MAGEA10-specific T cell receptor and its use

Abstract

The present invention relates to an isolated T cell receptor (TCR) specific for a MAGEA10-derived peptide and a polypeptide comprising a functional portion of a TCR. The present invention further relates to a multivalent TCR complex, a nucleic acid encoding a TCR, a cell expressing a TCR, and a pharmaceutical composition comprising a TCR. The present invention also relates to TCR for use as a drug, in particular TCR for use in the treatment of cancer.

Description

Technical Field

[0001] The present invention relates to isolated T cell receptors (TCRs) specific for MAGEA10-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, particularly TCRs for treating cancer. Background Art

[0002] MAGEA10, also known as melanoma-associated antigen 10, 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.). MAGEA10 is a cytosolic / cytoplasmic protein, and peptides derived from MAGEA10 protein are presented in the context of MHC class I (i.e., on HLA molecules). More specifically, MAGEA10-derived epitopes are presented on HLA-A2 molecules, indicating its applicability as a target for cancer immunotherapy. A large number of clinical trials targeting MAGEA proteins have been conducted in the field of immunotherapy (clinicaltrials.gov). MAGEA10 is a promising target for T cell-mediated immunotherapy. The principle of cancer immunotherapy using adoptive cell transfer (ACT) can utilize the patient's own immune system for highly tumor-specific cancer treatment. ACT uses ex vivo amplified autologous (patient-derived) T cells that are genetically modified to express T cell receptors (TCRs) that are specific for defined epitopes derived from intracellular proteins (e.g., MAGEA10). Therefore, there is still a need for efficient TCRs that only target tumor-specific / restricted antigens MAGEA10 and therefore have special potential for cancer immunotherapy. Therefore, a specific TCR targeting MAGEA10 is needed. Summary of the invention

[0003] One object of the present invention is to provide a T cell receptor (TCR) specific for MAGEA10.

[0004] In a specific embodiment, the isolated TCR comprises: a TCRα 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 TCRβ 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 binding form of the amino acid sequence of SEQ ID NO: 1. 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 and HLA-A*02:04, and 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 fragment may be an antigenic sequence specific for the antigen, i.e. not present in another protein or peptide in a mammal, particularly a human. The fragment may be shorter than the sequence of the antigen, for example at least 5%, at least 10%, at least 30%, at least 50%, at least 70%, at least 90% shorter than the antigen. The fragment may have a length of at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more amino acids.

[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 MAGEA10.

[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 substitutions. In addition, the TCR of the present invention may be labeled with a detectable marker. 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 the 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] According to the TCR of the present invention, in particular the soluble form of the TCR, can be modified by connecting additional functional parts, for example, to reduce immunogenicity, increase hydrodynamic size (size in solution) solubility and / or stability (for example, 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 present 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 specific embodiments, the functional portion comprises a TCRα variable chain and / or a TCRβ 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 expressing the TCR of the present invention as a functional polypeptide or a functional multivalent polypeptide on effector cells, in particular immune effector cells, wherein upon binding to the HLA-A2 binding form of the amino acid sequence SEQ ID NO: 1, IFN-γ secretion is induced in the aforementioned effector cells 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 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.

[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 as described herein that mediates specificity for MAGEA10. In a specific embodiment, the portion of the TCR that mediates specificity for MAGEA10 comprises the alpha chain CDR3 of SEQ ID NO: 4 and / or the beta chain CDR3 of SEQ ID NO: 7. In some embodiments, the portion of the TCR that mediates specificity for MAGEA10 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 contain 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 DisplayMAGE-A10 SLL - Binding of MHC-multimers to CD8+ T cells transduced with MAGE-A10 reactive TCR. CD8 T cells were isolated from PBMC of healthy donors and transduced with MAGE-A10 SLL -TCR and does not recognize MAGE-A10 SLL The transduced CD8 T cells were enriched by FACS using the mouse constant β region as a transduction marker. After expansion of these cells, they were transduced with MAGE-A10 SLL -MHC-multimers (SLL-tetramers) and antibodies against CD8 and against the murine constant beta region (mCβ) were stained and analyzed by flow cytometry.

[0032] Figure 2 DisplayMAGE-A10 SLL -TCR-transgenic T cells recognize MAGE-A10 presented on HLA-A2 SLL -peptide. SLL -peptide-T2 cell co-culture. T2 cells loaded with control peptide were used as negative control. Target cell recognition was analyzed by measuring IFN-γ concentration in the co-culture supernatant by standard ELISA. Figure 2It was also shown that control TCRs did not recognize MAGE-A10 SLL Epitope.

[0033] Figure 3 A and 3B show MAGE-A10 SLL -TCR-transgenic T cells recognize MAGE-A10 present on tumor cell lines SLL The ability of transgenic T cells to express different MAGE-A10 SLL Co-culture of positive tumor cell lines. Two cell lines (Mel624.38 and 647V) that do not express MAGE-A10 were used as negative controls. (A) Cytokine release was analyzed by measuring IFN-γ concentrations in co-culture supernatants by standard ELISA. (B) Cytotoxicity against tumor cell lines stably transduced with fluorescent markers was measured using IncuCyte.

[0034] Figure 4 DisplayMAGE-A10 SLL - Recognition of MAGE-A family members by TCR. Transgenic T cells were co-cultured with K562 cells expressing different MAGE-A family members. Cytokine release was analyzed by measuring IFN-γ concentration in the co-culture supernatant by standard ELISA.

[0035] Figure 5 A and 5B show targeting of MAGE-A10 SLL -HLA fine typing of TCR and targeting MAGE-A10 SLL - Analysis of potential alloreactivity of TCR. MAGE-A10 was loaded with cells expressing different HLA-A types (A). SLL LCLs with (B) or without peptide loading of MAGE-A10 SLL 2. Peptide LCL Library with MAGE-A10 SLL -TCR-transgenic T cells or negative control TCR co-culture. As a positive control, T2 cells were externally loaded with MAGE-A10 SLL -peptide. Cytokine release was analyzed by measuring IFN-γ concentrations in co-culture supernatants by standard ELISA.

[0036] Figure 6 DisplayMAGE-A10 SLL - Analysis of TCR recognition of normal cells. Transgenic T cells were compared with normal cells and externally loaded with MAGE-A10 SLL -peptide-containing normal cells were co-cultured. Cytokine release was analyzed by measuring IFN-γ concentration in the co-culture supernatant by standard ELISA. DETAILED DESCRIPTION

[0037] Before describing the present invention in detail with respect to some preferred embodiments of the invention, the following general definitions are provided.

[0038] The invention illustratively described below suitably may be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

[0039] 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.

[0040] 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 a preferred embodiment of the term "comprising of". If hereinafter a group is defined as comprising at least a certain number of embodiments, this is also to be understood as disclosing a group that preferably consists of only these embodiments.

[0041] 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.

[0042] Unless specifically stated otherwise, where an indefinite or definite article is used when referring to a singular noun, for example "a", "an" or "the", this includes the plural form of the noun. The term "about" or "approximately" in the context of the present invention indicates an interval of accuracy 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.

[0043] 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 the terms are used.

[0044] TCR Background

[0045] TCR is composed of two different and independent protein chains, namely TCR alpha (ɑ) chain and TCR beta (β) chain. TCRɑ chain contains variable region (V), joining region (J) and constant region (C). TCRβ chain contains variable region (V), diversity region (D), joining region (J) and constant region (C). The rearranged V(D)J region of both TCRɑ chain and TCRβ chain contains hypervariable region (CDR, complementary determining region), among which CDR3 region determines specific epitope recognition. In the C-terminal region, both TCRɑ chain and TCRβ chain contain hydrophobic transmembrane domains and end with a short cytoplasmic tail.

[0046] Typically, TCR is a heterodimer of one α chain and one β chain. This heterodimer can bind to MHC molecules presenting peptides.

[0047] 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.

[0048] TCR loci and genes use 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.

[0049] Target

[0050] The target of the TCR described herein is the peptide SLL (SEQ ID NO: 1) derived from MAGEA10 (NCBI Reference Sequence: NM_001011543.2).

[0051] TCR-specific sequences

[0052] Some embodiments relate to an isolated TCR comprising a TCR alpha chain and a TCR beta chain, wherein

[0053] - the TCRα chain comprises a complementarity determining region 3 (CDR3) having a sequence of SEQ ID NO: 4,

[0054] - The TCR β chain comprises a CDR3 having the amino acid sequence of SEQ ID NO:7.

[0055] Particular embodiments relate to an isolated TCR comprising:

[0056] - a TCRα 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;

[0057] - 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.

[0058] In some embodiments, the TCR comprises a variable TCR ɑ 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 β 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.

[0059] 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.

[0060] The TCR of the present invention used in the embodiment comprises: a TCR alpha chain comprising a complementary 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.

[0061] As can be seen from the examples, the TCR according to the present invention is specific for MAGEA10 and exhibits only very low cross-reactivity to other epitopes or antigens.

[0062] Other embodiments relate to an isolated TCR comprising a TCRɑ 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 TCRβ 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.

[0063] Specific embodiments relate to a TCR comprising a TCR ɑ chain having an amino acid sequence of SEQ ID NO: 10 and a TCR β 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 MAGEA10 peptide of SEQ ID NO: 1 comprises a Vɑ chain encoded by the TRAV29 / DV5 gene and a Vβ gene encoded by the TRBV24-1 gene.

[0064] Other embodiments relate to an isolated TCR comprising a TCR alpha chain and a TCR beta chain, wherein

[0065] - a variable TCRα 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 a CDR3 region as set forth in SEQ ID NO:4;

[0066] -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.

[0067] 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 Advance TM10DNA and protein sequence analysis software. User's Manual, 2004, pp. 389-662). Percent identity was determined using standard parameters of the AlignX application.

[0068] 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.

[0069] In some embodiments, the amino acid sequence of a TCR may contain one or more phenotypically silent substitutions.

[0070] "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.

[0071] 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.

[0072] 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.

[0073] 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:

[0074] 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.

[0075] 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 mini-antibodies, 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 (Germany)).

[0076] 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.

[0077] 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.

[0078] TCR according to the present invention can be soluble or membrane-bound. The term "soluble" refers to a TCR in a soluble form (i.e., without a transmembrane or cytoplasmic domain), for example, used as a targeting agent for delivering a therapeutic agent to an antigen presenting cell. For stability, a soluble ɑβ 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 ɑβ heterodimer of the present invention can be truncated at one or both C-termini, for example, up to 15, or up to 10, or up to 8 or less amino acids. In order to be used in adoptive therapy, the ɑβ heterodimer TCR can, for example, be transfected into a full-length chain with both cytoplasmic and transmembrane domains. TCR can include a disulfide bond corresponding to that found between the corresponding ɑ and β constant domains in nature, and in addition or alternatively, a non-natural disulfide bond can be present.

[0079] Thus, TCRs according to the invention, in particular soluble forms of TCRs, can be modified by attaching additional functional moieties, for example to reduce immunogenicity, increase hydrodynamic size (size in solution) solubility and / or stability (e.g. by enhancing protection from proteolytic degradation) and / or prolong serum half-life.

[0080] 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).

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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 α chain or the TCR β chain can be modified to include an epitope tag.

[0085] 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, thus 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.

[0086] 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.

[0087] 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.

[0088] However, in preferred embodiments, the CDR regions of the TCR are not modified or affinity matured in vitro, such as for the TCR receptors in the examples. This means that the CDR regions have naturally occurring sequences. This may be advantageous because in vitro affinity maturation may lead to immunogenicity to the TCR molecule. This may lead to a reduction in therapeutic efficacy and treatment or the generation of ineffective anti-drug antibodies and / or cause side effects.

[0089] 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.

[0090] In theory, unpredictable TCR specificity with a risk of cross-reactivity may occur due to mispairing between endogenous and exogenous TCR chains. To avoid mispairing of TCR sequences, recombinant TCR sequences can be modified to contain the most murine Cɑ and Cβ regions, a technique that has been shown to be effective in enhancing the correct pairing of a variety of differently transduced TCR chains. Murinization of TCRs (i.e., exchanging the human constant regions in the ɑ and β chains with their murine counterparts) is a technique commonly used to improve cell surface expression of TCRs in host cells. Without wishing to be bound by a particular theory, it is believed that murinized TCRs associate more effectively with the CD3 co-receptor; and / or preferentially pair with each other and are less likely to form hybrid TCRs on human T cells that have been genetically modified ex vivo to express a TCR of the desired antigen specificity, but still retain and express their "original" TCR.

[0091] 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.

[0092] 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.

[0093] A suitable single-chain TCR format comprises a first segment consisting of an amino acid sequence corresponding to the variable TCR ɑ region, a second segment consisting of an amino acid sequence corresponding to the variable TCR β region fused to the N-terminus of an amino acid sequence corresponding to the extracellular sequence of the TCR β chain constant region, 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 be composed of an amino acid sequence corresponding to the variable region of the TCR β chain, and the second segment may be composed of an amino acid sequence corresponding to the variable region sequence of the TCR ɑ chain fused to the N-terminus of an amino acid sequence corresponding to the extracellular sequence of the TCR ɑ chain constant region. The above-mentioned single-chain TCR may also include a disulfide bond between the first chain and the second chain, 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 segment and the second segment 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 TCRα chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to an extracellular sequence of a TCRα chain constant region, the second segment may be composed of an amino acid sequence corresponding to a TCRβ chain variable region fused to the N-terminus of an amino acid sequence corresponding to an extracellular sequence of a TCRβ chain constant region, and a disulfide bond may be provided between the first chain and the second chain. The linker sequence may be any sequence that does not impair the function of the TCR.

[0094] In the context of the present invention, a "functional" TCR ɑ and / or β 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 ɑ and / or β chain of the TCR, this would mean that the two chains are still able to form a T cell receptor (with unmodified ɑ and / or β chain or with another fusion protein ɑ and / or β 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.

[0095] In a specific embodiment, the TCR can be modified to a functional T cell receptor (TCR) ɑ 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, the TCR can be modified to a functional T cell receptor (TCR) ɑ and / or β chain fusion protein, wherein the T cell receptor (TCR) ɑ and / or β chain fusion protein comprises two or more epitope-tags spaced apart or directly connected in series. The embodiment of the fusion protein may comprise 2, 3, 4, 5 or even more epitope-tags, as long as the fusion protein maintains its biological activity / multiple activities ("functionality").

[0096] 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, for example, 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.

[0097] TCR variants

[0098] 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.

[0099] The functional portion can mediate the binding of TCR to antigen, particularly to antigen-MHC complex.

[0100] In one embodiment, the functional portion comprises a TCRα variable chain and / or a TCRβ variable chain as described herein.

[0101] 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).

[0102] 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, the linker portion should be appropriately selected based on their pharmaceutical suitability, such as their immunogenicity. Examples of linker portions that meet the above-mentioned required standards are known in the art, such as the field of connecting antibody fragments.

[0103] 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.

[0104] One embodiment relates to a multivalent TCR complex wherein at least one of the TCRs is associated with a therapeutic agent.

[0105] Cytokine and chemokine release

[0106] Some embodiments relate to an isolated TCR as described herein, a polypeptide as described herein, a multivalent TCR complex as described herein, wherein the secretion of IFN-γ 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.

[0107] The IFN-γ secretion induced by the binding of the TCR of the present invention expressed on the 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 500 pg / ml, more preferably exceed 1000 pg / ml, most preferably exceed 2000 pg / 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: 22).

[0108] "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.

[0109] 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 its antigen-binding fragment with the amino acid sequence of SEQ ID NO:1 or its HLA-A*02, preferably or HLA-A*02:01 bound form, and determining whether the candidate TCR or its antigen-binding fragment binds to the target and / or mediates an immune response.

[0110] Whether a candidate TCR or antigen-binding 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 HLA-A2-transduced K562 cells (or other APCs transduced with HLA-A2) transfected with ivtRNA encoding the amino acid sequence SEQ ID NO: 1 are incubated with CD8+-enriched PBMCs expressing a TCR or a molecule comprising the TCR fragment to be studied.

[0111] The TCR of the present invention is particularly useful because it exhibits high tumor cell recognition ability.

[0112] Furthermore, the TCRs as described herein exhibit high tumor cell killing ability. Furthermore, the TCRs of the present invention have high functional avidity.

[0113] Furthermore, TCRs displayed favorable cytokine release patterns that favored efficient tumor regression.

[0114] Nucleic acid, vector

[0115] Another aspect of the invention relates to a nucleic acid encoding a TCR as described herein or a polynucleotide encoding a TCR as described herein.

[0116]

[0117] "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 purposes of this article, replication can be in vitro replication or in vivo replication. Nucleic acid can be constructed or commercially available (e.g., from Genscript, Thermo Fisher and similar companies) using procedures known in the art based on chemical synthesis and / or enzymatic ligation reactions. For example, see Sambrook et al., for example, naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or increase the physical stability of the duplex formed after hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides) can be used to chemically synthesize nucleic acids. The nucleic acid can comprise any nucleotide sequence encoding a recombinant TCR, polypeptide or protein, or any of its functional parts or functional variants.

[0118] The 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 MAGEA10.

[0119] 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.

[0120] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high stringency conditions. "High stringency conditions" refers 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 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.

[0121] As described elsewhere herein, the nucleic acid encoding the TCR can be modified. Useful modifications in the entire nucleic acid sequence can be codon optimized. 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 amino acid sequence of the TCR chain that do not affect function. Generally, additions and deletions should not be made in the CDR3 region.

[0122] Another embodiment relates to a vector comprising a nucleic acid encoding a TCR as described herein.

[0123] 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.

[0124] "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.

[0125] Preferably, the vector is an expression vector. More preferably, the vector is a retroviral vector, more particularly a γ-retroviral or lentiviral vector.

[0126] Cells and cell lines

[0127] Another aspect of the invention relates to a cell expressing a TCR as described herein.

[0128] In some embodiments, the cell is isolated or non-naturally occurring.

[0129] In specific embodiments, the cell may comprise a nucleic acid encoding a TCR as described herein or a vector comprising the nucleic acid.

[0130] 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.

[0131] 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.

[0132] Some embodiments relate to a cell comprising:

[0133] a) an expression vector comprising at least one nucleic acid as described herein, or

[0134] 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.

[0135] 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.

[0136] 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).

[0137] Antibodies targeting TCR

[0138] Another aspect of the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to a portion of a TCR that mediates MAGEA10 specificity as described herein. In one embodiment, the portion of a TCR that mediates MAGEA10 specificity comprises the alpha chain CDR3 of SEQ ID NO:4 and / or the beta chain CDR3 of SEQ ID NO:7.

[0139] The antibody or its antigen-binding fragment can modulate the activity of TCR. It can block or not block the binding of TCR to MAGEA10. It can be used to modulate the therapeutic activity of TCR or for diagnostic purposes.

[0140] Pharmaceutical compositions, drug treatments and kits

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] According to a preferred embodiment, the pharmaceutical composition is an infusion or an injection.

[0147] 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.

[0148] Typically, pharmaceutical compositions comprise at least one pharmaceutically acceptable carrier.

[0149] 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.

[0150] 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.

[0151] In one embodiment, the cancer is a hematological cancer or a solid tumor.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] Recombinant TCR as described herein or the viral vector particles comprising the nucleotide sequence of the recombinant TCR provided herein encoding can be packaged as a test kit. 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 pipes, 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, devices for applying compositions to the subject and devices for applying compositions to the subject.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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 compound of the kit will be compatible with the desired method of administration of the compound.

[0163] experiment

[0164] Example 1: MAGE-A10 SLL -TCR-transgenic T cells bind MAGE-A10SLL -Polymer

[0165] An in vitro priming approach was used to isolate MAGE-A10 SLL Responsive T cell cloning. The priming system uses mature dendritic cells (mDC) from HLA-A*02:01-positive donors as antigen-presenting cells and autologous CD8+-enriched T cells as responder cells. In vitro transcribed RNA (ivtRNA) encoding 31 amino acids of the human MAGE-A10 gene (PRAHAEIRKMSLLKFLAKVNGSDPRSFPL) is used as a source of specific antigen. After electroporation into mDC, ivtRNA encoding MAGE-A10 is translated into protein, which is subsequently processed by HLA-A*02:01 molecules on mDC and presented as a peptide. In vitro co-culture of T cells with ivtRNA-transfected mDC from the same donor results in de novo induction of antigen-specific T cells. Antigen-specific T cells are enriched by FACS-based single-cell sorting. MAGE-A10 SLL The sequences of the TCRα and TCRβ chains of the reactive T cell clones were identified by next generation sequencing. Both TCR constant regions were replaced with their murine counterparts. The nucleotide sequences of the murinized TCRs were analyzed using GeneOptimizer TM The algorithm (ThermoFisher) was codon optimized and cloned into the retroviral vector pES.12-6. PBMCs of healthy donors were separated by ficoll gradient centrifugation. CD8 T cells were enriched by negative magnetic sorting (Miltenyi) and stimulated with anti-CD3 and anti-CD28 mAbs (BD Pharmingen, Heidelberg, Germany) in non-tissue culture 24-well plates. Double-tropic retroviral particles were produced by transfecting HEK293T cells with retroviral plasmids encoding corresponding TCRs and two expression plasmids. On the second day after stimulation, CD8 T cells were transduced, and transduced CD8+ cells were enriched by FACS using mouse constant β region as a transduction marker on the 12th day, and then amplified by rapid amplification scheme (Riddell SR, Science, July 10, 1992; 257(5067): 238-41).

[0166] result:

[0167] Separated from MAGE-A10 SLL CD8 T cells were transduced with TCRs of reactive T cell clones and control TCRs that do not recognize MAGE-A10. SLL -MHC-multimers stained with antibodies against CD8 and against the murine constant β region. TCRβ chain at 74% MAGE-A10SLL -TCR was expressed on CD8 T cells and on 96% of negative control TCR. Most MAGE-A10 SLL -TCR transduced cells (84%) with MAGE-A10 SLL -MHC-multimer binding. No MAGE-A10 was observed for negative control TCR SLL -MHC-multimer staining. These results indicate that from MAGE-A10 SLL TCRs isolated from reactive T cell clones can be transgenically expressed in T cells from healthy donors ( Figure 1 ).

[0168] Example 2: MAGE-A10 SLL -TCR-transgenic T cells recognize MAGE-A10 SLL -Peptide

[0169] To ensure the functionality of the transgenic expressed recombinant TCR, TCR-transduced effector T cells were co-cultured with T2 cells (target cells) loaded with peptide. -5 T2 cells loaded with the MAGE-A10-derived SLL peptide (SEQ ID NO: 1) of M were used as positive targets, and T2 cells loaded with an irrelevant peptide (SEQ ID NO: 22) were used as negative targets. As effector cells, CD8-enriched T cells were transduced to stably express recombinant MAGE-A10 SLL -TCR or negative control TCR (TCR specific for an irrelevant peptide according to SEQ ID NO: 22). Effector cells and target cells were co-cultured at an E:T ratio of 2:1 in 96-well round-bottom plates. After about 20 hours of co-culture, supernatants were collected and analyzed by standard sandwich ELISA.

[0170] result:

[0171] Expression of MAGE-A10 SLL -TCR effector cells only expressed in cells loaded with MAGE-A10 SLL When T2 cells loaded with peptides were cultured together, IFN-γ was secreted, indicating specific recognition of the presented peptide:MHC complex. Negative targets, i.e., T2 cells loaded with control peptides, were recognized only by the negative control TCR ( Figure 2 ).

[0172] Example 3: MAGE-A10 SLL -TCR-transgenic T cells recognize and kill HLA-A*02:01 / MAGE-A10-positive tumor cell lines

[0173] To analyze the efficacy, specificity, and suitability of TCRs for clinical applications, a panel of tumor cell lines was tested for TCR-mediated recognition by measuring IFN-γ secretion and cytotoxicity when co-cultured with TCR-transduced effector cells.

[0174] For IFN-γ release assay, effector cells were stained with MAGE-A10 SLL -TCR or negative control TCR transduction and co-cultured with different tumor cell lines. Cell lines A375, NCI-H1755 and UACC-62 (HLA-A2+ / MAGEA10+) and HLA-A2-transfected cell line KYO-1 (HLA-A2+ / MAGEA10+) were used as positive targets. Cell lines Mel624.38 and 647-V (HLA-A2+ / MAGEA10-) were used as negative targets. The HLA-A2-expression of the cell lines was analyzed by flow cytometry, and MAGEA10-expression data was generated using qPCR or extracted from publicly available databases known to those skilled in the art (data not shown). Effector cells and target cells were seeded in 96-well round-bottom plates at an E:T of 2:1, supernatants were harvested after about 20 hours of co-culture, and IFN-γ levels were analyzed by standard sandwich ELISA. For cytotoxicity assays, IncuCyte TM Zoom device undergoes long-term lethality test over three days. IncuCyte TM The device is a microscope-based system that allows for live imaging of cells. A panel of tumor cell lines (HLA-A2+ / MAGEA10+: A375, NCI-H1755, and UACC-62, HLA-A2+ / MAGEA10-: Mel624.38 and 647-V) were seeded into 96-well flat-bottom plates (20,000 cells per well) and co-cultured with effector T cells expressing TCRs (100,000 cells per well). The tumor cell lines stably expressed the nuclear-restricted red fluorescent protein mKate2, allowing IncuCyte TM The Zoom system was able to determine the number of red fluorescent cells (expressed as RCU × μm) in each well at multiple time points over a total period of 72 hours. 2 / Image represents total integrated intensity, RCU=Red Calibration Unit). Over time, an increase in the number of cells per well indicates tumor cell growth, while a decrease in the number of cells per well indicates TCR-mediated cytotoxicity of fluorescent target cells.

[0175] result:

[0176] MAGE-A10 SLL-TCR-transgenic T cells released IFN-γ when co-cultured with HLA-A2+ / MAGEA10+ tumor cell lines A375, NCI-H1755, and KYO-1, but did not respond to HLA-A2+ / MAGEA10- cell lines Mel624.38 and 647-V. In addition, the A2+ / MAGEA10+ tumor cell line UACC62 was not recognized. T cells expressing the negative control TCR did not recognize any tumor cell line. The HLA-A2+ / MAGEA10+ tumor cell lines A375 and NCI-H1755 were killed by MAGE-A10-TCR-transgenic T cells, and the growth of UACC62 cells was inhibited. The HLA-A2+ / MAGEA10- cell lines Mel624.38 and 647-V were not killed, and T cells expressing the negative control TCR did not lyse any tumor cell line. These results indicate that MAGE-A10 SLL -TCR transgenic T cells can recognize MAGE-A10 on HLA-A*02 at physiological levels in tumor cell lines SLL -peptides, and they can mediate cytotoxicity against these cells ( Figure 3 A and 3B).

[0177] Example 4: Analysis of potential cross-recognition of other MAGE-A family members

[0178] Since other members of the MAGE-A family may contain SLL -peptide had highly similar peptide sequences, so other MAGE-A family members were further investigated for potential TCR-mediated cross-recognition. The expression of MAGE-A 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 clinical development of TCRs.

[0179] To study the recognition of endogenously processed and presented peptides derived from other MAGE-A family members, they were recombinantly expressed in HLA-A2 positive K562 cells. SLL -TCR-effector cell co-cultures were performed to analyze potential cross-recognition of peptides derived from MAGE family members.

[0180] HLA-A2 + K562 cells were transduced with constructs encoding the following MAGE family members: MAGE-A1 (NCBI Reference Sequence (Accession): NM_004988.5), MAGE-A2 (NM_005361.3), MAGE-A3 (NM_005362.4), MAGE-A4 (NM_001011548.1), MAGE-A5 (NM_021049.4), MAGE-A6 (NM_005363.3), MAGE-A8 (NM_001166400.1), MAGE-A9 (NM_005365.4), MAGE-A11 (NM_005366.4), MAGE-A12 (NM_001166386.3), and the cells were crossed with constructs expressing MAGE-A10. SLL T cells expressing negative control TCR or T cells expressing negative control TCR were co-cultured. The supernatants were collected after about 20 hours of co-culture and analyzed for secreted IFN-γ by standard sandwich ELISA.

[0181] result:

[0182] Expression of MAGE-A10 SLL -TCR T cells only recognize MAGE-A10 positive HLA-A2+K562 cells, but not HLA-A2+K562 cells transduced with any other MAGE-A family members ( Figure 4 ).

[0183] Example 5: MAGE-A10 SLL - Analysis of HLA fine typing and potential alloreactivity of TCR

[0184] Since TCR recognizes its specific peptide only when bound to a specific MHC molecule, not only the level of antigen expression but also the HLA type of the target cell defines the target as a positive or negative target. Depending on the HLA type, the patient may or may not be included in the treatment regimen of TCR-based ACT.

[0185] To evaluate which HLA molecules other than HLA-A*02:01 can present MAGEA10-derived SLL peptides and can be expressed by MAGE-A10 SLL Therefore, 53 lymphoblastoid cell lines (LCLs) covering the most common HLA allotypes in Europeans and North American-Caucasians were used as antigen-presenting cells. -5M; SEQ ID NO 1) were used for co-culture with effector cells expressing TCR. Co-culture was performed in a 2:1 E:T setting in a 96-well round-bottom plate. Supernatants were collected after about 20 hours and analyzed for IFN-γ levels by standard sandwich ELISA.

[0186] result:

[0187] MAGE-A10 SLL - Restriction analysis of the TCR showed the potential of the TCR to recognize MAGEA10-derived epitopes presented not only on HLA-A*02:01 but also on HLA-A*02:04. Since unloaded LCL expressing MAGE-A10 was not recognized SLL -T cells whose TCRs show no cross-recognition for any HLA ( Figure 5 A and 5B).

[0188] Example 6: Potential recognition analysis of normal cells

[0189] In order to investigate the safety properties of TCRs, TCR-mediated recognition of cells derived from healthy tissues of different origins needs to be investigated and excluded. Therefore, effectors expressing TCRs were co-cultured with normal cells from the kidney (renal cortical epithelial cells, HRCepC), lung (lung fibroblasts, NHLF) and iPS-derived hepatocytes, cardiomyocytes, astrocytes and endothelial cells (EC). The cells studied were endogenously negative for the antigen MAGE-A10, thus allowing the identification of potential MAGE-A10-independent off-target toxicities. Normal cells were thawed and cultured for one week prior to co-culture according to the supplier's instructions. Cells were seeded in 96-well flat-bottom plates and co-cultured with cells expressing MAGE-A10 SLL -TCR-expressing T cells or negative control T cells expressing TCR. HLA-A2 expression of target cells was confirmed by antibody staining and flow cytometry (data not shown). Unmodified target cells or overloaded (10 -5 M) MAGE-A10 SLL -peptide target cells served as a positive control. After about 20 hours of co-culture, supernatants were collected and analyzed for IFN-γ levels by standard sandwich ELISA.

[0190] result:

[0191] Expression of MAGE-A10 SLL -TCR of T cells responding to all loaded MAGE-A10 SLL -peptide cells release IFN-γ, indicating the potential of the target cells to appropriately present TCR epitopes on their cell surface. Unmodified normal cells are not expressed by the transgenic MAGE-A10 SLL- Effector recognition of TCRs, indicating that the TCRs studied have a favorable safety profile ( Figure 6 ).

[0192] This application also includes the following items:

[0193] Project 1: Isolated T cell receptor (TCR) specific for MAGEA10.

[0194] Item 2: The isolated TCR according to Item 1, wherein the TCR specifically recognizes the amino acid sequence SEQ ID NO: 1 or a fragment thereof.

[0195] Item 3: The isolated TCR according to Items 1 and 2, wherein the TCR specifically recognizes the HLA-A2 binding form of the amino acid sequence of SEQ ID NO:1.

[0196] Item 4: An isolated TCR according to any of the preceding items, 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 and HLA-A*02:04.

[0197] Item 5: An isolated TCR according to any of the preceding items, wherein the TCR specifically recognizes the amino acid sequence of SEQ ID NO: 1 presented by a molecule encoded by HLA-A*02:01.

[0198] Item 6: An isolated TCR according to any of the preceding items, wherein the TCR comprises

[0199] - 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,

[0200] - 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.

[0201] Item 7: An isolated TCR according to any of the preceding items, wherein the TCR comprises:

[0202] A variable TCR alpha region having an amino acid sequence at least 80% identical to SEQ ID NO:8; and

[0203] A variable TCR beta region having an amino acid sequence at least 80% identical to SEQ ID NO:9.

[0204] Item 8: An isolated TCR according to any one of the preceding embodiments, wherein the TCR comprises

[0205] a variable TCR alpha region having the amino acid sequence of SEQ ID NO: 8; and

[0206] The variable TCR β region has the amino acid sequence of SEQ ID NO:9.

[0207] Item 9: An isolated TCR according to any of the preceding items, wherein the TCR comprises

[0208] A TCR alpha chain having an amino acid sequence at least 80% identical to SEQ ID NO: 10; and

[0209] A TCR β chain having an amino acid sequence at least 80% identical to SEQ ID NO:11.

[0210] Item 10: An isolated TCR according to any of the preceding items, wherein the TCR comprises

[0211] TCR alpha chain having the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:10; and

[0212] The TCR β chain has the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:11.

[0213] Item 11: An isolated TCR according to any of the preceding items, wherein the TCR comprises a TCR alpha chain and a TCR beta chain, wherein

[0214] - 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,

[0215] 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.

[0216] Item 12: An isolated TCR according to any of the preceding items, wherein the TCR is purified.

[0217] Item 13: An isolated TCR according to any of the preceding items, wherein the amino acid sequence of the TCR comprises one or more phenotypically silent substitutions.

[0218] Item 14: An isolated TCR according to any of the preceding items, wherein the amino acid sequence of the TCR is modified to contain a detectable label, a therapeutic agent or a pharmacokinetic modifying portion.

[0219] Item 15: An isolated TCR according to Item 14, wherein the therapeutic agent is selected from the group consisting of immune effector molecules, cytotoxic agents and radionuclides.

[0220] Item 16: An isolated TCR according to Item 15, wherein the immune effector molecule is a cytokine.

[0221] Item 17: An isolated TCR according to any of the preceding items, wherein the TCR is soluble or membrane-bound.

[0222] Item 18: An isolated TCR according to Item 14, wherein the pharmacokinetic modifying portion is at least one polyethylene glycol repeating unit, at least one diol group, at least one sialic acid group, or a combination thereof.

[0223] Item 19: An isolated TCR according to any one of the preceding items, wherein the TCR is a single-chain type, wherein the TCRα chain and the TCRβ chain are connected by a linker sequence.

[0224] Item 20: An isolated TCR according to items 1 to 19, wherein the TCR ɑ chain or the TCR β chain is modified to comprise an epitope tag.

[0225] Item 21: An isolated polypeptide comprising a functional portion of the TCR of any one of items 1 to 20, wherein the functional portion comprises at least one of the amino acid sequences of SEQ ID NOs: 4 and 7.

[0226] Item 22: An isolated polypeptide comprising a functional portion of the TCR of any one of items 1 to 20, wherein the functional portion comprises the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 and 7.

[0227] Item 23: An isolated polypeptide according to Item 21, wherein the functional part comprises a TCRɑ variable chain and / or a TCRβ variable chain.

[0228] Item 24: A multivalent TCR complex comprising a TCR as embodied in any one of items 1 to 20.

[0229] Item 25: An isolated TCR according to items 1 to 20, a polypeptide according to items 21 to 23, or a multivalent TCR complex according to item 24, 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.

[0230] Item 26: Nucleic acid encoding the TCR according to any one of items 1 to 20 or encoding the polypeptide according to items 21 to 23.

[0231] Item 27: A vector comprising the nucleic acid of Item 26.

[0232] Item 28: The vector according to Item 27, wherein the vector is an expression vector.

[0233] Item 29: The vector according to Item 27 or 28, wherein the vector is a retroviral vector.

[0234] Item 30: The vector according to Item 27 or 28, wherein the vector is a lentiviral vector.

[0235] Item 31: A cell expressing a TCR according to items 1 to 20.

[0236] Item 32: A cell according to Item 31, wherein the cell is isolated or non-naturally occurring.

[0237] Item 33: The cell according to items 31 and 32, wherein the cell comprises the nucleic acid according to item 26 or the vector according to items 27 to 30.

[0238] Item 34: A cell according to Items 31 to 33, wherein the cell comprises:

[0239] a) an expression vector comprising at least one nucleic acid as embodied in item 26, or

[0240] b) a first expression vector comprising a nucleic acid encoding the alpha chain of the TCR as embodied in any one of items 1 to 20, and

[0241] A second expression vector comprising a nucleic acid encoding the β chain of the TCR as embodied in any one of items 1 to 20.

[0242] Item 35: A cell according to any one of Items 31 to 34, wherein the cell is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC).

[0243] Item 36: A cell according to any one of Items 31 to 35, wherein the cell is a T cell.

[0244] Item 37: An antibody or antigen-binding fragment thereof that specifically binds to the portion of the TCR according to items 1 to 20 that mediates MAGEA10 specificity.

[0245] Item 38: An antibody according to Item 37, wherein the portion of the TCR that mediates MAGEA10 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 ID 5, 6 and 7.

[0246] Item 39: A pharmaceutical composition comprising a TCR according to items 1 to 20, a polypeptide according to items 21 to 23, a multivalent TCR complex according to item 24, a nucleic acid according to item 26, a vector according to items 27 to 30, a cell according to any one of items 31 to 36, or an antibody according to items 37 to 38.

[0247] Item 40: A pharmaceutical composition according to Item 39, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

[0248] Item 41: The TCR according to items 1 to 20, the polypeptide according to items 21 to 23, the multivalent TCR complex according to item 24, the nucleic acid according to item 26, the vector according to items 27 to 30, the cell according to any one of items 31 to 36, or the antibody according to items 37 to 38, for use as a medicament.

[0249] Item 42: TCR according to items 1 to 20, polypeptide according to items 21 to 23, multivalent TCR complex according to item 24, nucleic acid according to item 26, vector according to items 27 to 30 or cell according to any one of items 31 to 36, for use in treating cancer.

[0250] Item 43: A TCR, polypeptide, multivalent TCR complex, nucleic acid, vector or cell according to Item 42, wherein the cancer is a blood cancer or a solid tumor.

[0251] Item 44: A TCR, polypeptide, multivalent TCR complex, nucleic acid, vector or cell according to Items 42 and 43, 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.

[0252] Item 45: The TCR, polypeptide, multivalent TCR complex, nucleic acid, vector or cell according to items 42 and 43, wherein the cancer is preferably selected from the group consisting of sarcoma or osteosarcoma. Sequence Listing <110> Medigene Immunotherapies GmbH <120> MAGEA10-specific T cell receptor and its use <130> M11465WO / DB <150> EP19185192 <151> 2019-07-09 <160> twenty three <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Homo sapiens <400> 1 Ser Leu Leu Lys Phe Leu Ala Lys Val 1 5 <210> 2 <211> 6 <212> PRT <213> Homo sapiens <400> 2 Asn Ser Met Phe Asp Tyr 1 5 <210> 3 <211> 7 <212> PRT <213> Homo sapiens <400> 3 Ile Ser Ser Ile Lys Asp Lys 1 5 <210> 4 <211> 13 <212> PRT <213> Homo sapiens <400> 4 Cys Ala Ala Asn Ser Asn Ser Gly Tyr Ala Leu Asn Phe 1 5 10 <210> 5 <211> 5 <212> PRT <213> Homo sapiens <400> 5 Lys Gly His Asp Arg 1 5 <210> 6 <211> 6 <212> PRT <213> Homo sapiens <400> 6 Ser Phe Asp Val Lys Asp 1 5 <210> 7 <211> 13 <212> PRT <213> Homo sapiens <400> 7 Cys Ala Thr Gly Asp Pro Leu Gln Glu Thr Gln Tyr Phe 1 5 10 <210> 8 <211> 139 <212> PRT <213> Homo sapiens <400> 8 Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro 1 5 10 15 Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln 20 25 30 Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn 35 40 45 Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys 50 55 60 Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys 65 70 75 80 Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala 85 90 95 Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala 100 105 110 Val Tyr Phe Cys Ala Ala Asn Ser Asn Ser Gly Tyr Ala Leu Asn Phe 115 120 125 Gly Lys Gly Thr Ser Leu Leu Val Thr Pro His 130 135 <210> 9 <211> 131 <212> PRT <213> Homo sapiens <400> 9 Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu Leu Gly Thr 1 5 10 15 Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr 20 25 30 Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His 35 40 45 Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser 65 70 75 80 Asp Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95 Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110 Gly Asp Pro Leu Gln Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu 115 120 125 Leu Val Leu 130 <210> 10 <211> 275 <212> PRT <213> Artificial Sequence <220> <223> With humanized constant region <400> 10 Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro 1 5 10 15 Asp Trp Val Asn Ser Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln 20 25 30 Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn 35 40 45 Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys 50 55 60 Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys 65 70 75 80 Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala 85 90 95 Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala 100 105 110 Val Tyr Phe Cys Ala Ala Asn Ser Asn Ser Gly Tyr Ala Leu Asn Phe 115 120 125 Gly Lys Gly Thr Ser Leu Leu Val Thr Pro His Ile Gln Asn Pro Glu 130 135 140 Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu 145 150 155 160 Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn Val Pro Lys Thr Met 165 170 175 Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr Val Leu Asp Met Lys Ala 180 185 190 Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser 195 200 205 Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser 210 215 220 Ser Asp Val Pro Cys Asp Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr 225 230 235 240 Asp Met Asn Leu Asn Phe Gln Asn Leu Ser Val Met Gly Leu Arg Ile 245 250 255 Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu 260 265 270 Trp Ser Ser 275 <210> 11 <211> 304 <212> PRT <213> Artificial sequence <220> <223> murinized constant region <400> 11 Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu Leu Gly Thr 1 5 10 15 Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr 20 25 30 Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His 35 40 45 Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser 65 70 75 80 Asp Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95 Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110 Gly Asp Pro Leu Gln Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu 115 120 125 Leu Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Thr Leu 130 135 140 Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg 195 200 205 Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln 210 215 220 Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser 225 230 235 240 Pro Lys Pro Val Thr Gln Asn Ile Ser Glu Ala Trp Gly Arg Ala 245 250 255 Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly Val Leu Ser Ala 260 265 270 Thr with Tyr Glu and Gly with Lys Fragment Thr with Tyr Fragment Val 275 280 285 Leu Will Ser Gly Leu Will Met Ala Met Will Lys Lys Asn Ser 290,295,300 <210> 12 <211> 18 <212> DNA <213> Homo sapiens <400> 12 aactctatgt tcgactac 18 <210> 13 <211> 21 <212> DNA <213> Homo sapiens <400> 13 randomization g 21 <210> 14 <211> 39 <212> DNA <213> Homo sapiens <400> 14 tgcgccgcca atagcaatag cggctacgcc ctgaatttc 39 <210> 15 <211> 15 <212> DNA <213> Homo sapiens <400> 15 aagggccacg accgg 15 <210> 16 <211> 18 <212> DNA <213> Homo sapiens <400> 16 agcttcgacg tgaaggac 18 <210> 17 <211> 39 <212> DNA <213> Homo sapiens <400> 17 tgcgccactg gcgaccctct gcaagagaca cagtatttc 39 <210> 18 <211> 415 <212> DNA <213> Homo sapiens <400> 18 atggctatgc tgctgggagc ctctgtgctg atcctgtggc tgcaacccga ttgggtcaac 60 agccagcaga agaacgacga ccagcaagtg aagcagaaca gccccagcct gagcgtgcaa 120 gagggcagaa tcagcatcct gaactgcgac tacaccaact ctatgttcga ctacttcctg 180 tggtacaaga agtaccccgc cgagggaccg accttcctga tcagcatcag cagcatcaag 240 gacaagaacg aggacggccg gttcaccgtg tttctgaaca agagcgccaa gcacctgagc 300 ctgcacatcg tgccttctca gcctggcgat agcgccgtgt atttctgcgc cgccaatagc 360 aatagcggct acgccctgaa tttcggcaag ggcacaagcc tgctggtcac ccctc 415 <210> 19 <211> 394 <212> DNA <213> Homo sapiens <400> 19 atggccagcc tgctgttctt ctgcggcgcc ttttatctgc tcggcaccgg ctctatggac 60 gccgacgtta cacagacccc tcggaacaga atcaccaaga ccggcaagcg gatcatgctg 120 gaatgcagcc agaccaaggg ccacgaccgg atgtactggt acagacagga tccaggactg 180 ggcctgagac tgatctacta cagcttcgac gtgaaggaca tcaacaaggg cgagatcagc 240 gacggctaca gcgtgtcaag acaggcccag gccaagttca gcctgagcct ggaaagcgct 300 atccccaacc agacagccct gtacttctgc gccactggcg accctctgca agagacacag 360 tatttcggcc ctggcaccag actgctggtt ctgg 394 <210> 20 <211> 825 <212> DNA <213> Artificial Sequence <220> <223> With murine constant region; codon optimized <400> 20 atggctatgc tgctgggagc ctctgtgctg atcctgtggc tgcaacccga ttgggtcaac 60 agccagcaga agaacgacga ccagcaagtg aagcagaaca gccccagcct gagcgtgcaa 120 gagggcagaa tcagcatcct gaactgcgac tacaccaact ctatgttcga ctacttcctg 180 tggtacaaga agtaccccgc cgagggaccg accttcctga tcagcatcag cagcatcaag 240 gacaagaacg aggacggccg gttcaccgtg tttctgaaca agagcgccaa gcacctgagc 300 ctgcacatcg tgccttctca gcctggcgat agcgccgtgt atttctgcgc cgccaatagc 360 aatagcggct acgccctgaa tttcggcaag ggcacaagcc tgctggtcac ccctcacatc 420 cagaatccgg agcccgccgt ataccagctg aaggacccta gaagccagga cagcaccctg 480 tgcctgttca ccgacttcga cagccagatc aacgtgccca agaccatgga aagcggcacc 540 ttcatcaccg acaagacagt gctggacatg aaggccatgg acagcaagtc caacggcgca 600 atcgcctggt ccaaccagac cagcttcaca tgccaggaca tcttcaaaga gacaaacgcc 660 acatacccca gcagcgacgt gccctgtgat gccaccctga cagagaagtc cttcgagaca 720 gacatgaacc tgaacttcca gaatctgtcc gtgatgggcc tgagaatcct gctgctgaag 780 gtggccggct tcaatctgct gatgaccctg cggctgtggt ccagc 825 <210> 21 <211> 912 <212> DNA <213> Artificial Sequence <220> <223> With humanized constant region; codon optimized <400> 21 atggccagcc tgctgttctt ctgcggcgcc ttttatctgc tcggcaccgg ctctatggac 60 gccgacgtta cacagacccc tcggaacaga atcaccaaga ccggcaagcg gatcatgctg 120 gaatgcagcc agaccaaggg ccacgaccgg atgtactggt acagacagga tccaggactg 180 ggcctgagac tgatctacta cagcttcgac gtgaaggaca tcaacaaggg cgagatcagc 240 gacggctaca gcgtgtcaag acaggcccag gccaagttca gcctgagcct ggaaagcgct 300 atccccaacc agacagccct gtacttctgc gccactggcg accctctgca agagacacag 360 tatttcggcc ctggcaccag actgctggtt ctggaagatc tccggaacgt gaccccccct 420 aaagtgaccc tgttcgaacc cagcaaggcc gagatcgcca acaagcagaa agccaccctc 480 gtgtgcctgg ccagaggctt cttccccgac catgtggaac tgtcttggtg ggtcaacggc 540 aaagaggtgc acagcggagt gtccaccgac cctcaggcct acaaagagag caactacagc 600 tactgcctga gcagcagact gcgggtgtcc gccaccttct ggcacaaccc ccggaaccac 660 ttcagatgcc aggtgcagtt tcacggcctg agcgaagagg acaagtggcc cgaaggctcc 720 cccaagcccg tgacccagaa tatctctgcc gaggcctggg gcagagccga ctgtggaatt 780 accagcgcca gctaccacca gggcgtgctg tctgccacca tcctgtacga gatcctgctg 840 ggcaaggcca ccctgtacgc cgtgctggtg tctggcctgg tgctgatggc catggtcaag 900 aagaagaaca gc 912 <210> 22 <211> 10 <212> PRT <213> Homo sapiens <400> 22 Lys Leu Tyr Gly Leu Asp Trp Ala Glu Leu 1 5 10 <210> 23 <211> 29 <212> PRT <213> Homo sapiens <400> 23 Pro Arg Ala His Ala Glu Ile Arg Lys Met Ser Leu Leu Lys Phe Leu 1 5 10 15 Ala Lys Val Asn Gly Ser Asp Pro Arg Ser Phe Pro Leu 20 25

Claims

1. An isolated T cell receptor (TCR) specific for MAGEA10, wherein the TCR comprises: - a TCRα 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 β 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.

2. The isolated TCR according to claim 1, wherein the TCR specifically recognizes the amino acid sequence SEQ ID NO:

1.

3. 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.

4. 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 and HLA-A*02:

04.

5. The isolated TCR of claim 1, wherein the TCR comprises: 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.

6. An isolated polypeptide comprising a functional portion of the TCR according to any one of claims 1 to 5, wherein the functional portion comprises: - a TCRα 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 β 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, wherein the functional portion mediates binding of the TCR to the antigen MAGEA10.

7. The isolated TCR according to any one of claims 1 to 5 or the polypeptide according to claim 6, 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.

8. Nucleic acid encoding the TCR according to any one of claims 1 to 5 or encoding the isolated polypeptide according to claim 6.

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 5.

11. Use of the TCR according to any one of claims 1 to 5, the polypeptide 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 and melanoma.

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