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WO2025056659A1 - Variant du récepteur des lymphocytes t contre mkras7-16 g12v et g12c - Google Patents

Variant du récepteur des lymphocytes t contre mkras7-16 g12v et g12c Download PDF

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WO2025056659A1
WO2025056659A1 PCT/EP2024/075464 EP2024075464W WO2025056659A1 WO 2025056659 A1 WO2025056659 A1 WO 2025056659A1 EP 2024075464 W EP2024075464 W EP 2024075464W WO 2025056659 A1 WO2025056659 A1 WO 2025056659A1
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tcr
seq
cell
amino acid
acid sequence
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Giulia LONGINOTTI
Mario CATARINELLA
Kathrin DAVARI
Melanie SALVERMOSER
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Medigene Immunotherapies GmbH
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Medigene Immunotherapies GmbH
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4244—Enzymes
    • A61K40/4253—GTPases, e.g. Ras or Rho
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the present invention relates to a TCR polypeptide complex and/or a combination comprising a novel TCR characterized by its CDR sequences of the a chain variable region and the p chain variable region and by its antigenic specificity to (a) particular mutated KRAS epitope(s) or a fragment thereof presented in the context of a MHC class I molecule.
  • the present invention further relates to a nucleic acid, a vector as well as a host cell comprising said nucleic acid or said vector.
  • the present invention relates to a method for obtaining said TCR and said chimeric co-stimulatory receptor and to a pharmaceutical or diagnostic composition comprising the abovementioned.
  • the present invention further relates to the abovementioned for use as a medicament or for use in a method of diagnosing, detecting, preventing, and/or treating cancer. Furthermore, the present invention relates to a method of detecting the presence of a cancer in a subject in vitro, by applying within said method the abovementioned, and the use of the abovementioned for generating modified lymphocytes and a kit.
  • T lymphocytes which form a part of the cell mediated immune system play a major role in the eradication of pathogens.
  • T cells develop in the thymus and express T cell receptor molecules on their surface that allow the recognition of peptides presented on human leukocyte antigen (HLA) molecules which are expressed on nucleated cells (antigen presentation).
  • HLA human leukocyte antigen
  • Antigens of pathogens i.e. foreign antigens presented by HLA molecules will elicit a powerful T cell response whereas self-antigens usually do not lead to a T cell response due to a negative selection of self-antigen specific T cells in the thymus during the development of such T cells.
  • the immune system can thus discriminate between nucleated cells presenting foreign- or self-antigens or self, but aberrant antigens and specifically target and eradicate infected cells via potent cytokine release and cellular cytotoxicity mechanisms of the T cells.
  • ACT adoptive cell transfer
  • TME tumor microenvironment
  • KRAS tumor-specific antigens
  • KRAS has the highest mutation rate in a variety of frequently fatal solid cancers, including pancreatic ductal adenocarcinoma, endometrial cancer, non-small cell lung cancer, and colorectal cancer.
  • the worldwide incidence of solid tumors harboring KRAS mutations is estimated to exceed 300,000 patients (Alexandrov LB, et al. , Nature. 2020, 578(7793):94-101).
  • mutant KRAS has been considered “undruggable” (Huang L, et al., Signal Transduct Target Ther. 2021 ;6(1):386), except for G12C mutations in some targeted therapies, novel and efficient treatment options for mKRAS tumors are urgently needed. It is the object of the present invention to comply with the needs set out above.
  • TCR T-cell receptor
  • CDR Complementarity Determining Region
  • novel TCR is further characterized by its antigenic specificity to (a) particular mutated KRAS epitope(s) or a fragment thereof presented in the context of a major histocompatibility complex (MHC) class I molecule as outlined below.
  • MHC major histocompatibility complex
  • TCR-T Adoptive T Cell Receptor T cell
  • TEM solid tumor microenvironments
  • PD-L1 programmed death ligand 1
  • TME solid tumor microenvironments
  • PD-L1 signaling to T cells via PD-1 limits proliferation, cytokine secretion and killing, while exhaustion is induced by repetitive TCR signaling in the absence of T cell co-stimulation. This holds particularly true for advanced stage disease.
  • TCR-T cells both impairments in TCR-T cells can be mitigated by co-expression of the high-affinity TCR of the present invention with a particular chimeric costimulatory receptor, such as for example a PD1-41 BB costimulatory switch receptor (CSR), comprised of the extracellular domain of PD-1 at its N-terminus with the intracellular signaling domain of 4-1 BB (CD137) at its C-terminus.
  • a chimeric costimulatory receptor such as for example a PD1-41 BB costimulatory switch receptor (CSR)
  • CD137 intracellular signaling domain of 4-1 BB
  • TCR has additionally antigenic specificity to (an) particular mutated KRAS epitope(s) or a fragment thereof presented in the context of a MHC class I molecule.
  • it has dual specificity for the epitopes comprising the amino acid sequence of SEQ ID NO: 1 (also referred to as mKRAS G12V - 10mer) and SEQ ID NO: 34 (also referred to as mKRAS G12C - 10mer), meaning said TCR is capable of recognizing each epitope presented in the context of a MHC class I molecule in tumor cells or a combination of both (see Figure 5).
  • the invention relates to a TCR polypeptide complex and/or a combination comprising: a) a TCR; and b) a chimeric co-stimulatory receptor, wherein the TCR comprises: i) an a chain CDR 1 comprising the amino acid sequence of SEQ ID NO: 14; an a chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15; an a chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and ii) a p chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3; a p chain CDR3 comprising the amino acid sequence of SEQ ID NO: 4, wherein the TCR has antigenic specificity to (an) epitope(s) presented in the context of a MHC class I molecule, the epitope(s): a) comprising the amino acid sequence of VVVGAVGVGK (
  • the invention relates to fusion proteins comprising (a) an extracellular domain containing a polypeptide derived from PD-1 or CD40L at its N-terminus; (b) a transmembrane domain (e.g., derived from PD-1); and (c) an intracellular domain containing a polypeptide derived from 4-1 BB or CD28 at its C-terminus. Also, fusion proteins with CD28 at the N-terminus and CD40L at the C-terminus are envisaged.
  • the invention relates to a cell population comprising cells or a cell expressing: a) a TCR; and b) a chimeric co-stimulatory receptor, wherein the TCR comprises: i) an a chain CDR 1 comprising the amino acid sequence of SEQ ID NO: 14; an a chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15; an a chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and ii) a p chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2; a chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3; a p chain CDR3 comprising the amino acid sequence of SEQ ID NO: 4, wherein the TCR has antigenic specificity to (an) epitope(s) presented in the context of a MHC class I molecule, the epitope(s): a) comprising the amino acid sequence of VVVGAVGVGK (SEQ ID
  • the invention relates to a nucleic acid comprising a nucleotide sequence encoding the TCR as mentioned above and defined herein and a nucleotide sequence encoding the chimeric co-stimulatory receptor as mentioned above and defined herein.
  • the invention relates to a vector comprising the nucleic acid as mentioned above and defined herein.
  • the invention relates to a host cell comprising the nucleic acid or the vector as mentioned above and defined herein.
  • the present invention also relates to a method for obtaining the TCR and the chimeric co-stimulatory receptor as mentioned above, comprising (i) incubating the host cell as defined above under conditions causing expression of said TCR and of said chimeric co-stimulatory receptor; and (ii) purifying said TCR and said chimeric co-stimulatory receptor.
  • the invention relates to a pharmaceutical or diagnostic composition
  • a pharmaceutical or diagnostic composition comprising one or more of: (i) the complex and/or the combination; (ii) the cell population or the cell, (iii) the nucleic acid; (iv) the vector; and/or (v) the host cell as mentioned above and defined herein; and, optionally, pharmaceutically excipient(s).
  • the present invention relates to the complex and/or the combination, and/or the cell population or the cell, the nucleic acid, the vector, the host cell and/or the pharmaceutical composition as mentioned above and defined herein for use as a medicament and/or for use in therapy.
  • the present invention relates to the complex and/or the combination, and/or the cell population or the cell, the nucleic acid, the vector, the host cell and/or the pharmaceutical composition as mentioned above and defined herein for use in a method of detecting, diagnosing, prognosing, preventing and/or treating cancer.
  • the present invention also relates to a method of detecting the presence of a cancer in a subject in vitro, comprising (a) contacting a sample obtained from a subject and comprising one or more cells with (i) the complex and/or the combination; (ii) the cell population or the cell, (iii) the nucleic acid; (iv) the vector; (v) the host cell; and/or (vi) the pharmaceutical composition as mentioned above and defined herein, thereby forming a complex, and (b) detecting the complex, wherein detection of the complex is indicative of the presence of the cancer in the subject.
  • the present invention also relates to the use of the complex and/or the combination, and/or the cell population or the cell, the nucleic acid and/or the vector as mentioned above and defined herein for generating modified lymphocytes.
  • the present invention relates to a kit comprising the complex and/or the combination, and/or the cell population or the cell, the nucleic acid, the vector, the host cell, and/or the pharmaceutical or diagnostic composition as mentioned above and defined herein.
  • Fig. 1 shows retroviral transduction induces high and robust transgenic TCR and PD1- 41 BB CSR expression.
  • TCR in combination with PD1-41 BB (TCR + PD1-41 BB) was expressed into CD8 + T cells by retroviral transduction.
  • Untransduced CD8 + T cells (UT) were prepared in the same manner and used as controls.
  • CD8 + T cells were enriched using anti-CD8, anti-TCR Cpi and anti-PD1 antibodies by fluorescence-activated cell sorting (FACS).
  • T cell samples were stained with anti-TCR C
  • Fig. 2 shows high and exclusive mKRAS?-i6G12V (decamer peptide, 10-mer) multimer binding.
  • TCR + PD1-41 BB-transduced CD8 + T cells TCR + PD1-41 BB
  • mKRAS?-i6 G12V decamer peptide, 10-mer
  • HLA- A*11 :01 multimer HLA- A*11 :01 multimer and analyzed by flow cytometry.
  • Untransduced CD8 + T cells (UT) were stained and analyzed in parallel as internal controls. Populations shown are pre-gated on live single cells. One representative experiment is shown.
  • FIG. 3 shows that T cells exhibit exclusive mKRAS?-i6 G12V specificity with no KRAS wild type recognition.
  • TCR + PD1-41 BB-transduced CD8 + T cells (TCR + PD1-41 BB) were cocultured with PDL1 -transduced K562 cells expressing transgenic HLA-A*11 :01-encoding molecules (K562_A11_PDL1) loaded with either mKRAS?-i6 G12V peptide or KRASy- wild type (WT) peptide at high concentration (10' 5 M).
  • CD8 + T cell samples were also co-cultured with K562_A11_PDL1 cells transfected with either ivtRNA encoding a fragment of mKRAS G12V gene (-402 bp) or ivtRNA encoding a fragment of KRAS wild type gene (-402 bp).
  • K562_A11_PDL1 cells unloaded as well as electroporated (EP) with water served as target controls. Untransduced (UT) CD8 + T cells were included as negative controls.
  • an ELISA was performed to evaluate IFN-y secretion by T cells. The mean value of duplicates is shown with standard deviations. One representative experiment is shown.
  • Fig. 4 shows that T cells exhibit high peptide sensitivity for mKRASy-ie G12V (10-mer) epitope.
  • TCR + PD1-41 BB-transduced CD8 + T cells (TCR + PD1-41 BB) tested in a co-culture experiment using K562_A11_PDL1 cells loaded with graded amounts of either mKRASy-16 G12V (10-mer) peptide or mKRASs-ie G12V (9-mer) peptide (10 -5 M to 10' 12 M) for direct comparison of peptide sensitivity for the two peptide length variants.
  • an ELISA was performed to evaluate IFN-y secretion by T cells.
  • Fig. 5 shows that T cell recognition of mKRAS?-i6 G12C epitope is mediated by presentation by HLA-A*11 :01 molecules.
  • TOR + PD1-41 BB-transduced CD8 + T cell (TOR + PD1-41 BB) were tested in a co-culture experiment with K562_A11_PDL1 cells loaded with either mKRAS?-i6 G12V, mKRAS?-i6 G12D, mKRAS?-i6 G12C or mKRAS?-i6 G12R peptides at high concentration (10' 5 M).
  • Target cells loaded with KRASy-iewild type (WT) peptide as well as unloaded targets were included in the experiment as internal controls. Additionally, K562_A11_PDL1 cells were transfected with four ivtRNA constructs encoding the previously described KRAS mutations individually (-402 bp spanning the G12 mutation site). ivtRNA encoding KRASy-ie wild type epitope (WT) was also used to transfect target cells as internal control. Water electroporation (water EP) of target cells served as negative control. IFN-y secretion was evaluated by ELISA 20 h after setting up the co-culture. Shown is the mean value of duplicates with standard deviations. One representative experiment is shown.
  • Fig. 6 shows peptide-specific restriction to four different HLA-A*11 subtypes.
  • CD8 + T cells expressing either no transgenic TOR (UT) or the combination of TOR and PD1-41 BB CSR (TCR + PD1-41 BB) were co-cultured with a set of H LA-transduced K562 cell samples.
  • Each K562 sample expressed one of the following transgenic HLA molecules: HLA-A*11 :01 , HLA- A*11 :02, HLA-A*11 :03, HLA-A*11 :04.
  • Each H LA-transduced K562 sample was tested after mKRASy-16 G12V peptide loading (10' 5 M). IFN-y release was evaluated by ELISA 20 h after setting up the co-culture. Shown is the mean value of duplicates with standard deviations. One representative experiment is shown.
  • Fig. 7 shows that there are no signs of target peptide-independent cross-recognition of global frequent HLA allotypes.
  • TOR + PD1-41 BB-transduced CD8 + T cells (TOR + PD1-41 BB) and untransduced (UT) CD8 + T cells were co-cultured with two cellular libraries.
  • the first library comprised 70 lymphoblastoid cell lines (LCLs, LOL library, Figure 7A) and the second library comprised 31 K562 cell samples transduced with individual HLA molecules (K562 library, Figure 7B).
  • K562_A11 cells loaded with mKRASy-ie G12V peptide (10' 5 M) served as internal positive control (pos control). IFN-y secretion was evaluated by ELISA 20 h after setting up the co-culture. Shown is the mean value of duplicates with standard deviations. One representative experiment is shown.
  • Fig. 8 shows the successful de-risking of potential peptide off-target toxicity.
  • Mismatched peptides which were identified as recognized by TCR + PD1-41 BB in a screening assay performed with peptide loading of target cells at high concentrations (10' 6 M), were tested for their potential to induce IFN-y release when translated from ivtRNA and endogenously processed and presented by K562_A1 _PDL1 cells.
  • Midi-gene fragments were designed spanning the respective peptide-coding region and linked to GFP reporter gene.
  • Fig. 9 shows that no signs of off-target toxicity were identified using a panel of healthy cell samples.
  • HLA-A*11 :01-positive primary healthy cells and induced pluripotent stem cell (iPS)-derived cell lines representing essential organs were tested for recognition by TCR + PD1-41 BB-transduced CD8 + T cells (TCR + PD1-41 BB).
  • Untransduced (UT) CD8 + T cells served as internal controls.
  • Target cells loaded with mKRAS?-i6 G12V peptide (10' 5 M) were included as internal positive controls.
  • IFN-y secretion was evaluated by ELISA 20 h after setting up the co-culture. Shown is the mean value of duplicates with standard deviations. Shown one representative experiment.
  • HRCEpC Human Renal Cortical Epithelial Cells
  • NHLF Normal Human Lung Fibroblasts
  • HCF-c Human Cardiac Fibroblasts- Atrial
  • Fig. 10 shows high and specific IFN-y secretion in response to tumor cells expressing endogenous levels of mKRAS G12V antigen.
  • Tumor cell lines derived from various indications expressing either mKRAS G12V at different levels (DAN-G, SW527, NCI-H441 , SW480) or only KRAS wild type (DU145, Mel624.38) were selected for this co-culture experiment.
  • DAN-G, SW527, NCI-H441 , SW480 or only KRAS wild type (DU145, Mel624.38) were selected for this co-culture experiment.
  • All selected tumor cell lines were transduced with PDL1 and tested both with and without PDL1 transduction.
  • the co-culture experiment was set up by using the described tumor cell lines and TCR + PD1-41 BB-transduced CD8 + T cells (TCR + PD1-41 BB).
  • Untransduced (UT) CD8 + T cells served as negative control.
  • Fig. 11 shows that a strong and specific cytotoxic response occurs against tumor cells expressing endogenous levels of mKRAS G12V antigen even after multiple tumor challenges.
  • Tumor cell lines derived from various indications expressing either mKRAS G12V at different levels (DAN-G, SW527, NCI-H441 , SW480) or only KRAS wild type (DU145, Mel624.38) were selected for this co-culture experiment.
  • DAN-G, SW527, NCI-H441 , SW480 or only KRAS wild type (DU145, Mel624.38) were selected for this co-culture experiment.
  • All selected tumor cell lines were transduced with PDL1 and tested both with and without PDL1 transduction. All tumor cells stably expressed a red fluorescent protein for cell tracking.
  • TCR + PD1-41 BB-transduced CD8 + T cells TCR + PD1-41 BB-transduced CD8 + T cells
  • UT untransduced CD8 + T cells.
  • Cytotoxicity mediated by T cells was determined by reduction in the absolute number of red fluorescent tumor cells over time using a live-cell imaging system. Absolute cell numbers of red fluorescent tumor cells per well at every point of measurement were calculated using the IncuCyte® software. Shown are the cell numbers (target cells/well) as mean of three replicates. One representative experiment is shown ( Figure 11A).
  • PDL1 -transduced tumor cells served to further challenge T cells and to assess cytotoxic activity mediated by TCR + PD1-41 BB-transduced CD8 + T cells in the presence of high levels of PDL1.
  • Cytotoxicity mediated by T cells was determined by the reduction of red fluorescence signal over time using a live-cell imaging system. Shown is the red fluorescence integrated intensity (Red Calibrated Unit (RCU)/pm 2 /image) at every point of measurement as mean of three replicates which was calculated using the IncuCyte® software. One representative experiment is shown ( Figure 11 B).
  • Fig. 12 shows signs of TCR functionality in CD4 + T cells.
  • TCR in combination with PD1- 41 BB CSR was transduced in CD3 + T cells derived from healthy donors (mixed population containing both CD4 + and CD8 + T cells) and utilized as effector samples in a co-culture experiment.
  • CD3 + T cells derived from healthy donors (mixed population containing both CD4 + and CD8 + T cells) and utilized as effector samples in a co-culture experiment.
  • Corresponding untransduced (UT) CD3 + T cells served as controls.
  • Co-culture experiments were established using as targets K562_A11_PDL1 cells loaded with either KRAS 7 -i 6 wild type (10‘ 5 M) or mKRAS 7 -i 6 G12V peptide (10’ 5 M and 10’ 7 M), K562_A11 cells transduced with mKRAS G12V-encoding construct and mKRAS G12V-positive DAN- G_A11 tumor cells. After overnight incubation in the presence of Brefeldin A, co-culture samples were assessed for CD3, CD8, CD4, Cpi surface expression as well as for IFN-y intracellular staining of by flow cytometry. Populations were analyzed to detect IFN-y-positive cells contained in CD8 + and in CD4 + T cells.
  • Fig. 13 shows that IFN-y release and cytotoxic response against tumor cells are mediated by TCR + PD1-41 BB-transduced CD4 + T cells.
  • TCR in combination with PD1-41 BB CSR was expressed in both CD8 + T cells and CD4 + T cells.
  • CD8 + and CD4 + TCR-expressing T cells were then enriched by fluorescence-activated cell sorting (FACS). After enrichment, T cell samples were stained with mKRAS 7 -ieG12V (10-mer) HLA-A*11 :01 multimer and analyzed by flow cytometry. Populations shown are pre-gated on live single cells. Shown one representative experiment. ( Figure 13A).
  • Enriched T cells samples were co-cultured with either mKRAS G12V-positive (DAN-G) or KRAS wild type (DU 145) which were labelled with a red fluorescent protein to allow tumor cell tracking over time.
  • mKRAS?-i6 G12V peptide-loaded tumor cell lines were used as internal positive controls.
  • Cytotoxicity mediated by T cells was determined by reduction in the absolute number of red fluorescent tumor cells over time using a live-cell imaging system. Absolute cell numbers of red fluorescent tumor cells per well at every point of measurement were calculated using the IncuCyte® software. Shown are the cell numbers (target cells/well) as mean of three replicates. Shown one representative experiment ( Figure 13C).
  • Fig. 14 shows a higher polyfunctionality of T cells co-expressing the transgenic TCR and PD1-41 BB CSR compared to T cells expressing only the transgenic TCR.
  • TCR-transduced CD8 + T cells with (TCR+PD1-41 BB) and without (TCR) co-expression of PD1-41 BB CSR were analyzed after 24 hours of co-culture with mKRAS G12V-positive DAN-G_A11_PD-L1 tumor cells using single-cell proteomic analysis of a panel of 32 secreted cytokines, chemokines, and cytotoxic molecules (IsoLight® technology). Cells secreting 2 or more cytokines were considered polyfunctional (Figure 14A).
  • Polyfunctional Strength Index (PSI) of the displayed samples was defined as the number of T cells secreting more than 2 effector molecules per cell (polyfunctional T cells) multiplied by mean fluorescence intensity (MFI) of the proteins secreted by the respective cells and categorized in proteins associated with effector, stimulatory, chemo-attractive, regulatory and inflammatory properties ( Figure 14B).
  • TCR3 PD1-41 BB The TCR of the invention can also be referred to in the Figures as “TCR3 PD1-41 BB”.
  • TCR as used herein includes native TCRs as well as TCR variants, fragments and constructs as defined herein.
  • the term thus includes heterodimers comprising TCR alpha and beta chains as well as multimers and single chain constructs; optionally comprising further domains and/or moieties.
  • the TCR In its native form, the TCR exists as a complex of several proteins on the surface of T cells.
  • the T cell receptor is composed of two (separate) protein chains, which are produced from the independent T cell receptor alpha and beta (TCR a and TCR
  • Each chain of the TCR possesses one N-terminal immunoglobulin-like (Ig)-variable (V) domain/region, one Ig-constant-like (C) domain/region, a transmembrane/cell membrane-spanning region anchoring the chain in the plasma membrane, and a short cytoplasmic tail at the C-terminal end.
  • variable regions of the alpha and beta chain are hypervariable or complementarity determining regions (CDR1alpha/beta, CDR2alpha/beta and CDR3 alpha/beta) surrounded by framework (FR) regions.
  • CDR3 is the prime determinant of antigen recognition and specificity (i.e. the ability to recognize and interact with a specific antigen), whereas CDR1 and CDR2 mainly interact with the MHC molecule presenting the antigenic peptide.
  • the TCR of the present invention is characterized by comprising i) an a chain CDR3 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 16; and/or ii) a p chain CDR3 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 4.
  • TCR comprising an a chain CDR3 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 16.
  • TCR comprising an a chain CDR3 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 4.
  • the present invention also comprise the TCR as defined herein being characterized as comprising i) an a chain CDR3 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 16 and ii) a chain CDR3 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 4.
  • the CDR3 of the present invention can in principle be combined with any of the CDR1 depicted in SEQ ID NO: 14 and/or 2 and/or with any of the CDR2 depicted in SEQ ID NO: 15 and/or 3, preferably provided that the TCR retains its ability to recognize its epitope as defined elsewhere herein, to a similar, the same or even a higher extent as the TCR evaluated in the Examples.
  • the TCR of the invention may further be provided in “isolated” or “substantially pure” form.
  • “Isolated” or “substantially pure” when used herein means that the TCR have been identified separated and/or recovered from a component of its production environment, such that the “isolated” TCR is free or substantially free of other contaminant components from its production environment that might interfere with its therapeutic or diagnostic use. Contaminant components may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. “Isolated” TCR will thus be prepared by at least one purification step removing or substantially removing these contaminant components. The aforementioned definition is equally applicable to “isolated” polynucleotides/nucleic acids, mutatis mutandis.
  • the TCR of the present invention as defined herein may further be characterized as comprising at least one (e.g. 1 , 2, 3, or 4) of the following: i) an a chain CDR 1 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 14; ii) an a chain CDR2 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83,
  • a p chain CDR1 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 15; and/or iv) a chain CDR2 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 2; and/or iv) a chain CDR2 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92
  • the present invention may also comprise the TCR as defined above being further characterized as comprising an a chain CDR 1 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined above being further characterized as comprising an a chain CDR 2 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84,
  • the present invention may also comprise the TCR as defined above being further characterized as comprising a p chain CDR1 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 2.
  • the present invention may also comprise the TCR as defined above being further characterized as comprising a p chain CDR2 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85,
  • the present invention may also comprise the TCR as defined herein further being characterized as comprising i) an a chain CDR 1 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 14; ii) an a chain CDR2 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 14; ii) an a chain CDR2 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86
  • the present invention may also comprise the TCR as defined herein further being characterized as comprising i) a p chain CDR1 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 2; ii) a p chain CDR2 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity to SEQ ID NO: 3; and iii) a chain CDR3 comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative or highly conservative amino acid substitutions as defined herein, preferably corresponding to position 1 , 2, 3, 4, 5, 6, and/or 7 of SEQ ID NO: 14; position 1 , 2, 3, 4, 5, 6, 7, and/or 8 of SEQ ID NO: 15; position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and/or 13 of SEQ ID NO: 16; position 1 , 2, 3, 4, and/or 5 of SEQ ID NO: 2; position 1 , 2, 3, 4, 5, and/or 6 of SEQ ID NO: 3; and/or position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, and/or 16 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein and further being characterized, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative or highly conservative amino acid substitutions as defined herein corresponding to position 1 , 2, 3, 4, 5, 6, and/or 7 of SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined herein, wherein the a least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 1 of SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 2 of SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 3 of SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 4 of SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 5 of SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 6 of SEQ ID NO: 14.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 7 of SEQ ID NO: 14.
  • the present invention may also comprise the TOR as defined herein and further being characterized, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 1 of SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 2 of SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 3 of SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 4 of SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 5 of SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 6 of SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 7 of SEQ ID NO: 14.
  • the present invention may also comprise the TCR as defined herein and further being characterized, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative or conservative amino acid substitutions as defined herein corresponding to position 1 , 2, 3, 4, 5, 6, 7, and/or 8 of SEQ ID NO: 15.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 1 of SEQ ID NO: 15.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 2 of SEQ ID NO: 15.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 3 of SEQ ID NO: 15.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 4 of SEQ ID NO: 15.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 5 of SEQ ID NO: 15.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 6 of SEQ ID NO: 15.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 7 of SEQ ID NO: 15.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 8 of SEQ ID NO: 15.
  • the present invention may also comprise the TCR as defined herein and further being characterized, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 1 of SEQ ID NO: 15.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 2 of SEQ ID NO: 15.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 3 of SEQ ID NO: 15.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 4 of SEQ ID NO: 15.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 5 of SEQ ID NO: 15.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 6 of SEQ ID NO: 15.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 7 of SEQ ID NO: 15.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96,
  • the present invention may also comprise the TCR as defined herein and further being characterized, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative or conservative amino acid substitutions as defined herein corresponding to position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and/or 13 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 1 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97,
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 3 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 4 of SEQ ID NO: 16.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 5 of SEQ ID NO: 16.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 6 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 7 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 8 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 9 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 10 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 11 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 12 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 13 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein and further being characterized, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 1 of SEQ ID NO: 16.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 2 of SEQ ID NO: 16.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 3 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 4 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 5 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 6 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 7 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 8 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 9 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 10 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 11 of SEQ ID NO: 16.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96,
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 13 of SEQ ID NO: 16.
  • the present invention may also comprise the TOR as defined herein and further being characterized, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative or conservative amino acid substitutions as defined herein corresponding to position 1 , 2, 3, 4, and/or 5 of SEQ ID NO: 2.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 1 of SEQ ID NO: 2.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 2 of SEQ ID NO: 2.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97,
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 4 of SEQ ID NO: 2.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 5 of SEQ ID NO: 2.
  • the present invention may also comprise the TCR as defined herein and further being characterized, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 1 of SEQ ID NO: 2.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 2 of SEQ ID NO: 2.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 3 of SEQ ID NO: 2.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 4 of SEQ ID NO: 2.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 5 of SEQ ID NO: 2.
  • the present invention may also comprise the TCR as defined herein and further being characterized, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative or conservative amino acid substitutions as defined herein corresponding to position 1 , 2, 3, 4, 5 and/or6 of SEQ ID NO: 3.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 1 of SEQ ID NO: 3.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 2 of SEQ ID NO: 3.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 3 of SEQ ID NO: 3.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 4 of SEQ ID NO: 3.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 5 of SEQ ID NO: 3.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 6 of SEQ ID NO: 3.
  • the present invention may also comprise the TCR as defined herein and further being characterized, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 1 of SEQ ID NO: 3.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 2 of SEQ ID NO: 3.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 3 of SEQ ID NO: 3.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 4 of SEQ ID NO: 3.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 5 of SEQ ID NO: 3.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96,
  • the present invention may also comprise the TCR as defined herein and further being characterized, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative or conservative amino acid substitutions as defined herein corresponding to position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, and/or 16 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 1 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97,
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 3 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 4 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 5 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 6 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 7 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 8 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 9 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 10 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 11 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 12 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 13 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 14 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86,
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 15 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises conservative amino acid substitutions as defined herein corresponding to position 16 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein and further being characterized, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 1 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 2 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87,
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 3 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 4 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 5 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 6 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 7 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 8 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 9 of SEQ ID NO: 4.
  • the present invention may also comprise the TOR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 10 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 11 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 12 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 13 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 14 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 15 of SEQ ID NO: 4.
  • the present invention may also comprise the TCR as defined herein, wherein the at least 80%, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99% or even 100% identity comprises highly conservative amino acid substitutions as defined herein corresponding to position 16 of SEQ ID NO: 4.
  • the present invention comprises the TCR as defined herein, further comprising i) an a chain CDR 1 comprising the amino acid sequence of SEQ ID NO: 14; ii) an a chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15; iii) an a chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16; iv) a p chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2; v) a chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and vi) a p chain CDR3 comprising the amino acid sequence of SEQ ID NO: 4 (see Table 2). [0063] Complete variable region:
  • the present invention may further provide a TCR comprising a TCR a chain variable region and/or a TCR p chain variable region. Said a and chain variable region sequences are also shown in Table 2.
  • the present invention may further comprise the TCR as defined herein comprising i) an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or even 100% identity to SEQ ID NO: 20; and/or ii) an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or even 100% identity to SEQ ID NO: 8.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or even 100% identity to SEQ ID NO: 20.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or even 100% identity to SEQ ID NO: 8.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 85 % identity to SEQ ID NO: 20.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 85 % identity to SEQ ID NO: 8.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 90 % identity to SEQ ID NO: 20.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 90 % identity to SEQ ID NO: 8.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 95 % identity to SEQ ID NO: 20.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 95 % identity to SEQ ID NO: 8.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 98 % identity to SEQ ID NO: 20.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 98 % identity to SEQ ID NO: 8.
  • the present invention comprises the TCR as defined herein, further comprising i) an a chain variable region comprising the amino acid sequence of SEQ ID NO: 20, and/or ii) a p chain variable region comprising the amino acid sequence of SEQ ID NO: 8.
  • the TCR may further comprise a constant (C) region in its a and/or p chain.
  • the constant region can be a human constant region or derived from another species, yielding a “chimeric” TCR.
  • human alpha and/or beta chains can be replaced by their murine counterparts (“murinization”) which has been found to enhance surface expression of human TCRs by supporting preferential pairing of the TCR a and p chains, and a more stable association with the CD3 co-receptor.
  • the present invention may comprise the TCR as defined herein further comprising a (minimally) murinized constant region in its a and/or chain.
  • the present invention may further comprise the TCR as defined herein comprising i) an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or even 100% identity to SEQ ID NO: 22; and/or ii) an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or even 100% identity to SEQ ID NO: 10.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or even 100% identity to SEQ ID NO: 22.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or even 100% identity to SEQ ID NO: 10.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 85 % identity to SEQ ID NO: 22.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 85 % identity to SEQ ID NO: 10.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 90 % identity to SEQ ID NO: 22.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 90 % identity to SEQ ID NO: 10.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 95 % identity to SEQ ID NO: 22.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 95 % identity to SEQ ID NO: 10.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 98 % identity to SEQ ID NO: 22.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 98 % identity to SEQ ID NO: 10.
  • the present invention comprises the TCR as defined herein, further comprising i) an a chain constant region comprising the amino acid sequence of SEQ ID NO: 22, and/or ii) a p chain constant region comprising the amino acid sequence of SEQ ID NO: 10 (see Table 2).
  • the present invention may further comprise the TCR as defined herein comprising i) an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or even 100% identity to SEQ ID NO: 24; and/or ii) an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or even 100% identity to SEQ ID NO: 12.
  • the present invention may further comprise the TOR as defined herein comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or even 100% identity to SEQ ID NO: 24.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 80 %, such as at least 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or even 100% identity to SEQ ID NO: 12.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 85 % identity to SEQ ID NO: 24.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 85 % identity to SEQ ID NO: 12.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 90 % identity to SEQ ID NO: 24.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 90 % identity to SEQ ID NO: 12.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 95 % identity to SEQ ID NO: 24.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 95 % identity to SEQ ID NO: 12.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 98 % identity to SEQ ID NO: 24.
  • the present invention may further comprise the TCR as defined herein comprising an amino acid sequence having at least 98 % identity to SEQ ID NO: 12.
  • the present invention comprises the TCR as defined herein, further comprising i) an a chain comprising the amino acid sequence of SEQ ID NO: 24; and/or ii) a p chain comprising the amino acid sequence of SEQ ID NO: 12 (see Table 2).
  • sequence identity indicates the extent to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is often expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared.
  • KRAS also referred to as GTPase KRas, V-Ki-Ras2 Kirsten rat sarcoma viral oncogene, or KRAS2
  • KRAS variant A There are two transcript variants of KRAS: KRAS variant A and KRAS variant B.
  • references to "KRAS" refer to both variant A and variant B, unless specified otherwise. Without being bound to a particular theory or mechanism, it is believed that, when mutated, KRAS may be involved in signal transduction early in the oncogenesis of many human cancers. A single amino acid substitution may activate the mutation.
  • mutated KRAS When activated, mutated KRAS binds to guanosine-5 '-triphosphate (GTP) and converts GTP to guanosine 5 '-diphosphate (GDP).
  • GTP guanosine-5 '-triphosphate
  • GDP guanosine 5 '-diphosphate
  • the mutated KRAS protein product may be constitutively activated.
  • Mutated KRAS protein may be expressed in any of a variety of human cancers such as, for example, pancreatic (e.g., pancreatic carcinoma), colorectal, lung (e.g., lung adenocarcinoma), endometrial, ovarian (e.g., epithelial ovarian cancer), and prostate cancers.
  • the TOR of the present invention may further have antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 1 (VVVGAVGVGK).
  • Such epitope is mutated and refers to a mutated KRAS peptide which generally corresponds to positions 7-16 of the unmutated, wild type (WT) KRAS protein amino acid sequence of SEQ ID NO: 37 with the exception that for SEQ ID NO: 1 , the glycine at position 12 of SEQ ID NO: 37 (which refers to position 6 in SEQ ID NO: 1) is substituted with valine, respectively.
  • WT wild type
  • the mutated KRAS amino acid sequence SEQ ID NO: 1 is also referred to herein as "mKRAS G12V - 10mer.”
  • the TOR may further have antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 34 (VVVGACGVGK).
  • epitope is mutated and refers to a mutated KRAS peptide which generally corresponds to positions 7-16 of the unmutated, wild type (WT) KRAS protein amino acid sequence of SEQ ID NO: 37 with the exception that for SEQ ID NO: 34, the glycine at position 12 of SEQ ID NO: 37 (which refers to position 6 in SEQ ID NO: 34) is substituted with cysteine, respectively.
  • the mutated KRAS amino acid sequence SEQ ID NO: 34 is also referred to herein as "mKRAS G12C - 10mer.”
  • the TCR of the present invention may be capable of recognizing and binding either mKRAS G12V or mKRAS G12C or both in combination, but not the WT KRAS peptide.
  • the TCR may further have antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of a fragment of the amino acid sequence of SEQ ID NO: 1 , or a variant of the amino acid sequence of SEQ ID NO: 1 comprising one or more (e.g.
  • the TCR may further have antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of a fragment of the amino acid sequence of SEQ ID NO: 34, or a variant of the amino acid sequence of SEQ ID NO: 34 comprising one or more (e.g. one or two) conservative amino acid substitutions (preferably corresponding to position 1 , 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of SEQ ID NO: 34).
  • Such mutated epitope refers to a mutated KRAS peptide which generally corresponds to positions 8-16 of the unmutated, wild type (WT) KRAS protein amino acid sequence of SEQ ID NO: 37 with the exception that for SEQ ID NO: 35, the glycine at position 12 of SEQ ID NO: 37 (which refers to position 5 in SEQ ID NO: 35) is substituted with valine, respectively.
  • the mutated KRAS amino acid sequence SEQ ID NO: 35 is also referred to herein as "mKRAS G12V - 9mer.”
  • the fragment as defined herein may also comprise the mutated epitope comprising the amino acid sequence of VVGACGVGK (SEQ ID NO: 36).
  • Such mutated epitope refers to a mutated KRAS peptide which generally corresponds to positions 8-16 of the unmutated, wild type (WT) KRAS protein amino acid sequence of SEQ ID NO: 37 with the exception that for SEQ ID NO: 36, the glycine at position 12 of SEQ ID NO: 37 (which refers to position 5 in SEQ ID NO: 36) is substituted with cysteine, respectively.
  • the mutated KRAS amino acid sequence SEQ ID NO: 36 is also referred to herein as "mKRAS G12C - 9mer.”
  • variant may refer to a polypeptide having specific activity as described herein comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.) positions.
  • a “variant thereof” as used herein, may particularly comprise the mutated KRAS peptides as mentioned above with one or more positions being substituted.
  • substitution means replacement of the amino acid occupying a position with a different amino acid
  • a deletion means removal of the amino acid occupying a position
  • an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.
  • “silent” mutations mean base substitutions within a nucleic acid sequence which do not change the amino acid sequence encoded by the nucleic acid sequence. “Conservative or equivalent” substitutions (or mutations) mean substitutions as listed as “Exemplary Substitutions” in Table 1 below. “Highly conservative” substitutions as used herein mean substitutions as shown under the heading “Preferred Substitutions” in Table 1 below. [0082] Table 1 : Amino acid substitutions.
  • amino acid typically refers to an amino acid having its art recognized definition such as an amino acid selected from the group consisting of: alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gin or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (He or I): leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); pro line (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Vai or V), although modified, synthetic, or rare amino acids may be used
  • polypeptide variants (or mutants) of the invention may be referred to by the use of the following nomenclature: original amino acid(s): position(s): substituted amino acid(s). According to this nomenclature, for instance the substitution of G (glycine) for V (valine) in position 12 can be shown as “G12V”.
  • position when used in accordance with the disclosure means the position of either an amino acid within an amino acid sequence depicted herein or the position of a nucleotide within a nucleic acid sequence depicted herein.
  • corresponding as used herein also includes that a position is not only determined by the number of the preceding nucleotides/amino acids, but is rather to be viewed in the context of the circumjacent portion of the sequence. Accordingly, the position of a given amino acid or nucleotide in accordance with the disclosure may vary due to deletion or addition of amino acids or nucleotides elsewhere in the sequence.
  • the TCR may have further antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 35. In a further embodiment of the invention, the TCR may have further antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 36.
  • the TCR may have further antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 1 comprising one conservative amino acid substitution, preferably corresponding to position 1 , 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of SEQ ID NO: 1.
  • the TCR may have further antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 1 comprising one highly conservative amino acid substitution, preferably corresponding to position 1 , 2, 3, 4, 5, 6, 7, 8,
  • the TCR may have further antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 1 comprising one conservative amino acid substitution corresponding to position 1 , 2, 3, 4, 5, 7, 8, 9 and/or
  • the TCR may have further antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 1 comprising one highly conservative amino acid substitution corresponding to position 1 , 2, 3, 4, 5, 7, 8, 9 and/or 10 of SEQ ID NO: 1.
  • the TCR may further have antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 1 comprising two conservative amino acid substitutions, preferably corresponding to position 1 , 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of SEQ ID NO: 1.
  • the TCR may further have antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 1 comprising two highly conservative amino acid substitutions, preferably corresponding to position 1 , 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of SEQ ID NO: 1.
  • the TOR may have further antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 1 comprising two conservative amino acid substitutions corresponding to position 1 , 2, 3, 4, 5, 7, 8, 9 and/or 10 of SEQ ID NO: 1.
  • the TCR may have further antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 1 comprising two highly conservative amino acid substitutions corresponding to position 1 , 2, 3, 4, 5, 7, 8, 9 and/or 10 of SEQ ID NO: 1.
  • the TCR may further have antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 34 comprising one conservative amino acid substitution, preferably corresponding to position 1 , 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of SEQ ID NO: 34.
  • the TCR may further have antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 34 comprising one highly conservative amino acid substitution, preferably corresponding to position 1 , 2, 3, 4, 5, 6, 7, 8,
  • the TCR may further have antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 34 comprising one conservative amino acid substitution corresponding to position 1 , 2, 3, 4, 5, 7, 8, 9 and/or
  • the TCR may further have antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 34 comprising two conservative amino acid substitutions, preferably corresponding to position 1 , 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of SEQ ID NO: 34.
  • the inventive TCR has antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 34 comprising two highly conservative amino acid substitutions, preferably corresponding to position 1 , 2, 3, 4, 5, 6, 7, 8, 9 and/or 10 of SEQ ID NO: 34.
  • the TCR may further have antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 34 comprising two conservative amino acid substitutions corresponding to position 1 , 2, 3, 4, 5, 7, 8, 9 and/or 10 of SEQ ID NO: 34.
  • the TCR may further have antigenic specificity to an epitope presented in the context of a MHC class I molecule, the epitope comprising or consisting of the amino acid sequence of SEQ ID NO: 34 comprising two highly conservative amino acid substitutions corresponding to position 1 , 2, 3, 4, 5, 7, 8, 9 and/or 10 of SEQ ID NO: 34.
  • antigenic specificity means that the TCR can specifically bind to and immunologically recognize mutated target, e.g., mutated KRAS peptide as defined herein, with high avidity.
  • Effector host cells expressing the TCR as described herein are envisaged to bind to their antigenic target (i.e. the epitope(s) as defined herein) with a high functional avidity.
  • functional avidity refers to the capability of TCR expressing cells (in particular T-cells expressing native TCRs as described herein) to respond in vitro to a given concentration of a ligand and is thought to correlate with the in vivo effector capacity of TCR expressing cells.
  • TCR expressing cells with high functional avidity respond in in vitro tests to very low antigen doses, while such cells of lower functional avidity require higher amounts of antigen before they mount an immune response similar to that of high-avidity TCR expressing cells.
  • the functional avidity can be therefore considered as a quantitative determinant of the activation threshold of a TCR expressing cell. It is determined by exposing such cells in vitro to different amounts of cognate antigen. TCR expressing cells with high functional avidity respond to low antigen doses.
  • a TCR expressing cell will typically be considered to bind with “high” functional avidity to its antigenic target (having antigenic specificity for the mutated epitope(s)) if a) it secretes at least about 200 pg/mL or more (e.g., 200 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, 5,000 pg/mL or more, 7,000 pg/mL or more, 10,000 pg/mL or more, or 20,000 pg/mL or more, or a range defined by any two of the foregoing values) of interferon gamma (IFN-gamma) upon co-culture with antigennegative HLA-A*11 expressing target cells loaded with a low concentration of the mutated target peptide ranging
  • Cells expressing the TCR of the invention may also secrete IFN-y upon coculture with antigen-negative HLA-A1T target cells pulsed with higher concentrations of mutated target peptide.
  • Other methods to determine specific binding of the TOR of the invention may include the 51Cr-release assay described by Gertner-Dardenne et al. J Immunol 188(9): 4701-4708, CD107a/b mobilization described by Leisegang et al., Clin. Cancer Res 2010. 16: 2333-2343 and peptide: MHC multimer binding analyses described by Wilde et al., J Immunol 2012; 189:598-605.
  • a TCR may be considered to have "antigenic specificity" for a mutated target if T cells expressing the TCR secrete at least twice as much IFN-y upon co-culture with (a) antigen-negative HLA-A1T target cells pulsed with a low concentration of mutated target peptide or (b) antigen-negative HLA-A1 T target cells into which a nucleotide sequence encoding the mutated target has been introduced such that the target cell expresses the mutated target as compared to the amount of IFN-y expressed by a negative control.
  • the negative control may be, for example, (i) T cells expressing the TCR, co-cultured with (a) antigen-negative HLA-A1T target cells pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide with a different sequence from the mutated target peptide) or (b) antigen-negative HLA-A11 + target cells into which a nucleotide sequence encoding an irrelevant peptide has been introduced such that the target cell expresses the irrelevant peptide, or (ii) untransduced T cells (e.g., derived from PBMC, which do not express the TCR) co-cultured with (a) antigen-negative HLA-A11 + target cells pulsed with the same concentration of mutated target peptide or (b) antigen-negative HLA-A1T target cells into which a nucleotide sequence encoding the mutated target has been introduced such that the target cell expresses the mutated target.
  • a TCR may be considered to have "antigenic specificity" for a mutated target if at least twice as many of the numbers of T cells expressing the TCR secrete IFN-y upon co-culture with (a) antigen-negative HLA-A1T target cells pulsed with a low concentration of mutated target peptide or (b) antigen-negative HLA-A11 + target cells into which a nucleotide sequence encoding the mutated target has been introduced such that the target cell expresses the mutated target as compared to the numbers of negative control T cells that secrete IFN-y.
  • concentration of peptide and the negative control may be as described herein.
  • the numbers of cells secreting IFN-y may be measured by methods known in the art such as, for example, ELISPOT.
  • the TCR of the present invention specifically binds to the abovementioned epitopes.
  • the term “specific(ally) binding” generally indicates that a TCR binds via its antigen binding site more readily to its intended epitope than to a random, unrelated non-target antigen.
  • the term “specifically binds” indicates that the binding specificity of the TCR will be at least about 5-fold, preferably 10-fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for its epitope than its binding specificity for a non-target antigen.
  • each embodiment concerning the antigenic specificity for the abovementioned epitope(s) as defined herein may also be applicable to the TCR of the invention being able to recognize said mutated epitope(s) in the context of a MHC class II molecule.
  • the TCR of the invention is able to recognize said mutated epitope (antigenic target), e.g., mutated KRAS, in a MHC class I and/or class Il-dependent manner, meaning the epitope(s) is/are presented in the context of a MHC class I and/or class II molecule.
  • MHC class l-dependent manner means that the TCR elicits an immune response upon binding to a mutated target, e.g., mutated KRAS peptide as defined herein, 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.
  • MHC class I molecule covers any HLA-A, HLA-B and/or HLA- C molecules.
  • MHC class Il-dependent manner means that the TCR elicits an immune response upon binding to a mutated target, e.g., mutated KRAS peptide as defined herein, within the context of an MHC class II molecule.
  • the TCR as defined herein may further have antigenic specificity for the mutated epitope as defined above presented in the context of an HLA-A molecule. In a more preferred embodiment of the invention, the TCR as defined herein may further have antigenic specificity for the mutated epitope as defined above presented in the context of an HLA-A*03 molecule. In an even more preferred embodiment of the invention, the TCR as defined herein may further have antigenic specificity for the mutated epitope as defined above presented in the context of an HLA-A*11 molecule.
  • the TCR as defined herein may further have antigenic specificity for the mutated epitope as defined above presented in the context of at least any one of an HLA-A*11 :01 molecule, an HLA-A*11 :02 molecule, an HLA-A*11 :03 molecule, or an HLA- A*11 :04 molecule.
  • novel TCR that recognizes its cognate peptide as defined above in the context of HLA-A*11 :01 might also recognize the peptide presented by another HLA-A*11 subtype such as HLA-A*11 :02, HLA-A*11 :03, HLA- A*11 :04 and/or by other H LA alleles of the HLA-A*03 superfamily, where HLA-A*11 is one of many members from (see Figure 6).
  • HLA-A*03 molecule may also refer to an “any HLA-molecule of the HLA- A*03 super family, such as HLA-A*11”.
  • epitope may refer to a part of an antigen (antigenic target) that is recognized by the immune system, specifically by antibodies, B cells, or T cells.
  • the epitope is the specific piece of the antigen to which the TCR binds to (particularly with the CDR3 as the prime determinant of antigen recognition and specificity) and is defined herein as mentioned elsewhere above.
  • human leukocyte antigen or “HLA” may refer to any of various (polymorphic) proteins that are encoded by genes of the major histocompatibility complex in humans and are found on the surface of many cell types (such as white blood cells).
  • HLA class I alleles include HLA-A, HLA-B and HLA-C alleles (http://hla.alleles.org/genes/index.html).
  • MHC human major histocompatibility complex
  • class II centromeric
  • class III centromeric
  • class I telomeric
  • MHC major histocompatibility complex
  • TCR T-cell receptor
  • the term “allele” may refer to any of the alternative forms of a gene (e.g., human HLA gene) that may occur at a given locus. Each HLA allele name has a unique number corresponding to up to four sets of digits separated by colons. The length of the allele designation is dependent on the sequence of the allele and that of its nearest relative. [00103] HLA-A alleles may be clustered into superfamilies on the basis of structural motifs and peptide binding specificity. These superfamilies reflect the broad characteristics of anchor motifs present in the peptides that they bind and present.
  • an HLA allele name comprises the following elements: “HLA” prefix, hyphen (“-“) used to separate gene name from HLA prefix, gene name (e.g., “A”, “B”, “C”), separator “*”, field 1 depicting allele group (e.g., 03, 11 , 01 , 26, 24, 32, 02, 07, 15, 58, 40, 35, 08, 04, 07, 16, 03, 07 or 06 etc.), field separator field 2 depicting specific HLA protein (e.g., 01 or 02 etc.).
  • Exemplary human class I HLA genes of the present invention include HLA-A (Class I a-chain), HLA-B (Class I a-chain) and HLA-C (Class I a-chain).
  • human class I HLA alleles of the present invention include HLA-E, HLA-F, HLA-G alleles as well as pseudogenes HLA-H, HLA-J, HLA-K, HLA-L, HLA-N, HLA-P, HLA-S, HLA-T, HLA-U, HLA-V, HLA-W and HLA-Y (http://hla.alleles.org/alleles/class1.html).
  • Exemplary alleles of the present invention include any human class I and class II HLA alleles, e.g., selected from the group consisting of class I HLA-A, HLA-B and HLA-C alleles.
  • Such alleles may be selected from the following exemplary human HLA allele groups: HLA-A*03, HLA-A*11, HLA-A*01, HLA-A*26, HLA-A*24, HLA-A*32 and HLA-A*02; HLA-B*07, HLA-B*15, HLA-B*58, HLA-B*40, HLA-B*35 and HLA-B*08; HLA-C*04, HLA-C*07, HLA-C*16, HLA-C*03, HLA-C*07 and HLA-C*06 alleles.
  • Exemplary human HLA alleles of the present invention include but are not limited to: HLA-A*03:01, HLA-A*30:01, HLA-A*31 :01, HLA-A*33:01, HLA- A*68:01 , HLA-A*11 :01 , HLA-A*11 :02, HLA-A*11 :03, HLA-A*11 :04 alleles.
  • HLA-A, HLA-B or HLA-C allele Any human class I HLA allele may be within the meaning of the present invention.
  • HLA-A, HLA-B or HLA-C allele Any human class II HLA allele may be within the meaning of the present invention.
  • Exemplary class II HLA alleles of the present invention further include but are not limited to alleles of HLA- DRA, HLA-DRB1 , HLA-DRB2-9, HLA-DQA1 , HLA-DQA2, HLA-DQB1, HLA-DPA1 , HLA- DPB1 , HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB proteins (e.g., http://hla.alleles.org/alleles/class2.html). Further human class I and class II HLA alleles may also be queried at the IPD-IMGT/HLA Database as described above using “Allele Query Tool”.
  • HLA-A3 superfamily is of high importance within the HLA-A allotype.
  • HLA-A*11 forms a part of the HLA-A3 superfamily which further comprises, but is not limited to, HLA-A*03:01 , HLA-A*30:01 , HLA-A*31 :01 , HLA- A*33:01 and HLA-A*68:01 , characterized by recognition of peptides with small or aliphatic residues in position 2 and basic residues at their C-terminus. All currently classified HLA-A*11 serotypes fall into the A3 superfamily (Sidney et al., 2008, BMC Immunology; 9(1)).
  • the term “functionally expressed” may refer to HLA allele/s that is/are expressed (e.g., on cell surface) and functioning as HLA protein/s, e.g., presenting peptides derived from endogenous protein (e.g., from inside the cell).
  • the term “functionally expressed” may in particularly refer to HLA allele/s that is/are expressed (e.g., on cell surface, e.g., of the mammal of the present invention) and functioning as HLA protein/s, e.g., presenting peptides derived from endogenous protein (e.g., from inside the cell, e.g., of the mammal of the present invention) in that the corresponding MHC I polypeptides are expressed on the surface of cells, e.g., of the mammal of the present invention, and present MHC antigens to which the non-human mammal providing an antigen-specific CD8 + T cell response and, optionally, providing an antigen-specific CD4 + T cell response.
  • the present invention may further comprise the TCR as defined herein, wherein the TCR has said antigenic specificity for said epitope(s) as defined herein in the absence of the CD8 co-receptor.
  • the CD8 co-receptor is known to directly bind to HLA class I molecules and to be critical for the development of CD8 + T cells.
  • CD8 co-receptor stabilizes the binding of a TCR to the HLA: peptide complex and facilitates early events of the TCR signaling cascade.
  • the present inventors showed that the TCR of the invention as defined elsewhere herein restricted to MHC class I molecules can induce T cell activation upon antigen encounter without the need of CD8-costimulation (see Figure 12).
  • Chimeric co-stimulatory receptor(s) may refer to a chimeric co-stimulatory molecule(s) or chimeric receptor(s), which may be synthetic and/or engineered cell-surface receptor(s) designed to enhance the activation and/or function of immune cell(s), particularly T-cells, preferably in the context of cancer immunotherapy.
  • These receptors are chimeric in nature because they combine components, fragments and/or portions from different receptors (e.g., naturally occurring, recombinant and/or synthetic) to create a novel receptor with specific properties (e.g., therapeutic properties).
  • the structure of a chimeric co-stimulatory receptor of the present invention comprises three main components: (1) Extracellular Domain: this portion may be responsible for recognizing and/or binding to a specific target molecule, e.g., on cancer cell(s) and/or other cells of interest. It can be derived from various receptors (e.g., PD-1), such as antibodies and/or ligands, that are capable of binding to the target.
  • PD-1 Protein 1
  • SEQ ID NO: 28 a cell surface receptor protein that plays a role in the regulation of the immune system.
  • the extracellular domain might be derived from a monoclonal antibody targeting a cancer-specific antigen.
  • the extracellular domain might be derived from CD40L;
  • Transmembrane Domain (TMD) this region can be derived from a natural cell surface receptor and/or can anchor the chimeric receptor in the cell membrane;
  • TMD of the present invention might be derived from a PD-1 and further preferably comprising or consisting of SEQ ID NO: 30;
  • Intracellular Domain the intracellular domain is where the co-stimulatory signalling activity is conferred.
  • the intracellular domain of the present invention might be derived from 4-1 BB protein and further preferably comprising or consisting of SEQ ID NO: 32.
  • the intracellular domain of the present invention can be derived from a co-stimulatory receptor, such as CD28 and/or 4-1 BB (CD137), which can provide activation signals to T cells.
  • a co-stimulatory receptor such as CD28 and/or 4-1 BB (CD137)
  • CD137 co-stimulatory receptor
  • the chimeric receptor of the present invention can be designed to provide strong activation signals to the immune cell upon binding its target.
  • the present invention relates to fusion proteins comprising (a) an extracellular domain containing a polypeptide derived from PD-1 or CD40L at its N-terminus; (b) a transmembrane domain; and (c) an intracellular domain containing a polypeptide derived from 4-1 BB or CD28 at its C-terminus. Also, fusion proteins with CD28 at the N-terminus and CD40L at the C- terminus are envisaged.
  • the present invention relates to a fusion protein comprising: (a) an extracellular domain (ECD) containing a polypeptide derived from PD-1 or CD40L at its N-terminus; (b) a transmembrane domain (TMD); and (c) an intracellular domain (ICD) containing a polypeptide derived from 4-1 BB or CD28 at its C-terminus.
  • ECD extracellular domain
  • TMD transmembrane domain
  • ICD intracellular domain
  • the extracellular domain (ECD) contains a polypeptide derived from PD-1 at its N-terminus
  • the intracellular domain (ICD) contains a polypeptide derived from 4-1 BB at its C-terminus and vice versa.
  • the intracellular domain contains a polypeptide derived from CD28 at its C-terminus and vice versa.
  • the ICD is located N-terminally of the TMD, while the TMD is located at the very C-terminus of the fusion protein.
  • the present invention relates to a fusion (i.e., chimeric) protein comprising the extracellular domain (ECD) of CD40L and the intracellular signaling domain (ICD) of CD28.
  • ECD extracellular domain
  • ICD intracellular signaling domain
  • a chimeric co-stimulatory receptor-expressing T cell encounters a cell expressing the target molecule (e.g., a cancer cell)
  • the receptor's extracellular domain binds to the target, and the intracellular domain delivers co-stimulatory signals to the T cell.
  • This activation can enhance the immune cell's ability to recognize, attack, and/or eliminate the target cells, particularly in the context of cancer immunotherapy.
  • PD1-41 BB may refer to a fusion polypeptide comprising a PD- 1-derived polypeptide (e.g., wherein said PD-1 is also known as Programmed Cell Death Protein 1 , e.g., having UniProt Accession Number: Q15116) and a 4-1 BB-derived polypeptide (e.g., wherein said 4-1 BB is also known as CD137 or TNFRS9, which can be used interchangeably, e.g., having UniProt Accession Number: Q07011).
  • PD- 1-derived polypeptide e.g., wherein said PD-1 is also known as Programmed Cell Death Protein 1 , e.g., having UniProt Accession Number: Q15116
  • 4-1 BB-derived polypeptide e.g., wherein said 4-1 BB is also known as CD137 or TNFRS9, which can be used interchangeably, e.g., having UniProt Accession Number: Q07011.
  • the term "derived from” may particularly mean that the polypeptide contained in the extracellular domain comprises at least a part of PD-1 protein (e.g., human PD-1), preferably the extracellular domain of PD-1 , respectively.
  • the chimeric co-stimulatory receptor comprising an extracellular domain derived from PD-1 has binding activity for PD-L1 , PD-L2 or other inhibitory ligands of PD-1.
  • the term “derived from” PD-1 also allows that up to 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids are substituted, deleted, and/or inserted compared to a native (or parent) sequence of PD-1 (e.g.
  • the term "derived from” may particularly mean that the polypeptide contained in the intracellular domain comprises at least a part of 4-1 BB protein (e.g., human 4-1 BB), preferably the intracellular domain of 4-1 BB, respectively.
  • the chimeric co-stimulatory receptor comprising an intracellular domain derived from 4-1 BB is capable of increasing the proliferation rate of a T cell expressing said chimeric co- stimulatory receptor upon stimulation with PD-L1 , PD-L2 or another inhibitory ligand of PD-1 and/or is capable of increasing the effector function (such as increased IFN-y release and/or increased cytotoxicity) of a T cell expressing said chimeric co-stimulatory receptor compared to a corresponding T cell not expressing the chimeric co-stimulatory receptor.
  • 4-1 BB also allows that up to 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids are substituted, deleted, and/or inserted compared to a native (or parent) sequence of 4-1 BB (human or murine, preferably human 4-1 BB) or part thereof (e.g., intracellular domain).
  • a PD-1 -4-1 BB fusion polypeptide as described herein, also referred to as PD1- 41 BB fusion, can refer to a chimeric protein resulting from a fusion of the PD-1 and 4-1 BB proteins and/or fragment(s) and/or portion(s) thereof.
  • a fusion protein can be associated and/or used with/for cancer immunotherapy and/or is designed to harness the immune system to target cancer cells more effectively.
  • the PD-1 portion of the fusion protein may allow the fusion polypeptide of the present invention to bind to PD-L1 and/or PD-L2 ligands on the surface of cancer cells and/or antigen-presenting cells.
  • PD-1 binding to its ligands normally sends inhibitory signals to T cells, preventing them from attacking these cells.
  • this interaction may be modified.
  • the 4-1 BB portion of the fusion protein may provide a co- stimulatory signal to T cells when engaged.
  • This co-stimulatory signal may help activate and/or enhance the immune response against the cancer cells, overcoming the inhibitory effects of the PD-1 portion.
  • the fusion protein of the present invention may combine the checkpoint-blocking capability of PD-1 with the immune-activating potential of 4-1 BB.
  • a PD-1-4-1 BB fusion of the present invention is capable of enhancing the antitumor immune response, e.g., by simultaneously blocking the inhibitory signals mediated by PD-1 and providing a strong co-stimulatory signal through 4-1 BB.
  • This approach is advantageous for use in immunotherapy (e.g., for cancer treatment), as it may overcome some of the resistance mechanisms that cancer cells often use to evade the immune system.
  • fusion proteins comprising or consisting of the extracellular domain (ECD) derived from PD-1 , a transmembrane domain (TMD, e.g., derived from PD-1), and the intracellular domain (ICD) derived from 4-1 BB are also referred to herein as “PD-1 :4-1 BB” or “PD-1 :BB”.
  • fusion proteins comprising or consisting of the extracellular domain (ECD) derived from PD-1 , a transmembrane domain (TMD), and the intracellular domain (ICD) derived from CD28 are also referred to herein as “PD-1 :CD28”.
  • CD28 may refer to another cell surface receptor found on T cells, and it plays a co-stimulatory role in the activation of T cells.
  • CD28 signalling helps promote T cell activation and proliferation when engaged by its ligands, such as B7-1 (CD80) and B7-2 (CD86), on antigen-presenting cells.
  • fusion proteins comprising or consisting of the extracellular domain (ECD) derived from CD40L (e.g., having UniProt Accession Number: P29965), a transmembrane domain (TMD), and the intracellular domain (ICD) derived from CD28 are also referred to herein as “CD40L:CD28”.
  • ECD extracellular domain
  • TMD transmembrane domain
  • ICD intracellular domain
  • PD-1 e.g., having UniProt Accession Number: Q15116
  • Q15116 UniProt Accession Number: Q15116
  • PD-1 binds to its ligands PD-L1 or PD-L2, which can be found on other cells, it sends inhibitory signals to the T cell, essentially putting the brakes on the immune response. This mechanism helps prevent the immune system from attacking healthy cells and tissues, but it can also be exploited by cancer cells to evade immune surveillance.
  • 4-1 BB also known as CD137 or TNFRS9, which can be used interchangeably, e.g., having UniProt Accession Number: Q07011
  • 4-1 BB is another cell surface receptor, but it has a different role than PD-1.
  • 4-1 BB is typically found on activated T cells and natural killer (NK) cells.
  • NK natural killer
  • extracellular domain also known as the “extracellular region” or “extracellular portion” may refer to a structural component of protein(s), which can be found at the outer surface of cells and/or within the extracellular matrix. This region of a protein is preferably located outside the cell membrane and/or the confines of a cellular compartment, e.g., where it may play a role in various biological functions, including cell signalling, recognition, and/or interaction with other molecules.
  • the extracellular domain of the present invention is a polypeptide derived from the PD-1 (e.g., human PD-1 , e.g., comprising or consisting of SEQ ID NO: 28 or a variant thereof).
  • TMD transmembrane domain
  • Integral membrane proteins are a class of proteins that are embedded within the lipid bilayer of a biological membrane, such as the cell membrane (e.g., plasma membrane) and/or the membranes of organelles like the endoplasmic reticulum and/or mitochondria.
  • TMD of the present invention is a polypeptide derived from the PD-1 (e.g., human PD-1, e.g., comprising or consisting of SEQ ID NO: 30 or a variant thereof).
  • intracellular domain which can also be referred to as “cytoplasmic domain”, may refer to a structural component of integral membrane proteins. It may refer to the portion of the protein that is located on the cytoplasmic side of a biological membrane, such as the cell membrane (e.g., plasma membrane) and/or the membranes of organelles like the endoplasmic reticulum, Golgi apparatus, and/or mitochondria.
  • the intracellular domain of the present invention is a polypeptide derived from 4-1 BB (e.g., human 4-1 BB, e.g., comprising or consisting of SEQ ID NO: 32 or a variant thereof).
  • polypeptide complex which can be used interchangeably with “protein complex” may refer to a group of two or more individual protein or polypeptide molecules (e.g., a TCR and a chimeric co-stimulatory receptor as defined herein) that are bound together, preferably physically and functionally, to perform a specific biological function and/or task within a cell (e.g., T-cell) or organism.
  • the term “combination” may refer to the coexistence and/or interaction (e.g., functional interaction) of a group of two or more individual protein or polypeptide molecules (e.g., a TCR and a chimeric co-stimulatory receptor as defined herein) within a cellular context (e.g., on/in a cell, preferably a T-cell; or population of cells as define herein, e.g., population of T cells) or biological system.
  • a cellular context e.g., on/in a cell, preferably a T-cell; or population of cells as define herein, e.g., population of T cells
  • the present invention provides/relates to a T-cell receptor (TCR) polypeptide complex and/or a combination comprising: a TCR; and a chimeric co- stimulatory receptor, wherein the TCR comprises: i) an a chain Complementarity Determining Region 3 (CDR3) comprising an amino acid sequence having at least 80 % identity to SEQ ID NO: 16; and/or ii) a chain Complementarity Determining Region 3 (CDR3) comprising an amino acid sequence having at least 80 % identity to SEQ ID NO: 4.
  • TCR comprises: i) an a chain Complementarity Determining Region 3 (CDR3) comprising an amino acid sequence having at least 80 % identity to SEQ ID NO: 16; and/or ii) a chain Complementarity Determining Region 3 (CDR3) comprising an amino acid sequence having at least 80 % identity to SEQ ID NO: 4.
  • the present invention may further encompass the TCR as defined herein, wherein the TCR is selected from the group consisting of a native TCR, a TCR variant, a TCR fragment, and a TCR construct.
  • TCR encompasses TCR variants, which include TCR sequence variants, fragments and constructs. All TCR variants are envisaged to be functional variants of the TCR of the invention.
  • functional variant refers to a TCR, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent TCR, its variable regions or its antigen-binding regions and shares its biological activity, i.e. its ability to specifically bind to the antigenic target for which the parent TCR of the invention has antigenic specificity to a similar, the same or even a higher extent as the TCR disclosed herein.
  • TCR variants includes “sequence variants” of the TCR disclosed herein, i.e. variants substantially comprising the amino acid sequence of the TCR as described above (also referred to as the “parent” TCR) but containing at least one amino acid modification (i.e. a substitution, deletion, or insertion) as compared to the “parent” I “native” TCR amino acid sequence, provided that the variant preferably retains the antigenic specificity of the inventive “patent” TCR as shown in the Examples.
  • TCR sequence variants of the invention are typically prepared by introducing appropriate nucleotide changes into the nucleic acids encoding the “parent” TCR, or by peptide synthesis.
  • the aforementioned amino acid modifications may be introduced into, or present in, the variable region or the constant region of the TCR, and may serve to modulate properties like binding strength and specificity, post-translational processing (e.g. glycosylation), thermodynamic stability, solubility, surface expression or TCR assembly.
  • amino acid modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequences of the parent TCR.
  • exemplary insertional variants of a TCR of the invention include fusion products of said TCR and an enzyme or another functional polypeptide.
  • Exemplary substitutional variants of a TCR of the invention are those including amino acid substitutions in variable regions or CDRs of the alpha and/or beta chain, the framework region or the constant region.
  • Murinization of TCRs is a technique that is commonly applied in order to improve cell surface expression of TCRs in host cells. Without wishing to be bound by specific theory, it is thought that murinized TCRs associate more effectively with CD3 coreceptors; and/or that preferentially pair with each other and are less prone to form mixed TCRs on human T cells engineered ex vivo to express the TCRs of desired antigenic specificity, but still retaining and expressing their “original” TCRs. [00129] Recently nine amino acids responsible for the improved expression of murinized
  • TCRs have been identified (Sommermeyer and llckert, J Immunol. 2010 Jun 1 ; 184(11):6223- 31) and it is envisaged to substitute one or all of the amino acid residues in the TCRs alpha and/or beta chain constant region for their murine counterpart residues.
  • This technique is also referred to as “minimal murinization” and offers the advantage of enhancing cell surface expression while, at the same time, reducing the number of “foreign” amino acid residues in the amino acid sequence and, thereby, the risk of immunogenicity.
  • TCR sequence variants are envisaged to comprise at least one of the CDR1 , CDR2, CDR3, alpha chain variable regions, beta chain variable regions, alpha chains and/or beta chains as disclosed herein, or comprising or consisting of an amino acid sequence that is at least about 80%, about 85%, about 90%, about 95% , about 96%, about 97%, about 98%, about 99%, or identical to the amino acid sequences disclosed herein, provided that said variants exhibit comparable, the same or improved binding characteristics as compared to the TCR evaluated in the Examples.
  • TCR as used herein further comprises TCR constructs.
  • constructs includes proteins or polypeptides comprising at least one antigen binding domain of the TCR of the invention, but do not necessarily share the basic structure of a native TCR (i.e. variable regions incorporated into a TCR alpha chain and a TCR beta chain forming a heterodimer).
  • TCR constructs and fragments are typically obtained by routine methods of genetic engineering and are often artificially constructed to comprise additional functional protein or polypeptide domains.
  • TCR constructs and fragments of the invention are envisaged to comprise at least one CDR3alpha and/or at least one CDR3beta as disclosed elsewhere herein.
  • constructs and fragments comprising at least one CDR1 alpha, CDR2alpha, CDRI beta, CDR2beta, alpha chain variable region, beta chain variable region, alpha chain and/or beta chain, or combinations thereof, optionally in combination with further protein domains or moieties as exemplified herein.
  • the TCR constructs and fragments provided herein may further be envisaged to be capable of specifically binding to the same antigenic target as the TCR described above and evaluated in the Examples.
  • the present invention may also comprise the TCR construct as defined herein comprising at least one TCR alpha-chain(s) and at least one TCR beta-chain(s) covalently linked to each other to form TCR heterodimers or multimers.
  • TCR construct encompasses heterodimers and multimers in which at least one TCR alpha chain variable region or TCR alpha-chain and at least one TCR betachain variable region are covalently linked to each other.
  • a multivalent TCR construct according to the invention comprises a multimer of two or three or four or more TCRs associated (e. g. covalently or otherwise linked) with one another, preferably via a linker molecule.
  • Suitable linker molecules include, but are not limited to, multivalent attachment molecules such as avidin, streptavidin, neutravidin and extravidin, each of which has four binding sites for biotin.
  • biotinylated TCRs can be formed into multimers having a plurality of TCR binding sites.
  • the number of TCRs in the multimer will depend upon the quantity of TCR in relation to the quantity of linker molecule used to make the multimers, and also on the presence or absence of any other biotinylated molecules.
  • Exemplary multimers are dimeric, trimeric, tetrameric or pentameric or higher-order multimer TCR constructs.
  • Multimers of the invention may also comprise further functional entities such as labels or drugs or (solid) carriers.
  • TCR construct also encompasses TCR molecules which are linked via a suitable linker to a spheric body, preferably a uniform bead, more preferably a polystyrene bead, most preferably a bio-compatible polystyrene bead.
  • TCR constructs can also be comprised of the TCR as defined herein and a bead having a pre-defined fluorescence dye incorporated into the bead.
  • TCR fusion proteins [00137]
  • the present invention may also comprise the TCR as defined herein, further comprising one or more fusion component(s) selected from the group consisting of a Fc receptor; a Fc domain, including IgA, IgD, IgG, IgE, and IgM; a cytokine, including IL-2 or IL- 15; a toxin; an antibody or an antigen-binding fragment thereof, including anti-CD3, anti-CD28, anti-CD5, anti-CD 16 or an anti- CD56 antibody or an antigen-binding fragment thereof; or a CD247 (CD3-zeta), CD28, CD137, or a CD134 domain, or combinations thereof, optionally further comprising at least one linker.
  • fusion protein are called a “fusion protein”.
  • TCR construct may also relate to such fusion proteins or polypeptides comprising at least one TCR alpha chain, TCR alpha chain variable region or CDR3alpha and/or at least one TCR beta chain, TCR beta chain variable region or CDR3beta; and further one or more fusion component(s).
  • Useful components include Fc receptors; Fc domains (derived from IgA, IgD, IgG, IgE, and IgM); cytokines (such as IL-2 or IL-15); toxins; antibodies or antigen-binding fragments thereof (such as anti-CD3, anti-CD28, anti-CD5, antiCD 16 or anti- CD56 antibodies or antigen-binding fragments thereof); CD247 (CD3-zeta), CD28, CD137, CD134 domains; or any combinations thereof.
  • Exemplary antibody fragments that can be used as fusion components include fragments of full-length antibodies, such as (s)dAb, Fv, Fd, Fab, Fab’, F(ab')2 or “r IgG” (“half antibody”); modified antibody fragments such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv- zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies, tandem diabodies (Tandab’s), tandem di-scFv, tandem tri-scFv, minibodies, multibodies such as triabodies or tetrabodies, and single domain antibodies such as nanobodies or single variable domain antibodies comprising only one variable domain, which might be VHH, VH or VL.
  • modified antibody fragments such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scF
  • TCR constructs of the invention may be fused to one or more antibody or antibody fragments, yielding monovalent, bivalent and polyvalent/multivalent constructs and thus monospecific constructs, specifically binding to only one target antigen as well as bispecific and polyspecific/multispecific constructs, which specifically bind more than one target antigens, e.g. two, three or more, through distinct antigen binding sites.
  • a linker may be introduced between the one or more of the domains or regions of the TCR construct of the invention, i.e. between the TCR alpha chain CDR3, TCR alpha chain variable region, and/or a TCR alpha chain, the TCR beta chain CDR3, TCR beta chain variable region, and/or a TCR beta chain, and/or the one or more fusion component(s) described herein.
  • Linkers are known in the art and have been reviewed, inter alia, by Chen et al. Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369. In general, linkers include flexible, cleavable and rigid linkers and will be selected depending on the type of construct and intended use/application.
  • non-immunogenic, flexible linkers are often preferred in order to ensure a certain degree of flexibility or interaction between the domains while reducing the risk of adverse immunogenic reactions.
  • Such linkers are generally composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids and include “GS” linkers consisting of stretches of Gly and Ser residues.
  • TCR constructs envisaged in accordance with the invention are those comprising at least one TCR alpha chain, TCR alpha chain variable region or CDR3 alpha as defined herein, at least one TCR beta chain, TCR beta chain variable region or CDR3 beta as defined herein, optionally linked to each other and fused, optionally via a liker, to at least one antibody or an antibody fragment (such as a single chain antibody fragment (scFv)) directed against an antigen or epitope on the surface of lymphocytes, such as a T cell.
  • scFv single chain antibody fragment
  • Useful antigenic targets recognized by the antibody or antibody fragment include CD3, CD28, CD5, CD 16 and CD56.
  • Said construct can in general have any structure as long the “TCR portion” (i.e. TCR alpha and beta chain or variable regions or CDR3s thereof) retains its ability to recognize the antigenic target defined herein, and the “antibody portion” binds to the desired surface antigen or epitope, thereby recruiting and targeting the respective lymphocyte to the target cell.
  • TCR portion i.e. TCR alpha and beta chain or variable regions or CDR3s thereof
  • the “antibody portion” binds to the desired surface antigen or epitope, thereby recruiting and targeting the respective lymphocyte to the target cell.
  • Such constructs may advantageously serve as “adapters” joining an antigen presenting cell displaying the antigenic target (such as a tumor cell) and a lymphocyte (such as a cytotoxic T cell or NK cell) together.
  • a TCR construct of the invention may comprise at least one TCR antigen binding domain as described herein (for instance a TCR variable alpha and variable beta chain fused to each other) linked to a scFv (or other binding domain) of the desired binding specificity, e.g. CD3 or CD56.
  • the scFv (or other binding domain) binds to T cells such as via the CD3 receptor or to CD56 for NK cell activation, and the other to a tumor cell via the antigenic target as defined herein specifically expressed on the tumor cell.
  • tribodies comprising at least one TCR antigen binding domain as described herein, an scFv (or other binding domain) and a further domain e.g. for targeting the construct to a site of action within the body (e.g. an Fc domain).
  • the present invention may also comprise the TCR as defined herein, further comprising at least one label.
  • the TCR of the invention can be labelled.
  • Useful labels are known in the art and can be coupled to the TCR or TCR variant using routine methods, optionally via linkers of various lengths.
  • label or “labelling group” refers to any detectable label.
  • labels fall into a variety of classes, depending on the assay in which they are to be detected - the following examples include, but are not limited to: isotopic labels, which may be radioactive or heavy isotopes, such as radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 89 Zr, 90 Y, 99 Tc, 111 ln, 125 l, 131 l); magnetic labels (e.g., magnetic particles); redox active moieties; optical dyes (including, but not limited to, chromophores, phosphors and fluorophores) such as fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), chemiluminescent groups, and fluorophores which can be either "small molecule" fluorophores or proteinaceous fluorophores; enzymatic groups (e.g.
  • TCR horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase; biotinylated groups; or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.). Labelling is particularly envisaged when the TCR, TCR variants or especially soluble TCR constructs (such as those comprising at least one TCR alpha and/or TCR beta chain as described herein) are intended for diagnostic use.
  • a secondary reporter e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.
  • the TCR of the invention can be modified by attaching further functional moieties, e.g. for reducing immunogenicity, increasing hydrodynamic size (size in solution) solubility and/or stability (e.g. by enhanced protection to proteolytic degradation) and/or extending serum half-life.
  • further functional moieties e.g. for reducing immunogenicity, increasing hydrodynamic size (size in solution) solubility and/or stability (e.g. by enhanced protection to proteolytic degradation) and/or extending serum half-life.
  • Exemplary functional moieties for use in accordance with the invention include peptides or protein domains binding to other proteins in the human body (such as serum albumin, the immunoglobulin Fc region or the neonatal Fc receptor (FcRn), polypeptide chains of varying length (e.g., XTEN technology or PASylation®), non-proteinaceous polymers, including, but not limited to, various polyols such as polyethylene glycol (PEGylation), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, or of carbohydrates, such as hydroxyethyl starch (e.g., HESylation®) or polysialic acid (e.g., PolyXen® technology).
  • PEGylation polyethylene glycol
  • polypropylene glycol polyoxyalkylenes
  • copolymers of polyethylene glycol and polypropylene glycol or of carbohydrates, such as hydroxyethyl starch (e.g
  • Other useful functional moieties include “suicide” or “safety switches” that can be used to shut off effector host cells carrying the TOR in a patient’s body.
  • suicide or “safety switches” that can be used to shut off effector host cells carrying the TOR in a patient’s body.
  • An example is the inducible Caspase 9 (iCasp9) “safety switch” described by Gargett and Brown Front Pharmacol. 2014; 5: 235.
  • effector host cells are modified by well-known methods to express a Caspase 9 domain whose dimerization depends on a small molecule dimerizer drug such as AP1903/CI P, and results in rapid induction of apoptosis in the modified effector cells.
  • the system is for instance described in EP2173869 (A2).
  • HSV-TK Herpes Simplex Virus thymidine kinase
  • TCR as defined herein with an altered glycosylation pattern may also be envisaged herein.
  • glycosylation patterns can depend on the amino acid sequence (e.g., the presence or absence of particular glycosylation amino acid residues, discussed below) and/or the host cell or organism in which the protein is produced.
  • Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • N-linked glycosylation sites to the binding molecule is conveniently accomplished by altering the amino acid sequence such that it contains one or more tri-peptide sequences selected from asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline).
  • O-linked glycosylation sites may be introduced by the addition of or substitution by, one or more serine or threonine residues to the starting sequence.
  • glycosylation of TCRs is by chemical or enzymatic coupling of glycosides to the protein.
  • the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.
  • deglycosylation i.e., removal of carbohydrate moieties present on the binding molecule
  • deglycosylation may be accomplished chemically, e.g. by exposing the TCR to trifluoromethanesulfonic acid, or enzymatically by employing endo- and exoglycosidases.
  • the TCR of the invention can be modified to introduce additional domains which aid in identification, tracking, purification and/or isolation of the respective molecule (tags).
  • tags comprise peptide motives known as Myc-tag, HAT-tag, HA- tag, TAP-tag, GST-tag, chitin binding domain (CBD-tag), maltose binding protein (MBP-tag), Flag-tag, Strep-tag and variants thereof (e.g. Strep ll-tag), His-tag, CD20, Her2/neu tags, myc- tag, FLAG-tag, T7-tag, HA(hemagglutinin)-tag, or GFP-tags.
  • peptide motives known as Myc-tag, HAT-tag, HA- tag, TAP-tag, GST-tag, chitin binding domain (CBD-tag), maltose binding protein (MBP-tag), Flag-tag, Strep-tag and variants thereof (e.g. Strep ll-tag), His-tag, CD
  • Epitope tags are useful examples of tags that can be incorporated into the TCR of the invention.
  • Epitope tags are short stretches of amino acids that allow for binding of a specific antibody and therefore enable identification and tracking of the binding and movement of soluble TCRs or host cells within the patient’s body or cultured (host) cells. Detection of the epitope tag, and hence, the tagged TCR, can be achieved using a number of different techniques. Examples of such techniques include: immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting ("Western"), and affinity chromatography.
  • the epitope tags can for instance have a length of 6 to 15 amino acids, in particular 9 to 11 amino acids. It is also possible to include more than one epitope tag in the TCR of the invention.
  • Tags can further be employed for stimulation and expansion of host cells carrying the TCR by cultivating the cells in the presence of binding molecules (antibodies) specific for said tag.
  • the TCR of the present invention can be provided in soluble form.
  • Soluble TCRs are useful as diagnostic tools, and carriers or “adapters” that specifically target therapeutic agents or effector cells to, for instance, a cancer cell expressing the antigenic target recognized by the soluble TCR.
  • Soluble TCRs will typically be fragments or constructs comprising TCR alpha and/or beta chains, or variable regions or CDRs thereof and optionally stabilized via disulfide bonds or covalently linked via a suitable linker molecule, e.g. as described above in the context of TCR constructs of the invention. They will typically not comprise e.g. a transmembrane region.
  • amino acid modifications in the polypeptide sequence may be introduced in order to enhance solubility of the molecules, and/or correct folding and pairing of the alpha and beta chains (if desired), in particular when produced in a recombinant host that does not provide for the aforementioned features.
  • folding and pairing of the TCR alpha and beta chains is typically accomplished in vitro.
  • TCR according to the invention may therefore for instance comprise additional cysteine residues, as described elsewhere herein.
  • cysteine bridges include, for instance, the addition of leucine zippers and/or ribosomal skipping sequences, e.g. sequence 2A from picorna virus as described in Walseng et al. (2015), PLoS ONE 10(4): e0119559 to increase folding, expression and/or pairing of the TCR alpha and/or beta chains.
  • leucine zippers and/or ribosomal skipping sequences e.g. sequence 2A from picorna virus as described in Walseng et al. (2015), PLoS ONE 10(4): e0119559 to increase folding, expression and/or pairing of the TCR alpha and/or beta chains.
  • the present invention further comprises a nucleic acid comprising a nucleotide sequence encoding the TCR as defined elsewhere herein.
  • polynucleotide or “nucleic acid” as used herein comprises a sequence of polyribonucleotides and polydeoxribonucleotides, e.g. modified or unmodified RNA or DNA, each in single-stranded and/or double-stranded form linear or circular, or mixtures thereof, including hybrid molecules.
  • the nucleic acids according to this invention thus comprise DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations thereof or derivatives (such as PNA) thereof.
  • a polynucleotide may comprise a conventional phosphodiester bond or a non- conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
  • the polynucleotides of the invention may also contain one or more modified bases, such as, for example, tritylated bases and unusual bases such as inosine. Other modifications, including chemical, enzymatic, or metabolic modifications, are also conceivable, as long as a binding molecule of the invention can be expressed from the polynucleotide.
  • the polynucleotide may be provided in isolated form as defined elsewhere herein.
  • a polynucleotide may include regulatory sequences such as transcription control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding site, introns, or the like.
  • the nucleic acid can comprise any nucleotide sequence which encodes the TCR of the invention.
  • the present invention may comprise a nucleic acid comprising or consisting of a nucleotide sequence encoding the TCR of the invention of at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 9 and/or SEQ ID NO: 21 (see Table 2).
  • the present invention may comprise a nucleic acid comprising or consisting of a nucleotide sequence encoding the TCR of the invention of at least about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 9.
  • the present invention may comprise a nucleic acid comprising or consisting of a nucleotide sequence encoding the TCR of the invention of at least about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 21.
  • the present invention may also comprise a nucleic acid comprising or consisting of a nucleotide sequence encoding the TCR of the invention of at least about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 13 and/or SEQ ID NO: 25 (see Table 2).
  • the present invention may comprise a nucleic acid comprising or consisting of a nucleotide sequence encoding the TCR of the invention of at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 13.
  • the present invention may comprise a nucleic acid comprising or consisting of a nucleotide sequence encoding the TCR of the invention of at least about 80%, about 81 %, about 82%, about 83%, about 84%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 25.
  • the nucleic acid described above may or may not comprise additional or altered nucleotide sequences encoding e.g., altered amino acid residues, a signal peptide to direct secretion of the encoded TCR, constant regions or other heterologous polypeptides as described herein. Such nucleic acids may thus encode fusion polypeptides, fragments, variants and other derivatives of the binding molecules described herein.
  • the nucleotide sequences of the present invention may be codon-optimized for optimal expression in the desired host cell, e.g. a human lymphocyte; or for expression in bacterial, yeast or insect cells that are particularly envisaged for the expression of the soluble TCR of the invention.
  • Codonoptimization refers to the exchange in a sequence of interest of codons that are generally rare in highly expressed genes of a given species by codons that are generally frequent in highly expressed genes of such species, such codons encoding the same amino acids as the codons that are being exchanged. Selection of optimum codons thus depends on codon usage of the host genome and the presence of several desirable and undesirable sequence motifs.
  • polypeptide is equally used herein with the term "protein”. Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise one or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids).
  • polypeptide(s) as used herein describes a group of molecules, which, for example, consist of more than 30 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc.
  • An example for a heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains.
  • polypeptide and protein also refer to naturally modified polypeptides/proteins wherein the modification is affected e.g. by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.
  • vector comprising one or more of the nucleic acids as described herein.
  • a “vector” is a nucleic acid molecule used as a vehicle to transfer (foreign) genetic material into a host cell where it can for instance be replicated and/or expressed.
  • vector encompasses, without limitation plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors, and adenovirus-associated vectors (AAV)), phages, phagemids, cosmids and artificial chromosomes (including BACs and YACs).
  • viral vectors including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma virus vectors, and adenovirus-associated vectors (AAV)
  • phages phagemids
  • cosmids and artificial chromosomes including BACs and YACs.
  • the vector itself is generally a nucleotide sequence, commonly a DNA sequence that comprises an insert (transgene) and a larger sequence that serves as the “backbone” of the vector.
  • Engineered vectors typically comprise an origin for autonomous replication in the host cells (if stable expression of the polynucleotide is desired), selection markers, and restriction enzyme cleavage sites (e.g. a multiple cloning site, MCS).
  • Vector may additionally comprise promoters, genetic markers, reporter genes, targeting sequences, and/or protein purification tags.
  • suitable vectors are provided in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012).
  • the vector of the present invention can be a targeting vector.
  • Targeting vectors can be used to integrate a polynucleotide into the host cell’s chromosome by methods known in the art, such as described by J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012). Briefly, suitable means include homologous recombination or use of a hybrid recombinase that specifically targets sequences at the integration sites. Targeting vectors are typically circular and linearized before used for homologous recombination.
  • the foreign polynucleotides may be DNA fragments joined by fusion PCR or synthetically constructed DNA fragments which are then recombined into the host cell. It is also possible to use heterologous recombination which results in random or non-targeted integration.
  • the vector of the present invention can also be an expression vector.
  • “Expression vectors” or “expression constructs” can be used for the transcription of heterologous polynucleotide sequences, for instance those encoding the TCR of the invention, and translation of their mRNA in a suitable host cell.
  • regulatory sequence refers to a nucleotide sequence necessary for the expression of an operably linked coding sequence of a (heterologous) polynucleotide in a particular host organism or host cell and thus include transcriptional and translational regulatory sequences.
  • regulatory sequences required forexpression of heterologous polynucleotide sequences in prokaryotes include a promoter(s), optionally operator sequence(s), and ribosome binding site(s).
  • promoters, polyadenylation signals, enhancers and optionally splice signals are typically required.
  • specific initiation and secretory signals also may be introduced into the vector in order to allow for secretion of the polypeptide of interest into the culture medium.
  • Exemplary regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • the expression vectors may also include origins of replication and selectable markers.
  • vectors of the invention may further comprise one or more selection markers.
  • Suitable selection markers for use with eukaryotic host cells include, without limitation, the herpes simplex virus thymidine kinase (tk), hypoxanthine-guanine phosphoribosyltransferase (hgprt), and adenine phosphoribosyltransferase (aprt) genes.
  • Other genes include dhfr (methotrexate resistance), gpt (mycophenolic acid resistance) neo (G-418 resistance) and hygro (hygromycin resistance).
  • Vector amplification can be used to increase expression levels.
  • the selection marker gene can either be directly linked to the polynucleotide sequences to be expressed or introduced into the same host cell by cotransformation.
  • the present invention thus further provides one or more of the nucleotide sequences described herein inserted into (i.e. comprised by) a vector.
  • the invention provides (replicable) vectors comprising a nucleotide sequence encoding the TCR of the invention, or an alpha or beta chain thereof, or an alpha or beta variable region, or any CDR alpha or CDR beta operably linked to a promoter.
  • suitable expression vectors are viral vectors, such as retroviral vectors e.g. MP71 vectors or retroviral SIN vectors; and lentiviral vectors or lentiviral SIN vectors.
  • viral vectors such as retroviral vectors e.g. MP71 vectors or retroviral SIN vectors; and lentiviral vectors or lentiviral SIN vectors.
  • Viral vectors comprising polynucleotides encoding the TCR of the invention are for instance capable of infecting lymphocytes, which are envisaged to subsequently express the heterologous TCR.
  • Another example for a suitable expression vector is the Sleeping Beauty (SB) transposon transposase DNA plasmid system, SB DNA plasmid.
  • SB Sleeping Beauty
  • the nucleic acids and/or in particular expression vectors of the invention can also be transferred into cells by transient RNA transfection.
  • a cell population comprising cells may refer to a group and/or collection of individual cells of the same or similar type that share certain characteristics and/or properties within a specific biological context.
  • Cell populations of the present invention can be defined based on various criteria, such as cell type (e.g., T cells, preferably CD8 + T cell(s)), developmental stage, location within a tissue or organ, functional characteristics (e.g., immune cells, preferably CD8 + T cell(s)) and/or genetic markers (e.g., expressing a TCR and a chimeric co-stimulatory receptor as defined herein).
  • a cell population comprising cells of the present invention is a population of immune cells (e.g., comprising T cells, preferably CD8 + T cell(s)), preferably a population of T cells (e.g., comprising T cells, preferably CD8 + T cell(s)).
  • the cell population of the present invention may comprise a significant percentage of cells, such as 0.1 %, 0.2 %, 0.3 %, 0.4 %, 0.5 %, 1 %, 1.5 %,2 %, 3 %, 4 %, 4.5 % cells.
  • a cell population of the present invention as defined herein may comprise cells which secrete at least two, (e.g. two, three, four or more) proteins.
  • the cell as defined herein may secrete at least two, (e.g. two, three, four or more) proteins.
  • such cell population as defined herein comprises cells which secrete at least two proteins which are selected from the group consisting of effector proteins (effector PSI; e.g. Gzm-B, IFN-y, MIP-1a, TNF-a), stimulatory cytokines (stimulatory PSI; e.g. GM-CSF, IL-5, IL-9), chemo-attractive cytokines (chemoattractive PSI; e.g.
  • inflammatory cytokines inflammatory PSI; e.g. IL-6
  • inflammatory PSI e.g. IL-6
  • effector PSI e.g. Gzm- B, IFN-y, MIP-1a, TNF-a
  • stimulatory cytokines e.g. GM-CSF, IL-5, IL-9
  • chemo-attractive cytokines chemoattractive PSI; e.g. MIP-1 P
  • such cell as defined herein secretes at least two proteins which are selected from the group consisting of effector proteins (effector PSI; e.g.
  • Gzm-B IFN-y, MIP-1a, TNF-a
  • stimulatory cytokines (stimulatory PSI; e.g. GM-CSF, IL-5, IL-9), chemo-attractive cytokines (chemoattractive PSI; e.g. MIP-1 P) and inflammatory cytokines (inflammatory PSI; e.g. IL-6), preferably at least two of effector proteins (effector PSI; e.g. Gzm-B, IFN-y, MIP-1a, TNF-a), stimulatory cytokines (stimulatory PSI; e.g. GM-CSF, IL-5, IL-9), and/or chemo-attractive cytokines (chemoattractive PSI; e.g. MIP-1 P).
  • effector PSI e.g. Gzm-B, IFN-y, MIP-1a, TNF-a
  • stimulatory cytokines (stimulatory PS
  • cells of the present invention may refer to a basic structural and functional unit of life in all living organisms.
  • Preferred cells of the present invention are immune cells, preferably T cells.
  • a cell e.g. T cell
  • a cell population e.g. T-cell population
  • co-expressing the TCR as defined herein and the chimeric costimulatory receptor such as PD1-41 BB CSR as further defined herein thus shows a higher polyfunctionality (secretion of 2 or more proteins) compared to a cell or a cell population as defined herein expressing only the TCR of the invention, but not the chimeric costimulatory receptor (see Figure 14).
  • T cells (or T lymphocytes) as defined herein are a type of white blood cells.
  • T cells There are several types of T cells including: helper T cells (CD4+ T cells), cytotoxic T cells (CD8+ T cells) and regulatory T cells (Tregs).
  • a T cell(s) of the invention may be selected from the group consisting of: helper T cell(s) (CD4+ T cell(s)), cytotoxic T cell(s) (CD8+ T cell(s)) and regulatory T cell(s) (Tregs), preferably CD8 + T cell(s).
  • such cell as defined herein secretes at least two proteins which are selected from the group consisting of effector proteins (effector PSI), stimulatory cytokines (stimulatory PSI), chemo-attractive cytokines (chemoattractive PSI) and inflammatory cytokines.
  • the present invention further comprises a host cell comprising the TCR, the nucleic acid or the vector as described herein.
  • a host cell comprising the TCR, the nucleic acid or the vector as described herein.
  • a variety of host cells can be used in accordance with the invention.
  • the term “host cell” encompasses cells which can be or has/have been recipients of polynucleotides or vectors described herein and/or express (and optionally secreting) the TCR of the present invention.
  • the terms “cell” and “cell culture” are used interchangeably to denote the source of a TCR unless it is clearly specified otherwise.
  • the term “host cell” also includes “host cell lines”.
  • the term “host cell” includes prokaryotic or eukaryotic cells, and also includes without limitation bacteria, yeast cells, fungi cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g., murine, rat, macaque or human cells.
  • the term “host cell” may also encompass any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • the invention thus provides, inter alia, host cells comprising a polynucleotide or a vector, e.g. an expression vector comprising a nucleotide sequence encoding the TCR or a TCR construct as described herein.
  • Polynucleotides and/or vectors of the invention can be introduced into the host cells using routine methods known in the art, e.g. by transfection, transformation, or the like.
  • the present invention thus further provides host cells comprising at least one TCR, nucleic acid and/or vector as described herein.
  • a host cell may be chosen that modulates the expression of the inserted nucleotide sequences, and/or modifies and processes the gene product (i.e. RNA and/or protein) as desired.
  • Such modifications e.g., glycosylation
  • processing e.g., cleavage
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the product.
  • eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • production host cells in particular in soluble form
  • effector host cells host cells expressing the TCR of the invention and having effector function
  • effector host cells are particularly useful for therapeutic applications and are envisaged for administration to a subject in need thereof.
  • Preferred “effector host cells” include lymphocytes such as cytotoxic T lymphocytes (CTLs), CD8 + T cells, CD4 + T cells, natural killer (NK) cells, natural killer T (NKT) cells, gamma/delta-T-cells.
  • the present invention may also comprise the host cell as defined herein, which is selected from lymphocytes including but not limited to cytotoxic T lymphocytes (CTLs), CD8 + T cells, CD4 + T cells, natural killer (NK) cells, natural killer T (NKT) cells, or gamma/ delta-T-cells.
  • Said effector host cells can be modified using routine methods to comprise a nucleotide sequence encoding the TCR of the invention, and are envisaged to express the TCR described herein, in particular on the cell surface.
  • modified host cells expressing the TCR of the invention generally refers to (effector or production) host cells treated or altered to express the TCR according to the present invention, for instance by RNA transfection. Other methods of modification or transfection or transduction may also be envisaged.
  • modified host cell thus includes “transfected”, “transduced” and “genetically engineered” host cells preferably expressing the TCR of the present invention.
  • such “(modified) effector host cells” are capable of mediating effector functions through intracellular signal transduction upon binding of the TCR to its specific epitope.
  • effector functions include for instance the release of perforin (which creates holes in the target cell membrane), granzymes (which are proteases that act intracellularly to trigger apoptosis), the expression of Fas ligand (which activates apoptosis in a Fas-bearing target cell) and the release of cytokines, preferably Th1/Tc1 cytokines such as IFN-y, IL-2 and TNF-a.
  • an effector host cell engineered to express the TCR of the invention that is capable recognizing and binding to its epitope in the subject to be treated is envisaged to carry out the above-mentioned effector functions, thereby killing the target (e.g. cancer) cells.
  • Cytolysis of target cells can be assessed e.g. with the CTL fluorescent killing assay (CTL, USA) detecting the disappearance of fluorescently labeled target cells during co-culture with TCR-transfected recipient T cells.
  • effector host cells preferably express a functional TCR, i.e. that typically comprises a TCR alpha and beta chain described herein; and also the signal transducing subunits CD3 gamma, delta, epsilon and zeta (CD3 complex). Moreover, expression of co-receptors CD4 or CD8 may also be desired.
  • lymphocytes harboring the required genes involved in antigen binding, receptor activation and downstream signalling e.g. Lek, FYN, CD45, and/or Zap70
  • T cells are particularly suitable as effector host cells.
  • effector host cells expressing the TCR of the invention as a “binding domain” without the CD3 signal transducing subunit and/or aforementioned downstream signalling molecules (i.e. being capable of recognizing the antigenic target described herein, but without effecting functions mediated by CD3 and/or the aforementioned downstream signalling molecules) are also envisaged herein.
  • Such effector cells are envisaged to be capable of recognizing the epitope described herein, and optionally of effecting other functions not associated with CD3 signalling and/or signalling of the aforementioned downstream signalling molecules.
  • Examples include NK or NKT cells expressing the TCR and being capable of e.g. releasing cytotoxic granules upon recognition of their antigenic target.
  • Effector host cells in particular lymphocytes such as T cells can be autologous host cells that are obtained from the subject to be treated and transformed or transduced to express the TCR of the invention. Techniques for obtaining and isolating the cells from the patient are known in the art.
  • “Production host cells” used for the expression of the TCR of the invention are preferably capable of expressing high amounts of recombinant protein.
  • Exemplary mammalian host cells that can be used for as “production host cells” include Chinese Hamster Ovary (CHO cells) including DHFR minus CHO cells such as DG44 and DLIXBI 1 , NSO, COS (a derivative of CVI with SV40 T antigen), HEK293 (human kidney), and SP2 (mouse myeloma) cells.
  • exemplary host cell lines include, but are not limited to, HELA (human cervical carcinoma), CVI (monkey kidney line), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), P3x63-Ag3.653 (mouse myeloma), BFA-lcIBPT (bovine endothelial cells), and RAJI (human lymphocyte). Host cell lines are typically available from commercial services, the American Tissue Culture Collection (ATCC) or from published literature.
  • ATCC American Tissue Culture Collection
  • Non-mammalian cells such as bacterial, yeast, insect or plant cells are also readily available and can also be used as “production host cells” as described above.
  • Exemplary bacterial host cells include enterobacteriaceae, such Escherichia coli, Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenza.
  • Other host cells include yeast cells, such as Saccharomyces cerevisiae, and Pichia pastoris. Insect cells include, without limitation, Spodoptera frugiperda cells.
  • conceivable expressions systems include microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors; yeast (e.g., Saccharomyces , Pichia) transformed with recombinant yeast expression vectors; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid).
  • bacteria e.g., E. coli, B. subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
  • yeast e.g., Saccharomyces , Pichia transformed with recombinant yeast expression vector
  • metallothionein promoter e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter, the cytomegalovirus (CMV) major immediate-early promoter (Ml EP) promoter
  • CMV cytomegalovirus
  • Ml EP major immediate-early promoter
  • Suitable mammalian host cells can be selected from known cell lines (e.g., COS, CHO, BLK, 293, 3T3 cells), however it is also conceivable to use lymphocytes such as cytotoxic T lymphocytes (CTLs), CD8 + T cells, CD4 + T cells, natural killer (NK) cells, natural killer T (NKT) cells, gamma/ delta-T-cells.
  • CTLs cytotoxic T lymphocytes
  • NK natural killer
  • NKT natural killer T
  • gamma/ delta-T-cells gamma/ delta-T-cells.
  • the present invention also comprises a method for producing or obtaining the TCR as described herein comprising the steps of (i) incubating the host cell (i.e., a production host cell) under conditions causing expression of said TCR and (ii) purifying said TCR.
  • the host cells harboring the expression vector are grown under conditions appropriate to the production of the TCR defined herein, in particular alpha chains and/or beta chains as described elsewhere herein, and assayed for alpha and/or beta chain protein synthesis.
  • vectors encoding both the alpha and beta chains may be co- expressed in the host cell for expression of the entire molecule.
  • a TCR of the invention may be purified by any purification method known in the art, for example, by chromatography (e.g., ion exchange chromatography (e.g. hydroxylapatite chromatography), affinity chromatography, particularly Protein A, Protein G or lectin affinity chromatography, sizing column chromatography), centrifugation, differential solubility, hydrophobic interaction chromatography, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange chromatography (e.g. hydroxylapatite chromatography), affinity chromatography, particularly Protein A, Protein G or lectin affinity chromatography, sizing column chromatography
  • centrifugation e.g. hydroxylapatite chromatography
  • differential solubility e.g. sizing column chromatography
  • hydrophobic interaction chromatography e.g., hydrophobic interaction chromatography
  • TCR TCR
  • nucleic acid the nucleic acid
  • vector the vector
  • host cell any definitions regarding the TCR, the nucleic acid, the vector, or the host cell can be applied for the production method as defined herein, where necessary.
  • composition [00210]
  • the present invention further comprises a pharmaceutical composition
  • a pharmaceutical composition comprising one or more active agents, namely the TCR, the nucleic acid, the vector and/or the host cell as described herein, and, optionally, one or more pharmaceutically excipient(s).
  • active agents namely the TCR, the nucleic acid, the vector and/or the host cell as described herein, and, optionally, one or more pharmaceutically excipient(s).
  • composition particularly refers to a composition suitable for administering to a subject, preferably to a mammal, most preferably to a human.
  • compositions suitable for administration to non-human mammal are generally also encompassed by the term.
  • compositions and its components are preferably pharmaceutically acceptable, i.e. capable of eliciting the desired therapeutic effect without causing any undesirable local or systemic effects in the recipient.
  • Pharmaceutically acceptable compositions of the invention may for instance be sterile.
  • pharmaceutically acceptable may mean approved by a regulatory agency or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.
  • the active agent described in the foregoing is preferably present in the pharmaceutical composition in a therapeutically effective amount.
  • therapeutically effective amount is meant an amount of the active agent that elicits the desired therapeutic effect.
  • Therapeutic efficacy and toxicity can be determined by standard procedures, e.g. in cell culture or in test animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • ED50 the dose therapeutically effective in 50% of the population
  • LD50 the dose lethal to 50% of the population.
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, ED50/LD50.
  • Pharmaceutical compositions that exhibit large therapeutic indices are preferred.
  • TCR TCR
  • nucleic acid nucleic acid
  • vector vector and/or host cell
  • Suitable dosages provide sufficient amounts of the active agent of the invention and are preferably therapeutically effective, i.e. elicit the desired therapeutic effect.
  • Suitable dosage ranges for instance for the soluble TCR as described herein, can be determined using data obtained from cell culture assays and animal studies and may include the ED50. Typically, dosage amounts may vary from 0.1 to 100000 micrograms, up to a total dose of about 2 g, depending upon the route of administration.
  • Exemplary dosages of the active agent of the invention may be in the range from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 0.01 mg/kg to about 1 mg/kg, or from about 0.1 mg/kg to about 1 mg/kg.
  • Guidance as to particular dosages and methods of delivery is provided in the literature. It is recognized that treatment may require a single administration of a therapeutically effective dose or multiple administrations of a therapeutically effective dose of the active agent of the invention.
  • the pharmaceutical composition may optionally comprise one or more excipients and/or additional active agents.
  • excipient includes fillers, binders, disintegrants, coatings, sorbents, antiadherents, glidants, preservatives, antioxidants, flavoring, coloring, sweeting agents, solvents, co-solvents, buffering agents, chelating agents, viscosity imparting agents, surface active agents, diluents, humectants, carriers, diluents, preservatives, emulsifiers, stabilizers and tonicity modifiers. It is within the knowledge of the skilled person to select suitable excipients for preparing the desired pharmaceutical composition of the invention.
  • Exemplary carriers for use in the pharmaceutical composition of the invention include saline, buffered saline, dextrose, and water.
  • suitable excipients will inter alia depend on the active agent used, the disease to be treated, and the desired formulation of the pharmaceutical composition.
  • the present invention further provides pharmaceutical compositions comprising one or more of the active agents specified above (for instance a host cell or the TCR of the invention), and one or more additional active agents that are suitable for treatment and/or prophylaxis of the disease to be treated.
  • the active agents specified above for instance a host cell or the TCR of the invention
  • additional active agents that are suitable for treatment and/or prophylaxis of the disease to be treated.
  • additional active agents suitable for combinations include known anti-cancer drugs such as cis-platin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II, temozolmide, topotecan, trimetreate glucuronate, auristatin E vincristine and doxorubicin; and peptide cytotoxins such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNAase and RNAase; radionuclides such as iodine 131 , rhenium 186, indium 111 , yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs, such as antibody directed enzyme pro-drugs; immuno-stimuls,
  • a variety of routes are applicable for administration of the pharmaceutical composition according to the present invention. Typically, administration will be accomplished parenterally. Methods of parenteral delivery include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.
  • the pharmaceutical composition of the invention can be formulated in various forms, depending inter alia on the active agent used (e.g., the TCR), e.g. in solid, liquid, gaseous or lyophilized form and may be, inter alia, in the form of an ointment, a cream, transdermal patches, a gel, powder, a tablet, solution, an aerosol, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tincture or fluid extracts or in a form which is particularly suitable for the desired method of administration.
  • Processes known per se for producing medicaments are indicated in 22nd edition of Remington's Pharmaceutical Sciences (Ed.
  • a pharmaceutical composition comprising, for instance, host cells or the TCR as described herein will typically be provided in a liquid form, and preferably comprise a pharmaceutically acceptable buffer.
  • a pharmaceutical composition of the invention After a pharmaceutical composition of the invention has been prepared it can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would for instance include amount, frequency and method of administration.
  • the present invention further comprises a diagnostic composition
  • a diagnostic composition comprising one or more diagnostic agents, namely the TCR, the nucleic acid, the vector and/or the host cell as described herein, and optionally, one or more diagnostically acceptable excipient(s).
  • said diagnostic agent will comprise means for detecting its binding to its epitope, for instance a label as described in the context of the TCR of the invention.
  • the host cell it is for instance conceivable to use modified host cells comprising a dye or a contrast agent that is released (instead of cytotoxic granules) upon antigen recognition.
  • diagnostic composition when used herein refers to a composition comprising one or more diagnostic agents of the present invention as defined herein, which can be applied for use in diagnosis in vitro.
  • Said diagnostically acceptable excipient includes also any excipient that does not itself elicit an adverse reaction, which would be harmful when used in in vitro and in vivo diagnosis.
  • Suitable excipients are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and lipid aggregates such as, e.g. oil droplets or liposomes.
  • the excipient used in combination with the one or more diagnostic agents of the present invention may be water-based and forms an aqueous solution.
  • An oil-based excipient solution containing the one or more diagnostic agents of the present invention is an alternative to the aqueous carrier solution.
  • Either aqueous or oil-based solutions further contain thickening agents to provide the composition with the viscosity of a liniment, cream, ointment, gel, or the like. Suitable thickening agents are well known to those skilled in the art.
  • Diagnostically acceptable excipients include, by the way of illustration and not limitation, diluent, disintegrants, binding agents, adhesives, wetting agents, polymers, lubricants, gliands, substances added to mask or counteract a disagreeable texture, taste or odor, flavors, dyes, fragrances, and substances added to improve appearance of the composition.
  • the diagnostic composition can additionally include preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, buffers, coating agents, surfactants or antioxidants.
  • the present invention also comprises the TCR, nucleic acid, vector, host cell and/or the pharmaceutical composition as described herein for use as a medicament and/or for use in therapy.
  • the TCR, nucleic acid, vector, host cell and/or pharmaceutical composition can in general be employed for treatment detection, diagnosis, prognosis, prevention and/or treatment of diseases or disorders.
  • treatment in all its grammatical forms includes therapeutic or prophylactic treatment of a subject in need thereof.
  • a “therapeutic or prophylactic treatment” comprises prophylactic treatments aimed at the complete prevention of clinical and/or pathological manifestations or therapeutic treatment aimed at amelioration or remission of clinical and/or pathological manifestations.
  • treatment thus also includes the amelioration or prevention of diseases.
  • subject or “patient” are used interchangeably herein to refer to any subject, particularly a mammal, for whom therapy is desired.
  • Mammalian subjects generally include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.
  • the TCR, nucleic acid, vector, host cell and/or pharmaceutical composition provided herein are especially envisaged for treatment of human subjects.
  • the TCR of the invention, the nucleic acid, the vector (such as viral vectors), the host cell and/or the pharmaceutical composition of the invention can be administered directly to the subject in need thereof.
  • the present invention also comprises the TCR, the nucleic acid, the vector, the host cell, and/or the pharmaceutical composition as defined herein for use in a method of detecting, diagnosing, prognosing, preventing and/or treating cancer.
  • the invention may also comprise the TCR, the nucleic acid, the vector, the host cell and/or the pharmaceutical composition for the use as defined herein, wherein prevention and/or treatment of cancer comprises administering at least any one of the following: the TCR, the nucleic acid, the vector, the host cell, or the pharmaceutical composition to a subject in need thereof.
  • Said method can also comprise the steps of (a’) providing one or more of the TCR, the nucleic acid, the vector, the host cell, and/or the pharmaceutical composition of the present invention; and (b’) administering one or more of the abovementioned to the subject in need thereof.
  • the method can comprise a further step of cancer therapy, e.g. radiation, or administration of one or more anti-cancer agents.
  • the present invention may also comprise the TCR, the nucleic acid, the vector, the host cell and/or the pharmaceutical composition for the use as defined herein, wherein prevention and/or treatment of cancer comprises (a) introducing at least any one of the following: the TCR, the nucleic acid, the vector, the host cell or the pharmaceutical composition into lymphocytes comprised by a sample obtained from a subject as defined herein, thereby obtaining modified lymphocytes, and (b) administering the modified lymphocytes of step (a) to a subject in need thereof.
  • the treatment according to the invention may also comprise the steps of (a’) providing a sample of a subject, said sample comprising lymphocytes; (b’) providing one or more of the TCR, nucleic acid, vector, host cell and/or pharmaceutical composition of the invention; (o’) introducing of one or more of the abovementioned of step (b’) into the lymphocytes of step (a’) and, thereby, obtaining modified lymphocytes; (d’) administering the modified lymphocytes of step (o’) to a subject in need thereof.
  • the lymphocytes provided in step (a’) are particularly envisaged to be “effector host cells” as described in the foregoing and are advantageously selected from T cells, NK cells and/or NKT cells, especially CD8 + T cells; and can be obtained in a previous step (a”) from a sample -in particular a blood sample- of the subject by routine methods known in the art. It is however also conceivable to use other lymphocytes that are preferably capable of expressing the TCR of the present invention and exert the desired biological effector functions as described herein. Moreover, said lymphocytes will typically be selected for compatibility with the subject’s immune system, i.e. they will preferably not elicit an immunogenic response.
  • step (o’) can be carried out by introducing a nucleic acid or vector described herein via electroporation into the lymphocytes, or by infecting the lymphocytes with a viral vector, such as a lentiviral or retroviral vector as described previously in the context of the effector host cell.
  • a viral vector such as a lentiviral or retroviral vector as described previously in the context of the effector host cell.
  • transfection reagents such as liposomes, or transient RNA transfection.
  • the transfer of antigen-specific TCR genes into (primary) T cells by e.g.
  • TCR any definitions regarding the TCR, the nucleic acid, the vector, the host cell or the pharmaceutical composition can be applied for the treatments as defined herein, where necessary.
  • the present invention envisages use of the diagnostic agents described in the foregoing for detecting, diagnosing and/or prognosing cancer in a subject which can be accomplished in vivo or in vitro.
  • the invention provides a diagnostic composition for use in detecting, diagnosing and/or prognosing cancer in a subject in vivo, said composition comprising, as a diagnostic agent, the TCR, the nucleic acid, the vector, the host cell and/or the pharmaceutical composition of the invention.
  • the method will typically comprise (a) administering said diagnostic agent to the subject and (b) detecting binding of said diagnostic agent to its antigenic target.
  • the invention provides a method of detecting, diagnosing and/or prognosing cancer in a subject in vitro.
  • the present invention comprises a method of detecting the presence of a cancer in a subject in vitro, comprising (a) contacting a sample obtained from a subject and comprising one or more cells with the TCR, the nucleic acid, the vector, the host cell, and/or the pharmaceutical composition, thereby forming a complex, and (b) detecting the complex, wherein detection of the complex is indicative of the presence of the cancer in the subject.
  • the TCR as defined elsewhere herein comprises a tag as defined herein.
  • the present invention may also encompass a method of detecting the presence of a cancer in a subject in vitro, comprising the steps of (a’) providing a sample of a subject, said sample comprising one or more cells; (b’) contacting said sample with the TCR, the nucleic acid, the vector, the host cell, and/or the pharmaceutical composition of the invention; thereby forming a complex, and (o’) detecting the complex.
  • Said complex is envisaged to be indicative for binding of the diagnostic agent as defined herein to its antigenic target and is of the presence of a (cancer) cell expressing said antigenic target.
  • binding of the diagnostic agent to its antigenic target is detectable by using routine methods known in the art and will inter alia depend on the specific diagnostic agent used.
  • Suitable labels that can be coupled to the diagnostic agent of the invention are exemplified in the section relating to labeled TCR constructs.
  • the present invention also comprises the use of the TCR, the nucleic acid, and/or the vector as described elsewhere herein for generating modified lymphocytes as defined herein.
  • Means and methods for introducing, e.g. a nucleic acid and a vector into the lymphocytes have been described elsewhere herein.
  • TCR any definitions regarding the TCR, the nucleic acid, the vector, the host cell or the pharmaceutical composition can be applied for the uses, where necessary.
  • Kit [00248]
  • the present invention also comprises a kit comprising the TCR, the nucleic acid, the vector, the host cell, and/or the pharmaceutical or diagnostic composition as defined herein.
  • a kit comprises the TCR per se
  • said TCR may be provided in a vial or a container.
  • the other components such as the nucleic acid, the vector, the host cell, and/or the pharmaceutical or diagnostic composition.
  • Said kit may comprise the component as described above, preferably in a vial or container, in dried form, such as a lyophilized, air-dried, or spray-dried form (in form of a powder), for later reconstitution into a liquid form or other form prior to use. Further, said kit may also comprise the component, preferably in a vial or container, in a frozen state, being thawed prior to use.
  • the kit comprising the component may further comprise a diagnostically or pharmaceutically acceptable excipient as defined elsewhere herein.
  • said excipient may also be comprised in one or more containers or vials in said kit, meaning said kit additionally comprising either one vial or container comprising said excipient as a mixture or said kit additionally comprising for each component such as the excipient separate vials or containers.
  • the term "at least" preceding a series of elements is to be understood to refer to every element in the series.
  • the term “at least one” refers to one, two, three or more such as four, five, six, seven, eight, nine, ten and more.
  • the term “about” means plus or minus 20%, preferably plus or minus 10%, more preferably plus or minus 5%, most preferably plus or minus 1 %.
  • the present invention further relates to the following items:
  • CDR3 p chain Complementarity Determining Region 3
  • a cell population (preferably a T-cell population) comprising cells (e.g., isolated and/or recombinant cells, e.g., T-cells) or a cell (e.g., isolated and/or single and/or recombinant cell, e.g., T-cell) expressing: a) a TCR; and b) a chimeric co-stimulatory receptor, preferably said chimeric co-stimulatory receptor is a fusion polypeptide comprising one or more (e.g., two) PD-1-derived polypeptide (e.g., wherein said PD-1 is also known as Programmed Cell Death Protein 1 , e.g., having UniProt Accession Number: Q15116) and 4-1 BB-derived polypeptide (e.g., wherein said 4-1 BB is also known as CD137 or TNFRS9, which can be used interchangeably, e.g., having UniProt Accession Number: Q07011); further preferably said
  • CDR3 p chain Complementarity Determining Region 3
  • TCR further comprises at least one (e.g., 1 , 2, 3, or 4) of the following: i) an a chain Complementarity Determining Region 1 (CDR 1) comprising an amino acid sequence having at least 80 % (e.g.
  • CDR2 a chain Complementarity Determining Region 2 (CDR2) comprising an amino acid sequence having at least 80 % (e.g.
  • a p chain CDR2 comprising an amino acid sequence having at least 80 % (e.g. at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO: 3.
  • the TCR has antigenic specificity to (an) epitope(s) presented in the context of a MHC class I molecule, the epitope(s): a) comprising the amino acid sequence of VVVGAVGVGK (SEQ ID NO: 1), or a fragment thereof (e.g., SEQ ID NO: 35) or a variant thereof comprising one or more (e.g.
  • the complex and/or the combination, and/or the cell population or the cell of any one of the preceding items, wherein the at least 80% (e.g. at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity comprises conservative or highly conservative amino acid substitutions, preferably corresponding to position 1 , 2, 3, 4, 5, 6, and/or 7 of SEQ ID NO: 14; position 1 , 2, 3, 4, 5, 6, 7, and/or 8 of SEQ ID NO: 15; position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, and/or 13 of SEQ ID NO: 16; position 1 , 2, 3, 4, and/or 5 of SEQ ID NO: 2; position 1 , 2, 3, 4, 5, and/or 6 of SEQ ID NO: 3; and/or position 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, and/or 16
  • the TCR comprises at least one (e.g., 1 , 2, 3, 4, 5, or 6) of the following: i) an a chain CDR 1 comprising the amino acid sequence of SEQ ID NO: 14; ii) an a chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15; iii) an a chain CDR3 comprising the amino acid sequence of SEQ ID NO: 16; iv) a p chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2; v) a chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3; and/or vi) a p chain CDR3 comprising the amino acid sequence of SEQ ID NO: 4.
  • the TCR comprises: i) an a chain variable region comprising the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 80 % (e.g. at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO: 20; and/or ii) a p chain variable region comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having at least 80 % (e.g.
  • TCR further comprises: i) an a chain constant region comprising the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 80 % (e.g.
  • the TCR comprises: i) an a chain comprising the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 80 % (e.g. at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO: 24; and/or ii) a chain comprising the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80 % (e.g.
  • TCR construct comprises at least one TCR alpha-chain(s) and at least one TCR beta-chain(s) covalently linked to each other to form TCR heterodimers or multimers.
  • the TCR further comprising one or more fusion component(s) selected from the group consisting of a Fc receptor; a Fc domain, including IgA, IgD, IgG, IgE, and IgM; a cytokine, including IL-2 or IL-15; a toxin; an antibody or an antigenbinding fragment thereof, including anti-CD3, anti-CD28, anti-CD5, anti-CD 16 or an anti- CD56 antibody or an antigen-binding fragment thereof; and a CD247 (CD3-zeta), CD28, CD137, or a CD134 domain, or combinations thereof, optionally further comprising at least one linker.
  • a Fc receptor a Fc domain, including IgA, IgD, IgG, IgE, and IgM
  • a cytokine including IL-2 or IL-15
  • a toxin an antibody or an antigenbinding fragment thereof, including anti-CD3, anti-CD28, anti-CD5, anti-CD 16 or an
  • the chimeric co-stimulatory receptor comprises: i) an extracellular domain, preferably comprising a polypeptide derived from PD- 1 , further preferably at its N-terminus (e.g., SEQ ID NO: 28); ii) a transmembrane domain, preferably comprising a polypeptide derived from PD-1 (e.g., SEQ ID NO: 30); and/or iii) an intracellular domain, preferably comprising a polypeptide derived from 4- 1 BB, further preferably at its C-terminus (e.g., SEQ ID NO: 32).
  • said extracellular domain comprising a polypeptide derived from PD-1 comprises an amino acid sequence with 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or insertions compared to SEQ ID NO: 28; ii) said extracellular domain comprising a variant of the parent extracellular PD-1 polypeptide of SEQ ID NO: 28, wherein said variant comprising an alteration (e.g., a substitution, deletion and/or insertion) at one or more positions corresponding to positions of the parent polypeptide, wherein said variant having at least 80% (e.g.
  • transmembrane domain comprising a polypeptide derived from PD-1 comprises an amino acid sequence with 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or insertions compared to the amino acid sequence of SEQ ID NO: 30; iv) said transmembrane domain comprising a variant of the parent PD-1 transmembrane polypeptide of SEQ ID NO: 30, wherein said variant comprising an alteration (e.g., a substitution, deletion and/or insertion) at one or more positions corresponding to positions of the parent polypeptide, wherein said variant having at least 80% (e.g.
  • said intracellular domain comprising a polypeptide derived from 4-1 BB comprises an amino acid sequence with 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and/or insertions compared to SEQ ID NO: 32; and/or vi) said intracellular domain comprising a variant of the parent 4-1 BB intracellular polypeptide of SEQ ID NO: 32, wherein said variant comprising an alteration (e.g., a substitution, deletion and/or insertion) at one or more positions corresponding to positions of the parent polypeptide, wherein said variant having at least 80% (e.g.
  • CTLs cytotoxic T lymphocytes
  • NK natural killer
  • NKT natural killer T
  • gamma/ delta-T- cells gamma/ delta-T- cells.
  • a nucleic acid comprising a nucleotide sequence encoding the TCR (or fragment thereof, e.g., as depicted in Table 2) as defined by any one of items 1-24 and/or a nucleotide sequence encoding the chimeric co-stimulatory receptor as defined by any one of items 1-24 (or fragment thereof, e.g., as depicted in Table 2, preferably one or more of SEQ ID NO: 27, 29, 31 and/or 33).
  • a vector comprising the nucleic acid of item 29.
  • a host cell e.g., isolated and/or recombinant host cell comprising the nucleic acid of item 29, or the vector of item 30.
  • a pharmaceutical or diagnostic composition comprising one or more of:
  • the complex and/or the combination, and/or the cell population or the cell of any one of items 1-28, the nucleic acid of item 29, the vector of item 30, the host cell of item 31 and/or the pharmaceutical composition of item 33 for use in a method of detecting, diagnosing, prognosing, preventing and/or treating cancer.
  • the complex and/or the combination, and/or the cell population or the cell, the nucleic acid, the vector, the host cell and/or the pharmaceutical composition for the use of item 35, wherein prevention and/or treatment of cancer comprises: administering at least any one of the following (i) to (vi):
  • a method of detecting the presence of a cancer in a subject in vitro comprising:
  • (b) detecting the complex wherein detection of the complex is indicative of the presence of the cancer in the subject.
  • a kit comprising the complex and/or the combination, and/or the cell population or the cell of any one of items 1-28, the nucleic acid of item 29, the vector of item 30, the host cell of item 31 , and/or the pharmaceutical or diagnostic composition of item 33.
  • Table 2 TCR sequences and epitopes.
  • mKRAS?-i6 G12V-specific TCR Identification and isolation of mKRAS?-i6 G12V-specific TCR from T cell repertoires of a healthy donor using in vitro high-throughput T cell priming technology.
  • This invention describes a TCR with high specificity and sensitivity for mKRAS?- 16 G 12V epitope isolated from T cell repertoires of a healthy donor using in vitro high-throughput T cell priming technology as follows.
  • the priming system employed used monocyte-derived mature dendritic cells (mDCs) isolated from peripheral blood mononuclear cells (PBMC) of HLA-A*11 :01-positive healthy donor as antigen-presenting cells and autologous CD8-enriched T cells as responding cells.
  • mDCs monocyte-derived mature dendritic cells isolated from peripheral blood mononuclear cells (PBMC) of HLA-A*11 :01-positive healthy donor as antigen-presenting cells and autologous CD8-enriched T cells as responding cells.
  • ivtRNA In vitro transcribed RNA (ivtRNA) encoding a fragment of the human mKRAS protein containing G12V mutation (amino acid sequence GLLKMTEYKLWVGAVGVGKSALTIQLIQ, see SEQ ID NO: 38) served as the source of specific antigen.
  • the mKRAS G12V-encoding ivtRNA was translated into a polypeptide, which was subsequently processed and presented as peptides by HLA-A*11 :01 molecules endogenously expressed by mDCs.
  • co-cultures of T cells with ivtRNA-transfected mDCs from the same donor led to de novo induction of antigen-specific T cells that served as the source of corresponding TCRs.
  • mKRAS G12V-specific T cells were identified using HLA-A*11 :01 mKRAS G12V fluorescently labelled multimers and separated by single cell sorting using fluorescence-activated cell sorting (FACS) technology.
  • FACS fluorescence-activated cell sorting
  • specific T cell clones of interest were pre-screened for differential mKRAS G12V vs. wildtype (wt) KRAS recognition on tumor target cells, and further selected using a broader tumor cell panel with multiple mKRAS G12V-endogenously positive tumor cell lines, mKRAS G12V-overexpressing tumor cell lines and wtKRAS tumor cell lines as negative controls.
  • NGS next-generation sequencing
  • TCR sequencing and cloning TCR sequencing and cloning.
  • TCR-a and TCR-p chains of mKRAS?-i6 G12V-specific T cell clones were identified by next-generation sequencing using an established standard protocol for analysis with the MiSeq system (Illumina, San Diego, CA, USA). For sample preparation, the manufacturer’s recommendations were followed using the Dynabeads mRNA DIRECT Kit (Thermo Fisher), SMART Scribe reverse transcriptase (Takara Bio, Montain View, CA, USA), and the MiSeq V3 Kit (Illumina). The constant regions of all TCR chains were minimally murinized to increase the stability of the TCRs (Sommermeyer et al. 2010, J.
  • the chimeric co-stimulatory receptor PD1-41 BB consisting of the extracellular and transmembrane region of PD-1 (CD279, amino acids (aa) 21-191) and the intracellular domain of 4-1 BB (CD137, aa 214-255), was added to the vector 5' of the TCR and separated by a T2A element.
  • the sequence was codon-optimized (GeneArt, Thermo Fisher) (Scholten et al. 2006, Clin. Immunol; 119, 135-145), ordered from GeneArt (Thermo Fisher), and cloned into the pES.12-6 self-inactivating gamma-retrovirus vector.
  • PBMCs Peripheral blood mononuclear cells
  • BIOSELL synthetic copolymer Biocoll®
  • Leucosep tubes Greiner Bio-One
  • CD8 + T cells were enriched starting from PBMCs by MACS® technology.
  • the CD8 + T cell Isolation kit (Miltenyi) was used to isolate untouched CD8 + T cells, avoiding direct labeling surface molecules on the desired cells that could interfere with subsequent applications. The MACS separation was performed according to the manufacturer’s protocol.
  • CD8 + T cells carrying endogenous Cp2-TCRs were isolated by depleting T cells carrying endogenous Cpi-TCRs by MACS® technology in combination with biotinylated anti-human Cpi TCR antibody (JOVI.1 , Ancell).
  • CD8 + endogenous Cp2-TCR + T cells were activated using DynabeadsTM Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Gibco).
  • Cell culture medium RPMI Medium 1640; Gibco
  • IL-2 PROLEUKIN® S, Clinigen
  • Retroviral vector plasmids containing the TCR in combination with PD1-41 BB CSR were co-transfected in HEK293FT cells with helper plasmids encoding Moloney MLV gag/pol and the GALV env gene to produce amphotropic retroviruses by using the TranslT®-LT1 transfection reagent (Mirus).
  • Retroviral supernatants were harvested 48 h after transfection and 2 mL of filtered viral supernatant were added per well to Retronectin (Takara Bio)-coated 24-well plates that were centrifuged at 1000* g at 32 °C for 90 min. For transduction, 0.25-0.5 x 10 6 activated T cells were added per 24-well.
  • T cells were transduced in the same manner a second time approximately 24 h after the first transduction; 18 to 24 h after transduction, T cells underwent a 10-day-phase of expansion after transfer into G-Rex flasks (Wilson Wolf, Saint Paul, MN, USA), during which they were supplied with fresh IL-2 at a final concentration of 50U/ml twice a week.
  • the cells were harvested from the G-Rex flasks, and transduction rates were determined before the cells were enriched to obtain transgenic TCR + PD1-41 BB + high-purity T cell populations.
  • Enriched cells were frozen in small aliquots and 10 days prior each functional assay cells were thawed and expanded by a rapid expansion protocol by addition of a restimulation cocktail, as previously described (Riddell and Greenberg 1990, J. Immunol. Methods;128, 189-201).
  • PBMCs were isolated from blood samples by density gradient centrifugation using a separating solution containing the synthetic copolymer Biocoll® (BIOSELL) and Leucosep tubes (Greiner Bio- One).
  • CD3 + T cells were enriched starting from PBMCs by MACS® technology.
  • the Pan T cell Isolation Kit (Miltenyi) was used to isolate untouched CD3 + T cells, avoiding direct labeling surface molecules on the desired cells that could interfere with subsequent applications. The MACS separation was performed according to the manufacturer’s protocol.
  • CD3 + T cells carrying endogenous Cp2-TCRs were isolated by depleting T cells carrying endogenous Cpi-TCRs by MACS® technology in combination with biotinylated anti-human Cpi TCR antibody (JOVI.1 , Ancell).
  • CD3 + endogenous Cp2-TCR + T cells were activated using DynabeadsTM Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Gibco).
  • Cell culture medium RPMI Medium 1640; Gibco
  • 10% human serum 1x non-essential amino acids (NEAA), 2mM L-glutamine, and 1mM sodium-pyruvate, 1x Penicillinstreptomycin, 10 mM HEPES and 50 pM 2-Mercaptoethanol was supplemented with 50 U/ml IL-2 (PROLEUKIN® S, Clinigen).
  • Retroviral vector plasmids containing the TCR in combination with PD1-41 BB CSR were co-transfected in HEK293FT cells with helper plasmids encoding Moloney MLV gag/pol and the GALV env gene to produce amphotropic retroviruses by using the TranslT®-LT1 transfection reagent (Mirus). Retroviral supernatants were harvested 48 h after transfection and 2 mL of filtered viral supernatant were added per well to Retronectin (Takara Bio)-coated 24-well plates that were centrifuged at 1000* g at 32 °C for 90 min. For transduction, 0.25-0.5 x 10 6 activated T cells were added per 24-well.
  • T cells were transduced in the same manner a second time approximately 24 h after the first transduction; 18 to 24 h after transduction, T cells underwent a 10-day-phase of expansion after transfer into G-Rex flasks (Wilson Wolf, Saint Paul, MN, USA), during which they were supplied with fresh IL-2 at a final concentration of 50U/ml twice a week. The cells were harvested from the G-Rex flasks, and transduction rates were determined before the cells were used to evaluate intracellular IFN-y stain in response to target stimulation.
  • G-Rex flasks Wang Wolf, Saint Paul, MN, USA
  • CD8 + and CD4 + T cell populations expressing the transgenic TCR and PD1-4 BB CSR were separated by FACS from transduced CD3 + T cells. Enriched cells were expanded 10 days prior the functional assay by a rapid expansion protocol by addition of a restimulation cocktail, as previously described (Riddell and Greenberg 1990, J. Immunol. Methods;128, 189-201).
  • Tumor cell lines were transduced with HLA-A*11 :01-encoded molecules to allow surface expression of the desired HLA molecules. Additionally, to obtain robust and stable PDL1 expression, all selected tumor cell lines were transduced with PDL1.
  • K562 (ATCC, CCL-243) transduced with either HLA-A*11 only or HLA-A*11 in combination with PDL1
  • Dan-G (DSMZ, ACC 249) transduced with either HLA-A*11 only or HLA-A*11 in combination with PDL1
  • NCI-H441 (ATCC, HTB-174) transduced with either HLA- A*11 only or HLA-A*11 in combination with PDL1
  • DU145 (ATCC, HTB-81) transduced with either HLA-A*11 only or HLA-A*11 in combination with PDL1 and Mel624.38 (Kind gift of M.
  • HLA-A*11 panelli, National Institutes of Health, Bethesda, MD, USA) transduced with either HLA-A*11 only or HLA-A*11 in combination with PDL1 were all maintained in RPMI 1640 containing 10% FBS, 2mM L-glutamine, 1 %NEAA and 1% Penicillin-Streptomycin at 5% CO2.
  • SW527 ATCC, CRL-7940 were transduced with either HLA-A*11 only or HLA-A*11 in combination with PDL1 were maintained in DMEM containing 10% FBS, 2mM L-glutamine, 1% NEAA and 1% Penicillin-Streptomycin at 5% CO2.
  • SW480 (ATCC, CCL-228) transduced with either HLA-A*11 only or HLA-A*11 in combination with PDL1 were maintained in Leibovitz L-15 Medium containing 10% FBS and 1% Penicillin-Streptomycin at 0% CO2.
  • HLA-A*11 01-positive normal human renal cortical epithelial cells (HRCEpC, PromoCell, Heidelberg, Germany), primary HLA-A*11 :01-positive normal human cardiac fibroblasts (HCF-c, PromoCell), primary HLA-A*11 :01-positive normal human atrial cardiac fibroblasts (NHCF-A, Lonza, Basel, Switzerland), primary HLA-A*11 :01-positive normal human lung fibroblasts (NHLF, Lonza), induced pluripotent stem cell-derived HLA- A*11 :01 -transfected iCell Cardiomyocytes (iCardio, Fujifilm Cellular Dynamics, Madison, Wl, USA), induced pluripotent stem cell-derived HLA-A*11 :01-transduced endothelial cells (iEC, Fujifilm Cellular Dynamics), induced pluripotent stem cell-derived HLA-A*11 :01-transfected astrocytes (iA
  • Flow cytometric analysis was performed on a MACSQuant X (Miltenyi Biotec) or a BD LSRFortessa Flow Cytometer (BD).
  • FlowJo 10.2 software FlowJo, Ashland, OR, USA
  • Multimer staining was performed by using fluorochrome-labeled custom-synthesized tetramers (mKRAS 7 -i6 G12V peptide/HLA-A*11 :01 and mKRAS 8 -i6 G12V peptide/HLA-A* 11 :01 , ImmunAware) according to manufacturer’s instructions.
  • Cell sorting was conducted using either FACSAria Fusion flow cytometer (BD) or an SH800S cell sorter (Sony).
  • IFN-y release by T cells in response to different target cells was assessed in coculture supernatants after 20 h of stimulation by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • co-culture experiments were performed using different panels of target cells comprising tumor cell lines, mKRAS?-i6 G12V peptide-loaded targets, mKRASs-16 G12V peptide-loaded targets, KRASs-16 wild type peptide-loaded targets, LCL library, K562 library, mismatched peptide-loaded targets, in vitro transcribed RNA (/vtRNA)- transfected target cells and a panel of healthy cells.
  • Peptides were ordered at Peptides&Elephants GmbH.
  • mMESSAGE mMACHINETM T7 kit (Thermo Fischer Scientific) was used to produce /vtRNA according to manufacturer’s instructions.
  • target cells were harvested, counted and adjusted to 1x10 6 cells/ml using appropriate medium in a tube. Subsequently, the required amount of peptide was added to the cells to reach the desired concentration. Cells were incubated for 1h and 30 minutes at 37°C. After incubation, the appropriate medium was added to a final volume of 15 ml. Cells were centrifuged (350 x g for five minutes) and the supernatant was discarded to remove unbound peptides. Cells were resuspended in fresh medium at the required concentration for the subsequent applications.
  • K562 target cells were harvested, counted and washed once with RPMI1640 medium without serum. Cells were adjusted at 2 - 3 x 10 6 cells/200 pl in medium without serum and 200 pl of the cell suspension were transferred into a pre-cooled electroporation cuvette. Subsequently, either 20 pg of RNA or 20 pl of water (negative control) were added into the cuvette. The suspension was shortly mixed by pipetting and then quickly electroporated using the Gene Pulser XcellTM device (BioRad, Hercules, CA, USA). Electroporation was performed using an exponential protocol at 300 V and 300 pF. Immediately after electroporation, cells were transferred into the appropriate fresh medium and placed into the incubator. After three hours, cells were counted and resuspended at the adequate concentrations for further applications.
  • Tumor cell lysis was assessed using either IncuCyte S3® or Zoom® device (Sartorius, Goettingen, Germany) and following manufacturer’s recommendations for real-time quantitative live-cell imaging.
  • tumor cells were transduced with NucLightRed (Sartorius) and seeded into 96-well flat-bottom plates 24 h prior to the addition of TCR + PD1- 41 BB-transduced T cells at E:T ratio of 2:1 (2 x 10 4 effectors 1 96-well).
  • mKRAS?-i6 G12V peptide was added at the desired concentration into the wells containing pre-seeded tumor cell lines and incubated for 1h 30 minutes at 37°C. Lysis of tumor cells was monitored by imaging the plates at regular intervals (every 4 h). The number of NucLightRed-labeled tumor cells over time was calculated using IncuCyte software (Sartorius).
  • TCR-transduced CD3 + T cells (0.2 x106 cells/well) were co-cultured for 13-15 h with peptide-loaded (10-5/10-7 M), target gene-transduced or antigen-positive target cells at an E:T ratio of 1 :1 in 96-well round bottom wells in the presence of Brefeldin A solution (10pg/ml, Invitrogen).
  • Frequency of IFN-y + CD8 + and IFN-y + CD4 + T cells was determined by multi-color flow cytometry using combinations of fluorochrome-conjugated antibodies antihuman CD3 PE-Cy7 (BD), anti-human CD4 FITC (BD), anti-human CD8 PerCP (BD), antihuman CP1 TCR-PE (JOVI.1 , BD), IFN-y V450 (BD) and live/dead dye FVS780 (BD). After coculture, cells were washed with PBS and incubated with antibodies at 2-8°C for 30 minutes.
  • Example 1 High and robust transgenic TCR and PD1-41BB CSR expression.
  • TCR costimulatory switch receptor
  • CD8 + T cells were enriched using anti-TCR Cpi antibody and anti-PD1 antibody by fluorescence-activated cell sorting (FACS). After enrichment and rapid expansion protocol, CD8 + T cells were stained with anti-TCR Cpi , anti-PD1 and anti-CD8 antibodies and analyzed by flow cytometry to assess transduction efficiency and expression levels of the transgenes. Binding of TCR Cpi antibody and PD1 antibody to TCR + PD1 -41 BB- transduced CD8 + T cells indicated efficient transduction with high expression levels of the transgenic TCR which correlated with the expression of the transgenic PD1 -41 BB CSR ( Figure 1).
  • Example 2 High mKRAS?-i6 G12V (10-mer) multimer binding.
  • Multimer binding is first proof of transgenic TCR functionality. Therefore, after an enrichment and rapid expansion protocol, TCR + PD1-41 BB-transduced CD8 + T cells were stained with mKRAS?-i6 G12V (10-mer) HLA-A*11 :01 multimer and analyzed by flow cytometry. Results showed high and robust 10-mer multimer binding for TCR + PD1-41 BB-transduced T cells ( Figure 2). These findings indicated binding of TCR + PD1-41 BB CSR to HLA-A*11 :01 multimer loaded with mKRAS?-i6 G12V 10-mer peptide.
  • a 10-mer length peptide variant has been described as the most abundant and stable mKRAS G12V peptide presented on HLA-A*11 :01 molecules in tumor cells by mass spectrometry analysis (Jaewon Choi et al. 2021 , Cell Rep Methods; Adham S. Bear et al. 2021 , Nature Communications).
  • Example 3 Exclusive mKRAS?-i6 G12V specificity with no KRAS wild type recognition.
  • TCR + PD1- 41 BB-transduced CD8 + T cells were co-cultured with PDL1 -transduced K562 cells expressing transgenic HLA-A*11 :01-encoding molecules (hereafter designated as K562_A11_PDL1) loaded with either mKRAS?-i6 G12V peptide or KRAS7-16 wild type peptide at high concentration (10' 5 M).
  • CD8 + T cell samples were also co-cultured with K562_A11_PDL1 cells transfected with either ivtRNA encoding a fragment of mKRAS G12V gene (-402 bp) or ivtRNA encoding a fragment of KRAS wild type gene (-402 bp).
  • ivtRNA-transfected targets served to assess the capacity of TCR + PD1-41 BB CSR-transduced CD8 + T cells to recognize KRAS epitopes when processed and presented by internal cellular pathways. Unloaded and untransfected K562_A11_PDL1 cells were included as negative target controls. Untransduced CD8 + T cells served as internal effector controls. After 20 hours (h) of co-culture, an ELISA was performed to evaluate IFN-y secretion by T cells.
  • TCR + PD1-41 BB-transduced CD8 + T cells recognized only K562_A11_PDL1 cells when loaded with either mKRASy-16 G12V peptide or transfected with mKRAS G12V ivtRNA. No IFN-y release was observed in response to target cells expressing KRAS7-16 wild type ( Figure 3). These results showed exclusive mKRASy-16 G12V peptide specificity of TCR + PD1-41 BB CSR-transduced CD8 + T cells (no recognition of KRAS wild type) and the capacity to recognize the epitope when processed and presented by cellular internal pathways.
  • Example 4 High peptide sensitivity for mKRASy-16 G12V (10-mer) epitope.
  • Functional avidity refers to the accumulated strength of multiple affinities of individual non-covalent binding interactions, such as the transgenic TCR and the peptide-HLA complex. As such the functional avidity of TCR-transduced T cells serves as a measure of peptide sensitivity. TCRs conferring high peptide sensitivity can recognize lower amounts of peptide.
  • Example 5 Recognition of mKRASy-16 G12C epitope presented by HLA- A*11 :01 molecules.
  • TCR + PD1- 41 BB-transduced CD8 + T cell were tested in a co-culture experiment with K562_A11_PDL1 cells loaded with either mKRASy-16 G12V, mKRASy-16 G12D, mKRASy-16 G12C or mKRASy-16 G12R peptides at high concentration (10' 5 M).
  • Target cells loaded with KRASy-16 wild type peptide as well as unloaded targets were included in the experiment as internal controls.
  • K562_A11_PDL1 cells were transfected with four ivtRNA constructs encoding the previously described KRAS mutations individually (-402 bp spanning the G12 mutation site).
  • ivtRNA-transfected targets served to assess the capacity of TCR + PD1-41 BB-transduced CD8 + T cells to recognize alternative KRAS mutated epitopes when processed and presented by internal cellular pathways.
  • ivtRNA encoding KRASy-16 wild type epitope was also used to transfect target cells, serving as an internal control. Water electroporation of target cells served as a negative control. IFN-y secretion was evaluated by ELISA 20 h after setting up the coculture.
  • TCR + PD1-41 BB-transduced CD8 + T cells recognized mKRASy-16 G12V epitope at high levels, both as loaded peptide and as an ivtRNA-encoded epitope.
  • mKRASy-16 G12C epitope was slightly recognized by TCR + PD1-41 BB- transduced CD8 + T cells ( Figure 5).
  • Example 6 Peptide-specific restriction to four different HLA-A*11 subtypes.
  • HLA-A*11 :01 HLA- A*11 :02, HLA-A*11 :03, HLA-A*11 :04
  • HLA class I molecules can be clustered into groups (designated as HLA supertype families) representing sets of molecules that share largely overlapping peptide binding specificity.
  • HLA-A*11 :01 has been described as a member of the HLA-A*03 superfamily together with HLA-A*03:01 , HLA-A*30:01 , HLA-A*31 :01 , HLA-A*33:01 and HLA- A*68:01.
  • a specific TCR that recognizes its cognate peptide in the context of HLA- A*11 :01 might also recognize the peptide presented by another HLA-A*11 subtype and/or by other HLA alleles of the HLA-A*03 superfamily.
  • TCR was characterized in the context of the HLA-A*11 subtypes and HLA-A*03 superfamily members.
  • CD8 + T cells expressing either no transgenic TCR or the combination of TCR and PD1-41 BB CSR were co-cultured with H LA-transduced K562 cell samples.
  • Each K562 sample expressed individual transgenic HLA molecules previously described and was tested after mKRAS?-i6 G12V peptide loading (10' 5 M). IFN-y release was evaluated by ELISA 20 h after setting up the co-culture.
  • TCR + PD1-41 BB-transduced CD8 + T cells recognized mKRAS?-i6 G12V peptide presented by HLA molecules encoded by HLA-A*11 :02, HLA-A*11 :03 and HLA- A*11 :04 at similar levels compared to HLA-A*11 :01 ( Figure 6).
  • TCR + PD1-41 BB-transduced CD8 + T cells presented peptide-specific restriction to four different HLA-A*11 subtypes.
  • Example 7 No signs of target peptide-independent cross-recognition of global frequent HLA allotypes.
  • the first library comprised 70 lymphoblastoid cell lines (LCLs, LCL library) covering the most frequent HLA-A, -B and -C alleles in Asian and USA/European Caucasian populations.
  • HLA-A, -B and -C alleles exhibiting HLA frequencies of more than 0.5% were covered by at least one LCL sample and HLA-A, -B and -C alleles exhibiting HLA frequencies over 5% were covered by at least two LCL samples.
  • the second library comprised 31 K562 cell samples transduced with individual HLA molecules (K562 library) covering HLA-A alleles exhibiting HLA frequencies over2.5% in Asian populations and HLA-B and HLA-C alleles most frequent in Asian populations not or not fully covered in the LCL library.
  • TCR + PD1-41 BB-transduced CD8 + T cells and untransduced CD8 + T cells were co-cultured with the two libraries.
  • K562_A11 cells loaded with mKRAS?-i6 G12V peptide (10‘ 5 M) served as an internal positive target control.
  • IFN-y secretion was evaluated by ELISA 20 h after setting up the co-culture.
  • Example 8 Successful de-risking of potential peptide off-target toxicity.
  • a co-culture experiment was performed using K562_A11_PDL1 cells loaded with the selected 299 mismatched peptides individually (10 -5 M, 10' 6 M, 10' 7 M).
  • Target cells loaded with mKRAS?-i6 G12V peptide served as positive controls while unloaded targets served as negative controls.
  • TCR + PD1-41 BB-transduced CD8 + T cells as well as untransduced CD8 + T cells were utilized as effector cells. IFN-y secretion was assessed by ELISA 20 h after co-culture.
  • Cross-recognized peptides can be de-risked if they are not processed from the original protein, fail to be loaded on HLA molecules endogenously after overexpression in target cells or are not able to trigger activation of TCR + PD1-41 BB-transgenic CD8 + T cells.
  • Example 9 No signs of off-target toxicity were identified using a panel of healthy cell samples.
  • HLA-A*11 01-positive primary healthy cells and induced pluripotent stem cell (iPS)-derived cell lines representing essential organs were tested for recognition by TCR + PD1-41 BB-transduced CD8 + T cells.
  • iPS induced pluripotent stem cell
  • Example 10 High and specific IFN-y secretion in response to tumor cells.
  • T cells expressing the candidate TCR are able to specifically recognize tumor cells expressing the target antigen.
  • a standard way to assess tumor cell recognition by TCR-transgenic T cells is the evaluation of IFN-y release by T cells upon target antigen-specific stimulation.
  • Tumor cell lines derived from various cancers pancreatic, colorectal, lung and breast tumors expressing mKRAS G12V at different levels (three homozygous cell lines and one heterozygous cell line) were selected as target cells for this co-culture experiment.
  • two tumor cell lines expressing only KRAS wild type served as mKRAS G12V- negative tumor target cells. All tumor cell lines were transduced with HLA-A*11 :01 -encoded molecules to allow surface expression of the desired HLA molecules. Expression of the transgenic HLA-A*11 molecules was confirmed by staining with anti-HLA-A11 antibody and subsequent flow cytometry analysis.
  • the co-culture experiment was set up by using the described tumor cell lines and TCR + PD1-41 BB-transduced CD8 + T cells. Untransduced CD8 + T cells served as negative control. IFN-y secretion was assessed 20 h after setting up the co-culture.
  • IFN-y release was observed only when TCR + PD1-41 BB-transduced CD8 + T cells were stimulated with mKRAS G12V-positive tumor cells (Figure 10). Highest IFN-y release was observed for co-cultures with mKRAS G12V-positive tumor cells transduced with PDL1 , showing the maximum effect of PD1-41 BB CSR to transform in stimulation the natural inhibitory PDL1 signal for each tumor cell line. Importantly, no recognition of any KRAS wild type tumor cells was observed. These findings confirmed specificity of TCR + PD1-41 BB and its capacity to be activated and subsequently drive release of IFN-y in response to tumor cells expressing endogenous levels of mKRAS G12V. Additionally, they demonstrated that the specificity of the TCR-transgenic CD8 + T cells was not affected by PD1-41 BB CSR coexpression.
  • Example 11 Strong and specific cytotoxic response against tumor cells even after multiple tumor challenges.
  • Example 12 Signs of TCR functionality in CD4 + T cells.
  • the CD8 co-receptor is known to directly bind to HLA class I molecules and to be critical for the development of CD8 + T cells.
  • the CD8 co-receptor stabilizes the binding of a TCR to the MHC-peptide complex and facilitates early events in the TCR signaling cascade.
  • TCR in combination with PD1-41 BB CSR was transduced in CD3 + T cells derived from healthy donors (mixed population containing both CD4 + and CD8 + T cells) and utilized as effector samples in a co-culture experiment. Corresponding untransduced CD3 + T cells served as controls.
  • TCR + PD1-41 BB CSR expressed in CD8 + T cells was activated and resulted in a high and robust fraction of IFN-y-positive cells after co-culture with all targets expressing mKRASy- G12V epitope (peptide-loaded and mKRAS G12V-transduced targets) (Figure 12).
  • TCR + PD1-41 BB CSR expressed in CD4 + T cells also recognized mKRASy-16 G12V peptide-loaded K562_A11_PDL1 cells (both concentrations), mKRAS G12V- transduced K562_A11 cells and mKRAS G12V-positive Dan-G tumor cells, showing that the functionality of the transgenic TCR + PD1-41 BB was not dependent on the presence of the CD8 co-receptor.
  • Example 13 IFN-y release and cytotoxic response against tumor cells mediated by TCR + PD1-41 BB-transduced CD4 + T cells.
  • TCR in combination with PD1-41 BB CSR was expressed in both CD8 + T cells and CD4 + T cells.
  • CD8 + and CD4 + TCR- expressing T cells were then enriched by fluorescence-activated cell sorting (FACS). After enrichment and rapid expansion protocol, CD8/CD4 purity and transgenic TCR expression (anti-Cb1 antibody) was verified by flow cytometry analysis. Additionally, T cell samples were stained with mKRAS?-i6 G12V (10-mer) HLA-A*11 :01 multimer and analyzed by flow cytometry. The results showed high and robust 10-mer multimer binding by TCR + PD1-41 BB when expressed not only in CD8 + T cells but also in CD4 + T cells, confirming signs of TCR functionality in CD4 + T cells (Figure 13A).
  • Enriched CD8 + and CD4 + T cell samples were co-cultured with either mKRAS G12V-positive or KRAS wild type tumor cell lines, which were characterized as previously described.
  • mKRAS?-i6 G12V peptide-loaded tumor cell lines were used as internal positive controls. All tumor cells expressed a red fluorescent protein for cell tracking over time. Red fluorescent tumor cells were seeded one day prior to the start of the co-culture experiment. After addition of effector cells, the co-culture plates were monitored over time by a live-cell imaging system to evaluate the reduction of red fluorescent tumor cells, indicating in turn cytotoxicity mediated by TCR + PD1-41 BB-transgenic T cells ( Figure 13C). Additionally, an aliquot of each co-culture supernatant was collected to assess IFN-y secretion by ELISA 20 h after setting up the co-culture.
  • KRAS wild type tumor cells were not recognized by CD8 + T cells nor by CD4 + T cells expressing TCR + PD1-41 BB.
  • Example 14 T cells co-expressing the transgenic TCR and PD1-41BB CSR showed a higher polyfunctionality compared to T cells expressing only the transgenic TCR.
  • TCR-transgenic CD8 + T cells with or without PD1-41 BB CSR were analyzed at single-cell level to evaluate polyfunctionality (release of 2 or more proteins) using the IsoLight® technology (PhenomeX). So called “polyfunctional” T cells can secrete multiple proteins (in particular cytokines and other effector proteins such as Gzm-B), thereby enabling a whole variety of effector functions and a highly effective immune response.
  • TCR-transduced T cells co-expressing PD1-41 BB CSR showed a higher percentage of poly-functional T cells, meaning T cell secreting 2 or more proteins, compared to TCR-transduced T cells lacking PD1-41 BB CSR ( Figure 14A).
  • TCR + PD1-41 BB-transduced T cells showed a higher polyfunctional strength index (PSI) compared to T cells expressing only the transgenic TCR ( Figure 14B).
  • PSI polyfunctional strength index
  • Classification of the various cytokines/lytic proteins released revealed a high contribution of effector (Gzm-B, IFN-y, MIP-1a, TNF-a) and stimulatory (GM-CSF, IL-5, IL-9) cytokines/lytic proteins to the superior PSI, followed by chemo-attractive (MIP-1 P) cytokines.
  • Regulatory cytokines were not released and inflammatory cytokine (IL-6) only to a lower extent.

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Abstract

La présente invention concerne un complexe polypeptidique TCR et/ou une combinaison comprenant un nouveau TCR caractérisé par ses séquences CDR de la région variable de chaîne α et de la région variable de chaîne β et par sa spécificité antigénique à (a) un ou plusieurs épitope(s) KRAS muté(s) particulier(s) ou un fragment correspondant présenté dans le contexte d'une molécule du CMH de classe I. La présente invention concerne en outre un acide nucléique, un vecteur ainsi qu'une cellule hôte comprenant ledit acide nucléique ou ledit vecteur. La présente invention concerne un procédé d'obtention dudit TCR et dudit récepteur de costimulation chimérique et une composition pharmaceutique ou diagnostique comprenant les éléments susmentionnés. La présente invention concerne en outre les éléments susmentionnés aux fins d'une utilisation en tant que médicament ou aux fins d'une utilisation dans une méthode de diagnostic, de détection, de prévention et/ou de traitement du cancer. En outre, la présente invention concerne une méthode de détection de la présence d'un cancer chez un sujet in vitro, par application, dans ladite méthode, des éléments susmentionnés, et l'utilisation des éléments susmentionnés pour générer des lymphocytes modifiés et un kit.
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2173869A2 (fr) 2007-06-21 2010-04-14 Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Protéine de fusion comportant un domaine de caspase et un domaine associé au récepteur d'hormone nucléaire et procédés et utilisations associés
US20190247432A1 (en) * 2017-12-12 2019-08-15 The Trustees Of The University Of Pennsylvania Genetically Modified Immune Cells Targeting NY-ESO-1 and Methods of Use Thereof
US20190330306A1 (en) * 2016-12-22 2019-10-31 Windmil Therapeutics, Inc. Compositions and Methods for Modulating the Immune System
WO2020082130A1 (fr) * 2018-10-25 2020-04-30 The Council Of The Queensland Institute Of Medical Research Récepteurs de lymphocytes t et utilisations associées
WO2021097365A2 (fr) * 2019-11-15 2021-05-20 Gritstone Oncology, Inc. Protéines de liaison à l'antigène ciblant des néoantigènes partagés
WO2022038115A1 (fr) 2020-08-18 2022-02-24 Medigene Immunotherapies Gmbh Enrichissement de lymphocytes t au moyen d'un anticorps anti-css
WO2022060904A1 (fr) * 2020-09-16 2022-03-24 Obsidian Therapeutics, Inc. Compositions et procédés pour l'expression de récepteurs de lymphocytes t avec cd40l régulé par petites molécules dans les lymphocytes t

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2173869A2 (fr) 2007-06-21 2010-04-14 Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Protéine de fusion comportant un domaine de caspase et un domaine associé au récepteur d'hormone nucléaire et procédés et utilisations associés
US20190330306A1 (en) * 2016-12-22 2019-10-31 Windmil Therapeutics, Inc. Compositions and Methods for Modulating the Immune System
US20190247432A1 (en) * 2017-12-12 2019-08-15 The Trustees Of The University Of Pennsylvania Genetically Modified Immune Cells Targeting NY-ESO-1 and Methods of Use Thereof
WO2020082130A1 (fr) * 2018-10-25 2020-04-30 The Council Of The Queensland Institute Of Medical Research Récepteurs de lymphocytes t et utilisations associées
WO2021097365A2 (fr) * 2019-11-15 2021-05-20 Gritstone Oncology, Inc. Protéines de liaison à l'antigène ciblant des néoantigènes partagés
WO2022038115A1 (fr) 2020-08-18 2022-02-24 Medigene Immunotherapies Gmbh Enrichissement de lymphocytes t au moyen d'un anticorps anti-css
WO2022060904A1 (fr) * 2020-09-16 2022-03-24 Obsidian Therapeutics, Inc. Compositions et procédés pour l'expression de récepteurs de lymphocytes t avec cd40l régulé par petites molécules dans les lymphocytes t

Non-Patent Citations (30)

* Cited by examiner, † Cited by third party
Title
"UniProt", Database accession no. Q15116
ADHAM S. BEAR ET AL., NATURE COMMUNICATIONS, 2021
ALEXANDROV LB ET AL., NATURE, vol. 578, no. 7793, 2020, pages 94 - 101
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUCLEIC ACIDS RES, vol. 25, 1997, pages 3389 - 3402
CHEN ET AL., ADV DRUG DELIV REV, vol. 65, no. 10, 15 October 2013 (2013-10-15), pages 1357 - 1369
GERTNER-DARDENNE ET AL., J IMMUNOL, vol. 189, no. 9, 2012, pages 4701 - 4708
HABEL ET AL., PLOS PATHOG, vol. 18, no. 3, 2022
HUANG L ET AL., SIGNAL TRANSDUCT TARGET THER, vol. 6, no. 1, 2021, pages 386
JAEWON CHOI ET AL., CELL REP METHODS, 2021
JARAVINE VMÖSCH ARAFFEGERST S ET AL., BMC CANCER, vol. 17, no. 1, 2017, pages 892
KIEBACK ET AL., PROC NATL ACAD SCI U SA., vol. 105, no. 2, 15 January 2008 (2008-01-15), pages 623 - 8
KOBOLD S ET AL: "Impact of a New Fusion Receptor on PD-1-Mediated Immunosuppression in Adoptive T Cell Therapy", JOURNAL OF THE NATIONAL CANCER INSTITUTE, OXFORD UNIVERSITY PRESS, GB, vol. 107, no. 8, 23 June 2015 (2015-06-23), pages djv146 - 1, XP002743492, ISSN: 0027-8874, [retrieved on 20150801], DOI: 10.1093/JNCI/DJV146 *
KUBALL J ET AL., BLOOD, vol. 109, no. 6, 15 March 2007 (2007-03-15), pages 2331 - 8
LEE, J.K. ET AL., PRECIS. ONC., vol. 6, 2002, pages 91
LEISEGANG ET AL., CLIN. CANCER RES, vol. 16, 2010, pages 2333 - 2343
LEISEGANG ET AL., MOL. MED, vol. 86, 2008, pages 573 - 583
RIDDELLGREENBERG, J. IMMUNOL. METHODS, vol. 128, 1990, pages 189 - 201
SAILER NADJA ET AL: "T-Cells Expressing a Highly Potent PRAME-Specific T-Cell Receptor in Combination with a Chimeric PD1-41BB Co-Stimulatory Receptor Show a Favorable Preclinical Safety Profile and Strong Anti-Tumor Reactivity", CANCERS, vol. 14, no. 8, 14 April 2022 (2022-04-14), pages 1998, XP093001527, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9030144/pdf/cancers-14-01998.pdf> DOI: 10.3390/cancers14081998 *
SCHMITT ET AL., HUM GENE THER, vol. 20, no. 11, November 2009 (2009-11-01), pages 1240 - 1248
SCHOLTEN ET AL., CLIN. IMMUNOL, vol. 119, 2006, pages 135 - 145
SIDNEY ET AL., BMC IMMUNOLOGY, vol. 9, no. 1, 2008
SMITH, J. MOL. BIOL., vol. 147, 1981, pages 195 - 197
SOMMERMEYER ET AL., J. IMMUNOL, vol. 184, 2010, pages 6223 - 6231
SOMMERMEYERUCKERT, J IMMUNOL., vol. 184, no. 11, 1 June 2010 (2010-06-01), pages 6223 - 31
WALSENG ET AL., PLOS ONE, vol. 10, no. 4, 2015, pages 0119559
WILDE ET AL., BLOOD, vol. 114, 2009, pages 2131 - 2139
WILDE ET AL., ONCOIMMUNOLOGY, vol. 1, 2012, pages 129 - 140
XUE ET AL., CLIN EXP IMMUNOL., vol. 139, no. 2, February 2005 (2005-02-01), pages 167 - 172
YANG Y ET AL., J CLIN MED, vol. 12, no. 2, 2023, pages 709

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