Antigen-binding proteins specifically binding MAGE-a

Abstract

To provide new anti-cancer agents that specifically target molecules highly specific to cancer cells but not to normal cells.SOLUTION: The present invention concerns antigen-binding proteins specifically binding melanoma associated antigen A (MAGE-A) protein-derived antigens. The invention in particular provides antigen-binding proteins which specifically bind to the MAGE-A antigenic peptide comprising or consisting of a specific sequence in a complex with a major histocompatibility (MHC) protein. The antigen-binding proteins of the invention contain, in particular, the complementary determining regions (CDRs) of novel engineered T cell receptors (TCRs) that specifically bind to the MAGE-A peptide / MHC complex. The antigen-binding proteins of the invention are of use for the diagnosis, treatment and prevention of MAGE-A expressing cancerous diseases.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to antigen binding proteins that specifically bind to antigens derived from melanoma associated antigen A (MAGE-A) proteins. The present invention provides antigen binding proteins that specifically bind to MAGE-A antigenic peptides, in particular comprising or consisting of SEQ ID NO: 1 in complex with major histocompatibility (MHC) proteins. The antigen binding proteins of the present invention contain, in particular, complementarity determining regions (CDRs) of novel engineered T cell receptors (TCRs) that specifically bind to said MAGE-A peptide / MHC complexes. The antigen binding proteins of the present invention are useful for the diagnosis, treatment and prevention of MAGE-A manifesting cancerous diseases. Furthermore, nucleic acids encoding the antigen binding proteins of the present invention, vectors comprising these nucleic acids, recombinant cells expressing the antigen binding proteins, and pharmaceutical compositions comprising the antigen binding proteins of the present invention are provided. [Background technology]

[0002] The MAGE-A tumor antigen with the amino acid sequence "KVLEHVVRV" (SEQ ID NO: 1), also known as the MAG-003 peptide, binds to the HLA-A domains of MAGE-A4 (amino acids 286-294) and MAGE-A8 (amino acids 288-296). *02:01-restricted cytotoxic T lymphocyte (CTL) epitopes (Non-Patent Document 1). Both MAGE-A4 and MAGE-A8 are proteins and members of the MAGE-A gene family. The functions of MAGE-A8 and -A4 are unknown, but they may play a role in embryonic development and tumor transformation, or aspects of tumor progression. Multiple alternative splicing variants have been identified for these proteins. The MAGE-A antigen set forth in SEQ ID NO: 1 belongs to the cancer / testis (CT) antigens, which are expressed in tumors but not in normal tissues except testis and placenta. For example, MAGE-A4 protein and mRNA expression has been associated with the development and prognosis of various cancers. Peptides such as MAGE-A, particularly the MAGE-A4 and / or MAGE-A8 derived peptide "KVLEHVVRV", presented by major histocompatibility complex (MHC) molecules, may be bound by TCR and are therefore targets for, for example, T cell-based immunotherapy. Therefore, MAGE-A antigens and their epitope peptides have been used in tumor immunotherapy trials. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Zheng-Cai J.et al.Clin Dev.Immunol.2010,567594 Summary of the Invention [Problem to be solved by the invention]

[0004] While progress has been made in the development of molecularly targeted drugs for the treatment of cancer, there remains a need in the art to develop new anti-cancer agents that specifically target molecules that are highly specific to cancer cells, but not normal cells. [Means for solving the problem]

[0005] Thus, the present invention provides a novel antigen-binding protein specific for the tumor-expressed antigen MAGE-A of SEQ ID NO: 1 in complex with an MHC protein.

[0006] WO 2017 / 158103 discloses TCRs that bind to a complex of a MAGE-A antigen peptide having the amino acid sequence KVLEHVVRV of SEQ ID NO: 1 and an HLA class I molecule, more specifically natural TCRs such as the natural TCR R7P1D5, and their use in the diagnosis, treatment and prevention of cancer diseases. However, natural TCRs that specifically bind to cancer antigens presented on MHC have a lower affinity (K D =1-300 μM). Part of the explanation for this phenomenon seems to be that T cells developing in the thymus are negatively selected on self-peptide MHC ligands (tolerance induction), and T cells with too high affinity for such self-peptide MHC are deleted. This low affinity may be one possible explanation for tumor immune escape (Alek sic et al. 2012, Eur J Immunol. 2012 Dec; 42(12): 3174-9). It therefore seems desirable to design TCR variants that bind with higher affinity to cancer antigens for use as antigen recognition constructs in adoptive cell therapy (ACT) or as recognition modules in soluble approaches, i.e. using bispecific molecules (Hickman et al. 2016, J Biomol Screen. 2016 Sep; 21(8): 769-85).

[0007] However, increasing the affinity of TCRs may also increase the risk of side effects. As mentioned above, in nature, high affinity TCRs against tumor-associated antigens that are self-proteins are eliminated by thymic selection to avoid recognition of self-peptides present on normal tissues via cross-reactivity. Therefore, simply increasing the TCR affinity for its target sequence is likely to also increase affinity for similar non-cancer specific peptides, thus increasing the risk of cross-reactivity and undesirable cytotoxic effects on healthy tissues. That this is not just a theoretical risk has been painfully discovered with engineered TCRs targeting MAGE-A3. Notably, previously published results have shown fatal toxicity in two patients infused with T cells engineered to express TCRs targeting MAGE-A3, which cross-react with a peptide of the muscle protein titin, even though no cross-reactivity was predicted in preclinical studies (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.

[0008] Therefore, there is an unmet medical need to develop and provide TCR or antigen binding proteins that specifically bind to their targets with higher affinity while maintaining a high safety profile due to reduced or decreased cross-reactivity with off-target peptides (also referred to as "similar peptides"), and thus can target even tumor cells or cell lines with reduced expression of the target antigen peptide. In other words, the inventors have been able to develop antigen binding proteins that exhibit high binding affinity to their target peptides while maintaining high tumor selectivity.

[0009] Furthermore, as shown by the present inventors, the MAGE-A specific TCR molecule TCR R7P1D5 exhibits relatively low solubility either as a single chain construct or in a bispecific form termed TCER (hereinafter the "TCER™" molecule or "TCER™"), which comprises a portion that specifically binds to a surface molecule on a T cell and a portion that specifically binds to an MHC-peptide complex.

[0010] Thus, despite advances in TCR technology, there remains a need for additional cancer therapies, particularly those that efficiently target and kill cancer cells. In particular, there is a need to develop novel antigen-binding proteins that i) are selective and specific for the tumor-specific MAGE-A peptide of SEQ ID NO: 1 in complex with an MHC protein with the desired biological activity, ii) have favorable metabolic, pharmacokinetic, solubility, stability and / or safety profiles, and iii) can be manufactured on a large scale.

[0011] Thus, in the context of the present invention, the inventors have engineered several antigen binding proteins comprising CDR variants derived from said parent TCR R7P1D5, which have increased binding affinity to peptide-MHC complexes, increased stability and / or increased solubility, making them suitable for medical applications.

[0012] Moreover, as demonstrated by the present inventors, these novel antigen-binding proteins effectively bind to and kill low copy number target cancer cells, advantageously with low cross-reactivity to similar peptides and maintaining a high safety profile.

[0013] Furthermore, these novel antigen-binding proteins, especially in the form of TCER™ molecules, exhibit high cytotoxicity against tumor cells. For example, the novel antigen-binding proteins in the form of TCER™ molecules exhibit half-maximal effective concentrations (EC 50) is in the range of 1 to 300 pM, 1 pM to 100 pM, and more specifically, between 1 pM and 20 pM for cells. Furthermore, the EC 50 is 100-fold, preferably 1000-fold or more higher in cell lines of healthy tissues such as primary cell lines than in the tumor cell line Hs695T, demonstrating its high safety.

[0014] Furthermore, the inventors have demonstrated significant tumor growth inhibition in vivo in therapeutic mouse models for these novel antigen binding proteins, particularly the TCER™ molecule, even at low doses.

[0015] The present invention therefore relates to an antigen-binding protein specific for a MAGE-A derived antigenic peptide in complex with an MHC protein, said antigen-binding protein having increased stability, e.g. reduced aggregation during expression and / or purification, compared to the native TCR R7P1D5. Furthermore, said antigen-binding protein has high cytotoxicity, e.g. against the cell lines NCI-H175, Hs695T cells and U2OS cells.

[0016] In summary, the inventors' surprising discoveries provide, inter alia, the following advantages to the art: (i) reduced cross-reactivity of TCRs or antigen binding proteins with similar peptides on healthy tissues and maintaining high tumor selectivity; (ii) improved safety profile of TCRs or antigen binding proteins; (iii) reduced off-target and extra-tumor cytotoxicity of TCRs or antigen binding proteins; and (iv) provision of improved specific, selective and safe TCRs or antigen binding proteins.

[0017] definition The term "antigen-binding protein" herein refers to a polypeptide or binding protein capable of binding to at least one antigen, in particular an antigen as defined in the context of the present invention. In a preferred embodiment, said antigen-binding protein is capable of simultaneously binding to two different antigens, for example as known from bispecific antibodies. The antigen-binding protein of the present invention comprises at least six CDRs originally derived from a TCR, as defined in the context of the present invention. In a particular embodiment, the antigen-binding protein of the present invention comprises at least one variable α domain and at least one variable β domain originally derived from a TCR, as defined in the context of the present invention.

[0018] A "domain" is a region of a protein that is defined based on sequence homology and is often associated with a particular structural or functional entity. Examples of such domains are the variable light domain of an antibody light chain, the variable heavy domain of an antibody heavy chain, the C domain of an antibody heavy chain, and the C domain of an antibody light chain. H1 , C H2 , and C H3 domain (constant domain), the variable domain of the α chain of the TCR, or the variable domain of the β chain of the TCR.

[0019] The term "antigen" or "target antigen" as used herein refers to a molecule, or part of a molecule or complex, that can be bound by at least one antigen binding site, wherein said one antigen binding site is present, for example, in a conventional antibody, a conventional TCR, and / or an antigen binding protein of the invention.

[0020] The antigen which is specifically bound by the antigen-binding protein of the invention is a MAGE-A antigenic peptide which comprises or consists of the amino acid sequence "KVLEHVVRV" of SEQ ID NO:1, more specifically a MAGE-A antigenic peptide which comprises or consists of the amino acid sequence "KVLEHVVRV" of SEQ ID NO:1 in a complex with an MHC protein.

[0021] The term "epitope", as used herein, comprises 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 may be determined by methods known in the art, including X-ray crystallography or NMR analysis. Structural epitopes of antibodies typically comprise 20-30 amino acids. Structural epitopes of TCRs typically comprise 20-30 amino acids. A "functional epitope" is a subset of amino acids that form a structural epitope, comprising amino acids of an antigen that are important for interface formation with an antigen-binding protein of the invention, either directly by forming non-covalent bonds such as H-bonds, salt bridges, aromatic stacking or hydrophobic interactions, or indirectly by stabilizing the binding conformation of the antigen, as determined, for example, by mutational scanning. Typically, a functional epitope of an antigen to which an antibody binds comprises 4-6 amino acids. Typically, a functional epitope of a peptide-MHC complex comprises 2-6 amino acids of the peptide and 2-7 amino acids of the MHC molecule. Since MHCI-presented peptides typically have a length of 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. In the context of the present invention, an epitope, in particular a functional epitope bound by an antigen binding protein of the present invention, comprises or consists of a functional epitope comprising amino acids of the antigen required for the formation of the binding interface, thus at least three, preferably at least four amino acids of the MAGE-A antigenic peptide of SEQ ID NO:1.

[0022] The "MAGE-A" or "melanoma-associated antigen A" subfamily proteins were the first tumor-associated antigens identified at the molecular level (van der Bruggen P, et al. Science. 1991;254:1643-47). MAGE-A is a subfamily of 12 genes (MAGE-A1 to MAGE-A12) located in the q28 region of the X chromosome. Members of the MAGE-A subfamily proteins are normally expressed only in the testis or placenta, and their restricted expression suggests that they may function in germ cell development. MAGE-A proteins have also been detected in the early development of the central nervous system, spinal cord, and brainstem, revealing that MAGE-A proteins may also be involved in neurogenesis. Members of this family encode proteins with 50-80% sequence identity to each other, and all MAGE proteins share a common MAGE homology domain (MHD), a highly conserved domain consisting of approximately 170 amino acids. The biological functions and underlying regulatory mechanisms of MAGE-A protein expression in cancer remain incompletely understood.

[0023] "MAGE-A4" or "Melanoma Associated Antigen 4" protein is a member of the MAGE-A gene family and has Uniprot accession number P43358 with SEQ ID NO:97 (available as of July 8, 2019). The localization of MAGE-A4 has been described as cytoplasmic. However, MAGE-A4 staining has also been detected in the nucleus, with differential distribution between the nucleus and cytoplasm in well-differentiated versus poorly differentiated cancers (Sarcevic B et.al.,2003,Oncology 64,443-449). MAGE-A4 is used as a male germ cell marker. It is not expressed in spermatogonia, but is expressed in pre-spermatogonia and mature germ cells (Mitchell et al.,2014,Mod. Pathol.27,1255-1266). MAGE-A4 protein and mRNA expression have been associated with the development and prognosis of various cancers.

[0024] "MAGE-A8" or "melanoma associated antigen 8" protein is a member of the MAGE-A superfamily and has Uniprot accession number P43361, SEQ ID NO:98 (available as of July 8, 2019).

[0025] The "MAGE-A4" and "MAGE-A8" proteins share 72% sequence identity as determined by protein sequence alignment using the BLASTP 2.9.0 algorithm (Stephen F. et al. (1997) Nucleic Acids Res. 25:3389-3402). Furthermore, both "MAGE-A4" and "MAGE-A8" comprise the MAG-003 peptide, i.e., KVLEHVVRV (SEQ ID NO: 1).

[0026] The "major histocompatibility complex" (MHC) is a set of cell surface proteins essential for the adaptive immune system of vertebrates to recognize foreign molecules, thus determining histocompatibility. The main function of MHC molecules is to bind antigens derived from pathogens and present them on the cell surface for recognition by appropriate T cells. Human MHC is also referred to as HLA (human leukocyte antigen) complex (often 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 bearing the appropriate T cell receptor (TCR), while complexes of peptides and MHC class II molecules are recognized by CD4-positive helper T cells bearing the appropriate TCR. Identification and characterization of tumor-associated antigens and corresponding T cell receptors are important in the development of cancer immunotherapies, such as vaccines and cell therapies, since both types of responses, CD8-dependent and CD4-dependent, jointly and synergistically contribute to antitumor effects. The HLA-A gene is located on the short arm of chromosome 6 and encodes the larger α-chain of HLA-A. The diversity of HLA-Aα-chains is key to HLA function. This diversity promotes genetic diversity in a population. The more types of HLA, the more types of antigens are "presented" on the cell surface, since each HLA has a different affinity for peptides of a particular structure. Each individual can express up to two types of HLA-A, one type from each parent. Some individuals inherit identical HLA-A from both parents, reducing the diversity of individual HLAs; however, the majority of individuals inherit two different copies of HLA-A. All HLA groups follow this same pattern. In other words, each individual expresses only one or two of the 2432 known HLA-A alleles.

[0027] An MHC class I HLA protein in the context of the present invention is an HLA-A, HLA-B or HLA-C protein, preferably an HLA-A protein, more preferably an HLA-A * It could also be 02.

[0028] "HLA-A *"02" represents a specific HLA allele, in which the letter A represents the gene and the suffix " * "02" indicates the A2 serotype.

[0029] In an MHC class I-dependent immune response, not only can peptides bind to specific MHC class I molecules expressed by tumor cells, but they must also be subsequently recognized by T cells bearing specific T cell receptors (TCRs).

[0030] TCRs are heterodimeric cell surface proteins of the immunoglobulin superfamily that are associated with the invariant protein of the CD3 complex, which is involved in mediating signal transduction. TCRs exist in αβ and γδ forms, which are structurally similar but have rather different anatomical locations and possibly functions. The extracellular portions of the naturally occurring heterodimeric αβ and γδ TCRs each contain two polypeptides, each of which has a membrane-proximal constant domain and a membrane-distal variable domain. Each of the constant and variable domains contains intrachain disulfide bonds. The variable domains contain highly polymorphic loops, similar to the complementarity determining regions (CDRs) of antibodies. The use of TCR gene therapy overcomes several current hurdles. It typically endows the subject's (patient's) own T cells with the desired specificity, allowing sufficient numbers of T cells to be generated in a short period of time and avoiding their exhaustion. For example, TCRs can be transduced into potent T cells (e.g., central memory T cells or T cells with stem cell properties), which may ensure good persistence, storage, and function upon transfer. TCR-engineered T cells are infused into cancer patients rendered lymphopenic by chemotherapy or irradiation, allowing efficient engraftment but preventing immunosuppression.

[0031] The term "TCR" herein refers to TCRs and fragments thereof, as well as single chain TCRs and fragments thereof, in particular the variable α and β domains of single domain TCRs, as well as chimeric, humanized, bispecific or multispecific TCRs.

[0032] A "fragment of a TCR" comprises a portion of an intact or naturally occurring TCR, in particular the antigen binding or variable region of an intact or naturally occurring TCR. Examples of TCR fragments include the V α -C a or V β -C β Such fragments include fragments or portions of the α, β, δ, and γ chains, such as the corresponding hinge regions; or V α , V β , V δ , V γ single chain VαVβ fragments; bispecific and multispecific TCRs formed from TCR fragments. The TCR fragments perform the same function as naturally occurring full length TCRs, i.e. the fragments selectively and specifically bind to their target peptides.

[0033] "Single chain TCR (scTCR)" refers herein to a protein, in which the V α and V β , or V δ and V γ The variable domains of the TCR, such as, are located on one polypeptide. Typically, the variable domains are separated by a linker, wherein said linker typically comprises 5 to 20 amino acids, such as, for example, 5 to 15.

[0034] For example, "native" as used in the phrase "native TCR" refers to a wild-type TCR.

[0035] In the adoptive cell transfer (ACT) approach, the generated transgenic T cells may express a wild-type TCR with wild-type α and β chains and a recombinant TCR with α and β chains specific for the respective target antigen-MHC complex. Both the wild-type α and β chains and the recombinant α and β chains can usually still cross-pair with each other. This potential for unwanted pairing is called "TCR mispairing" and is a recognized problem in the field of TCR (gene) therapy. Mispairing and incorrect pairing between the recombinant TCR α or β chains and the endogenous TCR β or α chains, respectively, can result in reduced surface expression of the recombinant TCR αβ heterodimer, which reduces the functional avidity of the modified T cells. Furthermore, T cells expressing mispaired TCRs and grown under high IL-2 conditions have been demonstrated to induce graft-versus-host disease (GvHD) in preclinical models.

[0036] Some strategies to optimize the pairing of recombinant TCR α and β and avoid mispairing may include: 1. Mouserized TCR: In this approach, the human TCR α and β constant chains are replaced with the corresponding mouse domains. Although human and mouse TCR-C domains show a high degree of homology, small differences affect the stability of the TCR / CD3 interaction and thus the TCR surface expression level. 2. Cysteine-modified TCR: This approach introduces a cysteine ​​amino acid in a structurally favorable position, thus allowing the formation of at least one additional disulfide bridge and promoting the correct pairing between the two TCR chains. For example, site-directed mutations such as T48C in the TCR α constant chain and S57C in the TCR β constant chain result in a TCR heterodimer linked by two interchain bonds (i.e., an introduced disulfide bridge plus an integral transmembrane disulfide bridge (position 95 in the α constant domain and position 131 in the β constant domain). 3. Domain swapping: constant domains are swapped between the α and β chains of a tumor-specific T cell receptor to generate domain-swapped (ds) TCRs. These dsTCR chains, when correctly paired, retain all domains necessary to recruit the CD3 protein, be expressed on the surface of T cells, and mediate a functional T cell response upon binding to target antigen. In contrast, mispaired TCRs containing one dsTCR chain and one wild-type TCR chain fail to recruit, export, and signal transduce CD3. They lack the necessary critical domains and therefore cannot mediate deleterious autoimmunity. 4. Exclusive TCR heterodimers: This approach utilizes steric and electrostatic forces to promote correct pairing between TCRα and β transgenes while simultaneously suppressing pairing between exogenous and endogenous TCRα and β chains. In one example, site-directed mutagenesis is used to introduce S85R into the α constant domain and R88G into the β constant domain to obtain the necessary change in electrostatic charge, thus generating a "knob-into-hole" configuration with each other that is said to minimally distort the secondary and tertiary structures. 5. Use of chimeric TCR-CD3ζ chains, where each TCR chain is fused to a CD3ζ molecule. 6. Use of single chain TCRs, where the Vα chain of a defined TCR is fused to the β chain using a flexible peptide linker.7. Use of shRNA sequences or zinc finger nucleases to knock down the expression of endogenous TCR. Another approach, called "Velcro", allows the pairing of two TCR chains with each other due to favorable electrostatic interactions in the heterodimeric state. That is, the two peptides are mainly deployed in isolated form, but when mixed, they preferentially associate and form a stable parallel coiled-coil heterodimer. This approach may also be applied to generate soluble TCRs, in which the heterodimeric complex is favored by fusing peptides to truncated α and β chains, respectively.

[0037] Natural α-β heterodimeric TCRs have an α chain and a β chain. Each α chain comprises a variable region, a joining region, and a constant region; the β chain usually also contains a short diversity region between the variable and joining regions, but this diversity region is often considered part of the joining region. The constant regions or C 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). Each variable region, referred to herein as the α and β variable domains, comprises three "complementarity determining regions" (CDRs) embedded in framework sequences, one of which is a hypervariable region designated CDR3. The α variable domain CDRs are referred to herein as CDRa1, CDRa2, CDRa3, and the β variable domain CDRs are referred to herein as CDRb1, CDRb2, CDRb3. There are several types of α chain variable (Vα) regions and several types of β chain variable (Vβ) regions, which are distinguished by their frameworks, by CDR1 and CDR2 sequences, and by partially defined CDR3 sequences. The Vα types are referred to by unique TRAV numbers in the IMGT nomenclature, and the Vβ 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). For more information about immunoglobulin genes, see the International ImMunoGeneTics information system®, Lefranc M-Pbet al (Nucleic Acids Res. 2015 Jan;43(Database Publication):D413-22; and http: / / www.imgt.org / ).Thus, a conventional TCR antigen-binding site typically contains six CDRs, comprising a set of CDRs from each of the α and β chain variable regions, in which the CDR1 and CDR3 sequences are involved in recognition and binding of peptide antigens that bind to HLA proteins, and the CDR2 sequence is involved in recognition and binding of the HLA protein.

[0038] As with antibodies, "TCR framework region" (FR) refers to the amino acid sequences inserted between the CDRs, i.e., portions of the TCR alpha and beta chain variable regions that are conserved to some degree among different TCRs in a single species. The alpha and beta chains of a TCR each have four FRs, referred to herein as FR1-a, FR2-a, FR3-a, FR4-a, and FR1-b, FR2-b, FR3-b, FR4-b, respectively. Thus, the alpha chain variable domain may be referred to as (FR1-a)-(CDRa1)-(FR2-a)-(CDRa2)-(FR3-a)-(CDRa3)-(FR4-a), and the beta chain variable domain may be referred to as (FR1-b)-(CDRb1)-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)-(FR4-b).

[0039] In the context of the present invention, the CDR / FR definitions in the α or β chain or the γ or δ chain are to be 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 TCR-derived domain are indicated according to said IMGT definition. Preferably, the IMGT positions of the CDR / FR amino acid positions of the first variable domain are given in analogy with the IMGT numbering of TRAV5, and / or the IMGT positions of the CDR / FR amino acid positions of the second variable domain are given in analogy with the IMGT numbering of TRBV12-4. With respect to gamma / delta TCRs, the term "TCR gamma variable domain" as used herein refers to the junction of the TCR gamma V (TRGV) and TCR gamma J (TRGJ) regions without the leader region (L), and the term TCR gamma constant domain refers to the extracellular TRGC region or to a C-terminal truncated TRGC sequence. Similarly, the term "TCR delta variable domain" refers to the junction of the TCR delta V (TRDV) and TCR delta D / J (TRDD / TRDJ) regions without the leader region (L), and the term "TCR delta constant domain" refers to the extracellular TRDC region or to a C-terminal truncated TRDC sequence.

[0040] In an "antibody", also called an "immunoglobulin", 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 (l) and kappa (k). There are five major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. The light chains contain a variable domain (V L ) and constant domain (C L The heavy chain contains two domains or regions: a variable domain (V H ) and three constant domains (collectively C H It is called C. H1 , C H2 , and C H3) and four domains. L ) and heavy chain (V H The variable regions of both the light chain (C) and the light chain (D) determine antigen binding recognition and specificity. L ) and heavy chain (C H The constant region domains of the antibody are involved in antibody chain binding, secretion, placental transport, complement fixation, and F c Receptor (F c Fv fragments confer important biological properties such as binding to antigenic determinants (e.g., Fab fragments). Fv fragments are the N-terminal portion of the Fab fragments of immunoglobulins and consist of one light chain variable portion and one heavy chain variable portion. The specificity of an antibody resides in the structural complementarity between the antibody combining site (synonymous for antibody binding site) and an antigenic determinant. The antibody combining site is composed mainly of residues from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or FRs influence the overall domain structure and thus the binding combining site. CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, a conventional antibody antigen-binding site contains six CDRs, comprising a set of CDRs from each of the heavy and light chain V regions.

[0041] In the context of the present invention, an antibody or immunoglobulin is IgM, IgD, IgG, IgA or IgE.

[0042] "Antibody framework region" (FR) refers to the amino acid sequences inserted between the CDRs, i.e., the portions of the immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins of a single species. The light and heavy chains of an immunoglobulin 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 may be designated as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable domain may be designated as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H).

[0043] In the context of the present invention, F C The definition of CDR / FR in an immunoglobulin light or heavy chain other than the domain is to be determined based on the IMGT definition (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org). Thus, the amino acid sequences of CDR1, CDR2, and CDR3 and the amino acid sequences of FR1, FR2, FR3, FR4, and FR5 of a given variable chain are shown according to the IMGT definition.

[0044] As will be appreciated by those skilled in the art, the structure of an antibody, particularly the variable heavy and light chains of the antibody, is similar to the TCR alpha and beta variable domain structures. Thus, the CDRs of the TCRs of the present invention may be grafted into conventional antibodies, bispecific antibodies, or multispecific antibodies.

[0045] By knowing the amino acid sequence of the CDR of the antibody, TCR or antigen-binding protein of the present invention, a person skilled in the art can easily determine the framework region, such as the TCR framework region or the antibody framework region. However, if the CDR is not shown, a person skilled in the art can first determine the CDR amino acid sequence based on the IMGT definition of TCR or the IMGT definition of antibody, and then determine the amino acid sequence of the framework region.

[0046] As used herein, a "human framework region" is 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 antigen binding protein, such as a naturally occurring human antibody or human TCR.

[0047] The term "antibody" refers to antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular the variable heavy chains of single domain antibodies, and chimeric antibodies, humanized antibodies, bispecific or multispecific antibodies.

[0048] A "conventional antibody" as referred to herein is an antibody that has the same domain as an antibody isolated from nature and comprises CDRs and framework regions from the antibody. Similarly, a "conventional TCR" as referred to herein is a TCR that comprises the same domain as a natural TCR and CDRs and framework regions from a TCR.

[0049] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and that has been modified to replace 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 and C L It is a domain.

[0050] Many methods for humanizing antibody sequences are known in the art; see, for example, the review by Almagro & Fransson (2008) Front Biosci. 13:1619-1633. One commonly used method is CDR grafting, or antibody reshaping, which involves grafting the CDR sequences of a donor antibody, usually a mouse antibody, onto the framework scaffold of a human antibody of different specificity. Because CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of the CDR-grafted non-human antibody, back mutations may be introduced at selected positions of the CDR-grafted antibody to retain the binding specificity and affinity of the parent antibody. Identification of possible back mutation positions may be performed using information available in the literature and antibody databases. An alternative humanization technique to CDR grafting and back mutation is resurfacing, in which non-surface exposed residues of non-human origin are retained while surface residues are modified to human residues. Another alternative technique is known as "guided selection" (Jespers et al. (1994) Biotechnology 12, 899) and can be used to derive fully human antibodies, which maintain the epitope and binding properties of the parent antibody, for example from mouse or rat antibodies. A further method of humanization is the so-called 4D humanization. The 4D humanization protocol is described in US Patent Application No. 20110027266A1 (WO 2009032661A1) and involves the selection of the variable light chain (V L ) and heavy chain (V H An example of the application of 4D humanization for the humanization of a .) domain is illustrated below.

[0051] In chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences.

[0052] Although amino acid residues that are part of the CDRs are typically not modified in connection with humanization, in certain instances it may be desirable to modify individual CDR amino acid residues, for example to remove glycosylation sites, deamidation sites, isomerization sites, or undesired cysteine ​​residues. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X may be any amino acid except Pro. Removal of N-glycosylation sites may be achieved by mutating either the Asn or Ser / Thr residue to a different residue, particularly via conservative substitution. Deamidation of asparagine and glutamine residues may occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation when present primarily in the sequence Asn-Gly, and less so in other dipeptide sequences such as Asn-Ala. Thus, where such a deamidation site, particularly Asn-Gly, is present in a CDR sequence, it may be desirable to eliminate the site by removing one of the involved residues, typically by a conservative substitution. Substitutions in the CDR sequence to remove one of the involved residues are also intended to be encompassed by the present invention.

[0053] An "antibody fragment" comprises a portion of an intact antibody, in particular the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, and diabodies and multi-specific antibodies formed from antibody fragments. An antibody fragment may also be a heavy chain antibody or a single domain antibody such as a VHH.

[0054] The term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 daltons and antigen-binding activity, in which about the N-terminal half of the H chain and the entire L chain are bound together via disulfide bonds, among fragments obtained by treating IgG with a protease such as papain.

[0055] The term "bispecific" in reference to the antigen-binding protein of the present invention refers to an antigen-binding protein having at least two valencies and binding specificities for two different antigens, and thus comprising 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 having two binding sites. It should be noted that the term valency refers to the number of binding sites, in which the binding sites may bind to the same or different targets, i.e., a bivalent antigen-binding protein may be monospecific, i.e., bind to one target, or bispecific, i.e., bind to two different targets. The target may be an antigen, a target peptide, an off-target, i.e., a similar peptide, etc.

[0056] In the context of the present invention, it is preferred that at least one antigen-binding site is derived from a TCR, more particularly that at least one antigen-binding site comprises a CDR from a TCR as defined in the context of the present invention.

[0057] In the present context of the term "bispecific", it is preferred that at least one specificity of the antigen-binding site is derived from a TCR, more particularly that at least one antigen-binding site comprises a CDR from a TCR as defined in the present context. Thus, "bispecific" in the present context refers to an antigen-binding protein that combines at least one antigen-binding site comprising a CDR as defined in the present context, i.e. a CDR from a TCR, with at least one further antigen-binding site, in which said at least one further antigen-binding site may be derived from an antibody and therefore comprise an antibody CDR, or may be derived from a further TCR and therefore comprise a CDR of a further TCR. However, in a preferred embodiment, said further antigen-binding site is derived from an antibody and therefore comprises an antibody CDR.

[0058] The term "morphology" herein refers to an antigen binding protein that comprises a particular number and type of domains present in said antigen binding protein and their spatial organization.

[0059] Many different forms, such as bispecific forms, have been described in the art, typically in the context of antibodies, and such forms typically include bispecific antibodies, crossover dual variable domain (CODV) and / or dual variable domain (DVD) proteins. An overview of these different bispecific antibodies and methods for producing them is disclosed, for example, in Brinkmann U. and Kontermann EEMAbs. 2017 Feb-Mar;9(2):182-212. In particular, DVD forms are disclosed, for example, in the following scientific papers (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. Bispecific 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;PMID:11789029.

[0060] A "bispecific antibody" as used in the context of an antibody refers to a bivalent molecule composed of two chains, each comprising a VH and a VL domain, typically from the same or different antibodies. The two chains typically have the following configurations: VHA-VLB and VHB-VLA (where A and B represent two different specificities), or VLA-VHB and VLB-VHA.

[0061] In the context of the present invention, a "diabody (Db)" or a "bispecific antibody format" is defined herein as comprising a linker (L Db1 and L Db2 It refers to a bivalent molecule composed of two polypeptide chains comprising two variable domains linked by a VI (V1) and V2 (V3) in which two of the domains are the first and second domains (V1 and V2) as defined in the context of the present invention, and the other two domains are variable domains derived from a TCR or derived from an antibody (V A , V B The V1V2 domains may be located on two different polypeptides, and the V A V B The domains are located on two different polypeptides and the domains dimerize in a head-to-tail orientation. Thus, the orientation is V1-L. Db1 -V A and V B -L Db2 -V2, V2-L Db1 -V A and V B -L Db2 -V1, V1-L Db1 -V B and V A -L Db2 -V2, or V2-L Db1 -V B and V A -L Db2 -V1. To allow the domains to dimerize in a head-to-tail orientation, a linker, i.e., L Db1 and L Db1can be the same or different and are short linkers. Short linkers are typically linkers between 2-12, 3-13 amino acids long, such as 4, 5, 6, 7, 8, 9 amino acids long (Brinkmann U. and Kontermann EE (MAbs.2017 Feb-Mar;9(2):182-212) or 8 amino acids long, such as "GGGS" in SEQ ID NO:100, "GGGGS" in SEQ ID NO:101, or "GGGSGGGG" in SEQ ID NO:96.

[0062] The "dual variable domain immunoglobulin (DVD-Ig™)" format was first described in 2007 by Wu C. et al. (Nat Biotechnol. 2007 Nov;25(11):1290-7). In this format, the target-binding variable domain of a second monoclonal antibody (B) is typically a target-binding variable domain of a conventional antibody (A) (domain V LA and V HA In the conventional antibody (A), the light chain of the antibody (B) is thus fused to an additional light chain variable domain (V LB ), and the heavy chain of the conventional antibody (A) comprises an additional heavy chain variable domain (V HB A DVD-Ig™ as described in the art thus typically comprises a DVD-Ig™. HB -LV HA -C H1 -C H2 -C H3 and one heavy chain comprising V LB -LV LA -C L The domain pair V LA / V HA and V LA / V LA therefore involutes in parallel.

[0063] In the context of the present invention, a "dual variable domain Ig format" refers to a protein comprising two polypeptide chains each comprising two variable domains linked by a linker (L1, L3), in which two of the domains are the first and second domains (V1 and V2) as defined in the context of the present invention, and the other two domains are the heavy and light chain variable domains (V HA and V HB In the context of the present invention, in the DVD-Ig form, the polypeptide chain is, for example, V1-L1-V HA -L2-C H1 -C H2 -C H3 and V2-L3-V LA -L4-C L , or V2-L1-V HA -L2-C H1 -C H2 -C H3 and V1-L3-V LA -L4-C L The connecting linkers L1 and L3 are preferably between 5 and 20 amino acid residues in length, such as 5 to 15 amino acid residues, and / or the connecting linkers L2 and L4 may be present or absent.

[0064] A "crossover dual variable domain Ig-like protein" as described in the art in the context of antibodies is a protein that comprises two V H and two V's L The domain is variable V H -V L are linked in a manner that allows crossover pairing of the domains, HA -V HB and V LB -V LA order, or V HB -V HA and V LA -V LB This refers to a form in which the amino acid sequence is arranged in either of the following orders (from the N-terminus to the C-terminus):

[0065] In the context of the present invention, a "crossover dual variable domain Ig-like protein" refers to a protein comprising two polypeptide chains, each comprising two variable domains linked by a linker (L1, L2, L3 and L4), in which two domains are the first and second domains (V1 and V2) as defined in the context of the present invention, and the other two domains are antibody heavy and light chain variable domains (V HA , V HB In the context of the present invention, in the CDVD-Ig form, the polypeptide chain is, for example, V1-L1-V HA -L2-C H1 -C H2 -C H3 and V LA -L3-V2-L4-C L , V2-L1-V HA -L2-C H1 -C H2 -C H3 and V LA -L3-V1-L4-C L、 V HA -L1-V1-L2-C H1 -C H2 -C H3 and V2-L3-V LA -L DVD3 -C L , or V HA -L1-V2-L2-C H1 -C H2 -C H3 and V1-L3-V LA -L4-C LIn this CDVD form, the linkers (L1-L4) are typically of different lengths, including all-glycine linkers and those linkers described below in the Linker section of this specification. For example, 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.

[0066] "At least one" herein refers to one or more specific entities, such as 1, 2, 3, 4, 5, or 6 or more specific entities. For example, at least one binding site herein refers to 1, 2, 3, 4, 5, or 6 or more binding sites.

[0067] A sequence "having at least 85% identity to a reference sequence" is a sequence that has 85% or more, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity over its entire length to the entire length of the reference sequence.

[0068] In the context of this application, "percentage of identity" is 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 well 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) of two sequences considering their entire length. The Needle program is available, for example, on the World Wide Web 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 "Gap Open" parameters equal to 10.0, "Gap Extend" parameters equal to 0.5, and the Blosum62 matrix.

[0069] 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 comprise amino acid mutations, such as 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 derived from another species than the reference sequence.

[0070] "Amino acid substitutions" may be conservative or non-conservative. Preferably, the substitutions are conservative substitutions, in which one amino acid is replaced by another amino acid having similar structural and / or chemical properties.

[0071] In one embodiment, conservative substitutions may include those described by Dayhoff in "The Atlas of Protein Sequence and Structure. Vol. 5," Natl. Biomedical Research, the entire contents of which are incorporated by reference. For example, in one embodiment, 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 one aspect, 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.

[0072] In further embodiments, conservative amino acid substitutions may include the replacement of an amino acid with another amino acid of the same class, such as, for example, (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, such as: (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).

[0073] In another embodiment, conservative substitutions may 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 entire contents of which are incorporated by reference.

[0074] Table 1: Conservative Amino Acid Substitutions [Table 1]

[0075] In another embodiment, conservative substitutions may be those shown in Table 2 under the heading of "conservative substitutions." If such substitutions result in altered biological activity, more substantial changes, designated "exemplary substitutions" in Table 2, may be introduced and the products screened, if necessary.

[0076] Table 2: Amino acid substitutions [Table 2]

[0077] In some embodiments, the antigen binding protein may comprise a mutated antigen binding protein, in which said mutated antigen binding protein comprises a first polypeptide chain (such as an α chain) and a second polypeptide chain (such as a β chain) that comprise up to 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, preferably in the CDR regions of the first variable domain (such as the Vα domain) and the second variable domain (such as the Vβ domain), compared to the antigen binding protein from which the mutation is derived. In this regard, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions in each of the CDR regions of the antigen binding protein, or in all of the CDR regions of the first and / or second variable domains. The substitutions may be in the CDRs of either the first and / or second variable domain.

[0078] In one embodiment, the variant is a functional variant.

[0079] The term "functional variant" as used herein refers to an antigen binding protein that has substantial or significant sequence identity or similarity with a parent antigen binding protein, such as an antigen binding protein containing conservative amino acid substitutions, wherein said functional variant retains the biological activity of the parent antigen binding protein. In one aspect, functional variants encompass variants of the antigen binding proteins described herein (the antigen binding proteins described herein themselves are referred to as parent antigen binding proteins) that, for example, retain the ability to recognize target cells to about the same extent, to the same extent, or to a greater extent than the parent antigen binding protein. With respect to the parent antigen binding protein, the functional variant may have an amino acid sequence that is, for example, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence to the parent antigen binding protein.

[0080] A functional variant may, for example, comprise the amino acid sequence of a parent antigen-binding protein that includes at least one conservative amino acid substitution. Alternatively, or in addition, a functional variant may comprise the amino acid sequence of a parent antigen-binding protein with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent antigen-binding protein.

[0081] The modified TCRs, polypeptides, and antigen binding proteins (including functional portions and functional variants) of the present disclosure can be of any length, i.e., comprise any number of amino acids, provided that the modified TCRs, polypeptides, or proteins (or functional portions or functional variants thereof) retain their biological activity, such as, for example, the ability to specifically bind to an antigen, the ability to detect diseased cells in a host, or the ability to treat or prevent a disease in a host.

[0082] The antigen binding proteins (including functional portions and functional variants) of the present invention may comprise 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, aminocyclohexanecarboxylic 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 acids, 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-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine may be mentioned.

[0083] In one embodiment, the antigen binding proteins of the invention (including functional portions and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts, and / or optionally dimerized or polymerized, or conjugated.

[0084] The antigen binding proteins of the disclosure may be synthetic, recombinant, isolated, and / or purified.

[0085] "Covalent bond" herein refers to a disulfide bridge or a peptide bond, or a covalent bond, for example via a linker or linker sequence, such as a polypeptide linker.

[0086] The term "linker" as used herein refers to one or more amino acid residues that are inserted between two domains, such as between a first and a second variable domain, and optionally between a light chain variable domain and a heavy chain variable domain, of an antigen binding protein of the invention, for example in a single chain construct, to provide sufficient flexibility for the domains to fold correctly to form an antigen binding site, or, in the case of bispecific antigen binding proteins, to form the antigen binding site and at least one further antigen binding site, either in crossover pairing (in CODV format, or in some of the bispecific antibody formats) or in parallel pairing configuration (e.g. in DVD format) of the antigen binding protein of the invention.

[0087] In some embodiments, the linker consists of 0 amino acids, i.e., there is no linker. The linker is inserted at the transition between variable domains or between variable and constant domains, respectively, at the amino acid sequence level. The approximate sizes of immunoglobulin domains as well as TCR domains are well understood, so that the transition between domains can be identified. It is known by those skilled in the art that 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 identified or assumed using modeling or secondary structure prediction techniques. The term linker as used in the context of the present invention refers to, but is not limited to, linkers designated L1, L2, L3, L4, L5, and L6.

[0088] Unless otherwise specified in the respective context, L1, L2, L3, L4, L5, and L 6、Linkers such as L1, L2, L3, L4, L5, and L6 can be at least 1-30 amino acids in length. 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, L5, and L6 can be peptides no longer than 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids 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, less than 12, less than 10 amino acid residues in length, e.g., 5-24, 10-24, or 5-10. In some embodiments, the linkers are one or more amino acid residues in length, such as more than 1, more than 2, more than 5, more than 10, more than 20 amino acid residues in length, more than 22 amino acid residues in length.

[0089] L1, L2, L3, L4, L5L 6、Exemplary linkers such as TVAAP (SEQ ID NO:99), GGGS (SEQ ID NO:100), GGGGS (SEQ ID NO:101), GGSGG (SEQ ID NO:140), TVLRT (SEQ ID NO:102), TVSSAS (SEQ ID NO:103), GGGSGGGG (SEQ ID NO:96), GGGGSGT (SEQ ID NO:148), GGSGGGGSGG (SEQ ID NO:141), GGGGSGGGGS (SEQ ID NO:104), GGSGGGGSGGGGSGG (SEQ ID NO:143), GGGGSAAA (SEQ ID NO:105), GGGGSGGGGSGT (SEQ ID NO:155), GGGGSGGGGSGGGGGS (SEQ ID NO:106), GGGGSGGGGSGGGGSGT (SEQ ID NO:161), GGGGSGGGGSGGGGSGGGGSGT (SEQ ID NO:163), GGSGGGGSGGGGSGGGGSGG (SEQ ID NO:144), GGGGSGGGGSGGGGSG The amino acid sequence comprises or consists of an amino acid sequence selected from the group of amino acids consisting of GGGSGGGGSGT (SEQ ID NO:165), GGGGSGGGGSGGGGSGGGGSGGGGSGS (SEQ ID NO:107), GGSGGGGSGGGGSGGGGSGGGGSGG (SEQ ID NO:145), GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:108), GSADDAKKDAAKKDGKS (SEQ ID NO:146), GGQGSGGTGSGGQGSGGTGSGGQGS (SEQ ID NO:147), TVLSSAS (SEQ ID NO:109) and GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:110), in particular the amino acid sequences of GGGSGGGG (SEQ ID NO:96), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:110), and GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:108).

[0090] As used in the context of this invention, c The term "domain" refers to a naturally occurring F domain, as further defined below. c Domain and F c Includes variants and sequences of domains. c Mutant and natural F c As with the molecule, "F cThe term "domain" includes molecules in either monomeric or multimeric form, whether resolved from a whole antibody or generated by other means.

[0091] "Natural F c The term "as used herein" refers to a molecule that comprises the sequence of a non-antigen-binding fragment obtained from the digestion of an antibody or generated by other means, whether in monomeric or multimeric form, and that may contain a hinge region. c The original immunoglobulin source of the F is in particular of human origin and can be any immunoglobulin, preferably IgG1 or IgG2, most preferably IgG1. c The molecule is composed of monomeric polypeptides that can associate into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent bonds. c The 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 One example is the disulfide-linked dimer obtained from papain digestion of IgG. c The term " is used herein generally for monomeric, dimeric, and multimeric forms. c An example of an amino acid sequence is IGHG1 * 01 Natural F c This is the amino acid sequence of sequence number 111.

[0092] "Hinge" or "hinge region" or "hinge domain" typically refers to a C H1 Domain and C H2It refers to the flexible part of the heavy chain located between the domains. It is about 25 amino acids long and is divided into the "upper hinge", "middle hinge" or "core hinge", and the "lower hinge". The "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 below: IgG1:E 216 PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 114) IgG2:E 216 RKCCVECPPCPAPPVAGP (SEQ ID NO: 115) IgG3:ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPE 216 PKSCDTPPPCPRCPAPELLG (SEQ ID NO: 116) IgG4:E 216 SKYGPPCPSCPAPEFLG (sequence number 117).

[0093] In the context of the present invention, this means F c Reference is made to amino acid positions in a domain, and these amino acid positions or residues are designated according to the EU numbering system, for example as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).

[0094] "F c The term "mutant" as used herein refers to a naturally occurring F c Although modified from F, the salvage receptor c Rn (neonatal F c It refers to a molecule or sequence that still comprises a binding site for a target polypeptide (receptor). c Mutants and their interactions with salvage receptors are known in the art. c The term "mutant" refers to a non-human naturally occurring F c In addition, the natural F ccomprises regions that provide structural features or biological activity that are not required for the antigen binding proteins of the invention and therefore can be removed. c The term "variant" refers to one or more naturally occurring F c or comprising one or more F c Sites or residues may be modified to avoid (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity upon expression in the selected host cell, (4) glycosylation, (5) interaction with complement, or (6) F other than the salvage receptor. c (7) affecting or participating in binding to a receptor, or antibody-dependent cellular cytotoxicity (ADCC).

[0095] Thus, in one embodiment, F c1 and / or F c2 In one embodiment, the Fc-domain comprises a hinge domain. c -Domain is human IgGF c domain, preferably derived from human IgG1, IgG2, IgG3 or IgG4, preferably IgG1 or IgG2, more preferably IgG1.

[0096] In some embodiments, the antigen binding protein comprises two F c Domains, i.e., TCER™ forms (F c1 and F c2 etc.), two F c The domains may be of the same immunoglobulin isotype or isotype subclass, or of different immunoglobulin isotypes or isotype subclasses, and are preferably the same. Thus, in some embodiments, c1 and F c2 is IgG1 subclass, or IgG2 subclass, or IgG3 subclass, or IgG4 subclass, preferably IgG1 subclass, or IgG2 subclass, more preferably IgG1 subclass.

[0097] In some embodiments, F c The domain is a mutant F c domain and therefore comprises one or more of the amino acid substitutions described herein below.

[0098] In some embodiments, F c The domain comprises or further comprises a "RF" and / or a "knob-into-hole" mutation, preferably a "knob-into-hole".

[0099] "RF mutation" refers to the C mutation described by Jendeberg, L. et al. (1997, J. Immunological Meth., 201:25-34). H3 Amino acid substitutions in the F domain, such as H435R and Y436F c C in Domain H3 This generally refers to the amino acid substitution of amino acids HY to RF in the F domain, which has been described as advantageous for purification purposes since it abolishes binding to Protein A. c If the domain comprises an F domain, the RF mutation may be in one or both, preferably one F c It may be in the domain.

[0100] "Knob-in-two-hole" technology, also known as "knob-in-two-hole," is a H3 -C H3 It refers to the amino acid substitutions T366S, L368A, and Y407V (hole) and T366W (knob) that promote heteromultimer formation at the interface. These "knob-into-hole" mutations can be further stabilized by the introduction of additional cysteine ​​amino acid substitutions Y349C and S354C. The "knob-into-hole" technology, together with stabilizing cysteine ​​amino acid substitutions, is described in U.S. Pat. Nos. 5,731,168 and 8,216,805.

[0101] In the context of the present invention, a "knob" mutation comprises or consists of, for example, the amino acid sequence of SEQ ID NO: 112, together with the amino acid substitution S354C for cysteine. c domain and the "hole" mutation comprises or consists of the amino acid sequence of SEQ ID NO: 113 together with the cysteine ​​amino acid substitution Y349C. c Exists in the domain.

[0102] In some embodiments, for example, F c1 One F of a polypeptide such as c The domain is H3 The F domain comprises the amino acid substitution T366W (knob), e.g. c2 The Fc domain of other polypeptides such as H3 It comprises the amino acid substitutions T366S, L368A, and Y407V (hole) in the domain, and vice versa.

[0103] In some embodiments, for example, F c1 One F of a polypeptide such as c The domain is H3 and / or further comprising the amino acid substitution S354C in the F domain, e.g. c2 F of other polypeptides such as c The domain is H3 It may comprise or further comprise the amino acid substitution Y349C in the domain, or vice versa.

[0104] Thus, in some embodiments, for example, F c1 One F of a polypeptide such as c The domain is H3 The F domain comprises the amino acid substitutions S354C and T366W (knob), e.g. c2 F of other polypeptides such as c The domain is H3 It comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V (hole) in the domain, and vice versa.

[0105] This series of amino acid substitutions was reported by Wei et al. (Structural basis of a novel heterodimeric F c This can be further expanded by including the amino acid substitution K409A in one polypeptide and F405K in the other polypeptide, as described by G. Klein et al., J. Molecular Biology, 2017, for bispecific antibody production, Oncotarget. Thus, in some embodiments, e.g., c1 One F of a polypeptide such as c The domain is H3 and / or further comprising the amino acid substitution K409A in the F domain, e.g. c2 F of other polypeptides such as c The domain is H3 It may comprise or further comprise the amino acid substitution F405K in the domain, or vice versa.

[0106] In some cases, artificially introduced cysteine ​​bridges may improve the stability of the antigen-binding protein, optimally without interfering with the binding properties of the antigen-binding protein. Such cysteine ​​bridges may further improve heterodimerization.

[0107] Further amino acid substitutions, such as charge pair substitutions, to improve heterodimerization of the resulting protein are described in the art, for example in EP 2970484.

[0108] Thus, in one embodiment, for example, F c1 One F of a polypeptide such as c The domain comprises or further comprises the charge pair substitutions E356K, E356R, D356R, or D356K and D399K or D399R, e.g., F c2 F of other polypeptides such as cThe domain comprises or further comprises the charge pair substitutions R409D, R409E, K409E, or K409D and N392D, N392E, K392E, or K392D, or vice versa.

[0109] In a further embodiment, F on one or both, preferably both, of the polypeptide chains. c The domain is F c γ receptor (F c yR) binding. Such modifications may include L234A, L235A.

[0110] Hinge, C H2 and C H3 By including an Fc portion consisting of domains, or portions thereof, in an antigen-binding protein, more particularly in a bispecific antigen-binding protein, the F c :F c -γ receptor (F c The problem of non-specific immobilization of these molecules induced by γ-glucose (F c gR binds to different cell surface molecules (F c gRI, F c gRIIa, F c gRIIb, F c gRIII) bond, and F of the IgG molecule c Such non-specific (i.e., not induced by either of the two binding domains of the bispecific molecule) immobilization can have the following consequences: i) impact on the pharmacokinetics of the molecule, and ii) different Fc of immune effector cells. c -Unfavorable due to off-target activation of mutants, F c Mutations that eliminate gR binding 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,F c yRI and Fc The human IgG1 IgG1 binding (IL-1RIII binding) discloses the exchange of residues 233-236 of human IgG1 with the corresponding sequence from human IgG2, i.e., residues 233P, 234V, and 235A, in which there is no amino acid at position 236 and F c gRI binding is abolished, C1q binding is abolished, and F c GluRIII binding is reduced. EP 1075496 discloses c Antibodies and other Fs with diversity in the region (such as one or more of 233P, 234V, 235A, no residue or G at position 236, 327G, 330S, and 331S) c Containing molecules are disclosed in which the recombinant antibodies are capable of binding to a target molecule without inducing significant complement-dependent lysis or cell-mediated destruction of the target.

[0111] Thus, in some embodiments, F c The region comprises or further comprises one or more or deletions of amino acids 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 the amino acids 233P, 234V, 235A, 236 (no residues) or G; and one or more amino acids selected from the group consisting of 327G, 330S, 331S, and most preferably the Fc region comprises or further comprises the amino acids 233P, 234V, 235A, 236 (no residues) and 331S.

[0112] In a further embodiment, F c The domain comprises or further comprises the amino acid substitutions N297Q, N297G or N297A, preferably N297Q.

[0113] The amino acid substitutions "N297Q", "N297G", or "N297A" are c This refers to an amino acid substitution at position 297 that suppresses the native N-glycosylation site in the F domain.c This further prevents gamma receptor interactions and reduces variability in the final protein product, i.e., the antigen binding protein of the invention, due to sugar residues as described, for example, in Tao, MH and Morrison, SL (J Immunol. 1989 Oct 15;143(8):2595-601.).

[0114] A further embodiment, particularly without the light chain, is c The domain comprises or further comprises the amino acid substitution S220C. The amino acid substitution "S220C" is H1 The cysteines that form the CL disulfide bridges are deleted.

[0115] In some embodiments, F c The domain comprises or further comprises at least two additional cysteine ​​residues, such as, for example, S354C and Y349C or L242C and K334C, in which S354C is c1 F of one polypeptide such as c domain, and Y349C is F c2 F of other polypeptides such as c and / or in which L242C and K334C are present in one or both F domains of the polypeptide and form a heterodimer. c1 or F c2 Either of the same F c Located in the domain, it forms an intradomain CC bridge.

[0116] "Purified" and "isolated," when referring to a polypeptide (i.e., an antigen binding protein of the invention) or nucleotide sequence, mean that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" specifically means that at least 75%, 85%, 95%, or 98% by weight of the same type of biological macromolecules are present.

[0117] An "isolated" 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.

[0118] A "domain" may be any region of a protein, generally defined on the basis of sequence homology, and often associated with a particular structural or functional entity.

[0119] A "recombinant" molecule is a molecule prepared, expressed, produced, or isolated by recombinant means.

[0120] The term "gene" refers to a DNA sequence that codes for or corresponds to a specific sequence of amino acids comprising all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as, for example, promoter sequences, which determine the conditions under which the gene is expressed. Some genes that are not structural genes may 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 regulators of DNA transcription. In particular, the term gene may contemplate a genomic sequence that codes for a protein, i.e., a sequence comprising regulators, promoter, intron, and exon sequences.

[0121] "Affinity" is theoretically defined by the equilibrium binding between an antigen-binding protein and an antigen, in the context of the present invention, by the equilibrium binding between an antigen-binding protein and its antigen, i.e., a MAGE-A antigenic peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1 in complex with an MHC protein. Affinity can be measured, for example, by the half-maximal effective concentration (EC 50 ) or the equilibrium dissociation constant (K D ) may also be represented as:

[0122] "K D " is the equilibrium dissociation constant between the antigen-binding protein and its antigen, koff / k on is 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. Affinity, or K D Values ​​can be experimentally assessed by a variety of known methods, such as measuring association and dissociation rates using surface plasmon resonance (SPR) or biolayer interferometry (BLI), as described in detail below in the "Antigen Binding Proteins" section of this specification.

[0123] "EC 50 "Half-maximal effective concentration," also referred to as "EC," usually refers to the concentration of a molecule that elicits a response halfway between the baseline and maximum after a specified exposure time. 50 and affinity are inversely proportional, 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 maximum after a specified exposure time, and more specifically, refers to the concentration of an antigen binding protein of the invention that induces a response halfway between the baseline and maximum after a specified exposure time. EC 50 The value can be assessed experimentally by a variety of known methods, such as, for example, using an IFN-γ release assay or an LDH release assay, as described in detail below in the "Antigen Binding Proteins" section of this specification.

[0124] "CD3" is a protein complex, composed of four different chains. In mammals, the complex contains the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains bind to a molecule known as the T cell receptor (TCR) and the ζ chain, generating an activation signal in T lymphocytes.

[0125] "CD28" is also expressed on T cells and can provide 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.

[0126] "CD134" is also referred to as OX40. CD134 / OX40 is expressed 24-72 hours following activation and is thought to define a secondary costimulatory molecule.

[0127] "4-1BB" can bind to a 4-1BB ligand on antigen-presenting cells (APCs), thereby generating a costimulatory signal for T cells.

[0128] "CD5" is another example of a receptor found primarily on T cells, although CD5 is also found at lower levels on B cells.

[0129] "CD95" is a further example of a receptor that modulates T cell function and is also known as the Fas 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.

[0130] The "NK cell-specific receptor molecule" is, for example, CD16, low affinity F c receptor, and NKG2D.

[0131] 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 and NK cells.

[0132] In the context of the present invention, the "transmembrane region" referred to herein above in (ii) may for example be the TCR α or β transmembrane domain.

[0133] The "cytoplasmic signaling region" may be, for example, a TCR alpha or beta intracellular domain.

[0134] "Diagnostic agent" herein refers to a detectable molecule or substance that provides a signal (directly or indirectly), such as a fluorescent molecule, a radioactive molecule, or any other label known in the art.

[0135] "Fluorescent molecules" known in the art include fluorescein isothiocyanate (FITC), phycoerythrin (PE), fluorophores for use with blue lasers (e.g., PerCP, PE-Cy7, PE-Cy5, FL3 and APC or Cy5, FL4), fluorophores for use with red, violet, or UV lasers (e.g., Pacific Blue, Pacific Orange).

[0136] As a "radioactive molecule", I 123 ,I 124 , In 111 , Re 186 , Re 188 , Tc 99 Antigen binding proteins of the invention may also comprise spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0137] Such diagnostic agents may be directly bound (ie, physically linked) or indirectly bound to the antigen binding protein.

[0138] "Therapeutic agent" herein refers to an agent having a therapeutic effect. In one embodiment, such a therapeutic agent may be a growth inhibitory agent, such as a cytotoxic agent or a radioisotope.

[0139] "Growth inhibitory agent" or "anti-proliferative agent", which may be used indiscriminately, refers to a compound or composition that inhibits the growth of cells, particularly tumor cells, either in vitro or in vivo.

[0140] 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, such as small molecule toxins 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 analogue, such as DM1 or DM4, a small drug, tomaymycin or pyrrolobenzodiazepine derivative, cryptophycin derivative, leptomycin derivative, auristatin or dolastatin analogue, prodrug, topoisomerase II inhibitor, DNA alkylating agent, antitubulin agent, CC-1065 or CC-1065 analogue.

[0141] The term "radioisotope" means 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 suitable for cancer therapy, such as radioisotopes of thorium-227, thorium-227, and thorium-227, which emit alpha radiation. Such radioisotopes generally emit primarily beta radiation. In one embodiment, the radioisotope is an alpha-radioisotope, more precisely, thorium-227, which emits alpha radiation.

[0142] A "PK-modifying moiety" herein refers to a moiety that modifies the pharmacokinetics (PK) of the antigen-binding protein of the present invention. Thus, the moiety in particular alters the in vivo half-life and distribution of the antigen-binding protein of the present invention. In a preferred embodiment, the PK-modifying moiety extends the half-life of the antigen-binding protein. Examples of PK modification moieties include 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), Pasylations (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-terminal cleavage of antibodies and / or unstructured polypeptides. c moieties (Schellenberger et al., (2009) Nat Biotechnol. Dec;27(12):1186-90).

[0143] "Half-life (T 1 / 2 "Half-life" generally refers to the time required for half the amount of a drug or other substance deposited in a living body to be metabolized or eliminated by normal biological processes. Thus, the half-life in the context of the present invention refers to the time required for half the amount of an antigen-binding protein of the present invention to be eliminated, for example, in a mouse. Half-life can be determined by methods well known in the art. In one example, the half-life is determined as described in detail in Example 10. Thus, in one example, the half-life is determined, for example, by administering the antigen-binding protein of the present invention to a mouse, such as a highly immunodeficient NOG mouse, by taking plasma samples from the occipital plexus at different time points, such as 0 hours, 0.1 hours, 2 hours, 8 hours, 24 hours, 48 ​​hours, 120 hours, 240 hours, 360 hours, etc., after intravenous administration. The plasma levels of the antigen-binding protein are then measured using techniques known in the art, such as ELISA, to determine the F c-V L The plasma concentration of the sample is typically obtained by interpolation from the respective standard curve, and the half-life is typically determined by linear regression of the plasma levels taken at different time points.

[0144] Antigen-binding proteins Using the TCR R7P1D5 as disclosed in WO 2017 / 158103 as a starting point, the inventors designed, generated and tested TCR variable alpha (Valpha) and optionally variable beta (Vbeta) domain variants in single chain (scTCR) form, using yeast screening assays as described in Examples 1 and 2 herein, or in single chain bispecific TCR constructs, and further optimized the binding affinity, as well as the yield and stability of soluble bispecific scTCR constructs as described in Example 4 herein. In this way, the inventors identified different variants of the variable alpha and optionally beta domains, more specifically of the CDRa3 of the variable alpha domain (there are 5 variants), which are linked to the CDRa1, CDRa2, CDRb1, CDRb2 and / or CDRb3 of the parent TCR, or to the antigen binding protein of the invention targeted, i.e. in complex with an MHC protein, preferably HLA-A. * It may be advantageously combined with any of the CDR variants discovered for CDRa1 (one variant), CDRa2 (three variants), CDRb1 (one variant), CDRb2 (five variants) and / or CDRb3 (one variant), which variants are advantageous for binding with high affinity as well as high specificity to a MAGE-A antigenic peptide comprising the amino acid sequence of SEQ ID NO: 1 in a complex with 02.

[0145] The inventors have demonstrated in the examples that the CDRs may be used in TCRs of ACT, single chain TCR constructs as well as other bispecific formats, and thus the identified CDR variants can be used to generate TCRs, preferably HLA-A, in complex with MHC proteins, with high affinity as well as high specificity for MAGE-A antigenic peptides comprising the amino acid sequence of SEQ ID NO:1. * Proof-of-principle demonstrated that different antigen-binding proteins in complex with 02 could be generated.

[0146] Furthermore, the inventors have discovered that mutating the framework of the TCR variable alpha (Valpha) and variable beta (Vbeta) domains improves the binding specificity, as well as the yield and stability, of soluble bispecific scTCR constructs, as described herein in Example 4.

[0147] In Examples 5-8, the inventors have investigated the human TCR-CD3 complex and, for example, HLA-A * We have designed, expressed and successfully tested the in vitro and in vivo efficacy against tumor cell lines of an antigen binding protein in the form of a TCER™ that specifically binds to a peptide:MHC complex comprising the MAGE-A peptide of SEQ ID NO:1 bound to an MHC class I molecule such as 02:01. To target BMA031, we have used the TCR-CD3 complex V, derived from the CD3-specific humanized antibody hUCHT1 described by Zhu et al. (J Immunol, 1995, 155, 1903-1910). H and V L domains, or an α / β TCR specific antibody described by Shearman et al. (J Immunol, 1991, 147, 4366-73) was modified and used in these examples. Stability and affinity maturation of antigen binding proteins may be provided using Vα and Vβ domains comprising the CDRs disclosed herein to target the MAGE-A:MHC complex. The same VL and VH, Vα and Vβ domains may be combined with Fab domains (CDRs) as described in Example 4. H1 Hinge and CL The human single chain T cell receptor format was used, further comprising:

[0148] Thus, the present invention refers to an antigen binding protein that specifically binds to a MAGE-A antigenic peptide comprising or consisting of the amino acid sequence of SEQ ID NO:1 "KVLEHVVRV", wherein said antigenic peptide is in a complex with an MHC protein, and wherein the antigen binding protein (a) a first polypeptide chain comprising a first variable domain comprising three complementarity determining regions (CDRs) CDRa1, CDRa2, and CDRa3; and (b) a second polypeptide chain comprising a second variable domain comprising three complementarity determining regions (CDRs): CDRb1, CDRb2, and CDRb3. comprising CDRa1 comprises or consists of the amino acid sequence "X1SSSTY" SEQ ID NO: 72, where X1 is any amino acid, preferably D or E, more preferably E, or an amino acid sequence which differs from SEQ ID NO: 72 by at least one amino acid substitution, preferably by one, two or three amino acid substitutions, preferably by one or two amino acid substitutions, such as one amino acid substitution, -CDRa2 comprises or consists of the amino acid sequence "IX1SX2X3DX4" SEQ ID NO: 73, wherein X1 to X4 are any amino acid, wherein preferably X1 is F or Y, more preferably Y, X2 is N or S, preferably S, X3 is Q or M, preferably Q, and X4 is M, V, S or Q, preferably S or Q, more preferably Q; - CDRa3 comprises or consists of the amino acid sequence "CAEX1X2SX3SKIIF" SEQ ID NO: 77, where X1 to X3 are any amino acids, with the proviso that if CDRa1 comprises or consists of the amino acid sequence SEQ ID NO: 5 and CDRa2 comprises or consists of the amino acid sequence SEQ ID NO: 6, then CDRa3 does not comprise or consist of the amino acid sequence "CAEYSSASKIIF" (SEQ ID NO: 7), preferably X1 is A, F or M, more preferably M, preferably X2 is S, T or N, more preferably T, preferably X3 is E or A, preferably E, with the proviso that CDRa3 does not comprise or consist of the amino acid sequence "CAEYSSASKIIF" (SEQ ID NO: 7), - CDRa3 comprises or consists of the amino acid sequence "CAEX1X2SX3SKIIF" SEQ ID NO: 77, wherein X1 to X3 are any amino acid, preferably X1 is F, M or A, preferably M, preferably X2 is S, T or N, more preferably T, preferably X3 is E or A, preferably E, - CDRb1 comprises or consists of the amino acid sequence "X1GHD" SEQ ID NO: 78, where X1 is any amino acid, preferably S or P, more preferably P, or an amino acid sequence which differs from SEQ ID NO: 78 by at least one amino acid substitution, preferably one or two amino acid substitutions, such as one amino acid substitution, -CDRb2 comprises or consists of the amino acid sequence "FX1X2X3X4P" SEQ ID NO: 79, wherein X1 to X4 are any amino acid, preferably X1 is C or N, more preferably C, preferably X2 is Y or N, more preferably Y, preferably X3 is G or N, more preferably G, preferably X4 is H, V, T, A or M, preferably H or T, most preferably T, wherein preferably, when X1 is C, the FR3-b amino acid sequence comprises the amino acid 66C at amino acid position 66; - CDRb3 comprises or consists of the amino acid sequence "CASRAX1TGELFF" SEQ ID NO: 82, where X1 is any amino acid, preferably D or N, preferably D, or an amino acid sequence which differs from SEQ ID NO: 82 by at least one amino acid substitution, preferably by one, two or three amino acid substitutions, preferably by one or two amino acid substitutions, such as one amino acid substitution.

[0149] In one particular embodiment, the present invention refers to an antigen binding protein that specifically binds to a MAGE-A antigenic peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 1, wherein said antigenic peptide is in a complex with an MHC protein, and wherein the antigen binding protein comprises (a) a first polypeptide chain comprising a first variable domain comprising three complementarity determining regions (CDRs) CDRa1, CDRa2, and CDRa3; (b) a second polypeptide chain comprising a second variable domain comprising three complementarity determining regions (CDRs): CDRb1, CDRb2, and CDRb3. comprising CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence which differs from SEQ ID NO: 5 by at least one amino acid substitution, preferably by one, two or three amino acid substitutions, preferably by one or two amino acid substitutions, such as one amino acid substitution, CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence which differs from SEQ ID NO: 6 by at least one amino acid substitution, preferably by one, two or three amino acid substitutions, preferably by one or two amino acid substitutions, such as one amino acid substitution, - CDRa3 comprises or consists of the amino acid sequence "CAEX1X2SX3SKIIF" SEQ ID NO: 77, where X1 to X3 are any amino acids, with the proviso that if CDRa1 comprises or consists of the amino acid sequence SEQ ID NO: 5 and CDRa2 comprises or consists of the amino acid sequence SEQ ID NO: 6, then CDRa3 does not comprise or consist of the amino acid sequence "CAEYSSASKIIF" (SEQ ID NO: 7), preferably X1 is A, F or M, more preferably M, preferably X2 is S, T or N, more preferably T, and preferably X3 is E or A, preferably E, with the proviso that CDRa3 does not comprise or consist of the amino acid sequence "CAEYSSASKIIF" (SEQ ID NO: 7), - CDRa3 comprises or consists of the amino acid sequence "CAEX1X2SX3SKIIF" SEQ ID NO: 77, wherein X1 to X3 are any amino acid, preferably X1 is F, M or A, preferably M, preferably X2 is S, T or N, more preferably T, preferably X3 is E or A, preferably E, - CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence which differs from SEQ ID NO: 12 by at least one amino acid substitution, preferably by one, two or three amino acid substitutions, preferably by one or two amino acid substitutions, such as one amino acid substitution, - CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence which differs from SEQ ID NO: 13 by at least one amino acid substitution, preferably by one, two or three amino acid substitutions, preferably by one or two amino acid substitutions, such as one amino acid substitution, - CDRb3 comprises or consists of the amino acid sequence SEQ ID NO: 14 or an amino acid sequence which differs from SEQ ID NO: 14 by at least one amino acid substitution, preferably one or two amino acid substitutions, such as one, two, three or four amino acid substitutions, preferably one amino acid substitution.

[0150] In one particular embodiment, the present invention refers to an antigen binding protein, such as a soluble antigen binding protein, that specifically binds to a MAGE-A antigenic peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 1, wherein said antigenic peptide is in a complex with an MHC protein, and wherein the antigen binding protein is (a) a first polypeptide chain comprising a first variable domain comprising three complementarity determining regions (CDRs) CDRa1, CDRa2, and CDRa3; and (b) a second polypeptide chain comprising a second variable domain comprising three complementarity determining regions (CDRs): CDRb1, CDRb2, and CDRb3. comprising CDRa1 comprises or consists of the amino acid sequence "X1SSSTY" SEQ ID NO: 72, where X1 is any amino acid, preferably D or E, more preferably E, or an amino acid sequence which differs from SEQ ID NO: 72 by at least one amino acid substitution, preferably by one, two or three amino acid substitutions, preferably by one or two amino acid substitutions, such as one amino acid substitution, - CDRa2 comprises or consists of the amino acid sequence "IYSSQDX1" SEQ ID NO: 74, wherein X1 is any amino acid, preferably Q, V or S, preferably Q; CDRa3 comprises or consists of the amino acid sequence "CAEMTSESKIIF" SEQ ID NO: 35 or an amino acid sequence which differs from SEQ ID NO: 35 by at least one amino acid substitution, preferably one, two, three or four amino acid substitutions, preferably one or two amino acid substitutions, such as one amino acid substitution, - CDRb1 comprises or consists of the amino acid sequence "X1GHDY" SEQ ID NO: 78, where X1 is any amino acid, preferably S or P, more preferably P, or an amino acid sequence which differs from SEQ ID NO: 78 by at least one amino acid substitution, preferably by one, two or three amino acid substitutions, preferably by one or two amino acid substitutions, such as one amino acid substitution, - CDRb2 comprises or consists of the amino acid sequence "FCYGX1P" SEQ ID NO: 81, wherein X1 is any amino acid, preferably H, V, A or T, preferably T, or an amino acid sequence which differs from SEQ ID NO: 81 by at least one amino acid substitution, preferably by one or two amino acid substitutions, more preferably by one amino acid substitution, wherein preferably when CDRb2 comprises or consists of the amino acid sequence "FCYGX1P" SEQ ID NO: 81, the FR3-b amino acid sequence comprises the amino acid 66C at amino acid position 66, - CDRb3 comprises or consists of the amino acid sequence "CASRAX1TGELFF" SEQ ID NO: 82, where X1 is any amino acid, preferably D or N, preferably D, or an amino acid sequence which differs from SEQ ID NO: 82 by at least one amino acid substitution, preferably by one, two, three or four amino acid substitutions, preferably by one or two amino acid substitutions, such as one amino acid substitution.

[0151] As indicated herein above, in some embodiments, the CDRb2 amino acid sequence comprises the amino acid 57C at position 57, which preferably forms a disulfide bridge with the cysteine ​​preferably present at position 66 of the FR3b amino acid sequence. Thus, in a preferred embodiment, if the CDRb2 amino acid sequence comprises the amino acid 57C at position 57, then the FR3-b amino acid sequence preferably comprises the amino acid 66C at amino acid position 66, and vice versa. Thus, in some embodiments, the FR3b amino acid sequence preferably comprises an amino acid substitution at this position 66, and thus in some related embodiments the FR3b amino acid sequence comprises the amino acid substitution I66C.

[0152] In some embodiments, CDRa1 in the context of the present invention comprises or consists of the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:55.

[0153] In one embodiment, CDRa2 in the context of the present invention comprises or consists of the amino acid sequence: "IYSSQDX1" SEQ ID NO:74, wherein X1 is any amino acid, preferably Q, V or S, more preferably Q, or SEQ ID NO:6, or an amino acid sequence which differs from SEQ ID NO:74 or SEQ ID NO:6 by at least one amino acid substitution, such as one, two or three amino acid substitutions, more preferably one amino acid substitution; preferably CDRa2 comprises or consists of the amino acid sequence "IYSSQDX1" SEQ ID NO:74, wherein X1 is any amino acid, preferably Q, V or S, more preferably Q, or an amino acid sequence which differs from SEQ ID NO:74 by at least one amino acid substitution, such as one, two or three amino acid substitutions, more preferably one amino acid substitution.

[0154] In some embodiments, CDRa2 in the context of the present invention comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:6 and SEQ ID NO:56, 57, and 59, preferably SEQ ID NO:6 and SEQ ID NO:56,

[0155] In some embodiments, the CDRa3 comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:35, 36, 37, 38, 39, preferably SEQ ID NO:35.

[0156] In some embodiments, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO:12 or SEQ ID NO:62.

[0157] In some embodiments, CDRb2 has the amino acid sequence: "FNNNVP" SEQ ID NO: 13, or - "FCYGX1" sequence number 81 comprising or consisting of wherein X1 is any amino acid, preferably H, V, A or T, more preferably T (wherein preferably, when CDRb2 comprises or consists of the amino acid sequence "FCYGX1P" SEQ ID NO: 81, the FR3-b amino acid sequence comprises the amino acid 66C at amino acid position 66); or The amino acid sequence differs from SEQ ID NO: 13 or 81 by at least one amino acid substitution, such as one, two or three amino acid substitutions, more preferably by one amino acid substitution.

[0158] In some embodiments, CDRb2 in the context of the present invention comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequences SEQ ID NO: 13 and SEQ ID NO: 63, 64, 65, 66 and 70, preferably SEQ ID NO: 13 and SEQ ID NO: 65, wherein preferably, when CDRb2 comprises or consists of the amino acid SEQ ID NO: 63, 64, 65, 66 or 70, the FR3-b amino acid sequence preferably comprises the amino acid 66C at amino acid position 66.

[0159] In one embodiment, CDRb3 in the context of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO:71.

[0160] In one particular embodiment, the antigen binding protein of the invention comprises: (a) a first polypeptide chain comprising a first variable domain comprising three complementarity determining regions (CDRs) CDRa1, CDRa2, and CDRa3; (b) a second polypeptide chain comprising a second variable domain comprising three complementarity determining regions (CDRs): CDRb1, CDRb2, and CDRb3. comprising - CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 5, - CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO: 6, CDRa3 comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 35, 36, 37, 38, 39, preferably SEQ ID NOs: 35, 36, 37, 38, more preferably SEQ ID NO: 35 and 38, such as SEQ ID NO: 35, - CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 12, - CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 13, - CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0161] In one particular embodiment, the antigen binding protein is (a) a first polypeptide chain comprising a first variable domain comprising three complementarity determining regions (CDRs) CDRa1, CDRa2, and CDRa3; (b) a second polypeptide chain comprising a second variable domain comprising three complementarity determining regions (CDRs): CDRb1, CDRb2, and CDRb3. comprising - CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 55, preferably SEQ ID NO: 55, CDRa2 comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 56, 57 and 59, preferably SEQ ID NOs: 56 and 59, more preferably SEQ ID NO: 56, - CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 35, - CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO: 62, - CDRb2 comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 63, 64, 65, 66 and 70, preferably SEQ ID NO: 65, wherein preferably, when CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO: 63, 64, 65, 66 or 70, the FR3-b amino acid sequence preferably comprises the amino acid 66C at amino acid position 66, - CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 71, preferably SEQ ID NO: 71.

[0162] The present invention further refers to antigen binding proteins comprising variants of the CDR amino acid sequences disclosed in the context of the present invention, typically variants of CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and / or CDRb3, such variants may contain at least one, such as four, three, two or one, preferably one, two or three amino acid substitutions, within which the preferred number of amino acid substitutions preferably depends on the length of the respective CDR.

[0163] Thus, in some embodiments, the CDRa1 comprises or consists of an amino acid sequence that differs from the CDRa1 amino acid sequence disclosed herein by at least one amino acid substitution, preferably one, two or three amino acid substitutions, preferably one or two amino acid substitutions, or only one amino acid substitution, wherein the amino acid substitution is preferably at any of amino acid positions 28, 29, 36, 37 and 38, more preferably at position 28.

[0164] In some embodiments, the CDRa2 comprises or consists of an amino acid sequence that differs from a CDRa2 amino acid sequence disclosed herein by at least one amino acid substitution, preferably one, two or three amino acid substitutions, preferably one or two amino acid substitutions, or only one amino acid substitution, wherein the amino acid substitution is preferably at position 56 or 65, more preferably at position 65.

[0165] In some embodiments, the CDRa3 comprises or consists of an amino acid sequence that differs from a CDRa3 amino acid sequence disclosed herein by at least one amino acid substitution, preferably one, two, three or four amino acid substitutions, preferably one or two amino acid substitutions, or only one amino acid substitution, wherein the amino acid substitution is preferably at any of amino acid positions 105, 106, 107, 108, 109, 113, 114, 115, 116, and 117.

[0166] In some embodiments, CDRb1 comprises or consists of an amino acid sequence that differs from the CDRb1 amino acid sequence disclosed herein by at least one amino acid substitution, preferably one, two or three amino acid substitutions, preferably one or two amino acid substitutions, such as one amino acid substitution, wherein said amino acid substitutions are preferably at any of amino acid positions 27, 28, 29, 37 and 38, more preferably at positions 27 and 38.

[0167] In some embodiments, CDRb2 comprises or consists of an amino acid sequence that differs from the CDRb2 amino acid sequence disclosed herein by at least one amino acid substitution, preferably one, two or three amino acid substitutions, preferably one or two amino acid substitutions, or only one amino acid substitution, wherein said amino acid substitution is preferably at position 56.

[0168] In some embodiments, the CDRb3 comprises or consists of an amino acid sequence that differs from the CDRb3 amino acid sequence disclosed herein by at least one amino acid substitution, preferably one, two, three or four amino acid substitutions, preferably one or two amino acid substitutions, or only one amino acid substitution, wherein said amino acid substitution is preferably at any of amino acid positions 105, 106, 107, 108, 109, 113, 114, 115, 116, 117.

[0169] The amino acid substitutions referred to herein above are defined in the "Definitions" section herein above, and preferably, the amino acid substitutions are conservative amino acid substitutions. As will be appreciated by those skilled in the art, the first variable domain comprising the CDR amino acid sequence defined herein and the second variable domain comprising the CDR amino acid sequence defined herein together form an antigen binding site, in which said antigen binding site binds to a MAGE-A antigenic peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 1 in complex with an MHC protein. The three complementarity determining regions (CDRs), CDRa1, CDRa2, and CDRa3, are derived from the variable α domain of the TCR. Thus, as will be appreciated by those skilled in the art, in some embodiments, the first variable domain may also be referred to as a variable α domain. Similarly, the three complementarity determining regions (CDRs), CDRb1, CDRb2, and CDRb3, are derived from the variable β domain of said TCR, and thus, as will be appreciated by those skilled in the art, the second variable domain may also be referred to as a variable β domain. More specifically, a first variable domain comprising an alpha variable framework amino acid sequence and an alpha variable CDR as defined in the context of the present invention, such as the sequence (FR1-a)-(CDRa1)-(FR2-a)-(CDRa2)-(FR3-a)-(CDRa3)-(FR4-a), may be referred to as a variable alpha domain and / or a second variable domain comprising a beta variable framework amino acid sequence and a beta variable CDR as defined in the context of the present invention, such as the sequence (FR1-b)-(CDRb1)-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)-(FR4-b), may be referred to as a variable beta domain.

[0170] In some embodiments, the CDRs may be grafted onto antibody framework sequences. Thus, in one example, the CDRs defined in the context of a first variable domain may be grafted onto a light chain variable domain, and thus the first variable domain may comprise or consist of the sequence (FR1-L)-(CDRa1)-(FR2-L)-(CDRa2)-(FR3-L)-(CDRa3)-(FR4-L) and be referred to as a light chain variable domain. In the same example, the CDRs defined in the context of a second variable domain may be grafted onto a heavy chain variable domain, and thus the second variable domain may comprise or consist of the sequence (FR1-H)-(CDRb1)-(FR2-H)-(CDRb2)-(FR3-H)-(CDRb3)-(FR4-H) and be referred to as a heavy chain variable domain. However, it will be appreciated by the skilled artisan that CDRs defined in the context of a first variable domain may also be grafted into a heavy chain variable domain and CDRs defined in the context of a second variable domain may be grafted into a light chain variable domain, and the above considerations apply mutatis mutandis.

[0171] In the context of the present invention, the CDRs of the antigen binding proteins of the present invention differ from the CDRs of the natural and parent TCR R7P1D5 at least in the amino acid sequence of the CDRa3 of said parent TCR R7P1D5, as disclosed in WO 2017 / 158103. As the skilled person will appreciate, the CDRa3 as defined herein above does not comprise or consist of the amino acid sequence "CAEYSSASKIIF" (SEQ ID NO: 7), which is the CDRa3 of the parent TCR R7P1D5.

[0172] In one embodiment, the first variable domain and the second variable domain as defined herein in the context of the antigen binding protein of the invention may comprise an amino acid substitution at position 44 according to the IMGT numbering. In a preferred embodiment, said amino acid at position 44 is substituted with another suitable amino acid to improve pairing. In a particular embodiment, preferably when said antigen binding protein is a TCR, said amino acid substitution improves, for example, chain pairing (i.e., α and β chain pairing or γ and δ chain pairing in a preferred embodiment), and one or both of the amino acid present at position 44 in the first variable domain (v144), the amino acid present at position 44 in the second variable domain n(v244) are v144D / v244R, v1 44R / v244D, v1 The substitution is made in the amino acid pair v144 / v244 selected from the group of amino acid pairs consisting of: v144E / v244K, v144K / v244E, v144D / v244K, v144K / v244D, v144R / v244E; v144E / v244R, v144L / v244W, v144W / v244L, v144V / v244W, v144W / v244V.

[0173] Thus, in a further embodiment, the antigen binding protein is selected from the group consisting of: v1Q44D / v2Q44R; v1Q44R / v2Q44D; v1Q44E / v2Q44K; v1Q44K / v2Q44E; v1Q44D / v2Q44K; v1Q44K / v2Q44D; v1Q44E / v2Q44R; v1Q44R / v2Q44E; v1Q44L / v2Q44W; v1Q44W / v2Q44L; v1Q44V / v2Q44W; and v1Q44W / v2Q44V; v1W44D / v2Q44R; v1W44R / v2Q44D; v1W44E / v2Q44K; v1W44K / v2 Q44E;v1W44D / v2Q44K;v1W44K / v2Q44D;v1W44E / v2Q44R;v1W44R / v2Q44E;v1W44L / v2Q44W;v1W44 / v2Q44L;v1W44V / v2Q44W; and v1W44 / v2Q44V;v1H44D / v2Q44R;v1H44R / v2Q44D;v1H44E / v2Q44K;v1H44K / v2Q44E;v1H44D / v2Q44K ;v1H44K / v2Q44D;v1H44E / v2Q44R;v1H44R / v2Q44E;v1H44L / v2Q44W;v1H44W / v2Q44L;v1H44V / v2Q44W;and v1H44W / v2Q44V;v1K44D / v2Q44R;v1K44R / v2Q44D;v1K44E / v2Q44K;v1K44 / v2Q44E;v1K44D / v2Q44K;v1K44 / v2Q44D;v1K4 4E / v2Q44R;v1K44R / v2Q44E;v1K44L / v2Q44W;v1K44W / v2Q44L;v1K44V / v2Q 44W; and v1K44W / v2Q44V;v1E44D / v2Q44R;v1E44R / v2Q44D;v1E44 / v2Q44K;v 1E44K / v2Q44E;v1E44D / v2Q44K;v1E44K / v2Q44D;v1E44 / v2Q44R;v1E44R / v2Q44E;v1E44L / v2Q44W;v1E44W / v2Q44L;v1E44V / v2Q44W;and v1E44W / v2Q44 V;v1Q44D / v2R44;v1Q44R / v2R44D;v1Q44E / v2R44K;v1Q44K / v2R44E;v1Q44D / v2R44K;v1Q44K / v2R44D;v1Q44E / v2R44;v1Q44R / v2R44E;v1Q44L / v2R44W;v1Q44W / v2R44L;v1Q44V / v2R44W;and v1Q44W / v2R44V;v1W44D / v2R44;v1W44R / v2R44D;v1W44E / v2R44K;v1W44K / v2R44E;v1W44D / v2R44K;v1W44K / v2R44D;v1W44E / v2R44;v1W44R / v2R44E;v1W44L / v2R44W;v1W44 / v2R44L;v1W44V / v2R44W;and v1W44 / v2R44V;v1H44D / v2R44;v1H44R / v2R44D;v1H44E / v2R 44K;v1H44K / v2R44E;v1H44D / v2R44K;v1H44K / v2R44D;v1H44E / v2R44;v1H44R / v2R44E;v1H44L / v2R44W;v1H44W / v2R44L;v1H44V / v2R44W;and v1H44W / v2R44V;v1K44D / v2R44;v1K44R / v2R44D;v1K44E / v2R44K;v1K44 / v2R44E; v1K44D / v2R44K;v1K44 / v2R44D;v1K44E / v2R44;v1K44R / v2R44E;v1K44L / v2 R44W;v1K44W / v2R44L;v1K44V / v2R44W;and v1K44W / v2R44V;v1E44D / v2R44;v1E44R / v2R44D;v1E44 / v2R44K;v1E44K / v2R44E;v1E44D / v2R44K;v1E44K / v2R44D;v1E44R / v2R44E;v1E44L / v2R44W;v1E44W / v2R44L;v1E44V / v2R44W;and v1E44W / v2R44V;v1Q44D / v2K44R;v1Q44R / v2K44D;v1Q44E / v244K;v1Q 44K / v2K44E;v1Q44D / v244K;v1Q44K / v2K44D;v1Q44E / v2K44R;v1Q44R / v2K44E;v1Q44L / v2K44W;v1Q44W / v2K44L;v1Q44V / v2K44W;and v1Q44W / v2K44V ;v1W44D / v2K44R;v1W44R / v2K44D;v1W44E / v244K;v1W44K / v2K44E;v1W44D / v244K;v1W44K / v2K44D;v1W44E / v2K44R;v1W44R / v2K44E;v1W44L / v2K44W;v1W44 / v2K44L;v1W44V / v2K44W;and v1W44 / v2K44V;v1H44D / v2K44R;v1H44R / v2K44D;v1 H44E / v244K;v1H44K / v2K44E;v1H44D / v244K;v1H44K / v2K44D;v1H44E / v2K44R;v1H44R / v 2K44E;v1H44L / v2K44W;v1H44W / v2K44L;v1H44V / v2K44W;and v1H44W / v2K44V;v1K44D / v2K44R;v1K44R / v2K44D;v1K44E / v244K;v1K44 / v2K44E;v1K44D / v244K;v1K44 / v2K44D;v1K 44E / v2K44R;v1K44R / v2K44E;v1K44L / v2K44W;v1K44W / v2K44L;v1K44V / v2K44W;and v1K 44W / v2K44V;v1E44D / v2K44R;v1E44R / v2K44D;v1E44 / v244K;v1E44K / v2K44E;v1E44D / v2 v1E44V / v2K44W; and v1E44W / v2Q44V.

[0174] In the above, for example, "v1Q44R / v2Q44D" is intended to mean that in the first variable domain, Q44 is substituted with R, and in the second variable domain, Q44 is substituted with D. Additional substitutions and explanations are described in US Patent Application Publication No. 2018-0162922.

[0175] The inventors of the present invention have demonstrated that CDRs derived initially from TCR alpha and beta variable domains may be used in antigen binding proteins having a different conformation from the native TCR. For example, in the experimental section, the inventors used the CDRs of the TCR alpha and beta variable domains in the TCRE™ conformation, as described in detail herein below and shown in the Examples (i.e., Example 6).

[0176] Furthermore, the present inventors have demonstrated that in bispecific TCRs comprising scTCRs or H (CD3)-C H1 -Vα-L z a first polypeptide comprising Vβ and a V L (CD3)-C L The CDRs as defined in the context of the present invention have been used in single chain TCR constructs, such as Fab stabilized scTCRs, comprising a second polypeptide comprising V H (CD3), V L (CD3), Vα, and Vβ are defined above, and L z is a linker consisting of the amino acid sequence of SEQ ID NO: 108, GGGGSGGGGSGGGGSGGGGSGGGGGS; H1 is the constant domain of the heavy chain, L is the light chain constant region (Example 4).

[0177] Thus, the skilled artisan will appreciate from these experiments as proof of principle that the CDRs defined herein in the context of the present invention may be used in different antigen binding proteins of the present invention and therefore in different formats as disclosed herein. The skilled artisan will further appreciate that the binding properties of the antigen binding proteins of the present invention, demonstrated in the context of one antigen binding format, are preferably at least conserved when the format of the antigen binding protein is changed (i.e. the CDR amino acid sequences of at least the first and second variable domains, preferably the amino acids of the first and second variable domains are identical).

[0178] As already mentioned above, the binding functionality of the CDRs in the context of the present invention may be provided in the framework of an antibody. For example, the CDR amino acid sequences defined in the context of the present invention, possibly including 3, 2 or 1 additional N- and / or C-terminal framework residues, may be directly grafted onto an antibody variable heavy / light chain sequence. More specifically, the CDRa1, CDRa2 and CDRa3 domains defined in the context of the present invention may be grafted onto a variable heavy chain amino acid sequence, and the CDRb1, CDRb2 and CDRb3 domains defined in the context of the present invention may be grafted onto a variable light chain amino acid sequence, or vice versa.

[0179] It will also be appreciated that in some embodiments, within an antibody, the variable light chain domain of the antibody may be replaced by a first variable domain as defined in the context of the present invention, and the variable heavy chain domain may be replaced by a second variable domain as defined in the context of the present invention, or vice versa.

[0180] However, in a preferred embodiment, the antigen binding protein further comprises an alpha framework region and a beta framework region.

[0181] In one embodiment, said first variable domain further comprises one or more framework regions, preferably FR1-a, FR2-a, FR3-a, and FR4-a selected from the group consisting of: -FR1-a comprises or consists of the amino acid sequence "EDVEQSLFLSVREGDSSVINCTYT" SEQ ID NO: 83 or an amino acid sequence that is at least 85% identical to SEQ ID NO: 83; -FR2-a comprises or consists of the amino acid sequence of "LYWYKQEPGAGLQLLTY" SEQ ID NO: 84, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 84; -FR3-a comprises or consists of the amino acid sequence of SEQ ID NO: 85 "KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF" or an amino acid sequence that is at least 85% identical to SEQ ID NO: 85; - FR4-a comprises or consists of the amino acid sequence of "GSGTRLSIRP" SEQ ID NO: 86, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 86; The second variable domain further comprises one or more framework regions, preferably FR1-b, FR2-b, FR3-b, and FR4-b selected from the group consisting of FR1-b, FR2-b, FR3-b, and FR4-b, wherein -FR1-b comprises or consists of the amino acid sequence "DAGVIQSPRHEVTEMGQEVTLRCKPI" SEQ ID NO: 87 or an amino acid sequence that is at least 85% identical to SEQ ID NO: 87; -FR2-b comprises or consists of the amino acid sequence of "LFWYRQTMMRGLELLIY" SEQ ID NO: 88 or an amino acid sequence that is at least 85% identical to SEQ ID NO: 88; -FR3-b comprises or consists of the amino acid sequence "IDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYF" SEQ ID NO: 89, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 89; or -FR4-b comprises or consists of the amino acid sequence of "GEGSRLTVL" SEQ ID NO:90, or an amino acid sequence that is at least 85% identical to SEQ ID NO:90.

[0182] In one embodiment, said first variable domain further comprises one or more framework regions, preferably FR1-a, FR2-a, FR3-a, and FR4-a selected from the group consisting of: -FR1-a comprises or consists of the amino acid sequence of "EDVEQSLFLSVREGDSSVINCTYT" SEQ ID NO: 83, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 83. -FR2-a comprises or consists of the amino acid sequence of "LYWYKQEPGAGLQLLTY" SEQ ID NO: 84, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 84; -FR3-a comprises or consists of the amino acid sequence of SEQ ID NO: 85 "KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF" or an amino acid sequence that is at least 90% identical to SEQ ID NO: 85; - FR4-a comprises or consists of the amino acid sequence of "GSGTRLSIRP" SEQ ID NO: 86, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 86; The second variable domain further comprises one or more framework regions, preferably FR1-b, FR2-b, FR3-b, and FR4-b selected from the group consisting of: -FR1-b comprises or consists of the amino acid sequence "DAGVIQSPRHEVTEMGQEVTLRCKPI" SEQ ID NO: 87 or an amino acid sequence that is at least 90% identical to SEQ ID NO: 87; -FR2-b comprises or consists of the amino acid sequence of "LFWYRQTMMRGLELLIY" SEQ ID NO: 88 or an amino acid sequence that is at least 90% identical to SEQ ID NO: 88; -FR3-b comprises or consists of the amino acid sequence of "IDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYF" SEQ ID NO: 89, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 89; or -FR4-b comprises or consists of the amino acid sequence of "GEGSRLTVL" SEQ ID NO:90, or an amino acid sequence that is at least 90% identical to SEQ ID NO:90.

[0183] In one embodiment, said first variable domain further comprises one or more framework regions, preferably FR1-a, FR2-a, FR3-a, and FR4-a selected from the group consisting of: -FR1-a comprises or consists of the amino acid sequence "EDVEQSLFLSVREGDSSVINCTYT" SEQ ID NO: 83 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 83; -FR2-a comprises or consists of the amino acid sequence of "LYWYKQEPGAGLQLLTY" SEQ ID NO: 84, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 84; -FR3-a comprises or consists of the amino acid sequence of SEQ ID NO: 85 "KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF" or an amino acid sequence that is at least 95% identical to SEQ ID NO: 85; - FR4-a comprises or consists of the amino acid sequence of "GSGTRLSIRP" SEQ ID NO: 86, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 86; The second variable domain further comprises one or more framework regions, preferably FR1-b, FR2-b, FR3-b, and FR4-b selected from the group consisting of: -FR1-b comprises or consists of the amino acid sequence "DAGVIQSPRHEVTEMGQEVTLRCKPI" SEQ ID NO: 87 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 87; -FR2-b comprises or consists of the amino acid sequence of "LFWYRQTMMRGLELLIY" SEQ ID NO: 88 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 88; -FR3-b comprises or consists of the amino acid sequence of "IDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYF" SEQ ID NO: 89, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 89; or -FR4-b comprises or consists of the amino acid sequence of "GEGSRLTVL" SEQ ID NO: 90, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 95.

[0184] Thus, the antigen binding protein of the present invention preferably further comprises a constant domain, such as, for example, the alpha and / or beta constant domain, or the gamma and / or delta constant domain, as described herein above. Preferably, the alpha and beta constant domains and / or the gamma and delta constant domains comprise further mutations not present in the native TCR, enhancing the pairing of the two recombinant chains, i.e., the alpha and beta chains. For example, position 48 of TRAC and position 57 of TRBC1 or TRBC2 may be replaced by a cysteine ​​residue. Further examples of such modifications are described above. Examples of the alpha and beta constant domains comprised in the antigen binding protein of the present invention are SEQ ID NO: 8 (alpha constant domain of TCR R7P1D5) and SEQ ID NO: 15 (beta constant domain of TCR R7P1D5).

[0185] In one embodiment, the antigen binding molecule of the invention comprises a TCR molecule as defined herein above, such as a molecule comprising at least one alpha variable domain and one beta variable domain (alpha / beta TCR), or a molecule comprising at least one gamma or delta variable domain. In other words, such TCRs are heterodimeric molecules, i.e. alpha / beta and gamma / delta heterodimers.

[0186] For use in adoptive cell therapy (ACT), the α / β or γ / δ heterodimeric TCRs may be transfected into the patient's T cells as full-length chains with both cytoplasmic and transmembrane domains. The constant domains may comprise appropriate mutations, i.e. disulfide bonds, to improve the pairing of the desired recombinant α / β or γ / δ chains and to reduce the pairing of the endogenous α / β or γ / δ with the respective recombinant chains. Thus, the antigen-binding proteins of the present invention preferably further comprise a constant domain, such as, for example, the α constant domain and / or the β constant domain described herein above. Preferably, the α and β constant domains comprise further mutations not present in the native TCR to enhance the pairing of the two chains, i.e., the α and β chains, or the γ and δ chains, respectively. For example, position 48 of TRAC and position 57 of TRBC1 or TRBC2 are substituted by cysteine ​​residues. Examples of alpha and beta constant domains comprised in the antigen binding proteins of the invention are SEQ ID NO: 8 (alpha constant domain of TCR R7P1D5) and SEQ ID NO: 15 (beta constant domain of TCR R7P1D5).

[0187] In a preferred embodiment, the TCR comprises an α chain comprising an α variable domain comprising CDR1α, CDR2α, and CDR3α, and further comprising a constant domain; and a β chain comprising a β variable domain comprising CDR1β, CDR2β, and CDR3β, and further comprising a β constant domain. In a further preferred embodiment, the α variable domain comprises three complementarity determining regions (CDRs), CDRa1, CDRa2, and CDRa3, in which CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:5, CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO:6, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:35, 36, 37, 38, 39, preferably the amino acid sequences of SEQ ID NO:35, 36, 37, 38, more preferably the amino acid sequences of SEQ ID NO:35 and 38, such as SEQ ID NO:35; the β variable domain comprises three complementarity determining regions (CDRs), CDRb1, CDRb2, and CDRb3, in which CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO:12, CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO:13, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO:14.

[0188] In a preferred embodiment, the TCR comprises an α chain comprising an α variable domain comprising CDR1α, CDR2α, and CDR3α, and further comprising a constant domain; and a β chain comprising a β variable domain comprising CDR1β, CDR2β, and CDR3β, and further comprising a β constant domain. In a further preferred embodiment, the α variable domain comprises three complementarity determining regions (CDRs), CDRa1, CDRa2, and CDRa3, in which CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:5, CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO:6, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:35, and the β variable domain comprises three complementarity determining regions (CDRs), CDRb1, CDRb2, and CDRb3, in which CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO:12, CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO:13, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO:14.

[0189] In another preferred embodiment, the TCR comprises an α chain comprising an α variable domain comprising CDR1α, CDR2α, and CDR3α, and further comprising a constant domain; and a β chain comprising a β variable domain comprising CDR1β, CDR2β, and CDR3β, and further comprising a β constant domain. In a further preferred embodiment, the α variable domain comprises three complementarity determining regions (CDRs), CDRa1, CDRa2, and CDRa3, in which CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:5, CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO:6, and CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:38, and the β variable domain comprises three complementarity determining regions (CDRs), CDRb1, CDRb2, and CDRb3, in which CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO:12, CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO:13, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO:14.

[0190] In another preferred embodiment, the variable α and β domains of the TCR further comprise an α framework region and a β framework region, respectively.

[0191] In one embodiment, the first alpha variable domain further comprises one or more framework regions, preferably FR1-a, FR2-a, FR3-a, and FR4-a selected from the group consisting of FR1-a, FR2-a, FR3-a, and FR4-a, wherein FR1-a comprises or consists of the amino acid sequence of "EDVEQSLFLSVREGDSSVINCTYT" SEQ ID NO: 83, FR2-a comprises or consists of the amino acid sequence of "LYWYKQEPGAGLQLLTY" SEQ ID NO: 84, FR3-a comprises or consists of the amino acid sequence of "KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF" SEQ ID NO: 85, and FR4-a comprises or consists of the amino acid sequence of "GSGTRLSIRP" SEQ ID NO: 86; The second β variable domain further comprises one or more framework regions, preferably FR1-b, FR2-b, FR3-b, and FR4-b selected from the group consisting of FR1-b, FR2-b, FR3-b, and FR4-b, wherein FR1-b comprises or consists of the amino acid sequence of "DAGVIQSPRHEVTEMGQEVTLRCKPI" SEQ ID NO: 87, FR2-b comprises or consists of the amino acid sequence of "LFWYRQTMMRGLELLIY" SEQ ID NO: 88, FR3-b comprises or consists of the amino acid sequence of "IDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYF" SEQ ID NO: 89, or FR4-b comprises or consists of the amino acid sequence of "GEGSRLTVL" SEQ ID NO: 90.

[0192] In another preferred embodiment, the TCR comprises any of the variable domains defined above, further comprising a constant domain comprising or consisting of the amino acid sequence of SEQ ID NO: 8, or a sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 8; the β constant domain comprises or consists of the amino acid sequence of SEQ ID NO: 15, or a sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 15.

[0193] Variants of the antigen-binding proteins described herein are contemplated and are expressly referred to herein with the phrase "at least 85% identical to the reference sequence" as defined in the "Definitions" section above. For example, sequences FR1-a, FR2-a, FR3-a, and FR4-a, and FR1-b, FR2-b, FR3-b, and FR4-b may differ from the reference sequences SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90 by at least one amino acid substitution, in particular by at least one conservative amino acid substitution, and / or by substitution with a canonical residue, where appropriate. In particular, the sequences FR1-a, FR2-a, FR3-a, and FR4-a, and FR1-b, FR2-b, FR3-b, and FR4-b of the first and second variable domains may differ from the reference sequences of SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90 only by conservative amino acid substitutions.

[0194] Modifications and variations may be made to the amino acid sequences of the antigen binding proteins of the invention, and the corresponding DNA sequences, respectively, which still result in a functional antigen binding protein or polypeptide having the desired properties. Modifications may be made to the first or second variable domain, particularly to the framework regions, or to each CDR, or to all CDRs contained in the first and / or second variable domain.

[0195] The inventors of the present invention further discovered that by using the framework regions of the parent TCR R7P1D5, certain amino acid substitutions in the framework regions of the antigen binding protein have advantageous effects. In particular, the amino acid substitutions S19A or S19V, preferably S19V, are critical for the expression of the soluble single-chain TCR construct, and in addition, the CDRa2 amino acid sequence comprising a Y at its second amino acid position (i.e., amino acid 57 according to IMGT) is Y (57Y) in CDRa2. Furthermore, the inventors discovered that the amino acid substitutions A48K, L50P in FR2-a, and the amino acid substitutions M46P, M47G in FR2-b, used alone or in combination, increase the expression yield of the soluble antigen binding protein of the present invention. The inventors also showed that I54F in FR2-b increases the expression yield of the soluble antigen binding protein and increases the stability of the soluble antigen binding protein, especially in addition to the CDRb3 amino acid sequence comprising a D at position 109 according to IMGT. Furthermore, the present inventors found that the I54F substitution in FR2-b significantly improved the specificity of the soluble antigen-binding protein.

[0196] Thus, in one embodiment, the framework region of the antigen binding protein of the invention comprises: - an amino acid substitution at position 19 of FR1-a, said amino acid substitution being preferably S19A, S19V, more preferably S19V, - an amino acid substitution at position 48 and / or 50 of FR2-a (said amino acid substitution at position 48 is preferably A48K and said amino acid substitution at position 50 is preferably L50P), - an amino acid substitution at positions 46, 47 and / or 54 of FR2-b (said amino acid substitutions at positions 46, 47 and / or 54 are preferably M46P, M47G and I54F, respectively, and in case the CDRb3 amino acid sequence as defined above comprises the amino acid 109D at position 109, said amino acid at position 54 is preferably I54F), - an amino acid substitution at position 66 of FR3-b (said amino acid substitution at position 66 is preferably I66C, and in case the CDRb2 amino acid sequence as defined above comprises the amino acid 57C at position 57, said amino acid at position 66 is preferably 66C); At least one amino acid substitution, preferably 1 to 5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably 1 to 5, such as 2 to 5 amino acid substitutions, such as 3 or 4, selected from the group of amino acid substitutions comprising: Therein, the positions of substitutions are given according to the IMGT nomenclature, in which, optionally, said amino acid substitutions are given relative to the amino acid sequence of the corresponding framework amino acid sequence of the parent TCR R7P1D5.

[0197] In a preferred embodiment, the framework region of the antigen binding protein of the invention comprises the amino acid substitutions S19V, A48K, and I54F or S19V, A48K, preferably S19V, A48K, and I54F, in which the positions of the substitutions are given according to the IMGT nomenclature. In one embodiment, said combination of amino acid substitutions is advantageously further combined with a CDRb3 comprising or consisting of the amino acid sequence "CASRAX1TGELFF" SEQ ID NO: 82, in which X1 is D, or in which the CDRb3 amino acid comprises the amino acid 109D at position 109 according to the IMGT numbering. In a further preferred embodiment, the framework region of the antigen binding protein of the invention further comprises the amino acid substitution I66C, in which preferably FR3-b comprises the amino acid substitution I66C when the CDRb2 amino acid sequence defined above comprises the amino acid 57C at position 57.

[0198] Thus, in one embodiment, said first variable domain further comprises 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, wherein: - FR1-a comprises or consists of the amino acid sequence "LYWYKQEPGKGLQLLTY" SEQ ID NO: 91 or an amino acid sequence which is at least 85% identical to SEQ ID NO: 85, preferably said amino acid sequence which is at least 91% identical to SEQ ID NO: 85 comprises the amino acid 19V (underlined in SEQ ID NO: 91), -FR2-a comprises or consists of the amino acid sequence "LYWYKQEPGKGLQLLTY" SEQ ID NO: 92, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 85, preferably said amino acid sequence that is at least 92% identical to SEQ ID NO: 85, comprising the amino acid 48K (underlined in SEQ ID NO: 92) and optionally an amino acid substitution at position 50, preferably L50P; - FR3-a comprises or consists of the amino acid sequence of "KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF" SEQ ID NO: 85 or an amino acid sequence that is at least 85% identical to SEQ ID NO: 85; - FR4-a comprises or consists of the amino acid sequence of "GSGTRLSIRP" SEQ ID NO: 86, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 86; the second variable domain further comprises one or more framework regions, preferably multiple 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 "DAGVIQSPRHEVTEMGQEVTLRCKPI" SEQ ID NO: 87 or an amino acid sequence that is at least 85% identical to SEQ ID NO: 87; -FR2-b comprises or consists of the amino acid sequence of "LFWYRQTMMRGLELLFY" SEQ ID NO: 93 or an amino acid sequence that is at least 85% identical to SEQ ID NO: 93, wherein preferably said amino acid sequence is at least 85% identical to SEQ ID NO: 93 comprising the amino acid 54F and optionally an amino acid substitution at positions 46 and / or 47, wherein said amino acid substitution at positions 46 and / or 47 is preferably M46P and / or M47G, respectively; - FR3-b comprises or consists of the amino acid sequence of SEQ ID NO: 89 "IDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYF" or an amino acid sequence which is at least 85% identical to SEQ ID NO: 89, or the amino acid sequence of SEQ ID NO: 94 "CDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYF" or an amino acid sequence which is at least 85% identical to SEQ ID NO: 94, in which preferably said amino acid sequence is at least 85% identical to SEQ ID NO: 94 comprising the amino acid 66C, and in the case where the CDRb2 amino acid sequence as defined above comprises the amino acid 57C at position 57, preferably FR3-b comprises or consists of an amino acid sequence which is at least 85% identical to SEQ ID NO: 94 comprising the amino acid 66C, -FR4-b comprises or consists of the amino acid sequence of "GEGSRLTVL" SEQ ID NO:90, or an amino acid sequence that is at least 85% identical to SEQ ID NO:90.

[0199] Variants of the antigen-binding proteins described herein are contemplated and are expressly referred to herein with the expression "at least 85% identical to the reference sequence" as defined in the "Definitions" section above. For example, the sequences FR1-a, FR2-a, FR3-a, and FR4-a, and FR1-b, FR2-b, FR3-b, and FR4-b may differ from the reference sequences SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:93, and SEQ ID NO:89 or SEQ ID NO:94, SEQ ID NO:90, where appropriate, by at least one amino acid substitution, in particular by at least one conservative amino acid substitution, and / or by substitution with a canonical residue. In particular, the sequences FR1-a, FR2-a, FR3-a, and FR4-a, and FR1-b, FR2-b, FR3-b, and FR4-b of the first and second variable domains may differ from the reference sequences SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:93 and SEQ ID NO:89 or SEQ ID NO:94, SEQ ID NO:90 only by conservative amino acid substitutions.

[0200] Modifications and variations may be made to the amino acid sequences of the antigen binding proteins of the present invention, and the corresponding DNA sequences encoding them, which still result in a functional antigen binding protein or polypeptide possessing desirable properties.

[0201] Thus, in one embodiment, the invention refers to an antigen binding protein comprising: (i) “EDVEQSLFLSVREGDSSVINCTYT DSSSTY LYWYKQEPGAGLQLLTY IFSNMDM KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF CAEMTSESKIIF GSGTRLSIRP" sequence number 118, "EDVEQSLFLSVREGDSSVINCTYT DSSSTY LYWYKQEPGAGLQLLTY IFSNMDM KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF CAEFTSESKIIF GSGTRLSIRP" sequence number 119, "EDVEQSLFLSVREGDSSVINCTYT DSSSTY LYWYKQEPGAGLQLLTY IFSNMDM KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF CAEFNSESKIIF GSGTRLSIRP" sequence number 120, "EDVEQSLFLSVREGDSSVINCTYT DSSSTY LYWYKQEPGAGLQLLTYI FSNMDM KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF CAEFSSASKIIF GSGTRLSIRP" sequence number 121, "EDVEQSLFLSVREGDSSVINCTYT DSSSTY LYWYKQEPGAGLQLLTYI FSNMDM KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF CAEATSESKIIF a first polypeptide chain comprising a first variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 122 of "GSGTRLSIRP," preferably an amino acid sequence at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 118 and SEQ ID NO: 121, more preferably SEQ ID NO: 118, or an amino acid sequence at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 118 to 122 (wherein, in the case of SEQ ID NOs: 118 to 122, the amino acid sequence at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 118 to 122 preferably comprises the amino acid sequences of CDRa1 of SEQ ID NO: 5, CDRa2 of SEQ ID NO: 6, and CDRa3 of SEQ ID NOs: 35, 36, 37, 38, and 39, respectively); (ii) a second polypeptide chain comprising a second variable domain comprising the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 11, wherein said amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 11 preferably comprises the amino acid sequences of CDRb1, CDRb2, and CDRb3 of SEQ ID NOs: 12, 13, and 14, respectively; or (iii) preferably, the amino acid sequence “EDVEQSLFLSVREGDS V VINCTYT DSSSTY LYWYKQEPGK GLQLLTY IYSSQDQ KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF CAEMTSESKIIF GSGTRLSIRP" SEQ ID NO: 151 or "EDVEQSLFLSVREGDS V VINCTYT ESSSTY LYWYKQEPG K GLQLLTY IYSSQDQ KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF CAEMTSESKIIF GSGTRLSIRP" a first polypeptide chain comprising a first variable domain comprising SEQ ID NO: 152, or an amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 151 or 152 (wherein preferably, the amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 151 preferably comprises the amino acid sequence of CDRa1 of SEQ ID NO: 5, CDRa2 of SEQ ID NO: 56, and CDRa3 of SEQ ID NO: 35, wherein preferably, the amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 152 preferably comprises the amino acid sequence of CDRa1 of SEQ ID NO: 55, CDRa2 of SEQ ID NO: 56, and CDRa3 of SEQ ID NO: 35, preferably further comprising amino acids 19V and / or 48K, (iv) the amino acid sequence “DAGVIQSPRHEVTEMGQEVTLRCKPIPGHDYLFWYRQTMMRGLELL F YFCYGTP C DDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRANTGELFFGEGSRLTVL" SEQ ID NO: 149 or "DAGVIQSPRHEVTEMGQEVTLRCKPIPGHDYLFWYRQTMMRGLELL F YFCYGTP CDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRADTGELFFGEGSRLTVL" a second polypeptide chain comprising a second variable domain comprising SEQ ID NO: 150, wherein preferably the amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 149 preferably comprises the amino acid sequence of CDRb1 of SEQ ID NO: 62, CDRb2 of SEQ ID NO: 65, and CDRb3 of SEQ ID NO: 14, wherein preferably the amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 150 comprises the amino acid sequence of CDRb1 of SEQ ID NO: 62, CDRb2 of SEQ ID NO: 65, and CDRb3 of SEQ ID NO: 71, preferably further comprising amino acids 54F and / or 66C.

[0202] In one embodiment, the antigen binding protein is an antibody or a fragment thereof, or a bispecific antibody or a fragment thereof, or a T cell receptor (TCR) or a fragment thereof, or a bispecific T cell receptor (TCR) or a fragment thereof. Antibody, TCR and respective fragments are defined herein above in the "Definitions" section.

[0203] In one embodiment, the antigen binding protein is of human origin, which is understood to be generated from a human antigen locus and thus comprises human sequences, in particular human TCR or antibody sequences.

[0204] In one embodiment, the first and second polypeptides are linked together, particularly via a covalent bond.

[0205] In one embodiment, the antigen binding protein is a soluble protein.

[0206] A "covalent bond," "linker sequence" or "polypeptide linker" is defined herein above in the "Definitions" section (see "Linker").

[0207] In one embodiment, the antigen binding protein of the invention further comprises one or more of the following: (i) one or more additional antigen-binding sites; (ii) a transmembrane domain, optionally including a cytoplasmic signaling domain; (iii) diagnostic agents; (iv) a therapeutic agent; or (v) PK modification part.

[0208] As is known to those of skill in the art, for example, an antigen binding site in the context of an antibody is typically formed by the six CDRs, which bind the antigen.

[0209] In the context of the present invention, the one or more further antigen binding sites referred to herein above under (i) are preferably selected from the group consisting of the antigens CD0 (such as the CD1 gamma, CD2 delta and CD2 epsilon chains), CD2, CD4, CD5, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61, CD64, CD68, CD94, CD90, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG7D, GITR, F c The binding site is selected from those which bind to an antigen selected from the group consisting of εRI, TCR α / β and TCR γ / δ, HLA-DR, or to an antigen of an effector cell, preferably CD3, TCR α / β or CD28, more preferably CD3 and TCR α / β.

[0210] "CD3" and "CD28" are defined herein above in the "Definitions" section.

[0211] In some embodiments, one of the one or more further antigen-binding sites referred to in (i) is capable of binding to a T cell-specific receptor molecule and / or a natural killer cell (NK cell)-specific receptor molecule. In certain embodiments, the antigen-binding protein is in a complex with the CD3 T cell co-receptor.

[0212] In a preferred embodiment, one of the one or more further antigen-binding sites referred to in (i) is capable of binding to TCR α / β.

[0213] In some embodiments, the T cell specific receptor is a CD3 T cell co-receptor.

[0214] In some embodiments, the T cell specific receptor is CD28, CD134, 4-1BB, CD5 or CD95.

[0215] "CD134", "4-1BB", "CD5", "CD95", and "NK cell-specific receptor molecule" are defined herein above in the "Definitions" section. "Transmembrane domain", "Cytoplasmic signaling domain", "Diagnostic agent", "Therapeutic agent", and "PK modifying moiety" are defined herein above in the "Definitions" section.

[0216] In some embodiments, the antigen binding proteins of the present invention are covalently linked to at least one growth inhibitory agent, either directly or via a cleavable or non-cleavable linker. Such antigen binding proteins to which at least one growth inhibitory agent is attached may also be referred to as conjugates.

[0217] 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), and may be transferred by the skilled artisan to the preparation of such antigen-binding proteins of the invention having at least one growth inhibitory agent attached thereto.

[0218] "Linker" in the context of attachment of at least one growth inhibitory agent means a chemical moiety comprising a covalent bond or a chain of atoms, which covalently attaches the polypeptide to the drug moiety.

[0219] Conjugates may be prepared by in vitro methods. A linking group is used to attach a drug or prodrug to the antibody. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. Conjugation of the antigen-binding protein of the invention to a cytotoxic or growth inhibitory agent can be achieved using bifunctional derivatization of N-succinimidyl pyridyldithiobutyrate (SPDB), butanoic acid 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl ester (nitro-SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), N-succinimidyl (2-pyridyldithio)propionate (SPDP), succinimidyl (N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), imidoesters. This may be done using a variety of bifunctional protein binding agents, including but not limited to conductors (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al (1987). Carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for attaching radionucleotides to antibodies (WO 94 / 11026).

[0220] The linker may be a "cleavable linker" that facilitates release of the cytotoxic or growth inhibitory agent inside the cell. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase-labile linker, a photolabile linker, or a disulfide-containing linker (see, e.g., U.S. Pat. No. 5,208,020) may be used. The linker may also be a "non-cleavable linker" (e.g., an SMCC linker), which may provide better resistance in some cases.

[0221] Alternatively, a fusion protein comprising an antibody of the invention and a cytotoxic or growth inhibitory polypeptide may be produced by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

[0222] 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).

[0223] In one embodiment, the antigen binding protein of the invention further comprises one or more of an enzyme, a cytokine (such as human IL-2, IL-7 or IL-15), a nanocarrier, or a nucleic acid.

[0224] In a preferred embodiment, the antigen-binding protein is bispecific. Bispecific antigen-binding proteins are also referred to herein as "bispecific molecules."

[0225] Many different bispecific formats have been described in the art and are described herein above in the "Definitions" section under "Forms". Techniques for generating proteins in different forms are also disclosed in the art cited in the corresponding sections, so that the skilled person can easily use the CDRs or variable domains defined in the context of the present invention in the forms disclosed herein. The generation of antigen binding proteins such as scTCRs or soluble bispecific binding proteins such as TCER™ is also disclosed in the Examples section herein. The skilled person will understand that the light and heavy chain variable domains may be in parallel orientation, and the α and β variable domains may be in parallel orientation, as in the DVD form, or the light and heavy chain variable domains may be in cross orientation, and the α and β variable domains may be in cross orientation, as in the CODV form.

[0226] Thus, the present invention further refers to an antigen-binding protein comprising two polypeptide chains forming two antigen-binding sites (A and B), wherein the first polypeptide chain has the formula: V3-L1-V4-L2-C L [I] and having a structure represented by where V3 is a third variable domain; V4 is a fourth variable domain; L1 and L2 are linkers; L2 may be present or absent; L is a light chain constant domain or a portion thereof, which may or may not be present; wherein the second polypeptide chain has the formula: V5-L3-V6-L4-C H1 [II] having a structure represented by where V5 is a fifth variable domain; V6 is a sixth variable domain; L3 and L4 are linkers; L4 may be present or absent; H1 is heavy chain constant domain 1 or a portion thereof, present or absent; V3 or V4 is a first variable domain as defined herein above and V5 or V6 is a second variable domain as defined herein above, or V5 or V6 is a first variable domain as defined in the context of the present invention and V3 or V4 is a second variable domain as defined herein above, wherein V3 is a first variable domain and V5 is a second variable domain as defined herein above, V4 is a light chain variable domain and V6 is a heavy chain variable domain, or V4 is a heavy chain variable domain and V6 is a light chain variable domain, or V3 is the second variable domain as defined herein above and V5 is the first variable domain, V4 is a light chain variable domain and V6 is a heavy chain variable domain, or V4 is a heavy chain variable domain and V6 is a light chain variable domain, or V3 is a first variable domain and V6 is a second variable domain as defined herein above, V4 is a light chain variable domain and V5 is a heavy chain variable domain, or V4 is a heavy chain variable domain and V5 is a light chain variable domain, or V3 is the second variable domain and V6 is the first variable domain as defined herein above, V4 is a light chain variable domain and V5 is a heavy chain variable domain, or V4 is a heavy chain variable domain and V5 is a light chain variable domain, V4 is a first variable domain and V5 is a second variable domain as defined herein above, V3 is a light chain variable domain and V6 is a heavy chain variable domain, or V3 is a heavy chain variable domain and V6 is a light chain variable domain, or V4 is the second variable domain as defined herein above, V5 is the first variable domain, V3 is a light chain variable domain and V6 is a heavy chain variable domain; or V3 is a heavy chain variable domain and V6 is a light chain variable domain; In this, the light chain variable domain and the heavy chain variable domain together form one antigen-binding site B, and in this, the first and second variable domains as defined herein above form one antigen-binding site A. The linkers L1, L2, L3, L4 are defined herein above in the "Definitions" section. However, in some embodiments, some linker lengths may be preferred for certain forms. However, knowledge of the linker lengths and their amino acid sequences belongs to the general knowledge in the art, and different forms of linkers, as well as the sequences of linkers and amino acids, are part of the state of the art and are disclosed in the disclosures cited herein above.

[0227] It will be appreciated by those skilled in the art that the antigen binding site A forming the first and second variable domains defined herein above specifically binds to a MAGE-A peptide / MHC complex as described herein in the context of the present invention.

[0228] In a preferred embodiment, V3 is the first variable domain and V6 is the second variable domain as defined in the context of the present invention, V4 is a light chain variable domain and V5 is a heavy chain variable domain, or V3 is the first variable domain and V6 is the second variable domain as defined in the context of the present invention, V4 is the heavy chain variable domain and V5 is the light chain variable domain.

[0229] In one embodiment, the polypeptide of formula [I] comprises at the C-terminus a polypeptide of formula [I], a linker (L5), and F c domain or a portion thereof, and / or wherein the polypeptide of formula [II] comprises at the C-terminus a polypeptide of formula [II], a linker (L6), and F c It further comprises a domain or a part thereof.

[0230] F c Domains are defined herein above in the "Definitions" section.

[0231] In one embodiment, the antigen binding protein comprises two polypeptide chains forming two antigen binding sites (A and B), In the above, one polypeptide chain has the formula [III]: V3-L1-V4-L2-C L -L5-F C1 [III] having a structure represented by In the above, one polypeptide chain has the formula [IV]: V5-L3-V6-L4-C H1 -L6-F C2 [IV] having a structure represented by In the formula, V 3、 L1, V4, L2, C L , V5, L3, V6, L4, C H1 is as defined herein above, wherein L5 and L6 are linkers present or absent, and wherein F C1 and F C2 F c domain, where F C1 and F C2 are the same or different, preferably different. c Domains are defined herein above in the "Definitions" section.

[0232] In one embodiment, F C1 comprises or consists of the amino acid sequence of SEQ ID NO: 113 (hole); C2 comprises or consists of the amino acid sequence of SEQ ID NO: 112 (knob), or vice versa; More preferably, when V4 or V3 is a heavy chain variable domain, F C1 comprises or consists of the amino acid sequence of SEQ ID NO: 113, and accordingly, when V5 or V6 is a light chain variable domain, F C2 comprises or consists of the amino acid sequence of SEQ ID NO: 112; or When V4 or V3 is a light chain variable domain, F C1comprises or consists of the amino acid sequence of SEQ ID NO: 112, and accordingly, when V5 or V6 is a heavy chain variable domain, F C2 comprises or consists of the amino acid sequence of SEQ ID NO:113.

[0233] In one embodiment, the heavy chain variable domain (V H ) and the light chain variable domain (V L ) are CD3 (including CD3γ, CD3δ, and CD3ε chains), CD4, 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, CD94, CD90, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, F c Together they form one binding site B which binds to an antigen selected from the group consisting of εRI, TCR α / β and TCR γ / δ, HLA-DR and / or bind to an effector cell.

[0234] In a further embodiment, the heavy chain variable domain (V H ) and the light chain variable domain (V L ) together form a single binding site that binds to a T cell-specific receptor molecule and / or a natural killer cell (NK cell)-specific receptor molecule, wherein the T cell-specific receptor molecule and the natural killer cell (NK cell)-specific receptor molecule are defined herein above in the "Definitions" section.

[0235] In one embodiment, the light chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 153 and the heavy chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 154, or the light chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 153 and the heavy chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 156, or the light chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 157 and the heavy chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 158, or the light chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 159 and the heavy chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 160, or the light chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 153 and the heavy chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 162, or the light chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 153 and the heavy chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 164.

[0236] Preferably, the light chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 153 and the heavy chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 154, or the light chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 153 and the heavy chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 156, or the light chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 159 and the heavy chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 160, or the light chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 153 and the heavy chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 162, or the light chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 153 and the heavy chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 164, more preferably the light chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 159 and the heavy chain variable domain comprises or consists of the amino acid sequence of SEQ ID NO: 160. As can be seen from the examples, the inventors of the present invention have demonstrated as proof of principle the use of CDRs as defined in the context of the present invention, and more specifically the variable domains as defined herein above, in the TCER™ format (Examples).

[0237] Thus, in a preferred embodiment, the antigen binding protein comprises two polypeptide chains forming two antigen binding sites, In the above, one polypeptide chain has the formula [III]: V3-L1-V4-L2-C L -L5-F C1 [III] having a structure represented by One polypeptide chain has the formula [IV]: V5-L3-V6-L4-C H1 -L6-F C2 [IV] having a structure represented by In the formula, L2, CL , L5 and L4, C H1 , L6 does not exist In the formula, V 3、 L1, V4, V5, L3, V6 are as defined herein above; Preferably, V3 is a first variable domain as defined in the context of the present invention and V6 is a second variable domain, V4 is a light chain variable domain and V5 is a heavy chain variable domain, or preferably, V3 is a first variable domain as defined in the context of the present invention and V6 is a second variable domain, V4 is a heavy chain variable domain and V5 is a light chain variable domain, Preferably, L1 and L3 comprise or consist of the amino acid sequence "GGGSGGGG" of (SEQ ID NO: 96), Preferably F C1 comprises or consists of the amino acid sequence of SEQ ID NO: 113, C2 comprises or consists of the amino acid sequence of SEQ ID NO: 112, or vice versa; More preferably, when V4 is a heavy chain variable domain, F C1 comprises or consists of the amino acid sequence of SEQ ID NO: 113, and accordingly, when V5 is a light chain variable domain, F C2 comprises or consists of the amino acid sequence of SEQ ID NO: 112, or More preferably, when V4 is a light chain variable domain, F C1 comprises or consists of the amino acid sequence of SEQ ID NO: 112, and accordingly, when V5 is a heavy chain variable domain, F C2 comprises or consists of the amino acid sequence of SEQ ID NO: 113, the light chain variable domain and the heavy chain variable domain together form one antigen-binding site which binds to an antigen as defined herein above, preferably CD3, TCR alpha / beta or CD28, more preferably CD3 or TCR alpha / beta, Therein, the first and second variable domains form one antigen-binding site which specifically binds to a MAGE-A antigenic peptide as defined in the context of the present invention.

[0238] The antigen binding protein of this embodiment is a bispecific TCR, or c The antigen-binding protein of this embodiment may also be referred to as a TCER™.

[0239] "TCER™" is a bispecific T cell receptor (TCR) that is a soluble antigen-binding protein comprising two antigen-binding domains, a first and a second variable domain as defined in the context of the present invention, and a further antigen-binding domain formed by the heavy and light chain variable domains of an antibody, also called a recruiter, including the variable light and heavy chain variable domains directed against CD3 and against TCR alpha / beta.

[0240] In one preferred embodiment, the antigen binding protein, such as TCER™, comprises: - Amino acid sequence (SEQ ID NO: 135): EDVEQSLFLSVREGDSVVINCTYTDSSSTYLYWYKQEPGKGLQLLTYIYSSQDSKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP GGGSGGGG EVQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWMGYINPYNDVTKYAEKFQGRVTLTSDTSTSSTAYMELSSLRSEDTAVHYCARGSYYDYEGFVYWGQGTLVTVSS EPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP The formula is V3-L1-V4-L2-C L -L5-F C1 [III] A first polypeptide (In the formula, L2, C L , L5 is absent, V3 of SEQ ID NO: 138 (mature TCR derived α variable domain) (CDRa1, CDRa2, CDRa3 of SEQ ID NO: 5, SEQ ID NO: 59, and SEQ ID NO: 35 are shown in bold), L1 of SEQ ID NO: 96 (underlined), V4 of SEQ ID NO: 160 (anti-TCR αβ VH), and F of SEQ ID NO: 113. C1(italics and underlined); - Amino acid sequence (SEQ ID NO: 136): QIQMTQSPSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIK GGGSGGGG DAGVIQSPRHEVTEMGQEVTLRCKPIPGHDYLFWYRQTMMRGLELLFYFCYGTPCDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRADTGELFFGEGSRLTVL EPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP The formula is V5-L3-V6-L4-C H1 -L6-F c2 [IV] A second polypeptide (In the formula, L4, C H1 , L6 is absent, V5 (anti-TCR αβ VL) of SEQ ID NO: 159, L3 (underlined) of SEQ ID NO: 96, V6 (TCR derived from β variable domain) of SEQ ID NO: 150 (CDRa1, CDRa2, CDRa3 of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 71 shown in bold), and F of SEQ ID NO: 112. C2 (italics and underlined), wherein F C1 and F C2 The sequence does not require the light chain to be linked via a cysteine, so there is no amino acid substitution of cysteine ​​to serine (S shown in bold), F c Amino acid substitutions in the IgG1 hinge region similar to the IgG2 hinge that abolish γ-receptor interaction (PVA is shown in bold at positions 233-235 and 331S), F c The N-glycosylation site of the F c The N297Q amino acid substitution (Q in bold) abolishes the interaction between γ receptors, and F C1 hole mutations (Y349C, T366S, L368A, and Y407V) in the case of , and F C2 In the case of F, it comprises knob mutations (S354C and T366W). C1 is SEQ ID NO: 113, F C2is sequence number 112.

[0241] In another preferred embodiment, the antigen binding protein, such as TCER™, comprises: Amino acid sequence (SEQ ID NO:136): QIQMTQSPSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIK GGGSGGGG DAGVIQSPRHEVTEMGQEVTLRCKPIPGHDYLFWYRQTMMRGLELLFYFCYGTPCDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRADTGELFFGEGSRLTVL EPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP The formula is V3-L1-V4-L2-C L -L5-F C1 [III] A first polypeptide (In the formula, L2, C L , L5 is absent, V3 of SEQ ID NO: 159 (anti-TCR αβ VL), L1 of SEQ ID NO: 96 (underlined), V4 of SEQ ID NO: 150 (TCR derived from β variable domain) (CDRb1, CDRb2, CDRb3 of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 71 shown in bold), and F of SEQ ID NO: 112 C1 (italics and underlined); Amino acid sequence (SEQ ID NO:137): EDVEQSLFLSVREGDSVVINCTYTDSSSTYLYWYKQEPGKGLQLLTYIYSSQDQKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP GGGSGGGG EVQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWMGYINPYNDVTKYAEKFQGRVTLTSDTSTSSTAYMELSSLRSEDTAVHYCARGSYYDYEGFVYWGQGTLVTVSS EPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA SIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP The formula is V5-L3-V6-L4-C H1 -L1-FC2 [IV] A second polypeptide (In the formula, L4, C H1 , L6 is absent, V5 of SEQ ID NO: 151 (mature TCR derived from α variable domain) (CDRa1, CDRa2, CDRa3 of SEQ ID NO: 5, SEQ ID NO: 56, and SEQ ID NO: 35 are shown in bold), L3 of SEQ ID NO: 96 (underlined), V6 of SEQ ID NO: 160 (anti-TCR αβ VH), and F of SEQ ID NO: 113. C2 (italics and underlined), In the formula, F C1 and F C2 The sequence does not require the light chain to be linked via a cysteine, so there is no amino acid substitution of cysteine ​​to serine (S shown in bold), F c Amino acid substitutions in the IgG1 hinge region similar to the IgG2 hinge that abolish γ-receptor interaction (PVA is shown in bold at positions 233-235 and 331S), F c The N-glycosylation site of the F c The N297Q amino acid substitution (Q in bold) abolishes the interaction between γ receptors, and F C2 hole mutations (Y349C, T366S, L368A, and Y407V) in the case of , and F C1 In the case of F, it comprises knob mutations (S354C and T366W). C1 is SEQ ID NO: 112, F C2 is sequence number 113.

[0242] In another preferred embodiment, the antigen binding protein, such as TCER™, comprises: Amino acid sequence (SEQ ID NO:131): EDVEQSLFLSVREGDSVVINCTYTDSSSTYLYWYKQEPGKGLQLLTYIYSSQDQKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP GGGSGGGGDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYFCQQGQTLPWTFGQGTKVEIK EPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA SIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP The formula is V3-L1-V4-L2-C L -L5-F C1 [III] A first polypeptide (In the formula, L2, C L , L5 is absent, V3 (mature TCR derived from α variable domain) of SEQ ID NO: 151 (CDRa1, CDRa2, CDRa3 of SEQ ID NO: 5, SEQ ID NO: 56, and SEQ ID NO: 35 are shown in bold), L1 of SEQ ID NO: 96 (underlined), V4 of SEQ ID NO: 153 (anti-TCR αβ VL), and F of SEQ ID NO: 112. C1 (italics and underlined); Amino acid sequence (SEQ ID NO:130): EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYAQKFQDRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS GGGSGGGG DAGVIQSPRHEVTEMGQEVTLRCKPIPGHDYLFWYRQTMMRGLELLFYFCYGTPCDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRADTGELFFGEGSRLTVLEPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP The formula is V5-L3-V6-L4-C H1 -L6-F C2 [IV] A second polypeptide (In the formula, L4, C H1 , L6 is absent, V5 of SEQ ID NO: 154 (anti-TCR αβ VL), L3 of SEQ ID NO: 96 (underlined), V6 of SEQ ID NO: 151 (TCR derived from β variable domain) (CDRb1, CDRb2, CDRb3 of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 71 are shown in bold), and F of SEQ ID NO: 113 C2 (italics and underlined), In the formula, F C1 and F C2 The sequence does not require the light chain to be linked via a cysteine, so there is no amino acid substitution of cysteine ​​to serine (S shown in bold), F c Amino acid substitutions in the IgG1 hinge region similar to the IgG2 hinge that abolish γ-receptor interaction (PVA is shown in bold at positions 233-235 and 331S), F c The N-glycosylation site of the F c The N297Q amino acid substitution (Q in bold) abolishes the interaction between γ receptors, and F C2 hole mutations (Y349C, T366S, L368A, and Y407V) in the case of , and F C1 In the case of F, it comprises knob mutations (S354C and T366W). C1 is SEQ ID NO: 112, F C2 is sequence number 113.

[0243] In another preferred embodiment, the antigen binding protein, such as TCER™, comprises: Amino acid sequence (SEQ ID NO: 133): EDVEQSLFLSVREGDSVVINCTYTESSSTYLYWYKQEPGKGLQLLTYIYSSQDQKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP GGGSGGGG DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYFCQQGQTLPWTFGQGTKVEIK EPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA SIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP The formula is V3-L1-V4-L2-C L -L5-F C1 [III] The first polypeptide (In the formula, L2, C L , L5 is absent, V3 (mature TCR derived from α variable domain) of SEQ ID NO: 152 (CDRa1, CDRa2, CDRa3 of SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 35 are shown in bold), L1 of SEQ ID NO: 96 (underlined), V4 of SEQ ID NO: 154 (anti-TCR αβ VL), and F of SEQ ID NO: 112. C1 (italics and underlined); Amino acid sequence (SEQ ID NO:130): EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYKGVSTYAQKFQDRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS GGGSGGGG DAGVIQSPRHEVTEMGQEVTLRCKPIPGHDYLFWYRQTMMRGLELLFYFCYGTPCDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRADTGELFFGEGSRLTVLEPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP The formula is V5-L3-V6-L4-C H1 -L6-F C2 [IV] A second polypeptide (In the formula, L4, C H1, L6 is absent, V5 of SEQ ID NO: 154 (anti-TCR αβ VL), L3 of SEQ ID NO: 96 (underlined), V6 of SEQ ID NO: 151 (TCR derived from β variable domain) (CDRb1, CDRb2, CDRb3 of SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 71 are shown in bold), and F of SEQ ID NO: 113 C2 (italics and underlined), In the formula, F C1 and F C2 The sequence does not require the light chain to be linked via a cysteine, so there is no amino acid substitution of cysteine ​​to serine (S shown in bold), F c Amino acid substitutions in the IgG1 hinge region similar to the IgG2 hinge that abolish γ-receptor interaction (PVA is shown in bold at positions 233-235 and 331S), F c The N-glycosylation site of the F c The N297Q amino acid substitution (Q in bold) abolishes the interaction between γ receptors, and F C2 hole mutations (Y349C, T366S, L368A, and Y407V) in the case of , and F C1 In the case of F, it comprises knob mutations (S354C and T366W). C1 is SEQ ID NO: 112, F C2 is sequence number 113.

[0244] In one embodiment, the present invention refers to an antigen binding protein comprising: i) the formula V3-L1-V4-L2-C, which comprises or consists of the amino acid sequence of SEQ ID NO: 127; L -L5-F C1 [III], and the first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 128. H1 -L6-F c2 A second polypeptide of [IV], ii) Formula V3-L1-V4-L2-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 127 L -L5-F c1[III], and the first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 130. H1 -L1-F c2 A second polypeptide of [IV], iii) Formula V3-L1-V4-L2-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 131 L -L5-F c1 [III], and the first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 130. H1 -L6-F c2 A second polypeptide of [IV], iv) the formula V3-L1-V4-L2-C, which comprises or consists of the amino acid sequence of SEQ ID NO: 133 L -L5-F c1 [III], and the first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 130. H1 -L6-F c2 A second polypeptide of [IV], v) the formula V3-L1-V4-L2-C, which comprises or consists of the amino acid sequence of SEQ ID NO: 135; L -L5-F c1 [III], and the first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 136. H1 -L1-F c2 A second polypeptide of [IV], vi) Formula V3-L1-V4-L2-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 137 L -L5-F c1 [III], and the first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 136. H1 -L1-F c2 A second polypeptide of [IV], vii) Formula V3-L1-V4-L2-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 139 L -L5-Fc1 [III], and the first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 136. H1 -L1-F c2 A second polypeptide of [IV], viii) Formula V3-L1-V4-L2-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 131 L -L5-F c1 [III], and the first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 142. H1 -L1-F c2 A second polypeptide of [IV], ix) the formula V3-L1-V4-L2-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 133 L -L5-F c1 [III], and the first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 142. H1 -L6-F c2 A second polypeptide of [IV], x) the formula V3-L1-V4-L2-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 131; L -L5-F c1 [III], and the first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 136. H1 -L1-F c2 A second polypeptide of [IV], xi) Formula V3-L1-V4-L2-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 133 L -L5-F c1 [III], and the first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 136. H1 -L1-F c2 A second polypeptide of [IV], xii) the formula V3-L1-V4-L2-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 133L -L5-F c1 [III], and the first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 166; H1 -L6-F c2 A second polypeptide of [IV], Preferred are iii) to xi), and more preferred are iii) and iv).

[0245] In an even more preferred embodiment of the invention, the amino acid sequence of SEQ ID NO: 131 is selected from the formula V3-L1-V4-L2-C L -L5-F c1 [III] and the amino acid sequence of SEQ ID NO: 130; H1 -L6-F c2 [IV]; or the present invention refers to an antigen-binding protein comprising a second polypeptide of the formula V3-L1-V4-L2-C, consisting of the amino acid sequence of SEQ ID NO: 133. L -L5-F c1 [III] and the amino acid sequence of SEQ ID NO: 130; H1 -L6-F c2 [IV]; or the present invention refers to an antigen-binding protein comprising a second polypeptide of the formula V3-L1-V4-L2-C, consisting of the amino acid sequence of SEQ ID NO: 135. L -L5-F c1 [III] and the amino acid sequence of SEQ ID NO: 136, H1 -L6-F c2 [IV]; or the present invention refers to an antigen-binding protein comprising a second polypeptide of the formula V3-L1-V4-L2-C, consisting of the amino acid sequence of SEQ ID NO: 137. L -L5-F c1 [III] and the amino acid sequence of SEQ ID NO: 136, H1 -L6-F c2[IV]; or the present invention refers to an antigen-binding protein comprising a second polypeptide of the formula V3-L1-V4-L2-C, consisting of the amino acid sequence of SEQ ID NO: 139. L -L5-F c1 [III] and the amino acid sequence of SEQ ID NO: 136, H1 -L6-F c2 [IV] refers to an antigen-binding protein comprising a second polypeptide of

[0246] It may also be desirable to modify the antigen binding proteins of the invention with respect to effector function, for example to enhance or decrease the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antigen binding protein. This is referred to herein in the context of the antigen binding proteins of the invention as F. c F of antigen-binding proteins, also called mutant forms c Alternatively, or in addition, this may be achieved by introducing one or more amino acid substitutions into the region. c Cysteine ​​residues may be introduced into the region to allow interchain disulfide bond formation in this region. The heterodimeric antigen binding proteins thus generated may have improved or reduced internalization capability and / or increased complement-mediated cell killing and / or antibody-dependent cellular cytotoxicity (ADCC) (Caron P C. et al. 1992; and Shopes B. 1992).

[0247] Another type of amino acid modification of the antigen binding proteins of the invention may be useful to alter the native glycosylation pattern of the antigen binding protein, namely by deleting one or more carbohydrate moieties found in the antigen binding protein and / or adding one or more glycosylation sites not present in the antigen binding protein. The presence of either the tripeptide sequences asparagine-X-serine, and asparagine-X-threonine, where X is any amino acid except proline, creates a potential glycosylation site. The addition or deletion of glycosylation sites to the antigen binding protein is conveniently accomplished by altering the amino acid sequence (in the case of N-linked glycosylation sites) so that it contains one or more of the above tripeptide sequences.

[0248] Another type of modification involves the removal of sequences identified in silico or experimentally as potentially resulting in degradation products or heterogeneity of antigen-binding protein preparations. As an example, deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation when present primarily in the sequence Asn-Gly, and less so in other dipeptide sequences such as Asn-Ala. Thus, when such deamidation sites, particularly Asn-Gly, are present in the antigen-binding proteins of the invention, it may be desirable to remove one of the involved residues, typically by conservative substitution, to remove the site. Such substitutions in the sequence to remove one or more involved residues are also intended to be encompassed by the present invention.

[0249] Another type of covalent modification involves chemically or enzymatically conjugating glycosides to the antigen-binding protein. These procedures are advantageous in that they do not require production of the antigen-binding protein in a host cell that has glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling mode used, sugars may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. For example, such methods are described in WO 87 / 05330.

[0250] Removal of any carbohydrate moieties present on the antigen-binding protein may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the antigen-binding protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), but leaves the antigen-binding protein intact. Chemical deglycosylation is described by Sojahr H. et al. (1987) and Edge, A. S. et al. (1981). Enzymatic cleavage of carbohydrate moieties of antibodies can be accomplished by the use of various endo- and exoglycosidases, as described by Thotakura, N. R. et al. (1987).

[0251] Another type of covalent modification of an antigen binding protein comprises coupling the antigen binding protein to one of a variety of non-proteinaceous polymers, such as, for example, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner described in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0252] In one particular embodiment, the antigen binding protein is a TCR. In one particular embodiment, the TCR is a human TCR. Thus, in one embodiment, the first variable domain is comprised in a TCR alpha or gamma chain; and / or the second variable domain is comprised in a TCR beta or delta chain. In one embodiment, the TCR is an alpha-beta heterodimer, comprising an alpha chain TRAC constant domain sequence and a beta chain TRBC1 or TRBC2 constant domain sequence.

[0253] The following alpha chain TRAC constant domain sequences and beta chain TRBC1 or TRBC2 constant domains are also referred to as TCR constant domain sequences. In one embodiment, the TCR constant domain sequences may be derived from any suitable species, such as any mammal, such as human, rat, monkey, rabbit, donkey, or mouse, preferably human. In some preferred embodiments, the TCR constant domain sequences may be slightly modified, for example by the introduction of heterologous sequences, preferably mouse sequences, which may enhance the expression and stability of the TCR. In addition, further stabilizing mutations known from the state of the art (e.g. WO 2018 / 104407, PCT / EP2018 / 069151, WO 2011 / 044186, WO 2014 / 018863), such as the substitution of unfavorable amino acids in the variable regions and / or the introduction of disulfide bridges between the TCR C domains, and the removal of unpaired cysteines, may be introduced.

[0254] In particular, the TCR constant domain sequence may be modified by truncation or substitution to remove the native disulfide bond between Cys4 in exon 2 of TRAC and Cys2 in exon 2 of TRBC1 or TRBC2. The constant domain sequence of the α and / or β chain may also be modified by substituting Thr48 of TRAC and Ser57 of TRBC1 or TRBC2 with a cysteine ​​residue, which forms a disulfide bond between the TCR α and β constant domains. 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. The constant domain may additionally or alternatively contain further mutations, substitutions or deletions to the native TRAC and / or TRBC1 / 2 sequence. The term TRBC1 / 2 encompasses naturally occurring polymorphic variants, such as, for example, N to K at position 4 of TRAC (Bragado et al. in Immunol. 1994 Feb;6(2):223-30).

[0255] In one embodiment, the TCR is chimeric.

[0256] "Chimeric TCR" herein refers to a TCR in which the TCR chains comprise sequences from multiple species. Preferably, a TCR in the context of the present invention may comprise an alpha chain, for example comprising a human variable region of the alpha chain and a mouse constant region of a mouse TCR alpha chain.

[0257] In one embodiment, the antigen binding protein of the invention is a single chain TCR (scTCR) or a single chain bispecific antibody, in which the first and second polypeptides are covalently linked together, where covalent linkage is defined herein above in the "Definitions" section.

[0258] The scTCR may comprise a polypeptide of the variable region of a first TCR chain (e.g., an α chain) and a polypeptide of the entire (full-length) second TCR chain (e.g., a β chain), or vice versa. Additionally, the scTCR may optionally comprise one or more linkers that primarily link the two or more polypeptides. The linker may be, for example, a peptide that binds the two single chains together, as described herein. Also provided are scTCRs of the invention fused to a human cytokine, such as IL-2, IL-7, or IL-15.

[0259] In one embodiment, the single chain TCR is Vα-L t -Vβ, Vβ-L t -Vα, Vα-Ca-L t -Vβ, Vα-Cb-L t -Vβ, Vα-L t -Vβ-Cb, Vα-L t -Vβ-Ca, Vα-Ca-L t -Vβ-Cb, Vα-Cb-L t - Vβ-Ca, preferably Vα-L t -Vβ, Vβ-L t -Vα, in which Vα is the first variable domain as defined herein above, Vβ is the second variable domain as defined herein above, Ca and Cb are the TCR α and β constant regions, respectively, which may or may not be present, and L t is a linker, which may or may not be present, and is defined herein above in the "Definitions" section.

[0260] In one embodiment such a single chain TCR may further comprise at least one further variable domain, preferably one or two further variable domains, linked either at the C-terminus or N-terminus.

[0261] In one embodiment such further variable domains are k In a preferred embodiment, the linker L kis a linker as defined herein above or C of the amino acid sequence of SEQ ID NO: 114 H1 This is a hinge sequence.

[0262] The present invention also includes particles that display the antigen-binding protein of the present invention, particularly TCR, 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 generating 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).

[0263] Furthermore, the inventors of the present invention demonstrate in the Examples, particularly in Example 3, that the antigen binding proteins of the present invention bind to a target antigen, i.e. in a complex with an MHC protein, preferably HLA-A. * Highly specific MAGE-A antigenic peptides in complex with 02, i.e., antigen binding proteins, specifically bind to the identified epitopes.

[0264] Thus, the antigen binding protein of the present invention specifically binds to a MAGE-A antigenic peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 1, wherein said antigenic peptide is in a complex with an MHC protein, preferably HLA-A. *02. More particularly, in one embodiment, the antigen binding protein specifically binds to an epitope comprising or consisting of at least three or at least four amino acid positions of the MAGE-A antigenic peptide of SEQ ID NO: 1, preferably at least three or four, preferably three or four, preferably three amino acid positions selected from the group consisting of 1, 5, 7, and 8 or 1, 3, 5, 7, and 8, in particular 1, 5, and 7, of the amino acid sequence of SEQ ID NO: 1. The determination of the exact epitope may vary slightly depending on the method used and the cut-off value selected. Due mainly to the property of binding to amino acid positions 1, 5, and 7, the antigen binding protein of the invention does not significantly bind to the similar peptides NOMAP-1-0320, NOMAP-1-1223, and ODC-001.

[0265] In one embodiment, the antigen binding protein of the invention does not significantly bind to a MAGE-A antigenic peptide variant comprising or consisting of the amino acid sequence of SEQ ID NO:1, in which at least one of positions 1, 5 and 7, at least one of positions 1, 5, 7 and 8, or at least one of positions 1, 3, 5, 7 and 8 are substituted, preferably with alanine.

[0266] "Does not bind significantly" in the context of antigen binding proteins of the invention, such as T cell expressed antigen binding proteins, in particular TCRs or fragments thereof or bispecific TCRs and fragments thereof, typically means a response, such as a signal detected for an antigen peptide variant, that is less than 30%, less than 25%, less than 20%, less than 15%, preferably less than 30% of the response, i.e. signal, obtained from a MAGE-A peptide consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 1, preferably under the same experimental conditions, in a functional assay, such as a TCR activation assay, such as IFN-γ release as described above. "Does not bind significantly" in the context of antigen peptide variants and in the context of antigen binding proteins of the invention, in particular soluble antigen binding proteins of the invention, typically means a response, such as a signal detected for an antigen peptide variant, that is less than 30%, less than 25%, less than 20%, less than 15%, preferably less than 30% of the response, i.e. signal, obtained from a MAGE-A peptide consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 1, preferably under the same experimental conditions. D The K determined for the antigen peptide variant in a binding assay, e.g., biolayer interferometry, is increased by more than 3-fold, more than 3.5-fold, more than 4-fold, more than 4.5-fold, more than 5-fold, preferably more than 3. D wherein the signal obtained for the antigenic peptide variant is preferably a background signal, and wherein the antigenic peptide variant or the MAGE-A peptide is in a complex with an MHC molecule.

[0267] In accordance with the above, in one embodiment the antigen binding proteins of the invention, in particular soluble antigen binding proteins, bind to a specific antigen, preferably HLA-A, in a complex with an MHC protein, with reduced affinity compared to the affinity for the specific antigen. * 02, i.e. in a complex with an MHC protein, preferably HLA-A. * 02 in complex with the MAGE-A antigenic peptide described in the context of the present invention, and the respective K DThe affinity of a molecule may be increased by more than 3-fold, more than 3.5-fold, more than 4-fold, more than 4.5-fold, more than 5-fold, preferably more than 3-fold, such as 3-10-fold. D " is defined herein above in the "Definitions" section.

[0268] MHC proteins, especially MHC class I and HLA-A * 02 Protein is defined herein above in the "Definitions" section.

[0269] In one embodiment, the antigen binding proteins of the invention are characterised as affinity matured antigen binding proteins capable of specifically and optionally binding to MAGE-A antigenic peptide / MHC complexes.

[0270] The antigen binding proteins of the present invention specifically bind to MAGE-A antigenic peptide complexes as specified herein above, more particularly to the epitope of SEQ ID NO:1 as defined herein above, and are able to distinguish between their respective targets, i.e. MAGE-A antigenic peptides, and other similar peptides to which they do not significantly bind.

[0271] The terms "specificity" or "specifically binds" refer to the ability of an antigen-binding protein to distinguish its bound target peptide sequence ("epitope") from similar epitopes, peptides or proteins, i.e., the antigen-binding protein "specifically binds" to a first antigen if it is not significantly cross-reactive to a second antigen.

[0272] "Similar peptides" in the context of the present invention, sometimes also referred to as "off-targets", typically relate to peptides comprising 8-16 amino acids in length. Similar peptides in the context of the present invention are typically MHC presented. Furthermore, similar peptides in the context of the present invention comprise or consist of an amino acid sequence similar to that of a MAGE-A antigenic peptide, more specifically, an epitope in which the biochemical / biophysical properties of the amino acids are identical and / or similar compared to the amino acids constituting the epitope of the corresponding MAGE-A peptide, compared to the epitope of the MAGE-A antigenic peptide. Due to this sequence similarity, the similar peptide may be bound by an antigen binding protein, and in this scenario, for example, if the similar peptide is presented by an MHC protein and thus bound by an antigen binding protein, such as a TCR, the ability of a given antigen binding protein, such as a TCR, to bind the similar peptide may not result in a desired T cell response, but may also result in an adverse reaction. Such adverse reactions may be "extra-tumor" side effects such as cross-reactivity of specific TCRs that cross-react with the peptide in healthy tissues, as reported on page 7 of Lowdell et al., Cytotherapy, published December 4, 2018. Similar peptides in the context of the present invention may be, for example, peptides that are expressed by normal tissues, such as HLA-A, ... * The peptides were selected from a database of 02 binding peptides (XPRESIDENT database). Therefore, due to these adverse reactions, the antigen binding proteins of the present invention are engineered to avoid binding to similar peptides, particularly those listed herein below.

[0273] Thus, analogous peptides in the context of the present invention include RABGAP1L-001 consisting of the amino acid sequence of SEQ ID NO:23, AXIN1-001 consisting of the amino acid sequence of SEQ ID NO:24, ANO5-001 consisting of the amino acid sequence of SEQ ID NO:25, TPX2-001 consisting of the amino acid sequence of SEQ ID NO:26, SYNE3-001 consisting of the amino acid sequence of SEQ ID NO:27, MIA3-001 consisting of the amino acid sequence of SEQ ID NO:28, HERC4-001 consisting of the amino acid sequence of SEQ ID NO:29, PSME2-001 consisting of the amino acid sequence of SEQ ID NO:30, HEATR5A-001 consisting of the amino acid sequence of SEQ ID NO:31, CNOT1-003 consisting of the amino acid sequence of SEQ ID NO:32, TEP1-003 consisting of the amino acid sequence of SEQ ID NO:33, PITPNM3-001 consisting of the amino acid sequence of SEQ ID NO:34, ZFC consisting of the amino acid sequence of SEQ ID NO:129 -001, INTS4-002 consisting of the amino acid sequence of SEQ ID NO: 171, SAMH-001 consisting of the amino acid sequence of SEQ ID NO: 172, PPP1CA-006 consisting of the amino acid sequence of SEQ ID NO: 173, RPL-007 consisting of the amino acid sequence of SEQ ID NO: 174, SETD1A-001 consisting of the amino acid sequence of SEQ ID NO: 175, NOMAP-1-0320 consisting of the amino acid sequence of SEQ ID NO: 176, NOMAP-1-1223 consisting of the amino acid sequence of SEQ ID NO: 177, and ODC-001 consisting of the amino acid sequence of SEQ ID NO: 178, COL6A3-010 consisting of the amino acid sequence of SEQ ID NO: 179, FAM115A-001 consisting of the amino acid sequence of SEQ ID NO: 180, PHTF2-001 consisting of the amino acid sequence of SEQ ID NO: 181, and RPP1-001 consisting of the amino acid sequence of SEQ ID NO: 181.

[0274] In one embodiment, the antigen binding proteins of the invention, in particular TCRs or fragments thereof, such as T cell expressed antigen binding proteins, or bispecific TCRs and fragments thereof, when said mimetic peptide is in complex with an MHC protein, preferably HLA-A *When in complex with .02, it does not bind or does not significantly bind to at least 1, at least 2, at least 3, at least 4, at least 5, such as 1, 2, 3, 4, 5, such as preferably at least 3 or all similar peptides selected from the list consisting of RABGAP1L-001, AXIN1-001, ANO5-001, TPX2-001, SYNE3-001, MIA3-001, HERC4-001, PSME2-001, HEATR5A-001, CNOT1-003, TEP1-003, PITPNM3-001, preferably HEATR5A-001, HERC4-001, and CNOT1-003. This is an important advantage in the context of the present invention, since binding to similar peptides, i.e. off-targets, may increase the risk of side effects, and therefore the fact that the antigen binding protein of the present invention does not cross-react with the similar peptides listed herein makes the present invention a promising anti-cancer drug.

[0275] "Does not bind significantly", in the context of similar peptides and in the context of antigen binding proteins of the invention, particularly TCRs or fragments thereof such as T cell expressed antigen binding proteins, or bispecific TCRs and fragments thereof, may also be referred to as "does not cross-react", and refers herein to a functional response measured in a functional assay for example of an antigen binding protein to a similar peptide / MHC compared to MAGE-AMAGE-A, in which the response of the antigen binding protein to the similar peptide / MHC is less than 30%, less than 20%, less than 10%, less than 5%, preferably less than 5%, such as 8%, 6%, 5% of the response of the same antigen binding protein to a MAGE-A antigenic peptide / MHC complex in the same experimental setting. In one example, the response is a functional response of T cells expressing said antigen binding protein, as described herein above and in the context of similar peptides, as described in Example 3 and in Figure 5.

[0276] Furthermore, in one embodiment, the antigen binding proteins of the invention, in particular the soluble antigen binding proteins of the invention, when said analogous peptides are in a complex with an MHC protein, preferably HLA-A.* When in complex with 02, RABGAP1L-001, AXIN1-001, ANO5-001, TPX2-001, SYNE3-001, MIA3-001, HERC4-001, PSME2-001, HEATR5A-001, CNOT1-003, TEP1-003, PITPNM3-001, INTS4-002, SAMH-001, PPP1CA-006, RPL-007, SETD1A-001, NOMAP-1-0320, NOMAP-1-1223, ODC-001, COL6A3-002, and COL6A4-001 were significantly higher in the IL-10A complex than in the IL-10A complex. 10, FAM115A-001, PHTF2-001 and RPP1-001, preferably CNOT1-003, SYNE3-001, TPX2-001, PSME2-001, more preferably SYNE3-001, TPX2-001, PSME2-001, does not bind or does not significantly bind to at least one similar peptide, at least 2, at least 3, at least 4, at least 5, etc., such as 1, 2, 3, 4, 5, etc., preferably at least 3 or all similar peptides selected from the group of peptides consisting of FAM115A-001, PHTF2-001 and RPP1-001, preferably CNOT1-003, SYNE3-001, TPX2-001, PSME2-001, more preferably SYNE3-001, TPX2-001, PSME2-001.

[0277] In one example, the antigen binding proteins of the invention, in particular the soluble antigen binding proteins of the invention, when said analogous peptide is in a complex with an MHC protein, preferably HLA-A * When in a complex with .02, it does not bind or significantly bind to INTS4-002, SAMH-001, PPP1CA-006, RPL-007, SETD1A-001, NOMAP-1-0320, NOMAP-1-1223 and ODC-001, INTS4-002, SAMH-001, PPP1CA-006, RPL-007, SETD1A-001 or NOMAP-1-0320, NOMAP-1-1223, ODC-001, COL6A3-010, FAM115A-001, PHTF2-001 and RPP1-001, etc., PPP1CA-006, RPL-007 and SETD1A-001, etc.

[0278] In the context of analogous peptides, and also in the context of antigen binding proteins such as the soluble antigen binding proteins of the invention, "does not bind significantly" is herein characterized by reduced affinity, faster off-rates and / or lower binding signals, in particular faster off-rates and / or lower binding signals. For example, in the context of the present invention, the antigen binding protein has a binding response to at least one analogous peptide / MHC complex that is less than 50%, less than 45%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4% or less than 3% of the response of the same antigen binding protein binding to a MAGE-A antigenic peptide / MHC complex under the same experimental circumstances and at the same antigen binding protein concentration, and / or for example, in the context of the present invention, the antigen binding protein binds to at least one analogous peptide / MHC complex with reduced affinity compared to its affinity to a specific antigen, i.e. a MAGE-A antigenic peptide / MHC complex as described herein, in which the respective K D is increased by 5, 7, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000-fold, preferably 5, 7, 10, 15, 20, 30, 40, 50, 100-fold, preferably 20-100-fold, more preferably 30-100-fold, 40-100-fold, typically 40-50-fold. For example, when an antigen-binding protein binds to the MAG-003 / MHC complex at a K of 1 nM, D For example, an antigen-binding protein binds to the RABGAP1L-001 / MHC complex with a K of 100 nM. D When antigen-binding proteins bind at Dand thus binds to RABGAP1L-001 / MHC with a 100-fold reduced affinity. In these examples, the binding response, dissociation constant, and binding affinity are preferably measured using biolayer interferometry, e.g., as described in Examples 4 and 5. In some further examples, the antigen binding protein binds to at least one similar peptide / MHC complex with a reduced affinity compared to its affinity for a specific antigen, i.e., a MAGE-A antigenic peptide / MHC complex as described herein, in which, for example, the K D Values ​​are typically the K values ​​for CNOT1-003, INTS4-002, and SAMH-001 relative to the affinity for the specific antigen, i.e., the MAGE-A antigenic peptide / MHC complex, as measured by biolayer interferometry as described in Example 5. D increases by more than 1000-fold, and by more than 500-fold in COL6A3-010, PHTF2-001, RPP1-001, and FAM115A-001.

[0279] In a further example, in an MHC complex, an antigen binding protein such as 114-iso1-BMA (36) or 114-iso2-UCHT1 (17) has a binding response of less than 5%, less than 4%, less than 3%, less than 2%, e.g., less than 1.5%, to ODC-001, NOMAP-1-0320, and NOMAP-1-1223, as measured by biolayer interferometry as described in Example 5.

[0280] In one embodiment, the antigen binding protein of the present invention comprises an antigen binding protein having an amino acid sequence of SEQ ID NO: 1 and an HLA molecule, preferably HLA-A * and a MAGE-A antigen peptide comprising or consisting of: DThus, the antigen-binding proteins of the present invention specifically bind to HLA molecules, preferably HLA-A. * 02 in a complex with a MAGE-A antigen peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1, in particular an affinity (K D ) has "K D " and "affinity" are defined herein above in the "Definitions" section.

[0281] K D Methods for measuring affinity, such as affinity for a protein, are known to those skilled in the art and include, for example, surface plasmon resonance and biolayer interferometry. As known to those skilled in the art, the experimental conditions used in such experiments, such as the buffers used, the concentration of the protein, etc., may affect the results.

[0282] In a related embodiment, the antigen binding proteins of the invention, in particular TCRs or fragments thereof or bispecific TCRs or fragments thereof, such as T cell expressed antigen binding proteins, have the amino acid sequence of SEQ ID NO: 1 and an HLA molecule, preferably HLA-A * 02 and a complex of a MAGE-A antigen peptide, the affinity (K D ).

[0283] In a particular embodiment, the antigen binding protein of the present invention is soluble. Soluble antigen binding proteins may include, for example, without limitation, antibodies or fragments thereof, or bispecific antibodies or fragments thereof, TCR fragments or bispecific TCRs or scTCRs. In a related embodiment, the antigen binding protein, which is preferably soluble, has the amino acid sequence of SEQ ID NO: 1 and an HLA molecule, preferably HLA-A. *02, or consisting of a complex of MAGE-A antigen peptide, a K of less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 1 nM, for example, 10 pM to 100 nM, 10 pM to 50 nM, 10 pM to 10 nM, particularly 50 pM to 100 nM, 100 pM to 50 nM, 100 pM to 10 nM D Combine with.

[0284] Thus, in one example, the antigen binding protein of the present invention is expressed as, for example, a soluble TCER™ as described herein above, and binds to HLA-A * Antigen-binding proteins were analyzed for their binding affinity to 02 / MAG-003 monomer. Typically, measurements are performed, for example, on an Octet RED384 system, typically using settings recommended by the manufacturer. Briefly, binding kinetics were typically measured, for example, using PBS, 0.05% Tween-20, 0.1% BSA as buffer, at 30° C., with a shaking speed of, for example, 1000 rpm. Antigen-binding proteins were loaded onto a biosensor, such as FAB2G or AHC, or analyzed in solution.

[0285] In one embodiment, the antigen binding protein of the invention optionally has increased affinity compared to a reference protein.

[0286] "Reference protein" herein refers to a protein to which the antigen binding protein of the present invention is compared. In one embodiment, the reference protein is directed against the same target as the present protein, i.e., a MAGE-A antigen peptide in complex with an MHC protein, and more preferably, said reference protein is in the same form as the antigen binding protein to which it is compared. In a particular embodiment, the reference protein is in the same form as the antigen binding protein to which it is compared, and the reference protein comprises the CDRs or the α and β variable domains of the parent TCR R7P1D5.

[0287] As disclosed herein, the antigen binding protein of the present invention comprises the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 1 and an HLA molecule, preferably HLA-A. * The antigen binding protein of the present invention recognizes and / or binds to a complex of MAGE-A antigenic peptides comprising or consisting of MAG-003 and HLA molecules. Thus, MAG-003 and HLA molecules are present in the major histocompatibility complex (MHC) class I, and binding of the antigen binding protein to said complex may elicit an immune response upon binding. In some embodiments, the antigen binding protein of the present invention induces an increased immune response compared to the immune response induced by a reference protein.

[0288] Thus, in one embodiment, the antigen binding protein of the present invention induces an immune response, preferably characterized by an increase in interferon (IFN) gamma levels. Thus, in one example, the immune response is characterized by an increase in EC 50 It may be characterized by its value.

[0289] In one embodiment, the antigen binding protein has an EC 50 preferably lower than EC as determined by IFN-γ release 50 [nM] value, and preferably the antigen binding protein has an EC 50 EC50 / 2, preferably 2, 3, 4, 5, 6, 7, 8, e.g., 2-9 times lower than the [nM] value 50 [nM], in which EC 50 The value is MHC, preferably HLA-A * The half-maximal IFN-γ release concentration [nM] determined on EC 02 expressing cells, preferably cells loaded with the antigenic peptide of SEQ ID NO: 1. 50is typically determined by ELISA, for example after co-culture of electroporated CD8+ T cells with T2 cells loaded with serial dilutions of a MAGE-A antigenic peptide, preferably the MAGE-A antigenic peptide of SEQ ID NO: 1. Preferably, the reference protein is in the same form as the antigen binding protein to which it is compared, the reference protein comprising the CDRs or the α and β variable domains of the parent TCR R7P1D5, and 50 are preferably determined in the same experimental setting.

[0290] In one embodiment the antigen binding protein has an EC50 of less than 100 nM, 50 nM, less than 10 nM, less than 900 pM, less than 500 pM, less than 300 pM, less than 200 pM, less than 150 pM, less than 100 pM, less than 50 pM, less than 20 pM, less than 10 pM, between 0.1 nM and 20 nM, such as 0.5 nM to 15 nM, 0.8 nM to 12 nM, 0.8 nM to 10 nM, 0.8 nM to 10 nM, such as 1 pM to 150 pM, 1 to 100 pM, 1 to 50 pM, 1 to 20 pM, 1 to 20 pM, such as 1 pM to 150 pM, 1 to 100 pM, 1 to 50 pM, 1 to 20 pM, 1 to 20 pM, etc. 50 It has a value.

[0291] In one embodiment, the antigen binding protein is a TCR and has an EC of less than 12 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 6 nM, less than 4 nM, between 0.1 nM and 20 nM, such as 0.5 nM to 15 nM, 0.8 nM to 12 nM, 0.8 nM to 10 nM, 0.8 nM to 10 nM, 0.8 nM to 10 nM, etc., as determined by IFN-γ release, against MAGE-A / MHC complex presenting cells. 50 has.

[0292] In this embodiment, the EC 50is determined by determining the level of IFN-γ released using CD8+ T cells (20,000 cells / well) expressing different antigen binding proteins of the invention upon co-culture with T2 cells (20,000 cells / well) loaded with a dilution series of MAG-003 (SEQ ID NO:1).

[0293] In one embodiment, the antigen binding protein is a soluble molecule such as TCER™ and has an EC of less than 200 pM, less than 150 pM, less than 100 pM, less than 50 pM, less than 20 pM, less than 10 pM, between 1 pM and 150 pM, between 1 and 100 pM, between 1 and 50 pM, between 1 and 20 pM, between 1 and 20 pM for MAGE-A / MHC complex presenting cells. 50 In this embodiment, EC 50 is determined by determining the level of LDH released upon co-culture with tumor cell lines presenting endogenous levels of MAG-003 using CD8+ T cells (20,000 cells / well) expressing different antigen binding proteins of the invention, preferably the cell line is Hs695T which presents about 1071 MAG-003 peptides in complex with MHC proteins on its surface, preferably the cell line is NCI-H1755 which presents about 3918 MAG-003 peptides in complex with MHC proteins on its surface, or preferably the cell line is U2OS which presents about 125 MAG-003 peptides in complex with MHC proteins on its surface.

[0294] The antigen binding proteins of the present invention have a high safety profile.

[0295] "Safety profile" herein refers to the ability to distinguish tumor cells from healthy tissue cells, which is often determined by determining a safety margin.

[0296] As used herein, the term "safety margin" or "therapeutic margin" refers to the coefficient of the half-maximal concentration of a compound required to induce 100% cytotoxicity in a tumor cell line compared to the half-maximal concentration of a compound required to induce 100% cytotoxicity in healthy tissue cells. The EC determined for a tumor cell line for an antigen-binding protein of interest is 50 is 1 pM, e.g., the EC 50 EC50 for tumor cell lines when the value is 1000 pM 50 EC on primary cells 50 , so the margin of safety is 1000.

[0297] In one embodiment, the antigen binding protein of the present invention inhibits ECs against healthy cells. 50 EC for MAGE-A / MHC complex-presenting cells is 500 to 12,000, preferably 1,000 to 10,000 higher than the value 50 E.g., ECs against healthy cells 50 EC for MAGE-A / MHC complex-presenting cells that is 100 or more, 500 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 8000 or more, or 10000 or more higher than the value 50 has.

[0298] "MAGE-A / MHC complex-presenting cells" herein refers to cells that present MAGE-A peptide / MHC complexes on their surface, in which the copy number of said MAGE-A / MHC complexes can typically be determined by methods known to those skilled in the art. In one embodiment, the MAGE-A / MHC complexes are target cells, such as cancer cells, in which cancer is defined herein below in the section "Therapeutic Methods and Uses". In the context of the present invention, MAGE-A peptide / MHC complexes are over-presented on the cell surface of MAGE-A / MHC complex-presenting cells, in particular cancer cells, compared to the level of said complexes on the cell surface of normal (healthy) tissue. By "over-presented" is meant that the MAGE-A / MHC complexes are presented at a level of at least 1.2-fold the level of presentation in healthy tissue, preferably at least 2-fold the level of presentation in healthy tissue or cells, and more preferably 5-fold to 10-fold.

[0299] In one embodiment, the MAGE-A / MHC complex presenting cells have a MAGE-A / 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, more than 2000, preferably a MAGE-A / MHC copy number of 50-2000, such as 80-2000, such as 100-2000, for example 120-2000.

[0300] "Copy number" herein refers to the number of MAGE-A / MHC complexes, as defined in the context of the present invention, present on the cell surface of a cell, such as a MAGE-A / 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 using fluorescently labeled antigen binding proteins.

[0301] "Healthy cells" herein refer to cells that are not cancer cells, and preferably, healthy cells herein refer to cells of tissues surrounding MAGE-A / MHC presenting cells, cells of tissues surrounding MAGE-A / MHC complexes presenting cancer cells. However, in some cases, healthy cells may also express and present MAGE-A / MHC complexes on their surface. Typically, in healthy cells in the context of the present invention, MAGE-A / MHC complexes are present in lower amounts (copy numbers) than in cancer cells, as will be understood by those skilled in the art.

[0302] The healthy cells are preferably selected from the group consisting of astrocytes, GABA neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, coronary artery endothelial cells, dermal microvascular endothelial cells, mesenchymal stem cells, nasal epithelial cells, peripheral blood mononuclear cells, and pulmonary artery smooth muscle cells, preferably GABA neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, coronary artery endothelial cells, nasal epithelial cells, peripheral blood mononuclear cells, and pulmonary artery smooth muscle cells.

[0303] In one embodiment, the antigen binding protein is selected from the group consisting of astrocytes, GABA neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, coronary artery endothelial cells, dermal microvascular endothelial cells, mesenchymal stem cells, nasal epithelial cells, peripheral blood mononuclear cells, lung fibroblasts, tracheal smooth muscle cells, epidermal keratinocytes, renal cortical endothelial cells, and pulmonary artery smooth muscle cells, preferably astrocytes, GABA neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, coronary artery endothelial cells, dermal microvascular endothelial cells, mesenchymal stem cells, nasal epithelial cells, peripheral blood mononuclear cells, and pulmonary artery smooth muscle cells, more preferably GABA neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, coronary artery endothelial cells, nasal epithelial cells, peripheral blood mononuclear cells, and pulmonary artery smooth muscle cells. 50 EC values ​​against MAGE-A / MHC complex-presenting cells were 1000-fold, 15000-fold, and 9000-fold higher than the values 50 has.

[0304] In a preferred embodiment, the antigen binding protein is an EC 50EC against MAGE-A / MHC complex-presenting cells that were 9,000-fold or 10,000-fold higher than the values 50 wherein preferably said healthy cells are selected from the group consisting of iPSC-derived astrocytes, iGABA neurons, icardiomyocytes, osteoblasts, pulmonary fibroblasts, tracheal smooth muscle cells, and epidermal keratinocytes and renal cortical epithelial cells.

[0305] Thus, in one embodiment, a healthy cell has a MAGE-A / MHC complex copy number of less than 50, less than 20, less than 10, preferably less than 10, preferably a MAGE-A / MHC complex copy number of between 0 and 10.

[0306] In one embodiment, the antigen binding proteins of the invention are capable of inducing ECs to similar peptide / MHC presenting cells. 50 EC for MAGE-A / MHC complex-presenting cells is 500 to 12,000, preferably 1,000 to 12,000 higher than the value 50 EC against similar peptide / MHC presenting cells 50 EC for MAGE-A / MHC complex-presenting cells that is 100 or more, 500 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, or 6000 or more higher than the value 50 has.

[0307] "Analogous peptides" are defined herein above.

[0308] Thus, an analogous peptide / MHC presenting cell is a cell that presents an analogous peptide / MHC complex on its cell surface. In one embodiment, the analogous peptide / MHC presenting cell has a copy number of analogous peptide / MHC that is more than 10, more than 20, more than 40, more than 50, more than 80, more than 100, more than 120, more than 150, more than 300, more than 400, more than 500, more than 800, preferably 50-1000, 50-8000, such as 50-500, such as 100-500, etc.

[0309] The present invention provides antigen binding proteins capable of inducing tumor regression in vivo, more specifically complete regression under test conditions. Antigen binding proteins were prepared and evaluated at different doses against measurable HS695T tumor xenografts implanted in female NOG mice engrafted with human PBMCs (see Example 7). TCER™ targeting MAG-003 (SEQ ID NOs: 127 and 130, Group 3; SEQ ID NOs: 135 and 136, Group 4) reduced tumor size by 86 mm on the day of randomization in all mice. 3 (Group 3) and 80 mm 3 Compared to basal levels in (group 4), it induced complete remission of Hs695T tumors from day 18 to the end of the study on day 25.

[0310] Thus, in one embodiment, the antigen binding protein of the invention inhibits or reduces tumor growth, more specifically, the antigen binding protein of the invention reduces the mean tumor volume of a tumor by more than 80%, more than 85%, more than 90%, more than 92%, more than 94%, more than 96%, more than 98%, more than 99%, 95%-100%, 96%-100%, such as 98%-100%, such as 98%, 99% or 100%, preferably 100%, wherein said tumor is a cancer as defined herein below in the "Therapeutic Methods and Uses" section. In one example, the antigen binding protein induces a complete remission in a cancer tumor, wherein the cancer is as defined herein below, for example in the human tumor cell line HS695T.

[0311] In one embodiment, the half-life (T 1 / 2 ) is 6 to 7 days, such as 6 or 7 days.

[0312] As can be further seen from the examples, particularly Example 1 and Figure 1, native TCRs such as native TCR R7P1D5 could not be expressed in soluble form in either yeast or CHO cells. In contrast, the antigen binding proteins of the present invention, particularly the TCER™ molecules, preferably the preferred TCER™ molecules described herein, are not only soluble but can be transiently expressed in CHO cells in amounts of more than 10 mg / L (cell culture), more than 20 mg / L or even 40 mg / L. Thus, in one embodiment, the antigen binding proteins of the invention may be expressed at high yield in host cells, preferably where the host cells are CHO cells, in which preferably the yield is greater than 10, 15, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45 mg / L of cell culture, such as 5-50, 5,-45, 5-40, 5-35, 10-35, 10-30, 10-25, 10-20 mg / L of cell culture. In one example, the antigen binding proteins are expressed in transiently transfected CHO-S cells, in which the cells are incubated at 4x10 6 Cells were cultured at a density of 10000 / mL in a total volume of 320 mL of GE Healthcare™ medium at 37° C. After one day, feeds (CellBoost 7a and b) were added and the temperature was reduced to 32° C. Cells, and therefore antigen binding protein, were harvested after a total culture time of 12 days and a total of three feedings.

[0313] As can be further seen from the examples, the inventors have demonstrated, for example in Examples 4 and 5, that the antigen binding proteins of the invention have improved stability compared to a reference protein, for example compared to an antigen binding protein comprising the CDRs of native TCR R7P1D5 and preferably the framework regions of said native TCR R7P1D5.

[0314] Thus, in one embodiment, the antigen-binding protein of the present invention optionally has improved stability compared to the reference protein.In the context of the present invention, improved stability refers to increased physical stability, for example, when exposed to temperature stress.Thus, the newly developed antigen-binding protein of the present invention may better withstand stress conditions, especially temperature stress, than the reference antigen-binding protein.

[0315] The term "stability" in the context of the present invention refers to physical stability, which may be qualitatively and / or quantitatively assessed using various analytical techniques 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). In the context of the present invention, these methods refer in particular to the assessment of aggregate formation (e.g., using size exclusion chromatography, measuring turbidity, and / or by visual inspection). To measure stability, samples comprising the 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 optionally qualitative analysis of the chemical and physical stability using appropriate analytical techniques.

[0316] In one embodiment, the antigen binding proteins of the invention are physically stable when exposed to stress conditions for a certain period of time, such as, for example, when exposed to a temperature of 40° C. for, for example, 14 days.

[0317] "Physical stability", in the context of the present invention, refers to an antigen-binding protein that is substantially free of signs of aggregation, precipitation and / or denaturation.

[0318] Methods to access physical stability are, for example, size exclusion chromatography (SEC), dynamic light scattering (DLS), light obscuration (LO), and color and clarity, as determined, for example, by visual inspection.

[0319] "No signs of aggregation" means, for example, that after exposure to stress conditions, such as a temperature of 40°C for 14 days in a buffer such as PBS, a sample comprising the antigen binding protein has a monomer content of greater than 80%, greater than 86%, greater than 88%, greater than 90%, greater than 92%, greater than 94%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, such as a monomer content of 94%-99%, 95%-99%, 96%-99%, 97%-99% monomer content, when measured by SEC, such as SEC-HPLC, in a buffer such as PBS.

[0320] Thus, in one embodiment, an antigen binding protein of the invention exhibits reduced aggregation compared to a reference protein, for example when exposed to stress conditions.

[0321] In size exclusion chromatography (SEC), depending on the column, operating pressure and buffer velocity used, under the conditions tested, a difference of 1 or 2%, such as 1%, 2%, 3%, 4%, preferably 2%, in monomer content is considered to be a significant difference in the context of the present invention.

[0322] This means that 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 the same conditions.

[0323] In one embodiment, the antigen binding proteins of the invention have reduced aggregation when exposed to stress conditions, more specifically, the antigen binding proteins of the invention have less than 10%, less than 4%, less than 3%, less than 2% aggregation when stress induced aggregation is measured before and after exposure to said stress conditions.

[0324] Nucleic Acids, Vectors, and Recombinant Host Cells A further object of the present invention relates to an isolated nucleic acid sequence comprising or consisting of a sequence encoding an antigen binding protein of the invention as defined herein above.

[0325] Typically, the nucleic acid is a DNA or RNA molecule that may be comprised in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0326] "Vector," "cloning vector," and "expression vector" mean a vehicle by which DNA or RNA sequences (e.g., foreign genes) are introduced into a host cell, transforming the host and promoting expression (e.g., transcription or translation) of the introduced sequences.

[0327] Therefore, a further object of the present invention relates to a vector comprising a nucleic acid according to the invention.

[0328] Such vectors may comprise regulatory elements such as promoters, enhancers, terminators, etc., and may cause or direct the expression of said polypeptide when administered to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include SV40 early promoter and enhancer (Mizukami T. et al. 1987), Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), immunoglobulin H chain promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983).

[0329] Any expression vector for animal cells can be used as long as it can insert and express the gene encoding the human antibody C region. Examples of suitable vectors include pAGE107 (Miyaji H et al.1990), pAGE103 (Mizukami T et al.1987), pHSG274 (Brady G et al.1984), pKCR (O'Hare K et al.1981), pSG1 β d2-4- (Miyaji H et al.1990), etc. Other examples of plasmids include self-replicating plasmids that contain a replication origin, or integrative plasmids such as pUC, pcDNA, pBR, etc.

[0330] Other examples of viral vectors include adenovirus, retrovirus, herpes virus, and AAV vectors. Such recombinant viruses may be produced by transfection into packaging cells or by transient transfection with helper plasmids or viruses, by techniques known in the art. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and WO 94 / 19478.

[0331] 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 an exogenous nucleic acid. The vector and / or particle can be used to transfer any nucleic acid into cells, either in vitro or in vivo. Many forms of viral vectors are known in the art.

[0332] The term "virion" is used to refer to a single infectious virus particle. "Viral vector", "viral vector particle" and "virus particle" also refer to the complete virus particle that exists outside the cell, with DNA or RNA core and protein coat.For example, the viral vector can be selected from adenovirus, poxvirus, alphavirus, arenavirus, flavivirus, rhabdovirus, retrovirus, lentivirus, herpesvirus, paramyxovirus or picornavirus.

[0333] Viruses may refer to naturally occurring viruses as well as artificial viruses. Viruses according to some embodiments of the present invention may be either enveloped or non-enveloped viruses. Parvoviruses (such as AAV) are examples of non-enveloped viruses. In preferred embodiments, the virus may be an enveloped virus. In preferred embodiments, the virus may be a retrovirus, particularly a lentivirus. Viral envelope proteins that may facilitate viral infection of eukaryotic cells may include vesicular stomatitis virus (VSV-G), modified feline endogenous retrovirus (RD114TR), and HIV-1 derived lentiviral vectors (LV) pseudotyped with envelope glycoproteins (GP) from modified gibbon ape leukemia virus (GALVTR). These envelope proteins may efficiently facilitate the entry of other viruses, such as parvoviruses, including adeno-associated virus (AAV), thereby demonstrating their broad efficiency. For example, Moloney murine leukemia virus (MLV) 4070 env (as described in Merten et al., J. Virol. 79:834-840, 2005, the contents of which are incorporated herein by reference), RD114 env, chimeric envelope protein RD114pro or RDpro (RD114-HIV chimera constructed by replacing the R peptide cleavage sequence of RD114 with the HIV-1 matrix / capsid (MA / CA) cleavage sequence, as described in Bell et al. Experimental Biology and Medicine 2010;235:1269-1276, the contents of which are incorporated herein by reference), baculovirus GP64 env (as described in Wang et al. J. Virol. 81:10869-10878, 2007, the contents of which are incorporated herein by reference), or GALV env (as described in Merten et al., J. Virol. 81:10869-10878, 2007, the contents of which are incorporated herein by reference). Other viral envelope proteins may be used, including those described in (as described by L. et al., J. Virol. 79:834-840, 2005) or derivatives thereof.

[0334] A further object of the present invention relates to host cells transformed, transduced or transfected with the nucleic acids and / or vectors according to the invention.

[0335] The term "transformation" originally refers to the naturally occurring process of gene transfer into a host cell, which involves the absorption of genetic material, e.g., nucleic acid such as DNA or RNA, by the cell through the cell membrane, so that the host cell expresses the introduced gene or sequence and produces a desired substance, typically a protein or enzyme, encoded by the introduced gene or sequence. There are two types, called natural transformation and artificial or induced transformation. Artificial or induced transformation methods are performed under laboratory conditions and are typically called transfection.

[0336] A host cell that receives and expresses foreign nucleic acid, such as DNA or RNA, during the transformation process has been "transformed."

[0337] The term "transfection" refers to a mode of gene transfer that involves the creation of pores on the cell membrane of a host cell, allowing the host cell to receive foreign genetic material. Typically, transfection refers to the transformation of eukaryotic cells, such as insect or mammalian cells. Chemical-mediated transfection involves the use of, for example, calcium phosphate or cationic polymers or liposomes. Non-chemically mediated transfection methods are typically electroporation, sonoporation, impalefection, phototransfection, or hydrodynamic delivery. Particle-based transfection uses gene gun technology, which uses nanoparticles to transfer nucleic acid into host cells, or another method called magnetofection. Nucleofection and the use of heat shock are other evolved methods for successful transfection. A host cell that receives foreign nucleic acid via a transfection method is "transfected".

[0338] The term "transduction" is generally understood to relate to the transfer of foreign nucleic acid, such as DNA or RNA, into a cell by a virus or viral vector. A host cell that receives and expresses foreign nucleic acid, such as DNA or RNA, by a virus or viral vector has been "transduced."

[0339] In some embodiments, the cells may be transduced using the methods described in US20190216852, the entire contents of which are incorporated by reference herein.

[0340] The nucleic acids of the invention may be used to produce recombinant antigen binding proteins of the invention in a suitable expression system.

[0341] The term "expression system" means a host cell and a compatible vector under appropriate conditions for expression of a protein encoded by foreign DNA, for example, carried by the vector and introduced into the host cell.

[0342] 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 (such as bacteria) and eukaryotic cells (such as 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, etc.), primary or established mammalian cell cultures (e.g., generated 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 (hereinafter referred to as the "DHFR gene") (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc. In some embodiments, YB2 / 0 cells may be preferred since the ADCC activity of chimeric or humanized antibodies is enhanced when expressed in these cells.

[0343] The present invention also relates to a host cell comprising an antigen recognition construct according to the present invention. In particular, the host cell of the present invention comprises a nucleic acid or vector as described herein above. The host cell may be a eukaryotic cell, such as, for example, a plant, an animal, a fungus, or an algae, or a prokaryotic cell, such as, for example, a bacterium or a protozoan. The host cell may be a cultured cell or a primary cell, i.e., a cell directly isolated from an organism, such as, for example, a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in suspension. For the purpose of producing an antigen-binding protein, such as a recombinant TCR, polypeptide, or protein, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. The host cell may be any cell type, may be derived from any tissue type, and may be at any developmental stage, the host cell is preferably a peripheral blood leukocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell. The T cells can be any T cells, such as cultured T cells, e.g., primary T cells; or T cells from cultured T cell lines, e.g., Jurkat, SupT1; or T cells obtained from a mammal, preferably T cells or T cell precursors from a human patient. When obtained from a mammal, the T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells can also be enriched or purified. Preferably, the T cells are human T cells. More preferably, the T cells are T cells isolated from a human. The T cells can be any T cell type and at any stage of development, including, but not limited to, CD4 positive and / or CD8 positive, CD4 positive helper T cells, e.g., Th1 and Th2 cells, CD8 positive T cells (e.g., cytotoxic T cells), tumor infiltrating cells (TILs), memory T cells, naive T cells, and the like. Preferably, the T cells are CD8 positive T cells or CD4 positive T cells. The host cells are preferably tumour-reactive T cells specific for tumour cells expressing MAGEA4 and / or MAGEA8.

[0344] According to the above, in one embodiment the present invention refers to a host cell comprising an antigen binding protein of the invention, or a nucleic acid, or a vector of the invention as defined herein above, 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.

[0345] In particular, in the expression of some antigen-binding proteins of the present invention, the expression vector may be of either type, including a type in which a gene encoding a first polypeptide such as an antibody heavy chain and α chain and a gene encoding a second polypeptide such as an antibody light chain and β chain are present on separate vectors, and a type in which both genes are present on the same vector (tandem type). In terms of ease of construction of an antigen-binding protein expression vector, ease of introduction into animal cells, and balance between the expression levels of antibody H and L chains 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 (International Publication WO 97 / 10354), pEE18, etc.

[0346] In one embodiment, such recombinant host cells may be used for the production of at least one antigen binding protein of the invention. Methods of Producing Antigen Binding Proteins of the Invention The present invention also provides a. providing a suitable host cell; b. providing a genetic construct comprising a coding sequence encoding an antigen binding protein of the present invention; c. introducing said genetic construct into said suitable host cell; d. expressing said genetic construct by said suitable host cell; and optionally e. Selecting cells that express and / or secrete the antibody; The present invention also relates to a method for producing an antigen binding protein as defined herein above comprising the steps of:

[0347] The antigen binding proteins of the present invention are defined in the corresponding section herein above.

[0348] A "genetic construct" herein refers to a nucleic acid that allows for the expression of a coding region in a host, and thus refers to a nucleic acid such as a vector or RNA as described above.

[0349] In one embodiment, the method may further comprise the step of displaying said antigen recognition construct on the cell surface of said suitable host cell.

[0350] In another preferred embodiment, the genetic construct of b) comprises a nucleic acid encoding an antigen-binding protein of the invention, such a nucleic acid being as defined herein above in the section "Nucleic acids, vectors and recombinant host cells".

[0351] In a related embodiment, the genetic construct is an expression construct comprising a promoter sequence operably linked to said coding sequence.

[0352] In a related embodiment, the genetic construct is introduced into a suitable host using transformation, transduction or transfection, as defined herein above.

[0353] Desirably, the transfection system for introducing the genetic construct into said suitable host cell is a retroviral or lentiviral vector system as described herein above in the sections on nucleic acids, vectors and recombinant host cells. Such systems are well known to those skilled in the art.

[0354] In one embodiment, the method further comprises isolation and purification of the antigen binding protein from the host cell, and optionally, reconstitution of the antigen binding protein in T cells.

[0355] The antigen binding proteins of the present invention may be produced by any technique known in the art, including, without limitation, chemical, biological, genetic or enzymatic techniques, either alone or in combination.

[0356] In some specific embodiments, the antigen binding proteins of the invention have an antibody or immunoglobulin-like framework.

[0357] Standard techniques for producing polypeptides such as antibodies or fragments thereof and TCRs or fragments thereof are known in the art, and these techniques can be transferred to the antigen binding proteins of the invention by those skilled in the art. For example, they can be synthesized using well-known solid-phase methods, in particular using commercially available peptide synthesizers (such as those manufactured by Applied Biosystems, Foster City, Calif.), following the manufacturer's instructions. Alternatively, the antigen binding proteins of the invention, such as antibodies or fragments thereof and TCRs or fragments thereof, can be synthesized by recombinant DNA techniques well known in the art. For example, 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, the fragments can be obtained as DNA expression products, from which the fragments can then be isolated using well-known techniques.

[0358] In one example, i.e. in the case of TCER™ bispecific molecules, DNA sequences encoding various combinations of VH and VL, and variable alpha (Vα) and variable beta (Vβ), as well as sequences encoding linkers, may be obtained, for example, by gene synthesis. The obtained DNA sequences may contain, for example, the hinge region, C, C-terminal region ... H2 , and CH3 The C domains may be cloned in frame into expression vectors encoding each of the C domains and further engineered, with or without additional interchain disulfide bond stabilization. H3 To incorporate "knob-into-hole" mutations into the C domain H2 To remove the N-glycosylation site of F (e.g., N297Q mutation); c - to introduce silencing mutations; or to introduce additional disulfide bond stabilization into VL and VH, respectively, according to the method 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); manipulations may be performed to include mutations that enhance stability or manufacturability.

[0359] The antigen binding proteins of the invention are suitably separated from the culture medium by immunoglobulin purification procedures such as, for example, protein A-Sepharose, protein L-Sepharose, protein G-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0360] In one embodiment, recovering the expressed antigen binding protein or polypeptide as used herein refers to performing Protein A chromatography, Protein L chromatography, Kappa selection chromatography, and / or size exclusion chromatography, preferably Protein L chromatography and / or size exclusion chromatography, more preferably Protein L-chromatography and size exclusion chromatography.

[0361] Methods for producing the antigen binding proteins of the present invention involving recombinant DNA and gene transfection techniques are well known in the art (see Morrison SL. et al. (1984) and patent documents U.S. Pat. No. 5,202,238; and U.S. Pat. No. 5,204,244).

[0362] Furthermore, methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well 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 the antigen-binding proteins of the present invention.

[0363] In one example, vectors for the expression of recombinant antigen-binding proteins of the invention were designed as monocistronic, controlled, for example, by a pUC19 derivative, a promoter element from HCMV. Plasmid DNA was amplified, for example, in E. coli according to standard culture methods and subsequently purified using a commercially available kit (Macherey & Nagel). The purified plasmid DNA was used for transient transfection of CHO-S cells, for example, according to the manufacturer's instructions (ExpiCHO™ system; Thermo Fisher Scientific) or using an electroporation system (MaxCyte STX). The transfected CHO-cells were cultured, for example, at 32°C to 37°C for 6 to 14 days and fed 1 to 2 times with ExpiCHO™ Feed or Cellboost 7a and 7b (GE Healthcare™) solutions.

[0364] The adjusted cell supernatant was clarified by filtration (0.22 μm), for example, using Sartoclear Dynamics® Lab Filter Aid (Sartorius). The bispecific antigen-binding protein was purified, for example, using an Akta 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. 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), to obtain highly pure monomeric protein according to standard protocols. Protein concentration was determined, for example, on a NanoDrop system (Thermo Scientific) using the extinction coefficient calculated according to the predicted protein sequence. If necessary, the concentration was adjusted 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.

[0365] The quality of the purified bispecific antigen-binding proteins was determined by HPLC-SEC, e.g., 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 Vanquishu HPLC-System.

[0366] In a particular embodiment, the T cell as defined above is provided in step a), and the antigen binding protein is TCR or a fragment thereof.Accordingly, the antigen binding protein is expressed on the cell surface of the T cell.Accordingly, the method generates a T cell that expresses an antigen binding protein, such as a TCR, specific for a cancer cell on its surface, as defined in the context of the present invention.

[0367] Pharmaceutical Compositions The present invention further refers 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.

[0368] 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 of the invention for use as a medicament.

[0369] The present invention also relates to the use of an antigen-binding protein according to the invention or / and a pharmaceutical composition according to the invention in the manufacture of a medicament.

[0370] 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.

[0371] In some embodiments, a subject may also be referred to as a patient.

[0372] Such pharmaceutical compositions may comprise a therapeutically effective amount of the antigen binding protein of the invention, or an antigen binding protein further comprising a therapeutic agent, mixed with a pharma- ceutical or physiologically acceptable formulation selected for compatibility with the mode of administration.

[0373] The antigen binding proteins of the invention will normally be supplied as part of a sterile pharmaceutical composition, which will normally include a pharma- ceutically acceptable carrier.

[0374] "Pharmaceutically" or "pharmacologically acceptable" refers to molecules and compositions that do not produce adverse, allergic or other untoward reactions, as appropriate, when administered to a mammal, particularly a human. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0375] A "pharmacologically acceptable carrier" may also be referred to as a "pharmacologically acceptable diluent" or a "pharmacologically acceptable vehicle" and may include solvents, fillers, stabilizers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. Thus, in one embodiment, the carrier is an aqueous carrier.

[0376] In another aspect, the aqueous carriers, when combined with the antigen binding proteins described herein, can impart improved properties, such as, for example, improved solubility, efficacy, and / or improved immunotherapy.

[0377] The form, route of administration, dosage, and regimen of the pharmaceutical composition will necessarily depend on the condition being treated, the severity of the disease, the age, weight, and sex of the patient, the desired duration of treatment, and the like. The pharmaceutical composition may be in any suitable form (depending on the desired method of administering it to the patient). It may be provided in a unit dosage form, generally provided in a sealed container, and may be provided as part of a kit. Such a kit will usually (but need not) include instructions for use. It may include a plurality of said unit dosage forms.

[0378] Empirical considerations such as biological half-life generally contribute to the determination of dosage. The frequency of administration may be determined and adjusted during the course of treatment, and is based on reducing the number of cancer cells, maintaining the reduction of cancer cells, reducing the proliferation of cancer cells, or killing cancer cells. Alternatively, sustained sustained release formulations of antigen-binding proteins may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0379] In one embodiment, the dosage of the antigen binding protein can be empirically determined in individuals who have received one or more doses. Individuals are given increasing doses of the antigen binding protein. To evaluate the effectiveness of the antigen binding protein, markers of the state of cancer cells can be tracked. These include direct measurement of cancer cell proliferation and cell death by FACS, other imaging techniques; and improved health as assessed by such measurements, or improved quality of life as measured by recognized tests or prolonged survival. It will be clear to those skilled in the art that dosages will vary depending on the individual, the stage of the disease, and previous and concurrent treatments being used.

[0380] In particular, the pharmaceutical compositions contain pharma- ceutically acceptable vehicles for injectable preparations, which may in particular be isotonic, sterile, saline (such as mono- or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts), or dry, in particular lyophilized, compositions, which upon addition of sterile water or saline, allow the constitution of an injectable solution.

[0381] To prepare a pharmaceutical composition, 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.

[0382] Pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It 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.

[0383] Solutions of the active compounds as free bases or pharmacologically acceptable salts 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.

[0384] The antigen binding proteins of the present invention may be formulated into neutral or salt form compositions using pharma- ceutically acceptable salts.

[0385] Sterile injection is prepared by incorporating the active compound in the required amount in a suitable solvent together with various other ingredients listed above as necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains basic dispersion medium and other necessary ingredients listed above.In the case of aseptic powder for preparing sterile injection, the preferred preparation method includes vacuum drying and freeze-drying technology, which produces powder of active ingredient and any additional desired ingredient from the solution that has been previously sterile-filtered.

[0386] The preparation of more concentrated or highly concentrated solutions for direct injection is also contemplated, in which the use of DMSO as a solvent is envisioned to result in very rapid penetration, delivering high concentrations of active agent to small tumor areas.

[0387] 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 used.

[0388] Treatments and Uses The inventors have demonstrated in Example 6 of the in vitro experiments section the cytotoxic activity of several antigen binding proteins of the invention, in particular the soluble TCER™ molecules #114-iso0-UCHT1(17), #114-iso1-UCHT1(17), #114-iso2-UCHT1(17), #114-iso0-BMA031(36), #114-iso1-BMA031(36), and #114-iso2-BMA031(36) against MAG-003 positive cancer cell lines such as Hs695T, A375, and U2OS. * Only little lysis was induced by the bispecific antigen-binding protein in cell lines expressing MAG-003 but not displaying peptide MAG-003, further demonstrating that the cytotoxic activity was highly specific and restricted to MAG-003 positive cells.

[0389] Furthermore, the inventors have demonstrated the in vivo efficacy of antigen binding proteins of the invention such as TCER™ targeting MAG-003 (SEQ ID NOs: 127 and 130, Group 3; SEQ ID NOs: 135 and 136, Group 4) leading to complete remission of HS695T tumors in NOG mice, as described in detail in Example 7 and shown in FIG. 11.

[0390] As demonstrated by the inventors, the antigen binding proteins of the present invention are useful for generating an immune response in a subject, preferably a patient, in which tumor cells can be destroyed by this immune response. Since the peptide KVLEHVVRV (SEQ ID NO: 1) is not presented or over-presented on normal tissues in comparable copy numbers, the immune response resulting from such a therapeutic vaccination can be expected to be highly specific against tumor cells, preventing the risk of an unwanted autoimmune reaction against normal cells of the patient. Moreover, as indicated in the introduction of this specification, the antigen binding proteins of the present invention specifically bind to an epitope in KVLEHVVRV, in particular said peptide MAG-003 as defined herein, and therefore do not recognize or cross-react with other related peptide antigens (similar peptides) present on normal tissues. Thus, the antigen binding proteins of the present invention are useful for immunotherapy.

[0391] Thus, the antigen binding proteins of the present invention, particularly bispecific antigen binding proteins such as TCER™ molecules, may be used to treat cancer.

[0392] The antigen binding proteins of the present invention may be used for therapeutic purposes in humans and / or non-human mammals, particularly humans.

[0393] In one embodiment, the antigen binding proteins of the present invention can bind to tumor cells and reduce proliferation and / or kill tumor cells that present the peptide KVLEHVVRV (SEQ ID NO: 1) / MHC complex on their cell surface. It is understood that the antigen binding protein is administered at a concentration that promotes binding under physiological (e.g., in vivo) conditions.

[0394] Thus, in one embodiment, the antigen binding proteins of the present invention may be used in immunotherapy against tumor cells of different tissues such as colon, lung, breast, prostate, ovary, pancreas, kidney, etc. In another embodiment, the antigen binding proteins of the present invention may bind to tumor cells, reduce their proliferation and / or kill them.

[0395] Thus, 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 present invention, as defined herein above under the sections "Antigen binding protein", "Nucleic acid" or "Pharmaceutical composition".

[0396] In a particular embodiment, the present invention relates to a method of treating a subject with a proliferative disease comprising administering to said subject a T cell expressing an antigen binding protein of the present invention on its cell surface.

[0397] In a further embodiment, the present invention refers to a method of eliciting an immune response in a subject with a proliferative disease, comprising administering to said subject a composition comprising T cells expressing an antigen recognition construct of the present invention on their cell surface.

[0398] In one embodiment, the immune response referred to in the method is a cytotoxic T cell response.

[0399] The present invention further refers to an antigen binding protein of the invention, a nucleic acid of the invention or a vector of the invention, a host cell of the invention or a pharmaceutical composition of the invention for use in the diagnosis, prevention and / or treatment of a proliferative disease.

[0400] The present invention further refers to the use of an antigen binding protein of the invention, a nucleic acid of the invention or a vector of the invention, a host cell of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for the diagnosis, prevention and / or treatment of a proliferative disease.

[0401] In one embodiment the invention refers to the use of an antigen binding protein, a nucleic acid or vector, a host cell or a pharmaceutical composition according to the invention for treating or preventing a proliferative disease or disorder in a subject.

[0402] In a preferred embodiment, the T cells are autologous to the subject or allogeneic to the subject, preferably autologous.Preferably, the T cells can be obtained from tumor-infiltrating lymphocytes or peripheral blood mononuclear cells.Preferably, the T cells are administered in the form of a composition.In a preferred related embodiment, the composition further comprises an adjuvant, wherein the adjuvant is defined herein above in the pharmaceutical composition section.

[0403] The terms "subject" or "individual" are used interchangeably and may be, for example, a human or non-human mammal, preferably a human.

[0404] In the context of the present invention, the term "treat" or "treatment" refers to therapeutic use (i.e., in a subject having a given disease) and means to reverse, alleviate, or inhibit the progression of one or more symptoms of such disorder or condition.Thus, treatment refers not only to treatment that results in a complete cure of the disease, but also to treatment that slows the progression of the disease and / or extends the survival of the subject.

[0405] By "preventing" is meant prophylactic use (i.e., in a subject susceptible to a given disease).

[0406] In one embodiment, a "disease" or "disorder" is any medical 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.

[0407] The term "in need of treatment" refers to a subject already with the disorder, as well as a subject in which the disorder is to be prevented. Thus, in one embodiment, the subject is a patient.

[0408] "Proliferative disorders," such as cancer, involve the uncontrolled and / or inappropriate proliferation of cells.

[0409] In one embodiment, the proliferative disorder or disease is a tumor disease characterised by expression of MAGEA4 and / or MAGEA8, for example in cancer or tumor cells of said tumor disease.

[0410] Thus, particularly preferred cancers are MAGEA4 and / or MAGEA8 positive cancers.

[0411] In the context of the present invention, a cancer is considered to be "MAGEA4 and / or MAGEA8 "positive"" if the relevant peptide, such as, for example, the MAG-003 peptide, is present in 98% or more of all cancers according to guidelines by the NCI. In any other indications mentioned herein, a biopsy may be performed, as this is the standard in the treatment of these cancers, and peptides may be identified according to XPresident® and related methods (see WO 03 / 100432; WO 2005 / 076009; WO 2011 / 128448; WO 2016 / 107740; US Patent Nos. 7,811,828, 9,791,444, and 2016 / 0187351, the contents of each of which are incorporated herein by reference in their entirety). In one embodiment, the cancer is easily assayed (i.e., diagnosed), for example, by using the antigen binding proteins of the present invention. Methods for identifying antigens expressing cancer using antigen-binding proteins are known to those skilled in the art.It should be understood that the terms "cancer" and "carcinoma" are not used interchangeably herein, since carcinoma is a specific type of cancer that occurs in the skin or tissues covering body organs.However, the tissue samples used in Example 11 are derived from tumor patients, and the samples are assigned to the patient's disease.

[0412] In one embodiment, the MAGEA4 and / or MAGEA8 "positive" cancers, i.e. those that present target peptides, are selected from the group consisting of lung cancer, such as non-small cell lung cancer and small cell lung cancer, liver cancer, head and neck cancer, skin cancer, renal cell carcinoma, brain cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, leukemia, breast cancer, Merkel cell carcinoma, melanoma, ovarian cancer, bladder cancer, uterine cancer, gallbladder and bile duct cancer, osteosarcoma, and esophageal cancer.

[0413] In one embodiment, the MAGEA4 and / or MAGEA8 "positive" cancers, i.e. those that present the target peptides, are selected from the group consisting of lung cancer, such as non-small cell lung cancer and small cell lung cancer, liver cancer, head and neck cancer, skin cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, breast cancer, melanoma, ovarian cancer, bladder cancer, uterine cancer, gallbladder and bile duct cancer, and esophageal cancer.

[0414] In another embodiment, the lung cancer is non-small cell lung cancer (NSCLC), preferably non-small cell lung cancer adenocarcinoma or squamous cell non-small cell lung cancer (SNSCLC); or small cell lung cancer (SCLC). In another embodiment, the cancer is skin cancer, preferably melanoma. In another embodiment, the cancer is head and neck cancer, preferably head and neck squamous cell carcinoma (HNSCC). In another embodiment, the cancer is liver cancer, preferably hepatocellular carcinoma (HCC). In another embodiment, the cancer is esophageal cancer, preferably gastroesophageal junction cancer.

[0415] Texts that provide guidance in cancer treatment include Cancer, Principles and Practice of Oncology, 4th Edition, DeVita et al, Eds. J.B. Lippincott Co., Philadelphia, Pa. (1993). The appropriate treatment approach is selected depending on the particular type of cancer and other factors, such as the patient's general condition, as recognized in the relevant field. The antigen binding proteins of the present invention may be used by themselves or may be added to a treatment regimen using other anti-cancer drugs typically used to treat cancer patients.

[0416] Thus, in some embodiments, the antigen binding proteins of the invention may be administered simultaneously with, prior to, or subsequent to a variety of drugs and therapies commonly used in cancer treatment, such as, for example, chemotherapeutic agents, non-chemotherapeutic agents, anti-tumor agents, and / or radiation, preferably chemotherapeutic agents.

[0417] "Diagnosis" herein refers to a medical diagnosis and refers to determining which disease or condition explains a person's symptoms and signs.

[0418] A "therapeutically effective amount" of an antigen binding protein or pharmaceutical composition thereof means an amount of the 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 or monthly 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 amount level for any particular patient will depend on a variety of factors, including the disorder or disease being treated and the severity of the disorder; the activity of the specific antigen binding protein used; the specific composition used, the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and excretion rate of the specific polypeptide used; the duration of treatment; drugs used in combination or simultaneously with the specific polypeptide used; and similar factors well known in the medical arts. For example, it is well within the skill of the art to start administering a compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0419] In one embodiment, the efficacy of treatment with an antigen binding protein of the invention is assayed in vivo, e.g., in a mouse model of cancer, by measuring the change in tumor volume between treatment and control groups.

[0420] For example, pharmaceutical compositions, vectors, nucleic acids, and cells of the invention may be provided, for example, in a substantially pure form in which 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% by weight of the same type of antigen binding protein is present.

[0421] 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 practicable method.

[0422] As disclosed herein, in some embodiments, host cells, preferably lymphocytes such as T lymphocytes or T lymphocyte progenitors, e.g., CD4 or CD8 positive T cells, most preferably T cells, are used in the medical uses or treatment methods described herein.

[0423] Thus, the host cell of the present invention, preferably T cell, can be used as an active ingredient of therapeutic composition.Thus, the present invention also provides a method for killing target cells in a patient, comprising administering an effective number of host cells, preferably T cell, as defined above to the patient.In the context of this method, the host cell, once administered to the subject, preferably induces an immune response.

[0424] For purposes of the methods of the invention in which a host cell or population of cells is administered to a subject, the host cells can be allogeneic (from another subject) or cells that are autologous to the subject. Preferably, the cells are autologous to the subject.

[0425] Where the host cells are allogeneic and therefore from another subject, said other subject is healthy.

[0426] In some embodiments, T cells may be produced according to the methods described in U.S. Patent No. 20190175650, U.S. Patent Application No. 16 / 271,393, and U.S. Patent Application No. 16 / 361,043, the contents of which are incorporated by reference in their entireties.

[0427] 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.

[0428] In a particular example, the host cell is a T cell.Therefore, in the context of the present invention, when the T cell as defined herein above is used as a drug, the T cell is usually taken from the subject by apheresis.Then, the T cell is genetically engineered to express the antigen-binding protein of the present invention on their cell surface, and then the genetically engineered T cell is expanded and then reinfused into the subject.In this example, the antigen-binding protein is preferably TCR.

[0429] 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, a scTCR, or a soluble antigen binding protein as defined herein, more preferably a bispecific soluble antigen binding protein, such as TCER™ as described herein.

[0430] Thus, the host cell has been transformed, transduced or transfected with the nucleic acid and / or vector according to the invention, as described herein above in the section "Nucleic Acids, Vectors and Recombinant Host Cells".

[0431] Where a host cell is transformed, transduced or transfected to express an antigen binding protein of the invention, preferably the cell comprises an expression vector capable of expressing the antigen binding protein. Once the host cell expresses an antigen binding protein of the invention, the host cell may be referred to as an activated host cell.

[0432] Protocols for this so-called adoptive transfer of T cells are well known in the art and are reviewed 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).

[0433] Several methods may be used to generate T cells in vitro. For example, autologous tumor-infiltrating lymphocytes may be used to generate CTLs. Plebanski et al. (Plebanski, M. et al., Eur. J Immunol 25 (1995): 1783-1787) utilized autologous peripheral blood lymphocytes (PLB) in preparing T cells. B cells may also be used in generating autologous T cells.

[0434] Allogeneic cells may also be used in the preparation of T cells, the methods of which are described in detail in US Pat. No. 6,805,861, incorporated herein by reference. In one embodiment, TCR-induced immune response or T cell response may refer to the proliferation and activation of effector functions induced by binding to MAG-003 / MHC complex in vitro or in vivo.For example, in MHC class I-restricted cytotoxic T cells, effector functions may be lysis of peptide-pulsed, peptide precursor-pulsed, or naturally peptide-presenting target cells; secretion of cytokines, preferably interferon-γ, TNF-α, or IL-2, induced by peptide; secretion of effector molecules, such as granzymes or perforins, induced by peptide; or degranulation.

[0435] As described herein above, host cells such as T cells expressing an antigen binding protein of the invention that specifically binds to the MAG-003 / MHC complex are useful in therapy. Thus, a further aspect of the present invention provides activated host cells obtainable by the aforementioned method of the present invention.

[0436] The activated host cells produced by the above methods may selectively recognise target cells.

[0437] "Target cells" herein refer to the above-mentioned presenting cells, MAGE-A / MHC presenting cells as defined herein above in the section "Antigen binding proteins".

[0438] In one embodiment, the target cells are preferably cancer cells, wherein cancer is defined herein above.

[0439] In vivo, target cells for the CD8 positive T cells according to the invention may be tumor cells (which sometimes express MHC class II) and / or stromal cells surrounding the tumor (tumor cells) (which sometimes also express MHC class II; (Dengjel, J. et al., Clin Cancer Res 12(2006):4163-4170).

[0440] kit Finally, the present invention also provides a kit comprising at least one antigen binding protein of the present invention.

[0441] In one embodiment, the kit comprises: a) at least one antigen-binding protein of the invention as defined herein above under the section "Antigen-binding proteins"; b) optionally packaging materials, and c) optionally, a label or package insert contained within said packaging material indicating that said antigen binding protein is effective for treating or for use in the treatment of cancer. The present invention relates to a method for producing a semiconductor device comprising the steps of:

[0442] 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 dissolution syringes).

[0443] In one embodiment, the invention encompasses a kit for producing a single-dose administration unit.

[0444] 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 contained in a first container. The kit then further comprises a second container with an aqueous formulation.

[0445] Therefore, the kit is a) a first container comprising at least one dried antigen binding protein of the invention as defined herein above in the section "Antigen binding proteins", b) a second container comprising the aqueous formulation; c) optionally packaging materials, and d) optionally, a label or package insert contained within said packaging material indicating that said antigen binding protein is effective for treating or for use in the treatment of cancer. The present invention relates to a method for producing a semiconductor device comprising the steps of:

[0446] Aqueous formulations are typically aqueous solutions comprising a pharma- ceutical acceptable carrier, as defined herein above under the section "Pharmaceutical Compositions."

[0447] In related embodiments, the "first container" and "second" container refer to the chambers of a multi-chambered pre-filled syringe (e.g., a dissolution syringe).

[0448] Cancer is defined herein above in the context of the present invention.

[0449] Throughout this application, the term "and / or" is a grammatical conjunction that should be interpreted to encompass that one or more of the circumstances to which it is connected may occur. For example, the phrase "such native sequence proteins may be prepared using standard recombinant and / or synthetic methods" indicates that the native sequence proteins may be prepared using standard recombinant and synthetic methods, that the native sequence proteins may be prepared using standard recombinant methods, or that the native sequence proteins may be prepared using synthetic methods.

[0450] Moreover, throughout this application, the term "comprising" should be interpreted to encompass all specifically mentioned features as well as optional, additional, unspecified features. As used herein, use of the term "comprising" also discloses embodiments in which no features are present other than those specifically mentioned (i.e., "consisting of").

[0451] Moreover, the indefinite articles "a" or "an" do 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.

[0452] The present invention will now be described in more detail with reference to the following figures and examples. All documents and patents cited herein are incorporated herein by reference in their entirety. Although the present invention has been illustrated and described in detail in the foregoing description, the examples should be considered as illustrative or exemplary, and not restrictive.

[0453] Array Description [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Brief description of the drawings]

[0454] 1-18 show embodiments described herein. [Figure 1] Results of converting a TCR into a stabilized scTCR via yeast surface display. ScTCR molecules displayed on the surface of transformed Saccharomyces cerevisiae EBY100 were stained with FITC-labeled anti-Vβ8 antibody and PE-labeled HLA-A*02 / MAG-003 tetramer. Unmodified scTCR R7P1D5 (left panel, SEQ ID NO: 19) is compared to a scTCR variant carrying two stabilizing single point mutations (right panel, SEQ ID NO: 22) derived from a selection of a random mutation scTCR library. It can be seen that the scTCR variant carrying two stabilizing single point mutations shows increased HLA-A*02 / MAG-003 tetramer staining. [Diagram 2]Figure 2A: Yeast surface display results for scTCR affinity maturation of CDR3α. Stabilized scTCRs comprising unmodified and mature CDR3α were stained with HLA-A*02 / MAG-003 monomers at a concentration of 15 nM. Counterstaining with a mixture of 10 HLA-A*02 tetramers, each applied at a concentration of 10 nM, containing peptides (SEQ ID NO: 23-32) with high sequence similarity to MAG-003 (SEQ ID NO: 1). Stabilized scTCR (SEQ ID NO: 22) containing unmodified α-chain CDR3 sequence CAEYSSASKIIF (SEQ ID NO: 7) was compared with scTCR variants comprising affinity matured α-chain CDR3 sequences CAEFSSASKIIF (SEQ ID NO: 38), CAEMTSESKIIF (SEQ ID NO: 35), CAEFTSESKIIF (SEQ ID NO: 36), CAEFNSESKIIF (SEQ ID NO: 37), and CAEATSESKIIF (SEQ ID NO: 39), respectively. Yeast clones in which the CDRs α2 (SEQ ID NO: 56, 57, 59), β1 (SEQ ID NO: 62), and β2 (SEQ ID NO: 63, 64, 65, 66, 70) have been mutated also show strongly improved binding while specificity for HLA-A*02 / MAG-003 is largely maintained. Figure 2B: Results of yeast surface display of scTCR affinity maturation of CDR2α, CDR3α, CDR1β, and CDR2β, respectively. Stabilized scTCRs comprising unmodified and matured CDRs were stained with HLA-A*02 / MAG-003 monomer (targeted pHLA monomer) and counterstained with a mixture of 10 HLA-A*02 tetramers, each applied at a concentration of 10 nM, containing peptides (SEQ ID NO: 23-32) with high sequence similarity to MAG-003 (SEQ ID NO: 1). (a) Comparison of scTCR variants containing unmodified (sequence number 6) and mature α-chain CDR2 sequences IYSSQDQ (sequence number 56), IYSSQDV (sequence number 57), and IYSSQDS (sequence number 59), each stained with 40 nM HLA-A*02 / MAG-003 monomer.(b) Comparison of scTCR variants containing unmodified (SEQ ID NO: 7) and mature α chain CDR3 sequences CAEFSSASKIIF (SEQ ID NO: 38), CAEMTSESKIIF (SEQ ID NO: 35), CAEFTSESKIIF (SEQ ID NO: 36), CAEFNSESKIIF (SEQ ID NO: 37) and CAEATSESKIIF (SEQ ID NO: 39) stained with 15 nM of HLA-A*02 / MAG-003 monomer, respectively. (c) Comparison of scTCR variants containing unmodified (SEQ ID NO: 12) and mature β chain CDR1 sequence PGHDY (SEQ ID NO: 62) stained with 40 nM of HLA-A*02 / MAG-003 monomer, respectively. (d) Comparison of scTCR variants containing unmodified (SEQ ID NO: 13) and mature β chain CDR2 sequences FCYGHP (SEQ ID NO: 63), FCYGVP (SEQ ID NO: 64), FCYGTP (SEQ ID NO: 65), FCYGAP (SEQ ID NO: 66), FCYGMP (SEQ ID NO: 70) stained with 5 nM each of HLA-A*02 / MAG-003 monomer. Staining with HLA-A*02 / MAG-003 tetramer was significantly increased for all R7P1D5 CDRa3 mutants when compared to the parental TCR R7P1D5, indicating improved peptide-HLA binding of the mutant variants. [Diagram 3] Staining of mature R7P1D5 TCR variant expressing human CD8+ T cells with PE-labeled HLA-A*02 / MAG-003 tetramer and FITC-labeled anti-Vβ8 antibody. For control purposes, cells not expressing TCR (MOCK) or 1G4 TCR specific for NYESO1-001 were stained with PE-labeled HLA-A*02 / NYESO1-001 tetramer. [Figure 4]Figure 1 shows IFN-γ release of mature R7P1D5 TCR variants expressing human CD8+ T cells in response to MAG-003. For control purposes, no TCR (MOCK) or 1G4 TCR specific for NYESO1-001 were expressed. IFN-γ release was determined by ELISA after co-culture of electroporated CD8+ T cells with T2 cells loaded with serial dilutions of MAG-003. Baseline levels of IFN-γ (released by mock-electroporated CD8+ T cells upon co-culture with T2 cells loaded respectively) were subtracted. Compared to parental TCR R7P1D5, mature R7P1D5 CDRa3 variants 1-5 showed increased IFN-γ release in response to MAG-003. [Diagram 5] IFN-γ release of mature R7P1D5 TCR variants expressing human CD8+ T cells in response to MAG-003 and different analogous peptides. For control purposes, no TCR (MOCK) or 1G4 TCR specific for NYESO1-001 were expressed. IFN-γ release was determined by ELISA after co-culture of electroporated CD8+ T cells with T2 cells loaded with 10 μM of peptide. Baseline levels of IFN-γ (released by mock-electroporated CD8+ T cells upon co-culture with T2 cells loaded with each) were subtracted. Compared to the parental TCR R7P1D5, mature R7P1D5 CDRa3 variants 1-5 showed increased IFN-γ release in response to MAG-003 and no IFN-γ release with the analogous peptides tested. [Figure 6] Figure 1 shows IFN-γ release of mature R7P1D5 TCR variant expressing human CD8+ T cells in response to MAG-003 and the respective alanine substituted peptides. For control purposes, no TCR (MOCK) or 1G4 TCR specific for NYESO1-001 were expressed. IFN-γ release was determined by ELISA after co-culture of electroporated CD8+ T cells with 10 μM peptide-loaded T2 cells. Baseline levels of IFN-γ (released by mock-electroporated CD8+ T cells upon co-culture with the respective loaded T2 cells) were subtracted. [Figure 7]Figure 7A: Production yield and heat stress stability data of soluble scTCR-Fab antigen binding proteins comprising TCR variable domains with different affinity matured CDR and / or framework mutations. Figure 7B: Binding curves of soluble scTCR-Fab antigen binding proteins S, #1, #2, and I to MAG-003 in complex with HLA-A*02 measured by biolayer interferometry. Increasing concentrations of HLA-A*02 / MAG-003 monomer in solution were used for each interaction measurement and are shown in nM. Figure 7C: Binding curves of soluble scTCR-Fab antigen binding proteins I (grey curve) and #19 (black curve) to MAG-003 and normal tissue derived off-target peptides SYNE3-001, TPX2-001, PSME2-001, respectively, in complex with HLA-A*02. Biolayer interferometry was used for the measurements, and the concentration of scTCR-Fab in solution was 1 μM. [Figure 8]Figure 8A: Production yield and heat stress stability data of soluble TCER™ antigen binding proteins comprising TCR variable domains with selected affinity matured CDRs combined with different VH / VL sequences of humanized T cell recruiting antibody BMA031 (36) and different variants 17, 17opt, 21, 23 of humanized T cell recruiting antibody UCHT1. Figure 8B: Binding curves of TCER™ antigen binding proteins comprising TCR variable domain variants 114-iso0, 114-iso1, and 114-iso2 combined with UCHT1 (17) and BMA031 (36) of MAG-003 in complex with HLA-A*02. Measurements were performed using Biolayer Interferometry, applying increasing concentrations of TCER™ molecules in solution, and are given in nM. Figure 8C: Binding curves of 114-iso1-BMA(36) and 114-iso2-UCHT1(17)TCER™ molecules to MAG-003 and normal tissue-derived off-target peptides NOMAP-1-0320, NOMAP-1-1223, and ODC-001, respectively, in complex with disulfide-stabilized HLA-A*02. Measurements were performed using Biolayer Interferometry, with a TCER™ concentration of 1 μM in solution. [Figure 9]Figure 1 shows in vitro efficacy data of TCER™-mediated killing of HLA-A*02 positive tumor cell lines with detectable MAG-003 expression (Hs695T and A375) and without detectable MAG-003 expression (BV173). TCER™ molecules were tested comprising UCHT1 (17) antibody variable domains with variable domains of TCR variants #114-iso0 (SEQ ID NOs: 127 and 130), #114-iso1 (SEQ ID NOs: 131 and 130), and #114-iso2 (SEQ ID NOs: 133 and 130) (top panel). In addition, TCER™ molecules were tested comprising BMA031 (36) antibody variable domains with variable domains of TCR variants #114-iso0 (SEQ ID NOs: 135 and 136) and #114-iso2 (SEQ ID NOs: 139 and 136) (bottom panel). Tumor cells were co-incubated with human PBMCs from a healthy HLA-A*02+ donor (HBC-720) at a ratio of 1:10 and cytotoxic activity was analyzed by LDH-release assay with increasing concentrations of TCER™ molecules. After 48 hours, lysis of the target cell lines was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (PROMEGA). Efficient cytolysis of TCER™ molecules comprising UCHT1 (17) and BMA031 (36) antibodies is shown. [Figure 10]Figure 1 shows TCER™-mediated in vitro killing of HLA-A*02 positive tumor cell lines (Hs695T, A375, and U2OS) with detectable MAG-003 expression. TCER™ molecules comprising the UCHT1 (17) antibody variable domain with the variable domains of TCR variants #114-iso1 (SEQ ID NOs: 131 and 130) and #114-iso2 (SEQ ID NOs: 133 and 130) and TCER™ molecules comprising the variable domains of the BMA031 (36) antibody with the variable domains of TCR variants #114-iso1 (SEQ ID NOs: 137 and 136) were tested. Tumor cells were co-incubated with human PBMCs from a healthy HLA-A*02+ donor (HBC-982) in a ratio of 1:10, and cytotoxic activity was analyzed by LDH-release assay with increasing concentrations of TCER™ molecules. After 48 hours, lysis of tumor cell lines was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (PROMEGA). Efficient tumor cell lysis of TCER™ molecules comprising UCHT1 (17) and BMA031 (36) antibodies is shown. [Figure 11]In immunodeficient NOG mice transplanted with human PBMC against tumor xenografts of Hs695T cells, an HLA-A*02 positive tumor cell line, with detectable presentation of MAG-003 (SEQ ID NO: 1) and PRAME-004 (SEQ ID NO: 168). TCER™-mediated in vivo efficacy is shown. TCER™ comprising the UCHT1 (17) antibody variable domain with the variable domain of TCR variant #114-iso0 (SEQ ID NO: 127 and 130, group 3) was compared with TCER™ comprising the BMA031 (36) antibody variable domain with the variable domain of TCR variant #114-iso0 (SEQ ID NO: 135 and 136, group 4). As a control, TCER targeting PRAME-004 (SEQ ID NO: 169 and 170, group 2) was used. Group 1 corresponds to the vehicle-treated control group, in which one animal was sacrificed due to tumor ulceration on day 11. Each symbol connected by a line represents an individual animal and treatment days are indicated by arrows. A complete response is shown for TCER™ comprising UCHT1 (17) and BMA031 (36) antibodies for T cell recruitment. [Figure 12]The in vitro safety data of the TCER™ molecule 114-iso1-BMA(36)) (SEQ ID NOs: 137 and 136) is evaluated in an LDH killing experiment with 11 human HLA-A*02 positive normal tissue primary cells from different organs. The different cells were co-cultured with PBMC effector cells from healthy HLA-A*02+ donors at a ratio of 1:10 with increasing TCER™ concentrations. The cells were co-incubated in a 50% mixture of primary tissue cell specific medium and T cell medium. To determine the safety margin, the TCER™ molecule was co-incubated in the same setup with the MAG-003 positive tumor cell line Hs695T. The safety margin was defined based on the lowest observed effect level (LOEL), determined as the first TCER™ concentration showing a response above the cut-off value. The cut-off was defined as ([standard deviation from all triplicates x3] + control normal tissue sample without TCER) and is indicated by a dotted line on each cytotoxicity plot. For each normal tissue cell type, the safety window determined based on the LOEL was greater than 1,000-fold or 10,000-fold, suggesting a favorable safety profile of the 114-iso1-BMA(36)TCER™ molecule. [Figure 13] Figure 1 shows TCER™-mediated in vivo efficacy in immunodeficient NOG mice engrafted with human PBMC against tumor xenografts of Hs695T cells, an HLA-A*02 positive tumor cell line, with detectable presentation of MAG-003 (SEQ ID NO: 1). TCER™ comprising UCHT1 (17) antibody variable domains with variable domains of TCR variants #114-iso1 (SEQ ID NO: 131 and 167, group 3) was compared with TCER™ comprising BMA031 (36) antibody variable domains with variable domains of TCR variants #114-iso1 (SEQ ID NO: 137 and 136, group 2). Group 1 corresponds to the vehicle-treated control group. The graph shows the average tumor volumes from 6 (groups 2 and 3) to 10 (vehicle) mice per group. The TCER™ molecule, comprising the UCHT1 (17opt) antibody and the T cell-mobilizing BMA031 (36) antibody, administered iv at 0.01 mg / kg body weight once weekly, showed complete remission. [Figure 14] Figure 1 shows the pharmacokinetic profile of the 114-iso1-UCHT1(17)TCER™ molecule in NOG mice. Mice received a single intravenous injection of 2 mg / kg body weight. Plasma concentrations of TCER™ in samples taken at different time points were determined by ELISA detecting either the presence of specific protein domains (Fc-VL assay) or dual specific binding activity (CD3-pMHC assay). [Figure 15] The design of the TCER™ molecule is presented. The bispecific T cell binding receptor (TCER™) is a fusion protein containing the variable domains (Vα, Vβ) of an affinity matured TCR and the variable domains (VH, VL) of a humanized T cell engaging antibody. The molecule uses an Fc portion with suppressed effector functions, which also results in an extended half-life and improved stability / manufacturability properties. [Figure 16] The in vitro safety data of the TCER™ molecule 114-iso1-BMA(36)) (SEQ ID NOs: 137 and 136) is evaluated in an LDH killing experiment with 12 human HLA-A*02 positive normal tissue primary cells from different organs. The different cells were co-cultured with PBMC effector cells from healthy HLA-A*02+ donors in a ratio of 1:10 with increasing TCER™ concentrations. The cells were co-incubated in a 50% mixture of primary tissue cell specific medium and T cell medium. To determine the safety margin, the TCER™ molecule was co-incubated in the same setup with the MAG-003 positive tumor cell line Hs695T. The safety margin was defined based on the lowest observed effect level (LOEL), determined as the first TCER™ concentration showing a response above the cut-off value. The cut-off was defined as ([standard deviation from all triplicates x3] + control normal tissue sample without TCER) and is indicated by a dotted line on each cytotoxicity plot. For each normal tissue cell type, the safety window determined based on the LOEL was greater than 1,000-fold or 10,000-fold, suggesting a favorable safety profile of the 114-iso1-BMA(36)TCER™ molecule. [Figure 17]Cytotoxicity and IFN-γ release of mature R7P1D5 TCR variant expressing human CD8+ T cells in response to tumor cell lines displaying different copy numbers of HLA-A*02 / MAG-003. For control purposes, no TCR (MOCK) or 1G4 TCR specific for NYESO1-001 were expressed. After co-culture of electroporated CD8+ T cells with tumor cell lines, LDH and IFN-γ levels were determined in the supernatant. [Figure 18] Normal tissues and tumor samples analyzed for peptide presentation are grouped according to their organ of origin. Top: Median MS signal intensities from technical replicates and aliquot measurements are plotted as points for single HLA-A*02 positive normal (left side of figure) and tumor samples (right side of figure) in which peptides were detected. Box plots represent normalized signal intensities across multiple samples, defined in logarithmic space. Boxes indicate median, 25th and 75th percentile values. Whiskers extend to the lowest data point that is within 1.5 interquartile range (IQR) of the lower quartile and to the highest data point that is within 1.5 IQR of the upper quartile. Bottom: Relative peptide detection frequency in each organ is shown as a bar graph. Numbers below the panels indicate the number of samples in which peptides were detected out of the total number of samples evaluated for this analysis in each organ (N=581 normal samples, N=771 tumor samples). If a peptide was detected on a sample but could not be quantified for technical reasons, the sample is included in this detection frequency display but no dot is shown at the top of the figure.

[0455] Abbreviations used: adipose: adipose tissue; adrenal gl: adrenal gland; bladder: bladder; bloodvess: blood vessel; esoph: esophagus; gall bl: gallbladder; intest.la: large intestine; intest.sm: small intestine; nerve cent: central nervous system; nerve periph: peripheral nerve; parathyr: parathyroid gland; perit: peritoneum; pituit: pituitary gland; skel.mus: skeletal muscle; AML: acute myeloid leukemia; BRCA: breast cancer; CCC: cholangiocarcinoma; CLL: chronic lymphocytic leukemia; CRC: colorectal cancer; GBC: gallbladder cancer; GBM: glioblastoma; GC: gastric cancer; GEJC: gastroesophageal junction cancer; HCC: hepatocellular carcinoma; HNSCC: squamous cell carcinoma of the head and neck; MEL: melanoma; NHL: non-Hodgkin's lymphoma; NSCLCadeno: non-small cell lung cancer adenocarcinoma; NSCLCother: NSCLC samples that could not be clearly assigned to NSCLCadeno or NSCLCsquam; NSCLCsquam: squamous cell non-small cell lung cancer; OC: ovarian cancer; OSCAR: esophageal cancer; PACA: pancreatic cancer; PRCA: prostate cancer; RCC: renal cell carcinoma; SCLC: small cell lung cancer; UBC: bladder carcinoma; UEC: uterine and endometrial cancer. EXAMPLES

[0456] Cellular receptors (TCRs) for cancer antigens often have lower affinity when compared to TCRs targeting viral antigens, which may be one possible explanation for tumor immune escape (Aleksic et al. 2012, Eur J Immunol. 2012 Dec; 42(12): 3174-9). It is therefore desirable to generate TCR variants with higher affinity for use as cancer antigen targeting constructs in adoptive cell therapy or as recognition modules for soluble therapeutics, i.e. bispecific molecules (Hickman et al. 2016, J Biomol Screen. 2016 Sep; 21(8): 769-85). The present invention therefore relates to the modification and optimization of the T cell receptor R7P1D5, identified from the human natural TCR repertoire, based on its high avidity and selectivity for the tumor-associated peptide MAG-003 (SEQ ID NO: 1). TCR R7P1D5 comprises an alpha variable domain having the amino acid sequence of SEQ ID NO: 4 and a beta variable domain having the amino acid sequence of SEQ ID NO: 11, as disclosed in WO 2017 / 158103.

[0457] Example 1: Generation of stable scTCRs In the present invention, TCR R7P1D5 (SEQ ID NO: 2 and 9, full length) was converted into a single chain TCR construct (scTCR R7P1D5, SEQ ID NO: 19) using the variable α (SEQ ID NO: 4) and β (SEQ ID NO: 11) domains and an appropriate glycine-serine linker sequence. For TCR maturation via yeast surface display, DNA of the corresponding sequence was synthesized and transformed into Saccharomyces cerevisiae EBY100 (MATaAGA1::GAL1→AGA1::URA3ura3→52trp1leu2→δ200his3→δ200pep4::HIS3prbd1.6Rcan1GAL) (AT...

Claims

1. An antigen-binding protein that specifically binds to a MAGE-A antigenic peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" (SEQ ID NO: 1) in a complex with a major histocompatibility complex (MHC) protein, said antigen-binding protein comprising: (a) a first polypeptide chain comprising a first variable domain comprising three complementarity determining regions (CDRs) CDRa1, CDRa2, and CDRa3, wherein: - said CDRa1 comprises the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:55; - said CDRa2 comprises the amino acid sequence of SEQ ID NO: 6, 56, 57 or 59; - said CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO: 35, 36, 37, 38, or 39, with the proviso that said CDRa3 does not comprise or consist of the amino acid sequence "CAEYSSASKIIF" (SEQ ID NO: 7); (b) a second polypeptide chain comprising a second variable domain comprising three CDRs: CDRb1, CDRb2, and CDRb3, - said CDRb1 comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 62, - said CDRb2 comprises the amino acid sequence of SEQ ID NO: 13, 63, 64, 65, 66 or 70, - said CDRb3 comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 71; comprising wherein said antigen binding protein comprises respective CDR amino acid sequences with no more than one, two or three amino acid substitutions; wherein said first variable domain and said second variable domain form a first antigen-binding site.

2. The antigen-binding protein of claim 1, which specifically binds to the antigen peptide KVLEHVVRV (SEQ ID NO: 1) in a complex with an MHC protein, preferably wherein the MHC protein is a human leukocyte antigen (HLA) protein, preferably HLA-A*02.

3. The antigen-binding protein of claim 1 or 2, which binds to a complex of the MAGE-A antigen peptide comprising or consisting of the amino acid sequence of SEQ ID NO:1 and HLA-A*02 with a K D of 30 μM or less.

4. The antigen-binding protein of any one of claims 1 to 3, which is an antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof, or a T cell receptor (TCR) or antigen-binding fragment thereof, or a bispecific T cell receptor (TCR) or antigen-binding fragment thereof.

5. An antigen-binding protein according to any one of claims 1 to 4, which is a T cell receptor (TCR) or an antigen-binding fragment thereof.

6. The antigen-binding protein of any one of claims 1 to 5, wherein the first polypeptide and the second polypeptide are covalently or non-covalently bound to each other.

7. The first variable domain comprises one or more framework regions selected from the group consisting of FR1-a, FR2-a, FR3-a, and FR4-a, wherein: - FR1-a comprises or consists of an amino acid sequence of SEQ ID NO: 83 or an amino acid sequence which is at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 83; - FR2-a comprises or consists of an amino acid sequence of SEQ ID NO: 84 or an amino acid sequence which is at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 84; - FR3-a comprises or consists of an amino acid sequence of SEQ ID NO: 85 or an amino acid sequence which is at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 85; - FR4-a comprises or consists of an amino acid sequence of SEQ ID NO: 86 or an amino acid sequence which is at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 86, and The second variable domain comprises one or more framework regions selected from the group consisting of FR1-b, FR2-b, FR3-b, and FR4-b, wherein: - FR1-b comprises or consists of an amino acid sequence of SEQ ID NO: 87 or an amino acid sequence which is at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 87, FR2-b comprises or consists of an amino acid sequence of SEQ ID NO: 88 or an amino acid sequence which is at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 88; - FR3-b comprises or consists of an amino acid sequence of SEQ ID NO: 89 or an amino acid sequence which is at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 89, or FR4-b comprises or consists of an amino acid sequence of SEQ ID NO: 90 or an amino acid sequence which is at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 90; 7. An antigen-binding protein according to any one of claims 1 to 6.

8. The framework region is - an amino acid substitution at position 19 of FR1-a, wherein said amino acid substitution is preferably S19A, S19V, more preferably S19V; - an amino acid substitution at position 48 and / or 50 of FR2-a, wherein said amino acid substitution at position 48 is preferably A48K and said amino acid substitution at position 50 is preferably L50P; - an amino acid substitution at positions 46, 47 and / or 54 of FR2-b, wherein said amino acid substitutions at positions 46, 47 and 54 are preferably M46P, M47G, and I54F, respectively, and wherein, in the case where the CDRb3 amino acid sequence defined above comprises the amino acid 109D at position 109, said amino acid at position 54 is preferably I54F; - an amino acid substitution at position 66 of FR3-b, wherein said amino acid substitution at position 66 is preferably I66C, and wherein in case the CDRb2 amino acid sequence defined above comprises the amino acid 57C at position 57, said amino acid at position 66 is preferably 66C, wherein the position of said substitution is given according to the IMGT nomenclature, at least one amino acid substitution selected from the group of amino acid substitutions comprising 8. The antigen-binding protein of claim 7.

9. An antigen-binding protein according to any one of claims 1 to 8, wherein the first variable domain is part of a TCR alpha or gamma chain and / or the second variable domain is part of a TCR beta or delta chain. (i) one or more additional antigen-binding sites; (ii) a transmembrane domain, optionally including a cytoplasmic signaling domain; (iii) diagnostic agents; (iv) a therapeutic agent; or (v) PK modification part 10. The antigen-binding protein of any one of claims 1 to 9, further comprising one or more of: (i) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:5, CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO:6, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:35, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO:12, CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO:13, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO:14; or (ii) CDRa1 comprises or consists of the amino acid sequence of SEQ ID NO:5, CDRa2 comprises or consists of the amino acid sequence of SEQ ID NO:56, CDRa3 comprises or consists of the amino acid sequence of SEQ ID NO:35, CDRb1 comprises or consists of the amino acid sequence of SEQ ID NO:62, CDRb2 comprises or consists of the amino acid sequence of SEQ ID NO:65, and CDRb3 comprises or consists of the amino acid sequence of SEQ ID NO:71; 11. An antigen-binding protein according to any one of claims 1 to 10.

12. An isolated nucleic acid comprising a sequence encoding the antigen-binding protein of any one of claims 1 to 11, or a nucleic acid vector comprising said isolated nucleic acid.

13. A recombinant host cell comprising the antigen-binding protein of any one of claims 1 to 11, or the isolated nucleic acid or vector of claim 12, preferably a) a lymphocyte, such as a T lymphocyte or a T lymphocyte precursor, e.g., a CD4 or CD8 positive T cell, or b) Cells for recombinant expression, such as Chinese Hamster Ovary (CHO) cells A recombinant host cell.

14. A composition comprising an antigen-binding protein according to any one of claims 1 to 11, or an isolated nucleic acid according to claim 12.

15. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1 to 11, an isolated nucleic acid or vector according to claim 12, or a host cell according to claim 13, and a pharma- ceutically acceptable carrier, diluent, stabilizer and / or excipient. Claim 16: a. providing a suitable host cell; b. Providing a genetic construct comprising a coding sequence encoding an antigen binding protein according to any one of claims 1 to 11; c. introducing said genetic construct into said suitable host cell; and d. Expressing said genetic construct by said suitable host cell.

12. A method for producing an antigen-binding protein according to any one of claims 1 to 11, comprising:

17. The method of claim 16, further comprising isolating and purifying the antigen-binding protein from the suitable host cell.

18. An antigen-binding protein according to any one of claims 1 to 11, an isolated nucleic acid or vector according to claim 12, a host cell according to claim 13, or a pharmaceutical composition according to claim 15, for use in medicine.

19. An antigen binding protein according to any one of claims 1 to 11, an isolated nucleic acid or vector according to claim 12, a host cell according to claim 13, or a pharmaceutical composition according to claim 15 for use in the diagnosis, prevention, and / or treatment of a proliferative disease such as cancer, such as a MAGEA4 and / or MAGEA8 positive cancer.

20. Use of an antigen-binding protein according to any one of claims 1 to 11, an isolated nucleic acid or vector according to claim 12, a host cell according to claim 13, or a pharmaceutical composition according to claim 14 for the manufacture of a medicament.

21. Use of an antigen-binding protein according to any one of claims 1 to 11, an isolated nucleic acid according to claim 12, a vector, a host cell according to claim 13 or a pharmaceutical composition according to claim 14 for the manufacture of a medicament for the diagnosis, prevention and / or treatment of a proliferative disease such as cancer, such as a MAGEA4 and / or MAGEA8 positive cancer.

22. Use of the antigen binding protein, isolated nucleic acid, vector, host cell, or pharmaceutical composition of claim 21, wherein the cancer is selected from the group consisting of lung cancer, liver cancer, head and neck cancer, skin cancer, renal cell cancer, brain cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, leukemia, breast cancer, Merkel cell carcinoma, ovarian cancer, bladder cancer, uterine cancer, gallbladder cancer, bile duct cancer, osteosarcoma cancer, and esophageal cancer.

23. (i) the cancer is lung cancer, the lung cancer being non-small cell lung cancer (NSCLC), preferably non-small cell lung cancer adenocarcinoma or squamous cell non-small cell lung cancer (SNSCLC), or small cell lung cancer (SCLC); (ii) the cancer is skin cancer, and the skin cancer is melanoma; (iii) the cancer is head and neck cancer, and the head and neck cancer is head and neck squamous cell carcinoma (HNSCC); (iv) the cancer is liver cancer, the liver cancer being hepatocellular carcinoma (HCC); (v) the cancer is esophageal cancer, and the esophageal cancer is gastroesophageal junction cancer; 23. Use of the antigen binding protein, isolated nucleic acid, vector, host cell, or pharmaceutical composition of claim 22.

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