T cell receptor for prame-positive cancer and immunotherapy using same

Abstract

To provide a new anticancer agent specifically targeting a molecule highly specific to a cancer cell.SOLUTION: Antigen recognizing constructs are provided for tumor-associated antigens (TAA), particularly for preferentially expressed antigen of melanoma (PRAME).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to antigen recognition constructs against tumor-associated antigens (TAA), particularly against preferentially expressed melanoma antigens (PRAME). The present invention provides novel T cell receptor (TCR)-based molecules that are selective and specific for the antigens of the present invention. The TCRs of the present invention, and TAA-binding fragments derived therefrom, are useful for the diagnosis, treatment, and prevention of TAAs that manifest cancerous diseases. Additionally, nucleic acids encoding the antigen recognition constructs of the present invention, vectors comprising these nucleic acids, recombinant cells expressing the antigen recognition constructs, and pharmaceutical compositions comprising the compounds of the present invention are provided. [Background technology]

[0002] PRAME is encoded by the PRAME gene, which is expressed at high levels in a large proportion of tumors, including melanoma, non-small cell lung cancer, ovarian cancer, renal cell carcinoma (RCC), breast cancer, cervical cancer, colorectal cancer, sarcoma, neuroblastoma, and several types of leukemia. PRAME is the best-characterized member of the PRAME family of leucine-rich repeat (LRR) proteins. While mammalian genomes contain multiple members of the PRAME family, only one PRAME-like LRR protein has been identified in other vertebrate genomes. PRAME is a cancer / testis antigen that is expressed at very low levels in normal adult tissues except for the testis, but at high levels in a variety of cancer cells.

[0003] T cell-based immunotherapy targets represent peptide epitopes derived from tumor-associated or tumor-specific proteins presented by molecules of the major histocompatibility complex (MHC). These tumor-associated antigens (TAA) can be peptides derived from all protein classes, such as enzymes, receptors, and transcription factors, which are expressed in each tumor cell and are usually upregulated compared to unaltered cells of the same origin.

[0004] Specific elements of the cellular immune response can specifically recognize and destroy tumor cells. Isolation of T cells from tumor-infiltrating cell populations or from peripheral blood suggests that these cells play an important role in the innate immune defense against cancer. In particular, CD8+ T cells play a key role in this response, recognizing class I molecules of the major histocompatibility complex (MHC) bearing peptides, usually 8–10 amino acid residues long, derived from proteins or defective ribosomal products (DRiPs) located in the cytosol. In humans, MHC molecules are also called human leukocyte antigens (HLA).

[0005] There are two classes of MHC molecules: MHC class I and MHC class II. Peptide-MHC class I complexes are recognized by CD8-positive T cells bearing the appropriate T cell receptor (TCR), while peptide-MHC class II molecule complexes are recognized by CD4-positive helper T cells bearing the appropriate TCR. Because both CD8-dependent and CD4-dependent responses synergistically contribute to antitumor effects, the identification and characterization of tumor-associated antigens and corresponding T cell receptors are important in the development of cancer immunotherapies, such as vaccines and cell therapies.

[0006] In an MHC class I-dependent immune response, peptides must not only be able to bind to specific MHC class I molecules expressed by tumor cells, but they must also be subsequently recognized by T cells bearing specific T cell receptors (TCRs). Thus, TAAs are the starting point for the development of T cell-based therapeutics, including, but not limited to, tumor vaccines and cell therapies.

[0007] Approximately 90% of peripheral blood T cells express TCRs composed of α and β polypeptides. A small percentage of T cells (approximately 5% of all T cells) have been shown to express TCRs composed of γ and δ polypeptides. γδ T cells are most abundant in the intestinal mucosa, within a population of lymphocytes known as intraepithelial lymphocytes (IELs). The antigenic molecules that activate γδ T cells remain largely unknown. However, γδ T cells are not MHC-restricted and do not require peptides to be presented by MHC molecules on antigen-presenting cells. Rather, they appear to be able to recognize whole proteins, although some recognize MHC class IB molecules. Human Vγ9 / Vδ2 T cells, which constitute the major γδ T cell population in peripheral blood, are unique in that they respond specifically and rapidly to the isopentenyl pyrophosphate precursor, HMB-PP, a small, nonpeptidic microbial metabolite. Estimates of T cell percentages found in peripheral blood of healthy donors are as follows: CD3+ = 70.78% ± 4.71; CD3+CD4+ = 38.97% ± 5.66; CD3+CD8+ = 28.955% ± 7.43; CD3+CD56+ = 5.22% ± 1.74; CD3-CD56+ = 10.305% ± 4.7; CD3+CD45RA+ = 45.00% ± 7.19; and CD3+CD45RO+ = 27.21% ± 7.34.

[0008] Each chain of the T cell antigen receptor of a T cell clone consists of a unique combination of domains designated variable (V), diversity (D), joining (J), and constant (C). In each T cell clone, the combination of the V, D, and J domains of both the α and β chains or both the δ and γ chains is responsible for antigen recognition in a manner that is uniquely characteristic of that T cell clone and defines the unique binding site, also known as the idiotype, of the T cell clone. In contrast, the C domains are not involved in antigen binding.

[0009] TCRs are heterodimeric cell surface proteins of the immunoglobulin superfamily that are associated with the invariant protein of the CD3 complex, which mediates signal transduction. TCRs exist in αβ and γδ forms, which are structurally similar but have significantly different anatomical locations and likely functions. The extracellular portions of the naturally occurring heterodimeric αβ and γδ TCRs each contain two polypeptides, each with a membrane-proximal constant domain and a membrane-distal variable domain. Each of the constant and variable domains contains an intrachain disulfide bond. The variable domain contains highly polymorphic loops similar to the complementarity-determining regions (CDRs) of antibodies. The use of TCR gene therapy overcomes several current hurdles. It can confer the desired specificity to a patient's own T cells, allowing for the rapid generation of sufficient numbers of T cells and avoiding their depletion. TCRs can be transduced into potent T cells (e.g., central memory T cells or T cells with stem cell properties), which may ensure good persistence and function upon transfer. TCR-engineered T cells are infused into cancer patients who have become lymphopenic due to chemotherapy or irradiation, allowing for efficient engraftment but preventing immunosuppression. Summary of the Invention [Problem to be solved by the invention]

[0010] Despite progress in the development of molecularly targeted drugs for cancer treatment, there remains a need in the art to develop new anti-cancer agents that specifically target molecules highly specific to cancer cells. This specification addresses this need by providing novel PRAME TCRs, respective recombinant TCR constructs, nucleic acids, vectors, and host cells that specifically bind to the TAA epitopes as disclosed; and methods of using such molecules in the treatment of cancer. In the context of the present invention, the term TAA particularly relates to the following preferred proteins: PRAME, and fragments or analogs thereof, particularly fragments or analogs comprising or consisting of the antigenic peptide sequence set forth in SEQ ID NOS: 97-115, preferably SEQ ID NOS: 97-106, more preferably SEQ ID NO: 97. [Means for solving the problem]

[0011] The object of the present invention is solved in a first aspect by an antigen recognition construct comprising at least one complementarity determining region (CDR) 90 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or preferably 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 129, and 135.

[0012] In some embodiments, the antigen recognition construct of the present invention specifically binds to a TAA-peptide-HLA molecule complex, wherein the TAA peptide comprises, or alternatively consists of, a variant of a TAA that is at least 66%, preferably at least 77%, more preferably at least 88% homologous (preferably at least 88% or at least 77% identical) to the amino acid sequence of a TAA of the present invention, said variant binding to an HLA class I or class II molecule and / or inducing T cells that cross-react with said peptide or a pharmaceutically acceptable salt thereof, and wherein said peptide is not the underlying full-length polypeptide.

[0013] As used herein, the term "identical" or percent "identity," when used anywhere herein in the context of two or more nucleic acid or protein / polypeptide sequences, refers to two or more sequences or subsequences that have (or at least have) a specified percentage of identical amino acid residues or nucleotides (i.e., are at least about 60% identical, preferably at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, or 94% identical, more preferably at least about 95%, 96%, 97%, 98%, 99% or more identical over a specified region, preferably over their full-length sequences, when compared and aligned for maximum correspondence over a comparison window or designated region), as determined using a sequence comparison algorithm or by manual alignment and visual inspection (see, e.g., the NCBI website). In certain embodiments, for example, when the protein or nucleic acid sequence of an antigen recognition construct of the present invention is compared with other proteins / genes, the percentage identity can be determined by a BLAST search supported on the NCBI website; particularly for amino acid identity, BLASTP is used with the following parameters: expectation threshold 10; word length: 6; matrix: BLOSUM62; gap cost: presence: 11; extension: 1; neighbor word threshold: 11; composition adjustment: conditional composition score matrix adjustment.

[0014] In the context of the present invention, any embodiment referred to as "comprising" a particular feature of the invention is understood to include, in some more preferred embodiments, the more restrictive statement "consisting of" or "consisting essentially of" the exact same feature of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0015] In another additional or alternative embodiment, the antigen recognition construct may further comprise a CDR1 and / or CDR2, or more preferably, a CDR2bis domain sequence. Within the variable domain, CDR1 and CDR2 or CDR2bis are found in the variable (V) region of the polypeptide chain, and CDR3 comprises part of the V and all of the diversity (D) and joining (J) regions. CDR3 is the most variable and is the primary CDR involved in specific and selectively recognizing antigens. CDR1, CDR2, and CDR2bis sequences may be selected from the CDR sequences of human variable chain alleles.

[0016] Natural α-β heterodimeric TCRs have an α chain and a β chain. Each chain comprises a variable region, a joining region, and a constant region; the β chain usually also contains a short diversity region between the variable and joining regions, although this diversity region is often considered part of the joining region. Each variable region comprises three CDRs (complementarity-determining regions) embedded in a framework sequence, one of which is a hypervariable region designated CDR3. There are several types of α chain variable (Vα) regions and several types of β chain variable (Vβ) regions, distinguished by their frameworks, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. Vα types are designated by unique TRAV numbers in the IMGT nomenclature, and Vβ types are designated by unique TRBV numbers. (For more information about immunoglobulin antibodies and TCR genes, see the international ImMunoGeneTic information system®, Lefranc M-Petal, Nucleic Acids Res. 2015 Jan;43(Database issue):D413-22; and http: / / www.imgt.org / ).

[0017] Thus, in a further or alternative embodiment, the antigen recognition construct of the present invention comprises the combined CDR3, CDR2, CDR2bis, and CDR2 sequences as shown in Table 3 below, which together with the CDR1 sequence represent each variable chain allele. Thus, an antigen recognition construct of the present invention comprising at least one, and preferably all four CDR sequences CDR2, CDR2, CDR2bis, and CDR3 is preferred. Preferably, the antigen recognition construct of the present invention comprises each CDR1, CDR2bis, and CDR3 of one of the TCR variable regions of the present invention disclosed herein (see Table 1 and the Examples section below).

[0018] The terms "specificity" or "antigen specificity" or "specific for" a given antigen, as used herein, mean that an antigen recognition construct is capable of specifically binding to said antigen, preferably a TAA antigen, more preferably with high avidity, when said antigen is presented by HLA, preferably by HLA A2. For example, a TCR as an antigen recognition construct may be considered to have "antigenic specificity" for a TAA if, upon co-culture with target cells pulsed with a low concentration of the TAA antigen, such as the TAA epitopes and antigens provided below, T cells expressing the TCR and contacted with the TAA-presenting HLA secrete at least about 200 pg / ml or more (e.g., 250 pg / ml or more, 300 pg / ml or more, 400 pg / ml or more, 500 pg / ml or more, 600 pg / ml or more, 700 pg / ml or more, 1000 pg / ml or more, 2,000 pg / ml or more, 2,500 pg / ml or more, 5,000 pg / ml or more) of interferon gamma (IFN-γ) (e.g., about 10 -11 mol / L, 10 -10 mol / L, 10 -9 mol / L, 10 -8 mol / L, 10 -7 mol / L, 10 -6 mol / L, 10 -5Alternatively, or in addition, a TCR may be considered to have "antigen specificity" for a TAA if cells expressing the TCR secrete at least twice the non-transduced background level of IFN-γ when co-cultured with target cells pulsed with a low concentration of the TAA antigen. Such "specificity" may be analyzed, for example, using ELISA.

[0019] In an alternative or additional embodiment of the invention, the antigen recognition construct selectively binds to a TAA-derived antigenic peptide; preferably the TAA antigenic peptide is a protein epitope or peptide having the amino acid sequence set forth in SEQ ID NO: 97-115, most preferably SEQ ID NO: 97, or a variant thereof, wherein the variant is a deletion, addition, insertion or substitution of amino acids at no more than three, preferably at no more than two, most preferably at no more than one amino acid position.

[0020] The term "selectivity" or "selectively recognize / bind" is understood to refer to the property of an antigen recognition construct, such as a TCR or antibody, that preferably selectively recognizes or binds to only one specific epitope, and preferably exhibits no or substantially no cross-reactivity to other epitopes. Preferably, "selectivity" or "selectively recognizes / binds" refers to an antigen recognition construct (e.g., a TCR) that preferably selectively recognizes or binds to only one specific epitope, and preferably exhibits no or substantially no cross-reactivity to other epitopes, wherein said epitope is unique to one protein, such that the antigen recognition construct exhibits no or substantially no cross-reactivity to other epitopes and other proteins.

[0021] The antigen recognizing the construct according to the invention is preferably selected from an antibody, or a derivative or fragment thereof, or a T cell receptor (TCR), or a derivative or fragment thereof. An antibody or TCR derivative or fragment of the invention should preferably retain the antigen binding / recognition ability of the parent molecule, in particular its specificity and / or selectivity as explained above. Such binding function may be retained by the presence of the CDR3 region as defined herein.

[0022] In one embodiment of the present invention, the TCR of the present invention can recognize a TAA antigen in a major histocompatibility complex (MHC) class I-dependent manner. "MHC class I-dependent manner," as used herein, means that the TCR elicits an immune response upon binding to a TAA antigen within the context of an MHC class I molecule. The MHC class I molecule can be any MHC class I molecule known in the art, such as, for example, an HLA-A molecule. In a preferred embodiment of the present invention, the MHC class I molecule is an HLA-A2 molecule.

[0023] The present invention provides both single chain antigens that recognize the construct and two chain antigens that recognize the construct.

[0024] In one embodiment, the TCR alpha variable domain has at least one mutation relative to the TCR alpha domain shown in Table 1; and / or the TCR beta variable domain has at least one mutation relative to the TCR alpha domain shown in Table 1. In one embodiment, a TCR comprising at least one mutation in the TCR alpha variable domain and / or the TCR beta variable domain has at least twice the binding affinity and / or binding half-life for a TAA peptide-HLA molecule complex as a TCR comprising an unmutated TCR alpha domain and / or an unmutated TCR beta variable domain.

[0025] The TCR alpha chain herein may further comprise a TCR alpha transmembrane domain and / or a TCR alpha intracellular domain. The TCR beta chain herein may further comprise a TCR beta transmembrane domain and / or a TCR beta intracellular domain.

[0026] The present invention provides a TCR or a fragment or derivative thereof as an antigen-recognition construct. The TCR is preferably a human TCR, and is understood to originate from a human TCR locus and therefore comprise a human TCR sequence. Furthermore, the TCR of the present invention may be characterized by being of human origin and specifically recognizing the TAA antigen of the present invention.

[0027] Another embodiment of the present invention additionally or alternatively provides an antigen recognition construct as described above that induces an immune response, preferably an immune response characterized by increased levels of interferon (IFN) gamma.

[0028] The TCRs of the present invention may be provided as single chain α or β, or γ and δ, molecules, or alternatively as two-chain constructs composed of both α and β chains, or both γ and δ chains.

[0029] The antigen recognition construct of the present invention may comprise a TCR alpha or gamma chain; and / or a TCR beta or delta chain; wherein the TCR alpha or gamma chain comprises a CDR50 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 3, 15, 27, 39, 51, 63, 75, and 129, and / or the TCR beta or delta chain comprises a CDR9 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9, 21, 33, 45, 57, 69, 81, and 135.

[0030] Most preferably, in some additional embodiments where the present disclosure refers to antigen-recognition constructs comprising any one, two, three, or all of the CDR2, CDR2, CDR2bis, and CDR3 regions of the TCR chains disclosed herein (see Table 1), antigen-recognition constructs comprising each CDR sequence of the present invention with three or fewer, two, and preferably only one modified amino acid residue may be preferred. The modified amino acid residues may be selected from amino acid insertions, deletions, or substitutions. Most preferably, the three, two, and preferably only modified amino acid residues are the first or last amino acid residues of each CDR sequence. When the modification is a substitution, in some embodiments, it is preferred that the substitution is a conservative amino acid substitution.

[0031] When the antigen recognition construct of the present invention is composed of at least two amino acid chains or antigen-binding fragments thereof, such as a two-chain TCR, the antigen recognition construct is or the amino acid sequence set forth in SEQ ID NO: 3 in the first polypeptide chain and the amino acid sequence set forth in SEQ ID NO: 9 in the second polypeptide chain; or the amino acid sequence set forth in SEQ ID NO: 15 in the first polypeptide chain and the amino acid sequence set forth in SEQ ID NO: 21 in the second polypeptide chain; or the amino acid sequence set forth in SEQ ID NO: 27 in the first polypeptide chain and the amino acid sequence set forth in SEQ ID NO: 33 in the second polypeptide chain; or the amino acid sequence set forth in SEQ ID NO: 39 in the first polypeptide chain and the amino acid sequence set forth in SEQ ID NO: 45 in the second polypeptide chain; or the amino acid sequence set forth in SEQ ID NO: 51 in the first polypeptide chain and the amino acid sequence set forth in SEQ ID NO: 57 in the second polypeptide chain; or the amino acid sequence set forth in SEQ ID NO: 63 in the first polypeptide chain and the amino acid sequence set forth in SEQ ID NO: 69 in the second polypeptide chain; or the amino acid sequence set forth in SEQ ID NO: 75 in the first polypeptide chain and the amino acid sequence set forth in SEQ ID NO: 81 in the second polypeptide chain; or the amino acid sequence set forth in SEQ ID NO: 129 in the first polypeptide chain and the amino acid sequence set forth in SEQ ID NO: 135 in the second polypeptide chain. Any one of the above-mentioned dual-chain TCRs, or antigen-binding fragments thereof, is a preferred TCR of the present invention. In some embodiments, the CDR3 of the dual-chain TCR of the present invention may be mutated. Mutations in the CDR3 sequences provided above preferably include substitution, deletion, addition, or insertion of no more than three, preferably no more than two, and most preferably no more than one amino acid residue. In some embodiments, the first polypeptide chain may be a TCR α or γ chain, and the second polypeptide chain may be a TCR β or δ chain. A combination of αβ or γδ TCRs is preferred.

[0032] In some embodiments, the TCR, or antigen-binding fragment thereof, is composed of a TCR alpha and a TCR beta chain, or a gamma and a delta chain. Such dual-chain TCRs comprise a variable region within each chain, each comprising one CDR1, one CDR2, or more preferably one CDR2bis, and one CDR3 sequence. The TCR comprises the CDR1, CDR2, CDR2bis, and CDR3 sequences contained in the variable chain amino acid sequences of SEQ ID NOs: 4 and 10; or 16 and 22; or 28 and 34; or 40 and 46; or 52 and 58; or 64 and 70; or 76 and 82; or 130 and 136.

[0033] Some embodiments of the present invention relate to a TCR or fragment thereof, composed of a TCR alpha and a TCR beta chain, wherein the TCR comprises a variable region sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or preferably 100% sequence identity to an amino acid sequence selected from the alpha and beta chains set forth in SEQ ID NOs: 4 and 10; or 16 and 22; or 28 and 34; or 40 and 46; or 52 and 58; or 64 and 70; or 76 and 82; or 130 and 136.

[0034] In a particularly preferred embodiment, the present invention provides an improved TCR, designated R11P3D3_KE, composed of a TCR alpha chain and a TCR beta chain, said TCR comprising a variable region sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or preferably 100% sequence identity to an amino acid sequence selected from the alpha and beta chains set forth in SEQ ID NOs: 132 and 138. This TCR showed surprisingly improved functionality in terms of tumor cell recognition when compared to its parent receptor, designated herein as R11P3D3.

[0035] The TCRs of the present invention may further comprise a constant region derived from any suitable species, for example, any mammal, such as human, rat, monkey, rabbit, donkey, or mouse. In one embodiment of the present invention, the TCRs of the present invention further comprise a human constant region. In some preferred embodiments, the constant region of the TCRs of the present invention may be slightly modified, for example, by the introduction of heterologous sequences, preferably mouse sequences, which may increase TCR expression and stability. In some preferred embodiments, the variable region of the intervening TCR may be slightly modified, for example, by the introduction of single point mutations, to optimize TCR stability and / or enhance TCR chain pairing.

[0036] Some embodiments of the present invention relate to a TCR or fragment thereof, composed of a TCR alpha and a TCR beta chain, wherein the TCR comprises a constant region having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or preferably 100% sequence identity to an amino acid sequence selected from the alpha and beta chains set forth in SEQ ID NOs: 5 and 11; or 17 and 23; or 29 and 35; or 41 and 47; or 53 and 59; or 65 and 71; or 77 and 83; or 131 and 137.

[0037] The TCR α or γ chain of the present invention comprises a CDR1 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 13, 25, 37, 49, 61, 73, and 127; and / or a CDR2 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 2, 14, 26, 38, 50, 62, 74, and 128. , 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 196, 197, 198, 199, 200, 201, 202, and 204.

[0038] According to the present invention, the TCR β or δ chain has a CDR1 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 7, 19, 31, 43, 55, 67, 79, and 133; and / or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 8, 20, 32, 44, 56, 68, 80, and 134. and / or a CDR2bis having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 8, 20, 32, 44, 56, 68, 80, and 134.

[0039] The antigen recognition construct may, in a further embodiment, comprise a binding fragment of a TCR, said binding fragment being selected from the group consisting of SEQ ID NOs: 1, 2, 3, 196; or 7, 8, 9; or 13, 14, 15, 197; or 19, 20, 21; or 25, 26, 27, 198; or 31, 32, 33; or 37, 38, 39, 199; or 43, 44, 45; or 49, 50, 51, 200; or 55, 56 or 79, 80, 81; or 127, 128, 129, 204; or 133, 134, 135.

[0040] In a further embodiment of the invention, the antigen recognition construct described elsewhere herein is a TCR or fragment thereof, composed of at least one TCR alpha chain sequence and one TCR beta chain sequence, wherein said TCR alpha chain sequence comprises CDR1, CDR2, CDR2bis, and CDRR3 sequences having the amino acid sequences of SEQ ID NOs: 1-3, and 196, and said TCR beta chain sequence comprises CDR1-CDR3 sequences having the amino acid sequences of SEQ ID NOs: 7-9; or said TCR alpha chain sequence comprises CDR1-CDR3 sequences having the amino acid sequences of SEQ ID NOs: 13-15, and and 197, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 19 to 21; or the TCR α chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 25 to 27, and 198, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 31 to 33; or the TCR α chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: the TCR α chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 37 to 39, and 199, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 43 to 45; or the TCR α chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 49 to 51, and 200, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 55 to 57; or the TCR the α chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 61 to 63, and 201, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 67 to 69; or the TCR α chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 73 to 75, and 202, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 79 to 81;Alternatively, the TCR α chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 127 to 129 and 204, respectively, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 133 to 135.

[0041] In a further embodiment of the present invention, the antigen recognition construct described herein is a TCR or fragment thereof comprising at least one TCR alpha chain sequence and one TCR beta chain sequence, wherein the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 4 and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 10; or the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 16 and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 22. or the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:28, and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:34; or the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:40, and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:46; or the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:52, and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:58; or or the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 64, and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 70; or the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 76, and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 82; or the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 130, and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 136.

[0042] In a further embodiment of the present invention, the antigen recognition construct as hereinbefore described is a TCR or a fragment thereof, and further comprises a TCR constant region having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 131, and 137, preferably wherein the TCR is composed of at least one TCR alpha and one TCR beta chain sequence, and The Rα chain sequence comprises a constant region having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 5, 17, 29, 41, 53, 65, 77, and 131; the TCRβ chain sequence comprises a constant region having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 11, 23, 35, 47, 59, 71, 83, and 137.

[0043] Also disclosed is an antigen recognition construct as described herein above, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6 and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12. The present invention also provides a TCR comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18 and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 24. In a further embodiment, the present invention provides an antigen recognition construct that is a TCR, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 30, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 36. In a further embodiment, the present invention provides an antigen recognition construct that is a TCR, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 42, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 48. In a further embodiment, the present invention provides an antigen recognition construct that is a TCR, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 54, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 60.In a further embodiment, the present invention provides an antigen recognition construct that is a TCR, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 72. In a further embodiment, the present invention provides an antigen recognition construct that is a TCR, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 78, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84. In a further embodiment, the present invention provides an antigen recognition construct that is a TCR, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 132, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 138.

[0044] As used herein, the terms "mouse" or "human" when referring to an antigen-recognition construct, or a TCR, or any component of a TCR described herein (e.g., complementarity-determining regions (CDRs), variable region, constant region, α chain, and / or β chain), refer to a TCR (or component thereof) derived from a mouse or human unrearranged TCR locus, respectively.

[0045] In one embodiment of the invention, chimeric TCRs are provided, wherein the TCR chains comprise sequences from multiple species. Preferably, the TCRs of the invention may comprise an alpha chain comprising a human variable region of the alpha chain and a mouse constant region, for example, a mouse TCR alpha chain.

[0046] In one embodiment, the TCR of the present invention is a human TCR comprising a human variable region according to the above embodiments and a human constant region.

[0047] In some embodiments, the antigen recognition construct is murine or humanized, terms used when amino acid sequences of heterologous origin are introduced into the construct of the invention.

[0048] The TCRs of the present invention may be provided as single-chain TCRs (scTCRs). The scTCRs of the present invention must comprise a complete or partial α chain sequence and a complete or partial β chain sequence in one polypeptide chain, preferably linked via a peptide linker. The scTCR may comprise a polypeptide of the variable region of a first TCR chain (e.g., an α chain) and a polypeptide of the entire (full-length) second TCR chain (e.g., a β chain), or vice versa. Furthermore, the scTCR may optionally comprise one or more linkers linking two or more polypeptides together. The linker may be, for example, a peptide that binds two single chains together, as described herein. Also provided are scTCRs of the present invention fused to human cytokines such as IL-2, IL-7, or IL-15.

[0049] The antigen recognition constructs of the present invention can also be provided in the form of a multimeric complex comprising at least two scTCR molecules, each fused to at least one biotin moiety or other interconnecting molecule / linker, such that the scTCRs are interconnected by biotin-streptavidin interactions to form the multimeric complex. Similar approaches known in the art for generating multimeric TCRs are also possible and are included in the present disclosure. Higher order multimeric complexes comprising more than two scTCRs of the present invention are also provided.

[0050] For the purposes of this invention, a TCR is a moiety having at least one TCR α or γ and / or TCR β or δ variable domain. Generally, they comprise both a TCR α variable domain and a TCR β variable domain, or alternatively, they comprise both a TCR γ variable domain and a TCR δ variable domain. They may be αβ / γδ heterodimers or in single-chain form. For use in adoptive therapy, αβ or γδ heterodimeric TCRs may be transfected as full-length chains, e.g., having both cytoplasmic and transmembrane domains. If desired, an introduced disulfide bond may exist between residues of the respective constant domains.

[0051] In a preferred embodiment, the antigen recognition construct is a human TCR, a fragment or derivative thereof. A human TCR or a fragment or derivative thereof is a TCR comprising more than 50% of the corresponding human TCR sequence. Preferably, only a small portion of the TCR sequence is of artificial origin or derived from other species. However, chimeric TCRs, such as those derived from human origin and having mouse sequences in their constant domains, are known to be advantageous. Therefore, TCRs according to the present invention that contain mouse sequences in the extracellular part of their constant domains are particularly preferred.

[0052] Therefore, the antigen recognition construct of the present invention can be used in a human leukocyte antigen (HLA)-dependent manner, preferably in an HLA-A dependent manner. * It is also preferred that the antigen recognition construct can recognize the antigen in an HLA-dependent manner. The term "HLA-dependent manner" in the context of the present invention means that the antigen recognition construct binds to the antigen only if the antigen peptide is presented by said HLA.

[0053] The antigen recognition construct according to the present invention, in one embodiment, preferably induces an immune response, and preferably the immune response is characterized by an increase in interferon (IFN) gamma levels.

[0054] Also provided by the present invention are polypeptides comprising a functional portion of any of the TCRs described herein (or functional variants thereof), for example, any one of the TCRs selected from R11P3D3, R16P1C10, R16P1E8, R17P1A9, R17P1D7, R17P1G3, R17P2B6, and R11P3D3_KE as provided in the Examples section and Table 1. The term "polypeptide," as used herein, includes oligopeptides and refers to a single chain of amino acids linked by one or more peptide bonds. For polypeptides of the present invention, a functional portion can be any portion comprising consecutive amino acids of the TCR (or functional variant) of which it is a part, provided that the functional portion specifically binds to a TAA antigen, preferably as disclosed in Table 2 herein, and to peptides A1-A9 (SEQ ID NOS: 97, and 98-106), and to peptides T1-T9 (SEQ ID NOS: 107-115). The term "functional portion," when used in reference to a TCR (or functional variant thereof), refers to any portion or fragment of a TCR (or functional variant thereof) of the present invention, which portion or fragment retains the biological activity of the TCR (or functional variant thereof) of which it is a part (parent TCR or parent functional variant thereof). A functional portion, for example, retains the ability to specifically bind to a TAA antigen (in an HLA-dependent manner), or detect, treat, or prevent cancer, to a similar extent, the same extent, or to a greater extent than the parent TCR (or functional variant thereof) that encompasses the portion of the TCR (or functional variant thereof). With respect to a parent TCR (or functional variant thereof), a functional portion can comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the variable sequence of the parent TCR (or functional variant thereof).

[0055] A functional portion can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent TCR or functional variant thereof. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, e.g., the ability to specifically bind to a TAA antigen; and / or to detect, treat, or prevent cancer. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent TCR or functional variant thereof.

[0056] The polypeptide may comprise a functional portion of either or both of the α and β chains of a TCR of the present invention or a functional variant thereof, such as a functional portion comprising one or more of CDR1, CDR2, CDR2bis, and (preferably) CDR3 of the variable region of the α and / or β chain of a TCR of the present invention or a functional variant thereof. In one embodiment of the present invention, the polypeptide may comprise a functional portion comprising the amino acid sequence of SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 129, and 135 (CDR3 of the variable region of the TCR of the present invention), or a combination thereof. In one embodiment of the present invention, the polypeptide may comprise a variable region of a TCR of the present invention or a functional variant thereof, for example, comprising a combination of the above CDR regions. In this regard, the polypeptide may comprise the amino acid sequence of any of SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 130, and 136 (the variable region of the α or β chain of a TCR of the invention).

[0057] In some cases, the constructs of the present invention may comprise one or two polypeptide chains comprising the sequences set forth in any of SEQ ID NOS: 1-84, 127-138, 196-202, and 204 (CDR sequences, constant region, variable region, and full-length sequence), or functional fragments thereof, and may further comprise other amino acid sequences, such as amino acid sequences encoding immunoglobulins or portions thereof, such that the proteins of the present invention may be fusion proteins. In this regard, the present invention also provides fusion proteins comprising at least one of the polypeptides of the present invention described herein together with at least one other polypeptide. The other polypeptide may be present as a separate polypeptide in a fusion protein or as a polypeptide expressed in frame (tandem) with one of the polypeptides of the present invention described herein. The other polypeptide may be any peptide or protein molecule, or a portion thereof, including, but not limited to, immunoglobulins, CD3, CD4, CD8, MHC molecules, e.g., CD1 molecules such as CD1a, CD1b, CD1c, and CD1d.

[0058] A fusion protein may comprise one or more copies of a polypeptide of the present invention and / or one or more copies of another polypeptide. For example, a fusion protein may comprise one, two, three, four, five or more copies of a polypeptide of the present invention and / or another polypeptide. Suitable methods for producing fusion proteins are known in the art and include, for example, recombinant methods. In some embodiments of the present invention, the TCRs (and functional portions and functional variants thereof), polypeptides, and proteins of the present invention may be expressed as a single protein comprising a linker peptide linking the α chain and the β chain and the γ chain and the δ chain. In this regard, the TCRs (and functional variants and functional portions thereof), polypeptides, and proteins of the present invention comprise the amino acid sequence of the variable region of a TCR of the present invention and may further comprise a linker peptide. The linker peptide may advantageously facilitate expression of the recombinant TCRs (including functional portions and functional variants thereof), polypeptides, and / or proteins in a host cell. The linker peptide may comprise any suitable amino acid sequence. Linker sequences for single-chain TCR constructs are well known in the art. Such single-chain constructs may further comprise one or two constant domain sequences. Upon expression of a construct containing a linker peptide by a host cell, the linker peptide may also be cleaved to separate the α and β chains and the γ and δ chains.

[0059] As already mentioned above, the binding functionality of the TCRs of the present invention may be provided in an antibody framework. For example, the CDR sequences of the TCRs of the present invention, possibly including three, two, or one additional N- and / or C-terminal framework residues, may be directly grafted onto antibody variable heavy / light chain sequences. The term "antibody" in its various grammatical forms is used herein to refer to immunoglobulin molecules and to refer to immunologically active portions of immunoglobulin molecules, i.e., molecules containing antigen-binding sites or paratopes. Such molecules are also referred to as "antigen-binding fragments" of immunoglobulin molecules. The present invention further provides antibodies, or antigen-binding portions thereof, that specifically bind to the antigens described herein. The antibodies may be any type of immunoglobulin known in the art. For example, the antibodies may be of any isotype, such as IgA, IgD, IgE, IgG, IgM, etc. The antibodies may be monoclonal or polyclonal. The antibodies may be natural antibodies, such as antibodies isolated and / or purified from mammals, such as mice, rabbits, goats, horses, chickens, hamsters, and humans. Alternatively, the antibody may be a genetically engineered antibody, such as a humanized antibody or a chimeric antibody. The antibody may be in monomeric or polymeric form.

[0060] The term "antibody" includes, but is not limited to, genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies (e.g., produced by "CDR-grafting"), antibody fragments, and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, trispecific antibodies, tetraspecific antibodies, etc.). The term "antibody" includes cys-bispecific antibodies and miniantibodies. Thus, any and all embodiments provided herein relate to "antibodies" or "antibody-like constructs," and unless expressly indicated otherwise, bispecific antibody, diabody, scFv fragments, chimeric antibody receptor (CAR) constructs, bispecific antibody, and / or miniantibody embodiments are also contemplated. The term "antibody" includes polypeptides of the immunoglobulin family or polypeptides comprising fragments of immunoglobulins capable of binding in a noncovalent, reversible, and specific manner to a corresponding antigen, preferably a TAA of the present invention, as disclosed herein. An exemplary antibody structural unit comprises a tetramer. In some embodiments, a full-length antibody can be composed of two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain (linked via a disulfide bond). Antibody structures and isotypes are well known to those skilled in the art (e.g., Janeway's Immunobiology, 9th edition, 2016).

[0061] Recognized immunoglobulin genes in mammals include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. (For more information on immunoglobulin genes, see the International Im-MunoGeneTics information system®, Lefranc M-Petal, Nucleic Acids Res. 2015 Jan;43 (Database issue):D413-22; and http: / / www.imgt.org / .) For full-length chains, light chains are classified as either kappa or lambda. For full-length chains, heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively. The N-terminus of each chain defines a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of the light and heavy chains, respectively. As used herein, "antibody" encompasses all variants of antibodies and fragments thereof. Thus, within this concept, full-length antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies (scFv), Fab, Fab', and multimeric versions of these fragments (e.g., F(ab')2) have essentially the same or similar binding specificities. In some embodiments, antibodies specifically bind to the peptide TAA of the present invention. Preferred antigen-recognition constructs according to the present invention include an antibody heavy chain, preferably a variable domain thereof, or an antigen-binding fragment thereof, and / or an antibody light chain, preferably a variable domain thereof, or an antigen-binding fragment thereof. Similarly, disulfide-stabilized variable region fragments (dsFv) can be prepared by recombinant DNA technology, but the antibody fragments of the present invention are not limited to these exemplary types of antibody fragments. Antibodies, or antigen-binding portions thereof, can also be modified to comprise detectable labels, such as, for example, radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).In some cases, the TCR CDR3 sequences may be slightly modified, preferably by no more than three amino acid residues, preferably by only two and most preferably by only one amino acid position compared to the CDR3 sequences set out in SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 129 and 135. Preferably, the antibody comprises a CDR3, preferably a combination of all CDR1, CDR2, CDR2bis and CDR3 regions as shown for the TCRs of the invention in Table 1, optionally with, in each case independently, two or no more than two, preferably one amino acid substitution, insertion and / or deletion respectively, compared to these sequences.

[0062] Suitable methods for producing antibodies are known in the art. For example, standard hybridoma methods are described, for example, in Kohler and Milstein, Eur. J. Immunol, 5, 51 1-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA Janeway et al. (eds.), Immunobiology, 8th Ed., Garland Publishing, New York, NY (201)). Alternatively, other methods are known in the art, such as the EBV-hybridoma method (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), and Roder et al., Methods Enzymol, 121, 140-67 (1986)) and bacteriophage vector expression systems (see, e.g., Huse et al., Science, 246, 1275-81 (1989)). Additionally, methods for producing antibodies in non-human animals are described, for example, in U.S. Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. Patent Application Publication No. 2002 / 0197266.

[0063] Some embodiments of the present invention also relate to TCRs or functional fragments and polypeptides thereof, which are soluble TCRs. As used herein, the term "soluble T cell receptor" refers to a heterodimeric truncated variant of a native TCR, comprising the extracellular portions of the TCR α and β chains, linked, for example, by disulfide bonds, but lacking the transmembrane and cytosolic domains of the native protein. The terms "soluble T cell receptor α and β chain sequences" refer to TCR α and β chain sequences lacking the transmembrane and cytoplasmic domains. The soluble TCR α and β chain sequences (amino acid or nucleic acid) may be identical to the corresponding sequences in a native TCR, or may comprise mutated soluble TCR α and β chain sequences compared to the corresponding native TCR sequences. As used herein, the term "soluble T cell receptor" encompasses soluble TCRs having mutated or non-mutated soluble TCR α and β chain sequences. The mutations may be in the variable or constant regions of the soluble TCR α and β chain sequences and may include, but are not limited to, amino acid deletions, insertions, substitution mutations, and nucleic acid sequence changes that do not alter the amino acid sequence. In any event, the soluble TCRs of the present invention retain the binding function of their parent molecules.

[0064] The above problems are further solved by a nucleic acid encoding the antigen recognition construct of the present invention, or any of the above protein or polypeptide constructs. The nucleic acid preferably has (a) a strand encoding the antigen recognition construct of the present invention; (b) a strand complementary to the strand of (a); or (c) a strand that hybridizes to the molecule described in (a) or (b) under stringent conditions. Stringent conditions are known to those skilled in the art, particularly from Sambrook et al., "Molecular Cloning." In addition, the nucleic acid optionally contains additional sequences necessary for expressing the nucleic acid sequence corresponding to the protein, particularly for expression in mammalian / human cells. The nucleic acid used can be contained in a vector suitable for expressing the nucleic acid sequence corresponding to the peptide within the cell. However, the nucleic acid can also be used to transform antigen-presenting cells, which may not be limited to classical antigen-presenting cells such as dendritic cells, so that they themselves produce the corresponding protein on their cell surface.

[0065] In some embodiments, the polypeptide of the antigen recognition construct is encoded by a nucleic acid and can be expressed in vivo or in vitro. Thus, in some embodiments, a nucleic acid encoding an antigen recognition construct is provided. In some embodiments, the nucleic acid encodes a portion or monomer of the antigen recognition construct of the present invention (e.g., one of the two chains of a TCR of the present invention), and / or a separate nucleic acid encodes another portion or monomer of the antigen recognition construct of the present invention (e.g., the other of the two chains of a TCR). In some embodiments, the nucleic acid encodes two or more antigen recognition construct polypeptide chains, e.g., at least two TCR chains. Nucleic acids encoding multiple antigen recognition construct chains may include a nucleic acid cleavage site between at least two chain sequences, may encode transcription or translation initiation sites between two or more chain sequences, and / or may encode proteolytic target sites between two or more antigen recognition construct chains.

[0066] "Nucleic acid," as used herein, includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, synthesized or obtained from a natural source (e.g., isolated and / or purified), and may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides.

[0067] Preferably, the nucleic acids of the invention are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by linking natural or synthetic nucleic acid segments to a nucleic acid molecule capable of autonomous replication within a living cell, or (ii) a molecule resulting from replication as described in (i) above. For purposes herein, autonomous replication can be in vitro replication or in vivo replication. The nucleic acid can comprise any nucleotide sequence that encodes any of the TCRs, polypeptides, or proteins described herein, or functional portions or functional variants thereof.

[0068] The present invention further provides a vector comprising the nucleic acid according to the present invention. Desirably, the vector is an expression vector or a recombinant expression vector. The term "recombinant expression vector" in the context of the present invention refers to a nucleic acid construct that allows for the expression of mRNA, protein, or polypeptide in a suitable host cell. The recombinant expression vector of the present invention can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include vectors designed for propagation and expansion, or for expression, or both, such as plasmids and viruses. Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo. Preferably, the recombinant expression vector is a viral vector, such as a retroviral vector. The recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, specific for the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector is introduced and in which expression of the nucleic acid of the present invention may be carried out. Furthermore, the vector of the present invention may contain one or more marker genes that allow for the selection of transformed or transfected hosts. The recombinant expression vector may comprise a native or canonical promoter operably linked to the nucleotide sequence encoding the construct of the present invention, or to a nucleotide sequence complementary to or hybridizing with the nucleotide sequence encoding the construct of the present invention. Promoter options include, for example, strong promoters, weak promoters, inducible promoters, tissue-specific promoters, and development-specific promoters. The promoter may be a non-viral promoter or a viral promoter. The recombinant expression vector of the present invention may be designed for either transient expression, stable expression, or both. The recombinant expression vector may also be engineered for constitutive or inducible expression.

[0069] The present invention also relates to a host cell comprising an antigen recognition construct according to the present invention. Specifically, the host cell of the present invention comprises a nucleic acid or vector as described hereinabove. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or algae cell, or a prokaryotic cell, such as a bacterium or protozoan cell. The host cell can be a cultured cell or a primary cell, i.e., a cell directly isolated from an organism, such as a human. The host cell can be an adherent cell or a suspension cell, i.e., a cell that grows in suspension. For the purpose of producing a recombinant TCR, polypeptide, or protein, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. Although the host cell can be any cell type, can be derived from any species, and can be at any developmental stage, the host cell is preferably a peripheral blood leukocyte (PBL) or peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell. The T cells can be any T cells, e.g., cultured T cells, such as primary T cells; or T cells from cultured T cell lines, e.g., Jurkat, SupT1; or T cells obtained from a mammal, preferably T cells or T cell precursors from a human patient. When obtained from a mammal, the T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells can also be enriched or purified. Preferably, the T cells are human T cells. More preferably, the T cells are T cells isolated from a human. The T cells can be of any T cell type and at any developmental stage, including, but not limited to, CD4-positive and / or CD8-positive, CD4-positive helper T cells, e.g., Th1 and Th2 cells, CD8-positive T cells (e.g., cytotoxic T cells), tumor-infiltrating lymphocytes (TILs), memory T cells, naive T cells, etc. Preferably, the T cells are CD8-positive or CD4-positive T cells.

[0070] Preferably, the host cells of the present invention are lymphocytes, preferably T lymphocytes, such as CD4-positive or CD8-positive T cells. The host cells are more preferably tumor-reactive T cells specific for TAA-expressing tumor cells.

[0071] The object of the present invention is to a. providing a suitable host cell; b. providing a genetic construct comprising a coding sequence encoding an antigen recognition construct according to the invention disclosed herein; c. introducing said genetic construct into said suitable host cell; d. expressing said genetic construct by said suitable host cell; The above-mentioned problems are also solved by a method for producing a TAA-specific antigen-recognition construct or a method for producing a cell line expressing a TAA-specific antigen-recognition construct, comprising the steps of:

[0072] The method may further comprise the step of displaying the antigen recognition construct on the cell surface of the suitable host cell.

[0073] In another preferred embodiment, the gene construct is an expression construct comprising a promoter sequence operably linked to the coding sequence. Preferably, the antigen recognition construct is of mammalian origin, preferably human origin. Preferred suitable host cells for use in the methods of the present invention are mammalian cells, such as human cells, particularly human T lymphocytes. T cells for use in the present invention are described in detail herein above.

[0074] Also encompassed by the present invention are embodiments in which the antigen recognition construct is a modified TCR, and the modification is the addition of a functional domain, such as a label or a therapeutically active substance. Additionally, TCRs with alternative domains, such as alternative membrane anchor domains, in place of the integral transmembrane region are encompassed. Also encompassed are TCRs with point mutations in the TCR variable or constant domains to improve TCR expression or stability and / or chain pairing.

[0075] Preferably, the transfection system for introducing the genetic construct into said suitable host cells is a retroviral vector system. Such systems are well known to those skilled in the art.

[0076] In one embodiment, the additional method steps of isolation and purification of the antigen recognition construct from cells, and optionally, reconstitution of the translated antigen recognition construct fragment in T cells, are also included in the invention.

[0077] In an alternative embodiment of the present invention, T cells are provided, obtained, or obtainable by a method for producing a T cell receptor (TCR) specific for tumor cells and having high avidity, as described hereinabove, depending on the host cell used in the method of the present invention, e.g., a human or non-human T cell, preferably a human TCR.

[0078] The term "isolated," as used herein in the context of a polypeptide, such as an antigen recognition construct (an example of which may be an antibody), refers to a polypeptide that has been purified from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use. An antigen recognition construct according to the present invention may be a recombinant, synthetic, or modified (non-natural) antigen-binding construct. The term "isolated," as used herein in the context of a nucleic acid or cell, refers to a nucleic acid or cell that has been purified from DNA, RNA, proteins or polypeptides or other contaminants (such as other cells) that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use, or it refers to a recombinant, synthetic, or modified (non-natural) nucleic acid. In this context, a "recombinant" protein / polypeptide or nucleic acid is one that has been produced using recombinant technology. Methods and techniques for producing recombinant nucleic acids and proteins are well known in the art.

[0079] An additional aspect of the present invention relates to an antigen recognition construct, a nucleic acid, a vector, a pharmaceutical composition, and / or a host cell disclosed herein for use in medicine. In a preferred embodiment, the use in medicine includes use in the diagnosis, prevention, and / or treatment of tumor diseases, such as malignant or benign tumor diseases. The tumor disease is, for example, a tumor disease characterized by the expression of a TAA in cancer or tumor cells of the tumor disease.

[0080] In accordance with the present disclosure, with regard to the above-mentioned medical uses of antigen recognition constructs and other substances derived therefrom or encoding same, the disease to be treated and / or diagnosed can be any proliferative disease, preferably ameliorated by expression of the TAA or TAA epitope sequences of the present invention, for example, acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal or rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder or pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, The present invention is characterized by any cancer, such as chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omentomesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and bladder cancer.Preferred cancers are cancers of the cervix, oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis.Particularly preferred cancers are TAA-positive cancers, including ovarian cancer, leukemia, or melanoma.

[0081] The constructs, proteins, TCR antibodies, polypeptides and nucleic acids of the present invention are particularly suitable for use in immunotherapy, preferably adoptive T cell therapy. The administration of the compounds of the present invention may involve, for example, the infusion of T cells of the present invention into the patient. Preferably, such T cells are the patient's autologous T cells, transduced ex vivo with the nucleic acid or antigen-recognition construct of the present invention.

[0082] The antigen recognition constructs, TCRs, polypeptides, proteins (including functional variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof) of the invention, hereinafter collectively referred to as "TCR materials of the invention," can be formulated into compositions, such as pharmaceutical compositions. In this regard, the invention provides pharmaceutical compositions comprising any of the antigen recognition constructs, TCRs, polypeptides, proteins, functional portions, functional variants, nucleic acids, expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof) described herein, together with a pharmaceutically acceptable carrier, excipient, and / or stabilizer. Pharmaceutical compositions of the invention containing any of the TCR materials of the invention can comprise, for example, two or more TCR materials of the invention, such as polypeptides and nucleic acids, or two or more different TCRs (including functional portions and functional variants thereof). Alternatively, the pharmaceutical composition may comprise the TCR material of the invention in combination with another pharmaceutically active agent or drug, such as a chemotherapeutic agent, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. Preferably, the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier may be any of those conventionally used for the particular TCR material of the invention under consideration. Such pharmaceutically acceptable carriers are well known to those skilled in the art and are readily available to the public. Preferably, the pharmaceutically acceptable carrier is one that has no adverse side effects or toxicity under the conditions of use.

[0083] Thus, there is also provided a pharmaceutical composition comprising any of the products of the invention and TCR material of the invention, in particular any protein, nucleic acid or host cell described herein. In a preferred embodiment, the pharmaceutical composition is for immunotherapy, preferably adoptive cell therapy.

[0084] Preferably, the TCR material of the present invention is administered by injection, e.g., intravenously. When the TCR material of the present invention is a host cell expressing a TCR of the present invention (or a functional variant thereof), a pharmaceutically acceptable carrier for the injectable cells may include any isotonic carrier, such as, for example, saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOLR electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or lactated Ringer's solution. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum egg white.

[0085] For purposes of the present invention, the amount or dose of TCR material of the present invention (e.g., the number of cells if the TCR material of the present invention is one or more cells) administered may be sufficient to affect, e.g., a therapeutic or prophylactic response, in a subject or animal over a reasonable time frame. For example, a dose of TCR material of the present invention should be sufficient to bind to a cancer antigen or detect, treat, or prevent cancer for a period of about 2 hours or more, e.g., 12-24 hours or more, from the time of administration. In certain embodiments, the period may be longer. The dose will be determined by the efficacy of the particular TCR material of the present invention and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) being treated.

[0086] It is contemplated that the pharmaceutical compositions, antigen recognition constructs, TCRs (including functional variants thereof), polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or cell populations of the invention can be used in methods of treating or preventing cancer or TAA-positive precancer. The TCRs of the invention (and their functional variants) are believed to specifically bind to the TAAs of the invention, such that the TCRs (or related inventive polypeptides or proteins and their functional variants), when expressed by or on cells such as T cells, can mediate an immune response against target cells that express the TAA of the invention, preferably presenting the TAA peptide through MHC I or II on the surface of said target cells. In this regard, the present invention provides a method of treating or preventing a condition, in particular cancer, in a mammal comprising a pharmaceutical composition as described herein, in particular an antigen recognition construct that is a TCR (and functional variants thereof), polypeptide, or protein; any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCR (and functional variants thereof) and polypeptides and proteins described herein; or any host cell or population of cells comprising a nucleic acid or recombinant vector encoding any of the constructs (and functional variants thereof) or polypeptides or proteins of the invention described herein, in an amount effective to treat or prevent the condition in a mammal, wherein the condition is preferably a cancer such as a cancer that expresses a TAA of the invention.

[0087] Examples of pharmaceutically acceptable carriers or diluents useful in the present invention include stabilizers and buffers (e.g., phosphate buffers) such as SPGA, carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein-containing agents such as bovine whey or skim milk.

[0088] The terms "treat" and "prevent," and terms derived therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art recognizes as having potential benefit or therapeutic effect. In this regard, the methods of the present invention may provide any amount or level of treatment or prevention of a condition in a mammal. Furthermore, the treatment or prevention provided by the methods of the present invention can include treatment or prevention of one or more conditions or symptoms of a condition, such as, for example, cancer, being treated or prevented. For example, treatment or prevention can include promoting tumor regression. Also, for purposes of this specification, "prevention" can encompass delaying the condition or symptom or onset of the condition.

[0089] The present invention also relates to methods of treating cancer comprising administering the TCRs, nucleic acids, or host cells herein in combination with at least one chemotherapeutic agent and / or radiation therapy.

[0090] Another aspect of the present invention further relates to a method for detecting a TAA protein or a complex of MHC and a TAA protein (a protein epitope of a TAA) in a (biological) sample, such as one obtained from a subject or patient, comprising the steps of contacting the sample with an antigen recognition construct that specifically binds to said TAA peptide, or with a TAA peptide / MHC complex, and detecting binding between said antigen recognition construct and said TAA peptide, or binding to the TAA peptide / MHC complex. In some embodiments, the antigen recognition construct is a TCR or antibody, or similar construct, or preferably an antigen recognition construct according to the invention described herein. In some embodiments, the (biological) sample is a tumor or cancer sample (such as one described elsewhere herein), e.g., a sample comprising tumor or cancer cells.

[0091] a) isolating cells from said subject; b) transforming the cell with at least one vector encoding an antigen recognition construct of the present invention to produce a transformed cell; c) growing the transformed cells to generate a plurality of transformed cells; d) administering a plurality of transformed cells to said subject; Also provided is a method of treating cancer in a subject in need thereof, comprising:

[0092] a) isolating cells from a healthy donor; b) transforming the cell with a vector encoding the antigen recognition construct of the present invention to produce a transformed cell; c) growing the transformed cells to generate a plurality of transformed cells; d) administering a plurality of transformed cells to said subject; Also provided is a method of treating cancer in a subject in need thereof, comprising:

[0093] a) contacting a biological sample with an antigen recognition construct herein; b) detecting binding of the antigen recognition construct to the biological sample; Also provided is a method for detecting cancer in a biological sample, comprising:

[0094] In some embodiments, the method for detecting cancer is performed ex vivo, in vivo, or in situ.

[0095] Also provided is a method for detecting the presence of a pathology in a mammal, the method comprising the steps of (i) contacting a sample comprising one or more cells from the mammal with a TCR (and functional variants thereof), polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, antibody, or antigen-binding portion thereof, of the invention, or any of the pharmaceutical compositions described herein, thereby forming a complex, and detecting the complex, wherein detection of the complex is indicative of the presence of a pathology in the mammal, the pathology being cancer, such as a TAA-expressing malignancy.

[0096] For the methods of the present invention for detecting a disease state in a mammal, the cell sample can be a sample comprising a whole cell, a lysate thereof, or a fraction of the whole cell lysate, such as, for example, a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction.

[0097] For purposes of the detection methods of the present invention, the contacting can be performed in vitro or in vivo with respect to a mammal. Preferably, the contacting is in vitro.

[0098] In addition, the detection of the complex can be carried out through many methods known in the art.For example, the antigen recognition constructs (and their functional variants), polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or antibodies or TCRs, or antigen-binding portions thereof, as described herein, can be labeled with detectable labels such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0099] For purposes of the methods of the present invention in which a host cell or cell population is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0100] With respect to the aforementioned medical uses of the TCR material of the present invention, the cancers to be treated and / or diagnosed include acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal or rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder or pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoma The cancer may be any cancer, such as ovarian tumor, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oral pharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omentomesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and bladder cancer.Preferred cancers are cancers of the cervix, oral pharynx, anus, anal canal, anorectum, vagina, vulva, or penis.Particularly preferred cancers are TAA-positive cancers, such as cancers that express PRAME, such as ovarian cancer, melanoma, or leukemia.

[0101] Generally, the present invention provides a method for treating a subject suffering from a tumor or tumor disease, comprising administering an antigen recognition construct, a nucleic acid, a vector, a pharmaceutical composition and / or a host cell as disclosed by the present invention.Preferably, the subject is a subject in need of such treatment.In a preferred embodiment, the subject is a mammalian subject, preferably a human patient, suffering from a TAA-positive tumor or tumor disease.

[0102] In view of the disclosure herein, it will be appreciated that the present invention further relates to:

[0103] Item 1: An antigen recognition construct comprising at least one complementarity determining region (CDR) 15 having at least 50% sequence identity to an amino acid sequence selected from SEQ ID NOs: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 129, and 135.

[0104] Item 2: The antigen recognition construct according to Item 1, wherein the antigen recognition construct can specifically and / or selectively bind to a TAA of an antigenic peptide of the present invention.

[0105] Item 3: The antigen recognition construct according to item 1 or 2, wherein the antigen recognition construct is an antibody, or a derivative or fragment thereof, or a T cell receptor (TCR), or a derivative or fragment thereof.

[0106] Item 4: The antigen recognition construct according to any one of Items 1 to 3, wherein the antigen recognition construct binds to a human leukocyte antigen (HLA) that presents a TAA antigen peptide, and the HLA is optionally type A2.

[0107] Item 5: The antigen recognition construct according to any one of Items 1 to 4, wherein the construct specifically and / or selectively binds to an epitope having an amino acid sequence selected from SEQ ID NOs: 97 to 115, preferably SEQ ID NO: 97.

[0108] Item 6: The antigen-recognition construct according to any one of items 1 to 5, wherein the construct is an α / β-TCR or a fragment or derivative thereof, or the construct is a γ / δ-TCR or a fragment or derivative thereof.

[0109] Item 7: An antigen recognition construct according to any one of items 1 to 6, characterized in that the construct is of human origin and specifically and / or selectively recognizes a TAA antigen peptide.

[0110] Item 8: The antigen recognition construct of any one of Items 1 to 7, wherein the antigen recognition construct is capable of inducing an immune response in a subject, and optionally, the immune response is characterized by an increase in interferon (IFN) gamma levels.

[0111] Item 9: The antigen recognition construct of any one of Items 1 to 8, comprising a TCR α or γ chain; and / or a TCR β or δ chain, wherein the TCR α or γ chain comprises a CDR3 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 3, 15, 27, 39, 51, 63, 75, and 129, and / or the TCR β or δ chain comprises a CDR3 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 9, 21, 33, 45, 57, 69, 81, and 135.

[0112] Item 10: The antigen recognition construct of Item 9, wherein the TCR alpha or gamma chain further comprises a CDR1 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 13, 25, 37, 49, 61, 73, and 127; and / or a CDR2 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 2, 14, 26, 38, 50, 62, 74, 128, 196, 197, 198, 199, 200, 201, 202, and 204.

[0113] Item 11: The antigen recognition construct of Item 9 or 10, wherein the TCR β or δ chain further comprises a CDR1 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 7, 19, 31, 43, 55, 67, and 79; and / or a CDR2 having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 8, 20, 32, 44, 56, 68, and 80, and 134.

[0114] Item 12: The antigen recognition construct of any one of Items 1 to 11, comprising a TCR variable chain region having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 130, and 136.

[0115] Item 13: The antigen recognition construct according to any one of items 1 to 12, wherein the construct is humanized, chimeric, and / or murine.

[0116] Item 14: A binding fragment of a TCR, wherein the binding fragment is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 196; or 7, 8, 9; or 13, 14, 15, 197; or 19, 20, 21; or 25, 26, 27, 198; or 31, 32, 33; or 37, 38, 39, 199; or 43, 44, 45; or 49, 50, 51, 200; or 55, 56, 57; or 14. The antigen recognition construct according to any one of items 1 to 13, comprising CDR1 to CDR3 optionally selected from CDR1 to CDR3 sequences having the amino acid sequences of 61, 62, 63, 201; or 67, 68, 69; or 73, 74, 75, 202; or 79, 80, 81; or 127, 128, 129, 204; or 133, 134, 135.

[0117] Item 15: The construct is a TCR or a fragment thereof composed of at least one TCR α chain and one TCR β chain sequence, wherein the TCR α chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 1 to 3 and 196, and the TCR β chain sequence comprises CDR7 to CDR9 sequences having the amino acid sequences of SEQ ID NOs: 7 to 9, or the TCR α chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 13 to 15 and 197, and the TCR β chain sequence comprises CDR7 to CDR9 sequences having the amino acid sequences of SEQ ID NOs: 19 or the TCR α chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 25 to 27 and 198, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 31 to 33; or the TCR α chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 37 to 39 and 199, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 43 to 45. or the TCR α chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 49 to 51 and 200, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 55 to 57; or the TCR α chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 61 to 63 and 201, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 67 to 69; or the TCR α chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 15. The antigen recognition construct according to any one of items 1 to 14, wherein the TCR α chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 73 to 75 and 202, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 79 to 81; or the TCR α chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 127 to 129 and 204, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 133 to 135.

[0118] Item 16: The construct is a TCR or a fragment thereof consisting of at least one TCR alpha chain and one TCR beta chain sequence, wherein the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 4, and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 10; or the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 16, and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 22; or the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 28, and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 34; or the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 40, and the TCR beta chain sequence may have the amino acid sequence of SEQ ID NO: 46. 16. The antigen recognition construct according to any one of items 1 to 15, wherein the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 52 and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 58; or the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 64 and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 70; or the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 76 and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 82; or the TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 130 and the TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 136.

[0119] Item 17: The construct is a TCR or fragment thereof further comprising a TCR constant region having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 131, and 137, preferably wherein the TCR is composed of at least one TCR alpha and one TCR beta chain sequence, and the TCR alpha chain sequence is selected from SEQ ID NOs: 5, 17, 29, 41, 53, 65, 71, 77, 83, 131, and 137. 17. The antigen recognition construct of any one of items 1 to 16, wherein the TCR beta chain sequence comprises a constant region having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 11, 23, 35, 47, 59, 71, 83, and 137.

[0120] Item 18: The antigen recognition construct of any one of Items 1 to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6 and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12.

[0121] Item 19: The antigen recognition construct of any one of Items 1 to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0122] Item 20a: The antigen recognition construct of any one of Items 1 to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 30, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 36.

[0123] Item 20b: The antigen recognition construct of any one of items 1 to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 42, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 48.

[0124] Item 20c: The antigen recognition construct of any one of Items 1 to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 54, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 60.

[0125] Item 20d: The antigen recognition construct of any one of items 1 to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 72.

[0126] Item 20e: The antigen recognition construct of any one of items 1 to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 78, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84.

[0127] Item 20f: The antigen recognition construct of any one of items 1 to 17, comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 132, and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 138.

[0128] Item 21: A nucleic acid encoding the antigen-recognition construct according to any one of Items 1 to 20f.

[0129] Item 22: A vector comprising the nucleic acid according to Item 21.

[0130] Item 23: A host cell comprising the antigen recognition construct according to any one of Items 1 to 20, or the nucleic acid according to Item 21, or the vector according to Item 22.

[0131] Item 24: The host cell according to Item 23, wherein the host cell is a lymphocyte, preferably a T lymphocyte or a T lymphocyte precursor, more preferably a CD4 or CD8 positive T cell.

[0132] Item 25: A pharmaceutical composition comprising the antigen recognition construct according to any one of Items 1 to 20f, or the nucleic acid according to Item 21, or the vector according to Item 22, or the host cell according to Item 23 or 24, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0133] Item 26: The antigen recognition construct according to any one of Items 1 to 20f, or the nucleic acid according to Item 21, or the vector according to Item 22, or the host cell according to Item 23 or 24, or the pharmaceutical composition according to Item 25, for use in medicine.

[0134] Item 27: An antigen recognition construct, or a nucleic acid, or a vector, or a host cell, or a pharmaceutical composition for use according to Item 26, for use in the diagnosis, prevention and / or treatment of a proliferative disease comprising a malignant or benign tumor disease.

[0135] Item 28: The antigen recognition construct, or nucleic acid, or vector, or host cell, or pharmaceutical composition for use according to Item 27, wherein the tumor disease is characterized by expression of a TAA in tumor cells of the tumor disease.

[0136] Item 29: The antigen recognition construct, or nucleic acid, or vector, or host cell, or pharmaceutical composition for use according to any one of Items 26 to 28, wherein the use in medicine is use in immunotherapy, optionally comprising adoptive cell transfer, and the immunotherapy comprises adoptive autologous or xenogeneic T cell therapy.

[0137] Item 30: a. providing a suitable host cell; b. Providing a genetic construct comprising a coding sequence encoding the antigen recognition construct according to any one of items 1 to 20f; c. introducing said genetic construct into said suitable host cell; d. expressing said genetic construct by said suitable host cell.

[0138] Item 31: The method of Item 30, further comprising cell surface display of the antigen recognition construct.

[0139] Item 32: The method of Item 30 or 31, wherein the genetic construct is an expression construct comprising a promoter sequence operably linked to the coding sequence.

[0140] Item 33: The method according to any one of Items 30 to 32, wherein the antigen recognition construct is of mammalian origin, preferably human origin.

[0141] Item 34: The method of any one of Items 30 to 33, wherein the suitable host cell is a mammalian cell, optionally selected from a human cell or a human T lymphocyte.

[0142] Item 35: The method of any one of Items 30 to 34, wherein the antigen recognition construct is a modified TCR, and the modification comprises the addition of a functional domain comprising a label, or the addition of an alternative domain comprising a membrane anchor domain.

[0143] Item 36: The method of item 35, wherein the antigen recognition construct is an α / β TCR, a γ / δ TCR, or a single-chain TCR (scTCR).

[0144] Item 37: The method of any one of items 30 to 36, wherein the gene construct is introduced into the suitable host cell by retroviral transfection.

[0145] Item 38: The method of any one of items 30 to 37, further comprising isolating and purifying the antigen recognition construct from a suitable host cell and, optionally, reconstituting the antigen recognition construct in a T cell. [Brief explanation of the drawings]

[0146] The present invention will be further described in the following examples with reference to the accompanying figures and sequences, without however being limited thereto. For the purposes of the present invention, all references cited herein are incorporated by reference in their entirety. The figures and sequences show: [Figure 1] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R11P3D3 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 97), various PRAME-004 alanine or threonine substitution mutants at positions 1-9 (X1-X9) of SEQ ID NO: 97 (SEQ ID NOs: 98-115), or the control peptide NYESO1-001 (SEQ ID NO: 126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubated with unloaded target cells served as controls. Several different donors were analyzed for the alanine substitution mutants (Ala_TCRA-0017 and Ala_IFN-041) and the threonine substitution mutant (Thr_TCRA-0036). [Figure 2] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R16P1C10 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 97), various PRAME-004 alanine or threonine substitution mutants at positions 1-9 (X1-X9) of SEQ ID NO: 97 (SEQ ID NOs: 98-115), or the control peptide NYESO1-001 (SEQ ID NO: 126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubated with unloaded target cells served as controls. Several different donors were analyzed for the alanine substitution mutants (Ala_TCRA-0017 and Ala_IFN-041) and the threonine substitution mutant (Thr_TCRA-0036). [Figure 3]IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R16P1E8 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 97), various PRAME-004 alanine or threonine substitution mutants at positions 1-9 (X1-X9) of SEQ ID NO: 97 (SEQ ID NOs: 98-115), or the control peptide NYESO1-001 (SEQ ID NO: 126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubated with unloaded target cells served as controls. Several different donors were analyzed for the alanine substitution mutants (Ala_TCRA-0017 and Ala_IFN-041) and the threonine substitution mutant (Thr_TCRA-0036). [Figure 4] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R17P1A9 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 97), various PRAME-004 alanine substitution variants at positions 1-9 (X1-X9) of SEQ ID NO: 97 (SEQ ID NOs: 98-106), or the control peptide NYESO1-001 (SEQ ID NO: 126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubation with unloaded target cells served as controls. Different donors were analyzed for the alanine substitution variants (Ala_IFN-040 and Ala_IFN-041). [Figure 5]IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R17P1D7 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 97), various PRAME-004 alanine or threonine substitution mutants at positions 1-9 (X1-X9) of SEQ ID NO: 97 (SEQ ID NOs: 98-115), or the control peptide NYESO1-001 (SEQ ID NO: 126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubation with unloaded target cells served as controls. Different donors were analyzed for the alanine substitution mutants (Ala_TCRA-0017 and Ala_IFN-041) and the threonine substitution mutant (Thr_TCRA-0036). [Figure 6] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R17P1G3 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 97), various PRAME-004 alanine or threonine substitution mutants at positions 1-9 (X1-X9) of SEQ ID NO: 97 (SEQ ID NOs: 98-115), or the control peptide NYESO1-001 (SEQ ID NO: 126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubation with unloaded target cells served as controls. Different donors were analyzed for the alanine substitution mutants (Ala_TCRA-0017 and Ala_IFN-041) and the threonine substitution mutant (Thr_TCRA-0036). [Figure 7]IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R17P2B6 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 97), various PRAME-004 alanine or threonine substitution mutants at positions 1-9 (X1-X9) of SEQ ID NO: 97 (SEQ ID NOs: 98-115), or the control peptide NYESO1-001 (SEQ ID NO: 126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubation with unloaded target cells served as controls. Different donors were analyzed for the alanine substitution mutants (Ala_TCRA-0017 and Ala_IFN-041) and the threonine substitution mutant (Thr_TCRA-0036). [Figure 8] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R11P3D3 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar but unrelated peptides TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117), DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQ ID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122), ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQ ID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubated with unloaded target cells served as controls. Different donors were analyzed for IFN-040 and IFN-041. [Figure 9]IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R16P1C10 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar but unrelated peptides TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117), DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQ ID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122), ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQ ID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubated with unloaded target cells served as controls. Different donors were analyzed for IFN-046 and IFN-041. [Figure 10] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R16P1E8 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar but unrelated peptides TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117), DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQ ID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122), ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQ ID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubated with unloaded target cells served as controls. Different donors were analyzed for IFN-040 and IFN-041. [Figure 11] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R17P1A9 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar but unrelated peptides TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117), DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQ ID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122), ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQ ID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubated with unloaded target cells served as controls. Different donors were analyzed for IFN-040 and IFN-041. [Figure 12] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R17P1D7 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar but unrelated peptides TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117), DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQ ID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122), ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQ ID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubated with unloaded target cells served as controls. Different donors were analyzed for IFN-040 and IFN-041. [Figure 13] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R17P1G3 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar but unrelated peptides TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117), DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQ ID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122), ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQ ID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubated with unloaded target cells served as controls. Different donors were analyzed for IFN-046 and IFN-041. [Figure 14]IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R17P2B6 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or similar but unrelated peptides TMED9-001 (SEQ ID NO:116), CAT-001 (SEQ ID NO:117), DDX60L-001 (SEQ ID NO:118), LRRC70-001 (SEQ ID NO:119), PTPLB-001 (SEQ ID NO:120), HDAC5-001 (SEQ ID NO:121), VPS13B-002 (SEQ ID NO:122), ZNF318-001 (SEQ ID NO:123), CCDC51-001 (SEQ ID NO:124) or IFIT1-001 (SEQ ID NO:125) or control peptide NYESO1-001 (SEQ ID NO:126). IFNγ release data were obtained using CD8+ T cells from two different healthy donors. RNA-electroporated CD8+ T cells alone or co-incubated with unloaded target cells served as controls. Different donors were analyzed for IFN-040 and IFN-041. [Figure 15] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R11P3D3 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 97) at various peptide loading concentrations from 10 μM to 10 pM. IFNγ release data were obtained using CD8+ T cells from two different healthy donors. Different donors were analyzed for TCRA-0003 and TCRA-0017. [Figure 16] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R16P1C10 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 97) at various peptide loading concentrations from 10 μM to 10 pM. IFNγ release data were obtained using CD8+ T cells from two different healthy donors. Different donors were analyzed for TCRA-0003 and TCRA-0017. [Figure 17]IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R16P1E8 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 97) at various peptide loading concentrations from 10 μM to 10 pM. IFNγ release data were obtained using CD8+ T cells from two different healthy donors. Different donors were analyzed for TCRA-0003 and TCRA-0017. [Figure 18] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R17P1D7 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 97) at various peptide loading concentrations from 10 μM to 10 pM. IFNγ release data were obtained using CD8+ T cells from two different healthy donors. Different donors were analyzed for TCRA-0003 and TCRA-0017. [Figure 19] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R17P1G3 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 97) at various peptide loading concentrations from 10 μM to 10 pM. IFNγ release data were obtained using CD8+ T cells from two different healthy donors. Different donors were analyzed for TCRA-0003 and TCRA-0017. [Figure 20] IFNγ release from CD8+ T cells electroporated with α and β chain RNA of TCR R17P2B6 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 97) at various peptide loading concentrations ranging from 10 μM to 10 pM. IFNγ release data were obtained using CD8+ T cells from two different healthy donors. Different donors were analyzed for TCRA-0003 and TCRA-0017. [Figure 21]HLA-A*02 / PRAME-004 tetramer or HLA-A*02 / NYESO1-001 tetramer staining of CD8+ T cells electroporated with α- and β-chain RNA of TCR R16P1C10 (Table 1), respectively. CD8+ T cells electroporated with RNA of 1G4 TCR (SEQ ID NOs: 85-96) that specifically binds to the HLA-A*02 / NYESO1-001 complex; mock-electroporated CD8+ T cells served as controls. [Figure 22] 100 nM of PRAME-004 peptide (SEQ ID NO: 97) or similar (identical to PRAME-004 at positions 3, 5, 6, and 7) but unrelated peptides ACPL-001 (SEQ ID NO: 139), HSPB3-001 (SEQ ID NO: 140), UNC7-001 (SEQ ID NO: 141), SCYL2-001 (SEQ ID NO: 142), RPS2P8-001 (SEQ ID NO: 143), PCNXL3-003 ( IFNγ release from CD8+ T cells (D103805 and D191451) lentivirally transduced with TCR R11P3D3 (Table 1), or non-transduced cells (D103805NT and D191451NT), after co-incubation with T2 target cells loaded with SEQ ID NO: 144), AQP6-001 (SEQ ID NO: 145), PCNX-001 (SEQ ID NO: 146), AQP6-002 (SEQ ID NO: 147), TRGV10-001 (SEQ ID NO: 148), NECAP1-001 (SEQ ID NO: 149), or FBXW2-001 (SEQ ID NO: 150), or control peptide NYESO1-001 (SEQ ID NO: 126). IFNγ release data were obtained using CD8+ T cells, D103805 and D191451, derived from two different healthy donors. [Figure 23]IFNγ release from CD8+ T cells lentivirally transduced with TCR R11P3D3 (Table 1) after co-incubation with T2 target cells loaded with 100 nM PRAME-004 peptide (SEQ ID NO: 97) or a similar (identical to PRAME-004 in positions 3, 5, 6, and 7) but unrelated peptide (SEQ ID NOs: 151-195) or the control peptide NYESO1-001 (SEQ ID NO: 126). IFNγ release data were obtained using CD8+ T cells derived from two different healthy donors, TCRA-0087 and TCRA-0088. [Figure 24] IFNγ release from CD8+ T cells (D103805 and D191451) lentivirally transduced with TCR R11P3D3 (Table 1) or non-transduced cells (D8NT and D103805NT) after co-incubation with different primary cells (HCASMC (coronary artery smooth muscle cells), HTSMC (tracheal smooth muscle cells), HRCEpC (renal cortical epithelial cells), HCM (cardiomyocytes), HCMEC (cardiac microvascular endothelial cells), HSAEpC (peripheral airway epithelial cells), and HCF (cardiac fibroblasts)) and iPSC-derived cell types (HN (neurons), iHCM (cardiomyocytes), HH (hepatocytes), and HA (stellate cells)). The tumor cell lines UACC-257 (high PRAME-004), Hs695T (moderate PRAME-004), U266B1 (very low PRAME-004), and MCF-7 (absent PRAME-004) express different amounts of PRAME-004 per cell. T cells alone served as a control. IFNγ release data were obtained using CD8+ T cells, D103805 and D191451, derived from two different healthy donors. [Figure 25]TCR after co-incubation with primary cells (NHEK (epithelial keratinocytes), HBEpC (bronchial epithelial cells), HDMEC (dermal microvascular endothelial cells), HCAEC (coronary artery endothelial cells), HAoEC (aortic endothelial cells), HPASMC (pulmonary artery smooth muscle cells), HAoSMC (aortic smooth muscle cells), HPF (pulmonary fibroblasts), SkMC (skeletal muscle cells), HOB (osteoblasts), HCH (chondrocytes), HWP (white preadipocytes), hMSC-BM (mesenchymal stem cells), and NHDF (dermal fibroblasts). γ release from CD8+ cells lentivirally transduced with R11P3D3 (Table 1). The tumor cell lines UACC-257 (high PRAME-004), Hs695T (moderate PRAME-004), U266B1 (very low PRAME-004), and MCF-7 (absent PRAME-004) display different copy numbers of PRAME-004 per cell. T cells alone served as controls. IFNγ release data were obtained using CD8+ T cells, TCRA-0084 and TCRA-0085, derived from two different healthy donors. [Figure 26]100 nM of PRAME-004 peptide (SEQ ID NO: 97) or similar (identical to PRAME-004 at positions 3, 5, 6, and 7) but unrelated peptides ACPL-001 (SEQ ID NO: 139), HSPB3-001 (SEQ ID NO: 140), UNC7-001 (SEQ ID NO: 141), SCYL2-001 (SEQ ID NO: 142), RPS2P8-001 (SEQ ID NO: 143), PCNXL3-003 (SEQ ID NO: 144), IFNγ release from CD8+ T cells (D103805 and D191451) lentivirally transduced with the enhanced TCR R11P3D3_KE (Table 1), or non-transduced cells (D103805NT and D191451NT), following co-incubation with T2 target cells loaded with TCR R11P3D3_KE (Table 1), TCR R11P3D3_KE (Table 1), TCR R11P3D3_KE (Table 1), or TCR R11P3D3_KE (Table 1), or non-transduced cells (D103805NT and D191451NT) after co-incubation with T2 target cells loaded with TCR R11P3D3_KE (Table 1), TCR R11P3D3_KE (Table 1), or TCR R11P3D3_KE (Table 1), or non-transduced cells (D103805NT and D191451NT). IFNγ release data were obtained using CD8+ T cells, D103805 and D191451, derived from two different healthy donors. [Figure 27] IFNγ release from CD8+ T cells lentivirally transduced with the enhanced TCR R11P3D3_KE (Table 1) after co-incubation with T2 target cells loaded with 100 nM PRAME-004 peptide (SEQ ID NO: 97) or a similar (identical to PRAME-004 in positions 3, 5, 6, and 7) but unrelated peptide (SEQ ID NOs: 151-195) or the control peptide NYESO1-001 (SEQ ID NO: 126). IFNγ release data were obtained using CD8+ T cells derived from two different healthy donors, TCRA-0087 and TCRA-0088. [Figure 28]IFNγ release from CD8+ T cells (D103805 and D191451) lentivirally transduced with the enhanced TCR R11P3D3_KE (Table 1) or non-transduced cells (D191451NT and D103805NT) after co-incubation with different primary cells (HCASMC (coronary artery smooth muscle cells), HTSMC (tracheal smooth muscle cells), HRCEpC (renal cortical epithelial cells), HCM (cardiomyocytes), HCMEC (cardiac microvascular endothelial cells), HSAEpC (peripheral airway epithelial cells), and HCF (cardiac fibroblasts)) and iPSC-derived cell types (HN (neurons), iHCM (cardiomyocytes), HH (hepatocytes), and HA (stellate cells)). The tumor cell lines UACC-257 (high PRAME-004), Hs695T (moderate PRAME-004), U266B1 (very low PRAME-004), and MCF-7 (absent PRAME-004) express different amounts of PRAME-004 per cell. T cells alone served as a control. IFNγ release data were obtained using CD8+ T cells, D103805 and D191451, derived from two different healthy donors. [Figure 29] Enhanced TCR after co-incubation with primary cells (NHEK (epithelial keratinocytes), HBEpC (bronchial epithelial cells), HDMEC (dermal microvascular endothelial cells), HCAEC (coronary artery endothelial cells), HAoEC (aortic endothelial cells), HPASMC (pulmonary artery smooth muscle cells), HAoSMC (aortic smooth muscle cells), HPF (pulmonary fibroblasts), SkMC (skeletal muscle cells), HOB (osteoblasts), HCH (chondrocytes), HWP (white preadipocytes), hMSC-BM (mesenchymal stem cells), NHDF (dermal fibroblasts) IFNγ release from CD8+ cells lentivirally transduced with R11P3D3_KE (Table 1). The tumor cell lines UACC-257 (high PRAME-004), Hs695T (moderate PRAME-004), U266B1 (very low PRAME-004), and MCF-7 (absent PRAME-004) display different copy numbers of PRAME-004 per cell. T cells alone served as a control. IFNγ release data were obtained using CD8+ T cells, TCRA-0084 and TCRA-0085, derived from two different healthy donors. [Figure 30] IFNγ release from TCR R11P3D3 or enhanced TCR R11P3D3_KE (Table 1) lentivirally transduced CD8+ T cells, or non-transduced cells, after co-incubation with UACC-257 (high PRAME-004), Hs695T (moderate PRAME-004), U266B1 (very low PRAME-004), and MCF-7 (absent PRAME-004) tumor cell lines, which present different amounts of PRAME-004 per cell. T cells alone served as controls. IFNγ release from both TCRs correlated with PRAME-004 presentation, with R11P3D3_KE inducing higher responses compared to R11P3D3. [Figure 31] IFNγ release from CD8+ T cells lentivirally transduced with enhanced TCR R11P3D3_KE (Table 1) cells after co-incubation with T2 target cells loaded with various PRAME-004 alanine substitution mutants (SEQ ID NOS: 98-106) at positions 1-9 (A1-A9) of SEQ ID NOS: 97. IFNγ release data were obtained using CD8+ T cells derived from three different healthy donors. [Figure 32] Potency assay evaluating the cytolytic activity of lentivirally transduced T cells expressing TCR R11P3D3 or enhanced TCR 11P3D3_KE against PRAME-004 tumor cells. Cytotoxic responses of R11P3D3 and R11P3D3_KE transduced, and non-transduced (NT) T cells were measured against A-375 (PRAME-004 low) or U2OS (PRAME-004 moderate) tumor cells. Assays were performed in a 72-hour fluorescence microscopy-based cytotoxicity assay. Results are presented as fold tumor growth over time.

[0147] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

[0148] [Table 2-1] [Table 2-2] [Table 2-3] [Example]

[0149] Seven PRAME-specific TCRs against the PRAME-004 peptides disclosed herein (R11P3D3, R16P1C10, R16P1E8, R17P1A9, R17P1D7, R17P1G3, and R17P2B6, see Table 1), each encoding a tumor-specific TCR-α chain and TCR-β chain, were isolated and amplified from T cells of a healthy donor. Cells from the healthy donor were stimulated in vitro according to a previously described method (Walter et al., 2003 J Immunol., Nov 15;171(10):4974-8) and transfected with HLA-A. * Target-specific cells were single-cell sorted using 02 multimers and then used for subsequent TCR isolation. TCR sequences were isolated through 5' RACE using standard methods, as described, for example, in *Molecular Cloning a Laboratory Manual, Fourth Edition* by Green and Sambrook. The TCR α and β variable regions of R11P3D3, R16P1C10, R16P1E8, R17P1A9, R17P1D7, R17P1G3, and R17P2B6 were sequenced and cloned for further functional characterization.

[0150] R11P3D3, R16P1C10, R17P1D7, and R17P2B6 are HLA-A * Derived from an HLA-A negative donor (in an alloreactive situation), R16P1E8, R17P1A9, and R17P1G3 are HLA-A * Derived from a 02 positive donor.

[0151] Further disclosed herein is a mutant TCR R11P3D3_KE, which is an enhanced mutant of R11P3D3. The enhanced TCR mutant R11P3D3_KE was modified from the parent TCR, and the coding sequence was obtained by gene synthesis prior to functional characterization of the TCR, as described in PCT / EP2017 / 081745, which is specifically incorporated herein by reference, and in Example 8 below.

[0152] Example 1: T cell receptor R11P3D3 TCR R11P3D3 (SEQ ID NOs: 1-12 and 196) binds to HLA-A * 02 is constrained against presentation PRAME-004 (SEQ ID NO: 97) (see Figure 8).

[0153] R11P3D3 specifically recognizes PRAME-004, as observed when primary human CD8+ T cells were loaded with either the PRAME-004 peptide or alanine or threonine-substituted mutants of PRAME-004 (Fig. 1), or different peptides showing high sequence similarity to PRAME-004 (Fig. 8). * This TCR releases IFNγ again upon co-incubation with HLA-A 02+ target cells. The NYESO1-001 peptide is used as a negative control. The TCR R11P3D3 binds to HLA-A 02+ target cells. * 02 exhibited an EC50 of 0.74 nM (Figure 15) and a binding affinity (K D )

[0154] Re-expression of R11P3D3 in human primary CD8+ T cells is associated with HLA-A * TCR R11P3D3 did not respond to any of the 25 healthy, primary, or iPSC-derived cell types tested (Figures 24 and 25), resulting in selective recognition and killing of 02 / PRAME-004-presenting tumor cell lines (Figures 24, 25, 30, and 32). * In the context of 02, an additional 67 similar peptides (57 of which are identical to PRAME-004 at positions 3, 5, 6, and 7) were tested for cross-reactivity against unrelated peptides (Figures 8, 22, and 23).

[0155] Example 2: T cell receptor R16P1C10 TCR R16P1C10 (SEQ ID NOs: 13-24 and 197) binds to HLA-A * 02 is constrained against presentation PRAME-004 (SEQ ID NO: 97) (see Figure 9).

[0156] R16P1C10 specifically recognizes PRAME-004, as determined by HLA-A receptor agonists loaded with either the PRAME-21 peptide or alanine or threonine substitution mutants of PRAME-1 (Figure 2) or a different peptide showing high sequence similarity to PRAME-10 (Figure 9). * Upon co-incubation with O2+ target cells, this TCR releases IFNγ again, leading to HLA-A * 02 tetramer (Figure 21). The NYESO1-001 peptide is used as a negative control. TCR R16P1C10 has an EC50 of 9.6 nM (Figure 16).

[0157] Example 3: T cell receptor R16P1E8 TCR R16P1E8 (SEQ ID NOs: 25-36 and 198) binds to HLA-A * 02 is constrained against presentation PRAME-004 (SEQ ID NO: 97) (see Figure 10).

[0158] R16P1E8 specifically recognizes PRAME-004, as determined by HLA-A receptor agonists (HLA-A) expressing primary human CD8+ T cells loaded with either the PRAME-004 peptide or alanine or threonine substitution mutants of PRAME-004 (Figure 3) or different peptides showing high sequence similarity to PRAME-004 (Figure 10). * Upon co-incubation with 02+ target cells, this TCR releases IFNγ again. The NYESO1-001 peptide is used as a negative control. TCRR16P1E8 has an EC50 of approximately 1 μM (Figure 17).

[0159] Example 4: T cell receptor R17P1A9 TCR R17P1A9 (SEQ ID NOs: 37-48 and 199) binds to HLA-A * 02 is constrained against presentation PRAME-004 (SEQ ID NO: 97) (see Figure 11).

[0160] R17P1A9 specifically recognizes PRAME-004, as observed when primary human CD8+ T cells were loaded with either the PRAME-004 peptide or an alanine-substituted mutant of PRAME-004 (Figure 4) or a different peptide showing high sequence similarity to PRAME-004 (Figure 11) and expressed on HLA-A receptors. * Upon co-incubation with 02+ target cells, this TCR releases IFNγ again. The NYESO1-001 peptide is used as a negative control.

[0161] Example 5: T cell receptor R17P1D7 TCR R17P1D7 (SEQ ID NOs: 49-60 and 200) binds to HLA-A * 02 is constrained against presentation PRAME-004 (SEQ ID NO: 97) (see Figure 12).

[0162] R17P1D7 specifically recognizes PRAME-004, as observed when primary human CD8+ T cells were loaded with either the PRAME-004 peptide or alanine or threonine-substituted mutants of PRAME-004 (Figure 5), or different peptides showing high sequence similarity to PRAME-004 (Figure 12), expressing HLA-A * This TCR releases IFNγ again upon co-incubation with 02+ target cells. The NYESO1-001 peptide is used as a negative control. The TCR R17P1D7 has an EC50 of 1.83 nM (Figure 18).

[0163] Example 6: T cell receptor R17P1G3 TCR R17P1G3 (SEQ ID NOs: 61-72 and 201) binds to HLA-A * 02 is constrained against presentation PRAME-004 (SEQ ID NO: 97) (see Figure 13).

[0164] R17P1G3 specifically recognizes PRAME-004, as observed when primary human CD8+ T cells were loaded with either the PRAME-004 peptide or alanine or threonine-substituted mutants of PRAME-004 (Figure 6), or different peptides showing high sequence similarity to PRAME-004 (Figure 13), expressing HLA-A * This TCR releases IFNγ again upon co-incubation with 02+ target cells. The NYESO1-001 peptide is used as a negative control. The TCR R17P1G3 has an EC50 of 8.63 nM (Figure 19).

[0165] Example 7: T cell receptor R17P2B6 TCR R17P2B6 (SEQ ID NOs: 73-84 and 202) binds to HLA-A * 02 is constrained against presentation PRAME-004 (SEQ ID NO: 97) (see Figure 14).

[0166] R17P2B6 specifically recognizes PRAME-004, as observed when primary human CD8+ T cells were loaded with either the PRAME-004 peptide or alanine or threonine-substituted mutants of PRAME-004 (Figure 7), or different peptides showing high sequence similarity to PRAME-004 (Figure 14). * This TCR releases IFNγ again upon co-incubation with HLA-A 02+ target cells. The NYESO1-001 peptide is used as a negative control. TCR R17P2B6 has an EC50 of 2.11 nM (Figure 20) and binds to HLA-A 02+ target cells. * 02 exhibited a binding affinity (K ) of 13 μM for PRAME-004 D )

[0167] Example 8: Enhanced T cell receptor R11P3D3_KE The mutant "enhanced pairing" TCR R11P3D3_KE was introduced as a variant of R11P3D3 in which the α and β variable domains, naturally harboring αW44 / βQ44, were mutated to αK44 / βE44. The double mutation was selected from the list provided in PCT / EP2017 / 081745, specifically incorporated herein by reference. It was specifically designed to restore optimal interaction and shape complementarity to the TCR scaffold.

[0168] Compared to the parental TCR R11P3D3, the enhanced TCR R11P3D3_KE shows superior sensitivity in PRAME-004 recognition. The response against PRAME-004-presenting tumor cell lines is stronger with the enhanced TCR R11P3D3_KE compared to the parental TCR R11P3D3 (Figure 30). Furthermore, the cytolytic activity of R11P3D3_KE is stronger compared to R11P3D3 (Figure 32). The observed improved functional response of the enhanced TCR R11P3D3_KE is consistent with that of Example 1 (R11P3D3, K D = 18 to 26 μM) and Example 8 (R11P3D3_KE, K D = 5.3 μM), which is in good agreement with the increased binding affinity for PRAME-004.

Claims

1. (i) a TCR alpha variable domain comprising a complementarity determining region (CDR) 1 set forth in SEQ ID NO: 1, a CDR2 set forth in SEQ ID NO: 196 or SEQ ID NO: 2, and a CDR3 set forth in SEQ ID NO: 3; and (ii) a TCR β variable domain comprising a CDR1 set forth in SEQ ID NO: 7, a CDR2 set forth in SEQ ID NO: 8, and a CDR3 set forth in SEQ ID NO:

9. An antigen recognition construct comprising: The antigen-recognition construct is a T cell receptor (TCR) or an antigen-recognition fragment thereof, and the TCR or the antigen-recognition fragment thereof is capable of specifically binding to a melanoma preferentially expressed antigen (PRAME) antigenic peptide shown in SEQ ID NO:

97.

2. (i) the TCR alpha variable domain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 130; and (ii) the TCR β variable domain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 136; The antigen recognition construct according to claim 1.

3. (i) the TCR alpha variable domain comprises the amino acid sequence of the TCR alpha variable domain contained in SEQ ID NO: 130; and (ii) the TCR β variable domain comprises the amino acid sequence of a TCR β variable domain contained in SEQ ID NO: 136; The antigen recognition construct according to claim 1 or 2.

4. the antigen-recognizing construct is a TCR comprising a TCR α chain and a TCR β chain; (i) the TCR α chain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 132; and (ii) the TCR β chain comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 138; The antigen recognition construct according to any one of claims 1 to 3.

5. the antigen-recognizing construct is a TCR comprising a TCR α chain and a TCR β chain; (i) the TCR alpha chain comprises the amino acid sequence of the TCR alpha chain contained in SEQ ID NO: 132; and (ii) the TCR β chain comprises the amino acid sequence of the TCR β chain contained in SEQ ID NO: 138; The antigen recognition construct according to any one of claims 1 to 4.

6. (i) an antigen recognition construct according to any one of claims 1 to 5; (ii) the amino acid sequence set forth in SEQ ID NO: 130 and the amino acid sequence set forth in SEQ ID NO: 136; and / or (iii) the amino acid sequence shown in SEQ ID NO: 132 and the amino acid sequence shown in SEQ ID NO: 138 A nucleic acid or nucleic acids encoding

7. A vector or vectors comprising the nucleic acid or nucleic acids of claim 6.

8. (i) comprising an antigen recognition construct according to any one of claims 1 to 5, a nucleic acid or nucleic acids according to claim 6, or a vector or vectors according to claim 7; and / or (ii) A host cell expressing the antigen recognition construct according to any one of claims 1 to 5.

9. a. providing a suitable host cell; b. Providing a nucleic acid or nucleic acids according to claim 6 or a vector or vectors according to claim 7; c. introducing said nucleic acid(s) or vector(s) into said suitable host cell; d. expressing said nucleic acid or nucleic acids or said vector or vectors by said host cell; A method for producing a cell or cell line expressing a PRAME-specific antigen recognition construct, comprising:

10. 10. The method of claim 9, wherein step (c) is performed by retroviral transduction, preferably lentiviral transduction.

11. 11. The method of claim 9 or claim 10, wherein the host cells are T cells, preferably CD4-positive T cells and / or CD8-positive T cells.

12. A host cell obtained or obtainable by the method of any one of claims 9 to 11.

13. 13. The host cell of claim 8 or claim 12, wherein the host cell is a T cell, preferably a CD4-positive T cell and / or a CD8-positive T cell, more preferably a human CD4-positive T cell and / or a human CD8-positive T cell.

14. A T cell population comprising a plurality of T cells according to claim 12 or claim 13.

15. 15. A pharmaceutical composition comprising the antigen recognition construct of any one of claims 1 to 5, the nucleic acid or nucleic acids of claim 6, the vector or vectors of claim 7, the host cell of claim 8 or 12, the T cell of claim 13, or the T cell population of claim 14, and further comprising a pharmaceutically acceptable carrier, stabilizer and / or excipient.

16. An antigen recognition construct according to any one of claims 1 to 5, a nucleic acid or nucleic acids according to claim 6, a vector or vectors according to claim 7, a host cell according to claim 8 or 12, a T cell according to claim 13, a T cell population according to claim 14, or a pharmaceutical composition according to claim 15, for use as a medicament.

17. 16. An antigen recognition construct according to any one of claims 1 to 5, a nucleic acid or nucleic acids according to claim 6, a vector or vectors according to claim 7, a host cell according to claim 8 or 12, a T cell according to claim 13, a T cell population according to claim 14, or a pharmaceutical composition according to claim 15, for use in treating cancer in a patient.

18. Cancers include acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal or rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder or pleural cancer, nasal cancer, nasal cavity cancer, middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, 18. The antigen recognition construct, nucleic acid or nucleic acids, vector or vectors, host cell, T cell, T cell population, or pharmaceutical composition of claim 17, wherein the antigen recognition construct, nucleic acid or nucleic acids, vector or vectors, host cell, T cell, T cell population, or pharmaceutical composition is selected from melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omentomesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and bladder cancer.

19. 19. The T cell or T cell population of any one of claims 16 to 18, or a pharmaceutical composition comprising said T cell or T cell population, wherein the T cells are autologous or allogeneic to the patient being treated.

20. 1. A plurality of T cells for use in a method of treating cancer in a patient, the method comprising administering to the patient the plurality of T cells, the plurality of T cells comprising: (i) transducing one or more T cells obtained from a patient or a healthy donor with a nucleic acid or nucleic acids according to claim 6 or a vector or vectors according to claim 7, thereby obtaining one or more transduced T cells; and (ii) expanding said one or more transduced T cells to obtain said plurality of transduced T cells. A T cell obtained by a method comprising:

21. Use of an antigen recognition construct according to any one of claims 1 to 5, a nucleic acid or nucleic acids according to claim 6, a vector or vectors according to claim 7, a host cell according to claim 8 or 12, a T cell according to claim 13, a T cell population according to claim 14, or a pharmaceutical composition according to claim 15 for the manufacture of a medicament.

22. Use of an antigen recognition construct according to any one of claims 1 to 5, a nucleic acid or nucleic acids according to claim 6, a vector or vectors according to claim 7, a host cell according to claim 8 or 12, a T cell according to claim 13, a T cell population according to claim 14, or a pharmaceutical composition according to claim 15, for the manufacture of a medicament for use in the prevention and / or treatment of cancer.

23. Cancers include acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal or rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder or pleural cancer, nose cancer, nasal cavity cancer, middle ear cancer, oral cancer, vaginal cancer, vulva cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin's lymphoma, and 23. The use of claim 22, wherein the cancer is selected from pharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omentomesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and bladder cancer.

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