Antigen-binding protein that specifically binds to PRAME

Abstract

The present invention relates to an antigen binding protein for an antigen derived from PRAME protein. The present invention provides in particular an antigen binding protein specific for the tumor expressed antigen PRAME, which comprises or consists of SEQ ID NO: 50 and is present in a complex with a major histocompatibility complex (MHC) protein. The antigen binding protein of the present invention in particular comprises a novel engineered T cell receptor (TCR) complementarity determining region (CDR) that specifically binds to said PRAME peptide. The antigen binding protein of the present invention is used for the diagnosis, treatment and prevention of PRAME-expressing cancerous diseases. Nucleic acids encoding the antigen binding protein of the present invention, vectors comprising said nucleic acids, recombinant cells expressing said antigen binding protein, and pharmaceutical compositions comprising the antigen binding protein of the present invention are further provided.

Description

[Technical field]

[0001] The present invention relates to an antigen binding protein against an antigen derived from the PRAME protein. The present invention provides in particular an antigen binding protein specific for the tumor expressed antigen PRAME, which tumor antigen comprises or consists of SEQ ID NO: 50 and is present in a complex with a major histocompatibility complex (MHC) protein. The antigen binding protein of the present invention in particular comprises a novel engineered T cell receptor (TCR) complementarity determining region (CDR) that specifically binds to said PRAME peptide. The antigen binding protein of the present invention is used in the diagnosis, treatment and prevention of PRAME-expressing cancerous diseases. Nucleic acids encoding the antigen binding protein of the present invention, vectors comprising said nucleic acids, recombinant cells expressing the antigen binding protein, and pharmaceutical compositions comprising the antigen binding protein of the present invention are further provided. [Background technology]

[0002] PRAME refers to "Preferentially Expressed Antigen in Melanoma" and belongs to a family of germline-encoded antigens known as cancer-testis antigens. Cancer-testis antigens are targets for immunotherapeutic intervention. PRAME is expressed in many solid tumors, as well as leukemias and lymphomas. The peptide SLLQHLIGL (SEQ ID NO: 50), also referred to as PRAME-004, corresponds to amino acids 425-433 of the full-length PRAME protein (SEQ ID NO: 328), which is presented on the cell surface in complex with MHC molecules, particularly HLA-A*02 (Kessler et al., J Exp Med. 2001 Jan 1 ;193(1 ):73-88). TCRs can bind to peptide epitopes presented by MHC molecules.

[0003] Although progress has been made in the development of molecular targeted drugs for cancer treatment, there is still a need in the art to develop novel anti-cancer drugs that specifically target molecules that are highly specific to cancer cells but not to normal tissue cells.PRAME-004 peptide is specifically expressed on tumors and is therefore a target for T cell-based immunotherapy.

[0004] WO2018 / 172533 discloses a TCR (such as TCR R11P3D3) that binds to PRAME-004 peptide in complex with MHC protein complex, and the use of said TCR in the diagnosis, treatment and prevention of cancerous diseases that (over)express PRAME.However, this TCR does not have a CDR region designed to bind to target antigen with high affinity.

[0005] Native TCRs typically have low affinity (K DIn contrast to viral foreign antigens, which bind MHC-presented antigens with affinity >10 μM (=300 μM-1 μM) and therefore have well-established TCR binding affinities in the range of 1-10 μM, binding to MHC-presented cancer self-antigens with affinity >10 μM is rarely observed (Aleksic et al. 2012, Eur J Immunol. 2012 Dec;42(12):3174-9). Part of the explanation for this phenomenon is the following: T cells developing in the thymus are negatively selected on self-peptide-MHC ligands, thus eliminating T cells with excessively high affinity for such self-peptide-MHC (tolerance induction). This low affinity of TCR for cancer self-antigens could be one possible explanation for tumor immune evasion (Aleksic et al. 2012, Eur J Immunol. 2012 Dec;42(12):3174-9). Therefore, it would be a desirable strategy to design TCR mutants that bind with higher affinity to cancer self-antigens for use as antigen recognition constructs in adoptive cell therapy (ACT). Furthermore, the design of high-affinity TCR mutants that can be expressed as soluble proteins would be desirable for targeting cancer self-antigens with soluble therapeutics (i.e., using bispecific molecules) (Hickman et al. 2016, J Biomol Screen. 2016 Sep;21(8):769-85).

[0006] However, increasing the affinity of TCR may also increase the risk of side effects. As mentioned above, in nature, high affinity TCRs for tumor-associated antigens, which are self-proteins, are eliminated by thymic selection to avoid the recognition of self-peptides present on normal tissues due to cross-reactivity. Therefore, simply increasing the affinity of TCRs for target sequences may also increase the affinity for similar peptides that are not cancer-specific, thus increasing the risk of cross-reactivity and undesirable cytotoxic effects on healthy tissues. This is not just a theoretical risk, as has been found acutely with engineered TCRs targeting MAGE-A3. Notably, previously published results showed fatal toxicity in two patients who had been infused with T cells engineered to express a TCR targeting MAGE-A3, which cross-reacts with a peptide derived from the muscle protein titin, even though preclinical studies had not predicted this cross-reactivity (Linette GP et al. Blood 2013; 122:863-71, Cameron BJ, et al. Sci. Transl. Med. 2013; 5: 197-103). These patients demonstrated that TCR-engineered T cells can have severe and unpredictable off-target and organ-specific toxicity. Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is an unmet medical need to develop and provide antigen binding proteins that specifically bind to targets with higher affinity, so that they can target even tumor cells or cell lines with reduced expression of the target antigen peptide, while maintaining a high safety profile due to low or reduced cross-reactivity with potential off-target peptides (also called "similar peptides" or "SimPeps"). Such antigen binding proteins should also ideally have a good metabolic and / or pharmacokinetic profile and be suitable for large-scale production compatible with industrial implementation. [Means for solving the problem]

[0008] Therefore, the inventors have designed antigen binding proteins specific for the PRAME-004 peptide that contain CDR variants derived from the parent TCR R11P3D3. The antigen binding proteins provided herein have increased binding affinity to peptide-MHC complexes, increased stability (e.g., reduced aggregation during expression and / or purification), and / or increased solubility, making them more suitable for medical applications.

[0009] Furthermore, the antigen-binding proteins of the present invention (particularly, bispecific T cell engaging receptors (TCER®)) exhibit high cytotoxicity against PRAME-004-positive tumor cells (e.g., cell lines Hs695T and U2OS cells) and exhibit half-maximal effective concentrations (EC 50 ) is 1 to 1000 pM. EC 50 is 100-fold higher, preferably more than 1000-fold higher, than in PRAME-004-negative tumor cells (e.g., cell line T98G), indicating an increased stability of the antigen-binding protein of the invention.

[0010] Furthermore, the present inventors have demonstrated significant tumor growth inhibition at low doses in a therapeutic in vivo mouse model of the antigen binding protein of the present invention.

[0011] In summary, the surprising discoveries of the inventors provide, inter alia, the following advantages over the state of the art: (i) increased affinity for the target peptide while maintaining high tumor selectivity; (ii) increased specificity / reduced cross-reactivity resulting in reduced off-target and off-tumor cytotoxicity and an overall improved safety profile; (iii) increased stability; (iv) improved expression yields and solubility suitable for large-scale manufacturing; and (v) provision of antigen-binding molecules with reduced immunogenicity.

[0012] In a first aspect, the present invention provides an antigen binding protein which specifically binds to a PRAME antigenic peptide comprising or consisting of the amino acid sequence SLLQHLIGL of SEQ ID NO: 50 and present in complex with a major histocompatibility complex (MHC) protein, (a) variable domain V, including complementarity determining regions (CDRs) CDRa1, CDRa2, and CDRa3; A A first polypeptide comprising: said CDRa1 comprises or consists of the amino acid sequence VKEFQD (SEQ ID NO: 16), or an amino acid sequence which differs from SEQ ID NO: 16 by one, two or three amino acid mutations, preferably amino acid substitutions; The CDRa3 comprises or consists of the amino acid sequence ALYNNLDMR (SEQ ID NO: 33) or ALYNNYDMR (SEQ ID NO: 34), or an amino acid sequence which differs from SEQ ID NO: 33 or SEQ ID NO: 34 by one, two or three (preferably one or two) amino acid mutations, preferably amino acid substitutions. A first polypeptide; (b) variable domain V, including CDRb1, CDRb2, and CDRb3 B a second polypeptide comprising said CDRb1 comprises or consists of the amino acid sequence SGHNS (SEQ ID NO: 10) or an amino acid sequence which differs from SEQ ID NO: 10 by one or two amino acid mutations, preferably amino acid substitutions; The CDRb3 comprises or consists of the amino acid sequence ASSX1GX2X3DX4QY (SEQ ID NO: 327), where X1 is P, A, or T, X2 is A or S, X3 is T or I, and X4 is K or A, or an amino acid sequence which differs from SEQ ID NO: 327 by 1, 2, or 3 amino acid mutations, preferably amino acid substitutions. A second polypeptide; The present invention relates to an antigen-binding protein comprising the

[0013] In a second aspect, the present invention relates to an isolated nucleic acid comprising a sequence encoding an antigen-binding protein of the first aspect of the invention.

[0014] In a third aspect, the present invention relates to a vector comprising the nucleic acid of the second aspect of the invention.

[0015] In a fourth aspect, the present invention relates to a host cell comprising the antigen binding protein of the first aspect of the invention, the nucleic acid of the second aspect, or the vector of the third aspect.

[0016] In a fifth aspect, the present invention relates to a pharmaceutical composition comprising the antigen binding protein of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, or the host cell of the fourth aspect, and a pharma- ceutically acceptable carrier.

[0017] In a sixth aspect, the present invention provides a method of producing an antigen binding protein of the first aspect of the invention comprising the steps of: (a) providing a host cell; (b) providing a genetic construct comprising a coding sequence encoding the antigen binding protein of the first aspect of the invention; (c) introducing the genetic construct into the host cell; and (d) expressing the genetic construct by the host cell. The present invention relates to a method comprising the steps of:

[0018] In a seventh aspect, the present invention provides an antigen binding protein of the first aspect, a nucleic acid of the second aspect, a vector of the third aspect, a host cell of the fourth aspect or a pharmaceutical composition of the fifth aspect for use in medicine, in particular for use in the diagnosis, prevention and / or treatment of a proliferative disease. [Brief description of the drawings]

[0019] [Figure 1] Conversion of TCR into stabilized scTCR by yeast surface display. scTCR molecules displayed on the surface of transformed Saccharomyces cerevisiae EBY100 were stained with anti-Myc-FITC antibody to determine expression levels and with PE-labeled HLA-A*02 / PRAME-004 tetramer to examine functional binding. The unmodified scTCR P11P3D3 (left panel, SEQ ID NO: 5) is compared with the R11P1D3_stabilized scTCR variant (right panel, SEQ ID NO: 6) that harbors nine stabilizing framework mutations and three single point mutations in the CDRs, obtained from scTCR library selection. [Diagram 2] Affinity mutation of scTCR CDR1 alpha by yeast surface display. Stabilized scTCRs containing unmodified and mature CDR1 alpha were stained with HLA-A*02 / PRAME-004 monomer at a concentration of 10 nM. Counterstained with a mixture of HLA-A*02 / SimPep tetramers (each applied at a concentration of 10 nM) containing peptides (SEQ ID NO: 51-59) with high sequence similarity to PRAME-004 (SEQ ID NO: 50). The stabilized scTCR R11P3D3SD (SEQ ID NO: 6) with the unmodified alpha chain CDR1 sequence SSNFYN (SEQ ID NO: 13; bottom right panel) is compared to scTCR variants containing affinity matured alpha chain CDR1 sequences VKEFQD, NKEFQD, TREFQD, NREFQD, TSEFQD, TKEFQD, VREFQD, TAEFQD, VSEFQD, VAEFQD, IKEFQN, VREFQN, and TAEFQN (SEQ ID NOs: 16-28), respectively. SSNFYN (SEQ ID NO: 13) is the corresponding CDRa1 sequence of the stabilized scTCR R11P3D3SD. [Diagram 3] Binding of high affinity scTCR yeast clones to similar peptides. Yeast clones carrying stabilized scTCRs with mature CDRs (SEQ ID NOs: 79-87 and 89-92) were stained with 100 nM HLA-A*02 monomer containing the PRAME-004 target peptide or one of the seven similar peptides (SEQ ID NOs: 52-56 and 58-59). [Figure 4] Binding of high affinity scTCR yeast clones to similar peptides. Yeast clones carrying stabilized scTCRs with mature CDRs (SEQ ID NOs: 79-87 and 89-92) were stained with 100 nM HLA-A*02 monomers containing the PRAME-004 target peptide or one of 19 similar peptides (SEQ ID NOs: 51, 57, 60, 62-69, and 71-78). R16P1C10_CDR6_scTCR (SEQ ID NO: 357) was included as a reference, but for this clone only binding to PRAME-004 and IFT17-003 (SEQ ID NO: 60) was evaluated. [Figure 5-1] Determination of binding motifs with high affinity scTCR yeast clones. Yeast clones carrying stabilized scTCRs with mature CDRs (SEQ ID NOs: 79, 80, 82, 83, and 85-87) were stained with PRAME-004 and PRAME-004 peptide variants containing alanine substitutions (SEQ ID NOs: 318-324) in the presence of HLA-A*02 at concentrations of 10 nM, 3 nM, 1 nM, and 0.3 nM. [Figure 5-2] Determination of binding motifs with high affinity scTCR yeast clones. Yeast clones carrying stabilized scTCRs with mature CDRs (SEQ ID NOs: 79, 80, 82, 83, and 85-87) were stained with PRAME-004 and PRAME-004 peptide variants containing alanine substitutions (SEQ ID NOs: 318-324) in the presence of HLA-A*02 at concentrations of 10 nM, 3 nM, 1 nM, and 0.3 nM. [Figure 6-1]Screening of similar peptides for soluble scTCR-Fab molecules. Binding to 14 similar peptides associated with HLA-A*02 (SEQ ID NOs: 187, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, and 212) was analyzed at a concentration of 1 μM scTCR-Fab using biolayer interferometry. The top curve in each graph represents scTCR-Fab binding to the target HLA-A*02 / PRAME-004 monomer. [Figure 6-2] Screening of similar peptides for soluble scTCR-Fab molecules. Binding to 14 similar peptides associated with HLA-A*02 (SEQ ID NOs: 187, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, and 212) was analyzed at a concentration of 1 μM scTCR-Fab using biolayer interferometry. The top curve in each graph represents scTCR-Fab binding to the target HLA-A*02 / PRAME-004 monomer. [Figure 7] In vitro cytotoxicity of TCER® molecules against target-positive and target-negative tumor cell lines. PBMCs from healthy HLA-A*02 positive donors were incubated with increasing concentrations of TCER® at a ratio of 1:10 with either the target-positive tumor cell line Hs695T (●) or the target-negative but HLA-A*02 positive tumor cell line T98G (○). TCER®-induced cytotoxicity was quantified after 48 hours of co-culture by measuring released LDH. Results of experiments evaluating TPP-93 and TPP-79 are shown in the lower and upper panels, respectively. [Figure 8]In vitro cytotoxicity of the TCER® molecule TPP-105 against target-positive and target-negative tumor cell lines. PBMCs from healthy HLA-A*02 positive donors were incubated with increasing concentrations of TPP-105 at a ratio of 1:10 with the target-positive tumor cell line Hs695T (●) or the target-negative but HLA-A*02 positive tumor cell line T98G (○). TCER®-induced cytotoxicity was quantified after 48 hours of co-culture by measuring released LDH. [Figure 9] Summary of cytotoxicity data for TCER® Slot III molecules. EC50 values ​​of dose-response curves obtained in LDH release assays were calculated using nonlinear 4-point curve fitting. For each evaluated TCER® molecule, calculated EC50 values ​​are depicted against the target-positive tumor cell lines Hs695T (●), U20S (○), and the target-negative but HLA-A*02-positive tumor cell line T98G (*). Each symbol thus represents one assay utilizing PBMCs from different HLA-A*02-positive donors. For TPP-871 / T98G, the EC50 is an estimate since T98G was not recognized by TPP-871. [Figure 10-1] In vitro cytotoxicity of TCER® Slot III mutants against T2 cells loaded with different concentrations of target peptide. Cytotoxicity was determined by quantifying LDH released in the supernatant. Human PBMCs were used as effector cells at an E:T ratio of 5:1. Readings were performed after 48 hours. [Figure 10-2] In vitro cytotoxicity of TCER® Slot III mutants against T2 cells loaded with different concentrations of target peptide. Cytotoxicity was determined by quantifying LDH released in the supernatant. Human PBMCs were used as effector cells at an E:T ratio of 5:1. Readings were performed after 48 hours. [Figure 10-3]In vitro cytotoxicity of TCER® Slot III mutants against T2 cells loaded with different concentrations of target peptide. Cytotoxicity was determined by quantifying LDH released in the supernatant. Human PBMCs were used as effector cells at an E:T ratio of 5:1. Readings were performed after 48 hours. [Figure 11-1] Normal tissue cell safety analysis for selected TCER® Slot III variants. TCER®-mediated cytotoxicity against five different normal tissue cell types expressing HLA-A*02 was evaluated in comparison to cytotoxicity against PRAME-004 positive Hs695T tumor cells. PBMCs from healthy HLA-A*02+ donors were co-cultured with normal tissue cells or Hs695T tumor cells (in triplicate) at a 10:1 ratio in a 1:1 mixture of the respective normal tissue cell medium (4, 10a, or 13a) and T cell medium (LDH-AM) or in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was assessed by measuring LDH release (LDH-Glo™ Kit, Promega). [Figure 11-2] Normal tissue cell safety analysis for selected TCER® Slot III variants. TCER®-mediated cytotoxicity against five different normal tissue cell types expressing HLA-A*02 was evaluated in comparison to cytotoxicity against PRAME-004 positive Hs695T tumor cells. PBMCs from healthy HLA-A*02+ donors were co-cultured with normal tissue cells or Hs695T tumor cells (in triplicate) at a 10:1 ratio in a 1:1 mixture of the respective normal tissue cell medium (4, 10a, or 13a) and T cell medium (LDH-AM) or in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was assessed by measuring LDH release (LDH-Glo™ Kit, Promega). [Figure 11-3]Normal tissue cell safety analysis for selected TCER® Slot III variants. TCER®-mediated cytotoxicity against five different normal tissue cell types expressing HLA-A*02 was evaluated in comparison to cytotoxicity against PRAME-004 positive Hs695T tumor cells. PBMCs from healthy HLA-A*02+ donors were co-cultured with normal tissue cells or Hs695T tumor cells (in triplicate) at a 10:1 ratio in a 1:1 mixture of the respective normal tissue cell medium (4, 10a, or 13a) and T cell medium (LDH-AM) or in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was assessed by measuring LDH release (LDH-Glo™ Kit, Promega). [Figure 12-1] Normal tissue cell safety analysis for selected TCER® Slot IV variants. TCER®-mediated cytotoxicity against 10 different normal tissue cell types expressing HLA-A*02 was evaluated in comparison to cytotoxicity against PRAME-004 positive Hs695T tumor cells. PBMCs from healthy HLA-A*02+ donors were co-cultured with normal tissue cells or Hs695T tumor cells (in triplicate) at a 10:1 ratio in a 1:1 mixture of the respective normal tissue cell medium (3, 4, 8a, 10a, 13a, or 16a) and T cell medium (LDH-AM) or in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was assessed by measuring LDH release (LDH-Glo™ Kit, Promega). [Figure 12-2]Normal tissue cell safety analysis for selected TCER® Slot IV variants. TCER®-mediated cytotoxicity against 10 different normal tissue cell types expressing HLA-A*02 was evaluated in comparison to cytotoxicity against PRAME-004 positive Hs695T tumor cells. PBMCs from healthy HLA-A*02+ donors were co-cultured with normal tissue cells or Hs695T tumor cells (in triplicate) at a 10:1 ratio in a 1:1 mixture of the respective normal tissue cell medium (3, 4, 8a, 10a, 13a, or 16a) and T cell medium (LDH-AM) or in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was assessed by measuring LDH release (LDH-Glo™ Kit, Promega). [Figure 12-3] Normal tissue cell safety analysis for selected TCER® Slot IV variants. TCER®-mediated cytotoxicity against 10 different normal tissue cell types expressing HLA-A*02 was evaluated in comparison to cytotoxicity against PRAME-004 positive Hs695T tumor cells. PBMCs from healthy HLA-A*02+ donors were co-cultured with normal tissue cells or Hs695T tumor cells (in triplicate) at a 10:1 ratio in a 1:1 mixture of the respective normal tissue cell medium (3, 4, 8a, 10a, 13a, or 16a) and T cell medium (LDH-AM) or in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was assessed by measuring LDH release (LDH-Glo™ Kit, Promega). [Figure 12-4]Normal tissue cell safety analysis for selected TCER® Slot IV variants. TCER®-mediated cytotoxicity against 10 different normal tissue cell types expressing HLA-A*02 was evaluated in comparison to cytotoxicity against PRAME-004 positive Hs695T tumor cells. PBMCs from healthy HLA-A*02+ donors were co-cultured with normal tissue cells or Hs695T tumor cells (in triplicate) at a 10:1 ratio in a 1:1 mixture of the respective normal tissue cell medium (3, 4, 8a, 10a, 13a, or 16a) and T cell medium (LDH-AM) or in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was assessed by measuring LDH release (LDH-Glo™ Kit, Promega). [Figure 12-5] Normal tissue cell safety analysis for selected TCER® Slot IV variants. TCER®-mediated cytotoxicity against 10 different normal tissue cell types expressing HLA-A*02 was evaluated in comparison to cytotoxicity against PRAME-004 positive Hs695T tumor cells. PBMCs from healthy HLA-A*02+ donors were co-cultured with normal tissue cells or Hs695T tumor cells (in triplicate) at a 10:1 ratio in a 1:1 mixture of the respective normal tissue cell medium (3, 4, 8a, 10a, 13a, or 16a) and T cell medium (LDH-AM) or in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was assessed by measuring LDH release (LDH-Glo™ Kit, Promega). [Figure 13-1]Normal tissue cell safety analysis for selected TCER® Slot IV variants. TCER®-mediated cytotoxicity against six different normal tissue cell types expressing HLA-A*02 was evaluated in comparison to cytotoxicity against PRAME-004 positive Hs695T tumor cells. PBMCs from healthy HLA-A*02+ donors were co-cultured with normal tissue cells or Hs695T tumor cells (in triplicate) at a 10:1 ratio in a 1:1 mixture of the respective normal tissue cell medium (10a, 13a, or 16a) and T cell medium (LDH-AM). After 48 hours, lysis of normal tissue cells and Hs695T cells was assessed by measuring LDH release (LDH-Glo™ Kit, Promega). [Figure 13-2] Normal tissue cell safety analysis for selected TCER® Slot IV variants. TCER®-mediated cytotoxicity against six different normal tissue cell types expressing HLA-A*02 was evaluated in comparison to cytotoxicity against PRAME-004 positive Hs695T tumor cells. PBMCs from healthy HLA-A*02+ donors were co-cultured with normal tissue cells or Hs695T tumor cells (in triplicate) at a 10:1 ratio in a 1:1 mixture of the respective normal tissue cell medium (10a, 13a, or 16a) and T cell medium (LDH-AM). After 48 hours, lysis of normal tissue cells and Hs695T cells was assessed by measuring LDH release (LDH-Glo™ Kit, Promega). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] definition "PRAME" or (Preferentially Expressed Antigen in Melanoma) was originally identified as an antigen that is overexpressed in melanoma [Ikeda et al Immunity. 1997 Feb;6(2): 199-208]; it is also known as CT130, MAPE, OIP-4, and has Uniprot accession number P78395 (available as of Jan. 11, 2019). The protein functions as a receptor for retinoic acid receptor signaling [Epping et al., Cell. 2005 Sep 23; 122(6):835-47]. PRAME belongs to a family of germline-encoded antigens known as cancer-testis antigens. Cancer-testis antigens are attractive targets for immunotherapeutic intervention because they typically have limited or no expression in normal adult tissues. PRAME is expressed in many solid tumors, as well as leukemias and lymphomas [Doolan et al., Breast Cancer Res Treat. 2008 May; 109(2):359-65;Epping et al., Cancer Res. 2006 Nov 15;66(22): 10639-42;Ercolak et al., Breast Cancer Res Treat. 2008 May; 109(2):359-65;Matsushita et al., Leuk Lymphoma. 2003 Mar;44(3):439-44;Mitsuhashi et al., Int. J Hematol. 2014; 100(1 ):88-95;Proto-Sequeire et al., Leuk Res. 2006 Nov;30(11): 1333-9;Szczepanski et al., Oral Oncol. 2013 Feb;49(2): 144-51;Van Baren et al., Br J Haematol. 1998 Sep;102(5):1376-9].The PRAME targeted therapy of the present invention may be particularly suitable for the treatment of cancers including, but not limited to, acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, melanoma, amelanotic melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous cell non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine cancer and endometrial cancer, chronic lymphocytic leukemia, colorectal cancer, osteosarcoma and synovial sarcoma, preferably breast cancer, cholangiocarcinoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, squamous cell non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine cancer and endometrial cancer, and synovial sarcoma.

[0021] A "PRAME antigenic peptide" comprises or consists of the amino acid sequence SLLQHLIGL (SEQ ID NO:50), which corresponds to amino acids 425-433 of the full-length PRAME protein of the amino acid sequence of SEQ ID NO:328, accessible under Uniprot accession number P78395 (available as of Jan. 11, 2019). A PRAME-derived peptide comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO:50) is also referred to herein as PRAME-004. PRAME-004 peptides are peptide epitopes derived from tumor-associated or tumor-specific proteins and are presented on the cell surface by molecules of the major histocompatibility complex (MHC). More specifically, PRAME-004-derived peptides are presented on the cell surface in a complex with HLA-A*02. Med. 2001 Jan 1; 193(1):73-88. In the context of the present invention, the terms "PRAME antigenic peptide", "PRAME peptide" or "PRAME-004" are used interchangeably and refer to a peptide comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 50). Preferably, the PRAME peptide consists of the amino acid sequence SLLQHLIGL. In the event that the PRAME peptide comprises further amino acids in addition to the amino acid sequence SLLQHLIGL, it is preferred that the total length of the PRAME peptide does not exceed 12 amino acids.

[0022] The term "antigen" or "target antigen" as used herein refers to a molecule or a portion of a molecule or complex to which an antigen-binding site can bind, said antigen-binding site being present, for example, in an antibody, TCR, and / or other antigen-binding protein of the invention. An antigen relevant to the present invention is a PRAME peptide comprising or consisting of the amino acid sequence SLLQHLIGL of SEQ ID NO: 50, more specifically a PRAME peptide comprising or consisting of the amino acid sequence SLLQHLIGL of SEQ ID NO: 50 in complex with an MHC protein (e.g. an HLA protein, such as HLA-A*02).

[0023] A "domain" can be any region of a protein, generally defined on the basis of sequence homology, and is often associated with a particular structural or functional entity.

[0024] The term "immunoglobulin (Ig) domain" in the context of the present invention refers to a protein domain consisting of a two-layer sandwich of seven to nine antiparallel β-strands arranged in two β-sheets with a Greek key topology. Ig domains are perhaps the most frequently used "building blocks" in naturally occurring proteins. Proteins containing Ig domains are incorporated into the immunoglobulin superfamily, including, for example, antibodies, T-cell receptors (TCRs), and cell adhesion molecules. Examples of Ig domains are the variable and constant domains of antibodies and TCRs.

[0025] V related to the present invention α refers to the variable domain of the TCR α chain.

[0026] V related to the present invention β refers to the variable domain of the TCR β chain.

[0027] V related to the present invention γ refers to the variable domain of the TCR gamma chain.

[0028] V related to the present inventionδ refers to the variable domain of the TCR delta chain.

[0029] V related to the present invention A refers to the variable domain comprising the CDRs from a TCR, in particular CDR1a, CDR3a and, where appropriate, CDR2a from the alpha chain. The sequences surrounding the CDRs (i.e., framework sequences) may be derived from the variable domain of a TCR, i.e., from the variable domain of the TCR alpha, beta, gamma or delta chain, or from the variable domain of an antibody, preferably from the variable domain of the TCR alpha chain.

[0030] V related to the present invention B refers to the variable domain comprising the CDRs from a TCR, in particular CDR1b, CDR3b and optionally CDR2b from the β chain. The sequences surrounding the CDRs (i.e., framework sequences) may be derived from the variable domain of a TCR, i.e., from the variable domain of the TCR α, β, γ or δ chain, or from the variable domain of an antibody, preferably from the variable domain of the TCR β chain.

[0031] V related to the present invention L refers to the variable domain of an antibody light chain.

[0032] V related to the present invention H refers to the variable domain of an antibody heavy chain.

[0033] C related to the present invention L refers to the constant domain of an antibody light chain.

[0034] C related to the present invention H1 , C H2 , and C H3 refers to the constant domain of an antibody heavy chain, specifically an IgG heavy chain.

[0035] The term "epitope", also known as antigenic determinant, refers to a portion of an antigen that is recognized by the immune system. As used herein, the term epitope includes the terms "structural epitope" and "functional epitope". A "structural epitope" is an amino acid of an antigen (e.g., a peptide-MHC complex) that is covered by an antigen-binding protein when bound to the antigen. Typically, all amino acids of an antigen that are within 5 Å of any atom of an amino acid of the antigen-binding protein are considered to be covered. Structural epitopes of antigens can be determined by methods known in the art, including X-ray crystallography or NMR analysis. Structural epitopes of antibodies typically include 20-30 amino acids. Structural epitopes of TCRs typically include 20-30 amino acids. A "functional epitope" is a subset of amino acids that form a structural epitope, including amino acids of an antigen that are important for forming an interface with an antigen-binding protein of the invention or a functional fragment thereof, either directly by forming non-covalent interactions (e.g., H-bonds, salt bridges, aromatic stacking, or hydrophobic interactions) or indirectly by stabilizing the antigen-binding conformation, as determined, for example, by mutational scanning. In the context of the present invention, a functional epitope is also referred to as a "binding motif". Typically, a functional epitope of an antibody-bound antigen comprises 4-6 amino acids. Typically, a functional epitope of a peptide-MHC complex comprises 2-6 or 7 amino acids of the peptide and 2-7 amino acids of the MHC molecule. Since peptides presented by MHC I typically have 8-10 amino acids, only a subset of the amino acids of each given peptide are part of the functional epitope of the peptide-MHC complex. An epitope (particularly a functional epitope to which an antigen binding protein of the invention binds) comprises or consists of amino acids of an antigen that are required for the formation of a binding interface. In the context of the present invention, a functional epitope (i.e., a binding motif) comprises at least amino acids 3, 5, and 7, and preferably does not include amino acids 1 and 4, of the PRAME-004 antigenic peptide of SEQ ID NO:50.

[0036] The "major histocompatibility complex" (MHC) is a set of cell surface proteins essential for the adaptive immune system to recognize foreign molecules in vertebrates, thus determining histocompatibility. The main function of MHC molecules is to bind antigens from pathogens and present them on the cell surface for recognition by appropriate T cells. Human MHC is also called HLA (human leukocyte antigen) complex (or simply HLA). The MHC gene family is divided into three subgroups: class I, class II, and class III. Complexes of peptides and MHC class I molecules are recognized by CD8-positive T cells with the appropriate T cell receptor (TCR), whereas complexes of peptides and MHC class II molecules are recognized by CD4-positive helper T cells with the appropriate TCR. Both CD8- and CD4-dependent responses jointly and synergistically contribute to antitumor effects, so the identification and characterization of tumor-associated antigens and the corresponding T cell receptors are important in the development of cancer immunotherapies, such as vaccines and cell therapies. The HLA-A gene is located on the short arm of chromosome 6 and encodes the large α-chain, which is a component of HLA-A. Mutations in the HLA-A α-chain are important for the function of HLA. This mutation promotes genetic diversity in the population. Since each HLA has a different affinity for peptides of a certain structure, a large number of HLA types means a large number of antigens that are "presented" on the cell surface. The MHC class I HLA protein related to the present disclosure can be an HLA-A protein, an HLA-B protein, or an HLA-C protein, and preferably an HLA-A protein, such as HLA-A*02. In an MHC class I-dependent immune response, a peptide must not only be able to bind to a certain MHC class I molecule expressed by a tumor cell, but must then be recognized by T cells with a specific T cell receptor (TCR).

[0037] An "antigenic peptide in a complex with an MHC protein" herein refers to an antigenic peptide that is non-covalently bound to an MHC molecule. Specifically, the antigenic peptide is located in the "peptide binding groove" formed by the MHC molecule. A complex of an MHC molecule and an antigenic peptide is also referred to herein as a "peptide-MHC complex" or "pMHC complex." In the case of a PRAME antigenic peptide, this complex is also referred to as a "PRAME antigenic peptide-MHC complex" or "PRME-004:MHC complex."

[0038] "HLA-A*02" indicates a particular HLA allele, where the letter A indicates the allele, and the prefix "*02 prefix" indicates the A2 serotype.

[0039] The term "antigen binding protein" as used herein refers to a polypeptide comprising an antigen binding site capable of specifically binding to an antigen. The antigen binding protein of the present invention comprises a CDR from a TCR, in particular a variable domain V comprising CDRa1, CDRa3 and, where appropriate, CDRa2 from a TCR. A and a variable domain V comprising CDRb1, CDRb3, and optionally CDRb2 from a TCR. B In certain embodiments, V A Domain-wide and / or V B The entire domain is derived from the TCR, so V α Domains and V β Domain, or V γ Domains and V δ In the present context, the term antigen binding protein includes multiple TCR and antibody formats as defined below. In one example, the antigen binding protein comprises TCR-derived CDRs (specifically CDRa1, CDRa3, CDRb1, CDRb3, and, where appropriate, CDRa2 and CDRb2 from TCRs as defined in the claims) grafted onto the heavy and light chains of an antibody. In another example, the TCR-derived V α Whole domain and / or TCR-derived V βThe entire domain is grafted onto the heavy and light chains of an antibody. Those skilled in the art will recognize that such constructs represent hybrid antigen-binding proteins, which have the antigen specificity of the TCR from which the CDRs or variable domains are derived, but have the overall structure of an antibody, and therefore may be referred to as "antibody". The term antigen-binding protein further includes bispecific or multispecific antigen-binding proteins. The V-binding proteins include CDRa1, CDRa3, CDRb1, CDRb3, and CDRa2 and CDRb2, as appropriate, from the TCRs defined in the claims. A and V B In addition to the above, such bispecific or multispecific antigen binding proteins further comprise at least one variable domain and optionally a constant domain, where the variable and / or constant domain may be derived from an antibody or a TCR. Again, the skilled artisan will recognize that such constructs comprising elements of both an antibody and a TCR represent a hybrid format and may be referred to as "bispecific TCRs", "bispecific antibodies" or "bispecific TCR antibody molecules" depending on the composition of the antigen binding protein, but also depending on the perspective and / or focus of the skilled artisan. In some embodiments, the antigen binding proteins of the present invention comprise V-type CDRs comprising CDRa1, CDRa3, CDRb1, CDRb3 and optionally CDRa2 and CDRb2 from the TCR as defined in the claims. A and V B Including V A or V BThe antigen-binding protein further comprises an additional domain fused directly or indirectly to the TCR. Such antigen-binding proteins may be referred to as "TCR fusion proteins". Examples of additional domains contained in "TCR fusion proteins" are listed below. In a preferred embodiment, the antigen-binding protein is a bispecific TCR-antibody molecule as defined below, more preferably a bispecific T-cell engaging receptor [TCER®] as defined below. In such an embodiment, the antigen-binding protein comprises two different antigen-binding sites and is capable of specifically binding to two different antigens simultaneously, e.g. as known for bispecific antibodies.

[0040] In one embodiment, the antigen binding protein of the disclosure specifically binds to the PRAME antigenic peptide comprising or consisting of the amino acid sequence SLLQHLIGL of SEQ ID NO:50 and present in complex with a major histocompatibility complex (MHC) protein, the antigen binding protein comprising: (a) variable domain V, including complementarity determining regions (CDRs) CDRa1, CDRa2, and CDRa3; A A first polypeptide comprising: CDRa1 comprises the amino acid sequence VKEFQD (SEQ ID NO: 16), or an amino acid sequence that differs from SEQ ID NO: 16 by up to 1, up to 2, or up to 3 amino acid substitutions; CDRa3 comprises the amino acid sequence ALYNNLDMR (SEQ ID NO:33) or ALYNNYDMR (SEQ ID NO:34), or an amino acid sequence that differs from SEQ ID NO:33 or SEQ ID NO:34 by up to 1, up to 2, or up to 3 amino acid substitutions; CDRa2 comprises the amino acid sequence FGPYGKE (SEQ ID NO:32), or an amino acid sequence that differs from SEQ ID NO:32 by up to one, up to two, or at least three amino acid substitutions; A first polypeptide; (b) variable domain V, including CDRb1, CDRb2, and CDRb3 B A second polypeptide comprising: CDRb1 comprises the amino acid sequence SGHNS (SEQ ID NO: 10), or an amino acid sequence that differs from SEQ ID NO: 10 by up to one or up to two amino acid substitutions; CDRb3 comprises the amino acid sequence ASSX1GX2X3DX4QY (SEQ ID NO: 327), where X1 is P, A, or T, X2 is A or S, X3 is T or I, and X4 is K or A, or an amino acid sequence that differs from SEQ ID NO: 327 by up to 1, up to 2, or up to 3 amino acid substitutions; CDRb2 comprises the amino acid sequence FQNTAV (SEQ ID NO:36), or a CDRb2 amino acid sequence that differs from SEQ ID NO:36 by at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions; A second polypeptide; Includes.

[0041] In one embodiment, the antigen binding protein of the disclosure comprises: CDRa1 comprising SEQ ID NO: 16, CDRa2 comprising SEQ ID NO: 32, CDRa3 comprising SEQ ID NO: 33, CDRb1 comprising SEQ ID NO: 10, CDRb2 comprising SEQ ID NO: 36, and CDRb3 comprising SEQ ID NO: 327 Includes.

[0042] In one embodiment, the antigen binding protein of the disclosure comprises: CDRa1 comprising SEQ ID NO: 16, CDRa2 comprising SEQ ID NO: 32, CDRa3 comprising SEQ ID NO: 34, CDRb1 comprising SEQ ID NO: 10, CDRb2 comprising SEQ ID NO: 36, and CDRb3 comprising SEQ ID NO: 327 Includes.

[0043] In certain embodiments, the amino acid substitutions are conservative amino acid substitutions.

[0044] "At least one," as used herein, refers to one or more of a specified object, e.g., 1, 2, 3, 4, 5, or 6, or more of a specified object. For example, at least one binding site, as used herein, refers to 1, 2, 3, 4, 5, or 6, or more of a specified binding site.

[0045] The term "bispecific" in the context of the present invention refers to an antigen-binding protein that has at least two valencies and binding specificities for two different antigens, and therefore contains at least two antigen-binding sites. The term "valency" refers to the number of binding sites of an antigen-binding protein, for example, a bivalent antigen-binding protein relates to an antigen-binding protein that has two binding sites. The binding sites may bind to the same or different targets, i.e., a bivalent antigen-binding protein may be monospecific (i.e., binds to one target) or bispecific (i.e., binds to two different targets). The antigen-binding molecules of the present invention contain at least one antigen-binding site that comprises a CDR derived from a TCR. In a preferred embodiment, the antigen-binding molecules of the present invention contain at least one antigen-binding site derived from a TCR.

[0046] The term "TCR" as used herein is meant to include conventional / native TCRs, and engineered TCRs, in particular functional TCR fragments, single chain TCRs, and bispecific or multispecific TCRs.

[0047] "Native TCR" refers to a wild-type TCR that can be isolated from nature. A TCR that has a domain and domain arrangement similar to a native TCR and contains CDRs and framework regions from a TCR can also be referred to as a "conventional TCR". A native / conventional TCR is a heterodimeric cell surface protein of the immunoglobulin superfamily that associates with an invariant protein of the CD3 complex that is involved in mediating signal transduction. Native heterodimeric TCRs exist in αβ and γδ forms, which are structurally similar but differ in location and possibly function. The extracellular portion of native heterodimeric αβ and γδ TCRs comprises two polypeptides, each of which has a membrane-proximal constant domain (also referred to as the constant region) and a membrane-distal variable domain (also referred to as the variable region). In the context of the present invention, such a TCR is also referred to as a full-length TCR. A native αβ heterodimeric TCR has an α chain and a β chain. The α chain comprises a variable (V), joining (J) and constant (C) region, whereas the β chain comprises a V, J and C region, usually further comprising a short diversity (D) region between the variable and joining regions, which is often considered as part of the joining region. The constant regions of the TCR α and β chains are referred to as TRAC and TRBC, respectively (Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10). In the context of the present invention, the constant regions of the TCR α and β chains (TRAC and TRBC) comprise the transmembrane (TM) region. Each of the constant and variable regions (or domains) contains intrachain disulfide bonds. The variable domains contain highly polymorphic loops similar to the complementarity determining regions (CDRs) of antibodies.

[0048] Each TCR variable domain contains three "TCR complementarity determining regions" (CDRs) embedded in framework sequences, one hypervariable region designated CDR3. In the context of the present invention, CDRa1, CDRa2 and CDRa3 refer to the α-chain CDRs and CDRb1, CDRb2 and CDRb3 refer to the β-chain CDRs. There are several types of α-chain and β-chain variable domains, distinguished by the framework, CDR1 and CDR2 sequences, and the partially defined CDR3 sequences. Alpha chain variable domain types are referred to by unique TRAV numbers in the IMGT nomenclature, and beta chain variable domain types are referred to by unique TRBV numbers in the IMGT nomenclature (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1): 42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2): 83-96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). Further information on antibodies and TCR genes can be found in the international ImMunoGeneTics information system, Lefranc MP et al., (Nucleic Acids Res. 2015 Jan;43(Database issue):D413-22; and http: / / www.imgt.org / ). In the TCR, the CDR1 and CDR3 amino acid residues contact the antigen peptide, and the CDR2 amino acid residues primarily contact the HLA molecule (Stadinski et al., J Immunol. 2014 June 15; 192(12): 6071-6082; Cole et al., J Biol Chem. 2014 Jan 10;289(2):628-38). Thus, the antigen specificity of the TCR is defined by the CDR3 and CDR1 sequences. The CDR2 sequence is not necessary to determine antigen specificity, but may play a role in the overall affinity of the TCR for the peptide:MHC complex.

[0049] "TCR framework region" (FR) refers to the amino acid sequence flanked by the CDRs, i.e., the portion of the variable domain that is conserved to some degree among different TCRs. The α, β, γ, and δ chain variable domains each have four FRs, referred to herein as FR1-a, FR2-a, FR3-a, FR4-a (for the α or γ chain), and FR1-b, FR2-b, FR3-b, FR4-b (for the β or δ chain), respectively. Thus, an α or γ chain variable domain can be described as (FR1-a)-(CDRa1)-(FR2-a)-(CDRa2)-(FR3-a)-(CDRa3)-(FR4-a), and a β or δ chain variable domain can be described as (FR1-b)-(CDRb1)-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)-(FR4-b). In the context of the present invention, the CDR / FR sequences in an α, β, γ or δ chain variable domain are determined based on the IMGT definition (Lefranc et al., Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org). Thus, the CDR / FR amino acid positions when related to a TCR or a TCR-derived domain are indicated according to said IMGT definition. Preferably, the CDR / FR sequences in the variable domain V α The IMGT positions of the CDR / FR amino acid positions of the variable domain V are assigned similarly to the IMGT numbering of TRAV24*01. β The IMGT positions of the CDR / FR amino acid positions are assigned similarly to the IMGT numbering of TRBV12-3*01.

[0050] The term "α / β TCR / CD3 complex" in the context of the present invention refers to the T cell receptor complex as present on the surface of a T cell. Most T cells express the α / β TCR, composed of disulfide-linked α and β chains, which typically bind to the composite surface of antigenic peptides presented by MHC. The TCR does not signal by itself, but is constitutively associated with CD3, a protein complex that is called the T cell coreceptor and contains intracellular signaling motifs (Birnbaum et al.; PNAS vol. 11, no. 49; 17576-17581, 2014). The α / β TCR is non-covalently coupled to this conserved multi-subunit signaling apparatus, which includes CD3εγ, CD3εδ, and CD3ζζ dimers, which together form the α / β TCR / CD3 complex.

[0051] "CD3" is a protein complex and is composed of four different chains. In mammals, this complex contains the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains are associated with the TCR and the ζ chain, which generate activation signals in T lymphocytes.

[0052] Engineered TCRs (hence the term "TCR" when used in the context of the present invention) include, inter alia, functional TCR fragments, stable matured TCRs, affinity matured TCRs, single chain TCRs, chimeric, humanized, bispecific and multispecific TCRs). A "functional TCR fragment" includes (a) a fragment of a native or conventional TCR that retains the TCR-derived ability to bind to a target antigen, and (b) a recombinant / engineered antigen binding protein that comprises CDR sequences from a TCR, in particular CDR1, CDR3 and, where appropriate, CDR2 sequences. Since binding to a target antigen is defined by these CDR sequences, an antigen binding protein that comprises these CDR sequences retains the ability to bind to the target antigen of the TCR from which the CDRs are derived. The skilled artisan will recognize that the CDRs must be interspersed with framework regions (FRs), the particular amino acid sequences of which are not critical for target antigen specificity. Thus, a variable domain comprising a TCR-derived CDR and an antibody-derived FR may be considered a "functional TCR fragment". Further examples of functional TCR fragments include single variable domains, e.g., V α , V β , V δ , V γ , or fragments of the α, β, δ, and γ chains, e.g., “V α -C α " or "V β -C β ", or a portion thereof. Such fragments may also further comprise a corresponding hinge region. "Single chain TCR (scTCR)" as used herein refers to a TCR in which the variable domains of the TCR are located on a single polypeptide. Typically, the variable domains in a scTCR are separated by a linker, which typically comprises 10-30 amino acids, for example 25 amino acids.

[0053] "Chimeric TCR" refers herein to a TCR in which the TCR chain comprises sequences from more than one species. Preferably, the TCR in the context of the present invention may comprise an α chain comprising a human variable region of the α chain (e.g., a murine constant region of a murine TCR α chain). "Bispecific TCR" refers to a bispecific TCR-antibody molecule, in particular scTCR-F.ab or T cell engaging receptor [TCER®] as defined below.

[0054] The term "antibody", as used herein, is meant to include conventional / native antibodies and engineered antibodies, in particular functional antibody fragments, single chain antibodies, single domain antibodies, bispecific or multispecific antibodies.

[0055] "Native antibody" refers to a wild-type antibody that may be isolated from nature. An antibody that has domains and domain arrangements similar to a native antibody and contains CDR and FR sequences derived from the antibody may also be referred to as a "conventional antibody". In a native / conventional antibody, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IGE. Each chain contains different domains (also called regions). The light chain contains a variable domain (V L ) and the constant domain (C L The heavy chain contains two domains, the variable domain (V H ), as well as three or four constant domains (collectively C H It is called C H1 , C H2 , and C H3 , C as appropriate H4 ). Light (V L ) chain and heavy (V H The variable domains of both light (C) chains determine binding recognition and specificity to the antigen. L ) chain and heavy (C H The constant domains of the Fc chains are involved in antibody chain assembly, secretion, placental transport, complement fixation, and function as Fc receptors (F c R)

[0056] The specificity of an antibody resides in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed of residues mainly from the "antibody complementarity determining region" (CDR) or hypervariable region. Sometimes, residues from non-hypervariable or framework regions (FR) influence the structure of the entire domain and thus the binding site. CDR refers to the amino acid sequence that defines the binding affinity and specificity of the natural Fv region of the native antibody binding site. The light and heavy chains of an antibody each have three CDRs, called CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, the antigen binding site of an antibody contains six CDRs, including the CDR set from each of the heavy and light chain V regions. "Antibody framework region" (FR) refers to the amino acid sequence intervening between the CDRs, i.e., the portion of the variable region of the antibody light and heavy chains that is relatively conserved among different antibodies in a single species. The light and heavy chains of an antibody each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Thus, the light chain variable domain can be described as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable domain can be described as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). As used herein, a "human framework region" refers to a framework region that is substantially identical (about 85% or more, particularly 90%, 95%, 97%, 99%, or 100%) to the framework region of a naturally occurring human antibody. In the context of the present invention, the CDR / FR definitions in the variable domain of an antibody light or heavy chain are determined based on the IMGT definitions (Lefranc et al., Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org). Thus, the amino acid sequences of CDR1, CDR2, and CDR3, as well as the amino acid sequences of FR1, FR2, FR3, and FR4 of a given variable chain are indicated according to said IMGT definitions.

[0057] Engineered antibody formats include functional antibody fragments, single chain antibodies, single domain antibodies, and chimeric, humanized, bispecific, or multispecific antibodies. Engineered antibody formats further include constructs in which the CDRs from a TCR (optionally including an additional 3, 2, or 1 N- and / or C-terminal framework residues) or the entire variable domain from a TCR are grafted onto an antibody heavy or light chain. More specifically, CDRa1, CDRa3, and optionally CDRa2 can be grafted onto the variable heavy chain amino acid sequence, and CDRb1, CDRb3, and optionally CDRb2 can be grafted onto the variable light chain amino acid sequence, or vice versa. As another example, the light chain variable domain of an antibody can be replaced with the α chain variable domain of a TCR, and the heavy chain variable domain can be replaced with the β chain variable domain of a TCR, or vice versa. A "functional antibody fragment" refers to a portion of a full-length antibody that retains the ability to bind to a target antigen, and in particular to the antigen-binding or variable region of a full-length antibody. Examples of functional antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, and diabody. Functional antibody fragments may also be single domain antibodies such as heavy chain antibodies. The term "Fab" refers to an antibody fragment obtained by treating IgG with a protease (e.g., papain), in which about half of the N-terminal side of the H chain and the entire L chain are bound to each other via disulfide bonds, has a molecular weight of about 50,000 daltons, and has antigen-binding activity. The Fv fragment is the N-terminal portion of the Fab fragment of an antibody, and consists of one light chain and one variable portion of one heavy chain.

[0058] As used herein, the "format" of an antigen-binding protein designates a defined spatial arrangement of domains, particularly variable domains and, where appropriate, constant domains. Important features of such antigen-binding protein formats are: the number of polypeptide chains (single-, double-, or multiple-chain), the type and length of linkers connecting different domains, the number of variable domains (and thus the number of valencies), the number of different variable domains (and thus the number of specificities for different antigens, e.g., bispecific, multispecific), and the order and orientation of the variable domains (e.g., crossover, parallel).

[0059] Many different bispecific and multispecific formats have been described in the art in relation to antibodies, and as will be understood by those skilled in the art, such bispecific and multispecific formats can be used in relation to the present invention by replacing the antibody domains of these formats with the variable domains described in relation to the present invention. Such formats include, for example, diabodies, crossover dual variable domains (CODVs), and dual variable domain (DV) proteins. A summary of various bispecific antibody formats and their production methods is disclosed, for example, in Brinkmann U. and Kontermann EE MAbs. 2017 Feb-Mar; 9(2): 182-212. DVD formats are disclosed, for example, in the following scientific publications: Wu C et al. Nat Biotechnol 2007; 25:1290-7; PMID:17934452; Wu C. et al. MAbs 2009; 1:339-47; Lacy SE et al. MAbs 2015; 7:605-19; PMID:25764208; Craig RB et al. PLoS One 2012; 7:e46778; PMID:23056448; Piccione EC et al. MAbs 2015. CODV is disclosed, for example, in Onuoha SC et al. Arthritis Rheumatol. 2015 Oct; 67(10):2661-72 or, for example, in WO 2012 / 135345, WO 2016 / 116626.Diabodies are described, for example, in Holliger P et al. Protein Eng 1996; 9:299-305; PMID:8736497;Atwell JL et al. Mol Immunol 1996; 33:1301-12; PMID:9171890;Kontermann RE, Nat Biotechnol 1997; 15:629-31; PMID:9219263;Kontermann RE et al. Immunotechnology 1997; 3:137-44; PMID:9237098;Cochlovius B et al. Cancer Res 2000; 60:4336-41; PMID:10969772; and DeNardo DG et al. Cancer Biother Radiopharm 2001; 16:525-35; Explained in PMID:11789029.

[0060] "Diabody" refers to a bivalent molecule composed of two chains each containing two variable domains that can be derived from the same or different antibodies. When the antibodies are different, typically one antibody (V LX and V HX The variable domains of antibody X, which contains LY and V HY The variable domains of antibody Y, including V, VL and VL, are also located on two different polypeptide chains. These domains dimerize in a head-to-tail orientation. The two chains may have the following structure: HX -L Db1 -V LY and V HY -L Db2 -V LX , or V LX -L Db1 -V HY and V LY -L Db2 -V HX , or V HX -L Db1 -V HY and V LY -L Db2 -VLX , or V LX -L Db1 -V LY and V HY -L Db2 -V HX To allow head-to-tail dimerization of the domains, the two chains are separated by a linker (i.e., L Db1 and L Db2 ). The linker is preferably a short linker. A short linker is typically 2 to 12, 3 to 13, for example, 3, 4, 5, 6, 7, 8, 9 amino acids in length, for example, 4, 5 [Brinkmann U. and Kontermann EE (MAbs. 2017 Feb-Mar; 9(2): 182-212], or 8 amino acids in length, for example, "GGGS" in SEQ ID NO: 290, "GGGGS" in SEQ ID NO: 286, or "GGGSGGGG" in SEQ ID NO: 214.

[0061] In the "dual variable domain immunoglobulin" [DVD-Ig™] format, the target-binding variable domain (Domain V) of monoclonal antibody Y is LY and V HY ) is typically a conventional antibody X (domain V LX and V HX ) and thus the light chain of this conventional antibody X is fused to an additional light chain variable domain (V LY ), and the heavy chain of conventional antibody X comprises an additional heavy chain variable domain (V HY DVD-Ig™, as described in the art, typically comprises two polypeptide chains: HY -L1-V HX -L2-C H1 -C H2 -C H3 and one heavy chain comprising V LY -L3-V LX -L4-C L or one light chain containing V HX -L1-V HY -L2-C H1 -C H2 -CH3 and one heavy chain comprising V LX -L3-V LY -L4-C L It is composed of one light chain containing domain V HY / V LY and V HX / V LX are paired in parallel. The linking linkers L1 and L3 are preferably 5 to 20 amino acids, for example 5 to 15 amino acids, and / or the linking linkers L2 and L4 may or may not be present.

[0062] The "crossover dual variable domain" (CODV) format described in the art comprises a variable domain (V LX and V HX ) and the variable domain of antibody Y (V LY and V HY ) represent a format in which these variable domains are linked in a manner that allows crossover pairing.

[0063] In the CODV-Ig format relevant to the present invention, the polypeptide chain may, for example, have the following structure: HX -L1-V HY -L2-C H1 -C H2 -C H3 and V LY -L3-V LX -L4-C L , or V HY -L1-V HX -L2-C H1 -C H2 -C H3 and V LX -L3-V LY -L4-C L , or V HX -L3-V HY -L4-C H1 -C H2 -C H3 and V LY -L1-V LX -L2-C L , or VHY -L3-V HX -L4-C H1 -C H2 -C H3 and V LX -L1-V LY -L2-C L The linking linkers (L1-L4), which may also be referred to as full glycine linkers or serine-glycine linkers, typically vary in length. To allow crossover pairing, one chain (heavy or light) typically includes a longer linker than the other chain. For example, in the CODV structures listed above, L1 is 3-12 amino acid residues long, L2 is 3-14 amino acid residues long, L3 is 1-8 amino acid residues long, and L4 is 1-3 amino acid residues long, or L1 is 5-10 amino acid residues long, L2 is 5-8 amino acid residues long, L3 is 1-5 amino acid residues long, and L4 is 1-2 amino acid residues long, or L1 is 7 amino acid residues long, L2 is 5 amino acid residues long, L3 is 1 amino acid residue long, and L4 is 2 amino acid residues long.

[0064] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and that has been modified by replacing certain amino acids, particularly in the framework regions of the heavy and light chains, to avoid or minimize immune responses in humans. The constant domains of a humanized antibody are primarily composed of human C H Domain and C L Domains. Numerous methods for humanizing antibody sequences are known in the art; see, for example, the review by Almagro & Fransson (2008) Front Biosci. 13: 1619-1633.

[0065] As will be appreciated by those skilled in the art, the structure of an antibody, in particular the structure of the heavy and light chain variable domains of an antibody, is similar to the structure of the variable domains of the TCR α, β, γ or δ chains, facilitating the grafting of CDRs defined in the context of the present invention into antibodies (e.g. conventional, bispecific or multispecific antibodies).

[0066] By knowing the amino acid sequence of the CDR of the antibody, TCR, or antigen-binding protein of the present invention, one skilled in the art can easily determine the framework region (e.g., TCR framework region or antibody framework region). If the CDR is not shown, one skilled in the art can first determine the CDR amino acid sequence based on the IMGT definition for TCR or the IMGT definition for antibody, and then determine the amino acid sequence of the framework region.

[0067] Bispecific TCR-Antibody Format In a preferred embodiment, the antigen-binding protein of the invention is a bispecific molecule, in particular a bispecific TCR-antibody molecule, i.e. an antigen-binding protein comprising at least two antigen-binding sites, one derived from an antibody and the other from a TCR or comprising at least the CDRs derived from a TCR (in particular CDR1a, CDR3a, CDR1b, CDR3b and, where appropriate, CDR2a and CDR2b). The antigen-binding site derived from an antibody is preferably a bispecific TCR molecule, in particular ... L and V H Includes.

[0068] In such bispecific TCR-antibody molecules, the variable domains may be arranged, for example, as described for the various bispecific antibody formats described above. Techniques for producing such bispecific antibodies are also disclosed in the prior art cited above, so that the skilled person can easily generate and produce the antigen binding proteins of the present invention in the formats disclosed herein using the CDRs or variable domains defined herein. In addition, further formats are possible, such as a format in which on each chain the variable domains are separated by a constant domain that mediates dimerization, so that in the final molecule, two antigen binding sites are located on either side of the dimerization constant domain. The skilled person is fully capable of selecting the appropriate linker to ensure folding in the desired conformation.

[0069] In a most preferred embodiment, the antigen-binding protein of the present invention is a bispecific T-cell engaging receptor (TCER®), a soluble Fc-containing bispecific antigen-binding molecule comprising a TCR antigen-binding site and an antibody antigen-binding site. The antibody antigen-binding site is formed by the heavy and light chain variable regions of the antibody, and is also called a "recruiter" because it binds to and recruits effector cells (e.g., T cells) to tumors. TCER® comprises two polypeptide chains, and the antigen-binding sites are formed by the variable domains located on different polypeptide chains in a crossover orientation.

[0070] In the context of this application, a sequence that is "at least 85% identical to a reference sequence" refers to a sequence that has 85% or more, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity over the entire length of the reference sequence. A protein consisting of an amino acid sequence that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical" to a reference sequence may contain mutations (e.g., deletions, insertions, and / or substitutions) compared to the reference sequence. In the case of substitutions, a protein consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence may correspond to a homologous sequence from another species than the reference sequence.

[0071] In the context of this application, "percentage of identity" may be calculated using global pairwise alignment (i.e., two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are known in the art. For example, the "Needle" program may be used, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimal alignment (including gaps) when considering the entire length of two sequences. The Needle program is available, for example, at the ebi.ac.uk World Wide Web site and is further described in the following publications: EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277. The percentage of identity between two polypeptides according to the invention is calculated using the EMBOSS:Needle (Global) program with the "Gap Open" parameter of 10.0, the "Gap Extend" parameter of 0.5, and the Blosum62 matrix.

[0072] "Amino acid substitutions" may be conservative or non-conservative. Preferably, the substitutions are conservative, in which one amino acid is replaced with another amino acid that has similar structural and / or chemical properties.

[0073] In certain embodiments, conservative substitutions may include those described by Dayhoff in "The Atlas of Protein Sequence and Structure. Vol. 5", Natl. Biomedical Research, the contents of which are incorporated by reference in their entirety. For example, in certain embodiments, amino acids belonging to one of the following groups may be exchanged for one another, thus constituting conservative exchanges: Group 1: alanine (A), proline (P), glycine (G), asparagine (N), serine (S), threonine (T); Group 2: cysteine ​​(C), serine (S), tyrosine (Y), threonine (T); Group 3: valine (V), isoleucine (I), leucine (L), methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K), arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H); and Group 6: aspartic acid (D), glutamic acid (E). In certain embodiments, conservative amino acid substitutions may be selected from T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G, and / or T→S.

[0074] In further embodiments, conservative amino acid substitutions may include substitutions of amino acids of the same class with other amino acids, such as (1) non-polar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp; (2) uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) acidic: Asp, Glu; and (4) basic: Lys, Arg, His. Other conservative amino acid substitutions may also be made as follows: (1) aromatic: Phe, Tyr, His; (2) proton donor: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, for example, U.S. Patent No. 10,106,805, the entire contents of which are incorporated by reference).

[0075] In another embodiment, conservative substitutions can be made according to Table 1. Methods for predicting tolerance to protein modifications can be found, for example, in Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the contents of which are incorporated by reference in their entirety.

[0076] [Table 1]

[0077] In another embodiment, conservative substitutions can be those shown in Table 2 under the heading of "conservative substitutions." If such substitutions alter biological activity, more substantial changes designated as "exemplary substitutions" in Table 2 can be introduced and the products screened, if desired.

[0078] [Table 2]

[0079] The antigen binding proteins of the present invention can be of any length, i.e., contain any number of amino acids, so long as they retain biological activity (e.g., the ability to specifically bind to a target antigen, the ability to detect diseased cells in a host, or the ability to treat or prevent disease in a host, etc.).

[0080] The antigen binding proteins of the invention may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptane Carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0081] In one embodiment, the antigen binding proteins of the invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts, and / or dimerized or polymerized, or conjugated.

[0082] In further embodiments, the antigen binding proteins of the present invention are in the form of a salt, e.g., a pharma- ceutically acceptable salt. Suitable pharma-ceutically acceptable acid addition salts may include those derived from mineral acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid), as well as those derived from organic acids (e.g., tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, and arylsulfonic acids, such as p-toluenesulfonic acid).

[0083] "Covalent linkage" as used herein refers to a peptide linkage or a covalent linkage, for example via a disulfide bridge, or a linker or linker sequence, such as a polypeptide linker.

[0084] The term "linker", as used herein, refers to one or more amino acid residues inserted between domains or between a domain and an agent, for example to provide sufficient flexibility of the domain or element (e.g., the variable domain of an antigen binding protein of the invention) for the antigen binding protein to fold correctly to form an antigen binding site in a crossover pairing (in some of the CODV or diabody formats) or parallel pairing configuration (e.g., in a DVD format).

[0085] In some embodiments, the linker consists of 0 amino acids, which means that the linker is not present. The linker is inserted at the transition between variable domains or between variable and constant domains (or between dimerization domains) at the amino acid sequence level, respectively. The approximate sizes of antibody and TCR domains are well understood, so that the transition between domains can be identified. The exact location of the domain transition can be determined by locating peptide stretches that do not form secondary structure elements such as β-sheets or α-helices, as demonstrated by experimental data or as can be envisaged by modelling or secondary structure prediction techniques. The term linker as used in the context of the present invention includes, but is not limited to, the linkers designated L1, L2, L3, L4, L5 and L6.

[0086] Linkers such as L1, L2, L3, L4, L5, and L6 can be at least 1-30 amino acids in length, unless otherwise specified in the respective context. In some embodiments, linkers such as L1, L2, L3, L4, L5, and L6 can be 2-25, 2-20, or 3-18 amino acids in length. In some embodiments, linkers such as L1, L2, L3, L4, L5, and L6 can be peptides that are 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids or less in length. In other embodiments, linkers such as L1, L2, L3, L4, L5, and L6 can be 5-25, 5-15, 4-11, 10-20, or 20-30 amino acids in length. In other embodiments, linkers such as L1, L2, L3, L4, L5, and L6 can be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In certain embodiments, linkers such as L1, L2, L3, L4, L5, and L6 can be less than 24, less than 20, less than 16 amino acid residues in length, less than 12, less than 10, e.g., less than 5-24, less than 10-24, or less than 5-10 amino acid residues in length. In some embodiments, the linker is one or more amino acid residues in length, e.g., more than 1, more than 2, more than 5, more than 10, more than 20 amino acid residues in length, such as more than 22 amino acid residues in length.

[0087] Exemplary linkers, such as L1, L2, L3, L4, L5, and L6, comprise or consist of an amino acid sequence selected from the group consisting of GGGS (SEQ ID NO:290), GGGGS (SEQ ID NO:286), GGGAS (SEQ ID NO:287), GGGSGGGG (SEQ ID NO:214), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:61), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:70), GGSGG (SEQ ID NO:226), GGGGSGGGGSGGGGGS (SEQ ID NO:280), GGGGSAAA (SEQ ID NO:358), in particular, GGGSGGGG (SEQ ID NO:214), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:70), and GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:61).

[0088] "F c The term "domain" as used in the context of the present invention refers to a native F c and F c The present invention encompasses variants, whether digested from whole antibodies or produced by other means, including monomeric, dimeric and multimeric F c Includes both the domain.

[0089] "Native F c The term "antigen-binding fragment" as used herein refers to a molecule that includes the sequence of a non-antigen-binding fragment resulting from antibody digestion or generated by other means, whether in monomeric, dimeric, or multimeric form, and may include the hinge region. c The original antibody source of the native F is particularly of human origin and can be any antibody class, although IgG1 and IgG2 are preferred. c The molecule is composed of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. Native F cThe number of intermolecular disulfide bonds between the monomeric subunits of the molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). c An example of a native F is the disulfide-linked dimer that results from papain digestion of IgG. c An example of an amino acid sequence is SEQ ID NO:329.

[0090] "F c The term "variant" as used herein refers to a salvage receptor Fc that has been modified from the native Fc. c Rn (neonatal F c "Fc variants" refers to molecules or sequences that still contain the binding site for the salvage receptor. Exemplary Fc variants and their interactions with the salvage receptor are known in the art. c The term "mutant" refers to a non-human native F c In addition, the present invention may include molecules or sequences that have been humanized from the native F. c contains regions that can be removed because they confer structural features or biological activity that are not required for the antigen binding proteins of the invention. c The term "mutant" refers to one or more native F c Absent a site or residue or one or more F c The present invention includes molecules or sequences in which sites or residues have been modified that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) F other than the salvage receptor, c receptor binding, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0091] In one embodiment, the Fc domain is a human IgG Fc domain, preferably derived from human IgG1, IgG2, IgG3, or IgG4, preferably derived from IgG1 or IgG2, more preferably derived from IgG1.

[0092] In some embodiments, the antigen binding protein comprises two F c Domain(F c1 and F c2 For example, in the TCER® format used in the examples, these two F c The domains are of the same antibody isotype or isotype subclass. Thus, in some embodiments, F c1 and F c2 and both are of the IgG1 subclass, or the IgG2 subclass, or the IgG3 subclass, or the IgG4 subclass. c1 and F c2 Both are of the IgG1 or IgG2 subclass, more preferably of the IgG1 subclass.

[0093] In some embodiments, F c The region further comprises RF and / or "knob-into-hole" mutations as defined herein below.

[0094] "RF mutations" are generally c Mutation of amino acids HY to RF in the CH3 domain of the F domain, for example the mutations H435R and Y436F in the CH3 domain described in Jendeberg, L. et al., (1997, J. Immunological Meth., 201: 25-34) and described as advantageous for purification purposes, since they abolish the binding of Protein A. c In the case of a F domain, the RF mutation may be in one or both F c It may be present in one of the Fc domains, preferably in one of the Fc domains.

[0095] The "knobs-into-holes" technique is a method for promoting heteromultimer formation using C H3 -C H3 We refer to the mutations T366S, L368A, and Y407V at the interface, specifically both T366S (hole) and T366W (knob). This knob-into-hole mutation can be further stabilized by the introduction of additional cysteine ​​amino acid substitutions Y349C and S354C.

[0096] A "knob" mutation is, for example, F of SEQ ID NO: 149. c In the amino acid sequence, a "hole" mutation is present, for example, F c present in the amino acid sequence.

[0097] In some embodiments, the F of one polypeptide c Domain (e.g., F c1 ) is that C H3 It contains the amino acid substitution T366W (knob) in the F domain of the other polypeptide. c Domain (e.g., F c2 ) is that C H3 It contains the amino acid substitutions T366S, L368A, and Y407V (hole) in the domain, and vice versa.

[0098] In some embodiments, the F of one polypeptide c Domain (e.g., F c1 ) is that C H3 or further comprising the amino acid substitution S354C in the F domain of the other polypeptide. c Domain (e.g., F c2 ) is that C H3ド The amino acid substitution Y349C may be present in the ribosome or may further comprise the amino acid substitution Y349C in the ribosome, or vice versa.

[0099] Thus, in some embodiments, the F c Domain (e.g., F c1 ) is that C H3 It contains the amino acid substitutions S354C and T366W (knob) in the F domain of the other polypeptide. c Domain (e.g., F c2 ) is that C H3 The domain contains the amino acid substitutions Y349C, T366S, L368A, and Y407V (hole), and vice versa.

[0100] This set of amino acid substitutions can be further expanded by inclusion of the amino acid substitutions K409A in one polypeptide and F405K in the other polypeptide, as described by Wei et al. (Structural basis of a novel heterodimeric Fc for bispecific antibody production, Oncotarget. 2017). Thus, in some embodiments, the F of one polypeptide is c Domain (e.g., F c1 ) is that C H3 or further comprising the amino acid substitution K409A in the F domain of the other polypeptide. c Domain (e.g., F c2 ) is that C H3 It may also include or further include the amino acid substitution F405K in the domain, or vice versa.

[0101] In some cases, artificially introduced cysteine ​​bridges can improve the stability of antigen-binding proteins, optimally without interfering with their binding properties. Such cysteine ​​bridges can further improve heterodimerization.

[0102] Further amino acid substitutions (eg, charge pair substitutions) to improve heterodimerization of the resulting protein have been described in the art (eg, EP 2970484).

[0103] Thus, in one embodiment, the F c Domain (e.g., F c1 ) comprises or further comprises the charge pair substitutions E356K, E356R, D356R, or D356K, and D399K, or D399R, and the F of the other polypeptide c Domain (e.g., F c2 ) comprises or further comprises the charged pair substitutions R409D, R409E, K409E, or K409D and N392D, N392E, K392E, or K392D, or vice versa.

[0104] In a further embodiment, F on one or both, preferably both, polypeptide chains is c The domain is F c It may contain one or more alterations that inhibit gamma receptor (FcyR) binding. Such alterations may include L234A, L235A.

[0105] In a further embodiment, F on one or both, preferably both, polypeptide chains is c The domain is F c The amino acid sequence of the amino acid sequence of the present invention may include a N297Q mutation to remove an N-glycosylation site within the amino acid sequence of the present invention. c Abolishes gamma receptor interactions.

[0106] "Hinge", "hinge region" or "hinge domain" typically refers to a C H1 Domain and C H2 "Hinge" refers to the flexible portion of the heavy chain located between the "Hinge" and "Hinge-Hinge" domains. It is approximately 25 amino acids long and is divided into the "upper hinge", "middle hinge" or "core hinge", and the "lower hinge". "Hinge subdomain" refers to the upper hinge, the middle (or core) hinge, or the lower hinge. The amino acid sequences of the hinges of IgG1, IgG2, IgG3, and IgG4 molecules are shown herein below: IgG1:E 216 PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 330) IgG2:E 216 RKCCVECPPCPAPPVAGP (SEQ ID NO: 331) IgG3:ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPE 216 PKSCDTPPPCPRCPAPELLG (SEQ ID NO: 332) IgG4:E 216 SKYGPPCPSCPAPEFLG (sequence number 333).

[0107] In the context of the present invention, F c Reference is made to amino acid positions in a domain, where these amino acid positions or residues are indicated according to the EU numbering system as described, for example, in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).

[0108] Hinge, C H2 domain, and C H3 F consists of domains c The incorporation of this moiety, or a portion thereof, into an antigen-binding protein, particularly a bispecific antigen-binding protein, can be used to bind the Fc:Fc gamma receptor (F c Problems arose with non-specific immobilization of these molecules induced by γR interactions. c γR binds to various cell surface molecules (Fc) that bind with different affinities to epitopes presented by the Fc portion of IgG molecules. c γRI,F c γRIIa, F c γRIIb, F c Such non-specific immobilization (i.e., not induced by either of the two binding domains of the bispecific molecule) is undesirable due to i) its impact on the pharmacokinetics of the molecule, and ii) off-target activation of immune effector cells. c Various F to remove γR binding cMutants and mutations have been identified. In this regard, Morgan et al. 1995, Immunology (The N-terminal end of the C H2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, FcyRI and FcyRIII binding) c Loss of γRI binding, loss of C1q binding, and F c EP 1075496 discloses the replacement of residues 233-236 of human IgG1 with the corresponding sequence from human IgG2 (i.e., residues 233P, 234V, and 235A, and no amino acid at position 236), which results in reduced γRIII binding. c Antibodies and other Fs with regional diversity (e.g., 233P, 234V, 235A, and a non-existent residue or G at position 236, and one or more of 327G, 330S, and 331S) c Disclosed are containment molecules in which the recombinant antibodies are capable of binding to a target molecule without triggering significant complement-dependent lysis or cell-mediated destruction of the target.

[0109] Thus, in some embodiments, F c The region includes or further includes one or more amino acids or deletions selected from the group consisting of 233P, 234V, 235A, 236 (no residues) or G, 327G, 330S, 331S, and preferably F c The region comprises or further comprises one or more amino acids selected from the group consisting of amino acids 233P, 234V, 235A, 236 (no residues) or G, and 327G, 330S, 331S, and most preferably F. c The region includes or further includes amino acids 233P, 234V, 235A, 236 (no residues), and 331S.

[0110] In a further embodiment, F cThe domain comprises or further comprises the amino acid substitutions N297Q, N297G, or N297A, preferably N297Q.

[0111] The amino acid substitutions "N297Q", "N297G", or "N297A" are c This refers to an amino acid substitution at position 297 that abolishes the native N-glycosylation site in the domain. This amino acid substitution results in the formation of a sugar residue that results in the formation of the F domain, as described, for example, in Tao, MH and Morrison, SL (J Immunol. 1989 Oct 15;143(8):2595-601.). c This further prevents gamma receptor interactions and reduces the diversity of the final protein product (ie, the antigen binding protein of the invention).

[0112] In a further embodiment, particularly in the absence of a light chain, F c The domain comprises or further comprises the amino acid substitution C220S. The amino acid substitution "C220S" results in H1 -C L The cysteines that form disulfide bonds are missing.

[0113] In some embodiments, F c The domain may contain or further contain at least two additional cysteine ​​residues (e.g., S354C and Y349C, or L242C and K334C), where S354C is a cysteine ​​residue that is required to form a heterodimer. c1 F of one polypeptide, etc. c It is located in the F domain. c2 F of the other polypeptide, etc. c L242C and K334C are present in the F domain and / or form an intradomain C-C bond in one or both polypeptides. c1 or F c2 The same F in either c Located in the domain.

[0114] The antigen binding proteins of the present disclosure may be synthetic, recombinant, isolated, engineered, and / or purified.

[0115] "Purified," when referring to a polypeptide (e.g., an antigen binding protein of the invention) or a nucleotide sequence (e.g., a nucleotide sequence encoding an antigen binding protein or functional fragment thereof described herein), means that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified," particularly as used herein, means that at least 75%, 85%, 95%, or 98% by weight of the same type of biological macromolecules are present.

[0116] A purified nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may contain some additional bases or moieties that do not adversely affect the basic characteristics of the composition.

[0117] The term "isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials of the natural state is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-native environment, such as a host cell. An isolated antigen binding protein is substantially free of other antigen binding proteins with different antigen specificities (e.g., an antigen binding protein that specifically binds to PRAME is substantially free of antigen binding proteins that specifically bind to antigens other than PRAME). Additionally, an isolated antigen binding protein may be substantially free of other cellular material and / or chemicals.

[0118] A "recombinant" molecule is one that has been prepared, expressed, produced, or isolated by recombinant means. Recombinant molecules do not occur in nature.

[0119] The term "gene" refers to a DNA sequence that codes for or corresponds to a specific sequence of amino acids that constitutes all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences (e.g., promoter sequences) that determine, for example, the conditions under which the gene is expressed. Some genes that are not structural genes can be transcribed from DNA into RNA but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene can cover genomic sequences that code for proteins (i.e., sequences that include regulators, promoters, introns, and exon sequences).

[0120] "Affinity" in the context of the present invention is defined as the equilibrium binding between an antigen-binding protein and its antigen (i.e., the PRAME-004 peptide according to SEQ ID NO: 50 in complex with an MHC protein). Affinity can be measured, for example, by the half-maximal effective concentration (EC 50 ) or the parallel dissociation constant (K D In the context of the present invention, high affinity can be expressed as a K of ≦100 nM, ≦50 nM, ≦10 nM, or ≦5 nM. D This refers to bonding at

[0121] "K D " is the equilibrium dissociation constant (k off / k on (the ratio of K D and affinity are inversely related. D The value is related to the concentration of the antigen-binding protein, K D The lower the value, the higher the affinity of the antigen-binding protein. DThe K value may be experimentally assessed by a variety of known methods, such as measuring association and dissociation rates by surface plasmon resonance (SPR) or biolayer interferometry (BLI), as described in more detail in the "Antigen Binding Proteins" section herein below. D is preferably measured by biolayer interferometry (BLI). More preferably, the K D is determined by BLI at a temperature of 20° C. to 35° C., preferably 25° C. to 32° C., more preferably about 30° C., and a pH of 6.5 to 8.0, preferably 7.0 to 7.6. Even more preferably, the K D is determined by BLI at a temperature of 20° C. to 35° C., preferably 25° C. to 32° C., more preferably about 30° C., a pH of 6.5 to 8.0, preferably 7.0 to 7.6, and a salt concentration of 100 to 200 mM, preferably 120 to 175 mM, more preferably about 140 mM. Most preferably, the K of the antigen-binding protein for the PRAME antigen peptide is D is determined by BLI at 30 °C in a buffer consisting of or containing PBS, 0.05% Tween-20, and 0.1% BSA. In such measurements, the concentration of the antigen-binding protein is typically between 1.56 and 500 nM depending on the affinity of the interaction being measured. The K of an antigen-binding protein with respect to two different peptides (e.g., a target peptide and a potential off-target peptide / similar peptide) is D When comparing peptide-HLA binding strengths, if two peptides have similar HLA binding strengths, the peptide-HLA loading conditions are identical for both measurements and the range of antigen-binding protein concentrations is chosen taking into account the expected affinity.

[0122] "EC 50 The "half maximal effective concentration," also called the "maximal effective concentration," typically refers to the concentration of a molecule that elicits a response halfway between the baseline and maximum after a specific exposure time. EC 50 and affinity are inversely correlated, and EC 50 The lower the value, the higher the affinity of the molecule.50 " refers to the concentration of an antigen binding protein of the invention that induces a response halfway between the baseline and the maximum after a particular exposure time, and in particular refers to the concentration of an antigen binding protein of the invention that induces a response halfway between the baseline and the maximum after a particular exposure time. EC 50 Value may be assessed experimentally by a variety of known methods, for example using binding assays such as ELISA or flow cytometry, or functional assays such as IFN-gamma release assays or lactate dehydrogenase (LDH) release assays.

[0123] Antigen-binding proteins The antigen-binding proteins of the present invention are designed from a scaffold sequence into which many mutations have been introduced. The antigen-binding proteins of the present invention have a profile that is particularly suitable for therapeutic use. In general, identifying such antigen-binding proteins is not straightforward and typically has a high attrition rate.

[0124] First of all, the skilled person needs to identify a suitable starting (i.e. scaffold) sequence. In the present case, this is a TCR that has good affinity (e.g., 200 μM or more) for the target peptide-HLA complex; high level of target specificity, e.g., binds relatively weakly or does not bind to alternative peptide-HLA complex; and can be refolded and purified with high yield. Given the degenerate nature of TCR recognition, it is very difficult for the skilled person to determine whether a particular scaffold TCR sequence has a specificity profile that would make it eligible for design for therapeutic use (Wooldridge, et al., J Biol Chem. 2012 Jan 6;287(2): 1 168-77).

[0125] A particularly important step is to convert this TCR into a soluble format that can be stably expressed. Naturally occurring TCRs are membrane-bound and expressed only in complex with CD3. Besides antibodies, which are routinely expressed as single-chain variable fragment (scFv) molecules, the corresponding single-chain T cell receptor variable domain (scTv) constructs are prone to aggregation and misfolding (Richman, et al. Mol Immunol. 2009 Feb;46(5):902-16. doi: 10.1016 / j.molimm.2008.09.021. Epub 2008 Oct 29). This step is essential for the generation of active biological material, but will also be essential for further engineering steps described below. The process of scTv conversion and the generation of stable and soluble molecules typically involves the design of one or more specific mutations (such as, but not limited to, substitutions, insertions, and / or deletions) in the framework regions and / or CDRs into the TCR starting sequence to increase the expression and stability of the scTv. Each TCR has a different set of mutations depending on the combination of variable domains and the configuration of CDR3. The specific mutations and / or combinations of mutations that result in a significant increase in solubility and stability are unpredictable and have a high dropout rate. In many cases, it may not be possible to achieve a significant increase in solubility and stability with a given TCR starting sequence.

[0126] The next challenge is to engineer TCRs to have higher affinity for target antigens while retaining desirable properties such as specificity and yield. TCRs, when present in nature, have weaker affinity for target antigens (low micromolar range) compared to antibodies, and TCRs for cancer antigens typically have weaker antigen recognition compared to virus-specific TCRs (Aleksic, et al. Eur J Immunol. 2012 Dec;42(12):3174-9). This weaker affinity, coupled with HLA downregulation in cancer cells, means that therapeutic TCRs for cancer immunotherapy typically require engineering to increase affinity for target antigens and thus generate stronger responses. Such increased affinity is essential for soluble TCR-based reagents. In such cases, antigen-binding affinities in the nanomolar to picomolar range are desirable, along with binding half-lives of several hours. The affinity maturation process typically involves designing specific mutations in CDRs and / or combinations of mutations in CDRs (such as, but not limited to, substitutions, insertions, and / or deletions) into the starting TCR sequence to increase the strength of antigen recognition. To significantly increase the affinity of a given TCR to a given target, a person skilled in the art may have to design combinations of mutations in CDRs from a large pool of possible options. The specific mutations and / or combinations of mutations that significantly increase affinity are unpredictable and have a high dropout rate. In many cases, it may be impossible to achieve a significant increase in affinity with a given TCR starting sequence.

[0127] The affinity maturation process must also take into account the need to maintain TCR antigen specificity. Increasing the affinity of a TCR for its target antigen creates a substantial risk of revealing cross-reactivity with other unintended targets as a result of the inherent degeneracy of TCR antigen recognition (Wooldridge, et al., J Biol Chem. 2012 Jan 6;287(2): 1 168-77;Wilson, et al., Mol Immunol.2004 Feb;40(14-15): 1047-55;Zhao ef al., J Immunol. 2007 Nov 1 ;179(9):5845-54). At natural levels of affinity, the recognition of cross-reactive antigens may be too low to result in a reaction. If the cross-reactive antigen is expressed on normal healthy cells, there is a high possibility of off-target binding in vivo, which may manifest as clinical toxicity. Therefore, in addition to increasing antigen binding strength, the skilled person must also design mutations in CDRs and / or combinations of mutations in CDRs that allow TCR to retain high specificity for target antigen and therefore show good safety profile in preclinical trials. Again, suitable mutations and / or combinations of mutations are unpredictable. The dropout rate of this stage is even higher and in many cases may not be achieved at all from a given TCR starting sequence. Despite the above-mentioned difficulties, the inventors have identified antigen-binding proteins that contain TCRs derived from CDRs with particularly high affinity (low nanomolar range) and high antigen specificity.

[0128] Using as a starting point the TCR R11P3D3 disclosed in WO 2018 / 172533, which is incorporated herein by reference, the inventors have designed, produced and tested variants of the variable alpha and beta domains of R11P3D3 in single chain TCR (scTCR) and TCER® formats, optionally in combination with Fab fragments. In this way, the inventors have identified various CDRs (in particular CDRa1, CDRa3, CDRb1 and CDR3, and where appropriate CDRa2 and CDRb2) that are associated with high affinity and specificity binding of the antigen binding protein of the invention to its target (i.e. the RAME-004 peptide in complex with an MHC protein).

[0129] The inventors have designed bispecific TCER® molecules, single chain TCRs (scRCRs), and bispecific scTCR-Fab molecules. All constructs bind specifically to peptide-MHC complexes containing the PRAME-004 peptide. The bispecific constructs disclosed in the Examples further bind to effector cells (especially T cells) via antibody-derived "recruiters". Thus, the inventors have demonstrated that CDRs can be used in single chain TCR constructs and bispecific TCR-antibody molecules, and therefore also demonstrated that the identified CDRs can be used to produce a variety of antigen-binding proteins with high affinity and high specificity for the PRAME-004 peptide in complex with MHC proteins.

[0130] Thus, in a first aspect, the present invention provides an antigen binding protein which specifically binds to a PRAME antigenic peptide comprising or consisting of the amino acid sequence SLLQHLIGL of SEQ ID NO: 50 and present in complex with a major histocompatibility complex (MHC) protein, comprising Variable domain V, including complementarity determining regions (CDRs) CDRa1, CDRa2, and CDRa3 A and - CDRa1 comprises or consists of the amino acid sequence VKEFQD (SEQ ID NO: 16) or an amino acid sequence which differs from SEQ ID NO: 16 by one, two or three amino acid mutations, preferably amino acid substitutions; - CDRa3 comprises or consists of the amino acid sequence of ALYNNLDMR (SEQ ID NO: 33) or ALYNNYDMR (SEQ ID NO: 34), or an amino acid sequence which differs from SEQ ID NO: 33 or SEQ ID NO: 34 by 1, 2 or 3 (preferably 1 or 2) amino acid mutations, preferably amino acid substitutions; Variable domain V A and, Variable domain V, including CDRb1, CDRb2, and CDRb3 B and - CDRb1 comprises or consists of the amino acid sequence of SGHNS (SEQ ID NO: 10) or an amino acid sequence which differs from SEQ ID NO: 10 by 1, 2 or 3 (preferably 1 or 2) amino acid mutations, preferably amino acid substitutions; - CDRb3 comprises or consists of the amino acid sequence ASSX1GX2X3DX4QY, where X1 is P, A or T, preferably P, X2 is A or S, preferably A, X3 is T or I, and X4 is T, K or A, preferably K or A, more preferably K (SEQ ID NO: 327), or an amino acid sequence which differs from SEQ ID NO: 327 by 1, 2 or 3 amino acid mutations, preferably amino acid substitutions; Variable domain V B and The present invention relates to an antigen-binding protein comprising the

[0131] The specificity of an antigen-binding protein is determined by the amino acid sequences CDRa1, CDRa3, CDRb1, and CDRb3, and is independent of the amino acid sequences of CDRa2 and CDRb2.

[0132] In some embodiments, CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence that differs from SEQ ID NO: 32 by 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, and / or CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence that differs from SEQ ID NO: 36 by 1, 2, 3, 4, 5, or 6 (preferably no more than 5, more preferably no more than 4, even more preferably no more than 3) amino acid mutations, preferably amino acid substitutions.

[0133] In some embodiments the antigen binding protein comprises CDRa1, CDRb1 and, optionally, CDRa2, and CDRb2 as defined above, wherein CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence which differs from SEQ ID NO: 33 by one, two or three (preferably one or two) amino acid mutations, preferably amino acid substitutions, and CDRb3 comprises or consists of the amino acid sequence of ASSX1GX2X3DX4QY (SEQ ID NO: 327), wherein X1 is P, A or T, preferably P; X2 is A or S, preferably A; X3 is T or I; and X4 is T, K or A, preferably K or A, more preferably K, or an amino acid sequence which differs from SEQ ID NO: 327 by one, two or three amino acid mutations, preferably amino acid substitutions.

[0134] In some embodiments the antigen binding protein comprises CDRa1, CDRb1 and, optionally, CDRa2, and CDRb2 as defined above, wherein CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence which differs from SEQ ID NO: 34 by one, two or three (preferably one or two) amino acid mutations, preferably amino acid substitutions, and CDRb3 comprises or consists of the amino acid sequence of ASSX1GX2X3DX4QY (SEQ ID NO: 327), wherein X1 is P, A or T, preferably P; X2 is A or S, preferably A; X3 is T or I; and X4 is T, K or A, more preferably K or A, preferably K; or an amino acid sequence which differs from SEQ ID NO: 327 by one, two or three amino acid mutations, preferably amino acid substitutions.

[0135] In a preferred embodiment, CDRb3 comprises or consists of the amino acid sequence ASSPGX2X3DX4QY (SEQ ID NO: 364), where X2 is A or S, preferably A, X3 is T or I, and X4 is T, K, or A, preferably K or A, more preferably K, or an amino acid sequence that differs from SEQ ID NO: 364 by one, two or three amino acid mutations, preferably amino acid substitutions.

[0136] In a preferred embodiment, CDRb3 comprises or consists of the amino acid sequence ASSPGX2TDX4QY (SEQ ID NO: 363), where X2 is A or S, preferably A, and X4 is T, K, or A, preferably K or A, more preferably K, or an amino acid sequence that differs from SEQ ID NO: 363 by one, two or three amino acid mutations, preferably amino acid substitutions.

[0137] In other preferred embodiments, CDRb3 comprises or consists of the amino acid sequence of ASSPGAX3DX4QY (SEQ ID NO: 365), where X3 is T or I, preferably I, and X4 is K or A, preferably K, or an amino acid sequence that differs from SEQ ID NO: 365 by 1, 2 or 3 amino acid mutations, preferably amino acid substitutions.

[0138] In some embodiments, the antigen binding protein comprises CDRa1, CDRb1 and, optionally, CDRa2 and CDRb2 as defined above, wherein CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that differs from SEQ ID NO: 33 by 1, 2 or 3 (preferably 1 or 2) amino acid mutations, preferably amino acid substitutions, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence that differs from SEQ ID NO: 48 by 1, 2, 3 or 4 (preferably 1, 2 or 3, more preferably 1 or 2) amino acid mutations, preferably amino acid substitutions.

[0139] In some embodiments the antigen binding protein comprises CDRa1, CDRb1 and, optionally, CDRa2 and CDRb2 as defined above, wherein CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:33 or an amino acid sequence which differs from SEQ ID NO:33 by one, two or three (preferably one or two) amino acid mutations, preferably amino acid substitutions, and wherein CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO:48, or SEQ ID NO:283, or SEQ ID NO:281, or SEQ ID NO:297, or an amino acid sequence which differs from SEQ ID NO:48, SEQ ID NO:297, SEQ ID NO:281, or SEQ ID NO:283 by one, two or three (preferably one or two) amino acid mutations, preferably amino acid substitutions.

[0140] In preferred embodiments, CDRa1, CDRa3, CDRb1, CDRb3, and optionally CDRa2 and CDRb2, respectively, differ from SEQ ID NO: 16, SEQ ID NO: 33 or 34, SEQ ID NO: 10, SEQ ID NO: 327, SEQ ID NO: 32, and SEQ ID NO: 36 by no more than two (preferably no more than one) amino acid mutations, preferably amino acid substitutions. In preferred embodiments, the amino acid substitutions in the CDRs are conservative substitutions.

[0141] Variable domain V A and variable domain V B together form an antigen-binding site that binds to the PRAME-004 antigenic peptide complexed with an MHC protein, which may hereinafter also be referred to as the "first antigen-binding site."

[0142] CDRa1, CDRa2, and CDRa3 are derived from the TCR α chain variable domain, and CDRb1, CDRb2, and CDRb3 are derived from the TCR β chain variable domain.

[0143] In addition to CDR, V A and V B The V comprises a framework region (FR). The FR sequence may be derived from a TCR, i.e., from the variable domain of a TCR α, β, γ, or δ chain, or from an antibody variable domain. A comprises the FR sequences of an antibody light chain variable domain and can therefore be described as (FR1-L)-(CDRa1)-(FR2-L)-(CDRa2)-(FR3-L)-(CDRa3)-(FR4-L); B contains the FR sequences of an antibody heavy chain variable domain and can therefore be described as (FR1-H)-(CDRb1)-(FR2-H)-(CDRb2)-(FR3-H)-(CDRb3)-(FR4-H). A comprises the FR sequences of an α or γ (preferably α) chain variable domain and can therefore be described as (FR1-a)-(CDRa1)-(FR2-a)-(CDRa2)-(FR3-a)-(CDRa3)-(FR4-a); Bcomprises the FR sequence of a β or δ (preferably β) chain variable domain and can therefore be described as (FR1-b)-(CDRb1)-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)-(FR4-b). A If the entire domain is derived from the TCR α chain, then V A Also, V α It can also be called. V B If the entire domain is derived from the TCR β chain, then V b Also, V β It may also be referred to as.

[0144] In some embodiments, - position 27 of CDRa1 according to IMGT is V or substituted by an amino acid selected from L, I, M, F, A, T, N, Q, H, E, D and S (in particular selected from T, N, S and I); - position 28 of CDRa1 according to IMGT is K or substituted by an amino acid selected from R, Q, H, N, A, V, S, G, L, I and T (in particular selected from R, A and S); - position 38 of CDRa1 according to IMGT is D or substituted by an amino acid selected from E, N, Q, H, K and R (in particular N); - position 64 of CDRa2 according to IMGT is K or substituted by an amino acid selected from R, Q, H, N, T, V, A, L, I, M and F (in particular selected from R, T and V); - position 114 of CDRa3 according to IMGT is L or Y or is substituted by an amino acid selected from M, W, H, Q, A, I, K, R, V, D, E, F and N (particularly selected from H, Q, A, I, K, R, V, D, E, F and N, more particularly selected from H, Q, A and I); - position 56 of CDRb2 according to IMGT is F or substituted by an amino acid selected from Y, M, L, W, H, V, I and A (in particular selected from Y, M and L), - position 57 of CDRb2 according to IMGT is Q or substituted by an amino acid selected from N, R, D, E, Q, H, K and K (in particular N), with the proviso that if the amino acid at position 63 is T or S then the amino acid at position 57 is not N, - position 58 of CDRb2 according to IMGT is N or substituted by an amino acid selected from Q, H, D, K, R, S and T (particularly S); - position 63 of CDRb2 according to IMGT is T or substituted by an amino acid selected from S, V, A, D, Q and E (in particular selected from S and E), with the proviso that if the amino acid at position 57 is N then the amino acid at position 63 is not T or S, - position 64 of CDRb2 according to IMGT is A or substituted by an amino acid selected from V, L, I, S, G and T (particularly T), - position 65 of CDRb2 according to IMGT is V or is substituted by an amino acid selected from L, I, M, A, T, F and S (in particular selected from I, L and T), - position 108 of CDRb3 according to IMGT is P, A or T or is substituted by an amino acid selected from V, L, I, S, G, R, K, N and Q (in particular selected from R and S), with the proviso that if the amino acid at position 110 is T or S then the amino acid at position 108 is not N, - position 110 of CDRb3 according to IMGT is A or S or is substituted by an amino acid selected from V, L, I, G, T and C (in particular T), with the proviso that if the amino acid at position 108 is N then the amino acid at position 110 is not T or S, - position 113 of CDRb3 according to IMGT is T or I or is substituted by an amino acid selected from V, L, I, G and T; - position 115 of CDRb3 according to IMGT is T, K or A or is substituted by an amino acid selected from G, L, I, V, R, Q, N, Y, H, E and F (particularly selected from L, I, V, R, Q, N, Y, H, E and F, more particularly selected from L, I, V and R).

[0145] In some embodiments, - CDRa1 comprises or consists of the amino acid sequence X1X2EFQX3 (SEQ ID NO: 334), where X1 is V, T, N, I, or S, preferably V, T, or N, and most preferably V; X2 is K, R, S, or A, more preferably K or R, and most preferably K; and X3 is D or N, and preferably D; - CDRa2 comprises or consists of the amino acid sequence FGPYGX1E (SEQ ID NO: 335), where X1 is K, R, T or V, preferably K or R, and most preferably K; - CDRa3 comprises or consists of the amino acid sequence ALYNNX1DMR (SEQ ID NO: 336), where Xi is L, Y, H, Q, A, I, K, R, V, D, E, F, or N, preferably L, Y, H, Q, A, I, K, or R, more preferably L, Y, H, Q, or A, and most preferably L or Y; - CDRb1 preferably comprises or consists of the amino acid sequence SEQ ID NO: 10, - CDRb2 comprises or consists of the amino acid sequence X1X2X3X4X5X6, where X1 is F, Y, M or L, preferably F or Y, most preferably F, X2 is Q or N, preferably Q (if X2 is N then X3 is also N), X3 is N or S, preferably N, X4 is T, S or E, preferably T or S, most preferably T (if X4 is S then X2 is Q), X5 is A or T, preferably A, and X X is V, I, L or T, preferably V or I, most preferably V (SEQ ID NO: 337), and more preferably CDRb2 comprises or consists of the amino acid sequence X1QX3TX5X6 (SEQ ID NO: 359), wherein X1 is F, Y, M or L, preferably F or Y, most preferably F, X3 is N or S, preferably N, X5 is A or T, preferably A, X6 is V, I, L or T, preferably V or I, most preferably V; - CDRb3 comprises or consists of the amino acid sequence ASSX1GX2X3DX4QY (SEQ ID NO: 338), wherein Xi is P, R, A, T or S, preferably P, T or A, most preferably P, X2 is A or S, preferably A, X3 is T or I, preferably T, and X4 is K, A, L, I, V, R, Q, N, Y, T, H, E or F, preferably K, A, L, I, V, R, Q, N, or Y, more preferably K, A, L, I, V or R, most preferably K or A.

[0146] In some embodiments, - CDRa1 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; - CDRa2 comprises or consists of an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 32, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 29, - CDRa3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:227, SEQ ID NO:233, SEQ ID NO:219, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, and SEQ ID NO:9; - CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 10, - CDRb2 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 35; and / or - CDRb3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:292, SEQ ID NO:293, SEQ ID NO:294, SEQ ID NO:297, SEQ ID NO:298, SEQ ID NO:301, SEQ ID NO:302 and SEQ ID NO:271, and SEQ ID NO:269.

[0147] Preferably, the antigen binding protein comprises a CDRa1 of SEQ ID NO: 16, a CDRa3 of SEQ ID NO: 34, a CDRb1 of SEQ ID NO: 10, a CDRb3 of SEQ ID NO: 48 or 292, and, optionally, a CDRa2 of SEQ ID NO: 32, and a CDRb2 of SEQ ID NO: 36.

[0148] In a preferred example, the antigen binding protein comprises a CDRa1 of SEQ ID NO: 16, a CDRa3 of SEQ ID NO: 34, a CDRb1 of SEQ ID NO: 10, a CDRb3 of SEQ ID NO: 48, and optionally a CDRa2 of SEQ ID NO: 32, and a CDRb2 of SEQ ID NO: 36. Thus, an antigen binding protein may comprise a CDRa1 of SEQ ID NO: 16, a CDRa3 of SEQ ID NO: 34, a CDRb1 of SEQ ID NO: 10, a CDRb3 of SEQ ID NO: 48, a CDRa2 of SEQ ID NO: 32, and a CDRb2 of SEQ ID NO:36.

[0149] In another preferred example, the antigen binding protein comprises a CDRa1 of SEQ ID NO: 16, a CDRa3 of SEQ ID NO: 34, a CDRb1 of SEQ ID NO: 10, and a CDRb3 of SEQ ID NO: 292, and optionally a CDRa2 of SEQ ID NO: 32, and a CDRb2 of SEQ ID NO: 36. Thus, an antigen binding protein may comprise a CDRa1 of SEQ ID NO: 16, a CDRa3 of SEQ ID NO: 34, a CDRb1 of SEQ ID NO: 10, a CDRb3 of SEQ ID NO: 292, a CDRa2 of SEQ ID NO: 32, and a CDRb2 of SEQ ID NO:36.

[0150] Preferably, the antigen binding protein comprises, without modification, a CDRa1 of SEQ ID NO: 16, a CDRa3 of SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 9, a CDRb1 of SEQ ID NO: 10, a CDRb3 of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 47, SEQ ID NO: 281, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 301 or SEQ ID NO: 283, and optionally a CDRa2 of SEQ ID NO: 32, and a CDRb2 of SEQ ID NO: 36.

[0151] In some embodiments, the PRAME antigenic peptide consists of SEQ ID NO:50.

[0152] In some embodiments, the antigen binding protein specifically binds to the amino acid sequence of SEQ ID NO:50 in complex with an MHC protein.

[0153] In some embodiments, the MHC protein is an MHC class I HLA protein (e.g., HLA-A, HLA-B, or HLA-C), preferably HLA-A, and more preferably HLA-A*02.

[0154] In a preferred embodiment, the antigen binding protein specifically binds to a structural epitope of the PRAME-004 antigenic peptide of SEQ ID NO: 50. In a more preferred embodiment, the antigen binding protein specifically binds to a functional epitope of the PRAME-004 antigenic peptide of SEQ ID NO: 50.

[0155] The inventors carried out experiments to identify the residues of PRAME-004 that are relevant for binding by the antigen-binding protein of the present invention (Figure 5, Tables 4, 10, 12, 16). As a result, the inventors were able to identify that amino acids 3, 5, 6, 7, and 8 of SEQ ID NO:50 are relevant for binding. The amino acid at position 3 is strongly recognized by the antigen-binding protein of the present invention. The amino acid at position 5 is also strongly recognized. The amino acid at position 7 is most strongly recognized. The amino acids at positions 6 and 8 have a small contribution. Positions 3, 5, and 7 of SEQ ID NO:50, and, where appropriate, positions 6 and 8 are also referred to herein as the "binding motif" of PRAME-004. The skilled artisan will recognize that the determination of the exact epitope or functional epitope may vary slightly depending on the method used and the cutoff value selected.

[0156] In some embodiments, the antigen binding protein specifically binds to a functional epitope comprising or consisting of 3, 4, or 5 amino acid positions selected from the group consisting of positions 3, 5, 6, 7, and 8, particularly positions 3, 5, and 7, of SEQ ID NO: 50. In some embodiments, the antigen binding protein specifically binds to a functional epitope comprising amino acid positions 3, 5, and 7 of SEQ ID NO: 50. In some embodiments, the antigen binding protein specifically binds to a functional epitope consisting of amino acid positions 3, 5, and 7, or positions 3, 5, 6, and 7, or positions 3, 5, 7, and 8, or positions 3, 5, 6, 7, and 8 (preferably not amino acid positions 1 and 4 of SEQ ID NO: 50). In other words, the antigen binding protein preferably specifically binds to amino acid positions 3, 5, and 7, and optionally positions 6 and / or 8 (preferably not positions 1 or 4) of SEQ ID NO:50 in complex with an MHC protein (particularly an HLA protein, more particularly HLA-A, even more particularly HLA-A*02). In one embodiment, the antigen binding protein of the present disclosure specifically binds to a functional epitope comprising at least three amino acid positions selected from the group consisting of positions 3, 5, 6, 7, and 8 of SEQ ID NO:50, with the proviso that the antigen binding protein does not bind to amino acid positions 1 and 4 of SEQ ID NO:50. In one embodiment, the antigen binding protein specifically binds to a functional epitope comprising or consisting of at least six or seven amino acid positions selected from the group consisting of positions 1, 3, 4, 5, 6, 7, and 8 of SEQ ID NO:50.

[0157] An amino acid sequence according to SEQ ID NO: 50 in which at least one position is substituted is referred to in the context of the present specification as a "PRAME variant sequence". In particular, one position is substituted with alanine (SEQ ID NOs: 318-324). A peptide having a PRAME variant sequence is also referred to herein as a PRAME variant peptide. In one embodiment, an antigen-binding protein of the present invention exhibits low affinity, in particular a low K for binding to the PRAME antigenic peptide of SEQ ID NO: 50. D ≥2, ≥5, ≥10, ≥20, or ≥30 times higher K Dand binds to a PRAME mutant peptide in which at least one of positions 1, 3, 4, 5, 6, 7 and 8, in particular one of positions 3, 5, 6, 7 and 8, more particularly one of positions 3, 5 and 7, is substituted with alanine in a complex with an MHC protein. D is determined as set forth in the Definitions section above.

[0158] When used in the antigen binding proteins of the invention, particularly in bispecific antigen binding proteins, and more particularly in the TCER® format, the CDR amino acid sequences identified by the inventors increase the binding affinity, stability, and specificity of the antigen binding protein, particularly compared to a reference protein.

[0159] "Reference protein" herein refers to a protein to which the antigen binding protein of the invention is compared. Comparison of the antigen binding protein of the invention with the reference protein is preferably performed in parallel and under similar (preferably identical) experimental conditions. Such a reference protein may be an antigen binding protein comprising the CDRs of parent / wild type TCR R11P3D3 as disclosed in WO 2018 / 172533. The reference protein is preferably in the same format as the antigen binding protein to be compared. In the case where the antigen binding protein is a scTCR, a suitable reference protein is scTCR R11P3D3SD (SEQ ID NO: 6), which comprises the variable domain of TCR R11P3D3 comprising stabilizing mutations. For example, the reference protein may be TCER® as described herein, which comprises the CDRs of TCR R11P3D3. Alternatively, the reference protein is an antigen binding protein comprising the CDRs of "CDR6" [e.g. TCDR®]. The CDRs of "CDR6" are CDRa1 of the amino acid sequence DRGSQS (SEQ ID NO: 339), CDRa2 of the amino acid sequence IYSNGD (SEQ ID NO: 340), CDRa3 of the amino acid sequence AAVIDNDQGGILT (SEQ ID NO: 341), CDRb1 of the amino acid sequence PGHRA (SEQ ID NO: 342), CDRb2 of the amino acid sequence YVHGEE (SEQ ID NO: 343), and CDRb3 of the amino acid sequence ASSPWDSPNVQY (SEQ ID NO: 344). The reference protein can be CDR6 TCER® (TPP-1109) comprising a first polypeptide chain comprising or consisting of SEQ ID NO: 153 and a second polypeptide chain comprising or consisting of SEQ ID NO: 154. TPP-1109 comprises the UCHT1 (V17) recruiter corresponding to SEQ ID NOs: 108 and 109. Additionally, the reference protein can be a CDR6 scTCR having a polypeptide chain comprising or consisting of SEQ ID NO: 357. In the Examples, the inventors show that an antigen binding protein comprising the CDR of CDR6 [in particular TCER® TPP-1109] binds to amino acids at positions 5, 6, 7, and 8 of the PRAME-004 antigenic peptide, but not to amino acids at positions 2 and 3 (Table 16).Therefore, antigen binding proteins comprising the CDRs of CDR6 do not bind to amino acid 3 of the PRAME-004 antigenic peptide, which, in contrast, is strongly bound / recognized by antigen binding proteins of the invention.

[0160] affinity The antigen binding proteins of the invention comprise CDR sequences selected to enhance affinity for the PRAME-004:MHC complex (Figure 2, Table 3). As can be seen from the examples (Tables 4, 8, 10, 12, 15, and 16), the antigen binding proteins of the invention have high affinity (particularly K of ≦50 nM, ≦10 nM, ≦5 nM, or ≦3 nM). D ) and binds to the PRAME-004:MHC complex.

[0161] Thus, in one embodiment, the antigen binding protein of the invention has increased affinity, particularly compared to a reference protein.

[0162] In one embodiment, the antigen binding protein of the invention has a K of ≦100 nM, ≦50 nM, ≦10 nM, preferably ≦5 nM, more preferably ≦3 nM, for example 10 pM to 100 nM, 10 pM to 50 nM, 10 pM to 10 nM, 10 pM to 5 nM, 10 pM to 3 nM. D and binds to a complex of a PRAME peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 50 and an HLA molecule, preferably HLA-A*02.

[0163] In one example, the antigen-binding protein of the invention is a scTCR-Fab and has a K of ≦100 nM, ≦50 nM, ≦40 nM, ≦30 nM, ≦20 nM, ≦15 nM, preferably ≦15 nM, for example 10 pM to 100 nM, 10 pM to 50 nM, 10 pM to 20 nM, 5 nM to 20 nM. D and binds to a complex of a PRAME peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 50 and an HLA molecule, preferably HLA-A*02.

[0164] In a further example, the antigen-binding protein of the invention is TCER and has a K of ≦100 nM, ≦50 nM, ≦10 nM, preferably ≦5 nM, more preferably ≦3 nM, for example 10 pM to 100 nM, 10 pM to 50 nM, 10 pM to 10 nM, 10 pM to 5 nM, 10 pM to 3 nM. D and binds to a complex of a PRAME peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 50 and an HLA molecule, preferably HLA-A*02.

[0165] K D Methods for measuring affinity, etc. are known to those skilled in the art and include, for example, surface plasmon resonance (SPR) and biolayer interferometry. D Exemplary methods for determining are also described in the Examples section. As known to those of skill in the art, the experimental conditions used in these experiments (e.g., buffers used, protein concentrations) can have a strong influence on the results.

[0166] Thus, in one example, the antigen binding proteins of the present invention are expressed, for example, as TCER® and analyzed for binding affinity to HLA-A*02:PRAME-004 monomers. Typically, measurements are performed, for example, on an Octet RED384 system, typically using settings recommended by the manufacturer. Briefly, binding kinetics are typically measured at 30° C. and at a permeability of 1000 rpm, for example, using PBS, 0.05% Tween-20, 0.1% BSA as buffer. Peptide-HLA complexes (particularly HLA-A*02 / PRAME-004 complexes) are loaded onto a biosensor, such as HIS1K, before analyzing serial dilutions of TCER®.

[0167] As disclosed herein, the antigen binding protein of the present invention specifically binds to a complex of a PRAME antigenic peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 50 and an HLA molecule, preferably HLA-A*02. In the case where the antigen binding protein is a TCR expressed on a T cell, the binding of the antigen binding protein to said complex may induce an immune response upon binding. Thus, in one embodiment, the antigen binding protein of the present invention induces an immune response, preferably characterized by an increase in interferon gamma (IFNγ) levels.

[0168] yield The inventors have demonstrated in the examples that the antigen binding proteins have high final product yields, in particular yields of >1 mg / l, >1.5 mg / l, >2 mg / l, >5 mg / l, >10 mg / l, >15 mg / l, >20 mg / l, >30 mg / l, >40 mg / l, >50 mg / l, >60 mg / l (Tables 5, 6, 7, 9, 11, and 14).

[0169] The inventors demonstrate in the examples that the antigen binding proteins have high final product yields, in particular, increased yields compared to reference proteins, and more particularly, increased yields compared to antigen binding proteins comprising the CDR of "CDR6" expressed under identical conditions (Tables 5, 6, 7, 9, 11, and 14).

[0170] In one example, the antigen binding protein is a scTCR-Fab (described in Example 2) and has a product yield of ≧8 mg / l, ≧10 mg / l, ≧15 mg / l, ≧20 mg / l, ≧30 mg / l, ≧40 mg / l, ≧50 mg / l, ≧60 mg / l, ≧70 mg / l, for example, 8 mg / l to 85 mg / l, 10 mg / l to 85 mg / l, 14 mg / l to 85 mg / l, 50 mg / l to 85 mg / l.

[0171] In another, the antigen binding protein is a TCER comprising the VL and VH of Recruiter UCHT1V17 and has a product yield of ≧10 mg / l, ≧12 mg / l, ≧15 mg / l, ≧16 mg / l, ≧17 mg / l, ≧18 mg / l, preferably ≧15 mg / l, for example 10 mg / l to 30 mg / l, 15 mg / l to 25 mg / l, 15 mg / l to 30 mg / l, preferably 15 mg / l to 30 mg / l.

[0172] Final product yield is typically determined 10-11 days after transfection. Methods for measuring product yield are known to those of skill in the art. Exemplary procedures are described in the Examples section.

[0173] Thus, in one embodiment, the antigen binding proteins of the invention have improved yields when expressed under identical conditions, particularly compared to the reference protein.

[0174] stability The present inventors have demonstrated in the Examples that the antigen-binding protein has high stability (Tables 5, 6, 7, 9, 11, and 14).

[0175] The term "stability" in the context of the present invention refers to physical stability and may be assessed qualitatively and / or quantitatively using a variety of analytical techniques as described in the art and reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). To measure stability, a sample comprising an antigen binding protein of the present invention may be tested in a stability test in which the sample is exposed to stress conditions for a selected period of time, followed by quantitative and, where appropriate, qualitative analysis of chemical and physical stability using appropriate analytical techniques. In the context of the present invention, this method refers in particular to the assessment of aggregate formation [e.g., using size exclusion chromatography (SEC)] by measuring turbidity [e.g., dynamic light scattering (DLS) or light obscuration (LO)], and / or by visual inspection (e.g., by determining color and transparency). A sample is considered stable if there is only slight aggregation as defined below.

[0176] In the context of the present invention, improved stability refers to increased physical stability, for example when exposed to heat stress, such that the newly developed antigen-binding proteins of the present invention can better tolerate stress conditions, particularly heat stress, compared to the reference protein.

[0177] "Low aggregation" means, for example, that a sample comprising the antigen-binding protein typically has a monomer content of ≥80%, ≥85%, ≥90%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% (e.g., 94%-99%, 95%-99%, 96%-99%, 97%-99% monomer content) when measured by SEC, such as SEC-HPLC in a buffer such as PBS, after exposure to stress conditions (e.g., a temperature of 40°C for 14 days in a buffer such as PBS). In the case of SEC, a difference of 1%, 2%, 3%, 4%, preferably 1 or 2%, more preferably 1% in monomer content is considered a significant difference in the context of the present invention under test conditions that depend on the column, operating pressure, and buffer velocity used. In other words, if a reference antigen binding protein has a monomer content of 96% and an antigen binding protein of the invention has a monomer content of 97%, then the monomer content of the antigen binding protein of the invention is significantly different and therefore significantly increased compared to the reference antigen binding protein when measured under identical conditions.

[0178] The inventors have demonstrated that the antigen binding proteins are stable, in particular having monomer contents of ≧80%, ≧85%, ≧90%, ≧92%, ≧94%, ≧95%, ≧97% after 14 days at 40° C. in a buffer such as PBS (Tables 5, 6, 7, 9, 11, and 14).

[0179] In one embodiment, the antigen binding proteins of the invention are stable or have improved stability when exposed to stress conditions for a certain period of time (e.g., when exposed to a temperature of 40° C. for 14 days), particularly compared to a reference protein.

[0180] In one embodiment, the antigen binding proteins of the invention exhibit no or little or reduced aggregation when exposed to stress conditions for a certain period of time (e.g., when exposed to a temperature of 40° C. for 14 days), particularly when compared to a reference protein.

[0181] specificity The inventors demonstrate in the Examples (Figures 3 to 6, Tables 3, 4, 8, 13, 15) that the antigen-binding protein of the present invention binds with high specificity to a target antigen, i.e., a PRAME antigen peptide comprising SEQ ID NO:50 in a complex with an MHC protein (preferably, a complex with HLA-A*02).

[0182] As explained above, the inventors have identified amino acid positions 3, 5, 6, 7 and 8 of SEQ ID NO:50 as relevant for binding of antigen binding proteins of the invention to the PRAME-004 antigenic peptide (i.e. the "binding motif" of the PRAME-004 antigenic peptide), particularly positions 3, 5 and 7. The inventors have identified, for example, potential off-target peptides that are similar to PRAME-004 sequences and / or motifs and therefore increase the risk of antigen binding proteins binding to PRAME-004.

[0183] In the context of the present invention, the term "analogous peptide" refers herein to a potential off-target peptide, i.e., a peptide (e.g., but not limited to, a homologous sequence or a similar motif) to which the antigen-binding molecule of the present invention may potentially bind based on biochemical / biophysical properties. The analogous peptide is typically 8 to 11 amino acids in length. Analogous peptides related to the present invention are typically presented by MHC. Furthermore, analogous peptides related to the present invention include peptides that contain or consist of an amino acid sequence similar to the amino acid sequence of the PRAME-004 antigen peptide, and more particularly, peptides that contain an epitope in which some or all of the amino acids have the same and / or similar biochemical / biophysical properties as the amino acids that constitute the epitope of the PRAME-004 peptide, as compared with the epitope of the PRAME-004 antigen peptide. In some examples, the similar peptides investigated in the context of the present invention were selected from a database of HLA-A*02 binding peptides presented by tumor and normal tissues (XPRESIDENT® database) using similarity scoring within the binding-relevant positions of PRAME-004 and the requirement of at least one detection in normal tissue. Binding of antigen-binding proteins to similar peptides presented by MHC proteins may cause adverse reactions. Such adverse reactions may be "off-tumor" side effects, such as cross-reactivity of certain TCRs with similar peptides in healthy tissues, as reported in Lowdell et al., Cytotherapy, published on December 4, 2018.

[0184] In particular, the following peptides are similar peptides relevant to the present invention: TMED9-001 (SEQ ID NO: 51), CAT-001 (SEQ ID NO: 52), DDX60L-001 (SEQ ID NO: 53), LRRC70-001 (SEQ ID NO: 54), PTPLB-001 (SEQ ID NO: 55), HDAC5-001 (SEQ ID NO: 56), VPS13B-002 (SEQ ID NO: 57), ZNF318-001 (SEQ ID NO: 58), CCDC51-001 (SEQ ID NO: 59), IFT17-003 (SEQ ID NO: 60), DIAPH1-004 (SEQ ID NO: 62), FADS2-001 (SEQ ID NO: 63), FRYL-001 (SEQ ID NO: 64), 03 (SEQ ID NO: 64), GIMAP8-001 (SEQ ID NO: 65), HSF1-001 (SEQ ID NO: 66), KNT-001 (SEQ ID NO: 67), MAU-001 (SEQ ID NO: 68), MCM4-001 (SEQ ID NO: 69), MPPE1-001 (SEQ ID NO: 71), MYO1B-002 (SEQ ID NO: 72), PRR12-001 (SEQ ID NO: 73), PTRF-003 (SEQ ID NO: 74), RASGRP1-001 (SEQ ID NO: 75), SMARCD1-001 (SEQ ID NO: 76), TGM2-001 (SEQ ID NO: 77), VAV1-001 (SEQ ID NO: 78), VIM-009 (SEQ ID NO: 317)FARSA-001 (SEQ ID NO: 306), ALOX15B-003 (SEQ ID NO: 304), FAM114A2-002 (SEQ ID NO: 305), GPR56-002 (SEQ ID NO: 307), IGHD-002 (SEQ ID NO: 308), NOMAP-3-0972 (SEQ ID NO: 309), NOMAP-3-1265 (SEQ ID NO: 310), NOMAP-3-1408 (SEQ ID NO: 311), NOMAP-3-1587 (SEQ ID NO: 312), NOMAP-3-1768 (SEQ ID NO: 313), NOMAP-5-0765 (SEQ ID NO: 314), PDCD10-004 (SEQ ID NO: 315), TSN-001 (SEQ ID NO: 316), ARMC9-002 (SEQ ID NO: 187), CLI-001 (SEQ ID NO: 188), COPG1-001 (SEQ ID NO: 190), COPS7A-001 (SEQ ID NO: 192), EIF-009 (SEQ ID NO: 194), EXT2-006 (SEQ ID NO: 196), LMNA-001 (SEQ ID NO: 198), PKM-005 (SEQ ID NO: 200), PSMB3-002 (SEQ ID NO: 202), RPL-007 (SEQ ID NO: 204), SPATS2L-003 (SEQ ID NO: 206), SYNE1-002 (SEQ ID NO: 208), TGM2-002 (SEQ ID NO: 210) and TPR-004 (SEQ ID NO: 212).

[0185] The term "specificity" generally refers to the ability of an antigen-binding protein to distinguish a target peptide from similar peptides as defined above. In other words, an antigen-binding protein binds to the PRAME-004:MHC complex with high affinity, in particular with a K of less than 100 nM, less than 50 nM, less than 10 nM, preferably less than 5 nM. D but does not bind significantly to similar peptide:MHC complexes.

[0186] One of skill in the art will recognize that there will be similar peptides to which the antigen binding proteins of the invention do not bind to a detectable extent (e.g., peptides that give no detectable binding signal or functional response above background levels), where "background levels" refers to the binding signal or functional response observed for a non-homologous, "non-analogous" peptide or in the absence of the peptide.

[0187] In the case of other similar peptides, very low but insignificant binding is detectable. These latter similar peptides may also be described as "potentially relevant" similar peptides. The terms "insignificant binding" and "no significant binding" refer to the fact that the antigen-binding protein is 1) PRAME-004 peptide: K for binding to MHC complex D ≥25, ≥30, ≥40, ≥50, ≥75, or ≥100-fold increased K D binding at (e.g., binding to a similar peptide:MHC complex); 2) exhibit a significantly reduced "functional response" (e.g., a functional response to an analogous peptide:MHC complex) compared to the functional response to the PRAME-004 peptide:MHC complex; or 3) demonstrate significantly reduced detection by labeled analogous peptide:MHC multimers compared to detection by PRAME-004 peptide:MHC multimers; Shows.

[0188] Affinity (especially K D ) is measured, preferably using biolayer interferometry (BLI) as described in the Examples section. D K for binding to the MHC complex compared to similar peptides D The increase in D For example, the K for binding to the analogous peptide:MHC complex may be expressed as a ratio of D However, the K D If there is a 100-fold increase compared to K D The ratio "analogous peptide / PRAME-004" is 100. The skilled artisan will appreciate that affinity for the analogous peptide:MHC complex may not be measurable if binding is too weak.

[0189] "Functional response" refers to a response measured in a functional assay (e.g., an activation assay such as an IFN-gamma release assay) or a cytotoxicity assay such as the LDH release assay described in the experimental section herein below. The IFN-gamma release assay measures IFN-gamma released by T cells exposed to a particular peptide:MHC complex. The LDH release assay measures LDH released from target cells expressing a peptide:MHC complex on their surface and killed by T cells that specifically bind to the peptide:MHC complex. This binding may be direct, via a TCR expressed on the T cell, or indirect, via a soluble bispecific molecule that binds the peptide:MHC complex and also binds to the T cell (i.e., recruits the T cell). EC for binding to PRAME-004 peptide:MHC complex 50 Compared to E.C. 50 The functional response in the IFN-gamma release assay is considered to be significantly decreased if the EC for binding to the PRAME-004 peptide:MHC complex is increased by ≧25, ≧30, ≧40, ≧50, ≧75, or ≧100 fold, preferably by ≧200, ≧300, ≧500, or ≧1000 fold. 50 Compared to E.C. 50 The functional response in the LDH release assay is considered to be significantly decreased if there is a ≧25, ≧30, ≧40, ≧50, ≧75, or ≧100-fold increase, preferably a ≧200, ≧300, ≧500, or ≧1000-fold increase.

[0190] Detection with labeled analogous peptide:MHC multimers refers in particular to staining with analogous peptide:MHC tetramers, where the antigen-binding protein is expressed on the surface of cells, preferably yeast cells (Examples 1.1 and 1.2). If the number of positive (i.e. stained) cells is ≦5%, ≦3%, or ≦1% of the total number of cells, or if the number of positive cells is ≦10%, ≦5%, or ≦2.5% of the positive cells stained with PRAME-004 peptide:MHC tetramer, or if EC 50Detection is considered significantly reduced if the detection rate is reduced by 50-fold, 75-fold, 100-fold, 150-fold, or 200-fold.

[0191] The antigen-binding protein of the present invention is designed to have high affinity to the target peptide, and furthermore, it avoids binding to similar peptides. This is an important advantage of the antigen-binding protein of the present invention, because binding to similar peptides increases the risk of side effects when present in normal tissues. Therefore, the fact that the antigen-binding protein of the present invention only binds to similar peptides with low affinity makes this antigen-binding protein a promising anti-cancer treatment in terms of safety.

[0192] The inventors demonstrate that antigen-binding proteins (particularly TCER® molecules) induce cytolysis in T2 cells loaded with the target peptide PRAME-004 by LDH release assay (Table 17). The inventors further demonstrate that antigen-binding proteins (particularly TCER® molecules) induce cytolysis in PRAME-positive tumor cell lines by LDH release assay, whereas PRAME-negative tumor cell lines were not affected by co-culture with TCER® molecules (Figures 7-9). These in vitro experiments further demonstrate the safety of the antigen-binding proteins of the invention and demonstrate that the cytotoxic effect is highly selective for PRAME-positive tumor tissue. Thus, the molecules of the invention exhibit a beneficial safety profile.

[0193] In some embodiments, the antigen binding proteins of the invention are TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFT17-003, DIAPH1-004, FADS2-001, FRYL-001, ... 3, GIMAP8-001, HSF1-001, KNT-001, MAU-001, MCM4-001, MPPE1-001, MYO1B-002, PRR12-001, PTRF-00 3, RASGRP1-001, SMARCD1-001, TGM2-001, VAV1-001, VIM-009, FARSA-001, ALOX15B-003, FAM114A2-0 02, GPR56-002, IGHD-002, NOMAP-3-0972, NOMAP-3-1265, NOMAP-3-1408, NOMAP-3-1587, NOMAP-3-1768, NOMAP-5-0765, PDCD10-004, TSN-001, ARMC9-002, CLI-001, COPG1-001, COPS7A-001, EIF-009, EXT2-006, LMNA-001, PKM-005, PSMB3-002, RPL-007, SPATS2L-003, SYNE1-002, TGM2-002, and TPR-004.

[0194] In a preferred embodiment, the antigen binding proteins of the invention do not significantly bind to IFT17-003 in complex with MHC.

[0195] In some embodiments, the antigen binding proteins of the invention exhibit a functional response to the PRAME-004 peptide:MHC complex in comparison to TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFT17-003, DIAP H1-004, FADS2-001, FRYL-003, GIMAP8-001, HSF1-001, KNT-001, MAU-001, MCM4-001, MPPE1-001, MYO1B-00 2, PRR12-001, PTRF-003, RASGRP1-001, SMARCD1-001, TGM2-001, VAV1-001, VIM-009, FARSA-001, ALOX15B-0 03, FAM114A2-002, GPR56-002, IGHD-002, NOMAP-3-0972, NOMAP-3-1265, NOMAP-3-1408, NOMAP-3-1587, NO MAP-3-1768, NOMAP-5-0765, PDCD10-004, TSN-001, ARMC9-002, CLI-001, COPG1-001, COPS7A-001, EIF-009, or exhibits a significantly reduced functional response to at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, or all of the similar peptides selected from the group consisting of EXT2-006, LMNA-001, PKM-005, PSMB3-002, RPL-007, SPATS2L-003, SYNE1-002, TGM2-002, and TPR-004. In a preferred embodiment, the antigen binding protein of the present invention exhibits a significantly reduced functional response to IFT17-003 in complex with MHC compared to the functional response to the PRAME-004 peptide:MHC complex.

[0196] In some embodiments, the antigen binding proteins of the invention (particularly soluble bispecific antigen binding proteins, more particularly antigen binding proteins in the TCER® format) exhibit a K D≥25, ≥30, ≥40, ≥50, ≥75, or ≥100-fold increased K DIn complex with MHC, TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFT17-003, DIAPH1-004, FADS2-001, FRYL-003, GIMAP8-001, HSF1-001, KNT-001, MAU-001, MCM4-001, MPPE1-001, MYO1B-002, PRR12-001, PTRF-003, RASGRP1-001, SMARCD1-001, and SMARCD2-002 were identified. 001, TGM2-001, VAV1-001, VIM-009, FARSA-001, ALOX15B-003, FAM114A2-002, GPR56-002, IGHD-002, NOMAP-3-0972, NOMAP-3-1265, NOMAP-3-1408, N OMAP-3-1587, NOMAP-3-1768, NOMAP-5-0765, PDCD10-004, TSN-001, ARMC9-002, CLI-001, COPG1-001, COPS7A-001, EIF-009, EXT2-006, LMNA-001, PKM -005, PSMB3-002, RPL-007, SPATS2L-003, SYNE1-002, TGM2-002, and TPR-004, in particular GIMAP8-001, MYO1B-002, SMARCD1-001, VIM-009, FARSA-001, ALOX15B-003, FAM114A2-002, GPR56-002, IGHD-002, NOMAP-3-0972, NOMAP-3-1265, NOMAP-3-1408, NOMAP-3-1587, NOMAP-3-1768, NOMAP-5-07 65, PDCD10-004, TSN-001, and / or ARMC9-002, CLI-001, COPG1-001, COPS7A-001, EIF-009, EXT2-006, LMNA-001, PKM-005, PSMB3-002, RPL-007, SPATS2L-003, SYNE1-002, TGM2-002, and TPR-004.In a preferred embodiment, the antigen binding proteins of the invention (particularly soluble bispecific antigen binding proteins, more particularly antigen binding proteins in the TCER® format) exhibit a K for binding to the PRAME-004 peptide:MHC complex. D ≥25, ≥30, ≥40, ≥50, ≥75, or ≥100-fold increased K D and binds to IFT17-003 in a complex with MHC.

[0197] In some embodiments, the antigen binding proteins of the invention exhibit increased expression of TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFT17-003, DIAPH1-004, FADS2-001, FRYL-003, G, in complex with MHC, particularly when expressed on the cell surface, and more particularly when expressed on the yeast cell surface, as compared to detection with PRAME-004 peptide:MHC multimers. IMAP8-001, HSF1-001, KNT-001, MAU-001, MCM4-001, MPPE1-001, MYO1B-002, PRR12-001, PTRF-003, RASGRP1-001, SMARCD1-001, TGM2-001, VAV1- 001, VIM-009, FARSA-001, ALOX15B-003, FAM114A2-002, GPR56-002, IGHD-002, NOMAP-3-0972, NOMAP-3-1265, NOMAP-3-1408, NOMAP-3-1587, NOMA P-3-1768, NOMAP-5-0765, PDCD10-004, TSN-001, ARMC9-002, CLI-001, COPG1-001, COPS7A-001, EIF-009, EXT2-006, LMNA-001, PKM-005, PSMB3-002, RPL-007, SPATS2L-003, SYNE1-002, TGM2-002, and TPR-004, and in particular, TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB -001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFT17-003, DIAPH1-004, FADS2-001, FRYL-003, GIMAP8-001, HSF1-001, KNT-001, MAU-001, MCM4-001, MPPE1-001, MYO1B-002, PRR12-001, PTRF-003, RASGRP1-001, SMARCD1-001, TGM2-001, and VAV1-001, more particularlyLabeled analog peptides including IFT17-003: Showing significantly lower detection by MHC multimers.

[0198] The antigen binding proteins of the invention have a high safety profile.

[0199] "Safety profile" as used herein refers to the ability to distinguish between tumor cells (particularly PRAME-004:MHC complex-presenting tumor cells) and healthy cells. This ability is often expressed in terms of a safety window.

[0200] "Safety window" or "therapeutic window" herein refers to a parameter that compares the concentration of a compound required to induce a degree of cytotoxicity (e.g., 10%, 50%, 90%, or 100% cytotoxicity) against tumor cells (e.g., PRAME-004:MHC complex-presenting tumor cells) with the concentration required to induce cytotoxicity (preferably, the same degree of cytotoxicity, more preferably, the same degree of cytotoxicity) against healthy cells. For example, if the concentration of an antigen protein required to induce 90% cytotoxicity against a tumor cell line is 1 pM, and the concentration required to induce 90% cytotoxicity against, for example, healthy cells is 1000 pM, then the safety window is 1000, because the cytotoxic concentration required for the tumor cell line is 1000 times lower than that required for healthy cells.

[0201] In some embodiments, the safety window is defined as half-maximal (50%) cytotoxicity (EC 50 ) and the concentration of a compound required to induce half-maximal (50%) cytotoxicity (EC 50 The concentration of the compound required to induce the EC20 activity against the tumor cell line is compared with that of the antigen-binding protein. 50 is 1 pM, e.g., EC 50 If the EC value for the tumor cell line is 1000 pM, the safety window is 1000 because 50is 1000 times lower than in healthy cells.

[0202] In a preferred embodiment the antigen binding protein of the invention is characterized by a safety window of ≧100, ≧500, ≧1000, ≧2000, ≧3000, ≧4000, ≧5000, ≧6000, ≧8000, ≧10000, for example, 500-10000, preferably 1000-10000.

[0203] "PRAME-004:MHC complex-presenting cells" herein refers to cells that present PRAME antigen peptides on their surface in complex with MHC molecules, and the copy number of said PRAME-004:MHC complexes may be determined by methods known to those skilled in the art. In a preferred embodiment, the PRAME-004:MHC complex-presenting cells are tumor cells, and the tumor is preferably a cancer as defined herein below in the "Therapeutic Methods and Uses" section. In the context of the present invention, the PRAME-004:MHC complex is over-presented on the cell surface of the PRAME-004:MHC complex-presenting cells compared to the level of said complex on the surface of cells in normal (healthy) tissue (also referred to as "healthy cells"). "Over-presented" means that the PRAME-004:MHC complex is present at a level of at least 1.2 times the level present in healthy tissue, preferably at least 2 times, more preferably 5-10 times the level present in healthy tissue or cells.

[0204] In one embodiment, the PRAME-004:MHC complex-presenting cells have a PRAME-004:MHC complex copy number of more than 50, more than 80, more than 100, more than 120, more than 150, more than 300, more than 400, more than 600, more than 800, more than 1000, more than 1500, or more than 2000, preferably a PRAME-004:MHC copy number of 50 to 2000, for example, 80 to 2000, for example, 100 to 2000, for example, 120 to 2000.

[0205] "Copy number" as used herein refers to the number of PRAME-004:MHC complexes as defined in the context of the present invention that are present on the cell surface of a cell (e.g., a PRAME-004:MHC presenting cell, e.g., a cancer cell, or a healthy cell). Protein copy number can be determined by a variety of methods known in the art, including FACS analysis of diseased cells with fluorescently labeled antigen binding proteins.

[0206] "Healthy cells" or "normal tissue cells" herein refer to cells that are not tumor cells, and preferably healthy cells herein refer to cells of tissues surrounding PRAME-004:MHC presenting cells, and in particular cells of tissues surrounding PRAME-004:MHC complex presenting tumor cells. However, in some cases, healthy cells may also express and present PRAME-004:MHC complexes on their surface. Typically, in healthy cells relevant to the present invention, the PRAME-004:MHC complexes are present in lower amounts (copy numbers) than in tumor cells, as will be understood by those skilled in the art. Thus, in one embodiment, healthy cells have a PRAME-004:MHC complex copy number less than 50, less than 20, less than 10, preferably less than 10 PRAME-004:MHC complex copy number, and preferably 0-10 PRAME-004:MHC complex copy number.

[0207] The healthy cells are preferably selected from the group consisting of astrocytes, GABA neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, aortic endothelial cells, coronary artery endothelial cells, dermal microvascular endothelial cells, mesenchymal stem cells, nasal epithelial cells, peripheral blood mononuclear cells, pulmonary artery smooth muscle cells, pulmonary fibroblasts, epidermal keratinocytes, renal cortical epithelial cells and tracheal smooth muscle cells, preferably astrocytes, in particular iPSC-derived astrocytes, cardiomyocytes, in particular iPSC-derived cardiomyocytes, aortic endothelial cells, mesenchymal stem cells, and tracheal smooth muscle cells.

[0208] In a preferred embodiment, the concentration of an antigen binding protein of the invention required to achieve at least 90%, preferably 100% cytotoxicity in tumor cells (in particular PRAME-004:MHC complex-presenting tumor cells) is greater than or equal to 100% in astrocytes, GABA neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, aortic endothelial cells, coronary artery endothelial cells, dermal microvascular endothelial cells, mesenchymal stem cells, nasal epithelial cells, peripheral blood mononuclear cells, pulmonary artery smooth muscle cells, lung fibroblasts, epidermal keratinocytes, renal cortical epithelial cells and tracheal smooth muscle cells. At least 100-fold, 500-fold, 1000-fold, 5000-fold, or 10,000-fold less than the concentration required to achieve at least 10%, at least 50%, at least 90%, or 100% cytotoxicity in healthy cells, preferably astrocytes, particularly iPSC-derived astrocytes, cardiomyocytes, particularly iPSC-derived astrocytes, iPSC-derived cardiomyocytes, aortic endothelial cells, mesenchymal stem cells, and tracheal smooth muscle cells.

[0209] The inventors have demonstrated that the CDRs defined in the present claims can be used in antigen binding proteins with various formats. For example, in the experimental section, the inventors used these CDRs in single chain TCR constructs such as TCER® molecules and bispecific TCRs comprising scTCRs fused to Fab fragments (scTCR-Fabs).

[0210] Thus, one of skill in the art will appreciate from these experiments that the CDRs described herein can be used in a variety of antigen binding proteins of the present invention.

[0211] In one embodiment, the format of the antigen binding protein is altered while preserving the epitope and binding specificity.

[0212] In some embodiments, the antigen-binding protein is a TCR or an antibody. Those skilled in the art will recognize that when the antigen-binding protein is an antibody, this "antibody" at least comprises the sequence of CDR1, CDR3, and optionally CDR2 from a TCR as defined in the claims, and is therefore not a native antibody or a conventional antibody. However, an antigen-binding protein that comprises, for example, a CDR from a TCR, a framework region from an antibody, and a constant domain from an antibody will have the overall structure of a conventional antibody and can be referred to as an "antibody".

[0213] In some embodiments, the antigen binding protein is bispecific, in particular a bispecific TCR, bispecific antibody, or bispecific TCR antibody molecule. The skilled artisan will recognize that even when the antigen binding protein is a bispecific "antibody", one of the antigen binding sites will comprise the CDR1, CDR3, and optionally CDR2 sequences from a TCR as defined in the claims, and the other antigen binding site may be entirely antibody derived.

[0214] In one embodiment, the antigen binding protein is of human origin, which is understood to be generated from a human antigen locus and thus to contain human sequences (particularly human TCR or antibody sequences).

[0215] In one embodiment, the antigen binding protein is characterized as an affinity matured antigen binding protein, which is capable of specifically binding the PRAME-004 antigenic peptide (particularly the PRAME-004:MHC complex) with higher affinity than the parent molecule (particularly TCR R11P3D3).

[0216] In some embodiments, the antigen binding protein is A and a first polypeptide chain comprising: B and a second polypeptide chain comprising:

[0217] In some embodiments, the first and second polypeptides, such that V A and V Bare located on a single polypeptide chain. Such a single chain construct may be a single chain TCR (scTCR), a single chain antibody, or a single chain bispecific antigen binding protein, in particular a single chain bispecific antibody, a single chain bispecific TCR, or a single chain bispecific TCR antibody molecule. An example of a single chain TCR (scTCR) is the construct used in Example 1, which may also be referred to as a single chain TCR variable domain ("scTv") molecule. An example of a single chain "antibody" would be an scFv in which the CDRs are replaced by CDRs from a TCR. An example of a single chain bispecific antibody would be an antibody in which one binding site is from an antibody and the other binding site is from a TCR or at least comprises a CDR from a TCR. As discussed above, such a hybrid antigen binding protein may alternatively be referred to as a single chain bispecific TCR or a single chain bispecific TCR antibody molecule.

[0218] Framework Area The inventors of the present invention have further discovered that specific mutations in the framework regions of the antigen binding protein have advantageous effects compared to the parental TCR R11P3D3.

[0219] V A So, the advantageous mutations are: - N20K (removes a naturally occurring potential N-glycosylation site), - W44K(V B in combination with Q44E to improve variable domain pairing, affinity, and stability); - A52F, V55Y, K92T, and G93D (increasing the stability of the antigen-binding protein).

[0220] V B So, the advantageous mutations are: - A84D, A84E, A84Q, A84N, A84S, preferably A84D (increases affinity for peptide-MC complexes) - Q44E(V A(In combination with W44K in - M46P and R48Q (increases the stability of the antigen-binding protein).

[0221] Mutations are designated according to the IMGT nomenclature.

[0222] Thus, the antigen binding protein of the present invention preferably comprises A (R11P3D3's V α and one or more (preferably all) of N20K, W44K, A52F, V55Y, K92T, and G93D in the comparison with V B (R11P3D3's V β The sequence (compared to) includes one or more (preferably all) of A84D, Q44E, M46P, and R48Q.

[0223] The antigen-binding protein of the present invention is A (R11P3D3's V α and may further include one or more of L2M, L39I, and Q14K in the sequence (compared to V B (R11P3D3's V β The amino acid sequence may further include one or more of E11L, E11K, and R22H in the amino acid sequence (compared to the amino acid sequence).

[0224] Thus, the antigen binding protein of the present invention preferably comprises A and one or more (preferably all) of the following amino acids 20K, 44K, 52F, 55Y, 92T, and 93D in V B Among these are one or more (preferably all) of 84D, 44E, 46P, and 48Q.

[0225] The antigen-binding protein of the present invention is A and may further comprise one or more of the following amino acids: 2M, 39I, and 14K; and B It may further include one or more of 11L or 11K and 22H.

[0226] In one embodiment, V A further comprising one or more framework regions, preferably all of the framework regions, selected from the group consisting of FR1-a, FR2-a, FR3-a, and FR4-a; - FR1-a comprises or consists of the amino acid sequence of SEQ ID NO: 345 or SEQ ID NO: 346, or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 345, and preferably contains K or N at position 20, more preferably contains K and / or contains L or M at position 2; - FR2-a comprises or consists of the amino acid sequence of SEQ ID NO: 347 or SEQ ID NO: 348, or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 347, and preferably comprises L, I or M, more preferably comprises L or I, at position 39 comprises A or D, more preferably comprises A, at position 47 comprises K or W, preferably comprises K, at position 44 comprises F or A, preferably comprises F, and / or at position 55 comprises Y or V, preferably comprises Y; - FR3-a comprises or consists of the amino acid sequence of SEQ ID NO: 349 or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 349, and preferably comprises T or K at position 92, preferably comprises T, and / or comprises D or G at position 93, preferably comprises D; - FR4-a comprises or consists of the amino acid sequence of SEQ ID NO: 350 or an amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NO: 350; V B further comprising one or more framework regions, preferably all of the framework regions, selected from the group consisting of FR1-b, FR2-b, FR3-b, and FR4-b; - FR1-b comprises or consists of the amino acid sequence of SEQ ID NO: 351 or SEQ ID NO: 352, or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 351, and preferably comprises H or N, more preferably comprises H, at position 10 comprises E, L or K, preferably comprises E, at position 11 comprises R or H; - FR2-b comprises or consists of an amino acid sequence of SEQ ID NO: 353 or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 353, and preferably comprises R or K, more preferably comprises R, at position 43 comprises E or Q, preferably comprises E, at position 44 comprises M or P, more preferably comprises P, and / or at position 48 comprises R or Q, more preferably comprises Q; - FR3-b comprises or consists of the amino acid sequence of SEQ ID NO: 354 or SEQ ID NO: 355, or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 354, and preferably contains at position 84 D, A, E, R, K, Q, N or S, more preferably D, A, E, Q, N or S, more preferably D or A, even more preferably D; - FR4-b comprises or consists of the amino acid sequence of SEQ ID NO: 356 or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 356.

[0227] Variants of the antigen-binding proteins described herein are contemplated and explicitly referred to using the expression "at least 85% identical to the reference sequence" as defined in the "Definitions" section herein above. By way of example, the amino acid sequences of FR1-a, FR2-a, FR3-a, FR4-a, FR1-b, FR2-b, FR3-b, and FR4-b may differ from the reference sequences SEQ ID NO:345, SEQ ID NO:346, SEQ ID NO:347, SEQ ID NO:348, SEQ ID NO:349, SEQ ID NO:350, SEQ ID NO:351, SEQ ID NO:352, SEQ ID NO:353, SEQ ID NO:354, SEQ ID NO:355, or SEQ ID NO:356, as appropriate, by at least one amino acid substitution, in particular by at least one conservative amino acid substitution and / or substitution with a canonical residue. In particular, V A and V B The sequences FR1-a, FR2-a, FR3-a, and FR4-a, FR1-b, FR2-b, FR3-b, and FR4-b may differ from the reference sequences SEQ ID NO:345, SEQ ID NO:346, SEQ ID NO:347, SEQ ID NO:348, SEQ ID NO:349, SEQ ID NO:350, SEQ ID NO:351, SEQ ID NO:352, SEQ ID NO:353, SEQ ID NO:354, SEQ ID NO:355, or SEQ ID NO:356, as appropriate, by conservative amino acid substitutions only.

[0228] Modifications and changes can be made in the amino acid sequences of the antigen binding proteins of the present invention, and in the corresponding DNA sequences, respectively, and still obtain a functional antigen binding protein or polypeptide having the desired properties. A and / or V B Modifications may be made, particularly in the framework regions or CDRs.

[0229] V A and V B V preferably contains an amino acid substitution at position 44 according to the IMGT numbering compared to TCR R11P3D3. In embodiments where the antigen binding protein is a TCR, these substitutions improve chain pairing (i.e., α and β chain pairing, or γ and δ chain pairing). A or V BThe amino acid at position 44 in may be substituted with an amino acid selected from the group consisting of Q, R, D, E, K, L, W, and V. Preferred are those present in SEQ ID NO: 347, SEQ ID NO: 348 (FR2-a), and SEQ ID NO: 353 (FR2-b), respectively, and having the amino acid pair VA 44K / VA 44E, V A Substitutions in W44K and V B Other suitable combinations are VA 44Q / VB 44Q, VA 44D / VB 44R, VA 44R / VB 44D, VA 44E / VB 44K, VA 44D / VB 44K, VA 44K / VB 44D, VA 44R / VB 44E; VA 44E / VB 44R, VA 44L / VB 44W, VA 44W / VB 44L, VA 44V / VB 44W, and VA 44W / VB It is 44V.

[0230] Additional permutations and explanations may be found in U.S. Patent Application Publication No. 2018-0162922, the contents of which are incorporated by reference in their entirety.

[0231] Variable domain In one embodiment, V Acomprises or consists of the amino acid sequence of a TCR-derived variable domain contained in a polypeptide of SEQ ID NO: 100, 103, 105, 106, 111, 122, 124, 126, 128, 151, 155, 156, 157, 158, 159, 166, 167, 169, 171, 173, 175, 177, 178, 179, 180, 181, 183, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 285, 291, 295, 299, or 303; B are sequence numbers 101, 102, 104, 107, 110, 119, 121, 131, 133, 143, 152, 153, 160, 161, 162, 163, 164, 165, 168, 170, 172, 174, 176, 182, 184, 185, 186, 216, 218, 220, 222, 224, 228, 230, 232, 234 , 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 282, 284, 296 or 300. The skilled artisan is fully capable of distinguishing the amino acid sequence of the TCR-derived variable domain within the polypeptide chains of the above mentioned SEQ ID NOs.

[0232] In one embodiment, - V Acomprises or consists of the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 132, preferably a CDRa1 of SEQ ID NO: 16, a CDRa2 of SEQ ID NO: 32, and a CDRa3 of SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 9, and may further comprise K or N, preferably K, at position 20, may comprise L, M or I, preferably L or I, at position 39, may comprise K or W, preferably K, at position 44, may comprise F or A, preferably F, at position 52, may comprise Y or V, preferably Y, at position 55, may comprise T or K, preferably T, at position 92, and / or may comprise D or G, preferably D, at position 93; - V B comprises or consists of the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence which is at least 85%, 90% or 95% identical to SEQ ID NO: 134, preferably comprises CDRb1 of SEQ ID NO: 10, CDRb2 of SEQ ID NO: 36 and CDRb3 of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 47, SEQ ID NO: 281, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 301 or SEQ ID NO: 283, and may further comprise E, L or K, preferably E, at position 11, may comprise R or H, at position 22, may comprise E or Q, preferably E, at position 44, may comprise P or M, preferably P, at position 46, may comprise Q or R, preferably Q, and / or may comprise D, A, E, R, K, Q, N or S, more preferably D, A, E, Q, N or S, preferably D or A, at position 84.

[0233] - V Acomprises or consists of an amino acid sequence of SEQ ID NO: 132, or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 132, including a CDRa1 of SEQ ID NO: 16, a CDRa2 of SEQ ID NO: 32, and a CDRa3 of SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 9, and further optionally comprises K or N, preferably K, at position 20, optionally comprises L, M or I, preferably L or I, at position 39, optionally comprises K or W, preferably K, at position 44, optionally comprises F or A, preferably F, at position 52, optionally comprises Y or V, preferably Y, at position 55, optionally comprises T or K, preferably T, at position 92, and / or optionally comprises D or G, preferably D, at position 93; - V B comprises or consists of the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence which is at least 85%, 90% or 95% identical to SEQ ID NO: 134, comprises a CDRb1 of SEQ ID NO: 10, a CDRb2 of SEQ ID NO: 36 and a CDRb3 of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 47, SEQ ID NO: 281, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 301 or SEQ ID NO: 283, and may further comprise E, L or K, preferably E, at position 11, may comprise R or H, at position 22, may comprise E or Q, preferably E, at position 44, may comprise P or M, preferably P, at position 46, may comprise Q or R, preferably Q, and / or may comprise D, A, E, R, K, Q, N or S, more preferably D, A, E, Q, N or S, preferably D or A, at position 84. It is preferred.

[0234] In a preferred embodiment, - V A comprises or consists of the amino acid sequence of SEQ ID NO: 132, SEQ ID NO: 129, SEQ ID NO: 137, or SEQ ID NO: 142; -V B comprises or consists of the amino acid sequence of SEQ ID NO:134, SEQ ID NO:130, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, or SEQ ID NO:148.

[0235] -V A comprises or consists of the amino acid sequence of SEQ ID NO: 132, and B comprises or consists of the amino acid sequence of SEQ ID NO: 134; -V A comprises or consists of the amino acid sequence of SEQ ID NO: 132, and B comprises or consists of the amino acid sequence of SEQ ID NO: 135; -V A comprises or consists of the amino acid sequence of SEQ ID NO: 132, and B comprises or consists of the amino acid sequence of SEQ ID NO: 140; -V A comprises or consists of the amino acid sequence of SEQ ID NO: 132, and B comprises or consists of the amino acid sequence of SEQ ID NO: 136; -V A comprises or consists of the amino acid sequence of SEQ ID NO: 137, and B comprises or consists of the amino acid sequence of SEQ ID NO: 134; -V A comprises or consists of the amino acid sequence of SEQ ID NO: 137, and B comprises or consists of the amino acid sequence of SEQ ID NO: 135; or -V A comprises or consists of the amino acid sequence of SEQ ID NO: 137, and B comprises or consists of the amino acid sequence of SEQ ID NO: 134 It is particularly preferred.

[0236] Most preferably, V Acomprises or consists of the amino acid sequence of SEQ ID NO: 132, B comprises or consists of the amino acid sequence of SEQ ID NO: 134, 135 or 140 (particularly SEQ ID NO: 135). A may comprise or consist of the amino acid sequence of SEQ ID NO: 132, and V B may comprise or consist of the amino acid sequence of SEQ ID NO: 135. A may comprise or consist of the amino acid sequence of SEQ ID NO: 132, and V B may comprise or consist of the amino acid sequence of SEQ ID NO:140.

[0237] Variants of the antigen binding proteins described herein are contemplated and specifically referred to using the phrase "at least 85% identical to a reference sequence" as defined in the definitions section herein above. For example, A and V B The sequences of may differ from the reference sequences of SEQ ID NO: 132 and SEQ ID NO: 134, respectively, by at least one amino acid substitution, in particular by at least one conservative amino acid substitution and / or substitution with a canonical residue. A and V B may differ from the reference sequences of SEQ ID NO: 132 and SEQ ID NO: 134, respectively, only by conservative amino acid substitutions.

[0238] Modifications and changes can be made in the amino acid sequences of the antigen binding proteins of the present invention, and the corresponding DNA sequences, respectively, and still obtain a functional antigen binding protein or polypeptide possessing desirable properties.

[0239] In one embodiment, the antigen binding protein of the present invention has the structure: (i) one or more additional antigen-binding sites: (ii) a transmembrane region that may include a cytoplasmic signaling region; (iii) diagnostic agents; (iv) a therapeutic agent; or (v) PK modification portion The present invention further includes one or more of:

[0240] When the above listed components (i) to (v) are polypeptides fused to an antigen-binding protein of the present invention, the antigen-binding protein may also be referred to as a "TCR fusion protein."

[0241] The further antigen-binding site is preferably derived from an antibody.

[0242] A "transmembrane region" in the context of the present invention may be, for example, a TCR alpha or beta transmembrane domain.

[0243] The "cytoplasmic signaling region" can be, for example, a TCR alpha or beta intracellular domain.

[0244] "Diagnostic agent," as used herein, refers to a detectable molecule or substance (e.g., a fluorescent molecule, a radioactive molecule, or any other label known in the art to produce (directly or indirectly) a signal.

[0245] "Fluorescent molecules" known in the art include fluorescein isothiocyanate (FITC), phycoerythrin (PE), fluorophores used with blue lasers (e.g., PerCP, PE-Cy7, PE-Cy5, FL3, and APC or Cy5, FL4), fluorophores used with red, violet, or UV lasers (e.g., Pacific Blue, Pacific Orange).

[0246] As a "radioactive molecule", I 123 , I 124 , In 111 , Re 186 , Re 188 , Tc 99Antigen binding proteins of the invention may also include spin labels (e.g., iodine-123, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron) for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

[0247] Such diagnostic agents may be directly bound (ie, physically linked) or indirectly linked to the antigen binding protein.

[0248] "Therapeutic agent" as used herein refers to an agent that has a therapeutic effect. The terms therapeutic agent and therapeutic drug are used interchangeably herein. In one embodiment, a therapeutic agent can be a growth inhibitory agent, such as a cytotoxic agent or a radioisotope.

[0249] "Growth inhibitory agent" or "anti-proliferative agent" (these terms may be used interchangeably) refers to a compound or composition that inhibits the growth of cells, especially tumor cells, either in vitro or in vivo.

[0250] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of a cell and / or causes the destruction of a cell. The term "cytotoxic agent" is intended to include chemotherapeutic agents, enzymes, antibiotics, and toxins (e.g., small molecule or enzymatically active toxins of bacterial, fungal, plant, or animal origin), including fragments and / or variants thereof, as well as various antitumor or anticancer agents disclosed below. In some embodiments, the cytotoxic agent is a taxoid, vinca, taxane, maytansinoid or maytansinoid analog (e.g., DM1 or DM4), small drug, tomaymycin or pyrrolobenzodiazepine derivative, cryptophycin derivative, leptomycin derivative, auristatin or dolastatin analog, prodrug, topoisomerase II inhibitor, DNA alkylating agent, antitubulin agent, CC-1065, or CC-1065 analog.

[0251] The term "radioisotope" is intended to include radioisotopes suitable for the treatment of cancer, such as At 211 , Bi 212 , Er 169 , I 131 , I 125 , Y 90 , In 111 , P 32 , Re 186 , Re 188 , Sm 153 , Sr 89 The term "radioisotopes" is intended to include radioisotopes of Lu, Cr, and Lu. Such radioisotopes generally emit primarily beta radiation. In one embodiment, the radioisotope is an alpha-emitter isotope, more precisely, thorium-227, which emits alpha radiation.

[0252] In some embodiments, the antigen-binding protein of the present invention is covalently linked to at least one growth inhibitory agent, either directly or through a cleavable or non-cleavable linker. Such an antigen-binding protein with at least one growth inhibitory agent attached thereto may also be referred to as a conjugate. The cleavable linker facilitates the release of the cytotoxic agent or growth inhibitory agent from the antigen-binding protein in cells. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase-labile linker, a photolabile linker, or a disulfide-containing linker (see, for example, U.S. Patent No. 5,208,020) may be used. The linker may also be a "non-cleavable linker" (e.g., SMCC linker), which may provide better tolerance in some cases.

[0253] The preparation of such conjugates (e.g., immunoconjugates) is described in WO 2004 / 091668, or in Hudecz, F., Methods Mol. Biol. 298: 209-223 (2005) and Kirin et al., Inorg Chem. 44(15): 5405-5415 (2005), the contents of which are incorporated herein by reference in their entireties, and can be adapted by the skilled artisan to prepare such antigen-binding proteins of the invention having at least one growth inhibitory agent attached thereto.

[0254] Alternatively, a fusion protein comprising an antigen binding protein of the invention and a cytotoxic or growth inhibitory polypeptide may be produced by recombinant techniques or peptide synthesis. The length of DNA may include the respective regions encoding the two portions of the conjugate adjacent to each other or may include the respective regions encoding the two portions of the conjugate separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

[0255] The antigen binding proteins of the invention may also be used in dependent enzyme mediated prodrug therapy by conjugating the polypeptide to a prodrug activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81 / 01145) into an active anti-cancer drug (see, e.g., WO 88 / 07378 and U.S. Pat. No. 4,975,278).

[0256] "PK-modifying moiety" refers herein to a moiety that modifies the pharmacokinetics of the antigen-binding protein of the present invention. Thus, the moiety specifically modifies the in vivo half-life and distribution of the antigen-binding protein of the present invention. In a preferred embodiment, the PK-modifying moiety increases the half-life of the antigen-binding protein. Examples of PK-modifying moieties include, but are not limited to, PEG [Dozier et al., (2015) Int J Mol Sci. Oct 28;16(10):25831-64, and Jevsevar et al., (2010) Biotechnol J.Jan;5(1):113-28], PAS [Schlapschy et al., (2013) Protein Eng Des Sel. Aug;26(8):489-501], albumin [Dennis et al., (2002) J Biol Chem. Sep 20;277(38):35035-43)], F of antibodies and / or unstructured polypeptides. c Part [Schellenberger et al., (2009) Nat Biotechnol. Dec; 27(12):1186-90].

[0257] In one embodiment, the antigen binding protein of the invention further comprises one or more of an enzyme, a cytokine (e.g., human IL-2, IL-7, or IL-15), a nanocarrier, or a nucleic acid.

[0258] Second antigen-binding site In a preferred embodiment, the antigen binding protein comprises an antibody light chain variable domain (V L) and an antibody heavy chain variable domain (V H The variable domain V L and variable domain V H together form an antigen-binding site, which may hereinafter also be referred to as the "second antigen-binding site." L and V H The antigen-binding site formed by preferably binds to an antigen of an effector cell and recruits the effector cell to the tumor, and may therefore also be referred to as a "recruiter." In the context of the present invention, "effector cell" refers to a T cell or a natural killer cell (NK cell).

[0259] In a preferred embodiment, V H and V H are CD2, CD3 (e.g., CD3γ, CD3δ, and CD3ε chains), CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61, CD64, CD68, CD90, CD94, CD95, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, F c and / or V H and V L binds to an effector cell. "The combination" refers to a complex of two or more of said antigens (e.g., a TCRα / β CD3 complex). Preferably, the antigen is CD3, a TCRα / β CD3 complex, or CD28, more preferably, CD3, or a TCRα / β CD3 complex.

[0260] In the case of targeting the TCR-CD3 complex, the V-terminal fragment derived from the CD3-specific humanized antibody hUCHT1 (Zhu et al., Identification of heavy chain residues in a humanized anti-CD3 antibody important for efficient antigen binding and T cell activation. J Immunol, 1995, 155, 1903-1910) was used. H and V L Domains may be used, in particular V domains derived from UCHT1 mutants UCHT1-V17, UCHT1-V17opt, UCHT1-V21, or UCHT1-V23. H and V L The V domain may be used, preferably from UCHT1-V17. H and V L A V domain may be used, more preferably comprising or consisting of SEQ ID NO: 109. H and V comprising or consisting of SEQ ID NO: 108 L Alternatively, the V from antibody BMA031 and its humanized versions (Shearman et al., Construction, expression and characterization of humanized antibodies directed against the human alpha / beta T cell receptor, J Immunol, 1991, 147, 4366-73), which targets the TCRα / β CD3 complex, may be used. H and V L Domains may be used, in particular the V domains derived from the BMA031 variants BMA031(V36) or BMA031(V10). H and V L The V domain may be used, preferably from BMA031 (V36). H and V LThe domain may be used, comprising or consisting of SEQ ID NO: 112, or SEQ ID NO: 114 (A02), or SEQ ID NO: 115 (D01), or SEQ ID NO: 116 (A02_H90Y), or SEQ ID NO: 117 (D01_H90Y). H and V comprising or consisting of SEQ ID NO: 113 L Alternatively, V from the CD3ε-specific antibody H2C (described in EP 2155783) may be used. H and V L The domain may be used, in particular the V domain comprising or consisting of SEQ ID NO: 118, or SEQ ID NO: 123 (N100D), or SEQ ID NO: 125 (N100E), or SEQ ID NO: 127 (S101A). H and V comprising or consisting of SEQ ID NO: 120 L All positions and CDR definitions are according to the Kabat numbering scheme.

[0261] In some embodiments, V H and V L Both bind to the TCRα / β CD3 complex and V H teeth, - a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 381 (SYVMH), - HCDR2 comprising the amino acid sequence of YINPYNDVTKYX1X2KFX3G (SEQ ID NO: 382), wherein X1 is A or N; X2 is E or Q; and / or X3 is Q or K, - HCDR3, and - Heavy chain framework regions (HFR) 1-4 Including, V L teeth, - light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 383 (SATSSVSYMH) - LCDR2 comprising the amino acid sequence of SEQ ID NO: 384 (DTSKLAS), and - LCDR3 Including, - at least one amino acid of HCDR1 comprising the amino acid sequence of SEQ ID NO: 1 and / or at least one amine of HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, which is not positively charged, is replaced by a positively charged amino acid; and / or - at least one amino acid of LCDR1 comprising the amino acid sequence of SEQ ID NO: 3 and / or at least one amine of LCDR2 comprising the amino acid sequence of SEQ ID NO: 4, which is not positively charged, is replaced by a positively charged amino acid; and / or - HFR3 contains a tyrosine (Y) residue at position 90 according to the Kabat numbering.

[0262] Preferably, the antigen-binding polypeptide comprises In the heavy chain, - the positively charged amino acid at position 31 is R, K, or H; - the positively charged amino acid at position 53 is R, K, or H; and / or - the positively charged amino acid at position 54 is R or K; and / or In the light chain, - the positively charged amino acid at position 31 is R or K; and / or - the positively charged amino acid at position 56 is R or K;

[0263] In some embodiments, V H comprises a sequence selected from the group consisting of SEQ ID NOs: 112, 114-117, and 366-376; L comprises a sequence selected from the group consisting of SEQ ID NOs: 113, and 377 to 380. H comprises or consists of the amino acid sequence of SEQ ID NO: 112, or 114 to 117; L comprises or consists of the amino acid sequence of SEQ ID NO: 113 or 378 (preferably 113).

[0264] "CD28" is expressed on T cells and can produce costimulatory signals necessary for T cell activation. CD28 plays an important role in T cell proliferation and survival, cytokine production, and T helper type 2 development.

[0265] "CD134" is also referred to as Ox40. CD134 / OX40 is expressed 24-72 hours after activation and can be used to define a secondary costimulatory molecule.

[0266] "4-1BB" is capable of binding to 4-1 BB ligands on antigen-presenting cells (APCs), thereby generating a costimulatory signal for T cells.

[0267] "CD5" is another example of a receptor that is found primarily on T cells, and is also found at lower levels on B cells.

[0268] "CD95" is a further example of a receptor that modifies T cell function, also known as the Fac receptor, which mediates apoptotic signaling by Fas ligand expressed on the surface of other cells. CD95 has been reported to regulate TCR / CD3-driven signaling pathways in resting T lymphocytes.

[0269] "NK cell-specific receptor molecules" are, for example, CD16, low affinity Fc receptor, and NKG2D.

[0270] An example of a receptor molecule present on the surface of both T cells and natural killer (NK) cells is CD2, and further members of the CD2 superfamily. CD2 can function as a costimulatory molecule on T cells and NK cells.

[0271] Bispecific and multispecific antigen-binding proteins Thus, the antigen-binding protein of the present invention preferably comprises a V that forms a first antigen-binding site specific for the PRAME-004:MHC complex. A and V Band V forming a second antigen-binding site capable of binding to an effector cell (preferably a T cell). L and V H Includes: V A , V B , V L , and V H may be located on a single polypeptide chain or on several polypeptide chains, preferably two polypeptide chains. A , V B , V L , and V H In addition, the antigen binding proteins of the present invention may or may not comprise a dimerization domain (preferably a constant immunoglobulin domain).

[0272] In some embodiments, V A , V B , V L , and V H The variable domains are located on two polypeptide chains. Preferably, each polypeptide chain contains two variable domains. One polypeptide chain is A and the other polypeptide chain is V B Preferably, the compound contains V A A polypeptide chain containing V L and V H V B A polypeptide chain containing V L and V H V A A polypeptide chain containing V L and V H Includes both V B A polypeptide chain containing V L Also V H Another possibility is that one polypeptide chain contains no V A and one polypeptide chain is V B and the third polypeptide chain comprises V L and V H It includes:

[0273] In a preferred embodiment, the antigen binding protein comprises a first and a second polypeptide chain, The first polypeptide chain has the formula [Ia]: V1-L1-D1-L2-V2-L3-D2[Ia] is represented by The second polypeptide chain has the formula [IIa] V3-L4-D3-L5-V4-L6-D4[IIa] is represented by During the ceremony, V1, V2, V3, and V4 are variable domains; A One of V1 to V4 is V B and one is V L and one is V H and; - D1, D2, D3, and D4 are dimerization domains and may or may not be present, D1 and D3, and D2 and D4 specifically bind to each other, and at least one pair of D1 and D3 or D2 and D4 is present; - L1, L2, L3, L4, L5 and L6 are linkers, L1 and L4 are present and L2, L3, L5 and L6 are optional.

[0274] One of V1 and V2 is V A and one of V3 and V4 is V B and one of the remaining two variable domains is V L and the other is V H In other words, V A and V B are located on different polypeptide chains, and V L and V H are located on different polypeptide chains.

[0275] The dimerization domain is preferably a heterodimerization domain that mediates heterodimerization of a first polypeptide chain with a second polypeptide chain, but not homodimerization of two first polypeptide chains or two second polypeptide chains. In a preferred embodiment, a pair of dimerization domains (e.g., D1 and D3, and / or D2 and D4) are selected from immunoglobulin constant domains, e.g., C1 and C2 from an antibody. L and C H1 , or C L -F c and C H1 -F c , or TCR-derived C α and C β , or a pair of C H3 Domain, or a pair of F c domain, C H3 Domain and F c The domain preferably contains an introduced mutation that forces heterodimerization, such as knobs-into-holes.

[0276] In an even more preferred embodiment, the antigen binding protein comprises a first and a second polypeptide chain, The first polypeptide chain has the formula [Ib]: V1-L1-V2-L3-D2[Ib] is represented by The second polypeptide chain has the formula [IIb]: V3-L4-V4-L6-D4[IIb] is represented by During the ceremony, V1, V2, V3, V4 are variable domains, one of which is V A One is V B One is V L One is V H and; - D2 and D4 are dimerization domains that specifically bind to each other, preferably Fc domains; - L1, L3, L4 and L6 are linkers, L3 and L6 may be present or absent.

[0277] As described with respect to formulas Ia and IIA, V A and V B are located on different polypeptide chains, and V L and V H are preferably located on different polypeptide chains, and that the dimerization domain is a heterodimerization domain.

[0278] In a preferred embodiment, D2 and D4 are a pair of F c Domain F c1 and F c2 In particular, D2 is F c1 and D4 is F c2 where F c1 and F c2 are the same or different, preferably different, and preferably contain a mutation that forces heterodimerization. In one embodiment, F c1 comprises or consists of the amino acid sequence of SEQ ID NO: 150 (hole); c2 comprises or consists of the amino acid sequence (knob) of SEQ ID NO: 149, and vice versa. c1 But, V L It is located on a polypeptide chain containing F c2 But, V H If it is located on a polypeptide chain containing c1 comprises or consists of the amino acid sequence (knob) of SEQ ID NO: 149, c2 comprises or consists of the amino acid sequence of SEQ ID NO: 150 (hole); c1 But, V H It is located on a polypeptide chain containing F c2 But, V L If it is located on a polypeptide chain containing c1 comprises or consists of the amino acid sequence of SEQ ID NO: 150 (hole); c2 comprises or consists of the amino acid sequence (knob) of SEQ ID NO: 149.

[0279] In an antigen binding protein comprising first and second polypeptide chains represented by formulas Ia and IIa or Ib and IIb, respectively, V A and V B , and V L and V H It will be understood by those skilled in the art that the orientations may be parallel, as in the DVD format, or crossed, as in the CODV format.

[0280] In formulae Ia and IIa, or Ib and IIb, V A , V B , V L , and V H can have the following orientations: (1) V1 is V H And V2 is V B And V3 is V A and V4 is V L Is it; (2) V1 is V B And V2 is V H And V3 is V L and V4 is V A Is it; (3) V1 is V B And V2 is V L And V3 is V H and V4 is V A Is it; (4) V1 is V L And V2 is V B And V3 is V A and V4 is V H Is it; (5) V1 is V H And V2 is V B And V3 is V L and V4 is V A Is it; (6) V1 is V B And V2 is V H And V3 is V A and V4 is V L Is it; (7) V1 is VL And V2 is V B And V3 is V H and V4 is V A Is it; (8) V1 is V B And V2 is V L And V3 is V A and V4 is V H Is it; (9) V1 is V H And V2 is V L And V3 is V A and V4 is V B Is it; (10) V1 is V L And V2 is V H And V3 is V A and V4 is V B Is it; (11) V1 is V H And V2 is V L And V3 is V B and V4 is V A or (12) V1 is V L And V2 is V H And V3 is V B and V4 is V A It is.

[0281] V A , V B , V L , and V H It is preferable that the V has the orientation described in (1) to (8), i.e., A and V B are located on different polypeptide chains, and V L and V H are preferably located on different polypeptide chains. A , V B , V L , and V H has the orientation described in (1) to (4), i.e., V A and V B, and V L and V H have a crossed orientation.

[0282] Linkers L1, L2, L3, L4, L5 are defined in the "Definitions" section above. In some embodiments, certain linker lengths may be preferred in certain formats. However, knowledge of linker length and amino acid sequence belongs to the general knowledge of the art, and linkers and linker amino acid sequences for various formats are part of the art and are disclosed in the disclosures cited above.

[0283] It is particularly preferred that the antigen binding proteins of the present invention are in TCER® format. In a TCER® format embodiment, the antigen binding protein comprises a first polypeptide chain and a second polypeptide chain represented by formulas [IIa] and [IIb] as defined above, wherein: - V1 is V H And V2 is V B And V3 is V A and V4 is V L Is it; V1 is V B And V2 is V H And V3 is V L and V4 is V A Is it; V1 is V B And V2 is V L And V3 is V H and V4 is V A or V1 is V L And V2 is V B And V3 is V A and V4 is V H and; - L3 and L6 are absent; - L1 and L4 preferably comprise or consist of the amino acid sequence of SEQ ID NO: 214; - D2 and D4 are a pair of F c Domain F c1 and F c2 and F c1 and F c2 are distinct and contain a mutation (preferably a "knob-into-hole" mutation) that forces heterodimerization.

[0284] In a preferred embodiment, -V L comprises or consists of the amino acid sequence of SEQ ID NO: 108, and H comprises or consists of the amino acid sequence of SEQ ID NO: 109, or -V L comprises or consists of the amino acid sequence of SEQ ID NO: 113, and V H comprises or consists of the amino acids of SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117; or -V L comprises or consists of the amino acid sequence of SEQ ID NO: 120, and H comprises or consists of the amino acids of SEQ ID NO:118, SEQ ID NO:123, SEQ ID NO:125, or SEQ ID NO:127.

[0285] In a particularly preferred embodiment, the antigen binding protein comprises a first polypeptide chain selected from SEQ ID NOs: 100, 103, 105, 106, 111, 122, 126, 128, 151, 155, 156, 157, 158, 159, 166, 167, 169, 171, 173, 175, 177, 178, 179, 180, 181, 183, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 285, 291, 295, 299, and 303, and a second polypeptide chain selected from SEQ ID NOs: 101, 102, 104, 107 , 110, 119, 121, 131, 133, 143, 152, 160, 161, 162, 163, 164, 165, 168, 170, 172, 174, 176, 182, 184, 185, 186, 216, 218, 220, 222, 224, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 282, 284, 296, or 300.

[0286] In an even more preferred embodiment, the antigen binding protein is selected from SEQ ID NOs: 100, 103, 105, 151, 156, 158, 166, 167, 175, 178, 180, 183, 193, 285, 291, 295, 299, and 303, more preferably selected from SEQ ID NOs: 100, 103, 105, 167, 183, 193, 285, 291, 295, 299, and 303. and a second polypeptide chain selected from SEQ ID NO: 101, 102, 104, 160, 161, 162, 163, 164, 165, 170, 172, 174, 176, 182, 185, 186, 284, 296, or 300, more preferably selected from SEQ ID NO: 101, 102, 104, 160, 162, 176, 186, 284, 296, or 300.

[0287] In a most preferred embodiment, the antigen binding protein comprises: - a first polypeptide chain of SEQ ID NO: 100 and a second polypeptide chain of SEQ ID NO: 101, or - a first polypeptide chain of SEQ ID NO: 103 and a second polypeptide chain of SEQ ID NO: 102, or - a first polypeptide chain of SEQ ID NO: 105 and a second polypeptide chain of SEQ ID NO: 104, or - a first polypeptide chain of SEQ ID NO: 167 and a second polypeptide chain of SEQ ID NO: 160, or - a first polypeptide chain according to SEQ ID NO: 183 and a second polypeptide chain according to SEQ ID NO: 176, or - a first polypeptide chain according to SEQ ID NO: 193 and a second polypeptide chain according to SEQ ID NO: 186, or - a first polypeptide chain according to SEQ ID NO: 285 and a second polypeptide chain according to SEQ ID NO: 284, or - a first polypeptide chain according to SEQ ID NO: 291 and a second polypeptide chain according to SEQ ID NO: 284, or - a first polypeptide chain according to SEQ ID NO: 295 and a second polypeptide chain according to SEQ ID NO: 186, or - a first polypeptide chain according to SEQ ID NO: 295 and a second polypeptide chain according to SEQ ID NO: 296, or - a first polypeptide chain of SEQ ID NO: 299 and a second polypeptide chain of SEQ ID NO: 162, or - a first polypeptide chain of SEQ ID NO: 285 and a second polypeptide chain of SEQ ID NO: 300, or - a first polypeptide chain according to SEQ ID NO: 303 and a second polypeptide chain according to SEQ ID NO: 162, or - a first polypeptide chain according to SEQ ID NO: 291 and a second polypeptide chain according to SEQ ID NO: 300, or - a first polypeptide chain according to SEQ ID NO: 151 and a second polypeptide chain according to SEQ ID NO: 284, or - a first polypeptide chain according to SEQ ID NO: 156 and a second polypeptide chain according to SEQ ID NO: 162, or - a first polypeptide chain according to SEQ ID NO: 158 and a second polypeptide chain according to SEQ ID NO: 284, or - a first polypeptide chain of SEQ ID NO: 158 and a second polypeptide chain of SEQ ID NO: 300, or - a first polypeptide chain of SEQ ID NO: 303 and a second polypeptide chain of SEQ ID NO: 161, or - a first polypeptide chain of SEQ ID NO: 303 and a second polypeptide chain of SEQ ID NO: 163, or - a first polypeptide chain according to SEQ ID NO: 291 and a second polypeptide chain according to SEQ ID NO: 164, or - a first polypeptide chain according to SEQ ID NO: 291 and a second polypeptide chain according to SEQ ID NO: 170, or - a first polypeptide chain according to SEQ ID NO: 291 and a second polypeptide chain according to SEQ ID NO: 172, or - a first polypeptide chain according to SEQ ID NO: 291 and a second polypeptide chain according to SEQ ID NO: 174, or - a first polypeptide chain according to SEQ ID NO: 166 and a second polypeptide chain according to SEQ ID NO: 170, or - a first polypeptide chain according to SEQ ID NO: 166 and a second polypeptide chain according to SEQ ID NO: 172, or - a first polypeptide chain according to SEQ ID NO: 166 and a second polypeptide chain according to SEQ ID NO: 174, or - a first polypeptide chain according to SEQ ID NO: 291 and a second polypeptide chain according to SEQ ID NO: 182, or - a first polypeptide chain according to SEQ ID NO: 291 and a second polypeptide chain according to SEQ ID NO: 185, or - a first polypeptide chain according to SEQ ID NO: 175 and a second polypeptide chain according to SEQ ID NO: 186, or - a first polypeptide chain according to SEQ ID NO: 178 and a second polypeptide chain according to SEQ ID NO: 186, or - a first polypeptide chain according to SEQ ID NO: 180 and a second polypeptide chain according to SEQ ID NO: 186, especially, - a first polypeptide chain of SEQ ID NO: 100 and a second polypeptide chain of SEQ ID NO: 101, or - a first polypeptide chain of SEQ ID NO: 103 and a second polypeptide chain of SEQ ID NO: 102, or - a first polypeptide chain of SEQ ID NO: 105 and a second polypeptide chain of SEQ ID NO: 104, or - a first polypeptide chain of SEQ ID NO: 158 and a second polypeptide chain of SEQ ID NO: 300, or - a first polypeptide chain of SEQ ID NO: 167 and a second polypeptide chain of SEQ ID NO: 160, or - a first polypeptide chain according to SEQ ID NO: 183 and a second polypeptide chain according to SEQ ID NO: 176, or - a first polypeptide chain according to SEQ ID NO: 193 and a second polypeptide chain according to SEQ ID NO: 186, or - a first polypeptide chain according to SEQ ID NO: 285 and a second polypeptide chain according to SEQ ID NO: 284, or - a first polypeptide chain according to SEQ ID NO: 291 and a second polypeptide chain according to SEQ ID NO: 164, or - a first polypeptide chain according to SEQ ID NO: 291 and a second polypeptide chain according to SEQ ID NO: 284, or - a first polypeptide chain according to SEQ ID NO: 295 and a second polypeptide chain according to SEQ ID NO: 186, or - a first polypeptide chain according to SEQ ID NO: 295 and a second polypeptide chain according to SEQ ID NO: 296, or - a first polypeptide chain of SEQ ID NO: 299 and a second polypeptide chain of SEQ ID NO: 162, or - a first polypeptide chain of SEQ ID NO: 285 and a second polypeptide chain of SEQ ID NO: 300, or - a first polypeptide chain according to SEQ ID NO: 303 and a second polypeptide chain according to SEQ ID NO: 162, or - a first polypeptide chain according to SEQ ID NO: 291 and a second polypeptide chain according to SEQ ID NO: 300, Even more particularly, - a first polypeptide chain of SEQ ID NO: 158 and a second polypeptide chain of SEQ ID NO: 300, or - a first polypeptide chain according to SEQ ID NO: 291, and a second polypeptide chain according to SEQ ID NO: 164.

[0288] Thus, in a most preferred embodiment, the antigen binding protein may comprise a first polypeptide chain of SEQ ID NO:158 and a second polypeptide chain of SEQ ID NO:300.

[0289] scTCR In some embodiments, the first and second polypeptides, such that V A and V B are located on a single polypeptide chain. In such an embodiment, the antigen-binding protein of the present invention can be described as a single-chain TCR. However, depending on the FR sequence and constant domain contained in the antigen-binding protein, it can also be referred to as a single-chain antibody or a single-chain TCR antibody molecule, as described above.

[0290] The scTCR may comprise a variable domain derived from or comprising at least a CDR derived from a first TCR, a variable domain derived from or comprising at least a CDR derived from a second TCR, and a constant domain of the first or second TCR; in other words, a single chain TCR comprises a variable domain derived from one TCR (e.g., an α or γ chain) and an entire chain of another TCR (e.g., a β or δ chain), or vice versa. Furthermore, the scTCR may comprise one or more linkers (preferably peptide linkers) linking the domains together. Such scTCRs of the invention are also provided, which are fused to a human cytokine (e.g., IL-2, IL-7, or IL-15).

[0291] In one embodiment, the single chain TCR is A -L t -V B , V B -L t -V A , V A -C α -L t -V B , V A-C β -L t -V B , V A -L t -V B -C β , V A -L t -V B -C α , V A -C α -L t -V B -C β , V A -C b -L t -V B -C α (Preferably, V A -L t -V B , V B -L t -V A ), wherein V A is a first variable domain as defined herein above, B is the second variable domain as defined herein above, α and C β are the TCR alpha constant domain and the TCR beta constant domain, respectively, which are present or absent, and L t is a linker, present or absent, as defined in the definitions section herein above.

[0292] In a specific embodiment, the antigen-binding protein of the present invention is a scTCR comprising the amino acid sequence of any of SEQ ID NOs: 79-87 or 89-92, or an amino acid sequence that is at least 85% identical to SEQ ID NOs: 79-87 or 89-92, preferably comprising the amino acid sequence of SEQ ID NO: 87.

[0293] scTCR-Fab Single chain TCRs may include additional variable domains linked either to the C-terminus or to the N-terminus, in particular the V L and / or VH may include.

[0294] In one embodiment, such further variable domain is a linker L k In a preferred embodiment, the linker L k is a linker as defined herein above, or a hinge-C of the amino acid sequence of SEQ ID NO: 360 H1 It is an array.

[0295] In certain embodiments, the antigen binding protein of the present invention is A , V B , and V L or V H (Preferably, V H ), and a first polypeptide chain comprising V L and V H The other of (preferably V L and a second polypeptide chain comprising an amino acid sequence of any one of SEQ ID NOs: 94 to 98, or an amino acid sequence at least 85% identical to SEQ ID NOs: 94 to 98. Preferably, the scTCR-Fab comprises a first polypeptide chain comprising or consisting of an amino acid sequence of SEQ ID NO: 93, or an amino acid sequence at least 85% identical to SEQ ID NO: 93.

[0296] full length TCR In another embodiment, the antigen binding protein of the invention comprises two polypeptide chains and is A is contained in the (full-length) TCR α or γ chain; B is comprised in a (full-length) TCR beta or delta chain. In such embodiments, the antigen binding protein preferably has the structure of a conventional αβ TCR or γδ TCR as described above. In one embodiment, the TCR is an αβ TCR and comprises an α chain constant domain (TRAC) sequence according to SEQ ID NO: 361 and a β chain constant domain (TRBC1 or TRBC2) sequence according to SEQ ID NO: 362.

[0297] In one embodiment, the TCR constant domain sequence may be derived from any suitable species (e.g., any mammal, e.g., human, rat, monkey, rabbit, donkey, or mouse, preferably human). In some preferred embodiments, the TCR constant domain sequence may be slightly modified, e.g., by the introduction of heterologous sequences (preferably mouse sequences) that may increase the expression and stability of the TCR. Similarly, additional stabilizing mutations known in the art (e.g., WO 2018 / 104407, PCT / EP2018 / 069151, WO 2011 / 044186, WO 2014 / 018863, e.g., replacement of undesired amino acids in the variable region and / or introduction of disulfide bridges between TCR C domains and removal of unpaired cysteines) may be introduced.

[0298] In particular, the TCR constant domain sequence may be modified by truncation or substitution to delete the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The alpha and / or delta chain constant domain sequences may also be modified by substitution of cysteine ​​residues at Thr48 of TRAC and Ser57 of TRBC1 or TRBC2, which form a disulfide bond between the alpha and beta constant domains of the TCR. TRBC1 or TRBC2 may further comprise a cysteine ​​to alanine mutation at position 75 of the constant domain and an asparagine to aspartic acid mutation at position 89 of the constant domain. The constant domain may also or alternatively comprise further mutations, substitutions, or deletions to the native TRAC and / or TRBC1 / 2 sequence. The terms TRAC and TRBC1 / 2 encompass natural polymorphic variants (eg, N to K at position 4 of TRAC) (Bragado et al Int Immunol. 1994 Feb;6(2):223-30).

[0299] The present invention also includes particles that display antigen-binding proteins (especially TCRs) and the inclusion of said particles in a library of particles. Such particles include, but are not limited to, phage, yeast, ribosomes, or mammalian cells. Methods for producing such particles and libraries are known in the art (see, for example, WO 2004 / 044004; WO 01 / 48145, Chervin et al. (2008) J. Immuno. Methods 339.2: 175-184).

[0300] Nucleic Acids, Vectors, and Recombinant Host Cells In a second aspect, the present invention relates to an isolated nucleic acid comprising or consisting of a sequence encoding an antigen-binding protein of the first aspect of the invention.

[0301] The term "nucleic acid", in the context of the present invention, refers to a single- or double-stranded oligomer or polymer of deoxyribonucleotides, or ribonucleotide bases, or both. A nucleotide monomer is composed of a nucleobase, a five-carbon sugar (such as, but not limited to, ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, nucleic acids are formed by phosphodiester bonds between individual nucleotide monomers. In the context of the present invention, the term nucleic acid includes, but is not limited to, ribonucleic acid (RNA) molecules and deoxyribonucleic acid (DNA) molecules, as well as synthetic forms of nucleic acids containing other bonds [e.g., peptide nucleic acids, as described in Nielsen et al. (Science 254:1497-1500, 1991)]. Typically, nucleic acids are single- or double-stranded molecules, composed of naturally occurring nucleotides. The description of a single strand of a nucleic acid also defines (at least in part) the sequence of the complementary strand. A nucleic acid may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. The illustrated double-stranded nucleic acid molecules may have 3' or 5' overhangs, and thus are not necessarily, but likely to be, completely double-stranded over their entire length. The term nucleic acid includes chromosomes or chromosome segments, vectors (e.g., expression vectors), expression cassettes, naked DNA or RNA polymers, primers, probes, cDNA, genomic DNA, recombinant DNA, cRNA, mRNA, tRNA, microRNA (miRNA), or small interfering RNA (siRNA). A nucleic acid may be, for example, single-stranded, double-stranded, or triple-stranded, and is not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence includes or encodes complementary sequences in addition to any sequence explicitly indicated.

[0302] The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" if it has been purified from other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques.

[0303] The nucleic acid molecules of the present disclosure can be obtained using standard molecular biology techniques, including but not limited to methods of amplification and reverse transcription of RNA. For example, once a DNA fragment encoding a variable chain is obtained, the DNA fragment can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region gene into a full-length gene. In this manipulation, the DNA fragment encoding the variant is operably linked to another DNA molecule, or to a fragment encoding another protein (e.g., a constant region or a flexible linker). The term "operably linked" as used in this context is intended to mean that the two DNA fragments are functionally linked, for example, such that the amino acid sequences encoded by the two DNA fragments remain in frame, or such that a protein is expressed under the control of a desired promoter. The isolated DNA encoding a variable region (e.g., a variable alpha region and / or a variable beta region) can be converted into a full-length gene by operably linking the variable-encoding DNA to another DNA molecule encoding a constant region. For example, the sequences of human constant region genes for TCRs or antibodies are known in the art, and DNA fragments encompassing this region can be obtained by standard PCR amplification.

[0304] Typically, the nucleic acid is a DNA or RNA molecule, which may be contained in a suitable vector.

[0305] The first and second polypeptides described herein can be encoded by one nucleic acid molecule or by two separate nucleic acid molecules.

[0306] Accordingly, also provided herein are expression vectors and host cells for producing the antigen binding proteins, or functional fragments thereof, described herein.

[0307] In a third aspect, the present invention relates to a vector comprising the nucleic acid of the second aspect of the invention.

[0308] The terms "vector," "cloning vector," and "expression vector" refer to a vehicle by which DNA or RNA sequences (e.g., foreign genes) can be introduced into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequences.

[0309] Various expression vectors can be employed to express the polynucleotide that codes for antigen binding protein or its functional fragment.Both viral and non-viral expression vectors can be used to produce the antigen binding protein or its functional fragment described herein in mammalian host cells.Non-viral vectors and systems include multiple plasmids, plasmids, cosmids, episomes, artificial chromosomes, phages, or viral vectors.

[0310] Such vectors can contain regulatory elements (e.g., promoters, enhancers, terminators, and the like) to cause or induce the expression of said polypeptide when administered to a subject.Examples of promoters and enhancers used in animal cell expression vectors include SV40 early promoter and enhancer (Mizukami T. et al. 1987), Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), antibody heavy chain promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983), and the like.

[0311] For example, non-viral vectors useful for expressing the polynucleotides and polypeptides described herein in mammalian (e.g., human or non-human) cells include any suitable vector known in the art for expressing proteins. Other examples of plasmids include replicative plasmids that contain an origin of replication, or integrative plasmids (e.g., pUC, pcDNA, pBR, and the like).

[0312] The term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin, has the ability to be packaged into a viral vector particle, and encodes at least a foreign nucleic acid. The vector and / or particle can be utilized to introduce a nucleic acid of interest into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Useful viral vectors include vectors based on retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, SV40, papilloma viruses, Epstein-Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV) vectors. Recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, and the like. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, U.S. Pat. Nos. 5,882,877, 6,013,516, 4,861,719, 5,278,056, and WO 94 / 19478.

[0313] The nucleic acids encoding the first and second polypeptides described herein may be present in the same vector or in separate vectors. The first and second polypeptides described herein may be present in the same vector or in separate vectors.

[0314] In a fourth aspect, the present invention relates to a host cell comprising an antigen binding protein of the first aspect of the invention, a nucleic acid of the second aspect or a vector of the third aspect, which host cell may be transfected, infected or transformed with a nucleic acid and / or vector according to the invention.

[0315] The term "transformation" refers to the introduction of a "foreign" (i.e., exogenous) gene, DNA, or RNA sequence into a host cell, such that the host cell expresses the introduced gene or sequence to produce a desired substance (typically an antigen binding protein or functional fragment thereof as described herein). A host cell that receives and expresses the introduced DNA or RNA has been "transformed."

[0316] The nucleic acids of the invention can be used to produce recombinant antigen binding proteins of the invention in a suitable expression system. The term "expression system" refers to a host cell and a compatible vector under suitable conditions for the expression of a protein encoded by foreign DNA carried by the vector and introduced into the host cell.

[0317] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria), and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, yeast of the genus Kluyveromyces or Saccharomyces, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, HEK cells, etc.), and primary or established mammalian cell cultures (e.g., those produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662), and the like. In some embodiments, YB2 / 0 cells may be preferred because the ADCC activity of chimeric or humanized antibodies is enhanced when expressed in these cells.

[0318] According to the above, in one embodiment the invention refers to an antigen binding protein of the invention as defined herein above, or a nucleic acid encoding an antigen binding protein of the invention, or a vector encoding an antigen binding protein of the invention, wherein said host cell is preferably a) a lymphocyte, such as a T lymphocyte or a T lymphocyte precursor cell, such as a CD4 or CD8 positive T cell, or b) a cell for recombinant expression, such as a Chinese Hamster Ovary (CHO) cell.

[0319] In particular, for the expression of a part of the antigen-binding protein of the present invention (especially an antigen-binding protein comprising two unlinked polypeptides), the expression vector may be either a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain are present in separate vectors, or a type in which both genes are present in the same vector (tandem type). In terms of ease of constructing an antigen-binding protein expression vector, ease of introduction into animal cells, and balance between the expression levels of antibody H chain and L chain in animal cells, a tandem type humanized antibody expression vector is preferred (Shitara K et al. J Immunol Methods. 1994 Jan. 3; 167(1-2):271-8). Examples of tandem type humanized antibody expression vectors include pKANTEX93 (WO 97 / 10354), pEE18, and the like.

[0320] In one embodiment, such recombinant host cells may be used for the production of at least one antigen binding protein of the invention.

[0321] Pharmaceutical Compositions In a fifth aspect, the present invention relates to a pharmaceutical composition comprising an antigen-binding protein of the invention, a nucleic acid of the invention, a vector of the invention or a host cell of the invention and a pharma- ceutically acceptable carrier.

[0322] It has been shown that the antigen binding protein of the present invention can produce cytotoxicity against tumor cells. Therefore, the antigen binding protein of the present invention is useful for destroying tumor cells in patients. An immune response in a patient can be induced by direct administration of the described antigen binding protein to the patient, ideally in combination with an immunogenicity enhancing agent (i.e., an adjuvant). The immune response generated by such a therapeutic vaccination can be expected to be highly specific against tumor cells, since the peptide SLLQHLIGL (SEQ ID NO: 50) is not presented in the same copy number or in excess on normal tissues, preventing the risk of an undesired autoimmune response against normal tissue cells in the patient.

[0323] The present invention also relates to an antigen-binding protein according to the invention for use as a medicament.The present invention also relates to a pharmaceutical composition according to the invention for use as a medicament.

[0324] The term "pharmaceutical composition" or "therapeutic composition," as used herein, refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a subject.

[0325] In some embodiments, a subject may also be referred to as a patient.

[0326] Such therapeutic or pharmaceutical compositions may contain a therapeutically effective amount of the antigen binding protein of the invention, or an antigen binding protein further comprising a therapeutic agent, in a mixture with a pharma- ceutically or physiologically acceptable formulation selected to be compatible with the mode of administration.

[0327] The antigen binding proteins of the present invention will normally be supplied as part of a sterile pharmaceutical composition which will normally include a pharma- ceutically acceptable carrier and / or a pharma- ceutically acceptable carrier diluent.

[0328] "Pharmaceutically" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to a mammal, particularly a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary.

[0329] A "pharmaceutically acceptable carrier or excipient" may also be referred to as a "pharmaceutically acceptable diluent" or a "pharmaceutically acceptable vehicle" and may include physiologically compatible solvents, fillers, stabilizers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Thus, in one embodiment, the carrier is an aqueous carrier.

[0330] In another aspect, the aqueous carrier may impart improved properties (e.g., improved solubility, efficacy, and / or improved immunotherapy) when combined with the antigen binding proteins described herein.

[0331] The form, route of administration, dosage, and regimen of the pharmaceutical composition will, of course, depend on the condition to be treated, the severity of the disease, the age, weight, and sex of the patient, etc. The pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition may be provided in a unit dosage form, generally provided in a sealed container, and provided as part of a kit. Such a kit will usually (but not necessarily) include instructions for use. Such a kit may also include a plurality of said unit dosage forms.

[0332] Empirical considerations such as biological half-life generally contribute to the determination of dosage. The frequency of administration can be determined and adjusted during the treatment period, and is based on the reduction of the number of cancer cells, the maintenance of the reduction of cancer cells, the reduction of the proliferation of cancer cells, or the killing of cancer cells. Alternatively, sustained continuous release formulations of antigen-binding proteins may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0333] In one embodiment, the dosage of antigen-binding molecule can be empirically determined in an individual receiving one or more doses. The individual is administered increasing doses of antigen-binding protein. To evaluate the effectiveness of antigen-binding protein, markers of cancer cell status can be tracked. This includes direct measurement of cancer cell proliferation and cell death by FACS, other imaging techniques, improved health status as measured by such measurements, or improved quality of life or prolonged survival as measured by accepted tests. It will be clear to those skilled in the art that dosage will vary depending on the individual, stage of disease, and previous and concurrent treatments being used.

[0334] In particular, the pharmaceutical compositions comprise a pharma- ceutically acceptable medium for injectable preparations, which may in particular be isotonic sterile saline (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, and the like, or mixtures of such salts), or dry (in particular lyophilized) compositions, which, if necessary, may constitute the injection solution by addition of sterile water or saline.

[0335] The dose used for administration may be adapted as a function of various parameters, in particular as a function of the mode of administration used, as a function of the pathology involved or as a function of the desired duration of treatment.

[0336] To prepare pharmaceutical compositions, an effective amount of the antigen binding protein of the present invention may be dissolved or dispersed in a pharma- ceutically acceptable carrier or aqueous medium.

[0337] Suitable pharmaceutical forms for injection use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions.In all cases, the formulation must be sterile and must be fluid to the extent that it can be easily injected with a syringe.The formulation must be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0338] A solution of the active compound as a free base or a pharmacologically acceptable salt can be prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0339] The antigen-binding proteins of the present invention may be formulated in the composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) which are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid, etc.) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like). Salts formed with the free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides), and organic bases (e.g., isopropylamine, trimethylamine, glycine, histidine, procaine, and the like).

[0340] Sterile injection solution is prepared by incorporating the required amount of active compound into suitable solvent with various other components as listed above as necessary, followed by filtration sterilization.Generally, dispersion solution is prepared by incorporating various sterilized active ingredients into a sterile medium that contains basic dispersion medium and other necessary components from those listed above.In the case of sterile powder for preparing sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying technology, which obtains powder of active ingredient and any additional desired ingredients from the solution that has been previously sterilized and filtered.

[0341] The preparation of more or more concentrated solutions for direct injection is also contemplated, and it is envisioned that the use of DMSO as a solvent will result in very rapid penetration, delivering high concentrations of active agent to small tumor areas.

[0342] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above, although drug release capsules and the like may also be employed.

[0343] Methods for Producing Antigen-Binding Proteins In a sixth aspect, the present invention provides a method of producing an antigen binding protein of the first aspect of the invention comprising the steps of: (a) providing a host cell; (b) providing a genetic construct comprising a coding sequence encoding the antigen-binding protein; (c) introducing the genetic construct into the host cell; and (d) expressing the genetic construct by the host cell, and optionally (e) selecting cells that express and / or secrete said antigen binding protein. The present invention relates to a method comprising the steps of:

[0344] In one embodiment, the method further comprises the isolation and purification of the antigen-binding protein from the host cell and, if appropriate, the reconstitution of the antigen-binding protein in T cells. The skilled artisan is fully capable of selecting a suitable host cell for expressing the antigen-binding protein.

[0345] The antigen binding proteins of the present invention may be produced by any technique known in the art, including but not limited to any chemical, biological, genetic, or enzymatic techniques, alone or in combination.

[0346] The antigen binding proteins of the invention are suitably separated from the culture medium by antibody purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0347] In one embodiment, recovering the expressed antigen binding protein or polypeptide, as used herein, refers to performing Protein A chromatography, Kappa Select chromatography, and / or size exclusion chromatography, preferably Protein A chromatography and / or size exclusion chromatography, more preferably Protein A chromatography and size exclusion chromatography.

[0348] By knowing the amino acid sequence of the desired sequence, one skilled in the art can produce the antigen-binding protein of the present invention by standard techniques for the production of polypeptides. For example, the protein can be synthesized using known solid-phase methods, in particular using commercially available peptide synthesizers (e.g., those manufactured by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, the antibodies and antigen-binding proteins of the present invention can be produced by recombinant DNA and genetic transfection techniques known in the art (see Morrison SL. et al. (1984) and patent documents US5,202,238; and US5,204, 244). For example, a fragment can be obtained as a DNA expression product after incorporating a DNA sequence encoding the desired (poly)peptide into an expression vector and introducing such a vector into a suitable eukaryotic or prokaryotic host that expresses the desired polypeptide, and the fragment can then be isolated using known techniques.

[0349] In one example (i.e., in the case of TCER®), DNA sequences encoding various combinations of VH and VL, and variable alpha (Valpha) and variable beta (Vbeta), as well as linkers, can be obtained, for example, by gene synthesis. The resulting DNA sequences can be cloned in frame into expression vectors encoding, for example, the hinge region, CH2 domain, and CH3 domain from human IgG4 [Accession#: K01316] and IgG1 [Accession#: P01857], respectively, and further manipulated. According to the methods described by Reiter et al. (Stabilization of the Fv Fragments in Recombinant Immunotoxins by Disulfide Bonds Engineered into Conserved Framework Regions. Biochemistry, 1994, 33, 5451 - 5459), engineering can be performed to incorporate knob-into-hole mutations in the CH3 domain, with or without additional interchain disulfide bond stabilization; N-glycosylation sites in CH2 can be eliminated (e.g., N297Q mutation); and V, V, VV ... L and V H F c Silencing mutations may be introduced or additional disulfide bond stabilization may be introduced.

[0350] Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are known in the art (see, for example, Riechmann L. et al. 1988; Neuberger MS. et al. 1985) and can be readily applied to the production of antigen-binding proteins.

[0351] In one example, vectors for the expression of recombinant antigen-binding proteins of the invention were designed as monocistronic, controlled by, for example, a promoter element from HCMV, a pUC19 derivative. Plasmid DNA was, for example, propagated in E. coli according to standard culture methods and subsequently purified using a commercially available kit (Macherey & Nagel). Purified plasmid DNA was, for example, used for transient transfection of CHO-S cells according to the manufacturer's instructions (ExpiCHO™ System; Thermo Fisher Scientific). Transfected CHO cells were, for example, cultured at 32°C to 37°C for 6 to 14 days and fed 1 to 2 times with ExpiCHO™ Feed solution.

[0352] The conditioned cell supernatant was clarified by filtration (0.22 μm), for example, using a Sartoclear Dynamics® Lab Filter Aid (Sartorius). The bispecific antigen-binding protein was purified, for example, using an Aekta Pure 25 L FPLC system (GE Lifesciences) equipped to perform affinity and size-exclusion chromatography in-line. Affinity chromatography was performed, for example, on a Protein A or L column (GE Lifesciences) according to standard affinity chromatography protocols. For example, size-exclusion chromatography was performed immediately after elution (pH 2.8) from the affinity column, for example, using a Superdex 200 pg 16 / 600 column (GE Lifesciences) according to standard protocols, to obtain highly pure monomeric protein. Protein concentration was determined, for example, on a NanoDrop system (Thermo Scientific) using the calculated extinction coefficient according to the predicted protein sequence. Concentration was adjusted, if necessary, using a Vivaspin device (Sartorius). Finally, the purified molecule was stored, for example, in phosphate buffered saline at a concentration of approximately 1 mg / mL at a temperature of 2-8°C.

[0353] The quality of the purified bispecific antigen-binding proteins was determined by HPLC-SEC, e.g., on a MabPac SEC-1 column (5 μm, 7.8×300 mm) run in 50 mM sodium phosphate (pH 6.8) containing 300 mM NaCl in a Vanquish UHPLC-System.

[0354] Therapeutic Methods and Uses In a seventh aspect, the present invention provides an antigen binding protein of the first aspect, a nucleic acid of the second aspect, a vector of the third aspect, a host cell of the fourth aspect or a pharmaceutical composition of the fifth aspect for use in medicine, in particular for use in the diagnosis, prevention and / or treatment of a proliferative disease. For therapeutic use (i.e. prevention and / or treatment), the antigen binding protein comprises a first antigen binding site (i.e. V) that binds to a PRAME antigenic peptide in complex with MHC. A and V B and a second antigen-binding site (i.e., V L and V H For some bispecific compounds of the invention, the inventors show in the in vitro experimental section the cytotoxic activity of the constructs against PRAME-positive cancer cell lines such as Hs695T and U20S. The inventors further demonstrate that said cytotoxic activity is highly specific and restricted to PRAME-positive cells, since cell lines not presenting peptide PRAME-004 were only slightly lysed by the bispecific antigen-binding protein.

[0355] Thus, the antigen binding proteins of the present invention (especially bispecific antigen binding proteins such as TCER®) may be used to treat cancer. The antigen binding proteins of the present invention may be used for therapeutic purposes in humans and / or non-human mammals. In one embodiment, the antigen binding proteins of the present invention may bind to tumor cells and reduce the proliferation and / or kill tumor cells that present the peptide SLLQHLIGL (SEQ ID NO:50):MHC complex on the cell surface. It is understood that the antigen binding proteins are administered at a concentration that promotes binding under physiological (e.g., in vivo) conditions. In another embodiment, the antigen binding proteins may be used for immunotherapy against tumor cells in various tissues, such as lung, breast, ovary, or kidney. In another embodiment, the antigen binding proteins may bind to tumor cells alone and reduce the proliferation and / or kill the tumor cells.

[0356] Accordingly, the present invention relates to a method of treating or preventing a proliferative disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding protein, nucleic acid or vector, host cell or pharmaceutical composition according to the invention as defined herein above in the "Antigen binding protein", "Nucleic acid", or "Pharmaceutical composition" sections.

[0357] In a particular embodiment, the present invention relates to a method of treating a subject having a proliferative disease, comprising administering to said subject a T cell that expresses an antigen binding protein of the present invention on its cell surface.

[0358] In a further embodiment, the present invention refers to a method of inducing an immune response in a subject having a proliferative disease, comprising administering to said subject a composition comprising T cells expressing an antigen that recognizes a construct of the present invention on their cell surface.

[0359] In one embodiment, the immune response referred to in the method is a cytotoxic T cell response.

[0360] In one embodiment the antigen binding protein of the invention, the nucleic acid of the invention or the vector of the invention, the host cell of the invention or the pharmaceutical composition of the invention is for use in the diagnosis, prevention and / or treatment of a proliferative disease.

[0361] The present invention further refers to the use of the antigen binding protein, the nucleic acid or vector, the host cell or the pharmaceutical composition according to the invention for the preparation of a medicament for treating or preventing a proliferative disease or disorder in a subject.

[0362] In one embodiment, the present invention refers to a method of eliciting an immune response in a patient having a cancer that presents a peptide comprising or consisting of the amino acid sequence of SLLQHLIGL (SEQ ID NO:50) in complex with an MHC protein, comprising administering to the patient an antigen binding protein of the present disclosure, wherein the cancer is selected from the group of cancers consisting of acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, melanoma, amelanotic melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, osteosarcoma, chronic lymphocytic leukemia, colorectal cancer, and synovial sarcoma.

[0363] In one embodiment, the invention refers to the use of an antigen binding protein, a nucleic acid or a vector, a host cell or a pharmaceutical composition according to the invention for treating or preventing a disease or disorder in a subject.

[0364] The terms "subject" or "individual" are used interchangeably and can be, for example, a human or non-human mammal, preferably a human.

[0365] In the context of the present invention, the term "treat" or "treatment" refers to therapeutic use (i.e., for a subject having a given disease) and means reversing, alleviating, inhibiting the progression of one or more symptoms of such a disorder or condition. Thus, treatment refers not only to treatment in which the disease is completely cured, but also to treatment that slows the progression of the disease and / or extends the survival of the subject.

[0366] "Preventing" refers to prophylactic use (i.e., in a subject susceptible to developing a given disease).

[0367] In one embodiment, a "disease" or "disorder" refers to any condition that would benefit from treatment with an antigen binding protein of the invention. In one embodiment, this includes chronic and acute disorders or diseases, including pathological conditions that predispose a subject to the disorder in question. The term "in need of treatment" refers to subjects already with the disorder, as well as subjects in which the disorder is to be prevented.

[0368] In certain embodiments, the antigen binding proteins of the present invention are bispecific, more specifically, TCER® as described herein.

[0369] "Proliferative disorders," such as cancer, involve unregulated and / or inappropriate proliferation of cells.

[0370] Thus, in one embodiment, the proliferative disorder is cancer.

[0371] In a further embodiment, the cancer is one in which the PRAME antigen is overexpressed, mutated and / or in which PRAME-derived tumor-associated antigens are presented in association with MHC.

[0372] Thus, particularly preferred cancers are PRAME-positive cancers.

[0373] In the context of the present invention, a cancer is considered "PRAME positive" if the relevant peptide (e.g., PRAME-004 peptide) is present in more than 98% of all cancers according to guidelines by NCI. For all other indications listed here, a biopsy can be performed as is standard in the treatment of these cancers, and peptides can be identified according to XPresident® and related methods [WO 03 / 100432; WO 2005 / 076009; WO 2011 / 128448; WO 2016 / 107740, U.S. Patent Nos. 7,811,828, 9,791,444, and U.S. Patent Publication No. 2016 / 0187351 (the contents of each of which are incorporated herein by reference in their entirety)]. In one embodiment, for example, cancer is easily assayed (i.e., diagnosed) by using the antigen binding protein of the present invention. Methods of using antigen binding proteins to identify cancers that express an antigen are known to those of skill in the art. It should be understood that the terms "cancer" and "carcinoma" are not used interchangeably herein, as carcinoma is a specific type of cancer that appears in the skin or tissues that underpin or cover the organs of the body.

[0374] In one embodiment, the cancer that is PRAME "positive" (i.e., presents the target peptide) is selected from the group consisting of acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, melanoma, amelanotic melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous cell non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, osteosarcoma, chronic lymphocytic leukemia, colorectal cancer, and synovial sarcoma, and is preferably selected from the group consisting of breast cancer, cholangiocarcinoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, squamous cell non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, and synovial sarcoma.

[0375] In one embodiment, when the cancer is a cancer in which PRAME antigen is overexpressed, mutated, and / or tumor-associated antigen from PRAEM is presented in association with MHC, it is easily assayed, for example, by using the antigen binding protein of the present invention. Methods of using antigen binding protein to identify cancer expressing antigen are known to those skilled in the art.

[0376] A textbook providing guidelines for cancer therapy is Cancer, Principles and Practice of Oncology, 4th Edition, DeVita et al, Eds. JB Lippincott Co., Philadelphia, Pa. (1993). As recognized in the relevant field, appropriate therapeutic approaches are selected according to the particular type of cancer and other factors, such as the patient's general condition. The antigen binding proteins of the present invention may be used by themselves or may be added to therapeutic regimens using other anti-neoplastic agents in the treatment of cancer patients.

[0377] Thus, in some embodiments, for example, the antigen binding protein may be administered concurrently with, prior to, or following a variety of drugs and treatments commonly employed in cancer treatment, such as chemotherapeutic agents, non-chemotherapeutic agents, antineoplastic agents, and / or radiation.

[0378] In one embodiment, the invention relates to a method of treating a patient having a cancer that presents a peptide comprising or consisting of the amino acid sequence of SLLQHLIGL (SEQ ID NO:50) in complex with an MHC protein comprising administering to the patient an antigen binding protein of the disclosure, wherein the cancer is selected from the group of cancers consisting of acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, melanoma, amelanotic melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, osteosarcoma, chronic lymphocytic leukemia, colorectal cancer, and synovial sarcoma.

[0379] "Diagnosis" herein refers to a medical diagnosis and refers to determining which disease or condition explains the symptoms and signs in a human.

[0380] A "therapeutically effective amount" of an antigen binding protein or pharmaceutical composition thereof means an amount of antigen binding protein sufficient to treat said proliferative disease at a reasonable benefit / risk ratio applicable to any medical treatment. However, it will be understood that the total daily usage of the antigen binding protein, nucleic acid or vector, host cell or pharmaceutical composition of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose for any particular patient will depend on a variety of factors, including: the disorder being treated and the severity of the disorder; the activity of the specific antigen binding protein employed; the specific composition employed; the age, weight, general health, sex, and diet of the patient; the administration time, route of administration, and excretion rate of the specific polypeptide employed; the duration of treatment; drugs used in combination or simultaneously with the specific polypeptide employed; and similar factors known in the medical arts. For example, it is known to those skilled in the art to start the dose of the compound at a level lower than that required to obtain the desired therapeutic effect, and gradually increase the dosage until the desired effect is obtained.

[0381] In one embodiment, the effectiveness of treatment with an antigen binding protein of the invention is assayed in vivo, e.g., in a mouse model of cancer, e.g., by measuring the change in tumor volume between treated and control groups.

[0382] The pharmaceutical compositions, vectors, nucleic acids, and cells of the invention may be provided in a substantially pure form, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure form.

[0383] The antigen-binding protein of the invention, the nucleic acid of the invention or the vector of the invention, the host cell of the invention or the pharmaceutical composition of the invention may be administered by any feasible method.

[0384] As disclosed herein, in some embodiments, the host cell as defined herein above is used in the medical applications or treatment methods described herein. In the same embodiment, the host cell is preferably a) a lymphocyte, such as a T lymphocyte or a T lymphocyte precursor, such as a CD4 or CD8 positive T cell, and most preferably a T cell.

[0385] Thus, the host cells (preferably T cells) of the present invention can be used as an active ingredient of a therapeutic composition.Therefore, the present invention also provides a method for killing target cells in a patient in which the target cells abnormally express a polypeptide comprising the peptide SLLQHLIGL (SEQ ID NO: 50), comprising administering to the patient an effective number of host cells (preferably T cells).In connection with this method, the host cells preferably induce an immune response when administered to a subject.

[0386] In certain embodiments, a TCR-induced response, or T cell response, can refer to the augmentation and activation of effector functions induced by a peptide, such as SLLQHLIGL (SEQ ID NO:50), in vitro or in vivo. In the case of MHC class I-restricted cytotoxic T cells, for example, the effector functions can be lysis of peptide-pulsed, peptide precursor-pulsed, or naturally peptide-presenting target cells, peptide-induced secretion of cytokines (preferably interferon-gamma, TNF-alpha, or IL-2), peptide-induced secretion of effector molecules (e.g., granzymes or perforin), or degranulation.

[0387] Thus, a host cell as defined herein above may be derived from the subject (autologous) or from another individual, preferably said other individual being healthy.

[0388] By "healthy" it is meant that the subject is in generally good health, preferably has a competent immune system, and more preferably is free of any disease that can be easily tested for and detected.

[0389] In a specific example, the host cell is a T cell.Thus, in the context of the present invention, when T cells as defined herein above are used as a medicine, the T cells are usually harvested from the cell by apheresis.The T cells are then genetically engineered to express the antigen-binding protein of the present invention on their cell surface, and the genetically engineered T cells are then expanded and then reinfused into the subject.In this example, the antigen-binding protein is preferably a TCR.

[0390] In another approach, the host cell may be a stem cell, such as a mesenchymal stem cell, engineered to express an antigen binding protein of the invention, in this example, the antigen binding protein is a soluble protein, such as an antibody, scTCR, or diabody as defined herein above.

[0391] Thus, host cells are transfected, infected or transformed with the nucleic acids and / or vectors of the invention, as described herein above in the "Nucleic Acids, Vectors and Recombinant Host Cells" section.

[0392] When a host cell is transfected to express an antigen binding protein of the invention, the cell preferably contains an expression vector capable of expressing the antigen binding protein, and the host cell may then be referred to as an activated host cell.

[0393] This so-called adoptive transfer of T cells protocol is known in the art, and reviews can be found in Gattioni et al. and Morgan et al. [Gattinoni, L. et al., Nat. Rev. Immunol. 6 (2006): 383-393; Morgan, RA et al., Science 314 (2006): 126-129].

[0394] Many other methods can be used to generate T cells in vitro. For example, autologous tumor-infiltrating lymphocytes can be used in generating CTL. Plebanski et al. (Plebanski, M. et al., Eur.J Immunol 25 (1995): 1783-1787) used autologous peripheral blood lymphocytes (PLB) in preparing T cells. Similarly, B cells can be used in producing autologous T cells.

[0395] Allogeneic cells may also be used in the preparation of T cells, methods of which are described in detail in US Pat. No. 6,805,861, which is incorporated herein by reference.

[0396] Host cells expressing the antigen binding protein of the invention against peptide SLLQHLIGL (SEQ ID NO: 50) are useful for therapy. Thus, a further aspect of the present invention provides an activated host cell obtainable by the above-mentioned method of the invention.

[0397] The activated host cells produced by the above method are capable of specifically recognizing cells which aberrantly express a polypeptide comprising the peptide SLLQHLIGL (SEQ ID NO:50).

[0398] By "aberrantly expressed" we also mean that the polypeptide is overexpressed compared to expression levels in normal (healthy) tissue, or that the gene is silent in the tissue from which the tumor originates, but is expressed in the tumor. By "overexpressed" we mean that the polypeptide is present at a level at least 1.2-fold that present in normal tissue, preferably at least 2-fold, and more preferably at least 5-fold or 10-fold that present in normal tissue.

[0399] In one embodiment, the host cell (particularly the T cell) recognizes the cell by interacting with (e.g. binding to) the PRAME-004 complex via its antigen binding protein (particularly its TCR). The host cell is useful in a method of killing target cells in a patient in which the target cells aberrantly express a polypeptide comprising the peptide SLLQHLIGL (SEQ ID NO:50), to which an effective number of activated host cells are administered. The T cells administered to the patient may be derived from the patient and activated as described above (i.e., are autologous T cells). Alternatively, the T cells are not derived from the patient, but from another individual. Of course, this is preferred when the individual is a healthy individual. By "healthy individual" we mean that the individual is generally in good health, preferably has a competent immune system, and more preferably is not suffering from any disease that can be easily tested for and detected.

[0400] In vivo, target cells for the CD8 positive T cells of the present invention may be cells of the tumor (which may express MHC class II) and / or stromal cells surrounding the tumor (tumor cells) (which may also express MHC class II) [Dengjel, J. et al., Clin Cancer Res 12 (2006): 4163-4170].

[0401] Diagnostic Uses PRAME is expressed on the surface of PRAME-expressing cancers as defined herein above. The antigen PRAME constitutes a cancer marker and therefore has the potential to be used to indicate the effectiveness of anti-cancer treatment or to detect disease recurrence.

[0402] Thus, in another aspect, the present invention provides an antigen binding protein of the first aspect, a nucleic acid of the second aspect, a vector of the third aspect, a host cell of the fourth aspect or a pharmaceutical composition of the fifth aspect for use as a diagnostic, in particular for use as an in vivo diagnostic, in a preferred embodiment, the diagnostic is for the diagnosis of a proliferative disease. In a more preferred embodiment, the diagnostic agent is for the diagnosis of a cancer presenting a peptide comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 50) in complex with an MHC protein, preferably said cancer is selected from the group of cancers consisting of acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, melanoma, amelanotic melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous cell non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, osteosarcoma, chronic lymphocytic leukemia, colorectal cancer, and synovial sarcoma.

[0403] Those skilled in the art will appreciate that for diagnostic purposes, the antigen binding protein may be A and V B but preferably V L and V H I am aware that it does not include

[0404] In certain embodiments, the antigen binding proteins of the invention are used as components of therapeutically relevant assays targeting tumors expressing PRAME to determine the sensitivity of a patient to a therapeutic agent, to monitor the efficacy of an anti-cancer treatment, or to detect the recurrence of disease following treatment. A Domains and V BAntigen binding proteins containing the domains are used as components of diagnostic assays and bispecific antigen binding proteins are used as components of therapeutic agents.

[0405] A further object of the invention therefore relates to an antigen-binding protein according to the invention for use in detecting PRAME expression in a subject in vivo or for use in detecting PRAME expression ex vivo or in vitro in a biological sample of a subject. Said detection may in particular be intended to: a) diagnosing the presence of cancer in a subject; or b) To determine the sensitivity of patients with cancer to therapeutic agents that target PRAME; or c) Detecting the expression of the surface protein PRAME on tumor cells, in particular for bispecific treatment according to the invention, to monitor the effectiveness of anti-PRAME cancer treatment or to detect cancer recurrence after anti-PRAME cancer treatment.

[0406] In certain embodiments, the antigen binding proteins are intended for in vitro or ex vivo use.

[0407] kit Finally, the present invention also provides kits comprising at least one antigen binding protein of the present invention.

[0408] In one embodiment, the kit comprises: a) at least one antigen binding protein of the invention as defined in the "Antigen binding proteins" section herein above, b) Packaging materials, as appropriate; and c) optionally, a label or packaging insert included in said packaging material indicating that said antigen binding protein is effective for treating or for use in treating cancer. Includes.

[0409] In a related embodiment, at least one antigen binding protein of the present invention is contained in single and / or multi-chambered pre-filled syringes (eg, liquid syringes and lyosyringes).

[0410] In one embodiment, the invention encompasses a kit for producing a single dosage unit.

[0411] Thus, in one embodiment the at least one antigen binding protein of the invention referred to under a) of the kit of the invention is a dried antigen binding protein of the invention in a first container, which kit then further comprises a second container which contains an aqueous formulation.

[0412] Thus, in one embodiment, the kit comprises: a) a first container containing at least one dried antigen binding protein of the invention as defined herein above in the "Antigen binding proteins" section; b) a second container containing the aqueous formulation; c) Packaging materials, as appropriate; and d) optionally, a label or packaging insert included in said packaging material indicating that said antigen binding protein is effective for treating or for use in treating cancer. Includes.

[0413] Aqueous formulations are typically aqueous solutions that include a pharma- ceutically acceptable carrier as defined in the "Pharmaceutical Compositions" section herein above.

[0414] In related embodiments, the "first container" and "second" container refer to the chambers of a multi-chambered pre-filled syringe (eg, a lyophilization syringe).

[0415] Throughout this application, the term "and / or" is a grammatical conjunction that should be interpreted to include one or more of the cases in which it is connected. For example, the phrase "such native sequence proteins may be prepared using standard recombinant and / or synthetic methods" indicates that the native sequence protein may be prepared using standard recombinant and synthetic methods, or that the native sequence protein may be prepared using standard recombinant methods, or that the native sequence protein may be prepared using synthetic methods.

[0416] Moreover, throughout this application, the term "comprising" should be interpreted to encompass not only all features specifically mentioned, but also any additional, unspecified ones. As used herein, use of the term "comprising" also discloses embodiments in which no features are present (i.e., "consisting of") other features than those specifically mentioned.

[0417] Moreover, the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0418] The present invention will now be described in more detail with reference to the following figures and examples. All publications and patents cited herein are incorporated herein by reference. The present invention is shown and explained in detail in the following description, but the examples should be considered as illustrative and not limiting. EXAMPLES

[0419] [Example 1]

[0420] Single-chain TCR (scTCR format) [Example 1.1] Generation of stable scTCRs In the present invention, TCR R11P3D3 (SEQ ID NO: 1 and 2, full length) was converted into a single chain TCR construct (scTCR R11P3D3, SEQ ID NO: 5) using variable alpha (SEQ ID NO: 3) and beta (SEQ ID NO: 4) domains and an appropriate glycine-serine linker sequence (SEQ ID NO: 61). For TCR maturation by yeast surface display, DNA of the corresponding sequences was synthesized and transformed into Saccharomyces cerevisiae EBY100 (MATa AGA1::GAL1-AGA1::URA3 ura3-52 trp1 leu2-delta200 his3-delta200 pep4::HIS3 prbd1.6R can1 GAL) (ATCC® MYA-4941™) together with a pCT302-based yeast display vector (Boder and Wittrup, Methods Enzymol. 2000;328:430-44;). The fusion protein (SEQ ID NO: 325) obtained after homologous recombination in yeast contains the leader peptide (Boder and Wittrup, Nat Biotechnol. 1997 Jun;15(6):553-7) at the N-terminus of the Aga2p protein (SEQ ID NO: 88) and the protein of interest (i.e. scTCR R11P3D3 (SEQ ID NO: 5) or a mutant thereof and additional peptide tags [FLAG and Myc (SEQ ID NO: 99 and 288)] to determine the expression level of the fusion protein. Libraries of scTCR mutants were generated by PCR using degenerate primers and transformation of yeast cells was carried out as described in WO 2018 / 091396, with 10 nucleotides per library. 9 Yeast clones were obtained.

[0421] The selection process for yeast clones carrying mutant scTCR variants with improved binding to PRAME-004 in the context of HLA-A*02 was performed essentially as described by Smith et al. (Methods Mol Biol. 2015;1319:95-141). Expression determined by Myc-tag-FITC staining and, in particular, functional binding by HLA-A*02 / PRAME-004 tetramer staining were applied to select the most promising candidates (Figure 1). scTCR conversion by yeast surface display revealed nine framework mutations combined with three single-point CDR mutations, resulting in a stabilized scTCR R11P3D3SD (SEQ ID NO: 6) that showed improved expression and HLA-A*02 / PRAME-004 tetramer binding. [Example 1.2]

[0422] Affinity maturation, binding motif and specificity assessment of stabilized scTCRs To generate scTCR molecules with higher binding affinity to HLA-A*02 / PRAME-004, all CDRs were individually matured using the previously identified stabilized scTCR R11P3D3SD (SEQ ID NO: 6). The CDR residues were randomized using degenerate DNA oligo primers essentially as previously described (Smith et al., Methods Mol Biol. 2015;1319:95-141). The resulting DNA library was transformed as described in Example 1.

[0423] To select specific R11P3D3SD scTCR variants with enhanced affinity, decreasing concentrations of HLA-A*02 / PRAME-004 tetramers or monomers were used in each selection round. After four selection rounds, single scTCR clones were isolated and sequenced, yielding a number of affinity matured CDR sequences. As exemplarily shown for scTCRs with mature CDRa1 sequences (SEQ ID NOs: 16-28, FIG. 2), a strong improvement in binding of HLA-A*02 / PRAME-004 monomers could also be demonstrated for scTCRs with mature CDRa2 and CDRb2 (SEQ ID NOs: 29-32 and 35-45, Table 3). The selectivity of HLA-A*02 / PRAME-004 binding was retained during maturation, as evidenced by the low binding of scTCRs to mixes of HLA-A*02 tetramers containing peptides with a high degree of sequence similarity to the PRAME-004 peptide (SEQ ID NO: 50) (similar peptides or SimPep). All selected scTCR mutants showed substantial staining with HLA-A*02 / PRAME-004 monomer at a concentration of 10 nM, whereas the non-mature stabilized scTCR R11P3D3SD as a reference showed no staining (Figure 2 and Table 3). Furthermore, binding of mature scTCR to a mix of similar peptides applied in a high avidity format of HLA-A*02 tetramer at a concentration of 10 nM was either not detectable or showed only a low signal compared to HLA-A*02 / PRAME-004 monomer binding, confirming the ability of scTCR mature mutants to bind PRAME-004 target peptide with high specificity.

[0424] [Table 3]

[0425] To further increase the affinity of the scTCR clones, the mature CDRs identified in the CDR libraries described above were systematically combined into one DNA library and transformed into Saccharomyces cerevisiae EBY100 as described in Example 1.1. This library was selected using HLA-A*02 / PRAME-004 monomers, and scTCRs from single yeast clones were sequenced and analyzed for binding to HLA-A*02 monomers containing either the PRAME-004 target peptide or one peptide from a group of 26 peptides sharing sequence similarity with PRAME-004 (similar peptides) (SEQ ID NOs: 51-60, 62-69, and 71-78). All selected high affinity scTCR variants (SEQ ID NOs: 79-87, and 89-92) had binding EC values ​​in the low nanomolar or sub-nanomolar range as calculated by nonlinear four-point curve fitting. 50 values, and bound strongly to HLA-A*02 / PRAME-004 monomer (Table 4). With the exception of SMARCD1-001 (SEQ ID NO: 76), which induced a binding signal slightly above background (Figure 4), none of the scTCR variants (SEQ ID NO: 79-87 and 89-92) showed binding above background levels to any of the analogous peptides (SEQ ID NO: 51-60, 62-69, and 71-78) in the context of HLA-A*02 monomer applied at a concentration of 100 nM (Figure 3, Figure 4, Table 4). The presented data confirm the high binding specificity of the scTCR variants combined with CDR mutations, which binding characteristics were superior to the reference scTCR (R16P1C10_CDR6_scTCR, SEQ ID NO: 357), which showed strong binding to IFT17-003 (SEQ ID NO: 60) at a level indistinguishable from PRAME-004 binding (Figure 4).

[0426] The set of high affinity scTCRs selected from yeast surface display was further examined for functional epitopes on the target peptides relevant for HLA-A*02 presentation, referred to as binding motifs. This was addressed by evaluation of binding of scTCR-bearing yeast cells to single alanine substitutions at positions 1, 3, 4, 5, 6, 7, and 8 of the PRAME-004 target peptide (SEQ ID NOs: 318-324) and the respective PRAME-004 peptide variants relevant for HLA-A*02. Four concentrations (10 nM, 3 nM, 1 nM, 0.3 nM) of HLA-A*02 monomer and PRAME-004 or the respective alanine-substituted peptides were used to stain high affinity scTCR-bearing yeast cells, revealing extensive binding motifs for all scTCR variants that are strongly recognized at positions 3, 5, and 7, as evidenced by the lack of staining signal at all monomer concentrations tested. For positions 6 and 8 of the PRAME-004 peptide, a contribution to the binding motif could be assumed, since alanine replacement at these positions significantly reduced the staining signal, even if observed at lower stringency compared to positions 3, 5, and 7. For positions 1 and 4 of the PRAME-004 peptide, a small or no contribution to the binding motif could be determined, since alanine replacement resulted in staining intensities almost equivalent to those observed with the PRAME-004 target peptide (Figure 5 and Table 4).

[0427] For further analysis, five scTCR clones R11P3D3SDA7_A02_scTCR (sequence number 79), R11P3D3SDA7_A09_scTCR (sequence number 82), R11P3D3SDA7_A10_scTCR (sequence number 83), R11P3D3SDA7_B03_scTCR (sequence number 85), and R11P3D3SDA7_B06_scTCR (sequence number 87) were converted to a scTCR-Fab bispecific format for further protein characterization (see Examples below).

[0428] [Table 4] [Example 2]

[0429] Production and characterization of soluble scTCR-Fab molecules A TCR consisting of V alpha and V beta domains was designed, produced and tested in single chain (scTCR) format conjugated with the Fab fragment of the humanized UCHT1 antibody (Table 5 and Table 18). The vector for expression of the recombinant protein was designed as monocistronic controlled by the HCMV-derived promoter element pUC19 derivative. Plasmid DNA was amplified in E. coli according to standard culture methods and subsequently purified using a commercial kit (Macherey & Nagel). The purified plasmid DNA was used for transient transfection of CHO cells. The transfected CHO cells were cultured at 32°C to 37°C for 10 to 11 days.

[0430] The conditioned cell supernatant was clarified by filtration (0.22 μm) using a Sartoclear Dynamics® Lab Filter Aid (Sartorius). The bispecific molecules were purified using an Aekta Pure 25 L FPLC system (GE Lifesciences) equipped to perform affinity and size-exclusion chromatography in-line. Affinity chromatography was performed on a Protein L column (GE Lifesciences) following standard affinity chromatography protocols. Size-exclusion chromatography was performed immediately after elution (pH 2.8) from the affinity column using a Superdex 200pg 16 / 600 column (GE Lifesciences) following standard protocols to obtain highly pure monomeric proteins. Protein concentrations were determined with a NanoDrop system (Thermo Scientific) using the calculated extinction coefficient according to the predicted protein sequence. Concentrations were adjusted, if necessary, using a Vivaspin device (Sartorius). Finally, the purified molecules were stored in phosphate-buffered saline at a concentration of approximately 1 mg / mL at a temperature of 2-8 °C. The yield of the final product was calculated after completion of purification and formulation.

[0431] The quality of the purified bispecific molecules was determined by HPLC-SEC on a MabPac SEC-1 column (5 μm, 4×300 mm) run in 50 mM sodium phosphate (pH 6.8) containing 300 mM NaCl in a Vanquish uHPLC-System.

[0432] Stress stability studies were performed by incubation of the molecules formulated in PBS for up to 2 weeks at 40° C. Integrity, aggregate content, and monomer recovery were analyzed by HPLC-SEC analysis as described above.

[0433] [Table 5]

[0434] The scTCR-Fab molecules TPP-70 to TPP-74 were analyzed for their binding affinity to HLA-A*02 monomers containing the PRAME-004 target peptide by biolayer interferometry. Measurements were performed on an Octet RED384 system using the settings recommended by the manufacturer. The assays were performed with a sensor offset of 3 mm and an acquisition rate of 5 Hz. Binding kinetics were measured at 30 °C and 1000 rpm shaking speed using PBS, 0.05% Tween-20, 0.1% BSA as buffer. Serial dilutions of scTCR-Fab molecules were analyzed after loading His-tagged HLA-A*02 / PRAME-004 monomers onto the HIS1K biosensor. Data evaluation was performed using Octet Data Analysis HT Software. Strong binding affinities were observed with K values ​​ranging from 4 nM to 12 nM. D Values ​​were determined for the scTCR-Fab molecules (Table 4). Additionally, the scTCR-Fab variants were screened for binding to 14 similar peptides (SEQ ID NOs: 187, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, and 212). Screening with similar peptides was performed by biolayer interferometry essentially as described above, analyzing high concentrations of scTCR-Fab molecules, as high as 1 μM, to allow detection of weak binding signals. None of the mature scTCR variants bound to any of the similar peptides tested (Figure 6). The scTCR from TPP-74 was used for the generation of bispecific molecules in alternative formats, such as the TCER® format. [Example 3]

[0435] T-cell Engaging Receptor [TCER®] Format [Example 3.1] Production and characterization of soluble scTCRs in bispecific TCER® format For the construction of TCER® molecules, DNA sequences encoding VH and VL from either hUCHT1 (Var17), a novel humanized version of the anti-CD3 antibody UCHT1, BMA031 (V36), a humanized antibody that binds to the TCR / CD3 complex, or the anti-CD3 antibody ID4, as well as sequences encoding Valpha and Vbeta and their respective linkers, were obtained by gene synthesis. The resulting DNA sequences were cloned in frame into an expression vector encoding the hinge region, CH2 and CH3 domains from human IgG1 [Accession#: P01857]. The CH2 and CH3 domains were engineered to contain various mutations (including the N297Q mutation) to inhibit binding to Fc gamma receptors and complement, and to incorporate a knob-into-hole structure in the CH3 domain in which the interchain disulfide bonds are further stabilized. Production, purification, and characterization of TCER® molecules (Table 6, Table 18) were performed as outlined in Example 2.

[0436] [Table 6]

[0437] The functionality of TCER® molecules for killing HLA-A*02 positive tumor cell lines (e.g., Hs695T) that present the PRAME-004 targeting peptide on the cell surface was evaluated in an LDH release assay. In addition, HLA-A*02 positive but PRAME-004 negative tumor cell lines (e.g., T98G) were evaluated to characterize the non-specific or off-target activity of TCER® variants. Tumor cell lines were co-cultured with PBMCs from healthy HLA-A*02 positive donors at a ratio of 1:10 with increasing TCER® concentrations. TCER®-induced cytotoxicity was quantified after 48 hours of co-culture by measuring released LDH. EC of dose-response curves 50Values ​​were calculated using non-linear 4-point curve fitting. Results (Table 6, Table 18) representative of the three TCER® molecules are shown in Figures 7 and 8. The results demonstrate that all three TCER® molecules utilizing different recruiting antibody domains are functional and induce T cell-mediated cytotoxicity in a manner strictly dependent on PRAME-004. [Example 3.2]

[0438] Slot I TCER® molecules were constructed utilizing VH and VL domains from hUCHT1 (Var17) or BMA031 (V36), and the Valpha and Vbeta domains described above (Example 3.1). TCER® molecules (Table 7, Table 18) were produced, purified, and characterized as outlined in Example 2.

[0439] [Table 7]

[0440] TCER® Slot I mutants TPP-106, TPP-108 to TPP-129 were analyzed for binding affinity to the target peptide-HLA complex (HLA-A*02 / PRAME-004) by biolayer interferometry. Measurements were performed on an Octet RED384 system as described above. Strong binding affinities were observed with K values ​​ranging from 3 nM to 10 nM. D The binding affinity was determined by K values ​​(Table 8). These data show the further affinity-improving effect of the TCR mutations bA84D and aN114Y, whereas the mutations bT115L / K, bL11E, bP46M, bQ48R, and aN20K do not appear to affect the binding affinity. Furthermore, the binding affinity was determined for three selected similar peptides that serve as potential off-target peptides associated with HLA-A*02, and compared to the target peptide-HLA binding, K D The strongest TCER® binding to similar peptides was observed for GIMAP8-001, with a K DThe window ranged from 26x to 168x. K >25x D The window already provides a good therapeutic window.

[0441] [Table 8] [Example 3.3]

[0442] Slot II Additional TCER® molecules were constructed utilizing the VH and VL domains from BMA031 (V36) or ID4, as well as the Valpha and Vbeta domains described above (Example 3.1). The production, purification, and characterization of each TCER® molecule (Table 9, Table 18) was performed as outlined in Example 2, whereby all ID4-based molecules were purified using a MAbSelect SuRE column (GE Lifesciences).

[0443] [Table 9]

[0444] TCER® Slot II variants TPP-207-TPP-222 and TPP-227-TPP-230 were analyzed for their binding affinity to the target peptide-HLA complex (HLA-A*02 / PRAME-004) by biolayer interferometry. Measurements were performed on the Octet RED384 system described above. Strong binding affinities were observed with K values ​​ranging from 1 nM to 7 nM. D The same TCR variants (i.e., the same V A and V BAs observed for the TCER® molecules in slot I (Example 3.2), the affinity improving effect of the TCR mutations bA84D and aN114Y could also be confirmed for the TCER® mutants generated in slot II, whereas no effect on affinity was found for the mutations bT115L / K, bP46M, bQ48R, aN20K.

[0445] TCR binding motifs were evaluated for selected TCER® molecules. To determine the binding motifs, affinities were measured for the target peptide-HLA complex (HLA-A*02 / PRAME-004) and for complexes with PRAME-004 variants carrying alanine substitutions at peptide positions 1, 3, 4, 5, 6, 7, or 8. Affinity measurements were performed on the Octet RED384 or HTX system described above. A position of PRAME-004 was considered to be part of the TCR binding motif if a 2-fold or greater decrease in binding affinity or signal (measured at the highest concentration analyzed) was detected for the alanine-substituted peptide variant. All TCER® variants showed a broad binding motif recognizing at least four peptide positions of PRAME-004 (Table 10).

[0446] [Table 10] [Example 3.4]

[0447] Slot IIa Based on the data generated for previous TCER® variants (Example 3.3), new variants were generated in which selected TCR amino acid positions were systematically substituted, where a positive effect on the protein properties or binding properties could be detected in previous experiments. The production, purification and characterization of each TCER® molecule (Table 11 and Table 18) was carried out as outlined in Example 3.3. A summary of the productivity and stress stability data is summarized in Table 11.

[0448] [Table 11]

[0449] TCER® Slot IIa variants TPP-235 to -250, -252 to -268, -270, -277, -279 were analyzed for binding affinity to the target peptide-HLA complex (HLA-A*02 / PRAME-004) by biolayer interferometry. Measurements were performed on the Octet RED384 or HTX systems described above. Strong binding affinities were observed with K in the range of 2 nM to 15 nM. D The affinity of each mutant was found by the amino acid substitution (Table 12). At position bA84, the amino acid substitutions revealed that bA84D was the most favorable substitution. At position aN114, alternative amino acid substitutions (e.g., A, H, I, and L) were found with affinity equivalent to aN114Y. Alternatives to bT115K / with comparable affinity were identified, including R, A, I, and V. The introduction of the mutation bA110S slightly reduced the affinity of each mutant.

[0450] Binding motifs were evaluated for selected TCER® variants. To determine the binding motif, affinity was measured for the target peptide-HLA complex (HLA-A*02 / PRAME-004) and for complexes with PRAME-004 variants carrying alanine substitutions at peptide positions 1, 3, 4, 5, 6, 7, or 8, as described above. If a 2-fold or greater decrease in binding affinity or signal (measured at the highest concentration analyzed) was detected for the alanine-substituted peptide variant, the PRAME-004 position was considered to be part of the TCR binding motif. All TCER® variants tested showed a broad binding motif recognizing at least three peptide positions (Table 12).

[0451] In addition to the binding motif, the binding specificity of selected TCER® Slot II and IIa mutants was further analyzed by biolayer interferometry for binding to a set of 16 similar peptides potentially serving as off-target peptides. Measurements were performed on an Octet HTX system essentially as described above. To analyze, peptide-HLA complexes containing the PRAME-004 target peptide, individual peptides from the set of similar peptides, or control peptides were loaded onto a HIS1K biosensor and the binding of TCER® mutants was analyzed at a high TCER® concentration of 1 μM. The response signal at the end of the 5 min association phase was used to calculate the relative binding signal of the similar peptide compared to the PRAME-004 target peptide for selected TCER® mutants (Table 13). Under these conditions, binding events with very low affinity that could be described as insignificant (e.g., K for binding to the PRAME-004 peptide:MHC complex) were not observed. D Compared to K D 25-fold, 30-fold, 40-fold, 50-fold, 75-fold, or 100-fold increased binding) would be detected. Of the 16 analyzed similar peptides, 11 peptides did not show any binding to any of the selected TCER® variants. For 5 of the 16 similar peptides, binding with a lower signal compared to PRAME-004 was detected, and 4 of these peptides were analyzed in more detail with respect to the TCER® Slot III variants (e.g., binding with a higher K compared to the PRAME-004 target peptide). D window was measured).

[0452] [Table 12]

[0453] [Table 13] [Example 3.5]

[0454] Slot III Further TCER® were constructed utilizing the VH and VL domains and Valpha and Vbeta from BMA031 (V36) or its variants (A02 and D01), or ID4, as described above (Example 3.1). Further TCER® molecules based on the UCHT1-V17 recruiting antibody (TPP-1109) were generated as a reference. DNA constructs encoding the respective molecules were generated as outlined above. The resulting plasmids were used to transfect CHO-S cells by electroporation (MaxCyte) for the transient expression and production of the TCER® variants (Tables 14 and 18). Purification, formulation and initial characterization of the molecules were performed as outlined above in Example 3.3.

[0455] [Table 14]

[0456] The ability of TCER® molecules to kill HLA-A*02 positive tumor cell lines presenting various levels of PRAME-004 target peptide on the cell surface was evaluated in an LDH release assay. In addition, HLA-A*02 positive but PRAME-004 negative tumor cell lines (e.g., T98G) were evaluated to characterize the non-specific or off-target activity of TCER® variants. Tumor cell lines were co-cultured with PBMC effectors from healthy HLA-A*02 positive donors at a ratio of 1:10 with increasing TCER® concentrations. TCER®-induced cytotoxicity was quantified after 48 hours of co-culture by measuring released LDH. EC of dose-response curves 50 Values ​​were calculated using non-linear four-point curve fitting. EC for two PRAME-004-positive tumor cell lines (Hs695T and U20S) and a PRAME-004-negative tumor cell line (T98G) 50 Values ​​were determined in different experiments with different PBMC donors and are summarized graphically in FIG.

[0457] TCER® Slot III mutants TPP-214, -222, -230, -666, -669, -871, -872, -876, -879, -891, -894 were analyzed for binding affinity to the target peptide-HLA complex (HLA-A*02 / PRAME-004) by biolayer interferometry. Measurements were performed on an Octet HTX system at 30° C. Assays were performed on a HIS1K biosensor in 16 channel mode with a sensor offset of 3 mm and an acquisition rate of 5 Hz using PBS, 0.05% Tween-20, 0.1% BSA as assay buffer. The following sequence of assay steps was repeated to measure all binding affinities: regeneration (5 s, 10 mM glycine pH 1.5) / neutralization (5 s, assay buffer; one regeneration cycle consists of 4 repetitions of regeneration / neutralization), baseline (60 s, assay buffer), loading (120 s, 10 μg / ml peptide-HLA), baseline (120 s, assay buffer), association (300 s, 2-fold serial dilutions of TCER® ranging from 100 nM to 1.56 nM or 50 nM to 0.78 nM, assay buffer as reference), dissociation (300 s, assay buffer). Data evaluation was performed using Octet Data Analysis HT Software. A reference sensor subtraction was performed to subtract potential dissociation of peptide-HLA loaded on the biosensor (through biosensors loaded with peptide-HLA measured in buffer). Data traces were baseline-fitted (average of the last 5 seconds), step-by-step correction was performed for the dissociation step, Savitzky-Golay filtering was applied, and curves were fitted globally using a 1:1 binding model (Rmax not linked by the sensor). Strong binding affinities were determined with K values ​​ranging from 2 nM to 5 nM. D In addition, binding affinities were determined for four previously identified potential off-target peptides and compared to target peptide-HLA binding, with K DThe windows were calculated. Measurements were performed on an Octet RED384 or HTX system at 30° C. Assays were performed on a HIS1K biosensor in 16 channel mode with a sensor offset of 3 mm and an acquisition rate of 5 Hz using PBS, 0.05% Tween-20, 0.1% BSA as assay buffer. The following sequence of assay steps was repeated to measure all binding affinities: regeneration (5 s, 10 mM glycine pH 1.5) / neutralization (5 s, assay buffer; one regeneration cycle consists of 4 repetitions of regeneration / neutralization), baseline (60 s, assay buffer), loading (120 s, 10 μg / ml peptide-HLA), baseline (120 s, assay buffer), association (300 s, 2-fold serial dilutions of TCER® ranging from 500 nM to 7.81 nM, assay buffer as reference), dissociation (300 s, assay buffer). Data evaluation was performed using Octet Data Analysis HT Software. A reference sensor subtraction was performed to subtract potential dissociation of peptide-HLA loaded on the biosensor (through the respective peptide-HLA loaded biosensor measured in buffer). Data traces were baseline aligned (average of the last 5 seconds), step-by-step correction was performed for the dissociation step, Savitzky-Golay filtering was applied, and curves were fitted globally using a 1:1 binding model (Rmax not linked by the sensor). Overall, rather weak binding to potential off-target peptides compared to the target peptide was found for all variants, with at least 60-fold to completely no binding windows shown. Despite showing a relative binding signal comparable to VIM-009, NOMAP-3-1408 was significantly lower in K D For VIM-009, the smallest K D The window was >100-fold (Table 15). Therefore, binding to VIM-009 was deemed irrelevant and affinity determination of NOMAP-3-1408 binding was deemed unnecessary based on the comparable binding signal to VIM-009. A 50-fold K DA window was calculated. However, for this interaction and some other interactions, the Rmax value calculated by the fitting algorithm was too low, so the interaction was assumed to be weaker than calculated, leading to a larger window. The respective interactions are shown in Table 15. To further analyze the specificity of the different mutants, the binding motif was determined by measuring the affinity of the target peptide-HLA complex and alanine-substituted mutants for positions 1, 3, 4, 5, 6, 7, 8. Measurements were performed on an Octet HTX system at 30°C. The assay was performed on a HIS1K biosensor in 16 or 8 channel mode, with a sensor offset of 3 mm and an acquisition rate of 5 Hz, using PBS, 0.05% Tween-20, 0.1% BSA as assay buffer. The following sequence of assay steps was repeated to measure all binding affinities: regeneration (5 s, 10 mM glycine pH 1.5) / neutralization (5 s, assay buffer; one regeneration cycle consists of 4 repetitions of regeneration / neutralization), baseline (60 s, assay buffer), loading (120 s, 10 μg / ml peptide-HLA), baseline (120 s, assay buffer), association (150 s, 2-fold serial dilutions of TCER® ranging from 400 nM to 6.25 nM, assay buffer as reference), dissociation (300 s, assay buffer). Data evaluation was performed using Octet Data Analysis HT Software. A reference sensor subtraction was performed to subtract potential dissociation of peptide-HLA loaded on the biosensor (through each peptide-HLA loaded biosensor measured in buffer). Data traces were baseline-fitted (average of the last 5 seconds), step-by-step corrections were performed for the dissociation step, Savitzky-Golay filtering was applied, and curves were fitted globally using a 1:1 binding model (Rmax not linked by the sensor). A position was considered to be part of a binding motif if it reduced affinity or binding signal by more than 2-fold (measured at the highest concentration analyzed).All TCER® variants tested showed a broad binding motif recognizing at least four and up to all analyzed peptide positions (Table 16). For bA84, aN114L, and bA110S / bT115A, a positive effect on the binding motif was observed, which is in agreement with previous data. For comparison, the binding motif of an alternative PRAME-004-targeting TCER® reference molecule (TPP-1109) was analyzed. This TCER® recognizes positions 5-8 of the peptide, so binding is restricted to this peptide stretch, whereas the positions recognized by the TCER® Slot III variants are more evenly distributed throughout the peptide.

[0458] TCER® slot III mutants TPP-214, -222, -230, -666, -669, -871, -872, -876, -879, -891, -894 were further characterized for their ability to kill T2 cells loaded with various levels of target peptides. After loading of T2 cells with various concentrations of PRAME-004 for 2 hours, peptide-loaded T2 cells were co-cultured with human PBMCs at an E:T ratio of 5:1 with increasing concentrations of TCER® mutants for 48 hours. Levels of LDH released in the supernatant were quantified using CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega). All TCER® mutants showed sub-picomolar EC 50 The results showed potent killing of PRAME-004-loaded T2 cells at EC 50 The values ​​increased with decreasing levels of PRAME-004 loading, however, even at very low PRAME-004 loading concentrations of 10 pM, killing was induced by all TCER® mutants except TPP-214.

[0459] [Table 15]

[0460] [Table 16]

[0461] [Table 17]

[0462] [Table 18]

[0463] In Table 18, the term "α chain" refers to V, with the exception of TPP-70, TPP-71, TPP-72, TPP-73, and TPP74. α (i.e., a variable domain from a TCR α chain). β (i.e., a variable domain derived from a TCR beta chain). In the case of TPP-70, TPP-71, TPP-72, TPP-73, and TPP74, the "alpha chain" does not contain any TCR-derived variable domains, while the "beta chain" contains two TCR-derived variable domains, one from the TCR alpha chain and one from the TCR beta chain. [Example 3.6]

[0464] Safety Evaluation of Selected TCER® Slot III Candidates The safety profile of the TCER® molecules TPP-230, TPP-666, TPP-871, and TPP-891 (Tables 14-18) was evaluated in killing experiments with astrocytes and cardiomyocytes (derived from induced pluripotent stem cells), as well as aortic endothelial cells, mesenchymal stem cells, and tracheal smooth muscle cells. Figure 11 shows the results of co-culture of the above normal cell types (all expressing HLA-A*02) with PBMC effector cells from healthy HLA-A*02+ donors at a ratio of 1:10 (target cells:effector cells) with increasing TCER® concentrations. Cells were co-cultured in a 1:1 mixture of the respective normal tissue cell medium and T cell medium, or in T cell medium alone (LDH-AM). After 48 hours of co-culture, the supernatant was collected and the normal tissue cell lysis induced by TCER® was evaluated by measuring LDH release by LDH-Glo™ Kit (Promega).To determine the safety window, TCER® molecules were co-cultured in the same setting with PRAME-004 positive tumor cell line Hs695T in a 1:1 mixture of normal tissue cell medium and T cell medium, respectively, and LDH release was subsequently evaluated.

[0465] As shown in Figure 11, no cytotoxicity was observed against normal tissue cells for TPP-230 and TPP-871, even at the highest TCER® concentration of 100 nM. In the case of TPP-666 and TPP-891, some lysis of normal tissue cells was observed at 100 nM TCER® concentration, but no lysis was detected at 10 nM. Compared to Hs695T tumor cells, which showed significant lysis at 100 pM for all TCER® molecules tested, and even lysis at 10 pM for some molecules, lysis of normal tissue cells at 100 nM concentration shows a 1,000-fold (TPP-666 and TPP-891) or larger (TPP-230 and TPP-871) safety window. [Example 3.7]

[0466] Slot IV Further TCER® were constructed utilizing the VH and VL domains from BMA031 (V36) or its modified variants (A02 and D01), or ID4, and the Valpha and Vbeta described above (Example 3.1). DNA constructs encoding the respective molecules were generated as outlined above. The resulting plasmids were used to transfect CHO-S cells by electroporation (MaxCyte) for transient expression and production of the TCER® variants (Tables 20 and 18). Purification, formulation, and initial characterization of the molecules were performed as outlined in Example 3.3 above.

[0467] [Table 19]

[0468] In Table 20, the term "α chain" is used α (i.e., a variable domain from a TCR α chain). β (i.e., a variable domain derived from a TCR β chain).

[0469] The ability of TCER® molecules to kill HLA-A*02 positive tumor cell lines presenting various levels of PRAME-004 target peptide on the cell surface was evaluated in an LDH release assay. In addition, HLA-A*02 positive but PRAME-004 negative tumor cell lines (e.g., T98G) were evaluated to characterize the non-specific or off-target activity of TCER® variants. Tumor cell lines were co-cultured with PBMC effectors from healthy HLA-A*02 positive donors at a ratio of 1:10 with increasing TCER® concentrations. TCER®-induced cytotoxicity was quantified after 48 hours of co-culture by measuring released LDH. EC of dose-response curves 50 Values ​​were calculated using non-linear four-point curve fitting. EC for PRAME-004 positive tumor cell line U20S and PRAME-004 negative tumor cell line (T98G)50 Values ​​were determined in different experiments with different PBMC donors and are summarized in FIG.

[0470] [Table 20]

[0471] TCER® Slot IV mutants TPP-1292, -1294 to -1298, -1300 to -1309, -1333, -1334 were analyzed for binding affinity to the target peptide-HLA complex (HLA-A*02 / PRAME-004) by biolayer interferometry. Measurements were performed on an Octet HTX system at 30° C. Assays were performed on a HIS1K biosensor in 16 channel mode with a sensor offset of 3 mm and an acquisition rate of 5 Hz using PBS, 0.05% Tween-20, 0.1% BSA as assay buffer. The following sequence of assay steps was repeated to measure all binding affinities: regeneration (5 s, 10 mM glycine pH 1.5) / neutralization (5 s, assay buffer; one regeneration cycle consists of 4 repetitions of regeneration / neutralization), baseline (60 s, assay buffer), loading (120 s, 10 μg / ml peptide-HLA), baseline (120 s, assay buffer), association (300 s, 2-fold serial dilutions of TCER® ranging from 100 nM to 1.56 nM or 50 nM to 0.78 nM, assay buffer as reference), dissociation (300 s, assay buffer). Data evaluation was performed using Octet Data Analysis HT Software. A reference sensor subtraction was performed to subtract potential dissociation of peptide-HLA loaded on the biosensor (through biosensors loaded with peptide-HLA measured in buffer). Data traces were baseline-fitted (average of the last 5 seconds), step-by-step correction was performed for the dissociation step, Savitzky-Golay filtering was applied, and curves were fitted globally using a 1:1 binding model (Rmax not linked by the sensor). Strong binding affinities were observed with K values ​​ranging from 2 nM to 15 nM. DIn addition, binding affinities were determined for two previously identified potential off-target peptides and compared to target peptide-HLA binding, with K DThe windows were calculated. Measurements were performed on an Octet RED384 or HTX system at 30° C. Assays were performed on a HIS1K biosensor in 16 channel mode with a sensor offset of 3 mm and an acquisition rate of 5 Hz using PBS, 0.05% Tween-20, 0.1% BSA as assay buffer. The following sequence of assay steps was repeated to measure all binding affinities: regeneration (5 s, 10 mM glycine pH 1.5) / neutralization (5 s, assay buffer; one regeneration cycle consists of 4 repetitions of regeneration / neutralization), baseline (60 s, assay buffer), loading (120 s, 10 μg / ml peptide-HLA), baseline (120 s, assay buffer), association (300 s, 2-fold serial dilutions of TCER® ranging from 500 nM to 7.81 nM, assay buffer as reference), dissociation (300 s, assay buffer). Data evaluation was performed using Octet Data Analysis HT Software. A reference sensor subtraction was performed to subtract potential dissociation of peptide-HLA loaded on the biosensor (through the respective peptide-HLA loaded biosensor measured in buffer). Data traces were baseline aligned (average of the last 5 seconds), step-by-step corrections were made for the dissociation steps, Savitzky-Golay filtering was applied, and curves were fitted globally using a 1:1 binding model (Rmax not linked by the sensor). Overall, rather weak binding to potential off-target peptides compared to the target peptide was found for all variants, with at least 10-fold to completely no binding windows being shown. The respective interactions are shown in Table 22. To further analyze the specificity of variants TPP-1294, -1295, -1298, -1333, -1334, binding motifs were determined by measuring the affinity of the target peptide-HLA complex and alanine-substituted variants for positions 1, 3, 4, 5, 6, 7, 8. Measurements were performed on an Octet HTX system at 30°C.Assays were performed on HIS1K biosensors in 16 or 8 channel mode with a sensor offset of 3 mm and an acquisition rate of 5 Hz using PBS, 0.05% Tween-20, 0.1% BSA as assay buffer. The following sequence of assay steps was repeated to measure all binding affinities: regeneration (5 s, 10 mM glycine pH 1.5) / neutralization (5 s, assay buffer; one regeneration cycle consists of 4 repetitions of regeneration / neutralization), baseline (60 s, assay buffer), loading (120 s, 10 μg / ml peptide-HLA), baseline (120 s, assay buffer), association (150 s, 2-f...

Claims

1. An antigen-binding protein that specifically binds to a PRAME antigen peptide comprising or consisting of the amino acid sequence SLLQHLIGL of SEQ ID NO: 50 and present as a complex with a major histocompatibility complex (MHC) protein, (a) a first polypeptide comprising a variable domain V containing complementarity-determining regions (CDRs) CDRa1, CDRa2, and CDRa3, Ａ wherein, said CDRa1 comprises or consists of the amino acid sequence VKEFQD (SEQ ID NO: 16), or an amino acid sequence different from SEQ ID NO: 16 by 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, said CDRa3 comprises or consists of the amino acid sequence ALYNNLDMR (SEQ ID NO: 33) or ALYNNYDMR (SEQ ID NO: 34), or an amino acid sequence different from SEQ ID NO: 33 or SEQ ID NO: 34 by 1, 2, or 3 (preferably 1 or 2) amino acid mutations, preferably amino acid substitutions, optionally, said CDRa2 comprises or consists of the amino acid sequence FGPYGKE (SEQ ID NO: 32), or an amino acid sequence different from SEQ ID NO: 32 by 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, and the first polypeptide, (b) a second polypeptide comprising a variable domain V containing CDRb1, CDRb2, and CDRb3, Ｂ wherein, said CDRb1 comprises or consists of the amino acid sequence SGHNS (SEQ ID NO: 10), or an amino acid sequence different from SEQ ID NO: 10 by 1 or 2 amino acid mutations, preferably amino acid substitutions, said CDRb3 comprises the amino acid sequence ASSX １ GX ２ X ３ DX ４ QY (SEQ ID NO: 327) (wherein X １ is P, A, or T, X ２ is A or S, X ３ is T or I, X ４is K or A), or comprises or consists of an amino acid sequence that differs from SEQ ID NO: 327 by 1, 2, or 3 amino acid mutations, preferably amino acid substitutions, Optionally, said CDRb2 comprises or consists of the amino acid sequence FQNTAW (SEQ ID NO: 36), or a CDRb2 amino acid sequence that differs from SEQ ID NO: 36 by 1, 2, 3, 4, 5, or 6 amino acid mutations, preferably amino acid substitutions, and a second polypeptide and comprises an antigen-binding protein. **Claim 2** The antigen-binding protein is multispecific, for example, quadrispecific, trispecific, or bispecific, preferably bispecific, and in particular, the antigen-binding protein is a bispecific TCR, a bispecific antibody, or a bispecific TCR antibody molecule, and / or the first and the second polypeptides are contained in a single polypeptide chain or two polypeptide chains, preferably, VA is contained in the first polypeptide chain, and VB is contained in the second polypeptide chain; and / or - the variable domain VA is a Vα domain or a Vγ domain, and the variable domain VB is a Vβ domain or a Vδ domain. The antigen-binding protein according to claim 1. **Claim 3** V Ａ further comprises one or more framework regions selected from the group consisting of FR1-a, FR2-a, FR3-a, and FR4-a, preferably all framework regions, - FR1-a comprises or consists of the amino acid sequence of SEQ ID NO: 345 or SEQ ID NO: 346, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 345, preferably contains K or N at position 20, more preferably contains K, and / or contains L or M at position 2, more preferably contains L; - FR2-a comprises, or consists of, the amino acid sequence of SEQ ID NO: 347 or SEQ ID NO: 348, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 347, preferably contains L, I, or M at position 39, more preferably contains L or I, contains A or D at position 47, more preferably contains A, contains K or W at position 44, preferably contains K, contains F or A at position 52, preferably contains F, and / or contains Y or V at position 55, preferably contains Y; - FR3-a comprises, or consists of, the amino acid sequence of SEQ ID NO: 349, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 349, preferably contains T or K at position 92, more preferably contains T, and / or contains D or G at position 93, preferably contains D; - FR4-a comprises, or consists of, the amino acid sequence of SEQ ID NO: 350, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 350; V Ｂ further comprises one or more framework regions selected from the group consisting of FR1-b, FR2-b, FR3-b, and FR4-b, preferably all framework regions, - FR1-b comprises, or consists of, the amino acid sequence of SEQ ID NO: 351 or SEQ ID NO: 352, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 351, preferably contains H or N at position 10, more preferably contains H, contains E, L, or K at position 11, preferably contains E, and / or contains R or H at position 22; - FR2-b comprises, or consists of, the amino acid sequence of SEQ ID NO: 353, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 353, preferably contains R or K at position 43, more preferably contains R, contains E or Q at position 44, preferably contains E, contains M or P at position 46, more preferably contains P, and / or contains R or Q at position 48, more preferably contains Q; - FR3-b comprises, or consists of, the amino acid sequence of SEQ ID NO: 354 or SEQ ID NO: 355, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 354, and preferably contains D, A, E, R, K, Q, N, or S at position 84, more preferably contains D, A, E, Q, N, or S, more preferably contains D or A, and even more preferably contains D; - FR4-b comprises, or consists of, the amino acid sequence of SEQ ID NO: 356, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO:

356. The antigen-binding protein according to claim 1.

4. - V Ａ comprises, or consists of, the amino acid sequence of SEQ ID NO: 132, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 132, and preferably contains CDRa1 of SEQ ID NO: 16, CDRa2 of SEQ ID NO: 32, and CDRa3 of SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO:

9. Further, it contains K or N at position 20, preferably K, contains L, M, or I at position 39, preferably L or I, contains K or W at position 44, preferably K, contains F or A at position 52, preferably F, contains Y or V at position 55, preferably Y, contains T or K at position 92, preferably T, and / or contains D or G at position 93, preferably D. In particular, V Ａ comprises, or consists of, the amino acids of SEQ ID NO: 132, SEQ ID NO: 129, SEQ ID NO: 137, or SEQ ID NO: 142; - V Ｂcomprises, or consists of, the amino acid sequence of SEQ ID NO: 134, or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 134, preferably comprising CDRb1 of SEQ ID NO: 10, CDRb2 of SEQ ID NO: 36, and CDRb3 of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 47, SEQ ID NO: 281, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 301, or SEQ ID NO: 283, further comprising E, L, or K at position 11, preferably E, R or H at position 22, E or Q at position 44, preferably E, P or M at position 46, preferably P, Q or R at position 48, preferably Q, and / or D, A, E, Q, N, or S at position 84, preferably D or A, particularly, V Ｂ comprises, or consists of, the amino acid sequence of SEQ ID NO: 134, SEQ ID NO: 130, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, or SEQ ID NO: 148 The antigen-binding protein according to claim 1.

5. Antibody light chain variable domain (V Ｌ ) and antibody heavy chain variable domain (V Ｈ ), preferably V Ｌ and V Ｈ are CD2, CD3, particularly CD3γ, CD3δ, and / or CD3ε, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61, CD64, CD68, CD90, CD94, CD95, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, F ｃbinds to an antigen selected from the group consisting of εR1, TCRα / β and TCRγ / δ, HLA-DR, and 4-1BB, or a combination thereof, and / or binds to effector cells, in particular, binds to T cells or natural killer cells. The antigen-binding protein according to claim 1.

6. The antigen-binding protein includes a first and a second polypeptide chain. The first polypeptide chain has the formula [Ia]: V １ -L １ -D １ -L ２ -V ２ -L ３ -D ２ [Ia] is represented by The second polypeptide chain has the formula [IIa] V ３ -L ４ -D ３ -L ５ -V ４ -L ６ -D ４ [IIa] is represented by In the formula, - V １ , V ２ , V ３ , and V ４ are variable domains, and one of V １ to V ４ is V Ａ , one is V Ｂ , one is V Ｌ , and one is V Ｈ ; - D １ , D ２ , D ３ , and D ４ are dimerization domains and may or may not be present. D １ and D ３ , and D ２ and D ４ specifically bind to each other. D １ and D ３ or D ２ and D ４ At least one pair of which exists, - L １ , L ２ , L ３ , L ４ , L ５ , and L ６ and L are linkers, １ and L ４ exist, ２ , L ３ , L ５ , and L ６ may or may not exist, The antigen-binding protein according to claim 5.

7. The antigen-binding protein comprises a first and a second polypeptide chain, wherein the first polypeptide chain has the formula [Ib]: VL - L1 - VB - Fc1 → [Ib] represented by the second polypeptide chain has the formula [IIb]: VA - L4 - VH - Fc2 → [IIb] represented by wherein - VA consists of the amino acid sequence of SEQ ID NO: 132, and VB consists of the amino acid sequence of SEQ ID NO: 135; - L1 and L4 are linkers, preferably L1 and L4 consist of the amino acid sequence of SEQ ID NO: 214; - VL and VH are antibody variable domains that together form an antigen-binding site that binds to the CD3 or TCRα / βCD3 complex; - Fc1 and Fc2 are a pair of Fc domains, preferably, Fc1 consists of the amino acid sequence of SEQ ID NO: 149 (knob), and Fc2 consists of the amino acid sequence of SEQ ID NO: 150 (hole), the antigen-binding protein according to claim 6. **Claim 8**: A first polypeptide chain that is at least 85% identical, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, to a sequence selected from SEQ ID NOs: 100, 103, 105, 106, 111, 122, 126, 128, 151, 155, 156, 157, 158, 159, 166, 167, 169, 171, 173, 175, 177, 178, 179, 180, 181, 183, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 285, 291, 295, 299 and 303, and - A second polypeptide chain that is at least 85% identical, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, to a sequence selected from SEQ ID NOs: 101, 102, 104, 107, 110, 119, 121, 131, 133, 143, 152, 160, 161, 162, 163, 164, 165, 168, 170, 172, 174, 176, 182, 184, 185, 186, 216, 218, 220, 222, 224, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 282, 284, 296 or 300 The antigen-binding protein according to claim 1, comprising **Claim 9**: The antigen-binding protein according to claim 1, comprising a first polypeptide chain as set forth in SEQ ID NO: 158 and a second polypeptide chain as set forth in SEQ ID NO:

300. **Claim 10** An isolated nucleic acid comprising a sequence encoding the antigen-binding protein according to claim 1. **Claim 11** A host cell comprising the antigen-binding protein according to any one of claims 1 to 9 or the nucleic acid according to claim 10, preferably, the host cell is a lymphocyte, preferably a T lymphocyte or a T lymphocyte progenitor cell, or a cell for recombinant expression, for example, a Chinese hamster ovary (CHO) cell or a yeast cell.

12. A pharmaceutical composition comprising the antigen-binding protein according to any one of claims 1 to 9 or the nucleic acid according to claim 10 and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition comprising the host cell according to claim 11 and a pharmaceutically acceptable carrier.

14. The pharmaceutical composition according to claim 12, wherein the antigen-binding protein is dissolved or dispersed in a pharmaceutically acceptable carrier or an aqueous medium.

15. A method for producing the antigen-binding protein according to claim 1, comprising: a. preparing a host cell; b. preparing a gene construct comprising a coding sequence encoding the antigen-binding protein according to claim 1; c. introducing the gene construct into the host cell; and d. expressing the gene construct by the host cell and further comprising: e. optionally, isolating and purifying the antigen-binding protein from the host cell. A method further comprising the above steps.

16. The antigen-binding protein produced by the method according to claim 15.

17. For use in medicine, in particular for use in the diagnosis, prevention, and / or treatment of proliferative diseases such as cancer, said cancer being selected from the group of cancers consisting of acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, amelanotic melanoma, non-Hodgkin lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, osteosarcoma, chronic lymphocytic leukemia, colorectal cancer, and synovial sarcoma, the antigen-binding protein according to any one of claims 1 to 9 or 15, or the nucleic acid according to claim 10.

18. Use in the manufacture of a medicament of the antigen-binding protein according to any one of claims 1 to 9 or 15, or the nucleic acid according to claim 10, in particular for use in the diagnosis, prevention, and / or treatment of proliferative diseases such as cancer, said cancer being selected from the group of cancers consisting of acute myeloid leukemia, breast cancer, cholangiocarcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, amelanotic melanoma, non-Hodgkin lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, uterine and endometrial cancer, osteosarcoma, chronic lymphocytic leukemia, colorectal cancer, and synovial sarcoma.

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