally believed that the conditions can be made more stringent by adding an increased amount of formamide.

[0127] As described elsewhere herein, the nucleic acid encoding the TCR can be modified. Useful modifications in the entire nucleic acid sequence can be codon optimization. Conservative substitution changes in the amino acid sequence that result in expression can be made. These changes can be made in the complementary determining regions and non-complementary determining regions of the TCR chain amino acid sequence that do not affect function. Generally, additions and deletions should not be made in the CDR3 region.

[0128] Another embodiment relates to a vector comprising a nucleic acid encoding a TCR as described herein.

[0129] The vector is preferably a plasmid, a shuttle vector, a phagemid, a cosmid, an expression vector, a retroviral vector, an adenoviral vector or a particle and / or a vector for gene therapy.

[0130] "Vector" is any molecule or composition capable of carrying a nucleic acid sequence into a suitable host cell, in which the synthesis of the encoded polypeptide can occur. Typically and preferably, a vector is a nucleic acid that has been engineered using recombinant DNA techniques known in the art to introduce a desired nucleic acid sequence (e.g., nucleic acid of the present invention). The vector may comprise DNA or RNA and / or comprise liposomes. The vector may be a plasmid, a shuttle vector, a phagemid, a cosmid, an expression vector, a retroviral vector, a lentiviral vector, an adenoviral vector, or a particle and / or a vector for gene therapy. The vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. The vector may also include one or more selectable marker genes and other genetic elements known to those of ordinary skill in the art. The vector is preferably an expression vector comprising a nucleic acid according to the present invention, and the nucleic acid is operably linked to a sequence that allows the expression of the nucleic acid.

[0131] Preferably, the vector is an expression vector. More preferably, the vector is a retroviral vector, more particularly a γ-retroviral or lentiviral vector.

[0132] Cells and cell lines

[0133] Another aspect of the invention relates to a cell expressing a TCR as described herein.

[0134] In some embodiments, the cell is isolated or non-naturally occurring.

[0135] In specific embodiments, the cell may comprise a nucleic acid encoding a TCR as described herein or a vector comprising the nucleic acid.

[0136] In the cell, the above-mentioned vector containing the nucleic acid sequence encoding the above-mentioned TCR can be introduced or the ivtRNA encoding the TCR can be introduced. The cell can be a peripheral blood lymphocyte, such as a T cell. The cloning and exogenous expression methods of TCR are described, for example, in Engels et al. (Relapse or eradication of cancer is predicted by peptide-majorhistocompatibility complex affinity. Cancer Cell, 23 (4), 516–26. 2013). For example, transduction of primary human T cells with lentiviral vectors is described in Cribbs' "simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells" BMC Biotechnol. 2013; 13: 98.

[0137] The terms "transfection" and "transduction" are interchangeable and refer to the process of introducing exogenous nucleic acid sequences into host cells (e.g., eukaryotic host cells). It should be noted that the introduction or transfer of nucleic acid sequences is not limited to the above methods, but can be achieved by any number of means, including electroporation, microinjection, gene gun delivery, lipofection, superfection, and the mentioned infection by retrovirus or other viruses suitable for transduction or transfection.

[0138] Some embodiments relate to a cell comprising:

[0139] a) an expression vector comprising at least one nucleic acid as described herein, or

[0140] b) a first expression vector comprising a nucleic acid encoding the alpha chain of a TCR as described herein, and a second expression vector comprising a nucleic acid encoding the beta chain of a TCR as described herein.

[0141] In some embodiments, the cell is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The cell can be a natural killer cell or a T cell. Preferably, the cell is a T cell. The T cell can be a CD4+ or CD8+ T cell. In some embodiments, the cell is a stem cell-like memory T cell.

[0142] Stem cell-like memory T cells (TSCM) are subpopulations of CD8+ or CD4+ T cells with a low degree of differentiation, characterized by the ability to self-renew and long-term existence. Once these cells encounter their antigens in vivo, they will further differentiate into central memory T cells (TCM), effector memory T cells (TEM) and terminally differentiated effector memory T cells (TEMRA), some of which TSCM remain static (Flynn et al., Clinical & Translational Immunology (2014). These remaining TSCM cells show the ability to establish persistent immune memory in vivo, and are therefore considered to be important T cell subsets for adoptive T cell therapy (Lugli et al., Nature Protocols 8, 33–42 (2013) Gattinoni et al., Nat. Med. 2011 Oct; 17 (10): 1290–1297). Immunomagnetic selection can be used to limit T cell pools to stem cell memory T cell subtypes (see Riddell et al. 2014, Cancer Journal 20 (2): 141–44).

[0143] Antibodies targeting TCR

[0144] Another aspect of the invention relates to an antibody or antigen-binding fragment thereof that specifically binds to a portion of a TCR that mediates MAGEA1 specificity as described herein. In one embodiment, the portion of a TCR that mediates MAGEA1 specificity comprises the α chain CDR3 of SEQ ID NO:4 and / or the β chain CDR3 of SEQ ID NO:7.

[0145] The antibody or its antigen-binding fragment can modulate the activity of TCR. It may or may not block the binding of TCR to MAGEA1. It can be used to modulate the therapeutic activity of TCR or for diagnostic purposes.

[0146] Pharmaceutical compositions, drug treatments and kits

[0147] Another aspect of the present invention relates to a pharmaceutical composition comprising a TCR as described herein, a polypeptide comprising a functional portion of the TCR, a multivalent TCR complex as described herein, a nucleic acid encoding the TCR, a vector comprising the nucleic acid, a cell comprising the TCR, or an antibody that specifically binds to a portion of the TCR as described herein.

[0148] Those active ingredients of the present invention are preferably used in a pharmaceutical composition that can treat or at least alleviate a disease in a dosage mixed with an acceptable carrier or carrier material. Such a composition can (in addition to the active ingredient and the carrier) comprise filler materials, salts, buffers, stabilizers, solubilizers and other materials known in the art.

[0149] The term "pharmaceutically acceptable" is defined as a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The choice of carrier depends on the application.

[0150] The pharmaceutical composition may contain additional ingredients that enhance the activity of the active ingredient or supplement the therapy. Such additional ingredients and / or factors may be part of the pharmaceutical composition to achieve a synergistic effect or to minimize adverse or undesirable effects.

[0151] Techniques for formulation or preparation and application / administration of the active ingredients of the present invention are disclosed in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition. Suitable applications are parenteral applications, such as intramuscular, subcutaneous, intramedullary injections, and intrathecal, direct intraventricular, intravenous, intranodal, intraperitoneal or intratumoral injections. Intravenous injection is the preferred method of treatment for patients.

[0152] According to a preferred embodiment, the pharmaceutical composition is an infusion or an injection.

[0153] Injectable compositions are pharmaceutically acceptable fluid compositions comprising at least one active ingredient, such as a population of amplified T cells expressing TCR (e.g., autologous or allogeneic for the patient to be treated). The active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition may additionally comprise a small amount of one or more non-toxic auxiliary substances, such as emulsifiers, preservatives, and pH buffers, etc. Such injectable compositions that can be used for use with the fusion proteins of the present disclosure are conventional; suitable formulations are well known to those of ordinary skill in the art.

[0154] Typically, pharmaceutical compositions comprise at least one pharmaceutically acceptable carrier.

[0155] Therefore, another aspect of the present invention relates to a TCR as described herein, a polypeptide comprising a functional portion of the TCR, a multivalent TCR complex as described herein, a nucleic acid encoding the TCR, a vector comprising the nucleic acid, a cell comprising the TCR, or an antibody that specifically binds to a portion of the TCR as described herein, for use as a medicament.

[0156] Some embodiments relate to a TCR as described herein, a polypeptide comprising a functional portion of the TCR, a multivalent TCR complex as described herein, a nucleic acid encoding the TCR, a vector comprising the nucleic acid, a cell comprising the TCR, for use in treating cancer.

[0157] In one embodiment, the cancer is a hematological cancer or a solid tumor.

[0158] Hematological cancers, also called blood cancers, do not form solid tumors and are therefore dispersed throughout the body. Examples of hematological cancers are leukemias, lymphomas or multiple myeloma. There are two main types of solid tumors, sarcomas and carcinomas. Sarcomas are tumors of, for example, blood vessels, bones, fatty tissue, ligaments, lymphatic vessels, muscles or tendons.

[0159] In one embodiment, the cancer is selected from the group consisting of prostate cancer, uterine cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, cervical cancer, colorectal cancer, gastric adenocarcinoma, bile duct cancer, breast cancer, bladder cancer, myeloid leukemia and acute lymphoblastic leukemia, sarcoma or osteosarcoma.

[0160] Also contemplated herein are pharmaceutical compositions and kits comprising one or more of the following: (i) an isolated TCR as described herein; (ii) a viral particle comprising a nucleic acid encoding a recombinant TCR; (iii) an immune cell modified to express a recombinant TCR as described herein, such as a T cell or NK cell; (iv) a nucleic acid encoding a recombinant TCR as described herein. In some embodiments, the disclosure provides a composition comprising a lentiviral vector particle containing a nucleotide sequence encoding a recombinant TCR as described herein (or a T cell modified to express a recombinant TCR using a vector particle as described herein). Such a composition can be administered to a subject in the methods of the disclosure as further described herein.

[0161] Compositions comprising modified T cells as described herein can be used in methods and compositions for adoptive immunotherapy according to known techniques, or variations thereof that will be apparent to those skilled in the art based on this disclosure.

[0162] In some embodiments, the cells are formulated by first harvesting them from their culture medium, then washing and concentrating the cells in a culture medium and container system suitable for administration in therapeutically effective amounts (a "pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or Ringer's lactate may also be used. The infusion medium may be supplemented with human serum albumin.

[0163] The number of cells in the composition for effective treatment is generally greater than 10 cells, up to 10 6 up to and including 10 8 or 10 9 cells, and can exceed 10 10 The number of cells will depend on the intended end use of the composition and the type of cells contained therein. For the purposes provided herein, the volume of cells is typically one liter or less, and may be 500 ml or less, or even 250 ml or 100 ml or less. Thus, the desired cell density is typically greater than 10 6 cells / ml and usually greater than 10 7 cells / ml, usually 10 8 Clinically relevant numbers of immune cells can be distributed into multiple infusions that cumulatively equal or exceed 10 9 , 10 10 or 10 11 cells. The pharmaceutical compositions provided herein can be in various forms, such as solid, liquid, powder, aqueous or lyophilized forms. Examples of suitable pharmaceutical carriers are known in the art. Such carriers and / or additives can be formulated by conventional methods and can be administered to a subject at a suitable dose. Stabilizers such as lipids, nuclease inhibitors, polymers and chelating agents can prevent the composition from degrading in vivo. In compositions intended to be administered by injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers and isotonic agents may be included.

[0164] Recombinant TCR as described herein or the viral vector particles comprising the nucleotide sequence encoding the recombinant TCR provided herein can be packaged as a test kit. The test kit can optionally include one or more components, such as instructions for use, devices and additional reagents, and components for implementing the method, such as tubes, containers and syringes. Exemplary test kits can include nucleic acids, recombinant TCR polypeptides or viruses encoding recombinant TCR provided herein, and can optionally include instructions for use, devices for detecting viruses in the subject, and devices for applying compositions to the subject.

[0165] Also contemplated herein are kits comprising polynucleotides encoding a target gene (e.g., a recombinant TCR). Also contemplated herein are kits comprising a viral vector encoding a target sequence (e.g., a recombinant TCR) and an optional polynucleotide sequence encoding an immune checkpoint inhibitor.

[0166] The kit considered herein also includes a kit for implementing the method of detecting the presence of the polynucleotides encoding any one or more TCR disclosed herein. In particular, such a diagnostic kit may include a component for performing deep sequencing to detect the appropriate amplification and detection primers and other related reagents of the polynucleotides encoding the TCR disclosed herein. In other embodiments, the kit herein may include a reagent for detecting the TCR disclosed herein, such as an antibody or other binding molecules. The diagnostic kit may also include instructions for determining the presence of the polynucleotides encoding the TCR disclosed herein or for determining the presence of the TCR disclosed herein. The kit may also include instructions. Instructions generally include describing the following tangible expressions: the components and the method of administration included in the kit, including determining the appropriate state of the subject, the appropriate dosage and the method of appropriate administration. The instructions may also include the guidance of monitoring the subject within the duration of the treatment time.

[0167] The kits provided herein may also include a device for administering the compositions described herein to a subject. Any of a variety of devices known in the art for administering drugs or vaccines may be included in the kits provided herein. Exemplary devices include, but are not limited to, hypodermic needles, intravenous needles, catheters, needleless injection devices, inhalers, and liquid dispensers, such as droppers. Typically, the device for administering the virus of the kit will be compatible with the virus of the kit; for example, a needleless injection device such as a high-pressure injection device may be included in a kit in which the virus is not destroyed by high-pressure injection, but is generally not included in a kit in which the virus is destroyed by high-pressure injection.

[0168] The kit provided herein may also include a device for administering a compound such as a T cell activator or stimulator or a TLR agonist such as a TLR4 agonist to a subject. Any of a variety of devices known in the art for administering a drug to a subject may be included in the kit provided herein. Exemplary devices include a subcutaneous needle, an intravenous needle, a catheter, a needle-free injection device, but are not limited to a subcutaneous needle, an intravenous needle, a catheter, a needle-free injection device, an inhaler, and a liquid dispenser, such as a dropper. Typically, the device for administering the kit compound will be compatible with the desired method of administration of the compound.

[0169] experiment

[0170] 1. Experiment: Isolation of MAGEA1-specific TCR

[0171] To isolate T cells that are specific for MAGEA1-derived epitopes and restricted to any MHC molecule of interest, an in vitro priming approach was used. The priming system uses mature dendritic cells (mDCs) from HLA-A*02:01-negative donors as antigen-presenting cells to initiate antigen-specific T cell responses and autologous CD8 + Amplification of T cells. An in vitro transcribed RNA (ivtRNA) encoding a minigene (SEQ ID NO: 27) and translated into an amino acid sequence comprising the MAGEA1-derived peptide KVLEYVIKV (such as reference SEQ ID NO: 1) is used as a source of specific antigen. At the same time, human HLA-A*02:01 encoding ivtRNA is used as a source of restriction elements transfected into mDC to establish allogeneic priming according to this dedicated HLA allele (as described in WO2007 / 017201). After electroporation into mDC, the minigene-encoded ivtRNA is translated into protein, which is then processed and presented as a peptide by the transgenic HLA-A*02:01 molecule expressed by the co-transfected mDC. In vitro co-culture of T cells with ivtRNA-transfected mDC from the same donor results in the de novo induction of antigen-specific T cells as a source of the corresponding TCR. After amplification, antigen-specific T cells can be enriched and single-cell cloned by limiting dilution or FACS-based single-cell sorting.

[0172] 1.1 Experimental layout

[0173] Dendritic cell priming of T cells for isolation and identification of high affinity TCRs was accomplished using peptide presentation via allogeneic HLA-A*02:01 molecules according to the following protocol:

[0174] According to Jonuleit et al. (Jonuleit et al. 1997, Eur. J. Immunol. 1997, 27: 3135-3142), mature dendritic cells were generated within 8 days using an appropriate maturation mixture of DCs. Antigen presenting cells (8-day mDCs) were derived from healthy donors and transfected with 20 μg of ivtRNA encoding MG_X1 and 20 μg of ivtRNA encoding allogeneic HLA molecules (HLA-A*02:01) by electroporation. The prepared mDCs were then incubated with autologous CD8-rich cells from healthy donors. + PBMCs were co-cultured at a ratio of 1:20 in an appropriate cell culture medium supplemented with IL-7 (5 ng / ml on day 0) and IL-2 (50 U / ml every two to three days) at 37°C (6% CO2) for about 14 days. 278-286 Identification of MAGEA1 by ProImmune 278-286Specific cells were then isolated by single cell sorting using FACS technology.

[0175] 1.2 Results

[0176] The described in vitro priming approach led to the identification of a T cell clone expressing the candidate TCR T15.8-4.3-83. The TCR sequence was identified using NGS analysis, reconstituted and transgenic expressed in appropriate effector T cells to fully characterize the TCR in terms of functionality and safety.

[0177] 2. Experiment: Epitope Specificity

[0178] To ensure the functionality of the transgenic expressed recombinant TCR, TCR-transduced effector T cells were co-cultured with peptide-loaded T2 cells (target cells). TCR-mediated recognition of specific peptide:MHC complexes leads to activation of TCR-expressing T cells and secretion of certain cytokines (e.g., IFN-γ). To analyze secreted IFN-γ in the co-culture supernatant, ELISA was performed.

[0179] 2.2 Experimental layout

[0180] T2 cells expressing HLA-A*02 were loaded with a saturating amount of peptide (10 -5 M). T2 cells loaded with MAGEA1-derived KVL peptide (SEQ ID NO: 1) were used as positive targets, and T2 cells loaded with unrelated ASTN1 peptide (SEQ ID NO: 28) were used as negative targets. As effector cells, CD8-rich T cell populations from two different donors were transduced to stably express recombinant TCR T15.8-4.3-83. Effectors were prepared to express two different versions of TCR T15.8-4.3-83, one version with mouse C region (murC) and the other version with the most mouse humanized C region (mmC). The control MAGEA1-TCR (benchmark TCR) was also expressed in T cells from two different donors and analyzed.

[0181] Effector cells and target cells were co-cultured in 96-well round-bottom plates at an E:T ratio of 2.5: 1. After about 20 hours of co-culture, supernatants were harvested and analyzed by ELISA (standard sandwich ELISA, BD human IFN-γ ELISA kit).

[0182] 2.3 Results

[0183] Effector cells expressing the TCR secreted IFN-γ only after co-culture with T2 cells loaded with the KVL peptide, indicating specific recognition of the presented peptide:MHC complex mediated by the transgenic TCR T15.8-4.3-83 and demonstrating its functionality. Negative targets (T2 cells loaded with an irrelevant ASTN1 peptide (SEQ ID NO: 28, KLYGLDWAEL)) were not recognized ( Figure 1 ). The transgenic TCR mediated specific target recognition and IFN-γ secretion, which was independent of the C region of the TCR.

[0184] 3. Experiment: Restriction Analysis

[0185] Since TCR can recognize its specific epitope only when combined with a specific MHC molecule, not only the level of antigen expression but also the HLA type of the target cell (in vitro and in vivo) defines the target as a positive or negative target. In other words, depending on the HLA type, the patient can be included or excluded from the TCR-based ACT treatment regimen.

[0186] To evaluate which HLA molecules, in addition to HLA-A*02:01, could be loaded with the MAGEA1-derived KVL peptide and recognized by T cells expressing the TCR T15.8-4.3-83, a detailed restriction analysis was performed. Thus, 53 LCL cell lines (EBV-transformed B cells) covering the most common HLA allotypes in Europeans and North American Caucasians were used as APCs (antigen presenting cells) and co-cultured with TCR-expressing effector cells that were either unloaded or loaded with the KVL peptide ( Figure 2 ).

[0187] 3.1 Experimental layout:

[0188] 53 LCL cell lines were loaded with saturating amounts of MAGEA1-derived KVL peptide (10 -5 M; SEQ ID NO: 1) for about 1.5 hours, washed and then co-cultured with effectors expressing TCR T15.8-4.3-83 from different effector preparations. Unloaded LCLs were used as controls. Co-cultures were set up in 96-well round-bottom plates at an E:T of 1:1, supernatants were collected after about 20 hours, and the levels of IFN-γ were analyzed by ELISA (standard sandwich ELISA, BD human IFN-γ ELISA assembly).

[0189] 3.2 Results

[0190] Restriction analysis of TCR T15.8-4.3-83 demonstrated the potential of the TCR to recognize its MAGEA1-derived epitopes, which are presented not only on HLA-A*02:01 but also to varying degrees on HLA-A*02:04, HLA-A*02:16, and HLA-A*02:17. Figure 2 Only data for those LCLs (out of 53 tested) that resulted in specific recognition of the peptide above background levels detected for unloaded LCLs are shown.

[0191] 4. Experiment: Tumor Cell Identification

[0192] To analyze the potency, specificity and suitability of T cell receptors for clinical applications, a panel of tumor cell lines was tested for TCR-mediated recognition by measuring IFN-γ secretion after co-culture with TCR-transduced effector cells.

[0193] 4.1 Experimental layout Figure 3 ):

[0194] Effector cells from 3 different donors were transduced with TCR T15.8-4.3-83 and benchmark Ctrl.MAGEA1-TCR. TCR-transduced or non-transduced effectors were co-cultured with different tumor cell lines. Cell lines U266, NCI-H1703, UACC-62, Saos-2 and Mel624.38 (HLA-A2+ / MAGEA1+) and HLA-A2-transfected cell lines KYO-1 and OPM-2 (HLA-A2+ / MAGEA1+) were used as positive targets. Cell lines NCI-H1755, 647-V and CMK (HLA-A2+ / MAGEA1-), unmodified OPM-2 and KYO-1 (HLA-A2- / MAGEA1+) were used as negative targets. HLA-A2 expression of each cell line was analyzed by FACS (data not shown), and MAGEA1 expression data were generated using qPCR or extracted from publicly available databases known to those skilled in the art. As a control, all effectors were also loaded with saturating concentrations (10 -5 T2 cells were co-cultured with MAGEA1-derived KVL peptide (SEQ ID NO: 1) or an irrelevant ASTN1 peptide (SEQ ID NO: 28). For co-culture, effectors and targets were seeded in a 96-well round-bottom plate at an E:T ratio of 2.5:1, and supernatants were collected after about 20 hours of co-culture and analyzed for IFN-γ levels by ELISA.

[0195] 4.2 Results Figure 3 ):

[0196] The potential of TCRs to effectively recognize tumor cells was assessed by co-culturing T15.8-4.3-83-transduced effectors with a panel of different tumor cell lines. Figure 3 As shown, both T15.8-4.3-83 and Ctrl MAGEA1-TCR mediated specific recognition of HLA-A2-positive and MAGEA1-positive tumor cell lines U266, NCi-H1703, UACC-62, Saos-2, and Mel624.38. KYO-1 and OPM-2 cells could only be recognized after transfection with ivtRNA encoding HLA-A2.

[0197] Untransfected KYO-1 and OPM-2 (HLA-A2- / MAGEA1-positive) and NCI-H1755, 647-V and CMK (HLA-A2+ / MAGEA1-) served as negative controls. Whenever the antigen MAGEA1 was not expressed or the required restriction element was not present, no negative control was recognized. This effective recognition of only MAGEA1-positive and HLA-A2-positive cell lines demonstrates the efficient and specific tumor cell recognition ability of the TCR.

[0198] 4.3 Experimental layout Fig.10 )

[0199] 20,000 TCR transgenic effector T cells were co-cultured with 20,000 tumor cells in 96-well round-bottom plates. Standard IFN-γ ELISA was performed after 20 hours of co-culture with melanoma cell line UACC-257, melanoma cell line UACC-62, or osteosarcoma cell line SAOS-2. Values ​​above 4000 pg were extrapolated using a cubic polynomial.

[0200] 4.5 Results Fig.10 ):

[0201] The ability of TCR transgenic effector T cell populations to produce IFN-γ in response to MAGEA1 and HLA-A*02:01 double-positive tumor cell lines was evaluated. Compared with TCR FH1, FH2, FH3 and FH4 disclosed in WO2018 / 170338 and R37P1C9 disclosed in WO2018 / 104438, T15.8-4.3-83 showed enhanced tumor cell recognition ability for all three cell lines UACC-257, UACC-62 or SAOS-2. UACC-62 was only recognized by T15.8-4.3-83.

[0202] 5. Experiment: Tumor Cell Killing

[0203] The ability of TCRs to not only recognize but also effectively lyse and thereby kill tumor cells is of great importance and clinical significance. To analyze the killing ability of TCR T15.8-4.3-83, IncuCyte TM The Zoom device was tested for 6 days of long-term lethality. TM The device is a microscope-based system that allows real-time imaging of cells. A panel of tumor cell lines were seeded into wells of a 96-well flat-bottom plate and co-cultured with TCR-expressing effectors from three different donors. The tumor cell lines stably expressed the nuclear-restricted red fluorescent protein mKate2, allowing IncuCyte TM The Zoom System is able to determine the exact number of red fluorescent cells in each well. An increase in the number of cells per well over time would mean tumor cell growth, while a decrease in the number of cells per well would indicate TCR-mediated killing.

[0204] 5.1 Experimental layout Figures 4A-4D )

[0205] The IncuCyte TM NucLight TM Red Lentivirus Reagent was used to stably transduce tumor cell lines 647-V (bladder urothelial carcinoma), UACC-62 (melanoma), Saos-2 (bone osteosarcoma), and NCI-H1703 (lung adenocarcinoma) with the nuclear-restricted red fluorescent protein mKate2. Tumor cells were seeded in 96-well flat-bottom plates and allowed to adhere to the plastic overnight. As a positive control, tumor cells were loaded with a saturating concentration of the MAGEA1-derived KVL peptide (10 -5 M) for about 1.5 hours, washed, and then used for co-culture. Tumor cells loaded with KVL or unmodified were cultured alone or co-cultured with effectors transduced with TCR T15.8-4.3-83 from 3 different donors. Co-culture with untransduced effectors served as a negative control. IncuCyte TM Zoom System takes pictures and uses IncuCyte TM The data were analyzed using Zoom software (version 2016B).

[0206] 5.2 Results Figures 4A-4D )

[0207] Figures 4A-4DTCR-mediated antigen-specific lysis of red fluorescent target cell lines 647-V (HLA-A2+ / MAGEA1-), UACC-62 (HLA-A2+ / MAGEA1+), Saos-2 (HLA-A2+ / MAGEA1+), and NCI-H1703 (HLA-A2+ / MAGEA1+) is shown respectively. Although the untransduced effector cells had no effect on the growth of any tumor cells (the number of cells increased over time), the effector cells transduced with TCR T15.8-4.3-83 effectively killed tumor cells NCI-H1703, UACC-62, and Saos-2 expressing the antigens MAGEA1 and HLA-A2 (the number of cells decreased over time). MAGEA1-negative 647-V cells were not killed. When the artificial KVL peptide was used as a control, all tumor cells were killed. Tumor cells cultured alone without or with KVL peptide were also analyzed and their growth is shown in each figure as a reference.

[0208] 5.3 Experimental layout Fig.11 )

[0209] 20,000 TCR transgenic effector T cells were combined with 7,500 Co-culture of SAOS-2 cells transduced with NucLight Red lentivirus. Tumor cells were seeded one day before the start of co-culture. After adding effector cells, the culture plate was transferred to the IncuCyte The red fluorescent cells were monitored for 72 hours at 37°C and 6% CO2, with images taken every 4 hours. The software calculated the cell count of red fluorescent tumor cells in each well at each measurement point (1 / mm 2 ). Each measurement point represents the average of 3 technical replicates.

[0210] 5.4 Results Fig.11 )

[0211] SAOS-2 cells cultured in the presence of untransduced T cells showed strong proliferation. Co-culture of different TCR transgenic T cells induced a decrease in the number of SAOS-2 cells. Interestingly, the speed and extent of killing varied between different TCR transgenic effector cells. Compared with TCR FH1, FH2, FH3 and FH4 disclosed in WO2018 / 170338 and R37P1C9 disclosed in WO2018 / 104438, TCR T15.8-4.3-83 showed the highest ability to lyse MAGEA1 and HLA-A*02:01 double-positive tumor cell lines.

[0212] 6. Experiment: TCR epitope recognition motif

[0213] In order to analyze the specific epitope recognition motifs of the TCR in detail, threonine substitution scanning and / or serine scanning were performed. By replacing the original amino acid of the epitope with threonine (or with serine, respectively), the positions within the epitope that are essential for TCR-mediated recognition can be determined. This recognition depends on the correct binding of the peptide to the HLA molecule and on the interaction of the peptide:MHC complex with the TCR itself, both of which can be affected by a single amino acid substitution.

[0214] 6.1 Experimental layout Figure 5 and Figure 6 )

[0215] To specifically define the epitope recognition motif of TCR T15.8-4.3-8, effectors expressing TCR T15.8-4.3-83 and effectors expressing the benchmark MAGEA1-TCR were prepared from three different donors. T cells were co-cultured with T2 cells loaded with the TCR-specific epitope KVL (SEQ ID NO: 1) or loaded with a peptide in which each individual amino acid residue was continuously replaced by threonine (threonine replacement scanning). T2 cells were loaded with saturating concentrations (10 -5 M) peptides, washed and co-cultured with effectors at an E:T ratio of 1: 1. The supernatants were collected after about 20 hours of co-culture and analyzed for secreted IFN-γ by ELISA.

[0216] 6.2 Results Figure 5 and Figure 6 )

[0217] Transduced with TCR T15.8-4.3-83 (eg Figure 6 ) or transduced with baseline Ctrl.MAGEA1-TCR (as Figure 5 The results of threonine substitution scanning of effectors (shown in Figure 1) demonstrated the different specificities of each TCR for its epitope and epitope-derived derivatives. As a reference, recognition of the MAGEA1-derived KVL epitope is shown on the left side of each figure. The baseline Ctrl.MAGE A1-TCR was unable to recognize peptides with threonine substitutions at positions 4 and 7 (E and I, Figure 5 ).

[0218] In contrast, TCR T15.8-4.3-83 appears to be highly sensitive to amino acid substitutions at positions 1, 3, and 5 (K, L, and Y), as indicated by the lack of peptide recognition ( Figure 6 ).

[0219] Exchanges of a single amino acid within the epitope significantly perturb recognition mediated by both TCRs, demonstrating their highly selective recognition modes and that the positions critical for recognition are clearly different for the two TCRs analyzed.

[0220] 6.3. Experimental layout Fig.13 )

[0221] 20,000 TCR transgenic T cells were mixed with 20,000 loaded 10 -5 T2 cells were co-cultured with M peptide. Standard IFN-γ ELISA was performed after 20 h of co-culture (values ​​above 4000 pg were extrapolated using a cubic polynomial). Instead of the above threonine residues, serine residues were systematically replaced with MAGEA1 KVL Individual amino acids in the peptide (serine scanning).

[0222] 6.4 Results Fig.13 )

[0223] T cells transduced with the T15.8-4.3-83 TCR displayed a different recognition motif (based on serine scanning) at a different fixed position than T cells transduced with the FH1 or R37P1C9 TCR.

[0224] A combination of threonine and serine scanning indicated that the first position in the epitope (lysine) and the fifth position in the epitope (tyrosine) appeared to be particularly important for the T15.8-4.3-83 TCR.

[0225] 7 Experiment: MAGE family members

[0226] Since 13 members of the MAGE family contain peptide sequences highly similar to the MAGEA1-derived KVL peptide (only 2–4 mismatched amino acids), in-depth analyses were performed to further investigate potential TCR T15.8-4.3-83-mediated cross-recognition. The expression of MAGE family members has been described not only in cancer and testis but also in vital organs, making potential cross-recognition of protein-derived peptides an exclusion criterion for TCRs used in clinical development.

[0227] To investigate the recognition of endogenously processed and presented peptides derived from other MAGE family members, all 13 members of the MAGE family containing peptide sequences similar to the MAGEA1 epitope KVLEYVIKV were recombinantly expressed in three cell lines of different origin. Potential cross-recognition of protein-derived MAGE peptides was analyzed by co-culturing cell lines expressing recombinant MAGE family members with effectors expressing TCR T15.8-4.3-83.

[0228] 7.1 Experimental Layout

[0229] IvtRNAs encoding the following MAGE family members were generated: MAGEA8 (NCBI Reference Sequence (Accession): NM_001166400.1), MAGEA9 (NM_005365.4), MAGEA11 (NM_005366.4), MAGEB1 (NM_002363.4), MAGEB2 (NM_002363.4), MAGEB3 (NM_002365.4), .4), MAGEB5 (NM_001271752.1), MAGEB16 (NM_001099921.1), MAGEB17 (NM_001277307.1), MAGEB18 (NM_173699.3), MAGEC2 (NM_016249.3), MAGED2 (NM_014599.5) and MAGEE2 (NM_138703.4). IvtRNA encoding MAGEA1 (NM_004988.4) and ASTN1 peptide KLY were generated as positive and negative control RNAs. Tumor cell lines HEK 293T (HLA-A2+ / MAGE-), LCL (HLA-A2+ / MAGE-), and HLA-A2-transduced K562 (MAGE-) were transfected with 20 μg of ivtRNA encoding one of the 13 MAGE family members, MAGEA1, or the ASTN1 peptide KLY. Transgenic expression of MAGE family members was analyzed by FACS (data not shown). As a control, T2 cells and the three cell lines were additionally loaded with the MAGEA1-derived KVL peptide (10 -5 M) or ASTN1-derived KLY peptide (10 -5 M). Peptide-loaded control cells and transfected cell lines were seeded into 96-well round-bottom plates 3 hours after transfection and co-cultured with untransduced or TCR T15.8-4.3-83-transduced effectors from two donors at an E:T ratio of 1:1. Supernatants were collected after approximately 20 hours of co-culture and analyzed for secreted IFN-γ by ELISA.

[0230] 7.2 Results

[0231] Figure 7The recognition of recombinant MAGE family members expressed in HEK 293T cells (A), K562 cells (B) and LCL cells (C) mediated by TCR T15.8-4.3-83 is shown. The recognition of T2 cells and HEK293T, K562 or LCL cells with KVL peptide or ASTN1 peptide in the external load is used as a control. Although the effector of non-transduction does not show any specific recognition, the effector specific recognition of TCR T15.8-4.3-83 transduction from 2 donors is transfected with all 3 cell lines of full-length MAGEA1 encoding ivtRNA. This shows that MAGEA1 encoding RNA is effectively translated into the proteasome processing of MAGEA1 protein and protein-derived KVL peptide, and then loaded and presented epitopes by the HLA-A2 molecules on the cell surface. No cell line transfected with ASTN1 peptide is recognized, nor is any recombinantly expressed MAGE family member above background level recognized. The peptides derived from MAGE family members that can be recognized after artificial loading of target cells (as described above) are not processed by endogenously expressed MAGE proteins, nor loaded onto MHC molecules or presented on the cell surface. Therefore, these peptides do not qualify as immunogenic T cell epitopes and are not considered clinically relevant targets for TCR T15.8-4.3-83-mediated cross-recognition.

[0232] 8. Experiment: Normal Cell Analysis

[0233] To investigate the safety of TCRs, TCR-mediated recognition of cells from healthy tissues of different origins must be investigated and excluded. Therefore, TCR-expressing effectors were co-cultured with normal cells from kidney (renal cortical epithelial cells obtained by PromoCell), lung (lung fibroblasts obtained by Lonza), and iPS-derived hepatocytes, cardiomyocytes, and endothelial cells (obtained by CellularDynamics). The cells investigated were endogenously negative for the antigen MAGEA1, thus allowing identification of potential MAGEA1-unrelated off-target toxicities.

[0234] 8.1 Experimental Layout

[0235] Normal cells were thawed and cultured for one week prior to co-culture as specified by the supplier. Cells were seeded in 96-well flat-bottom plates and co-cultured with TCR T15.8-4.3-83 transduced or untransduced effectors (data not shown) from three different donors. HLA-A2 expression of target cells was confirmed by antibody staining and FACS (data not shown), and unmodified or overloaded (10 -5M) target cells with MAGEA1-derived KVL (SEQ ID NO: 1) peptide were used as a control. As a control, effector cells and target cells were inoculated separately, and T2 cells were loaded with the TCR epitope KVL (10 -5 M; SEQ ID NO: 1). After about 20 hours of co-culture, the supernatant was collected and analyzed by ELISA. After about 48 hours of co-culture, the IncuCyte TM Phase contrast images were taken with a Zoom device (Essen BioScience Inc.) to visualize potential toxic effects in terms of lysis and detachment of adherent target cells.

[0236] 8.2 Results

[0237] like Figure 8 As shown, co-culture of effectors transduced with TCR T15.8-4.3-83 from 3 donors resulted in recognition of only cells artificially loaded with KVL peptide, representing the potential of target cells to correctly present TCR epitopes on their cell surface in the context of MHC molecules. Unmodified normal cells were not recognized by effectors expressing transgenic TCR T15.8-4.3-83, indicating a good safety profile for the studied receptors.

[0238] To visualize potential toxic effects on target cells mediated by TCR-transduced effectors, phase contrast images were taken of different target cells cultured alone, co-cultured with TCR T15.8-4.3-83-transduced effectors, or KVL peptide-loaded target cells co-cultured with TCR-expressing effectors. Fig. 9 Representative images of co-cultures with effector cells from one of three donors are shown.While all KVL-loaded target cells had clear TCR-mediated lysis (complete disruption of the cell layer), effector cells expressing the TCR did not lyse unmodified normal cells.

[0239] 9 Experiment: Functional Affinity

[0240] To measure the functional avidity of the different KVL peptide-specific TCRs, the half-maximal relative IFN-γ release (EC50 values) was determined when co-cultured with T2 cells loaded with graded amounts of KVL peptide.

[0241] Functional avidity refers to the cumulative strength of the multiple affinities of individual non-covalent binding interactions between the transgenic TCR and pMHC complex.

[0242] 9.1 Experimental Layout ( Fig.12 )

[0243] The functional avidity of the TCR transgenic T cell population was determined by loading the cells with graded amounts of KVL peptide (10 -4 M to 10-12 M) Half maximal relative IFN-γ release (EC50 value) of T2 cells during co-culture. Standard IFN-γ ELISA was performed after 20 hours of co-culture (values ​​greater than 4000 pg were extrapolated using a cubic polynomial).

[0244] 9.2 Results Fig.12 )

[0245] Different KVL-specific TCRs have different functional affinities for T2 cells loaded with KVL peptides. TCRT15.8-4.3-83 showed a lower EC50 value, i.e., high functional affinity for 2 cells loaded with KVL peptides compared to TCRs FH1, FH2, FH3 and FH4 disclosed in WO2018 / 170338 and R37P1C9 disclosed in WO2018 / 104438.

[0246] 10 Experiment: Cytokine Secretion

[0247] The TCR secretion patterns of TH2-specific cytokines IL-4, IL-5, and IL-13 were compared between T cells transduced with different TCRs. Th2-type inflammation has been proposed to promote tumor growth (Jager MJ, Desjardins L, T, Damato BE (eds): Current Concepts in Uveal Melanoma. Dev Ophthalmol. Basel, Karger, 2012, Vol. 49, pp. 137–149, J Immunother. 2018 Oct; 41(8): 369-378). Therefore, low or undetectable secretion levels of Th2 may be beneficial for tumor regression.

[0248] 10.1 Experimental layout (Figure 14)

[0249] 20,000 TCR transgenic effector T cells were co-cultured with 20,000 tumor cells in a round-bottom 96-well plate. MAP Kit The protocol in the system analyzes secreted cytokines in cell-free supernatants.

[0250] 10.2 Results (Figure 14)

[0251] T cells transduced with T1367 TCR secrete a variety of TH2 cytokines, especially IL-4, IL-5 and IL- 13. T cells transduced with 15.8-4.3-83 TCR did not show equivalent secretion of TH2 cytokines, while T1367 disclosed in WO2014 / 118236 showed significant secretion of these cytokines.

[0252] This application also includes the following implementation schemes:

[0253] Embodiment 1: Isolated T cell receptor (TCR) specific for MAGEA1.

[0254] Embodiment 2: The isolated TCR according to embodiment 1, wherein the TCR specifically recognizes the amino acid sequence SEQ ID NO: 1 or a fragment thereof.

[0255] Embodiment 3: An isolated TCR according to embodiments 1 and 2, wherein the TCR specifically recognizes the HLA-A2 and / or HLA-A26 binding form of the amino acid sequence of SEQ ID NO: 1, preferably the HLA-A2 binding form.

[0256] Embodiment 4: An isolated TCR according to any one of the preceding embodiments, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 1 presented by a molecule encoded by a gene selected from the group consisting of HLA-A*02:01, HLA-HLA-A*02:04, HLA-A*02:16 and HLA-A*02:17.

[0257] Embodiment 5: An isolated TCR according to any one of the preceding embodiments, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 1 presented by a molecule encoded by a gene selected from the group consisting of HLA-A*02:01, HLA-A*02:04, HLA-A*02:16 and HLA-A*02:17.

[0258] Embodiment 6: An isolated TCR according to any one of the preceding embodiments, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 1 presented by a molecule encoded by HLA-A*02:01.

[0259] Embodiment 7: An isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

[0260] - a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 2, a CDR2 having the amino acid sequence of SEQ ID NO: 3, and a CDR3 having the amino acid sequence of SEQ ID NO: 4,

[0261] - A TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO:5, a CDR2 having the amino acid sequence of SEQ ID NO:6, and a CDR3 having the amino acid sequence of SEQ ID NO:7.

[0262] Embodiment 8: An isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

[0263] A variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:8 and a variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:9.

[0264] Embodiment 9: An isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

[0265] The variable TCR alpha region has the amino acid sequence of SEQ ID NO:8 and the variable TCR beta region has the amino acid sequence of SEQ ID NO:9.

[0266] Embodiment 10: An isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

[0267] A TCR alpha chain having an amino acid sequence at least 80% identical to SEQ ID NO:10 and a TCR beta chain having an amino acid sequence at least 80% identical to SEQ ID NO:11.

[0268] Embodiment 11: An isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

[0269] A TCR alpha chain having the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:12 and a TCR beta chain having the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:13.

[0270] Embodiment 12: An isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises a TCR alpha chain and a TCR beta chain, wherein

[0271] - the variable TCR alpha region has an amino acid sequence that is at least 80% identical to SEQ ID NO:8, and comprises a CDR3 having the amino acid sequence shown in SEQ ID NO:4,

[0272] The variable TCR beta region has an amino acid sequence that is at least 80% identical to SEQ ID NO:9, and comprises a CDR3 having the amino acid sequence shown in SEQ ID NO:7.

[0273] Embodiment 13: An isolated TCR according to any one of the preceding embodiments, wherein the TCR is purified.

[0274] Embodiment 14: An isolated TCR according to any one of the preceding embodiments, wherein the amino acid sequence of said TCR comprises one or more phenotypically silent substitutions.

[0275] Embodiment 15: An isolated TCR according to any of the preceding embodiments, wherein the amino acid sequence of the TCR is modified to comprise a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety.

[0276] Embodiment 16: The isolated TCR according to Embodiment 15, wherein the therapeutic agent is selected from the group consisting of an immune effector molecule, a cytotoxic agent, and a radionuclide.

[0277] Embodiment 17: An isolated TCR according to embodiment 16, wherein the immune effector molecule is a cytokine.

[0278] Embodiment 18: An isolated TCR according to any of the preceding embodiments, wherein the TCR is soluble or membrane-bound.

[0279] Embodiment 19: An isolated TCR according to embodiment 15, wherein the pharmacokinetic modifying moiety is at least one polyethylene glycol repeating unit, at least one diol group, at least one sialic acid group, or a combination thereof.

[0280] Embodiment 20: An isolated TCR according to any one of the preceding embodiments, wherein the TCR is a single-chain type, wherein the TCR α chain and the TCR β chain are connected by a linker sequence.

[0281] Embodiment 21: The isolated TCR according to embodiments 1 to 20, wherein the TCR alpha chain or the TCR beta chain is modified to comprise an epitope tag.

[0282] Embodiment 22: An isolated polypeptide comprising a functional portion of the TCR of any one of embodiments 1 to 21, wherein the functional portion comprises at least one of the amino acid sequences of SEQ ID NOs: 4 and 7.

[0283] Embodiment 23: An isolated polypeptide comprising a functional portion of the TCR of any one of embodiments 1 to 21, wherein the functional portion comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 and 7.

[0284] Embodiment 24: An isolated polypeptide according to embodiment 21, wherein the functional part comprises a TCR alpha variable chain and / or a TCR beta variable chain.

[0285] Embodiment 25: A multivalent TCR complex comprising a TCR as embodied in any one of embodiments 1 to 21.

[0286] Embodiment 26: An isolated TCR according to embodiments 1 to 21, a polypeptide according to embodiments 22 to 24, a multivalent TCR complex according to embodiment 25, wherein IFN-γ secretion is induced by binding to the amino acid sequence of SEQ ID NO: 1 presented by a molecule encoded by HLA-A*02:01.

[0287] Embodiment 27: Nucleic acid encoding a TCR according to any one of embodiments 1 to 21 or encoding a polypeptide according to embodiments 22 to 24.

[0288] Embodiment 28: A vector comprising the nucleic acid of embodiment 27.

[0289] Embodiment 29: The vector according to embodiment 28, wherein the vector is an expression vector.

[0290] Embodiment 30: The vector according to embodiment 28 or 29, wherein the vector is a retroviral vector.

[0291] Embodiment 31: The vector according to embodiment 28 or 29, wherein the vector is a lentiviral vector.

[0292] Embodiment 32: A cell expressing a TCR according to embodiments 1 to 21.

[0293] Embodiment 33: A cell according to Embodiment 32, wherein the cell is isolated or non-naturally occurring.

[0294] Embodiment 34: The cell according to embodiments 32 and 33, wherein the cell comprises the nucleic acid according to embodiment 27 or the vector according to embodiments 28 to 31.

[0295] Embodiment 35: The cell of any one of embodiments 32 to 34, wherein the cell comprises:

[0296] a) an expression vector comprising at least one nucleic acid as embodied in embodiment 27, or

[0297] b) a first expression vector comprising a nucleic acid encoding the α chain of the TCR as embodied in any one of embodiments 1 to 21, and a second expression vector comprising a nucleic acid encoding the β chain of the TCR as embodied in any one of embodiments 1 to 21.

[0298] Embodiment 36: A cell according to any one of embodiments 32 to 35, wherein the cell is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC).

[0299] Embodiment 37: The cell according to any one of Embodiments 32 to 36, wherein the cell is a T cell.

[0300] Embodiment 38: An antibody or antigen-binding fragment thereof that specifically binds to the portion of the TCR according to embodiments 1 to 21 that mediates specificity for MAGEA1.

[0301] Embodiment 39: An antibody according to Embodiment 38, wherein the portion of the TCR that mediates MAGEA1 specificity comprises at least one of the CDRs shown in SEQ ID NOs: 2, 3, 4, 5, 6 and 7, preferably the α chain CDR3 of SEQ ID NO: 4 and / or the β chain CDR3 of SEQ ID NO: 7, more preferably the α chain CDRs shown in SEQ ID NOs: 2, 3 and 4, and the β chain CDRs of SEQ IDs 5, 6 and 7.

[0302] Embodiment 40: A pharmaceutical composition comprising a TCR according to embodiments 1 to 21, a polypeptide according to embodiments 22 to 24, a multivalent TCR complex according to embodiment 25, a nucleic acid according to embodiment 27, a vector according to embodiments 28 to 31, a cell according to any one of embodiments 32 to 37, or an antibody according to embodiments 38 to 39.

[0303] Embodiment 41: The pharmaceutical composition according to embodiment 40, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

[0304] Embodiment 42: A TCR according to embodiments 1 to 21, a polypeptide according to embodiments 22 to 24, a multivalent TCR complex according to embodiment 25, a nucleic acid according to embodiment 27, a vector according to embodiments 28 to 31, a cell according to any one of embodiments 32 to 37, or an antibody according to embodiments 38 to 39, for use as a medicament.

[0305] Embodiment 43: A TCR according to embodiments 1 to 21, a polypeptide according to embodiments 22 to 24, a multivalent TCR complex according to embodiment 25, a nucleic acid according to embodiment 27, a vector according to claims 28 to 31 or a cell according to any one of embodiments 32 to 37, for use in the treatment of cancer.

[0306] Embodiment 44: The TCR, polypeptide, multivalent TCR complex, nucleic acid, vector or cell according to Embodiment 43, wherein the cancer is a hematological cancer or a solid tumor.

[0307] Embodiment 45: A TCR, polypeptide, multivalent TCR complex, nucleic acid, vector or cell according to embodiments 43 and 44, wherein the cancer is selected from the group consisting of: prostate cancer, uterine cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, cervical cancer, colorectal cancer, gastric adenocarcinoma, bile duct cancer, breast cancer, bladder cancer, myeloid leukemia and acute lymphoblastic leukemia, sarcoma or osteosarcoma.

[0308] Embodiment 46: The TCR, polypeptide, multivalent TCR complex, nucleic acid, vector or cell according to embodiments 43 and 44, wherein the cancer is preferably selected from the group consisting of sarcoma or osteosarcoma. Sequence Listing <110> Medigene Immunotherapies GmbH <120> MAGEA1-specific T cell receptor and its use <130> M11421 <150> EP19167440 <151> 2019-04-04 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Homo sapiens <400> 1 Lys Val Leu Glu Tyr Val Ile Lys Val 1 5 <210> 2 <211> 7 <212> PRT <213> Homo sapiens <400> 2 Thr Arg Asp Thr Thr Tyr Tyr 1 5 <210> 3 <211> 8 <212> PRT <213> Homo sapiens <400> 3 Arg Asn Ser Phe Asp Glu Gln Asn 1 5 <210> 4 <211> 15 <212> PRT <213> Homo sapiens <400> 4 Cys Ala Leu Ser Glu Val Ala Ser Gly Gly Ser Tyr Ile Pro Thr 1 5 10 15 <210> 5 <211> 6 <212> PRT <213> Homo sapiens <400> 5 Gly Thr Ser Asn Pro Asn 1 5 <210> 6 <211> 5 <212> PRT <213> Homo sapiens <400> 6 Ser Val Gly Ile Gly 1 5 <210> 7 <211> 14 <212> PRT <213> Homo sapiens <400> 7 Cys Ala Trp Ser Gly Ser Gly Gly Asn Gln Pro Gln His Phe 1 5 10 <210> 8 <211> 138 <212> PRT <213> Homo sapiens <400> 8 Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val 1 5 10 15 Val Ser Ser Met Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser 20 25 30 Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg 35 40 45 Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu 50 55 60 Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile 65 70 75 80 Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn 85 90 95 Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys 100 105 110 Ala Leu Ser Glu Val Ala Ser Gly Gly Ser Tyr Ile Pro Thr Phe Gly 115 120 125 Arg Gly Thr Ser Leu Ile Val His Pro Tyr 130 135 <210> 9 <211> 130 <212> PRT <213> Homo sapiens <400> 9 Met Leu Cys Ser Leu Leu Ala Leu Leu Leu Gly Thr Phe Phe Gly Val 1 5 10 15 Arg Ser Gln Thr Ile His Gln Trp Pro Ala Thr Leu Val Gln Pro Val 20 25 30 Gly Ser Pro Leu Ser Leu Glu Cys Thr Val Glu Gly Thr Ser Asn Pro 35 40 45 Asn Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Arg Gly Leu Gln Leu Leu 50 55 60 Phe Tyr Ser Val Gly Ile Gly Gln Ile Ser Ser Glu Val Pro Gln Asn 65 70 75 80 Leu Ser Ala Ser Arg Pro Gln Asp Arg Gln Phe Ile Leu Ser Ser Lys 85 90 95 Lys Leu Leu Leu Ser Asp Ser Gly Phe Tyr Leu Cys Ala Trp Ser Gly 100 105 110 Ser Gly Gly Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser 115 120 125 Ile Leu 130 <210> 10 <211> 278 <212> PRT <213> Synthetic Sequence <220> <223> Minimal Murine Constant Region <400> 10 Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val 1 5 10 15 Val Ser Ser Met Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser 20 25 30 Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg 35 40 45 Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu 50 55 60 Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile 65 70 75 80 Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn 85 90 95 Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys 100 105 110 Ala Leu Ser Glu Val Ala Ser Gly Gly Ser Tyr Ile Pro Thr Phe Gly 115 120 125 Arg Gly Thr Ser Leu Ile Val His Pro Tyr Ile Gln Asn Pro Asp Pro 130 135 140 Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys 145 150 155 160 Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp 165 170 175 Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met 180 185 190 Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe 195 200 205 Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe 210 215 220 Phe Pro Ser Ser Asp Val Pro Cys Asp Val Lys Leu Val Glu Lys Ser 225 230 235 240 Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly 245 250 255 Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr 260 265 270 Leu Arg Leu Trp Ser Ser 275 <210> 11 <211> 307 <212> PRT <213> Artificial sequence <220> <223> Most mouse humanized constant region <400> 11 Met Leu Cys Ser Leu Leu Ala Leu Leu Leu Gly Thr Phe Phe Gly Val 1 5 10 15 Arg Ser Gln Thr Ile His Gln Trp Pro Ala Thr Leu Val Gln Pro Val 20 25 30 Gly Ser Pro Leu Ser Leu Glu Cys Thr Val Glu Gly Thr Ser Asn Pro 35 40 45 Asn Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Arg Gly Leu Gln Leu Leu 50 55 60 Phe Tyr Ser Val Gly Ile Gly Gln Ile Ser Ser Glu Val Pro Gln Asn 65 70 75 80 Leu Ser Ala Ser Arg Pro Gln Asp Arg Gln Phe Ile Leu Ser Ser Lys 85 90 95 Lys Leu Leu Leu Ser Asp Ser Gly Phe Tyr Leu Cys Ala Trp Ser Gly 100 105 110 Ser Gly Gly Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser 115 120 125 Ile Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe 130 135 140 Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln Lys Ala Thr Leu Val 145 150 155 160 Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp 165 170 175 Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro 180 185 190 Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser 195 200 205 Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe 210 215 220 Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr 225 230 235 240 Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp 245 250 255 Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val 260 265 270 Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu 275 280 285 Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg 290 295 300 Lys Asp Phe 305 <210> 12 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> Murine Constant Region <400> 12 Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val 1 5 10 15 Val Ser Ser Met Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser 20 25 30 Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg 35 40 45 Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu 50 55 60 Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile 65 70 75 80 Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn 85 90 95 Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys 100 105 110 Ala Leu Ser Glu Val Ala Ser Gly Gly Ser Tyr Ile Pro Thr Phe Gly 115 120 125 Arg Gly Thr Ser Leu Ile Val His Pro Tyr Ile Gln Asn Pro Glu Pro 130 135 140 Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys 145 150 155 160 Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu 165 170 175 Ser Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met 180 185 190 Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe 195 200 205 Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser 210 215 220 Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp 225 230 235 240 Met Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu 245 250 255 Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 260 265 270 Ser Ser <210> 13 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> Murine Constant Region <400> 13 Met Leu Cys Ser Leu Leu Ala Leu Leu Leu Gly Thr Phe Phe Gly Val 1 5 10 15 Arg Ser Gln Thr Ile His Gln Trp Pro Ala Thr Leu Val Gln Pro Val 20 25 30 Gly Ser Pro Leu Ser Leu Glu Cys Thr Val Glu Gly Thr Ser Asn Pro 35 40 45 Asn Leu Tyr Trp Tyr Arg Gln Ala Ala Gly Arg Gly Leu Gln Leu Leu 50 55 60 Phe Tyr Ser Val Gly Ile Gly Gln Ile Ser Ser Glu Val Pro Gln Asn 65 70 75 80 Leu Ser Ala Ser Arg Pro Gln Asp Arg Gln Phe Ile Leu Ser Ser Lys 85 90 95 Lys Leu Leu Leu Ser Asp Ser Gly Phe Tyr Leu Cys Ala Trp Ser Gly 100 105 110 Ser Gly Gly Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser 115 120 125 Ile Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Thr Leu Phe 130 135 140 Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val 145 150 155 160 Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp 165 170 175 Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala 180 185 190 Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val 195 200 205 Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val 210 215 220 Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro 225 230 235 240 Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp 245 250 255 Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser Ala Thr 260 265 270 Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu 275 280 285 Val Ser Gly Leu Val Leu Met Ala Met Val Lys Lys Lys Asn Ser 290 295 300 <210> 14 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Codon optimization <400> 14 acacgggaca ccacctacta c 21 <210> 15 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Codon optimization <400> 15 cggaacagct tcgacgagca gaac 24 <210> 16 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Codon optimization <400> 16 tgcgccctga gcgaagtggc cagcggcggc tcttacatcc ctaca 45 <210> 17 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Codon optimization <400> 17 ggcaccagca atcccaac 18 <210> 18 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Codon optimization <400> 18 agcgtcggca tcggc 15 <210> 19 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Codon optimization <400> 19 tgtgcttgga gtggcagcgg cggcaatcag cctcagcact tt 42 <210> 20 <211> 414 <212> DNA <213> Artificial sequence <220> <223> Codon optimization <400> 20 atgctgacag cctctctgct gagagccgtg atcgccagca tctgtgtggt gtctagcatg 60 gcccagaaag tgacacaggc ccagaccgag atcagcgtgg tggaaaaaga agatgtgacc 120 ctggactgcg tgtacgagac acgggacacc acctactacc tgttctggta caagcagcct 180 cctagcggcg agctggtgtt cctgatcaga cggaacagct tcgacgagca gaacgagatc 240 tccggccggt acagctggaa cttccagaag tccaccagca gcttcaactt caccatcacc 300 gccagccagg tggtggatag cgccgtgtat ttttgcgccc tgagcgaagt ggccagcggc 360 ggctcttaca tccctacatt tggcagaggc accagcctga tcgtgcaccc ttat 414 <210> 21 <211> 390 <212> DNA <213> Artificial sequence <220> <223> Codon optimization <400> 21 atgctgtgtt ctctgctggc tctgctgctg ggcacctttt ttggcgtcag aagccagacc 60 atccaccagt ggcctgctac actggtgcag cctgttggaa gccctctgag cctggaatgt 120 accgtggaag gcaccagcaa tcccaacctg tactggtaca gacaggccgc tggaagagga 180 ctgcagctgc tgttttacag cgtcggcatc ggccagatca gcagcgaggt tccacagaat 240 ctgagcgcca gcagacccca ggacagacag tttatcctga gcagcaagaa gctgctgctg 300 agcgacagcg gcttctacct gtgtgcttgg agtggcagcg gcggcaatca gcctcagcac 360 tttggagatg gcacccggct gagcatcctg 390 <210> 22 <211> 837 <212> DNA <213> Artificial Sequence <220> <223> Minimal murine constant region, codon optimized <400> 22 atgctgacag cctctctgct gagagccgtg atcgccagca tctgtgtggt gtctagcatg 60 gcccagaaag tgacacaggc ccagaccgag atcagcgtgg tggaaaaaga agatgtgacc 120 ctggactgcg tgtacgagac acgggacacc acctactacc tgttctggta caagcagcct 180 cctagcggcg agctggtgtt cctgatcaga cggaacagct tcgacgagca gaacgagatc 240 tccggccggt acagctggaa cttccagaag tccaccagca gcttcaactt caccatcacc 300 gccagccagg tggtggatag cgccgtgtat ttttgcgccc tgagcgaagt ggccagcggc 360 ggctcttaca tccctacatt tggcagaggc accagcctga tcgtgcaccc ttatattcag 420 aaccccgatc ctgccgtgta ccagctgaga gacagcaaga gcagcgacaa gagcgtgtgt 480 ctgttcaccg acttcgacag ccagaccaac gtgtcccaga gcaaggacag cgacgtgtac 540 atcaccgaca agaccgtgct ggacatgcgg agcatggact tcaagagcaa cagcgccgtg 600 gcctggtcca acaagagcga tttcgcctgc gccaacgcct tcaacaatag cattatcccc 660 gaggacacat tcttccccag ctccgatgtg ccctgcgacg tgaagctggt ggaaaagagc 720 ttcgagacag acaccaacct gaacttccag aacctgagcg tgatcggctt cagaatcctg 780 ctgctgaagg tggccggctt caatctgctg atgaccctga gactgtggtc cagctga 837 <210> 23 <211> 924 <212> DNA <213> Artificial Sequence <220> <223> Minimally murine constant region, codon optimized <400> 23 atgctgtgtt ctctgctggc tctgctgctg ggcacctttt ttggcgtcag aagccagacc 60 atccaccagt ggcctgctac actggtgcag cctgttggaa gccctctgag cctggaatgt 120 accgtggaag gcaccagcaa tcccaacctg tactggtaca gacaggccgc tggaagagga 180 ctgcagctgc tgttttacag cgtcggcatc ggccagatca gcagcgaggt tccacagaat 240 ctgagcgcca gcagacccca ggacagacag tttatcctga gcagcaagaa gctgctgctg 300 agcgacagcg gcttctacct gtgtgcttgg agtggcagcg gcggcaatca gcctcagcac 360 tttggagatg gcacccggct gagcatcctg gaagatctga acaaggtgtt ccctccagag 420 gtggccgtgt tcgagccttc taaggccgag attgcccaca cacagaaagc cacactcgtg 480 tgcctggcta ccggcttctt tcctgaccac gtggaactgt cttggtgggt caacggcaaa 540 gaggtgcaca gcggcgtcag cacagatccc cagcctctga aagaacagcc cgctctgaac 600 gacagccggt actgtctgag cagcagactg agagtgtccg ccacattctg gcagaacccc 660 agaaaccact tcagatgcca ggtgcagttc tacggcctga gcgagaacga tgagtggacc 720 caggatagag ccaagcctgt gacacagatc gtgtctgccg aagcctgggg cagagccgat 780 tgtggaatta ccagcgccag ctaccatcag ggcgtgctgt ctgccacaat cctgtacgag 840 atcctgctgg gcaaagccac tctgtacgcc gtgctggtgt ctgccctggt gctgatggcc 900 atggtcaaga gaaaggactt ttga 924 <210> 24 <211> 825 <212> DNA <213> Artificial Sequence <220> <223> Mouse Constant Region, Codon Optimized <400> 24 atgctgacag cctctctgct gagagccgtg atcgccagca tctgtgtggt gtctagcatg 60 gcccagaaag tgacacaggc ccagaccgag atcagcgtgg tggaaaaaga agatgtgacc 120 ctggactgcg tgtacgagac acgggacacc acctactacc tgttctggta caagcagcct 180 cctagcggcg agctggtgtt cctgatcaga cggaacagct tcgacgagca gaacgagatc 240 tccggccggt acagctggaa cttccagaag tccaccagca gcttcaactt caccatcacc 300 gccagccagg tggtggatag cgccgtgtat ttttgcgccc tgagcgaagt ggccagcggc 360 ggctcttaca tccctacatt tggcagaggc accagcctga tcgtgcaccc ttatatccag 420 aatccggagc ccgccgtata ccagctgaag gaccctagaa gccaggacag caccctgtgc 480 ctgttcaccg acttcgacag ccagatcaac gtgcccaaga ccatggaaag cggcaccttc 540 atcaccgaca agacagtgct ggacatgaag gccatggaca gcaagtccaa cggcgcaatc 600 gcctggtcca accagaccag cttcacatgc caggacatct tcaaagagac aaacgccaca 660 taccccagca gcgacgtgcc ctgtgatgcc accctgacag agaagtcctt cgagacagac 720 atgaacctga acttccagaa tctgtccgtg atgggcctga gaatcctgct gctgaaggtg 780 gccggcttca atctgctgat gaccctgcgg ctgtggtcca gctga 825 <210> 25 <211> 912 <212> DNA <213> Artificial sequence <220> <223> Mouse constant region <400> 25 atgctgtgtt ctctgctggc tctgctgctg ggcacctttt ttggcgtcag aagccagacc 60 atccaccagt ggcctgctac actggtgcag cctgttggaa gccctctgag cctggaatgt 120 accgtggaag gcaccagcaa tcccaacctg tactggtaca gacaggccgc tggaagagga 180 ctgcagctgc tgttttacag cgtcggcatc ggccagatca gcagcgaggt tccacagaat 240 ctgagcgcca gcagacccca ggacagacag tttatcctga gcagcaagaa gctgctgctg 300 agcgacagcg gcttctacct gtgtgcttgg agtggcagcg gcggcaatca gcctcagcac 360 tttggagatg gcacccggct gagcatcctg gaagatctcc ggaacgtgac ccccctaaa 420 gtgaccctgt tcgaacccag caaggccgag atcgccaaca agcagaaagc caccctcgtg 480 tgcctggcca gaggcttctt ccccgaccat gtggaactgt cttggtgggt caacggcaaa 540 gaggtgcaca gcggagtgtc caccgaccct caggcctaca aagagagcaa ctacagctac 600 tgcctgagca gcagactgcg ggtgtccgcc accttctggc acaacccccg gaaccacttc 660 aggtgccagg tgcagtttca cggcctgagc gaagaggaca agtggcccga aggctccccc 720 aagcccgtga cccagaatat ctctgccgag gcctggggca gagccgactg tggaattacc 780 agcgccagct accaccaggg cgtgctgtct gccaccatcc tgtacgagat cctgctgggc 840 aaggccaccc tgtacgccgt gctggtgtct ggcctggtgc tgatggccat ggtcaagaag 900 aagaacagct ga 912 <210> 26 <211> 395 <212> PRT <213> artificial sequence <220> <223> artificial sequence <400> 26 Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1 5 10 15 Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu 20 25 30 Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala 35 40 45 Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala 50 55 60 His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly Arg His Ser Leu 65 70 75 80 Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser His Ser Phe 85 90 95 Pro His Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg 100 105 110 Pro Gly Asp Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu 115 120 125 Pro Ala Glu Leu Thr Asp Ala Val Lys Val Met Asp Leu Pro Thr Gln 130 135 140 Glu Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile 145 150 155 160 Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu 165 170 175 His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val 180 185 190 Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr 195 200 205 Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln 210 215 220 Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro 225 230 235 240 Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr 245 250 255 Ile Val Ala Asn Pro His His His His His His Tyr Phe Ser Lys Glu 260 265 270 Glu Trp Glu Lys Met Lys Ala Ser Glu Lys Ile Phe Tyr Val Tyr Met 275 280 285 Lys Arg Lys Tyr Glu Ala Met Thr His His His His His His Asp Lys 290 295 300 Thr Gly Phe His Phe Cys Gly Gly Ser Leu Ile Ser Glu Asp Trp Val 305 310 315 320 Val Thr Ala Ala His Cys Gly Val Arg Thr Ser His His His His His 325 330 335 His Ser Ser Pro Gly Val Tyr Ala Arg Val Thr Lys Leu Ile Pro Trp 340 345 350 Val Gln Lys Ile Leu Ala Ala Asn His His His His His His Pro Arg 355 360 365 Ala Leu Ala Glu Thr Ser Tyr Val Lys Val Leu Glu Tyr Val Ile Lys 370 375 380 Val Ser Ala Arg Val Arg Phe Phe Phe Pro Ser 385 390 395 <210> 27 <211> 1188 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence <400> 27 atgtgggtcc cggttgtctt cctcaccctg tccgtgacgt ggattggtgc tgcacccctc 60 atcctgtctc ggattgtggg aggctgggag tgcgagaagc attcccaacc ctggcaggtg 120 cttgtggcct ctcgtggcag ggcagtctgc ggcggtgttc tggtgcaccc ccagtgggtc 180 ctcacagctg cccactgcat caggaaaa agcgtgatct tgctgggtcg gcacagcctg 240 tttcatcctg aagacacagg ccaggtattt caggtcagcc acagcttccc acacccgctc 300 tacgatatga gcctcctgaa gaatcgattc ctcaggccag gtgatgactc cagccacgac 360 ctcatgctgc tccgcctgtc agagcctgcc gagctcacgg atgctgtgaa ggtcatggac 420 ctgcccaccc aggagccagc actggggacc acctgctacg cctcaggctg gggcagcatt 480 gaaccagagg agttcttgac cccaaagaaa cttcagtgtg tggacctcca cgttatttcc 540 aacgacgtgt gtgcgcaagt tcaccctcag aaggtgacca agttcatgct gtgtgctgga 600 cgctggacag ggggcaaaag cacctgctcg ggtgattctg ggggcccact tgtctgtaac 660 ggtgtgcttc aaggtatcac gtcatggggc agtgaaccgt gtgccctgcc cgaaaggcct 720 tccctgtaca ccaaggtggt gcattaccgg aagtggatca aggacaccat cgtggccaac 780 ccccatcacc atcaccacca ctacttctct aaagaagt gggaaaagat gaaagcctcg 840 gagaaatct tctatgtgta tatgaagaga aagtatgagg ctatgactca tcaccatcac 900 caccacgaca aaaccggctt ccacttctgc gggggctccc tcatcagcga ggactgggtg 960 gtcaccgctg cccactgcgg ggtcaggacc tcccatcacc atcaccacca ctccagccct 1020 ggcgtgtacg cccgtgtcac caagctcata ccttgggtgc agaagatcct ggctgccaac 1080 catcaccatc accaccaccc aagggccctc gctgaaacca gctatgtgaa agtccttgag 1140 tatgtgatca aggtcagtgc aagagttcgc tttttcttcc catcctga 1188 <210> 28 <211> 10 <212> PRT <213> Homo sapiens <400> 28 Lys Leu Tyr Gly Leu Asp Trp Ala Glu Leu 1 5 10

Claims

1. An isolated T cell receptor (TCR) specific for MAGEA1, wherein the TCR comprises: - a TCR alpha chain comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 3, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, a TCR beta chain comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 7, The TCR specifically recognizes the amino acid sequence SEQ ID NO:

1.

2. The isolated TCR according to claim 1, wherein the TCR specifically recognizes the HLA-A2 binding form of the amino acid sequence of SEQ ID NO:

1.

3. The isolated TCR according to claim 1, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 1 presented by a molecule encoded by a gene selected from the group consisting of HLA-A*02:01, HLA-A*02:04, HLA-A*02:16 and HLA-A*02:

17.

4. The isolated TCR of claim 1, wherein the TCR comprises a variable TCR α region having an amino acid sequence at least 80% identical to SEQ ID NO: 8 and a variable TCR β region having an amino acid sequence at least 80% identical to SEQ ID NO:

9.

5. An isolated polypeptide comprising a functional portion of a TCR as claimed in any one of claims 1 to 4, wherein the functional portion comprises: - a TCR alpha chain comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 2, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 3, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 4, a TCR beta chain comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 5, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 6, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 7, The functional part mediates the binding of the TCR to the amino acid sequence SEQ ID NO:

1.

6. A multivalent TCR complex comprising at least two TCRs as described in any one of claims 1 to 4.

7. The isolated TCR according to any one of claims 1 to 4, the polypeptide according to claim 5, the multivalent TCR complex according to claim 6, wherein IFN-γ secretion is induced by binding to the amino acid sequence of SEQ ID NO: 1 presented by the molecule encoded by HLA-A*02:

01.

8. Nucleic acid encoding the TCR according to any one of claims 1 to 4 or encoding the isolated polypeptide according to claim 5.

9. A vector comprising the nucleic acid according to claim 8.

10. A cell expressing the TCR according to any one of claims 1 to 4.

11. Use of the TCR according to any one of claims 1 to 4, the polypeptide according to claim 5, the multivalent TCR complex according to claim 6, the nucleic acid according to claim 8, the vector according to claim 9 or the cell according to claim 10 in the preparation of a medicament for treating cancer, wherein the cancer is selected from the group consisting of lung adenocarcinoma, melanoma and osteosarcoma, wherein the treatment targets MAGEA1-positive tumor cells.

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