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T CELL RECEPTORS AND IMMUNE THERAPY USING THE SAME AGAINST PRAME POSITIVE CANCERS

20190256572 ยท 2019-08-22

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention pertains to antigen recognizing constructs against tumor associated antigens (TAA), in particular against Preferentially Expressed Antigen of Melanoma (PRAME). The invention in particular provides novel T cell receptor (TCR) based molecules which are selective and specific for the tumor expressed antigen of the invention. The TCR of the invention, and TAA binding fragments derived therefrom, are of use for the diagnosis, treatment and prevention of TAA expressing cancerous diseases. Further provided are nucleic acids encoding the antigen recognizing constructs of the invention, vectors comprising these nucleic acids, recombinant cells expressing the antigen recognizing constructs and pharmaceutical compositions comprising the compounds of the invention.

    Claims

    1. A method of treating a patient who has cancer, comprising administering to the patient a population of transformed cells expressing at least one vector encoding a T cell receptor (TCR), wherein the TCR comprises SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 9, or SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 19 and SEQ ID NO: 21, or SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 31 and SEQ ID NO: 33, or SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 43 and SEQ ID NO: 45, or SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 55 and SEQ ID NO: 57, or SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 67 and SEQ ID NO: 69, or SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 79 and SEQ ID NO: 81, or SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 133 and SEQ ID NO: 135, wherein each of SEQ ID NOs: 1, 3, 7, 9, 13, 15, 19, 21, 25, 27, 31, 33, 37, 39, 43, 45, 49, 51, 55, 57, 61, 63, 67, 69, 73, 75, 79, 81, 127, 129, 133, and 135 comprise at most one conservative amino acid substitution, wherein the TCR is capable of binding to a peptide consisting of the amino acid sequence of SLLQHLIGL (SEQ ID NO: 97) in a complex with an MHC class I molecule, and wherein the cancer is selected from acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladder cancer.

    2. The method of claim 1, wherein the population of transformed cells are produced by a method comprising isolating a cell from a subject, transforming the cell with at least one vector encoding the TCR to produce a transformed cell, and expanding the transformed cell to produce the population of transformed cells.

    3. The method of claim 2, wherein the subject is the patient.

    4. The method of claim 2, wherein the subject is a healthy donor.

    5. The method of claim 2, wherein the cell is a CD8+ T cell.

    6. The method of claim 1, wherein the TCR comprises an ? chain comprising the amino acid sequence of SEQ ID NO: 6 and a ? chain comprising the amino acid sequence of SEQ ID NO: 12, or an ? chain comprising the amino acid sequence of SEQ ID NO: 18 and a ? chain comprising the amino acid sequence of SEQ ID NO: 24, or an ? chain comprising the amino acid sequence of SEQ ID NO: 30 and a ? chain comprising of the amino acid sequence of SEQ ID NO: 36.

    7. The method of claim 1, wherein the MHC class I molecule is HLA-A*02.

    8. The method of claim 1, wherein the population of transformed cells are administered in the form of a pharmaceutical composition.

    9. The method of claim 8, wherein the pharmaceutical composition comprises a chemotherapeutic agent selected from the group consisting of asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and vincristine.

    10. The method of claim 1, wherein the TCR comprises: a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 1, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 2, a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 3. a CDR1? chain comprising the amino acid sequences of SEQ ID NO: 7, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 9, and wherein each of SEQ ID NOs: 2 and 8 comprises at most one conservative amino acid substitution.

    11. The method of claim 1, wherein the TCR comprises: a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 13, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 14, a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 15, a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 19, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 21, and wherein each of SEQ ID NOs: 14 and 20 comprises at most one conservative amino acid substitution.

    12. The method of claim 1, wherein the TCR comprises: a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 25, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 26, a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 27, a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 31, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 32, and a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 33, an wherein each of SEQ ID NOs: 26 and 32 comprises at most one conservative amino acid substitution.

    13. The method of claim 1, wherein the TCR comprises: a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 1, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 2, a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 3. a CDR1? chain comprising the amino acid sequences of SEQ ID NO: 7, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 9, and wherein each of SEQ ID NOs: 3 and 9 comprises at most one conservative amino acid substitution.

    14. The method of claim 1, wherein the TCR comprises: a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 13, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 14, a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 15, a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 19, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 21, and wherein each of SEQ ID NOs: 15 and 21 comprises at most one conservative amino acid substitution.

    15. The method of claim 1, wherein the TCR comprises: a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 25, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 26, a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 27, a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 31, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 32, and a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 33, an wherein each of SEQ ID NOs: 27 and 33 comprises at most one conservative amino acid substitution.

    16. The method of claim 1, wherein the TCR comprises: a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 1, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 2, a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 3. a CDR1? chain comprising the amino acid sequences of SEQ ID NO: 7, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 9.

    17. The method of claim 1, wherein the TCR comprises: a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 13, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 14, a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 15, a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 19, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 21.

    18. The method of claim 1, wherein the TCR comprises: a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 25, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 26, a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 27, a CDR1? chain comprising the amino acid sequence of SEQ ID NO: 31, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 32, and a CDR3? chain comprising the amino acid sequence of SEQ ID NO: 33.

    19. The method of claim 1, wherein the TCR comprises a CDR1? chain consisting of the amino acid sequence of SEQ ID NO: 1, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 2, a CDR3? chain consisting of the amino acid sequence of SEQ ID NO: 3, a CDR1? chain consisting of the amino acid sequence of SEQ ID NO: 7, a CDR2? chain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3? chain consisting of the amino acid sequence of SEQ ID NO: 9.

    20. The method of claim 1, wherein the TCR comprises a CDR1? chain consisting of the amino acid sequence of SEQ ID NO: 13, a CDR2? chain consisting of the amino acid sequence of SEQ ID NO: 14, a CDR3? chain consisting of the amino acid sequence of SEQ ID NO: 15, a CDR1? chain consisting of the amino acid sequence of SEQ ID NO: 19, a CDR2? chain consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR3? chain consisting of the amino acid sequence of SEQ ID NO: 21.

    Description

    FIGURES

    [0156] FIG. 1: IFN? release from CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R11P3D3 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or various PRAME-004 alanine- or threonine-substitution variants at positions 1-9 (X1-X9) of SEQ ID NO:97 (SEQ ID NO:98-115) or control peptide NYESO1-001 (SEQ ID NO:126). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Several different donors were analyzed with regard to alanine-substitution (Ala_TCRA-0017 and Ala_IFN-041) and threonine-substitution variants (Thr_TCRA-0036).

    [0157] FIG. 2: IFN? release from CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R16P1C10 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or various PRAME-004 alanine- or threonine-substitution variants at positions 1-9 (X1-X9) of SEQ ID NO:97 (SEQ ID NO:98-115) or control peptide NYESO1-001 (SEQ ID NO:126). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Several different donors were analyzed with regard to alanine-substitution (Ala_TCRA-0017 and Ala_IFN-041) and threonine-substitution variants (Thr_TCRA-0036).

    [0158] FIG. 3: IFN? release from CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R16P1E8 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or various PRAME-004 alanine- or threonine-substitution variants at positions 1-9 (X1-X9) of SEQ ID NO:97 (SEQ ID NO:98-115) or control peptide NYESO1-001 (SEQ ID NO:126). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Several different donors were analyzed with regard to alanine-substitution (Ala_TCRA-0017 and Ala_IFN-041) and threonine-substitution variants (Thr_TCRA-0036).

    [0159] FIG. 4: IFN? release from CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R17P1A9 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or various PRAME-004 alanine-substitution variants at positions 1-9 (X1-X9) of SEQ ID NO:97 (SEQ ID NO:98-106) or control peptide NYESO1-001 (SEQ ID NO:126). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Different donors were analyzed with regard to alanine-substitution variants (Ala_IFN-040 and Ala_IFN-041).

    [0160] FIG. 5: IFN? release from CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R17P1D7 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or various PRAME-004 alanine- or threonine-substitution variants at positions 1-9 (X1-X9) of SEQ ID NO:97 (SEQ ID NO:98-115) or control peptide NYESO1-001 (SEQ ID NO:126). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Different donors were analyzed with regard to alanine-substitution (Ala_TCRA-0017 and Ala_IFN-041) and threonine-substitution variants (Thr_TCRA-0036).

    [0161] FIG. 6: IFN? release from CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R17P1G3 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or various PRAME-004 alanine- or threonine-substitution variants at positions 1-9 (X1-X9) of SEQ ID NO:97 (SEQ ID NO:98-115) or control peptide NYESO1-001 (SEQ ID NO:126). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Different donors were analyzed with regard to alanine-substitution (Ala_TCRA-0017 and Ala_IFN-041) and threonine-substitution variants (Thr_TCRA-0036).

    [0162] FIG. 7: IFN? release from CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R17P2B6 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) or various PRAME-004 alanine- or threonine-substitution variants at positions 1-9 (X1-X9) of SEQ ID NO:97 (SEQ ID NO:98-115) or control peptide NYESO1-001 (SEQ ID NO:126). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Different donors were analyzed with regard to alanine-substitution (Ala_TCRA-0017 and Ala_IFN-041) and threonine-substitution variants (Thr_TCRA-0036).

    [0163] FIG. 8: IFN? release from CD8+ T-cells electroporated with alpha and beta 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 peptide 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). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Different donors were analyzed, IFN-040 and IFN-041.

    [0164] FIG. 9: IFN? release from CD8+ T-cells electroporated with alpha and beta 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 peptide 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). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Different donors were analyzed, IFN-046 and IFN-041.

    [0165] FIG. 10: IFN? release from CD8+ T-cells electroporated with alpha and beta 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 peptide 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). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Different donors were analyzed, IFN-040 and IFN-041.

    [0166] FIG. 11: IFN? release from CD8+ T-cells electroporated with alpha and beta 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 peptide 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). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Different donors were analyzed, IFN-040 and IFN-041.

    [0167] FIG. 12: IFN? release from CD8+ T-cells electroporated with alpha and beta 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 peptide 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). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Different donors were analyzed, IFN-040 and IFN-041.

    [0168] FIG. 13: IFN? release from CD8+ T-cells electroporated with alpha and beta 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 peptide 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). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Different donors were analyzed, IFN-046 and IFN-041.

    [0169] FIG. 14: IFN? release from CD8+ T-cells electroporated with alpha and beta 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 peptide 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). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. RNA electroporated CD8+ T-cells alone or in co-incubation with unloaded target cells served as controls. Different donors were analyzed, IFN-040 and IFN-041.

    [0170] FIG. 15: IFN? release from CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R11P3D3 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) in various peptide loading concentrations from 10 ?M to 10 pM. IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. Different donors were analyzed, TCRA-0003 and TCRA-0017.

    [0171] FIG. 16: IFN? release from CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R16P1C10 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) in various peptide loading concentrations from 10 ?M to 10 pM. IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. Different donors were analyzed, TCRA-0003 and TCRA-0017.

    [0172] FIG. 17: IFN? release from CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R16P1E8 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) in various peptide loading concentrations from 10 ?M to 10 pM. IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. Different donors were analyzed, TCRA-0003 and TCRA-0017.

    [0173] FIG. 18: IFN? release from CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R17P1D7 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) in various peptide loading concentrations from 10 ?M to 10 pM. IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. Different donors were analyzed, TCRA-0003 and TCRA-0017.

    [0174] FIG. 19: IFN? release from CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R17P1G3 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) in various peptide loading concentrations from 10 ?M to 10 pM. IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. Different donors were analyzed, TCRA-0003 and TCRA-0017.

    [0175] FIG. 20: IFN? release from CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R17P2B6 (Table 1) after co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO:97) in various peptide loading concentrations from 10 ?M to 10 pM. IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors. Different donors were analyzed, TCRA-0003 and TCRA-0017.

    [0176] FIG. 21: HLA-A*02/PRAME-004 tetramer or HLA-A*02/NYESO1-001 tetramer staining, respectively, of CD8+ T-cells electroporated with alpha and beta chain RNA of TCR R16P1C10 (Table 1). CD8+ T-cells electroporated with RNA of 1G4 TCR (SEQ ID: 85-96) that specifically binds to the HLA-A*02/NYESO1-001 complex and mock electroporated CD8+ T-cells served as controls.

    [0177] FIG. 22: IFN? release from CD8+ T-cells lentivirally transduced with TCR R11P3D3 (Table 1) (D103805 and D191451) or non-transduced cells (D103805 NT and D191451 NT) after co-incubation with T2 target cells loaded with 100 nM PRAME-004 peptide (SEQ ID NO:97) or similar (identical to PRAME-004 in 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), 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). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors, D103805 and D191451.

    [0178] FIG. 23: IFN? 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 similar (identical to PRAME-004 in positions 3, 5, 6 and 7) but unrelated peptides (SEQ ID NO:151-195) or control peptide NYESO1-001 (SEQ ID NO:126). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors, TCRA-0087 and TCRA-0088.

    [0179] FIG. 24: IFN? release from CD8+ T-cells lentivirally transduced with TCR R11P3D3 (Table 1) (D103805 and D191451) or non-transduced cells (D103805 NT and D191451 NT) 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 (Small airway epithelial cells), HCF (Cardiac fibroblasts)) and iPSC-derived cell types (HN (Neurons), iHCM (Cardiomyocytes), HH (Hepatocytes), HA (astrocytes)). Tumor cell lines UACC-257 (PRAME-004 high), Hs695T (PRAME-004 medium), U266B1 (PRAME-004 very low) and MCF-7 (no PRAME-004) present different amounts of PRAME-004 per cells. T-cells alone served as controls. IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors, D103805 and D191451.

    [0180] FIG. 25: IFN? release from CD8+ T-cells lentivirally transduced with TCR R11P3D3 (Table 1) after co-incubation with different primary cells (NHEK (Epidermal 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). Tumor cell lines UACC-257 (PRAME-004 high), Hs695T (PRAME-004 medium), U266B1 (PRAME-004 very low) and MCF-7 (no PRAME-004) present different copies of PRAME-004 per cells. T-cells alone served as controls. IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors, TCRA-0084 and TCRA-0085.

    [0181] FIG. 26: IFN? release from CD8+ T-cells lentivirally transduced with enhanced TCR R11P3D3_KE (Table 1) (D103805 and D191451) or non-transduced cells (D103805 NT and D191451 NT) after co-incubation with T2 target cells loaded with 100 nM PRAME-004 peptide (SEQ ID NO:97) or similar (identical to PRAME-004 in positions 3, 5, 6 and 7) but unrelated peptide 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), 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). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors, D103805 and D191451.

    [0182] FIG. 27: IFN? release from CD8+ T-cells lentivirally transduced with 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 similar (identical to PRAME-004 in positions 3, 5, 6 and 7) but unrelated peptides (SEQ ID NO:151-195) or control peptide NYESO1-001 (SEQ ID NO:126). IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors, TCRA-0087 and TCRA-0088.

    [0183] FIG. 28: IFN? release from CD8+ T-cells lentivirally transduced with enhanced TCR R11P3D3_KE (Table 1) (D103805 and D191451) or non-transduced cells (D103805 NT and D191451 NT) 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 (Small airway epithelial cells), HCF (Cardiac fibroblasts)) and iPSC-derived cell types (HN (Neurons), iHCM (Cardiomyocytes), HH (Hepatocytes), HA (astrocytes)). Tumor cell lines UACC-257 (PRAME-004 high), Hs695T (PRAME-004 medium), U266B1 (PRAME-004 very low) and MCF-7 (no PRAME-004) present different amounts of PRAME-004 per cells. T-cells alone served as controls. IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors, D103805 and D191451.

    [0184] FIG. 29: IFN? release from CD8+ T-cells lentivirally transduced with enhanced TCR R11P3D3_KE (Table 1) after co-incubation with different primary cells (NHEK (Epidermal 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). Tumor cell lines UACC-257 (PRAME-004 high), Hs695T (PRAME-004 medium), U266B1 (PRAME-004 very low) and MCF-7 (no PRAME-004) present different copies of PRAME-004 per cells. T-cells alone served as controls. IFN? release data were obtained with CD8+ T-cells derived from two different healthy donors, TCRA-0084 and TCRA-0085.

    [0185] FIG. 30: IFN? release from CD8+ T-cells lentivirally transduced with TCR R11P3D3 or enhanced TCR R11P3D3_KE (Table 1) or non-transduced cells after co-incubation with tumor cell lines UACC-257 (PRAME-004 high), Hs695T (PRAME-004 medium), U266B1 (PRAME-004 very low) and MCF-7 (no PRAME-004) present different amounts of PRAME-004 per cells. T-cells alone served as controls. IFN? release of both TCRs correlates with PRAME-004 presentation and R11P3D3_KE induces higher responses compared to R11P3D3.

    [0186] FIG. 31: IFN? 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 variants at positions 1-9 (A1-A9) of SEQ ID NO:97 (SEQ ID NO:98-106). IFN? release data were obtained with CD8+ T-cells derived from three different healthy donors.

    [0187] FIG. 32: Potency assay evaluating cytolytic activity of lentivirally transduced T cells expressing TCR R11P3D3 or enhanced TCR R11P3D3_KE against PRAME-004+ tumor cells. Cytotoxic response of R11P3D3 and R11P3D3_KE transduced and non-transduced (NT) T cells measured against A-375 (PRAME-004 low) or U2OS (PRAME-004 medium) tumor cells. The assays were performed in a 72-hour fluorescence microscopy-based cytotoxicity assay. Results are shown as fold tumor growth over time.

    [0188]

    TABLE-US-00001 TABLE1 TCRsequencesoftheinvention SEQ ID NO: TCR Chain Region Sequence 1 R11P3D3 alpha CDR1 SSNFYA 2 R11P3D3 alpha CDR2 MTL 3 R11P3D3 alpha CDR3 CALYNNNDMRF 4 R11P3D3 alpha variable MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETA domain KSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGT RLTVKP 5 R11P3D3 alpha constant NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domain AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 6 R11P3D3 alpha full- MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETA length KSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGT RLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDF KSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRI LLLKVAGFNLLMTLRLWSS 7 R11P3D3 beta CDR1 SGHNS 8 R11P3D3 beta CDR2 FNNNVP 9 R11P3D3 beta CDR3 CASSPGSTDTQYF 10 R11P3D3 beta variable MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGL domain ELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPG TRLTVL 11 R11P3D3 beta constant EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL domain KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 12 R11P3D3 beta full- MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFVVYRQTMMRGL length ELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPG TRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVS TDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVT QIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 13 R16P1C10 alpha CDR1 DRGSQS 14 R16P1C10 alpha CDR2 IY 15 R16P1C10 alpha CDR3 CAAVISNFGNEKLTF 16 R16P1C10 alpha variable MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG domain KSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAVISNFGNEKLTFGTG TRLTIIP 17 R16P1C10 alpha constant NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domain AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 18 R16P1C10 alpha full- MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG length KSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAVISNFGNEKLTFGTG TRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDF KSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRI LLLKVAGFNLLMTLRLWSS 19 R16P1C10 beta CDR1 SGHRS 20 R16P1C10 beta CDR2 YFSETQ 21 R16P1C10 beta CDR3 CASSPWDSPNEQYF 22 R16P1C10 beta variable MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGL domain QFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSPWDSPNEQYFG PGTRLTVT 23 R16P1C10 beta constant EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL domain KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 24 R16P1C10 beta full- MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGL length QFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSPWDSPNEQYFG PGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSG VSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP VTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 25 R16P1E8 alpha CDR1 NSAFQY 26 R16P1E8 alpha CDR2 TY 27 R16P1E8 alpha CDR3 CAMSEAAGNKLTF 28 R16P1E8 alpha variable MMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYS domain RKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSEAAGNKLTFGG GTRVLVKP 29 R16P1E8 alpha constant NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domain AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 30 R16P1E8 alpha full- MMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYS length RKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSEAAGNKLTFGG GTRVLVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSM DFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIG FRILLLKVAGFNLLMTLRLWSS 31 R16P1E8 beta CDR1 SGHAT 32 R16P1E8 beta CDR2 FQNNGV 33 R16P1E8 beta CDR3 CASSYTNQGEAFF 34 R16P1E8 beta variable MGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPK domain LLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSYTNQGEAFFGQG TRLTVV 35 R16P1E8 beta constant EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPL domain KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 36 R16P1E8 beta full- MGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPK length LLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSYTNQGEAFFGQG TRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVS TDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVT QIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 37 R17P1A9 alpha CDR1 DRGSQS 38 R17P1A9 alpha CDR2 IY 39 R17P1A9 alpha CDR3 CAVLNQAGTALIF 40 R17P1A9 alpha variable MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG domain KSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVLNQAGTALIFGKGTT LSVSS 41 R17P1A9 alpha constant NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domain AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 42 R17P1A9 alpha full- MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG length KSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVLNQAGTALIFGKGTT LSVSSNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK SNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL LLKVAGFNLLMTLRLWSS 43 R17P1A9 beta CDR1 SGDLS 44 R17P1A9 beta CDR2 YYNGEE 45 R17P1A9 beta CDR3 CASSAETGPWLGNEQFF 46 R17P1A9 beta variable MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGL domain QFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSAETGPWLGNEQFF GPGTRLTVL 47 R17P1A9 beta constant EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL domain KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 48 R17P1A9 beta full- MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGL length QFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSAETGPWLGNEQFF GPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHS GVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAK PVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 49 R17P1D7 alpha CDR1 TSESDYY 50 R17P1D7 alpha CDR2 QEAY 51 R17P1D7 alpha CDR3 CAYRWAQGGSEKLVF 52 R17P1D7 alpha variable MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPS domain RQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRWAQGGSEKLV FGKGTKLTVNP 53 R17P1D7 alpha constant YIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domain AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 54 R17P1D7 alpha full- MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPS length RQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRWAQGGSEKLV FGKGTKLTVNPYIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDM RSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS VIGFRILLLKVAGFNLLMTLRLWSS 55 R17P1D7 beta CDR1 MGHDK 56 R17P1D7 beta CDR2 SYGVNS 57 R17P1D7 beta CDR3 CATELWSSGGTGELFF 58 R17P1D7 beta variable MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELH domain LIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCATELWSSGGTGELFFGE GSRLTVL 59 R17P1D7 beta constant EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL domain KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 60 R17P1D7 beta full- MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELH length LIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCATELWSSGGTGELFFGE GSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV STDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 61 R17P1G3 alpha CDR1 DRGSQS 62 R17P1G3 alpha CDR2 IY 63 R17P1G3 alpha CDR3 CAVGPSGTYKYIF 64 R17P1G3 alpha variable MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG domain KSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVGPSGTYKYIFGTGT RLKVLA 65 R17P1G3 alpha constant NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domain AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 66 R17P1G3 alpha full- MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG length KSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVGPSGTYKYIFGTGT RLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDF KSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRI LLLKVAGFNLLMTLRLWSS 67 R17P1G3 beta CDR1 MNHEY 68 R17P1G3 beta CDR2 SMNVEV 69 R17P1G3 beta CDR3 CASSPGGSGNEQFF 70 R17P1G3 beta variable MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGL domain RQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSPGGSGNEQFFGP GTRLTVL 71 R17P1G3 beta constant EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL domain KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 72 R17P1G3 beta full- MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGL length RQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSPGGSGNEQFFGP GTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV STDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 73 R17P2B6 alpha CDR1 DRGSQS 74 R17P2B6 alpha CDR2 IY 75 R17P2B6 alpha CDR3 CAVVSGGGADGLTF 76 R17P2B6 alpha variable MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG domain KSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVVSGGGADGLTFGKG THLIIQP 77 R17P2B6 alpha constant YIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domain AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 78 R17P2B6 alpha full- MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSG length KSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVVSGGGADGLTFGKG THLIIQPYIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDF KSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRI LLLKVAGFNLLMTLRLWSS 79 R17P2B6 beta CDR1 PRHDT 80 R17P2B6 beta CDR2 FYEKMQ 81 R17P2B6 beta CDR3 CASSLGRGGQPQHF 82 R17P2B6 beta variable MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWY domain QQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLGR GGQPQHFGDGTRLSIL 83 R17P2B6 beta constant EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPL domain KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 84 R17P2B6 beta full- MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWY length QQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLGR GGQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWV NGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE WTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV KRKDF 85 1G4 alpha CDR1 DSAIYN 86 1G4 alpha CDR2 IQS 87 1G4 alpha CDR3 CAVRPTSGGSYIPTF 88 1G4 alpha variable METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLT domain SLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTS LIVHP 89 1G4 alpha constant YIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV domain AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 90 1G4 alpha full- METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLT length SLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTS LIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKS NSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILL LKVAGFNLLMTLRLWSS 91 1G4 beta CDR1 MNHEY 92 1G4 beta CDR2 SVGAGI 93 1G4 beta CDR3 CASSYVGNTGELFF 94 1G4 beta variable MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMG domain LRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGE GSRLTVL 95 1G4 beta constant EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL domain KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 96 1G4 beta full- MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMG length LRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGE GSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV STDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 127 R11P3D3_ alpha CDR1 SSNFYA KE 128 R11P3D3_ alpha CDR2 MTL KE 129 R11P3D3_ alpha CDR3 CALYNNNDMRF KE 130 R11P3D3_ alpha variable MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRKETAK KE domain SPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGTRL TVKP 131 R11P3D3_ alpha constant NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV KE domain AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA GFNLLMTLRLWSS 132 R11P3D3_ alpha full- MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRKETAK KE length SPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCALYNNNDMRFGAGTRL TVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKS NSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILL LKVAGFNLLMTLRLWSS 133 R11P3D3_ beta CDR1 SGHNS KE 134 R11P3D3_ beta CDR2 FNNNVP KE 135 R11P3D3_ beta CDR3 CASSPGSTDTQYF KE 136 R11P3D3_ beta variable MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRETMMRGL KE domain ELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPG TRLTVtext missing or illegible when filed 137 R11P3D3_ beta constant EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPL KE domain KEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEVVTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG 138 R11P3D3_ beta full- MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRETMMRGL KE domain ELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSPGSTDTQYFGPG TRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVS TDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVT 196 R11P3D3 alpha CDR2bi MTLNGDE 197 R16P1C10 alpha CDR2bi IYSNGD 198 R16P1E8 alpha CDR2bi TYSSGN 199 R17P1A9 alpha CDR2bi IYSNGD 200 R17P1D7 alpha CDR2bi QEAYKQQ 201 R17P1G3 alpha CDR2bi IYSNGD 202 R17P2B6 alpha CDR2bi IYSNGD 203 1G4 alpha CDR2bi IQSSQRE 204 R11P3D3__ alpha CDR2bi MTLNGDE text missing or illegible when filed indicates data missing or illegible when filed

    TABLE-US-00002 TABLE2 Peptidesequencesoftheinvention PeptideCode Sequence SEQIDNO: PRAME-004 SLLQHLIGL 97 PRAME-004_A1 ALLQHLIGL 98 PRAME-004_A2 SALQHLIGL 99 PRAME-004_A3 SLAQHLIGL 100 PRAME-004_A4 SLLAHLIGL 101 PRAME-004_A5 SLLQALIGL 102 PRAME-004_A6 SLLQHAIGL 103 PRAME-004_A7 SLLQHLAGL 104 PRAME-004_A8 SLLQHLIAL 105 PRAME-004_A9 SLLQHLIGA 106 PRAME-004_T1 TLLQHLIGL 107 PRAME-004_T2 STLQHLIGL 108 PRAME-004_T3 SLTQHLIGL 109 PRAME-004_T4 SLLTHLIGL 110 PRAME-004_T5 SLLQTLIGL 111 PRAME-004_T6 SLLQHTIGL 112 PRAME-004_T7 SLLQHLTGL 113 PRAME-004_T8 SLLQHLITL 114 PRAME-004_T9 SLLQHLIGT 115 TMED9-001 SILQTLILV 116 CAT-001 SLIEHLQGL 117 DDX60L-001 SLIQHLEEI 118 LRRC70-001 SLLKNLIYL 119 PTPLB-001 SLLNHLPYL 120 HDAC5-001 SLLQHVLLL 121 VPS13B-002 SLLQKQIML 122 ZNF318-001 SLSQELVGV 123 CCDC51-001 SVLGALIGV 124 IFIT1-001 VLLHHQIGL 125 NYESO1-001 SLLMWITQV 126 ACPL-001 LLLVHLIPV 139 HSPB3-001 IILRHLIEI 140 UNC7-001 KILLHLIHI 141 SCYL2-001 KVLPHLIPL 142 RPS2P8-001 SALVHLIPV 143 PCNXL3-003 NALVHLIEV 144 AQP6-001 VALGHLIGI 145 PCNX-001 NALVHLIEI 146 AQP6-002 WALGHLIGI 147 TRGV10-001 QALEHLIYI 148 NECAP1-001 ISLAHLILV 149 FBXW2-001 ETLDHLISL 150 ACCSL-001 ALLSHLICR 151 ACER1-001 KELRHLIEV 152 ADAMTS14-001 IALVHLIMV 153 ARHGAP17-001 CWLCHLIKL 154 ARSE-001 GKLTHLIPV 155 ATP-009 HLLMHLIGS 156 AUNI-001 TQLDHLIPG 157 C16orf96-001 QDLWHLIKL 158 CDC7-002 IALKHLIPT 159 CDC7-003 IALKHLILT 160 CHRNA1-001 LQLIHLINV 161 FASTKD5-001 SQLVHLIYV 162 FRYL-002 CLLPHLIQH 163 FTH1-001 MVLVHLIHS 164 HERC4-002 SDLFHLIGV 165 HPS5-001 KLLFHLIQS 166 HPS5-002 KLLLHLIQS 167 HTR2C-001 SFLVHLIGL 168 IPM-001 YGLKHLISV 169 KIF16-001 SELPHLIGI 170 KLHL33-001 YALSHLIHA 171 LAMA3-001 TLLGHLISK 172 LOC100128170-001 SQLSHLIAM 173 MAP2K7-001 FFLVHLICM 174 MON2-003 VSLHHLINA 175 OR2AK2-001 IMLIHLIRL 176 OR2AK2-002 ITLIHLIRL 177 OR2B6-001 SELFHLIPL 178 OR2B6-002 SVLFHLIPL 179 OTUD7A-001 AQLAHLILS 180 OVOS2-001 FLLGHLIPR 181 PIGC-002 MLLGHLIFF 182 RAD54L2-003 VLLFHLIEE 183 RASEF-001 VFLRHLITL 184 RASGRF1-003 TLLDHLIFK 185 RPS2P20-001 SVLVHLIPA 186 SACS-001 AKLEHLIYL 187 SPATA31D5-001 SLLPHLILS 188 TPST2-001 SILGHLICS 189 TRGV10-002 QSLEHLIYI 190 UGP-001 YILNHLINP 191 USP51-001 YKLLHLIWI 192 ZNF423-002 KLLCHLIEH 193 ZNF584-001 ALLDHLITH 194 ZNF99-001 FMLSHLIQH 195

    EXAMPLES

    [0189] Seven PRAME-specific TCRs directed to the herein disclosed PRAME-004 peptide (R11P3D3, R16P1C10, R16P1E8, R17P1A9, R17P1D7, R17P1G3 and R17P2B6, see Table 1), each encoding tumor specific TCR-alpha and TCR-beta chains, were isolated and amplified from T-cells of healthy donors. Cells from healthy donors were in vitro stimulated according to a method previously described (Walter et al., 2003 J Immunol., November 15; 171(10):4974-8) and target-specific cells were single-cell sorted using HLA-A*02 multimers and then used for subsequent TCR isolation. TCR sequences were isolated via 5 RACE by standard methods as described by e.g. Molecular Cloning a laboratory manual fourth edition by Green and Sambrook. The alpha and beta variable regions of TCRs R11P3D3, R16P1C10, R16P1E8, R17P1A9, R17P1D7, R17P1G3 and R17P2B6 were sequenced and cloned for further functional characterization.

    [0190] R11P3D3, R16P1C10, R17P1D7 and R17P2B6 are derived from HLA-A*02 negative donor (alloreactive setting) and R16P1E8, R17P1A9 and R17P1G3 are derived from a HLA-A*02 positive donor.

    [0191] Furthermore, the mutant TCR R11P3D3_KE, an enhanced variant of R11P3D3, is herein disclosed. Enhanced TCR variant R11P3D3_KE was modified from the parental TCR as described in PCT/EP2017/081745, herewith specifically incorporated by reference, and in example 8 below, and the coding sequence was obtained by gene synthesis prior to the functional characterization of the TCR.

    Example 1: T-Cell Receptor R11P3D3

    [0192] TCR R11P3D3 (SEQ ID NO:1-12 and 196) is restricted towards HLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 8).

    [0193] R11P3D3 specifically recognizes PRAME-004, as human primary CD8+ T-cells re-expressing this TCR release IFN? upon co-incubation with HLA-A*02+ target cells, loaded either with PRAME-004 peptide or alanine or threonine substitution variants of PRAME-004 (FIG. 1) or different peptides showing high degree of sequence similarity to PRAME-004 (FIG. 8). NYESO1-001 peptide is used as negative control. TCR R11P3D3 has an EC.sub.50 of 0.74 nM (FIG. 15) and a binding affinity (K.sub.D) of 18-26 ?M towards HLA-A*02-presented PRAME-004 (SEQ ID NO:97).

    [0194] Re-expression of R11P3D3 in human primary CD8+ T-cells leads to selective recognition and killing of HLA-A*02/PRAME-004-presenting tumor cell lines (FIGS. 24, 25 30 and 32). TCR R11P3D3 does not respond to any of the 25 tested healthy, primary or iPSC-derived cell types (FIGS. 24 and 25) and was tested for cross-reactivity towards further 67 similar peptides (of which 57 were identical to PRAME-004 in positions 3, 5, 6 and 7) but unrelated peptides in the context of HLA-A*02 (FIGS. 8, 22 and 23).

    Example 2: T-Cell Receptor R16P1C10

    [0195] TCR R16P1C10 (SEQ ID NO:13-24 and 197) is restricted towards HLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 9).

    [0196] R16P1C10 specifically recognizes PRAME-004, as human primary CD8+ T-cells re-expressing this TCR release IFN? upon co-incubation with HLA-A*02+ target cells and bind HLA-A*02 tetramers (FIG. 21), respectively, loaded either with PRAME-004 peptide or alanine or threonine substitution variants of PRAME-004 (FIG. 2) or different peptides showing high degree of sequence similarity to PRAME-004 (FIG. 9). NYESO1-001 peptide is used as negative control. TCR R16P1C10 has an EC.sub.50 of 9.6 nM (FIG. 16).

    Example 3: T-Cell Receptor R16P1E8

    [0197] TCR R16P1E8 (SEQ ID NO:25-36 and 198) is restricted towards HLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 10).

    [0198] R16P1E8 specifically recognizes PRAME-004, as human primary CD8+ T-cells re-expressing this TCR release IFN? upon co-incubation with HLA-A*02+ target cells, loaded either with PRAME-004 peptide or alanine or threonine substitution variants of PRAME-004 (FIG. 3) or different peptides showing high degree of sequence similarity to PRAME-004 (FIG. 10). NYESO1-001 peptide is used as negative control. TCR R16P1E8 has an EC.sub.50 of ?1 ?M (FIG. 17).

    Example 4: T-Cell Receptor R17P1A9

    [0199] TCR R17P1A9 (SEQ ID NO:37-48 and 199) is restricted towards HLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 11).

    [0200] R17P1A9 specifically recognizes PRAME-004, as human primary CD8+ T-cells re-expressing this TCR release IFN? upon co-incubation with HLA-A*02+ target cells, loaded either with PRAME-004 peptide or alanine substitution variants of PRAME-004 (FIG. 4) or different peptides showing high degree of sequence similarity to PRAME-004 (FIG. 11). NYESO1-001 peptide is used as negative control.

    Example 5: T-Cell Receptor R17P1D7

    [0201] TCR R17P1D7 (SEQ ID NO:49-60 and 200) is restricted towards HLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 12).

    [0202] R17P1D7 specifically recognizes PRAME-004, as human primary CD8+ T-cells re-expressing this TCR release IFN? upon co-incubation with HLA-A*02+ target cells, loaded either with PRAME-004 peptide or alanine or threonine substitution variants of PRAME-004 (FIG. 5) or different peptides showing high degree of sequence similarity to PRAME-004 (FIG. 12). NYESO1-001 peptide is used as negative control. TCR R17P1D7 has an EC.sub.50 of 1.83 nM (FIG. 18).

    Example 6: T-Cell Receptor R17P1G3

    [0203] TCR R17P1G3 (SEQ ID NO:61-72 and 201) is restricted towards HLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 13).

    [0204] R17P1G3 specifically recognizes PRAME-004, as human primary CD8+ T-cells re-expressing this TCR release IFN? upon co-incubation with HLA-A*02+ target cells, loaded either with PRAME-004 peptide or alanine or threonine substitution variants of PRAME-004 (FIG. 6) or different peptides showing high degree of sequence similarity to PRAME-004 (FIG. 13). NYESO1-001 peptide is used as negative control. TCR R17P1G3 has an EC.sub.50 of 8.63 nM (FIG. 19).

    Example 7: T-Cell Receptor R17P2B6

    [0205] TCR R17P2B6 (SEQ ID NO:73-84 and 202) is restricted towards HLA-A*02-presented PRAME-004 (SEQ ID NO:97) (see FIG. 14).

    [0206] R17P2B6 specifically recognizes PRAME-004, as human primary CD8+ T-cells re-expressing this TCR release IFN? upon co-incubation with HLA-A*02+ target cells, loaded either with PRAME-004 peptide or alanine or threonine substitution variants of PRAME-004 (FIG. 7) or different peptides showing high degree of sequence similarity to PRAME-004 (FIG. 14). NYESO1-001 peptide is used as negative control. TCR R17P2B6 has an EC.sub.50 of 2.11 nM (FIG. 20) and a binding affinity (K.sub.D) of 13 ?M towards HLA-A*02-presented PRAME-004.

    Example 8: Enhanced T-Cell Receptor R11P3D3_KE

    [0207] The mutated enhanced pairing TCR R11P3D3_KE is introduced as a variant of R11P3D3, where ? and ? variable domains, naturally bearing ?W44/?Q44, have been mutated to ?K44/?E44. The double mutation is selected among the list present in PCT/EP2017/081745, herewith specifically incorporated by reference. It is specifically designed to restore an optimal interaction and shape complementarity to the TCR scaffold.

    [0208] Compared with the parental TCR R11P3D3 the enhanced TCR R11P3D3_KE shows superior sensitivity of PRAME-004 recognition. The response towards PRAME-004-presenting tumor cell lines are stronger with the enhanced TCR R11P3D3_KE compared to the parental TCR R11P3D3 (FIG. 30). Furthermore, the cytolytic activity of R11P3D3_KE is stronger compared to R11P3D3 (FIG. 32). The observed improved functional response of the enhanced TCR R11P3D3_KE is well in line with an increased binding affinity towards PRAME-004, as described in example 1 (R11P3D3, K.sub.D=18-26 ?M) and example 8 (R11P3D3_KE, K.sub.D=5.3 ?M).