TW202506719A - Antigen binding proteins against mageb2 - Google Patents

Abstract

The present invention relates to antigen binding proteins that specifically bind to a tumor expressed melanoma-associated antigen (MAGE) B2 antigenic peptide in a complex with MHC. The antigen binding proteins are provided for use in the treatment of MAGEB2-expressing cancers. Further provided are nucleic acids encoding the antigen binding proteins, vectors comprising the nucleic acids, recombinant cells expressing the antigen binding proteins and pharmaceutical compositions comprising the antigen binding proteins.

Description

Antigen binding protein targeting MAGEB2

The present invention relates to antigen binding proteins that specifically bind to tumor-expressed melanoma-associated antigen (MAGE) B2 antigen peptides complexed with MHC. Antigen binding proteins for treating cancers expressing MAGEB2 are provided. Nucleic acids encoding the antigen binding proteins, vectors containing the nucleic acids, recombinant cells expressing the antigen binding proteins, and pharmaceutical compositions containing the antigen binding proteins are further provided.

TCR-based immunotherapy targets peptide epitopes derived from tumor-associated or tumor-specific proteins, which are presented by molecules of the major histocompatibility complex (MHC). These tumor associated antigens (TAAs) can be peptides derived from all protein classes, such as enzymes, receptors, transcription factors, etc., which are specifically expressed by cancer cells and/or upregulated in cancer cells. Unlike chimeric antigen receptor (CAR)-T cell therapy and current antibody-based approaches that can only target cell surface proteins, TCR-based immunotherapy enables the targeting of otherwise inaccessible intracellular proteins and thus significantly increases the number and diversity of targets derived from tumor-related or tumor-specific proteins.

Cancer-testis antigens (CTAs) are attractive targets for cancer immunotherapy due to their restricted expression in germ cells and aberrant reactivation in various cancers as well as their immunogenic properties. The melanoma antigen (MAGE) gene family includes intracellular cancer-testis antigens such as MAGEB2. MAGEB2 is normally expressed only in normal testes. MAGEB2, which has been found to enhance the ubiquitin ligase activity of the RING-type zinc finger containing E3 ubiquitin ligase, is aberrantly expressed in various human tumors such as lung cancer, breast cancer, melanoma and the like. Targeting MAGEB2 for immunotherapy was previously disclosed in WO2016/102272 A1, WO2017/097602 A1, WO2017/148888, WO2017/174822 A1, and WO2021/236638 A1. Many types of cancer still have high unmet medical needs, and patients need improved, effective and specific treatments. Therefore, it is necessary to develop new anticancer agents that specifically target intracellular proteins that are highly specific to cancer cells. The present invention addresses that need by providing novel antigen binding proteins that specifically bind to MAGEB2.

The present invention provides an antigen binding protein that specifically binds to a MAGEB2 antigen peptide complexed with a major histocompatibility complex (MHC) protein, wherein the MAGEB2 antigen peptide comprises or consists of the following: an amino acid sequence GVYDGEEHSV (SEQ ID NO: 1), wherein the antigen binding protein comprises a variable domain _VA comprising complementarity determining regions (CDRs) CDRa1, CDRa2, and CDRa3 and a variable domain _VB comprising CDRb1, CDRb2, and CDRb3, wherein CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 form an antigen binding domain A; preferably, wherein the variable domain _VA comprises or consists of the following: an amino acid sequence according to SEQ ID NO: 152 or a sequence of SEQ ID NO: 2 NO:152 has an amino acid sequence with at least 85%, 90%, 95%, 98%, or 99% identity, preferably, wherein the variable domain _VB comprises or consists of: an amino acid sequence according to SEQ ID NO:154 or an amino acid sequence with at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:154, preferably, wherein (1) CDRa1 comprises SEQ ID NO:2, CDRa3 comprises SEQ ID NO:3, CDRb1 comprises SEQ ID NO:5, and CDRb3 comprises SEQ ID NO:6; or (2) CDRa1 comprises SEQ ID NO:2, CDRa3 comprises SEQ ID NO:4, CDRb1 comprises SEQ ID NO:5, and CDRb3 comprises SEQ ID NO:6; wherein the CDRa1, CDRa3, CDRb1 and/or CDRb3 sequences may comprise one, two or three amino acid mutations.

The antigen binding proteins of the present invention are characterized by high stability, high affinity, high functional affinity, high potency, and high specificity. Therefore, the antigen binding proteins of the present invention are more effective and safer than the antigen binding proteins of the prior art, as demonstrated in the following exemplary section. In addition, these novel antigen binding proteins, particularly in the form of soluble antigen binding proteins (e.g., TCER® molecules), show high cytotoxicity to tumor cells.

In a second aspect, the invention provides nucleic acid(s) comprising a sequence encoding an antigen binding protein of the first aspect of the invention.

In a third aspect, the present invention provides (one or more) vectors, which (etc.) comprise (one or more) nucleic acids of the second aspect of the present invention.

In a fourth aspect, the present invention provides a host cell comprising the antigen-binding protein of the first aspect of the present invention, the nucleic acid(s) of the second aspect of the present invention, or the vector(s) of the third aspect of the present invention.

In a fifth aspect, the present invention provides a method for producing an antigen-binding protein according to the first aspect of the present invention.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising the antigen-binding protein of the first aspect of the present invention, (one or more) nucleic acids of the second aspect of the present invention, (one or more) vectors of the third aspect of the present invention, or the host cell of the fourth aspect of the present invention, and optionally, a pharmaceutically acceptable carrier.

In a seventh aspect, the present invention provides an antigen binding protein of the first aspect of the present invention, (one or more) nucleic acids of the second aspect of the present invention, (one or more) vectors of the third aspect of the present invention, a host cell of the fourth aspect of the present invention, or a pharmaceutical composition of the fourth aspect of the present invention for treating cancer, specifically expressing MAGEB2.

In an eighth aspect, the present invention provides a kit comprising the antigen binding protein of the first aspect of the present invention, (one or more) nucleic acids of the second aspect of the present invention, and/or (one or more) vectors of the third aspect of the present invention.

In order to facilitate the understanding of the present invention, some definitions of terms used in the present invention are described below.

As used herein, the term " amino acid " refers to one of the 20 naturally occurring amino acids or any non-natural analogs. Preferably, the term "amino acid" refers to one of the 20 naturally occurring amino acids.

As used herein, the term " polypeptide " or " protein " refers to a macromolecule composed of one or more sequences of amino acids. The protein may be a natural protein, i.e., a protein that exists in nature and is not produced by recombinant cells; or it may be produced by genetic engineering or recombinant cells and include a molecule having the amino acid sequence of a natural protein or a molecule having one or more amino acids deleted, added and/or substituted in the natural sequence.

As used herein, a " domain " can be any region of a protein, usually defined based on sequence homology and usually referring to a specific structural or functional entity. In the case of a variable domain of an antibody or TCR, the domain comprises CDR sequences derived from the antibody or TCR, also as disclosed below.

As used herein, the term " antigen " or " target antigen " refers to a molecule or a part of a molecule or a complex that can bind via an antigen binding site, wherein the antigen binding site is present in an antigen binding protein, preferably an antigen binding protein of the present invention. In the context of the present invention, the antigen is a melanoma-associated antigen (MAGE) B2 antigenic peptide, more specifically a MAGEB2 antigenic peptide, complexed with an MHC protein such as an HLA protein, such as HLA-A*02, which comprises or consists of the following amino acid sequence GVYDGEEHSV (SEQ ID NO: 1). The peptide GVYDGEEHSV (SEQ ID NO: 1) corresponds to amino acid residues 231-241 of the known MAGEB2 protein. In the case where the MAGEB2 antigenic peptide comprises additional amino acids in addition to the amino acid sequence GVYDGEEHSV (SEQ ID NO: 1), preferably, the total length of the MAGEB2 antigenic peptide does not exceed 30 or 20 amino acids, more preferably does not exceed 15 amino acids, and even more preferably does not exceed 12 amino acids. In the case where the MAGEB2 antigenic peptide comprises additional amino acids in addition to SEQ ID NO: 1, when the antigenic peptide is complexed with the MHC protein, the amino acids having SEQ ID NO: 1 are preferably located in the peptide binding groove of the MHC protein. It is known to those skilled in the art that the length of the antigenic peptide presented on MHC I is usually no more than 12 amino acids.

"MAGEB2:MHC complex presenting cell" herein refers to a cell presenting a MAGEB2 antigen peptide complexed with an MHC molecule on its surface. In a preferred embodiment, the cell presenting the MAGEB2:MHC complex is a tumor cell, wherein the tumor is preferably a cancer as defined herein below in the "treatment methods and uses" section. In the context of the present invention, the MAGEB2:MHC complex is over-presented on the cell surface of the cell presenting the MAGEB2:MHC complex compared to the level of the complex on the surface of cells of normal (healthy) tissue (also referred to as "healthy cells") or on the surface of control cells loaded with different antigen presenting peptides or without peptides. " Over - presented" means that the MAGEB2:MHC complex is present at a level of at least 2-fold, preferably 5-fold to 10-fold, that present in healthy tissue or control cells. For example, GVYDGEEHSV (SEQ ID NO: 1) was found to be over-presented in cancer, as disclosed in US20160250307, US20170165337, and US20170253633, respectively; the contents of the above are hereby incorporated by reference in their entirety.

In the context of the present invention, the term " immunoglobulin (Ig) domain" refers to a protein domain composed of a 2-layer sandwich of 7-9 antiparallel β strands arranged in two β pleats with a Greek key topology. Proteins containing Ig domains are classified into the immunoglobulin superfamily, including, for example, antibodies, T cell receptors (TCRs), and cell adhesion molecules. Examples of Ig domains are the variable and constant domains of antibodies and TCRs.

The term " antigen binding protein " (occasionally abbreviated as "ABP") herein refers to a polypeptide or a complex of two or more polypeptides that specifically binds to an antigen binding site of a MAGEB2 antigenic peptide in complex with a major histocompatibility complex (MHC) protein, wherein the MAGEB2 antigenic peptide comprises or consists of the amino acid sequence GVYDGEEHSV (SEQ ID NO: 1), and the polypeptide or the two or more polypeptides comprise CDRs as provided herein, such as CDRa1, CDRa3, and optionally, CDRa2, and CDRb1, CDRb3, and optionally, CDRb2. The two or more polypeptides of the antigen binding protein may be covalently or non-covalently linked together. As used in the context of the present invention, the term antigen binding protein includes a variety of different forms of antigen binding proteins as described below, including soluble antigen binding proteins, monovalent, bivalent and multivalent antigen binding proteins, monospecific, bispecific and multispecific antigen binding proteins, single-chain antigen binding proteins and antigen binding proteins comprising two or more chains, fusion proteins, and chimeric proteins. It is known to those skilled in the art that the CDRs provided herein can be included in the various forms disclosed in the prior art, such as Brinkmann U, Kontermann RE. The making of bispecific antibodies (MAbs. 2017 Feb/Mar; 9(2):182-212, doi:10.1080/19420862.2016.1268307) or WO2019/012138.

The term includes antigen binding proteins having the overall structure of a TCR, an antibody and/or a chimeric antigen receptor (CAR). The antigen binding proteins of the present invention comprise TCR-derived CDRs, specifically variable domain _VA comprising TCR-derived CDRa1, CDRa3 and, optionally, CDRa2, and variable domain _VB comprising TCR-derived CDRb1, CDRb3 and, optionally, CDRb2. The antigen binding proteins of the present invention comprise: variable domain _VA comprising complementary determining regions (CDRs) CDRa1, CDRa2, and CDRa3, e.g., on a first polypeptide, and variable domain _VB comprising CDRb1, CDRb2, and CDRb3, e.g., on a second polypeptide, wherein CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 form an "antigen binding domain A". "Antigen binding domain A" means a binding domain that binds to an antigenic peptide (in the context of the present invention, a MAGEB2 antigenic peptide) complexed with a major histocompatibility complex (MHC) protein. In a specific embodiment, the entire _VA domain and/or the entire _VB domain is TCR-derived and is therefore a TCR α, β, γ or δ variable domain (V _α , V _β , V _γ , or V _δ ). In a preferred embodiment, the antigen binding protein is a TCR or its (one or more) functional fragments of the variable domains _VA and _VB of a TCR, for example. In a specific embodiment, the antigen binding protein of the present invention comprises the CDRs as provided herein and, optionally, _VA and _VB , and comprises (one or more) further additional domains fused directly or indirectly to _VA or _VB . The additional domains may form (one or more) additional binding domains or (one or more) binding sites. For example and in specific embodiments, the additional binding domains may form antigen binding domain B. Additional binding domains may be included, for example, to form additional antigen binding domains, such as antigen binding domain C, etc. The additional/further domains contained in the antigen binding protein may also be additional proteins.

Such antigen binding proteins may also be referred to as " fusion proteins . " Examples of additional domains included in the antigen binding proteins of the present invention as fusion proteins are listed below.

If the antigen binding protein is a bispecific or multispecific antigen binding protein, it comprises, in addition to V _A and V _B as defined herein, at least one more binding domain/site, e.g., a variable domain, preferably two variable domains, and optionally, additional domains, such as (one or more) constant domains, wherein the variable domains and/or constant domains may be derived from antibodies or TCRs. Thus, in this aspect, the antigen binding protein may comprise two different antigen binding sites (one formed by V _A and V _B and one formed by at least one additional, preferably two variable domains) and is capable of specifically binding to two different antigens. In a specific embodiment, the antigen binding protein comprises TCR-derived V _A and V _B and two additional antibody-derived variable domains, specifically V _L and V _H . Such constructs comprising elements of both antibodies and TCRs represent hybrid forms and may be referred to, for example, as bispecific TCR-antibody fusion proteins or bispecific TCR molecules/proteins as used herein in the exemplary section. In bispecific fusion proteins, variable domains may be included in different forms and may be arranged in various orientations. The techniques used to produce such bispecific fusion proteins are known to those of ordinary skill in the art, and as disclosed herein below, they may therefore use variable domains as defined herein to generate and produce bispecific antigen-binding proteins in various forms. Those of ordinary skill in the art will be able to select suitable linkers to ensure folding in the desired configuration. The antigen binding proteins described herein may take the form of TCRs, antibodies, including engineered TCRs and antibodies, such as single chain fragment variable (scFv), disulfide-stabilized Fv (dsFv), scTV, Fab, Fab', F(ab')2, nanobodies, DARPins, Knottin, bifunctional antibodies, single chain bifunctional antibodies or oligomers thereof.

In the context of the present invention, the term " bispecific " refers to an antigen binding protein that has at least two valencies and has binding specificity for at least two different antigens (preferably two different antigens), and therefore comprises at least two antigen binding sites. The term " valence " refers to the number of binding sites of the antigen binding protein, for example a bivalent antigen binding protein refers to an antigen binding protein with two binding sites. The binding sites may bind to the same or different targets, i.e. a bivalent antigen binding protein may be monospecific, e.g. binding to one target, or bispecific, e.g. binding to two different targets. The bispecific antigen binding molecule of the present invention comprises at least one antigen binding domain A comprising TCR-derived CDRs. In a preferred embodiment, the antigen binding molecule of the present invention comprises at least one TCR-derived antigen binding site. The bispecific antigen binding protein of the present invention may be referred to as a bispecific TCR, or as a bispecific TCR/mAb bifunctional antibody. The antigen binding protein of this embodiment may also be referred to as a T cell engaging receptor or TCER®.

The bispecific antigen binding protein of the present invention, such as TCER®, may comprise a VH comprising CDRH1, CDRH2, and CDRH3 and a VL comprising CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 form an antigen binding domain B. Antigen binding domain B represents a binding domain that specifically binds to immune cells, preferably T cells or natural killer (NK) cells.

" TCER® " is a soluble antigen-binding protein comprising two antigen-binding domains, namely a first variable domain (VA) and a second variable domain (VB) as defined in the context of the present invention, and an additional antigen-binding domain formed by the heavy chain variable domain and the light chain variable domain of an antibody, also called a "recruiter", such as a variable heavy (VH) domain and a variable light (VL) domain directed against a T cell antigen (such as CD3) or against TCRα/β or a combination thereof.

In the context of the present invention, " _VA " or Va refers to a TCR variable domain comprising a TCR-derived CDR sequence and a TCR-derived framework sequence. The CDR sequences and framework sequences may be derived from the variable domain of the TCR α chain ( _Vα ), the variable domain of the TCR β chain ( _Vβ ), the variable domain of the TCR γ chain ( _Vγ ), or the variable domain of the TCR δ chain ( _Vδ ), preferably derived from Vα. The sequences surrounding the CDRs, i.e., the framework sequences, may be derived from the variable domain of the TCR, i.e., the variable domain of the TCR α chain, β chain, γ chain, or δ chain, or from the variable domain of an antibody, preferably derived from the variable domain of the TCR α chain. In the embodiments, various framework and CDR mutations/substitutions are shown.

In the context of the present invention, the CDR sequences and framework sequences of the V _A domain may not necessarily be derived from the same TCR chain. For example, CDRs derived from one TCR variable domain (of the donor TCR) may also be transplanted onto another TCR variable domain (of the recipient TCR). For example, the donor TCR may comprise a V _A encoded by, for example, TRAV5*01 and TRAJ31*01, and the recipient TCR may comprise a V _A encoded by, for example, TRAV14 and TRAJ33.

In the context of the present invention, " _VB " or Vb refers to a variable domain comprising a TCR-derived CDR sequence and a TCR-derived framework sequence. The CDR sequences and framework sequences may be derived from the variable domain ( _Vα ) of the TCR α chain, the variable domain ( _Vβ ) of the TCR β chain, the variable domain ( _Vγ ) of the TCR γ chain, or the variable domain ( _Vδ ) of the TCR δ chain, preferably derived from Vβ. The sequence surrounding the CDR, i.e., the framework sequence, may be derived from the variable domain of the TCR, i.e., the variable domain of the TCR α chain, β chain, γ chain, or δ chain, or derived from the variable domain of an antibody, preferably derived from the variable domain of the TCR β chain. In the embodiments, various framework and CDR mutations/substitutions are shown.

In the context of the present invention, the CDR sequences and framework sequences of the _VB domain may not necessarily be derived from the same TCR. For example, CDRs derived from one TCR variable domain (of the donor TCR) may be grafted onto another TCR variable domain (of the recipient TCR). For example, the donor TCR may comprise _VB encoded by, for example, TRBV29-1*01 and TRBJ1-2*01, and the recipient TCR may comprise _VB encoded by, for example, TRBV27 and TRBJ1-5.

In the context of the present invention, _Vα refers to the variable domain of the TCR α chain.

In the context of the present invention, _Vβ refers to the variable domain of the TCR β chain.

In the context of the present invention, _Vγ refers to the variable domain of the TCR γ chain.

In the context of the present invention, _Vδ refers to the variable domain of the TCR delta chain.

In the context of the present invention, _VL refers to the variable domain of the antibody light chain.

In the context of the present invention, _VH refers to the variable domain of the antibody heavy chain.

In the context of the present invention, _CL may refer to the constant domain of the antibody light chain.

In the context of the present invention, _CH1 , _CH2 , and _CH3 may refer to the constant domains of antibody heavy chains, specifically IgG heavy chains.

As disclosed herein, a constant domain may be included in an antigen binding protein and may further improve certain characteristics of the antigen binding protein and may therefore be present or absent. For example, the stability or half-life of the antigen binding protein may be increased, or purification of the antigen binding protein may be facilitated. The constant domain may also include additional binding domains. Exemplary constant domains may be, for example, a constant domain of an antibody, such as _CL or _CH , and/or an Fc domain/portion. The constant domain may also be albumin, an unstructured polypeptide, and/or a leucine zipper, etc.

The term "epitope" , also known as antigenic determinant , is the part of an antigen that is recognized by the immune system. As used herein, the term epitope includes the terms "structural epitope" and "functional epitope". " Structural epitopes " are those amino acids of an antigen, such as a peptide-MHC complex, that are covered by an antigen-binding protein when bound to an antigen. Generally, all amino acids of an antigen are considered to be covered within 5 Å of any atom of the amino acids of the antigen-binding protein. The structural epitope of an antigen can be determined by methods known in the art, including X-ray crystallography or NMR analysis. The structural epitope of an antibody generally comprises 20 to 30 amino acids. The structural epitope of TCR generally comprises 20 to 30 amino acids. " Functional epitope " as defined herein is a subset of those amino acids that form the structural epitope, and comprises amino acids of antigens that are crucial to forming an interface with the antigen-binding protein of the present invention or its functional fragment by directly forming non-covalent interactions such as H bonds, salt bridges, aromatic stacking or hydrophobic interactions, or by indirectly stabilizing the binding conformation of the antigen, and is determined, for example, by mutation scanning. In the context of the present invention, functional epitopes are also referred to as "binding motifs". In general, the functional epitope of an antigen bound by an antibody comprises between 4 and 6 amino acids (see Example 2 herein). Generally speaking, the functional antigenic determinant of the peptide-MHC complex comprises between 2 and 6 or 7 amino acids of the peptide and 2 to 7 amino acids of the MHC molecule. Since the length of peptides presented by MHC I is generally between 8 and 10 amino acids, only a subset of the amino acids of each given peptide is part of the functional antigenic determinant of the peptide-MHC complex. The antigenic determinant bound by the antigen binding protein of the present invention, specifically the functional antigenic determinant comprises or consists of the amino acids of the antigen required to form the binding interface.

The " Major Histocompatibility Complex " (MHC) is a tissue cell surface protein that is necessary for the acquired immune system in vertebrates to recognize foreign molecules and thus determine tissue compatibility. The main function of MHC molecules is to bind to antigens derived from pathogens and display them on the cell surface for recognition by appropriate T cells. Human MHC is also called human leukocyte antigen (HLA) complex (or just HLA). Therefore, in a preferred embodiment, MHC is 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 (MHC I) are usually recognized by CD8-positive T cells (CD8+ T cells) carrying the appropriate T cell receptor (TCR), while complexes of peptides and MHC class II molecules (MHC II) are usually recognized by CD4-positive helper T cells (CD4+ T cells) carrying the appropriate TCR. CD4 and CD8 usually serve as co-receptors for TCRs bound to MHC I and MHC II, respectively. In some exceptional cases, complexes of peptides and MHC I are recognized by CD8-negative (specifically CD8-negative, CD4-positive) T cells (Soto et al., 2013, Cancer Immunol Immunother. 2013 Feb; 62(2):359–369). Since the responses of CD8-positive and CD4-positive T cells jointly and synergistically promote anti-tumor effects, the recognition and characterization of tumor-associated antigens and corresponding T-cell receptors are crucial in the development of cancer immunotherapies such as vaccines and cell therapy. The HLA-A gene is located on the short arm of chromosome 6 and encodes the larger alpha chain component of HLA-A. Variation in the HLA-A alpha chain is key to HLA function. This variation promotes the genetic diversity of the population. Since each HLA has a different affinity for peptides with certain structures, a greater variety of HLAs means a greater variety of antigens to be "presented" on the cell surface. In the context of the present disclosure, MHC class I HLA proteins may be HLA-A, HLA-B, or HLA-C proteins, suitably HLA-A proteins, such as HLA-A*02. In an MHC class I-dependent immune response, peptides must not only be able to bind to certain MHC class I molecules expressed by tumor cells, they must then also be recognized by T cells carrying specific T cell receptors (TCRs).

The " antigenic peptide in a complex with an MHC protein " herein refers to an antigenic peptide that is non-covalently bound to an MHC molecule. Specifically, the antigenic peptide is located in the "peptide binding groove" formed by the MHC molecule. The complex of the MHC molecule and the antigenic peptide is also referred to herein as a "peptide-MHC complex" or a "pMHC complex". In the case of the MAGEB2 antigenic peptide, the complex is also referred to as a "MAGEB2 antigenic peptide-MHC complex" or a "MAGEB2:MHC complex".

The term " HLA-A*02 " refers to a specific HLA allele, wherein the letter A represents the allele and "*02" represents the A2 serotype.

" At least one " herein refers to one or more of the specified objects, such as 1, 2, 3, 4, 5 or 6 or more of the specified objects. For example, at least one binding site herein refers to 1, 2, 3, 4, 5 or 6 or more binding sites.

As used herein, the term " TCR " includes engineered TCRs.

" Native TCR " refers to wild-type TCR that can be isolated from nature. Native TCR is a heterodimeric cell surface protein of the immunoglobulin superfamily associated with an invariant protein of the CD3 complex that participates in mediating signal transduction. Native heterodimeric TCR exists in αβ and γδ forms, which are structurally similar but have different locations and possible functions. The native full-length αβ heterodimeric TCR is composed of an α chain and a β chain. The α chain contains a variable region (V region) encoded by the TRAV gene, a joining region (J region) encoded by the TRAJ gene, and a constant region (C region) encoded by the TRAC gene. The β chain includes a variable region (V region) encoded by the TRBV gene, a joining region (J region) encoded by the TRBJ gene, and a constant region (C region) encoded by the TRBC gene, and usually includes a short diversity region (D region) encoded by the TRBD gene between the V region and the J region, but this D region is usually considered as part of the J region (Lefranc, (2001), Curr Protoc Immunol Appendix 1:Appendix 10). Genes encoding the different α-chain and β-chain variable regions, joining regions, and constant regions are referred to by unique numbers in the IMGT nomenclature (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1):42-54; Scavner and Lefranc, (2000), Exp Clin Immunogenet 17(2):83-96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). Additional information on TCR genes can be found in the international ImMunoGeneTics information system®, Lefranc MP et al., (Nucleic Acids Res. 2015 Jan; 43(Database issue):D413-22; and http://www.imgt.org/).

The α-chain TRAC constant domain sequence and the β-chain TRBC1 or TRBC2 constant domain are also referred to as TCR constant domain sequences hereinafter. In one embodiment, the TCR constant domain sequence can 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 sequence can be slightly modified, for example, by introducing a heterologous sequence, preferably a mouse sequence, which can increase TCR expression and stability. Furthermore, further stabilizing mutations as known from the current state of the art (e.g., WO2018/104407, PCT/EP2018/069151, WO2011/044186, WO2014/018863), such as replacement of unfavorable amino acids in the variable regions and/or introduction of disulfide bonds between TCR C domains, and removal of unpaired cysteines, may be introduced.

At the protein level, TCR α, β, γ and δ chains contain two immunoglobulin domains, namely the variable domain and the constant domain. The variable domain corresponds to the V(D)J region. The constant domain corresponds to the C region. The constant domain is a membrane proximal domain and, in the context of the present invention, also includes a transmembrane (TM) domain and a short cytoplasmic tail. Each of the constant domain and the variable domain includes intrachain disulfide bonds. The variable domain ( _Vα and _Vβ in αβ TCR and _Vγ and _Vδ in γδ TCR) contains highly polymorphic loops that include complementary determining regions (CDRs).

Each TCR variable domain contains three " TCR complementation determining regions (CDRs) " embedded in framework sequences, one of which is a highly variable region named CDR3. In the context of the present invention, CDRa1, CDRa2, and CDRa3 represent alpha chain CDRs, and CDRb1, CDRb2, and CDRb3 represent beta chain CDRs. The sequences encoding CDRa1 and CDRa2 are contained in TRAV, the sequences encoding CDRa3 are contained in TRAV and TRAJ, the sequences encoding CDRb1 and CDRb2 are contained in TRBV, and the sequences encoding CDRb3 are contained in TRBV, TRBD, and TRBJ. In TCR, CDR1 and CDR3 amino acid residues contact antigenic peptides, while CDR2 amino acid residues mainly contact HLA molecules (Stadinski et al., J Immunol. 2014 June 15; 192(12):6071–6082; Cole et al., J Biol Chem. 2014 Jan 10; 289(2):628-38). Therefore, the antigenic specificity of TCR is defined by CDR3 and CDR1 sequences. CDR2 sequence is not necessary for determining antigenic specificity, but may play a role in the overall affinity of TCR for peptide:MHC complexes.

" TCR framework regions " (FR) refer to the amino acid sequences inserted between CDRs, i.e., those portions of the variable domains that are conserved to some extent among different TCRs. The α, β, γ, and δ chain variable domains each have four FRs, designated herein as FR1-a, FR2-a, FR3-a, FR4-a (for α or γ chain) and FR1-b, FR2-b, FR3-b, FR4-b (for β or δ chain), respectively. Thus, an alpha or gamma chain variable domain can be described as (FR1-a)-(CDRa1)-(FR2-a)-(CDRa2)-(FR3-a)-(CDRa3)-(FR4-a), and a beta or delta chain variable domain can be described as (FR1-b)-(CDRb1)-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)-(FR4-b). In the context of the present invention, the CDR/FR sequences in an alpha, beta-, gamma or delta chain variable domain are determined based on the IMGT definition (Lefranc et al., Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org). Thus, when referring to a TCR or TCR-derived domain, the CDR/FR amino acid positions are indicated according to the IMGT definition. Preferably, the IMGT positions of the CDR/FR amino acid positions of the variable domain Vα are given similarly to the IMGT numbers of TRAV5*01, and/or the IMGT positions of the CDR/FR amino acid positions of the variable domain Vβ are given similarly to the IMGT numbers of TRBV29-1*01.

An " engineered TCR " may be a protein that is similar to a natural TCR but comprises modifications in the variable and/or constant domains, such as a humanized TCR or a TCR with improved/altered characteristics (e.g., improved binding, heterodimerization or expression level), or may be a soluble TCR and/or a single-chain TCR, a monovalent, bivalent or multivalent TCR, a monospecific, bispecific or multispecific TCR, and/or a functional fragment of a TCR, or a functional fragment of one or more TCRs, or a fusion protein and/or a chimeric protein comprising a functional fragment of a TCR or a functional fragment of one or more TCRs.

As used in the context of the present invention, the term " _Fc domain _" encompasses native _Fc domains and _Fc domain variants as well as sequences as further defined herein below. Like _Fc variants and native _Fc molecules, the term " _Fc domain" includes molecules in monomeric or multimeric form, whether generated from intact antibody digestion or by other means.

As used herein, the term " native _{Fc "} refers to a molecule comprising a sequence of a non-antigen binding fragment, which is produced by antibody digestion or by other means, whether in monomeric or polymeric form, and may contain hinge regions. The original immunoglobulin source of native _Fc is specifically of human origin, and may be any of the immunoglobulins, preferably IgG1 or IgG2, and most preferably IgG1. Native _Fc molecules are composed of monomeric polypeptides that can be linked into dimer or polymeric forms by covalent (i.e., disulfide bonds) and non-covalent bonds. The number of intermolecular disulfide bonds between monomer subunits of native _Fc molecules 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). An example of a native _Fc is a disulfide-bonded dimer produced by papain digestion of IgG. As used herein, the term "native _Fc " is generic to monomeric, dimeric, and multimeric forms. Examples of native _Fc amino acid sequences are provided in WO2021023658 (A1).

" Hinge " or " hinge region " or " hinge domain " generally refers to the flexible portion of the heavy chain located between the _CH1 domain and the _CH2 domain. It has a length of about 25 amino acids and is divided into an "upper hinge", a "middle hinge" or "core hinge", and a "lower hinge". A "hinge subdomain" refers to an upper hinge, a middle (or core) hinge, or a lower hinge. The amino acid sequences of the hinges of IgG1, IgG2, IgG3, and IgG4 molecules are provided, for example, in WO2021023658 (A1).

In the context of the present invention, when referring to amino acid positions in the _Fc domain, these amino acid positions or residues are indicated according to the EU numbering system as described, for example, in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).

As used herein, the term " _Fc variant " refers to a molecule or sequence that is modified from a native _Fc , but still comprises a binding site for a salvage receptor _Fc Rn (neonatal _Fc receptor). Exemplary _Fc variants and their interactions with salvage receptors are known in the art. Thus, the term " _Fc variant" may include a molecule or sequence that is humanized from a non-human native _Fc . In addition, a native _Fc comprises _regions that may be removed because they provide structural features or biological activities that are not required for the antigen binding protein of the present invention. Thus, the term " _Fc variant" includes molecules or sequences that lack one or more native _Fc sites or residues, or in which one or more _Fc sites or residues have been modified to affect or participate in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity when expressed in a selected host cell, (4) glycosylation, (5) interaction with complements, (6) binding to _Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

Thus, in one embodiment, the Fc domain of, for example, _Fc1 and/or _Fc2 comprises a hinge domain.

In one embodiment, the _Fc domain is preferably derived from a human IgG _Fc domain of human IgG1, IgG2, IgG3, or IgG4, preferably IgG1 or IgG2, more preferably IgG1.

In some embodiments, in particular, when the antigen binding protein comprises two _Fc domains, for example, in the form of TCER® described herein below (such as _Fc1 and _Fc2 ), the two _Fc domains may be the same immunoglobulin isotype or isotype subclass, or different immunoglobulin isotypes or isotype subclasses, preferably the same. Therefore, in some embodiments, _Fc1 and _Fc2 are IgG1 subclass or IgG2 subclass or IgG3 subclass or IgG4 subclass, preferably IgG1 subclass or IgG2 subclass, more preferably IgG1 subclass.

In some embodiments, the _Fc domain is a variant _Fc domain, and thus comprises one or more of the amino acid substitutions described herein below.

In some embodiments, the _Fc domain comprises or further comprises a "RF" mutation and/or a "knob-and-hole" mutation, preferably a "knob-and-hole" mutation.

" RF mutation " generally refers to an amino acid substitution in _{the CH3 domain} of an Fc domain from amino acid HY to RF, such as the amino acid substitutions H435R and Y436F in the _CH3 domain, as described by Jendeberg, L. et al. (1997, J. Immunological Meth., 201:25-34), and described as being advantageous for purification purposes because it abolishes binding to protein A. In cases where the antigen binding protein comprises two _Fc domains, the RF mutation may be in one or both, preferably one, _Fc domain.

" Knob - in- mortise " or also known as "knob-in-mortise" technology refers to the amino acid substitutions T366S, L368A and Y407V (mortise), and T366W (knob), all of which are in the _CH3 - _CH3 interface to promote heteromultimer formation. These knob-in-mortise mutations can be further stabilized by introducing additional cysteine amino acid substitutions Y349C and S354C. Patents US5731168 and US8216805 describe the "knob-in-mortise" technology and stabilizing cysteine amino acid substitutions.

In the context of the present invention, the "knob" mutation and the cysteine amino acid substitution S354C are, for example, present in an _Fc domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24, and the "hole" mutation and the cysteine amino acid substitution Y349C are present in an _Fc domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25.

In some embodiments, the _Fc domain of one of the polypeptides, e.g., _Fc1 , comprises the amino acid substitution T366W (knob) in its _CH3 domain, and the Fc domain of the other polypeptide, e.g., _Fc2 , comprises the amino acid substitutions T366S, L368A, and Y407V (hole) in its _CH3 domain, or vice versa .

In some embodiments, the _Fc domain of one of the polypeptides, e.g., _Fc1 , comprises or further comprises the amino acid substitution S354C in its _CH3 domain, and the _Fc domain of the other polypeptide, e.g., _Fc2 , comprises or further comprises the amino acid substitution Y349C in its _CH3 domain, or vice versa .

Thus, in some embodiments, the _Fc domain of one of the polypeptides, e.g., _Fc1 , comprises the amino acid substitutions S354C and T366W (knob) in its _CH3 domain, and the _Fc domain of the other polypeptide, e.g., _Fc2 , comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V (hole) in its _CH3 domain, or vice versa .

This histamine substitution can be further expanded by including the amino acid substitution K409A on one polypeptide and F405K in another polypeptide, as described by Wei et al. (Structural basis of a novel heterodimeric F _c for bispecific antibody production, Oncotarget. 2017). Thus, in some embodiments, the F _c domain of one of the polypeptides, e.g., F _c1 , comprises or further comprises the amino acid substitution K409A in its _CH3 domain, and the F _c domain of the other polypeptide, e.g., F _c2 , comprises or further comprises the amino acid substitution F405K in its _CH3 domain, or vice versa .

In some cases, artificially introduced cysteine bridges can improve the stability of the antigen binding protein, and optimally do not interfere with the binding characteristics of the antigen binding protein. These cysteine bridges can further improve heterodimerization.

Further amino acid substitutions, such as charge pair substitutions, for improving the heterodimerization of the resulting proteins have been described in the state of the art, for example in EP 2 970 484.

Thus, in one embodiment, the _Fc domain of one of the polypeptides, e.g., _Fc1, comprises or further comprises a charged pair substitution E356K, E356R, D356R, or D356K, and D399K or D399R, and the _Fc domain of the other polypeptide, e.g., _Fc2 , comprises or further comprises a charged pair substitution R409D, R409E, K409E, or K409D, and N392D, N392E, K392E, or K392D, or vice versa .

In another embodiment, the _Fc domains on one or both, preferably both polypeptide chains, may contain one or more changes that inhibit _Fcγ receptor (FcγR) binding. Such changes may include L234A, L235A.

In the case of incorporating Fc parts consisting of the hinge _CH2 and _CH3 domains or parts thereof into antigen binding proteins, more particularly into bispecific antigen binding proteins, the problem of non-specific immobilization of these molecules induced by Fc:Fc-γ receptor (FcgR) interactions arises. FcgR consists of different cell surface molecules (FcgRI, FcgRIIa, FcgRIIb, FcgRIII) that bind with different affinities to antigenic determinants displayed by the Fc part of IgG molecules. Therefore, non-specific (i.e., not induced by either of the two binding domains of the bispecific molecule) immobilization is disadvantageous due to i) effects on the pharmacokinetics of the molecule and ii) off-target activation of immune effector cells. Various Fc variants and mutations have been identified that abrogate FcgR binding. In this context, Morgan et al. 1995, Immunology (The N-terminal end of the _CH2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, FcyRI and FcyRIII binding) revealed an exchange of residues 233-236 of human IgG1 with the corresponding sequences derived from human IgG2, namely residues 233P, 234V, and 235A, in which the amino acid was absent at position 236, resulting in abolished FcgRI binding, abolished C1q binding, and reduced FcgRIII binding. EP1075496 discloses antibodies and other Fc _- containing molecules having variants in the _Fc domain (such as one or more of 233P, 234V, 235A, and no residue or G in position 236, and 327G, 330S, and 331S), wherein the recombinant antibodies are capable of binding to target molecules without triggering significant complement-dependent lysis or cell-mediated destruction of the target.

Therefore, in some embodiments, the _Fc domain comprises or further comprises an amino acid selected from the group consisting of 233P, 234V, 235A, 236 (no residue) or G, 327G, 330S, 331S or one or more of the deletions, preferably, the _Fc domain comprises or further comprises amino acids 233P, 234V, 235A, 236 (no residue) or G, and one or more amino acids selected from the group consisting of 327G, 330S, 331S, and most preferably, the Fc domain comprises or further comprises amino acids 233P, 234V, 235A, 236 (no residue), and 331S.

In a further embodiment, the _Fc domain comprises or further comprises the amino acid substitution N297Q, N297G, or N297A, preferably N297Q.

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

In a further embodiment, particularly when there is no light chain, the _Fc domain comprises or further comprises the amino acid substitution S220C. The amino acid substitution "S220C" results in the deletion of the cysteine that forms the _CH1 - _CL disulfide bond.

In some embodiments, the _Fc domain comprises or further comprises at least two additional cysteine residues, e.g., S354C and Y349C or L242C and K334C, wherein S354C is in the _Fc domain of one polypeptide, such as _Fc1 , and Y349C is in the _Fc domain of another polypeptide, such as _Fc2 , to form a heterodimer, and/or wherein L242C and K334C are located in the same _Fc domain, in _Fc1 or _Fc2 of one or both polypeptides, to form an intradomain C-C bridge.

Additional replacements and descriptions may be found in U.S. Patent Application No. 20180162922, the contents of which are incorporated by reference in their entirety.

It may also be desirable to modify the antigen binding proteins of the invention with respect to effector function, for example to enhance or reduce antigen dependent cell mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antigen binding protein. This can be achieved by introducing one or more amino acid substitutions in the _Fc domain of the antigen binding protein, also referred to herein as _Fc variants in the case of the antigen binding proteins of the invention. Alternatively or in addition, (one or more) cysteine residues may be introduced into the _Fc domain, thereby allowing the formation of interchain disulfide bonds in this region. The heterodimeric antigen binding proteins thus generated may have improved or reduced internalization capacity and/or increased complement-mediated cytotoxicity and/or antibody-dependent cellular cytotoxicity (ADCC) (Caron PC. et al. 1992; and Shopes B. 1992).

Another type of amino acid modification of the antigen binding protein of the present invention can be used to change the original glycosylation pattern of the antigen binding protein, that is, by deleting one or more carbohydrate moieties present in the antigen binding protein and/or adding one or more glycosylation sites not present in the antigen binding protein. The presence of any of the tripeptide sequences aspartic acid-X-serine and aspartic acid-X-threonine, where X is any amino acid except proline, creates a potential glycosylation site. Adding glycosylation sites to the antigen binding protein or deleting such glycosylation sites is preferably achieved by altering the amino acid sequence so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites).

Another type of modification involves the removal of sequences identified in silico or experimentally as being likely to produce degradation products or heterogeneity in the antigen binding protein preparation. As an example, deamination of aspartic acid and glutamic acid residues can occur depending on factors such as pH and surface exposure. Aspartic acid residues are particularly susceptible to deamination, primarily when present in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such a deamination site, particularly Asn-Gly, is present in the antigen binding proteins of the invention, it may therefore be desirable to remove the site, generally by conservative substitution to remove one of the relevant residues. Such substitutions in a sequence to remove one or more of the relevant residues are also intended to be encompassed by the present invention.

Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antigen binding protein. The advantage of these procedures is that they do not require the production of the antigen binding protein in a host cell that has glycosylation capacity for N- or O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be linked to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as cysteine, (d) free hydroxyl groups such as serine, threonine or hydroxysuccinimide, (e) aromatic residues such as phenylalanine, tyrosine or tryptophan, or (f) amide groups of glutamine. Such methods are described, for example, in WO87/05330.

The removal of any carbohydrate moieties present on the antigen binding protein can be achieved chemically or enzymatically. Chemical deglycosylation requires exposure of the antigen binding protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment causes the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylglucosamine sugars), while leaving the antigen binding protein intact. Chemical deglycosylation is described by Sojahr H. et al. (1987) and Edge, AS. et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by using various endoglycosidases and exoglycosidases, as described by Thotakura, NR. et al. (1987).

Another type of covalent modification of the antigen binding protein includes linking the antigen binding protein to one of various nonprotein polymers such as 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.

" Functional fragment of a TCR " refers to a TCR fragment that retains or substantially retains the affinity, functional affinity and/or specificity of the TCR from which it is derived for the target antigen. Exemplary functional fragments such as _VA and _VB are demonstrated herein in the Examples below. Since binding to the target antigen peptide is defined by the CDR1 and CDR3 sequences and binding to the target antigen peptide-MHC complex is defined by CDR1, CDR2, and CDR3, an antigen binding protein comprising the CDR1 and CDR3 and, optionally, CDR2 sequences of a TCR retains the affinity, functional affinity and/or specificity of the parent TCR for the target antigen. It is known to those skilled in the art that CDRs are typically interspersed with framework regions (FRs), however, the specific amino acid sequences of the framework regions may not be directly involved in the target antigen specificity. Examples of functional TCR fragments include variable domains such as TCR α, β, γ or δ variable domains or fragments of α, β, δ or γ chains such as α, β, δ or γ chains without transmembrane domains and short cytoplasmic tails. As used herein, the term "fragment" refers to naturally occurring fragments (e.g., splice variants or peptide fragments) as well as artificially constructed fragments, particularly fragments obtained by genetic technology. Functional fragments of TCR can be included in the antigen-binding protein of the present invention in various forms.

A functional fragment of a TCR may retain or substantially retain affinity for the target antigen if the KD for binding to the target antigen, measured, for example, as outlined below, is the same as, or increased or decreased, preferably decreased, by no more than 10×, 5×, 3×, or 2×, compared to the KD of the TCR. A functional fragment as provided in the embodiments herein has improved affinity for the target antigen, for example, at least 100-fold, at least 500-fold, or at least 1000-fold, compared to the variable domain contained in the naturally occurring TCR from which the variable domain was derived.

A functional fragment of a TCR is considered to have retained or substantially retained the functional affinity for the target antigen if, for example, the functional affinity for the target antigen is the same as that of the TCR, or is increased or decreased, preferably decreased, by no more than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1%. In particular, a functional fragment of a TCR is considered to have retained or substantially retained the functional affinity for the target antigen if, for example, the cytotoxic activity of the functional fragment of the TCR in response to the target of the parent protein as measured in a cytotoxicity assay is the same as that of the TCR, or is increased or decreased, preferably decreased, by no more than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 5%, 3%, 2%, or 1%, preferably 10%, 8%, 5%, 3%, 2%, or 1%.

If a functional fragment of a TCR does not significantly bind to peptides other than the target antigen peptide of the TCR, the functional fragment of the TCR is considered to have retained or substantially retained the specificity for the target antigen (i.e., the ability to specifically bind to the target antigen).

In the case of antigenic peptide variants and in the case of antigen-binding proteins of the present invention, particularly soluble antigen-binding proteins of the present invention, "does not significantly bind " generally means, in a binding assay such as biointerferometry, that the relative response signal of the antigenic peptide variant is not higher than 30%, not higher than 25%, not higher than 20%, not higher than 15%, preferably not higher than 20% of the signal obtained for binding to the MAGEB2 peptide consisting of the amino acid sequence of SEQ ID NO: 1 under the same experimental conditions. For example, the relative response signal obtained for the analog peptide SP-02-1640 composed of the amino acid sequence "RLYDGLFKVI" of SEQ ID NO: 106 was no higher than 30% compared to the signal obtained for binding to the MAGEB2 peptide composed of the amino acid sequence of SEQ ID NO: 1 under the same experimental conditions.

The term " α/β TCR " or " γ/δ TCR " refers to a TCR comprising an α chain and a β chain or a γ chain and a δ chain, respectively, as described above. Such TCRs may also be described as " full-length TCRs " or "learned TCRs". An α/β TCR or a γ/δ TCR may be a natural TCR or may be an engineered TCR that retains the structure of a natural TCR, i.e., an engineered TCR that comprises minor modifications in the variable domain and/or the constant domain as described above, such as a humanized TCR.

As used herein, " single-chain TCR (scTCR) " refers to a TCR in which the variable domains of the TCR are located on a single polypeptide. Generally, the variable domains in a scTCR are separated by a linker, wherein the linker generally comprises 10 to 30 amino acids, such as 25 amino acids.

" Chimeric protein" herein refers to a protein comprising sequences from multiple species. "Chimeric TCR" herein refers to a TCR comprising sequences from multiple species. Preferably, in the context of the present invention, a chimeric TCR may comprise an α chain comprising at least one domain from human and one domain from mouse. More preferably, in the context of the present invention, a chimeric TCR may comprise an α chain comprising a variable domain of a human α chain and, for example, a constant domain of a mouse TCR α chain.

As used herein, the term " antibody " is intended to include natural antibodies and engineered antibodies. The term "engineered antibody" includes functional antibody fragments, single-chain antibodies, single-domain antibodies, bispecific antibodies or multispecific antibodies.

" Native antibodies " consist of two heavy chains and two light chains, where the 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, namely λ (lambda) and κ (kappa). There are five major classes (or isotypes) of heavy chains that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains different domains (also called regions). The light chain includes two domains, namely the variable domain ( _VL ) and the constant domain ( _CL ). The heavy chain consists of four or five domains, depending on the antibody isotype; one variable domain (VH) and three or four constant domains ( _CH1 , _CH2 and _CH3 and optionally, _CH4 , collectively referred to as _CH ). The variable domains, which have both the light ( _VL ) and heavy ( _VH ) chains, determine the binding recognition and specificity for the antigen. The constant domains, which have both the light ( _CL ) and heavy ( _CH ) chains, confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcRs).

The specificity of an antibody resides in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site consists of residues primarily from the "antibody complementation determining regions" (CDRs) or highly variable regions. Occasionally, residues from non-hypervariable regions or framework regions (FRs) affect the overall domain structure and, therefore, the binding site. CDR refers to the amino acid sequence that together defines the binding affinity and specificity of the native Fv region of a natural antibody binding site. The light and heavy chains of an antibody each have three CDRs, which are named CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, CDR3-H, respectively. Therefore, the antibody antigen binding site includes six CDRs, including a set of CDRs from each of the heavy and light chain V regions. "Antibody framework regions" (FR) refer to the amino acid sequences inserted between CDRs, that is, those portions of the antibody light and heavy chain variable regions that are relatively conserved among different antibodies in a single species. The light and heavy chains of an antibody each have four FRs, which are named FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Thus, the light chain variable domain can be described as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable domain can be described as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). As used herein, a "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to the framework region of a naturally occurring human antibody. In the context of the present invention, the definition of CDR/FR in the variable domain of the antibody light chain or heavy chain is determined based on the Kabat definition (Kabat et al ., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest", 1991). Therefore, the amino acid sequences of CDR1, CDR2, and CDR3 of a given variable chain and the amino acid sequences of the framework regions (e.g., FR1, FR2, FR3, and FR4) are indicated according to the Kabat definition in the disclosure provided herein.

Knowing the amino acid sequence of the CDR of the antibody, TCR or antigen binding protein of the present invention, one having ordinary skill in the art can easily determine the framework regions such as TCR framework regions or antibody framework regions. Where the CDRs are not indicated, one having ordinary skill in the art can first determine the CDR amino acid sequence based on the IMGT definition for TCRs or the Kabat definition for antibodies, and then determine the amino acid sequence of the framework region.

Engineered antibody formats include functional antibody fragments, single chain antibodies, single domain antibodies, and antibodies engineered with Fc and/or hinges, antibodies with silenced, enhanced or altered effector function, chimeric antibodies, humanized antibodies, bispecific or multispecific antibodies. Engineered antibody formats further include constructs in which the light chain variable domain of an antibody is replaced by the α chain variable domain of a TCR, and the heavy chain variable domain is replaced by the β chain variable domain of a TCR, or vice versa. " Functional antibody fragment " refers to a portion of a full-length antibody that retains the ability to bind to its target antigen, specifically affinity and/or specificity for its target antigen. Preferably, a functional antibody fragment comprises the antigen binding region or variable region of a full-length antibody. Examples of functional antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, nanobodies, DARPins, knottins, and bifunctional antibodies. Functional antibody fragments may also be single domain antibodies such as heavy chain antibodies. The term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 daltons and antigen binding activity, wherein in a fragment obtained by treating IgG with a protease such as papain, the H chain and about half of the N-terminal side of the entire L chain are bound together by disulfide bonds. The Fv fragment is the N-terminal portion of the Fab fragment of an antibody and is composed of a light chain and a variable portion of a heavy chain.

Antigen binding proteins and CDRs as provided herein can be incorporated in various formats as disclosed herein above. Additional exemplary antigen binding proteins comprising the CDRs of the invention in different formats are provided herein below.

The antigen binding protein may be a " bifunctional antibody ", or the binding sites provided herein may be included in a " bifunctional antibody format ". These terms refer to a bivalent molecule composed of two polypeptide chains, each polypeptide chain comprising two variable domains connected by a linker (e.g., L _Db1 and L _Db2 ), wherein two of the domains may be a first variable domain and a second variable domain as defined herein (e.g., V1 and V2), and the other two domains may be TCR-derived or antibody-derived variable domains (e.g., V3 and V4). Specifically, the _V1 and _V2 domains may be located on two different polypeptides, and the _V3 and _V4 domains may be located on two different polypeptides, and the domains dimerize in cross-recognition. The domains may also be located on additional polypeptide chains, for example, four polypeptide chains. L _Db1 and L _Db2 may be the same or different and may be short linkers. Short linkers are generally between 2 and 12, 3 and 13 amino acids in length, such as 3, 4, 5, 6, 7, 8, 9 amino acids, for example 4, 5 amino acids (Brinkmann U. and Kontermann RE (MAbs. 2017 Feb-Mar; 9(2):182–212) or a linker of 8 amino acids in length, such as "GGGSGGGG" in SEQ ID NO:26.

Another format of the antigen binding protein may be a " dual variable domain immunoglobulin (DVD-Ig™) " format, as described in 2007 by Wu C. et al. (Nat Biotechnol. 2007 Nov; 25(11): 1290-7). Thus, DVD-Ig™ as described in the art generally consists of two polypeptide chains, namely a heavy chain comprising VLVC _H1 - _CH2 - _CH3 and a light chain comprising VLVC _L. Thus, domain pairs V/V and V/V are paired in parallel. The CDRs and variable domains provided herein may be incorporated into this exemplary format.

" Dual-variable-domain Ig format " ( or " DVD-Ig format ") refers to a protein comprising two polypeptide chains, each polypeptide chain comprising two variable domains connected by a linker (L1 _{, L3} ), wherein two of the variable domains are a first variable domain and a second variable domain ( _VA and VB ₎ as defined in the context of the present invention, and the other two domains are derived from antibodies or TCRs.

As used herein, the " format " of an antigen binding protein specifies a defined spatial arrangement of domains, specifically variable domains and, optionally, constant domains. Such antigen binding protein formats are characterized by the number of polypeptide chains (single, double or multiple), the type and length of linkers connecting different domains, the number of variable domains (and thus the valency values), the number of different variable domains (and thus the values of specificity for different antigens, e.g., bispecificity, multispecificity), and the order and orientation of the variable domains (e.g., staggered, parallel). As indicated herein above and as shown in the Examples, the antigen binding domains of the present invention, e.g., as specified by the CDRs as provided herein, can be contained in various formats.

In the context of antigen binding proteins or antibodies, the term " humanized " refers to an antibody that is completely or partially of non-human origin and has been modified by replacing certain amino acids, specifically in the framework regions of the heavy and light chains, in order to avoid or minimize immune responses in humans. The constant domains of humanized antibodies are primarily human _CH and _CL domains. Many methods for humanizing antibody sequences are known in the art. For example, "humanized" antibodies can be made by introducing conservative substitutions, consensus sequence substitutions, germline substitutions and/or backmutations, see, for example, Teng et al., Proc. Natl. Acad. Sci. USA, 80:7308-7312, 1983; Kozbor et al., Immunology Today, 4:7279, 1983; Olsson et al., Meth. Enzymol., 92:3-16, 1982; and Almagro & Fransson (2008) Front Biosci. 13:1619-1633. One common approach is CDR transplantation or antibody reshaping, which involves transplanting the CDR sequences of a donor antibody, usually a mouse antibody, into a framework scaffold of a human antibody with different specificities. Since CDR transplantation may reduce the binding specificity and affinity and thus biological activity of the non-human antibody of the CDR transplantation, a back mutation can be introduced at the selected position of the CDR transplanted antibody in order to retain the binding specificity and affinity of the parent antibody. The position identification for possible back mutations can be performed using the information available in the literature and antibody database. The alternative humanization technology of CDR transplantation and back mutation is surface rearrangement, in which the non-surface exposed residues of non-human origin are retained, and the surface residues are changed to human residues. Another alternative technology is called "guided selection" (Jespers et al. (1994) Biotechnology 12, 899) and can be used to derive a fully human antibody that preserves the antigenic determinant and binding characteristics of the parent antibody from, for example, a mouse or rat antibody. An additional humanization method is the so-called 4D humanization. The 4D humanization process is described in patent application US20110027266 A1 (WO2009032661 A1) and is exemplified below in the context of applying 4D humanization to humanize the variable light (VL) and heavy ( _{VH )} domains of a rat antibody.

For chimeric antibodies, humanization generally involves modification of the framework regions of the variable region sequences.

The term " percentage of identity" refers to a quantitative measure of the degree of homology between two sequences, which in the context of the present invention are amino acid sequences. If the lengths of the two sequences to be compared are unequal, they must be aligned to give the best possible fit, thereby allowing for insertion of gaps or alternatively truncation of the ends of the nucleic acid sequence or amino acid sequence. A person of ordinary skill in the art will recognize that various means for comparing sequence identity are available (see below).

For example, in the context of the present application, a sequence that is "at least 85% identical to a reference sequence " is a sequence that has 85% or more, specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity over its entire length to a reference sequence (e.g., a variable domain disclosed herein). 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 include 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 other than the reference sequence.

In the context of the present application, " percent identity " can be calculated using a global pairwise alignment (i.e., comparing two sequences 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 can be used, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the best alignment of two sequences (including gaps) when the entire length of the two sequences is considered. The needle program is available, for example, on the ebi.ac.uk World Wide Web website and is further described in the following publication (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 percent identity between two polypeptides according to the present invention is calculated using the EMBOSS: needle (global) program and the Blosum62 matrix, wherein the "gap opening" parameter is equal to 10.0 and the "gap extension" parameter is equal to 0.5.

An " amino acid mutation " may be a deletion, insertion, or substitution. A mutation may also be a post-translational modification (one or more), for example at the N-terminus or C-terminus of a polypeptide chain. Post-translational modifications may also be included in the antigen binding protein after the antigen binding protein is formulated into a pharmaceutical composition.

" Amino acid substitutions " may be conservative or non-conservative. In one embodiment, the substitution is a conservative substitution, in which one amino acid is replaced by another amino acid with similar structural and/or chemical properties. Amino acid substitutions may also result from post-translational modifications. Thus, amino acid substitutions may also be included in the antigen binding protein after it is formulated into a pharmaceutical composition.

In one embodiment, conservative amino acid substitutions may include substitution of one amino acid with another amino acid of the same class, such as substitution of a non-polar amino acid with another non-polar amino acid.

In another embodiment, conservative substitutions can be made according to Table 1. Methods for predicting resistance to protein modification can be found, for example, in Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the contents of which are incorporated by reference in their entirety. Table 1. Conservative amino acid substitutions Amino Acids Replace (others known in the art) Ala Ser, Gly, Cys Arg Lys, Gln, His Asn Gln, His, Glu, Asp Asp Glu, Asn, Gln Cys Ser、Met、Thr Gln Asn, Lys, Glu, Asp, Arg Glu Asp, Asn, Gln Gly Pro、Ala、Ser His Asn, Gln, Lys Ile Leu, Val, Met, Ala Leu Ile, Val, Met, Ala Lys Arg, Gln, His Met Leu, Ile, Val, Ala, Phe Phe Met, Leu, Tyr, Trp, His Ser Thr、Cys、Ala Thr Ser, Val, Ala Trp Tyr、Phe Tyr Trp, Phe, His Val Ile, Leu, Met, Ala, Thr

The antigen binding proteins 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 may include, for example, aminocyclohexanecarboxylic acid, norleucine, α-aminodecanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxysuccinimide and trans-4-hydroxysuccinimide, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline- 2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, guanidine, 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.

In one embodiment, the antigen binding protein of the present invention or (one or more) amino acids of the antigen binding protein can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, for example, via disulfide bonds, or converted to acid addition salts and/or optionally, dimerized or polymerized, or conjugated. In a preferred embodiment, the glutamic acid of the antigen binding protein is cyclized to pyroglutamic acid. In addition, the N-terminal glutamic acid of the antigen binding protein can be cyclized to pyroglutamic acid.

As used herein, " covalent link " refers to a covalent link such as a disulfide bond or a peptide link or via a linker or linker sequence such as a polypeptide linker.

As used herein, the term " linker " refers to one or more amino acid residues inserted between multiple or one domain and an agent that provides sufficient mobility for, for example, a bispecific antigen binding variable domain to fold correctly to form an antigen binding site.

In some embodiments, the linker consists of 0 amino acids, which means that the linker is absent. The linker is inserted at the amino acid sequence level at the transition between variable domains or between a variable domain and a constant domain (or dimerization domain), respectively. The transition between domains can be recognized because the approximate size of antibody domains and TCR domains is well understood. The exact position of the domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta sheets or alpha helices, as confirmed by experimental data or as can be assumed by modeling or secondary structure prediction techniques.

Unless otherwise specified in the respective context, the length of the linker can be at least 1 to 30 amino acids. In some embodiments, the length of the linker can be 2-25, 2-20, or 3-18 amino acids. In some embodiments, the linker can be a peptide of no more than 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids in length. In other embodiments, the length of the linker can be 5-25, 5-15, 4-11, 10-20, or 20-30 amino acids. In other embodiments, the length of the linker 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 a specific embodiment, the length of the linker can be less than 24, less than 20, less than 16, less than 12, less than 10, such as 5 to 24, 10 to 24, or 5-10 amino acid residues. In some embodiments, the linker is equal to 1 or more amino acid residues in length, such as greater than 1, greater than 2, greater than 5, greater than 10, greater than 20 amino acid residues in length, greater than 22 amino acid residues in length. In a preferred embodiment, the linker is a glycine/serine linker, i.e., a linker consisting of or consisting essentially of glycine and serine residues.

The antigen binding proteins disclosed herein can be synthetic, recombinant, isolated, engineered, and/or purified. In certain aspects, the antigen binding protein is a non-naturally occurring antigen binding protein, such as when the antigen binding protein is a soluble bispecific antigen binding protein such as TCER®.

In the context of the present invention, an " engineered " antigen binding protein, specifically an engineered TCR, refers to a protein that is not naturally occurring or has been modified by biotechnology methods, specifically by introducing amino acid mutations/substitutions into the native protein sequence. Such biotechnology methods are well known to those of ordinary skill in the art.

When referring to a polypeptide, such as an antigen binding protein of the present invention, or a nucleotide sequence, such as one encoding an antigen binding protein described herein or a functional fragment thereof, " purified " means that the indicated molecule is present in the absence of substantially 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 biological macromolecules of the same type are present. As used herein, the term "purified" may further indicate that the antigen binding protein does not contain DNA, RNA, proteins, polypeptides, or cells that would interfere with its therapeutic, diagnostic, preventive, research, or other uses.

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

The term " isolated " means changed relative to or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials in its natural state is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-natural environment such as a host cell. An isolated antigen binding protein is substantially free of other antigen binding proteins with different antigenic specificities (for example, an antigen binding protein that specifically binds to MAGEB2 is substantially free of antigen binding proteins that specifically bind to antigens other than MAGEB2). In addition, an isolated antigen binding protein may be substantially free of other cellular materials and/or chemicals. Thus, "isolated" may refer to a protein that is removed from a cell culture and separated from components of the cell culture, for example, it may be separated from at least 90%, preferably at least 95%, of the components of the cell culture.

A "recombinant " molecule is one that is prepared, expressed, generated, or isolated by recombinant means. Recombinant molecules do not occur in nature. Thus, "recombinant" refers to a polypeptide or protein molecule that is produced using recombinant techniques, i.e., does not occur in nature. Methods and techniques for producing recombinant nucleic acids and polypeptides are well known in the art.

The term " gene " means a DNA sequence that encodes or corresponds to a specific sequence of amino acids that comprises all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences such as promoter sequences that determine the conditions under which the gene is expressed. Some genes that are not structural genes can be transcribed from DNA into RNA, but are not translated into amino acid sequences. Other genes can serve as regulatory factors for structural genes or as regulatory factors for DNA transcription. In particular, the term gene is intended to be used for the genome sequence that encodes a protein, that is, a sequence that includes regulatory factors, promoters, introns, and exon sequences.

In the context of the present invention, " affinity " is defined as the equilibrium binding between an antigen binding protein and its antigen, i.e., the MAGEB2 peptide in complex with an MHC protein. Affinity is usually expressed as the equilibrium dissociation constant ( _KD ).

" _KD " is the equilibrium dissociation constant, i.e., the ratio of _koff / _kon between an antigen binding protein and its antigen. _KD and affinity are inversely related. The _KD value is related to the concentration of the antigen binding protein, and the lower the _KD value, the higher the affinity of the antigen binding protein. _KD values can be experimentally evaluated by various known methods, such as measuring association and dissociation rates using surface plasmon resonance (SPR) or biolayer interferometry (BLI). Binding interactions can be measured under a wide range of settings using appropriate buffers that minimize nonspecific binding and maintain protein stability, including but not limited to a temperature range of 25°C to 37°C and a shaking speed range of 500 rpm to 1500 rpm. Examples of such buffers are phosphate buffered saline (PBS), Tris buffered saline (TBS), HEPES buffered saline (HBS) or other physiological buffers, with or without additives such as Tween, BSA, DMSO, or EDTA. The analyte can be immobilized on various sensors including but not limited to HIS1K sensor, NTA sensor, SA sensor, ProA sensor, ProG sensor, AHC sensor, FAB2G sensor at a concentration range including but not limited to 1 µg/ml to 100 µg/ml, and the duration range is 30 s to 300 s. _KD assays can be measured at various molar concentrations of analyte samples, including but not limited to the range of 25 nM to 500 nM, or up to 10 μM, thereby detecting possible off-target reactivity with high sensitivity. Exemplary assays of KD are provided herein in the Examples. For example, KD can be determined using BLI at 30°C and 1000 rpm shaking speed, using PBS, 0.05% Tween, 0.1% BSA as buffer, and HLA-A*02/MAGEB2-001 immobilized on a HIS1K sensor (at a concentration of 10 μg/ml for 120 s) prior to analyzing TCR variants at concentrations ranging from 25 nM to 200 nM.

In the context of the present invention, " efficacy " is defined as a parameter describing the ability of an antigen binding protein to activate effector cells, preferably T cells or NK cells, to kill cancer cells presenting target antigen peptides in complex with MHC on their surface. Efficacy can be determined in functional assays, in particular in cytotoxicity assays as described below.

In a " functional assay ", the antigen binding protein is co-cultured with " effector cells (E) " and " target cells (T) ", i.e., with cancer cells presenting the peptide:MHC complex. Therefore, the functional assay can also be described as a "co-culture assay". For all cell culture assays described herein, the cell culture temperature is preferably at about 37°C. The target cell can be a cell artificially loaded with the antigen peptide (e.g., T2 cells), or can be a cell endogenously presenting the target antigen peptide on its surface (e.g., a cancer cell expressing MAGEB2). Binding of the antigen binding protein to the peptide:MHC complex on the target cell and to the effector cell results in activation of the effector cell. Depending on the type of functional assay, there are different readouts for measuring the degree of activation. In a cytotoxicity assay, the killing of target cells by effector cells is determined, for example, by measuring a reduction in the proliferation of target cells, particularly cancer cells, or by measuring the release of intracellular proteins from target cells. Suitable intracellular proteins to be measured in a cytotoxicity assay may be endogenous proteins such as LDH.

The term " E:T ratio " refers to the ratio of effector cells (i.e., immune cells) to target cells. In some embodiments, the E:T ratio corresponds to the vaccination ratio, i.e., the ratio of the total number of immune cells to target cells.

In the example of lactate dehydrogenase (LDH) release assay, the effector cells are immune cells. These effector cells are co-cultured with tumor cells that endogenously express and present MAGEB2 antigenic peptides, and optionally, are additionally loaded with MAGEB2 antigenic peptides. In some embodiments, the tumor cells are SKMEL-5 cells, RPMI7951 cells, or SCC25 cells. In some embodiments of the LDH release assay, the inoculation ratio of total immune cells to target cells is 10:1. The efficacy of the antigen binding protein is considered high if in an LDH release assay as defined above, killing of tumor cells (as determined by the LDH release assay) is observed at an E:T ratio of 10:1. Alternatively, the efficacy of the antigen binding protein is considered high if in a cytotoxicity assay, preferably in an LDH release assay as defined above, the cytotoxic activity of the effector cells against the target cells at the highest concentration of the antigen binding protein tested is at least 50%, at least 60%, at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, or at least 95% of the cytotoxic activity of the control toxic agent. It is known to those skilled in the art that the cytotoxic activity may be higher than 100%. This is due to the fact that 100% cytotoxic activity is defined by a "maximum lysis control," which refers to the incubation of target cells with a toxic agent. In some embodiments, the toxic agent is a detergent that achieves target cell lysis, such as Triton-X100, Tween-20, Tween-80, or NP-40. In some specific embodiments, the maximum lysis control comprises the addition of a 0.9% Triton-X100 solution to the target cell culture. The cytotoxic activity of the toxic agent, i.e., the number of target cells killed by the toxic agent, is defined as 100%. Since target cells can still proliferate during the co-culture period, the number of target cells ultimately killed by the effector cells during the cytotoxicity assay can be higher than the number of target cells killed by the toxic agent during the maximum lysis control period. In these cases, the calculated cytotoxic activity will be greater than 100%.

" Half maximal effective concentration " is also referred to as " _EC50 ", and generally refers to the concentration of a molecule that induces a response that is intermediate between baseline and maximum after a specified exposure time. EC50 and affinity are inversely related, and the lower the EC50 value, the higher the affinity of the molecule. In one example, " _EC50 " refers to the concentration of an antigen-binding protein of the present invention that induces a response that is intermediate between baseline and maximum after a specified exposure time, and more specifically, refers to the concentration of an antigen-binding protein of the present invention that induces a response that is intermediate between baseline and maximum after a specified exposure time. EC50 values can be experimentally assessed by various known methods, such as binding assays using ELISA or flow cytometry or functional assays such as interleukin release assays or lactate dehydrogenase (LDH) release assays. In specific embodiments, " _EC50 " refers to the concentration of antigen binding protein that induces a response intermediate between baseline and maximum when target cells are co-cultured with effector cells in an LDH release assay as defined above. The functional affinity of the antigen binding protein is considered high if the _EC50 is less than about 500 pM, preferably less than about 200 pM, more preferably less than about 100 pM, or most preferably less than about 30 pM as determined in a cytotoxicity assay, preferably an LDH release assay as defined above, using a co-culture assay with tumor cells expressing MAGEB2 at a number of less than 200 copies of MAGEB2 per cell.

In the context of the present invention, the term " specificity " refers to the ability of an antigen binding protein to distinguish its target peptide from peptides having a different amino acid sequence, such as similar peptides as defined below. An antigen binding protein is considered specific for a target peptide if binding to the target peptide presented in an MHC molecule occurs with significantly higher affinity and/or higher functional affinity than binding to similar peptides. The specificity of an antigen binding protein is determined by the amino acid sequences CDRa1, CDRa3, CDRb1, and CDRb3. The amino acid sequences of CDRa2 and CDRb2 contact the MHC molecules and are not required for antigen specificity.

In the context of the present invention, " similar peptides " herein refer to possible off-target peptides, i.e., peptides that may be bound by the antigen binding protein of the present invention based on the biochemical/biophysical characteristics of the peptides, including but not limited to homologous sequences or similar motifs. The length of similar peptides generally comprises 8 to 12 amino acids. In the context of the present invention, similar peptides are generally presented by MHC, specifically MHC I. In addition, in the context of the present invention, similar peptides include the following peptides: comprising or consisting of an amino acid sequence similar to the amino acid sequence of the MAGEB2 antigenic peptide, more specifically, the following peptides: compared with the antigenic determinant of the MAGEB2 antigenic peptide, comprising an antigenic determinant in which some or all of the amino acids have the same and/or similar biochemical/biophysical characteristics as the amino acids constituting the antigenic determinant of the MAGEB2 antigenic peptide. In some examples, in the context of the present invention, the similar peptides studied are selected from a database of HLA-A*02 binding peptides presented by tumors and normal tissues ( ^XPRESIDENT® database), which uses similarity scores within binding-related positions of MAGEB2 and a requirement for at least one detection of normal tissues. Binding of antigen binding proteins to similar peptides presented by MHC proteins may lead to adverse reactions. These adverse reactions can be "off-tumor" side effects, such as cross-reactivity of specific TCRs with similar peptides in healthy tissues, as reported in Lowdell et al ., Cytotherapy, published on December 4, 2018).

In particular, in the context of the present invention, the following peptides are analogous peptides: SEQ ID NOs: 59-67, 106, and 133-151.

It is known to those skilled in the art that among similar peptides, there are some peptides that are not bound to a detectable degree by the antigen-binding protein of the present invention, for example, no binding signal above the background level is detected during affinity assays or no response above the background level is detected in functional assays. In this context, " background level " refers to the response observed in functional assays when target cells and effector cells are co-cultured at respective E:T ratios without the addition of bispecific TCR-antibody fusion protein.

For other similar peptides, low, but insignificant binding can be detected. These latter similar peptides can also be described as "possibly related" similar peptides. When binding to similar peptides and target antigen peptides is compared under similar, preferably identical experimental conditions, the antigen binding protein is considered to bind insignificantly to the similar peptide and to be specific for its target antigen peptide if at least one of the following applies: The cytotoxic activity in response to the similar peptide measured in the cytotoxicity assay as described above is 25% or less, 20% or less, 15% or less, 10% or less of the cytotoxic activity in response to the target antigen peptide MAGEB2.

The _EC50 of the analogous peptide measured in a functional assay, preferably a cytotoxicity assay as described above, is increased at least 50, at least 100, at least 200, or at least 500-fold compared to the _EC50 of the target antigen peptide MAGEB2.

The K _D of the analogous peptide is increased by at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100 times compared to the K _D of the target antigen peptide MAGEB2.

The relative response signal of the analogous peptide is no more than 30%, no more than 25%, no more than 20%, or no more than 15% of the response signal to the target antigen peptide.

" CD3 " is a protein complex and is composed of four different chains. In mammals, the complex contains one CD3γ chain, one CD3δ chain, and two CD3ε chains. These chains bind to molecules called T cell receptors (TCRs) and the ζ chain to generate activation signals in T lymphocytes.

CD28 is also expressed on T cells and can provide co-stimulatory signals required for T cell activation. CD28 plays an important role in T cell proliferation and survival, interleukin production, and type 2 T helper development.

" CD134 " is also known as OX40. CD134/OX40 is expressed 24 to 72 hours after activation and can be used to define secondary co-stimulatory molecules.

" 4-1BB " can bind to the 4-1BB ligand on antigen presenting cells (APC), thereby generating co-stimulatory signals for T cells.

" CD5 " is another example of a receptor that is primarily found on T cells. CD5 is also present at low levels on B cells.

" CD95 " is another example of a receptor that modifies T cell function and is also known as the Fas receptor that mediates apoptotic signaling through Fas ligand expressed on the surface of other cells. CD95 has been reported to regulate the TCR/CD3-driven signaling pathway in resting T lymphocytes.

“ NK cell specific receptor molecule ” includes, for example, CD16 and NKG2D, which are low-affinity Fc receptors.

" Copy number " herein refers to the number of MAGEB2/MHC complexes as defined in the context of the present invention present on the cell surface of cells such as MAGEB2/MHC presenting cells, such as cancer cells or healthy cells. The copy number of a protein can be determined by various methods known in the art, including FACS analysis of diseased cells using fluorescently labeled antigen binding proteins.

" Safety profile " in this context refers to the ability to distinguish tumor cells from healthy tissue cells, and this is usually measured by determining the safety window.

As used herein, " safety window " or " therapeutic window " refers to the factor of the half-maximal concentration of a compound required to induce 100% cytotoxicity in a tumor cell line when compared to the half-maximal concentration of the compound required to induce 100% cytotoxicity in healthy tissue cells. If, for an antigen binding protein of interest, the EC50 determined for a tumor cell line is 1 pM, and the EC50 value determined for, for example, primary cells is 1000 pM, then the safety window is 1000 because the EC50 for the tumor cell line is 1000-fold less than the EC50 for primary cells.

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

Nucleic acids can be present in whole cells, in cell lysates, or can be in partially purified or substantially pure form. Nucleic acids are "isolated" or "made substantially pure" when purified from other cellular components or other impurities, such as other cellular nucleic acids or proteins, by standard techniques.

The terms " vector " , " cloning vector " and " expression vector " refer to vehicles by which DNA or RNA sequences (e.g., foreign genes) can be introduced into host cells in order to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequences.

The term " viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and can be encapsulated into a viral vector particle and encodes at least one exogenous nucleic acid. The vector and/or particle can be used for the purpose of transferring the nucleic acid of interest into cells in vitro or in vivo. Many forms of viral vectors are known in the art. Useful viral vectors include vectors based on retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma viruses, Epstein Barr viruses, vaccinia virus vectors, and vectors based on Semliki Forest virus (SFV). Recombinant viruses can be produced by techniques known in the art, such as by transfecting encapsulated cells or by transient transfection with a helper plasmid or virus. Typical examples of virus encapsulated cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed procedures for producing such replication-defective recombinant viruses can be found, for example, in WO95/14785, WO96/22378, US5,882,877, US6,013,516, US4,861,719, US5,278,056, and WO94/19478.

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

The term " expression system " refers to host cells and compatible vectors under appropriate conditions, such as for expressing a protein encoded by foreign DNA carried by the vector and introduced into the host cell.

A " host cell " is a cell that can be used to express a nucleic acid, such as those disclosed herein.

As used herein, the term " pharmaceutical composition " or " therapeutic composition " refers to a compound or composition capable of inducing a desired therapeutic effect when appropriately administered to a subject.

The terms " subject " or " individual " are used interchangeably and include all mammals, preferably humans. In some embodiments, the subject to be treated may also be referred to as a "patient."

As used herein, a " therapeutic agent " refers to an agent having a therapeutic effect. In one embodiment, such a therapeutic agent may be a growth inhibitor such as a cytotoxic agent or a radioisotope.

The terms " growth inhibitory agent " or "anti-proliferative agent" may be used indiscriminately to refer to a compound or composition that inhibits the growth of cells, especially tumor cells, in vitro or in vivo.

As used herein, the term " cytotoxic agent " refers to a substance that inhibits or prevents cell function and/or causes cell destruction. The term " cytotoxic agent " is intended to include chemotherapeutic agents, enzymes, antibiotics, and small molecule toxins or enzymatic toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, as well as various antitumor agents or anticancer agents disclosed below. In some embodiments, the cytotoxic agent is a paclitaxel, a vinca roseus, a taxane, a maytansine or a maytansine analog such as DM1 or DM4, a small drug, a tolmemycin or pyrrolobenzodiazepine derivative, a scin derivative, a leptomycin derivative, an auristatin or a dolastatin analog, a prodrug, a topoisomerase II inhibitor, a DNA alkylating agent, an anti-tubulin agent, CC-1065 or a CC-1065 analog.

The term " radioactive isotope " is intended to include radioactive isotopes useful for treating cancer, such as At ²¹¹ , Bi ²¹² , Er ¹⁶⁹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , In ¹¹¹ , P ³² , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Sr ⁸⁹ , and Lu. Such radioisotopes generally emit primarily beta radiation. In one embodiment, the radioisotope is an alpha-emitter isotope, more specifically thrombium 227, which emits alpha radiation.

" Pharmaceutically " or " pharmaceutically acceptable " refers to molecular entities and compositions that do not produce adverse, allergic or other troublesome reactions when administered to mammals, particularly humans, as appropriate. A pharmaceutically acceptable carrier is a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material, or formulation aid of any type.

" Pharmaceutically acceptable carriers " may include physiologically compatible solvents, bulking agents, stabilizers, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents and the like. In one embodiment, the carrier is an aqueous carrier. In some embodiments, the aqueous carrier can impart improved properties, such as improved solubility, efficacy and/or improved immunotherapy, when combined with the antigen binding proteins described herein.

In the context of the present invention, the terms " treating " or "treatment" include therapeutic treatment (i.e., for a subject suffering from a given disease) and/or preventive/prophylactic treatment (i.e., for a subject susceptible to a given disease). Therapeutic treatment means reversing, alleviating and/or inhibiting the development of one or more symptoms of a disease or condition. Preventive treatment means preventing the occurrence of one or more symptoms of a disease 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, prevents or delays the onset of the disease and/or prolongs the survival of the subject.

A "therapeutically effective amount" of an antigen binding protein or pharmaceutical composition thereof means an amount of the antigen binding protein sufficient to treat the proliferative disease at a reasonable benefit/risk ratio applicable to any medical treatment. However, it should be understood that the total daily dosage 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 dosage level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific antigen binding protein used; the specific composition used, the patient's age, weight, general health, sex and diet; the time of administration, route of administration and excretion rate of the specific polypeptide used; the duration of treatment; drugs used in combination or concurrently with the specific polypeptide used; and similar factors well known in the medical field. For example, it is well known to those of ordinary skill in the art to start the compound at a dosage level lower than that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

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

" Proliferative diseases " such as cancer involve unregulated and/or inappropriate cell proliferation.

If, as defined herein, peptides related to the MAGEB2 antigenic peptide having SEQ ID NO: 1 are over-presented in a patient's cancer cells, the cancer is considered a " cancer expressing MAGEB2 " (also referred to as a MAGEB2 "positive" cancer). In all other indications noted herein, biopsies may be performed as this is standard in the treatment of such cancers and peptides may be identified according to ^XPresident® and related methods (according to WO03/100432; WO2005/076009; WO2011/128448; WO2016/107740, US7,811,828, US9,791,444, and US2016/0187351, the contents of each of which are hereby incorporated by reference in their entirety). In one embodiment, cancer is readily determined (i.e., diagnosed), for example, by using the antigen binding proteins of the present invention. Methods of using antigen binding proteins to identify cancers expressing antigens are known to those of ordinary skill in the art. It should be understood that the terms " cancer " and " carcinoma " are not used interchangeably herein because carcinomas are specific types of cancers that occur in the skin or in tissues that form a layer inside or cover an organ of the body.

The terms " first container " and " second container " refer to the chambers of a multi-chamber pre-filled syringe (e.g., a lyosyringe).

In the context of this specification, when referring to a specific value, the term " about " or " approximately " is intended to indicate that the value may deviate by ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%. It also includes a concrete value, for example, "about 50" includes the value "50".

Throughout this application, the terms " and / or " are grammatical conjunctions and are to be construed as covering the possibility that one or more of the cases they are connected to may exist.

Furthermore, throughout this application, the term " comprising " is to be interpreted as covering all specifically mentioned features as well as optional, additional, and unspecified features. As used herein, the use of the term " comprising " also discloses embodiments in which no features other than the specifically mentioned features are present ( i.e. , " consisting of" ). Therefore , both meanings are specifically intended and therefore separately disclosed embodiments according to the present invention.

Furthermore, the indefinite article " a /an " does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent clauses does not indicate that a combination of these measures cannot be used to advantage. Therefore, the article "a/an" preceding an element or component is intended to be non-restrictive with respect to the number of instances (i.e., occurrences) of an element or component. Thus, "a/an" should be read as including one or at least one, and unless the value clearly indicates a singularity, the singular form of an element or component also includes the plural.

As used herein, the term "of the invention" or "according to the invention" is intended to refer to all aspects and embodiments of the invention disclosed and/or claimed herein. Any aspect, item or embodiment referred to herein as "disclosed herein" or "described herein" should be understood as an aspect, item or embodiment of "of the invention" or "according to the invention".

The present invention will now be described in more detail with reference to the following figures and examples. All literature and patent documents cited herein are hereby incorporated by reference. Although the present invention has been described and illustrated in detail in the foregoing description, the examples are considered to be illustrative or exemplary rather than restrictive. Antigen Binding Protein

Exemplary antigen binding proteins are shown in Table 7. Therefore, the present invention also relates to (one or more) sequences as provided herein in Table 7. The residues marked in bold in Table 7 are exemplary residues that can undergo post-translational N-terminal modification, as otherwise specified herein. Such post-translational N-terminal modifications can, for example, generate pyroglutamic acid residues, as otherwise specified herein.

In a first aspect, the present invention provides an antigen binding protein that specifically binds to a MAGEB2 antigen peptide complexed with a major histocompatibility complex (MHC) protein, wherein the MAGEB2 antigen peptide comprises or consists of the following: an amino acid sequence GVYDGEEHSV (SEQ ID NO: 1), wherein the antigen binding protein comprises complementary determining regions (CDRs) CDRa1, CDRa2, and CDRa3 as provided herein, and comprises CDRb1, CDRb2, and CDRb3 as provided herein, and wherein CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 form an antigen binding domain A. For example, in one embodiment of the present invention, the antigen binding protein comprises: CDRa1 comprising the amino acid sequence of SEQ ID NO:2, CDRa3 comprising the amino acid sequence selected from the group consisting of SEQ ID NO:3 and 4, CDRb1 comprising the amino acid sequence of SEQ ID NO:5, and CDRb3 comprising the amino acid sequence of SEQ ID NO:6; wherein the CDRa1, CDRa3, CDRb1 and/or CDRb3 sequences may comprise one, two or three amino acid mutations. In another embodiment, the CDRa1, CDRa3, CDRb1 and/or CDRb3 sequences may each comprise at most one, at most two or at most three amino acid mutations.

In some embodiments of the antigen binding protein, CDRa2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and 8, and CDRb2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 and 10; wherein the CDRa2 and/or CDRb2 sequences may comprise one, two or three amino acid mutations. In another embodiment, the CDRa2 and/or CDRb2 sequences may each comprise at most one, at most two or at most three amino acid mutations.

In another aspect, the present invention provides an antigen binding protein that specifically binds to a MAGEB2 antigen peptide complexed with an MHC protein, wherein the MAGEB2 antigen peptide comprises or consists of the following amino acid sequence GVYDGEEHSV (SEQ ID NO: 1), wherein the antigen binding protein comprises: a first polypeptide comprising a variable domain _VA comprising complementary determining regions (CDRs) CDRa1, CDRa2, and CDRa3 and a second polypeptide comprising a variable domain _VB comprising CDRb1, CDRb2, and CDRb3, wherein CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 form an antigen binding domain A; wherein (1) CDRa1 comprises SEQ ID NO: 2, CDRa3 comprises SEQ ID NO: 3, CDRb1 comprises SEQ ID NO: 5, and CDRb3 comprises SEQ ID NO: 6; or (2) CDRa1 comprises SEQ ID NO: 7; NO:2, CDRa3 comprises SEQ ID NO:4, CDRb1 comprises SEQ ID NO:5, and CDRb3 comprises SEQ ID NO:6; wherein the CDRa1, CDRa3, CDRb1 and/or CDRb3 sequences may comprise one, two or three amino acid mutations.

In another aspect, the present invention provides an antigen-binding protein comprising a variable domain _VA comprising complementary determining regions (CDRs) CDRa1, CDRa2, and CDRa3 and a variable domain _VB comprising CDRb1, CDRb2, and CDRb3, wherein CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 form an antigen-binding domain A; wherein (1) CDRa1 comprises SEQ ID NO:2, CDRa3 comprises SEQ ID NO:3, CDRb1 comprises SEQ ID NO:5, and CDRb3 comprises SEQ ID NO:6; or (2) CDRa1 comprises SEQ ID NO:2, CDRa3 comprises SEQ ID NO:4, CDRb1 comprises SEQ ID NO:5, and CDRb3 comprises SEQ ID NO:6.

In some embodiments of the antigen binding protein, the CDRa1, CDRa3, CDRb1, and CDRb3 sequences comprise up to three amino acid mutations in each of CDRa1, CDRa3, CDRb1, and CDRb3.

In some embodiments of the antigen binding protein, (1) CDRa1 comprises SEQ ID NO:2, CDRa2 comprises SEQ ID NO:7, CDRa3 comprises SEQ ID NO:3, CDRb1 comprises SEQ ID NO:5, CDRb2 comprises SEQ ID NO:9, and CDRb3 comprises SEQ ID NO:6; (2) CDRa1 comprises SEQ ID NO:2, CDRa2 comprises SEQ ID NO:8, CDRa3 comprises SEQ ID NO:4, CDRb1 comprises SEQ ID NO:5, CDRb2 comprises SEQ ID NO:10, and CDRb3 comprises SEQ ID NO:6; (3) CDRa1 comprises SEQ ID NO:2, CDRa2 comprises SEQ ID NO:7, CDRa3 comprises SEQ ID NO:3, CDRb1 comprises SEQ ID NO:5, CDRb2 comprises SEQ ID NO:10, and CDRb3 comprises SEQ ID NO:6; or (4) CDRa1 comprises SEQ ID NO:2, CDRa2 comprises SEQ ID NO:8, CDRa3 comprises SEQ ID NO:4, CDRb1 comprises SEQ ID NO:5, CDRb2 comprises SEQ ID NO:9, and CDRb3 comprises SEQ ID NO:6; wherein the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may comprise one, two or three amino acid mutations. In another embodiment, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may each comprise at most one, at most two or at most three amino acid mutations.

In some embodiments of the antigen binding protein, (1) CDRa1 includes SEQ ID NO:2, CDRa2 includes SEQ ID NO:7, CDRa3 includes SEQ ID NO:3, CDRb1 includes SEQ ID NO:5, CDRb2 includes SEQ ID NO:9, and CDRb3 includes SEQ ID NO:6; (2) CDRa1 includes SEQ ID NO:2, CDRa2 includes SEQ ID NO:8, CDRa3 includes SEQ ID NO:4, CDRb1 includes SEQ ID NO:5, CDRb2 includes SEQ ID NO:10, and CDRb3 includes SEQ ID NO:6; (3) CDRa1 includes SEQ ID NO:2, CDRa2 includes SEQ ID NO:7, CDRa3 includes SEQ ID NO:3, CDRb1 includes SEQ ID NO:5, CDRb2 includes SEQ ID NO:10, and CDRb3 includes SEQ ID NO:6; or (4) CDRa1 includes SEQ ID NO:2, CDRa2 includes SEQ ID NO:8, CDRa3 includes SEQ ID NO:4, CDRb1 includes SEQ ID NO:5, CDRb2 includes SEQ ID NO:9, and CDRb3 includes SEQ ID NO:6; wherein the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may have one, two or three amino acid mutations.

In some embodiments of the antigen binding protein, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 sequences comprise up to three amino acid mutations in each of CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3.

In some embodiments of the antigen binding protein, the _VA comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, and 14, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 11 and comprising the CDRa1, the CDRa2, and the CDRa3 having SEQ ID NOs: 2, 7, and 3, respectively, an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 12 and comprising the CDRa1, the CDRa2, and the CDRa3 having SEQ ID NOs: 2, 8, and 4, respectively, an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 13 and comprising the CDRa1, the CDRa2, and the CDRa3 having SEQ ID NOs: 2, 7, and 3, respectively, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: NO:14 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 8, and 4, respectively; and the _VB comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, and 23, or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:15 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively, and has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:16 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively, having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 17 and comprising an amino acid sequence having the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs: 5, 10, and 6, respectively, having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 18 and comprising an amino acid sequence having the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs: 5, 10, and 6, respectively, having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 19 and comprising an amino acid sequence having the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs: 5, 10, and 6, respectively, having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 20 and comprising an amino acid sequence having the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs: 5, 10, and 6, respectively, NO:5, 10, and 6, having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:21 and comprising the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively, having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:22 and comprising the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 9, and 6, respectively, or having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:23 and comprising the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 9, and 6, respectively, Optionally, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may contain one, two or three amino acid mutations, preferably amino acid substitutions.

In some embodiments of the antigen binding protein, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 sequences comprise up to three amino acid mutations in each of CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3.

In some embodiments of the antigen binding protein, the _VA comprises the amino acid sequence of SEQ ID NO: 11 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 11 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs: 2, 7, and 3, respectively, and the _VB comprises the amino acid sequence of SEQ ID NO: 15 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 15 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs: 5, 9, and 6, respectively; the _VA comprises the amino acid sequence of SEQ ID NO: 12 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 12 and comprises the amino acid sequences of SEQ ID NOs: NO:2, 8, and 4, and the _VB comprises the amino acid sequence of SEQ ID NO:16 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:16 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; the _VA comprises the amino acid sequence of SEQ ID NO:13 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:13 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and the _VB comprises the amino acid sequence of SEQ ID NO:17 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:17 and comprises the amino acid sequences of SEQ ID NO: NO:5, 10, and 6 of the said CDRb1, the said CDRb2, and the said CDRb3; the said _VA comprises the amino acid sequence of SEQ ID NO:14 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:14 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively, and the said VB comprises the amino acid sequence of SEQ ID NO:18 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:18 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6 of the said CDRb1, the said CDRb2, and the said CDRb3; the said _VA comprises the amino acid sequence of SEQ ID NO:13 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:14 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively, and the said VB comprises the amino acid sequence of SEQ ID NO:18 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:18 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6 of the said CDRb1, the said CDRb2, and the said CDRb3; the said _VA comprises the amino acid sequence of SEQ ID NO:13 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:13 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 7, and 3, respectively, and the _VB comprises the amino acid sequence of SEQ ID NO:19 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:19 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; the _VA comprises the amino acid sequence of SEQ ID NO:11 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:11 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 7, and 3, respectively, and the _VB comprises the amino acid sequence of SEQ ID NO:19 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: NO:19 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; the _VA comprises the amino acid sequence of SEQ ID NO:14 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:14 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 8, and 4, respectively, and the _{VB comprises the amino acid sequence of SEQ ID NO:20 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:20 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3} of SEQ ID NOs:5, 10, and 6, respectively; the VA comprises the amino acid sequence of SEQ ID NO:14 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:14 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID _NOs :2, 8, and 4, respectively. NO:13 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:13 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 7, and 3, respectively, and the _{VB comprises the amino acid sequence of SEQ ID NO:21 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:21 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3} of SEQ ID NOs:5, 10, and 6, respectively; the _{VA comprises the amino acid sequence of SEQ ID NO:11 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:11 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 7, and 3, respectively, and the VB} comprises the amino acid sequence of SEQ ID NO:21 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:21 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID _NOs :5, 10, and 6, respectively. NO:21 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:21 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; the _VA comprises the amino acid sequence of SEQ ID NO:12 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:12 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 8, and 4, respectively, and the _VB comprises the amino acid sequence of SEQ ID NO:18 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:18 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; the V wherein _{the V A} comprises the amino acid sequence of SEQ ID NO: 13 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 13 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs: 2, 7, and 3, respectively, and the V _B comprises the amino acid sequence of SEQ ID NO: 22 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 22 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs: 5, 9, and 6, respectively; or the V _A comprises the amino acid sequence of SEQ ID NO: 14 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 14 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs: 2, 8, and 4, respectively, and the V _B comprises SEQ The amino acid sequence of SEQ ID NO:23 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:23 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 9, and 6, respectively; wherein optionally, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may comprise one, two or three amino acid mutations, preferably amino acid substitutions.

In some embodiments of the antigen binding protein, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 sequences comprise up to three amino acid mutations in each of CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3.

In all embodiments of the antigen-binding proteins of the present invention, the amino acid mutations (if any) within the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 sequences are preferably amino acid substitutions, more preferably conservative amino acid substitutions (see Table 1). Preferably, the CDR sequence comprises no more than two, preferably no more than one amino acid mutation. Further preferably, the (one or more) amino acid mutations (if any) are at the first position or the last position of the respective CDR sequence. In the best embodiment, the CDR sequence does not comprise any amino acid mutation.

In some embodiments of the antigen-binding protein, the first two positions and the last two positions of CDR1, CDR2 and/or CDR3 of VA and/or VB comprise conservative substitutions, and preferably CDRa1, CDRa3, CDRb1 and/or CDRb3 may comprise one, two or three amino acid mutations. In a specific embodiment of the antigen-binding protein, the first two positions and the last two positions of CDR3 of VA and/or VB domains comprise conservative substitutions, and preferably CDRa3 and/or CDRb3 may comprise one, two or three amino acid mutations.

Introducing mutations into known amino acid sequences is a standard procedure well known in the art and a routine task for those of ordinary skill in the art. Individual methods are known in the art (e.g., Stratagene's QuikChange Site Directed Mutagenesis Kit since 2007). Thus, those of ordinary skill in the art are well able to introduce specific mutations such as substitutions generally into amino acid sequences and specifically into CDR sequences.

Screening variants of a given CDR for binding to its target is also a procedure used by those of ordinary skill in the art. This application describes functional assays, including cytokine production assays for determining binding of the antigen binding protein of the present invention to the MAGEB2 antigen peptide. Binding of the antigen binding protein of the present invention to the MAGEB2 antigen peptide can also be determined by peptide MHC multimer staining or bio-interferometry.

Although the results of amino acid mutations in CDRs may not be easily predicted, one skilled in the art will be well able to generate and screen multiple mutants without undue burden. Thus, one skilled in the art will be able to generate antigen binding proteins carrying one, two or three amino acid mutations in their CDRs and subsequently identify antigen binding proteins having the same binding characteristics as an antigen binding protein comprising the CDR sequences of Table 7.

In some embodiments of the antigen binding protein, the _VA comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, and 14, and the _VB comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, and 23.

In a preferred embodiment of the antigen-binding protein, the _VA comprises the amino acid sequence of SEQ ID NO:11 and the _VB comprises the amino acid sequence of SEQ ID NO:15; the _VA comprises the amino acid sequence of SEQ ID NO:12 and the _VB comprises the amino acid sequence of SEQ ID NO:16; the _VA comprises the amino acid sequence of SEQ ID NO:13 and the _VB comprises the amino acid sequence of SEQ ID NO:17; the _VA comprises the amino acid sequence of SEQ ID NO:14 and the _VB comprises the amino acid sequence of SEQ ID NO:18; the _VA comprises the amino acid sequence of SEQ ID NO:13 and the _VB comprises the amino acid sequence of SEQ ID NO:19; the _VA comprises the amino acid sequence of SEQ ID NO:11 and the _VB comprises the amino acid sequence of SEQ ID NO:19; the _VA comprises the amino acid sequence of SEQ ID NO:14 and the _VB comprises the amino acid sequence of SEQ ID NO:20; The _VA comprises the amino acid sequence of SEQ ID NO:13 and the _VB comprises the amino acid sequence of SEQ ID NO:21; the _VA comprises the amino acid sequence of SEQ ID NO:11 and the _VB comprises the amino acid sequence of SEQ ID NO:21; the _VA comprises the amino acid sequence of SEQ ID NO:12 and the _VB comprises the amino acid sequence of SEQ ID NO:18; the _VA comprises the amino acid sequence of SEQ ID NO:13 and the _VB comprises the amino acid sequence of SEQ ID NO:22; or the _VA comprises the amino acid sequence of SEQ ID NO:14 and the _VB comprises the amino acid sequence of SEQ ID NO:23; Optionally, wherein the (one or more) framework regions comprise at least one amino acid substitution selected from the group consisting of: - an amino acid substitution at position 19 in FR1-a, wherein the amino acid substitution is a hydrophobic amino acid selected from the group consisting of: A, G, F, L, I, Y, W, V, M, and P, preferably A, F, I, L, V, and M, such as S19A, S19I, S19L, or S19V, more preferably S19A or S19V, even more preferably S19A, - an amino acid substitution at position 39 in FR2-a, wherein the amino acid substitution is L39F, L39I, or L39V, most preferably L39F, - an amino acid substitution at position 13 in FR1-b, wherein the amino acid substitution is C13V, C13G, C13T, C13S, or C13A, preferably C13V, - Amino acid substitutions at positions 94 and/or 95 in FR3-b, wherein the amino acid substitution at position 94 is M94V and/or the amino acid substitution at position 95 is S95E or S95D, preferably S95E, respectively; and wherein the positions of the substitutions are given according to the IMGT nomenclature.

In some embodiments, the antigen binding protein further comprises a constitutive domain, wherein the constitutive domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 and 25, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NOs: 24 and 25; and/or comprises a linker, wherein the linker comprises an amino acid sequence of SEQ ID NO: 26, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 26.

In some embodiments of the antigen binding protein, the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:27 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively; the first polypeptide comprises the amino acid sequence of SEQ ID NO:28 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:28 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 8, and 4, respectively; the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:29 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively. NO:2, 7, and 3 of the CDRa1, the CDRa2, and the CDRa3 amino acid sequence; The first polypeptide comprises the amino acid sequence of SEQ ID NO:30 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:30 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively; The first polypeptide comprises the amino acid sequence of SEQ ID NO:31 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:31 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively; The first polypeptide comprises the amino acid sequence of SEQ ID NO:32 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:32 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively. NO:2, 8, and 4 of the CDRa1, the CDRa2, and the CDRa3 amino acid sequence; or the first polypeptide comprises the amino acid sequence of SEQ ID NO:33 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:33 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:34 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:34 and comprises the amino acid sequences of SEQ ID NO:5, 9, and 6, respectively, CDRb1, the CDRb2, and the CDRb3 amino acid sequence; the second polypeptide comprises the amino acid sequence of SEQ ID NO:35 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:35 and comprises the amino acid sequences of SEQ ID NO:5, 9, and 6, respectively; NO:35 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; The second polypeptide comprises the amino acid sequence of SEQ ID NO:36 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:36 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; The second polypeptide comprises the amino acid sequence of SEQ ID NO:37 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:37 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; The second polypeptide comprises SEQ ID NO:38 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:38 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; The second polypeptide comprises the amino acid sequence of SEQ ID NO:39 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:39 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; The second polypeptide comprises the amino acid sequence of SEQ ID NO:40 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:40 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6, respectively. NO:5, 10, and 6 of the CDRb1, the CDRb2, and the CDRb3 amino acid sequence; The second polypeptide comprises the amino acid sequence of SEQ ID NO:41 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:41 and comprises the amino acid sequences of SEQ ID NO:5, 9, and 6, respectively; The second polypeptide comprises the amino acid sequence of SEQ ID NO:42 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:42 and comprises the amino acid sequences of SEQ ID NO:5, 9, and 6, respectively; The second polypeptide comprises the amino acid sequence of SEQ ID NO:43 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:43 and comprises the amino acid sequences of SEQ ID NO:5, 9, and 6, respectively; NO:43 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; The second polypeptide comprises the amino acid sequence of SEQ ID NO:44 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:44 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; or The second polypeptide comprises the amino acid sequence of SEQ ID NO:45 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:45 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; Optionally, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may contain one, two or three amino acid mutations, preferably amino acid substitutions.

In some embodiments of the antigen binding protein, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 sequences comprise up to three amino acid mutations in each of CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3.

In some embodiments of the antigen binding protein, the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:27 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:34 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:34 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 9, and 6, respectively; the first polypeptide comprises the amino acid sequence of SEQ ID NO:28 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:28 and comprises the amino acid sequences of SEQ ID NO: NO:2, 8, and 4 of the CDRa1, the CDRa2, and the CDRa3, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:35 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:35 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:29 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:36 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:37. NO:36 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; The first polypeptide comprises the amino acid sequence of SEQ ID NO:30 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:30 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 8, and 4, respectively, and The second polypeptide comprises the amino acid sequence of SEQ ID NO:37 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:37 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; The first polypeptide comprises SEQ ID NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:29 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:38 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:38 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:27 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and The second polypeptide comprises the amino acid sequence of SEQ ID NO:38 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:38 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; The first polypeptide comprises the amino acid sequence of SEQ ID NO:30 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:30 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 8, and 4, respectively, and The second polypeptide comprises the amino acid sequence of SEQ ID NO:39 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:39 and comprises the amino acid sequences of SEQ ID NOs:5, 10, and 6, respectively. NO:5, 10, and 6 of the CDRb1, the CDRb2, and the CDRb3 amino acid sequence; The first polypeptide comprises the amino acid sequence of SEQ ID NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:29 and comprises the amino acid sequences of SEQ ID NO:2, 7, and 3, respectively, and The second polypeptide comprises the amino acid sequence of SEQ ID NO:40 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:40 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6 of the CDRb1, the CDRb2, and the CDRb3, respectively; The first polypeptide comprises the amino acid sequence of SEQ ID NO:27 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:27 and comprises the amino acid sequences of SEQ ID NO:2, 7, and 3, respectively. NO:27 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 7, and 3, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:40 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:40 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; the first polypeptide comprises the amino acid sequence of SEQ ID NO:28 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:28 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 8, and 4, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:40 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:40 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; NO:37 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:37 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; The first polypeptide comprises the amino acid sequence of SEQ ID NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:29 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and The second polypeptide comprises the amino acid sequence of SEQ ID NO:41 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:41 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 9, and 6, respectively; The first polypeptide comprises the amino acid sequence of SEQ ID NO:30 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:30 and comprises the amino acid sequences of CDRa1, CDRa2, and CDRa3 of SEQ ID NOs:2, 8, and 4, respectively, and The second polypeptide comprises the amino acid sequence of SEQ ID NO:42 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:42 and comprises the amino acid sequences of CDRb1, CDRb2, and CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; The first polypeptide comprises the amino acid sequence of SEQ ID NO:31 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:31 and comprises the amino acid sequences of SEQ ID NO:5, 9, and 6, respectively. NO:2, 8, and 4 of the CDRa1, the CDRa2, and the CDRa3 amino acid sequence, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:43 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:43 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 9, and 6, respectively; the first polypeptide comprises the amino acid sequence of SEQ ID NO:32 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:32 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 8, and 4, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:44 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:44, respectively. NO:44 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; or the first polypeptide comprises the amino acid sequence of SEQ ID NO:33 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:33 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 8, and 4, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:45 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:45 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; Optionally, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may contain one, two or three amino acid mutations, preferably amino acid substitutions.

In a specific embodiment of the antigen binding protein, the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:27 and comprises the amino acid sequences of CDRa1, CDRa2, and CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:34 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:34 and comprises the amino acid sequences of CDRb1, CDRb2, and CDRb3 of SEQ ID NO:5, 9, and 6, respectively; or the first polypeptide comprises the amino acid sequence of SEQ ID NO:28 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:28 and comprises the amino acid sequences of SEQ ID NO: NO:2, 8, and 4 of the said CDRa1, the said CDRa2, and the said CDRa3 amino acid sequence, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:35 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:35 and comprises the said CDRb1, the said CDRb2, and the said CDRb3 amino acid sequence of SEQ ID NO:5, 10, and 6, respectively.

In some embodiments of the antigen binding protein, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 sequences comprise up to three amino acid mutations in each of CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3.

In some embodiments of the antigen-binding protein, the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:34; the first polypeptide comprises the amino acid sequence of SEQ ID NO:28 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:35; the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:36; the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:37; the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:38; the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:38; the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:39 amino acid sequence; the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:40; the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:40; the first polypeptide comprises the amino acid sequence of SEQ ID NO:28 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:37; the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:41; the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:42; the first polypeptide comprises the amino acid sequence of SEQ ID NO:31 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:43; the first polypeptide comprises the amino acid sequence of SEQ ID NO:32 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:44; or the first polypeptide comprises the amino acid sequence of SEQ ID NO:33 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:45.

In some specific aspects, the antigen binding protein comprises: The first polypeptide comprises the amino acid sequence of SEQ ID NO:27 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:34; or The first polypeptide comprises the amino acid sequence of SEQ ID NO:28 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:35.

In some embodiments, the antigen binding domain A is derived from a TCR, or is a TCR or fragment (or fragments) thereof.

In a preferred embodiment, the TCR is selected from the group consisting of: α/β TCR, γ/δ TCR, a functional fragment of TCR, and a fusion protein or chimeric protein comprising a functional fragment of TCR. In some embodiments, _VA and _VB are TCR variable domains, specifically TCR α, β, γ or δ variable domains. In some embodiments, _VA is TCR α, γ or δ variable domains and _VB is TCR β, γ or δ variable domains. Preferably, _VA is TCR α variable domains and _VB is TCR β variable domains, or _VA is TCR γ variable domains and _VB is TCR δ variable domains, or _VA is TCR α variable domains and _VB is TCR γ variable domains, or _VA is TCR δ variable domains and _VB is TCR β variable domains. In preferred embodiments, _VA and _VB are TCRα and TCRβ domains, respectively. In some embodiments, _VA is a TCRγ variable domain comprising CDR1 and CDR3 derived from a TCRα variable domain and, optionally, CDR2, and/or _VB is a TCRδ variable domain comprising CDR1 and CDR3 derived from a TCRβ variable domain and, optionally, CDR2.

In some embodiments, the MHC protein is an HLA protein, preferably HLA-A, more preferably HLA-A*02.

The antigen binding protein of the present invention has high specificity for the MAGEB2 antigen peptide (SEQ ID NO: 1), specifically increased specificity compared to a reference protein when measured under similar, preferably identical, experimental conditions. The inventors demonstrated in Example 6 that the antigen binding protein of the present invention binds to the target antigen, i.e., the MAGEB2 antigen peptide, in complex with an MHC protein with high specificity.

The inventors have identified potential off-target peptides that are, for example, similar in sequence and/or motif to MAGEB2 and therefore have an increased risk of binding to the antigen binding protein that binds to MAGEB2. However, the antigen binding proteins of the invention do not specifically bind to these peptides.

In some embodiments, the antigen binding protein does not specifically bind to at least one, at least two, at least three, at least four, at least five, or all similar peptides selected from the group consisting of SEQ ID NO: 59 (SP-02-1621), SEQ ID NO: 60 (SP-02-1622), SEQ ID NO: 61 (SP-02-1623), SEQ ID NO: 62 (SP-02-1624), SEQ ID NO: 63 (SP-02-1625), SEQ ID NO: 64 (SP-02-1626), SEQ ID NO: 65 (SP-02-1627), SEQ ID NO: 66 (SP-02-1628), SEQ ID NO: 67 (SP-02-1629), SEQ ID NO: 106 (SP-02-1640), SEQ ID NO: 133 (SP-01-0010), SEQ ID NO: 134 (SP-01-0011), SEQ ID NO: 135 (SP-01-0012), SEQ ID NO: 136 (SP-01-0013), SEQ ID NO: 137 (SP-01-0014), SEQ ID NO: 138 (SP-01-0015), SEQ ID NO: 139 (SP-01-0016), SEQ ID NO: 140 (SP-01-0017), SEQ ID NO: 141 (SP-01-0018), SEQ ID NO: 142 (SP-01-0019), SEQ ID NO: 143 (SP-01-0021), SEQ ID NO: 144 (SP-01-0022), SEQ ID NO: ID NO: 134 (SP-02-1631), SEQ ID NO: 135 (SP-02-1635), SEQ ID NO: 136 (SP-02-1641), SEQ ID NO: 137 (SP-02-1642), SEQ ID NO: 138 (SP-02-1643), SEQ ID NO: 139 (SP-02-1644), SEQ ID NO: 140 (SP-02-1647), SEQ ID NO: 141 (SP-02-1648), SEQ ID NO: 142 (SP-02-1650), SEQ ID NO: 143 (SP-02-1651), SEQ ID NO: 144 (SP-02-1652), SEQ ID NO: 145 (SP-02-1653), SEQ ID NO: 146 (SP-02-1655), SEQ ID NO:147 (SP-02-1656), SEQ ID NO:148 (SP-02-1664), SEQ ID NO:149 (SP-02-1673), SEQ ID NO:150 (SP-02-1722), and SEQ ID NO:151 (SP-02-1724).

In a preferred embodiment, the antigen binding protein is a soluble protein.

In some embodiments, the antigen binding protein is a multispecific antigen binding protein, particularly a bispecific antigen binding protein.

In certain embodiments, the antigen binding protein comprises an additional antigen binding domain or site B. In further embodiments, the antigen binding protein may comprise additional antigen binding sites/domains such as C, D, E, etc. Additional binding domains may, for example, aggregate or recruit additional binding moieties that are spatially proximal to the antigen binding protein, such as recruiters for T cells.

In some embodiments, the antigen binding protein further comprises a variable domain _VH comprising CDRH1, CDRH2, and CDRH3 and a _VL comprising CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 form an antigen binding domain B. The VH and the VL may be derived from an antibody. The antigen binding domain B may also be another TCR-derived binding domain, or another binding domain such as an Fc domain.

In certain embodiments, the antigen binding domain B specifically binds to immune cells, preferably T cells or natural killer (NK) cells.

In some embodiments, the antigen binding domain B specifically binds to an antigen selected from the group consisting of: CD2; CD3, specifically CD3γ, CD3δ and/or CD3ε; CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD44, C D45RA, CD49, CD55, CD56, CD61, CD64, CD68, CD69, CD89, CD90, CD94, CD95, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Ly-6.2C, Mel14, Nkp46, NKG2D, GITR, _FcεRI , TCRα/β, TCRγ/δ, HLA-DR and 4-1 BB, or a combination thereof.

Examples of receptor molecules present on the surface of both T cells and natural killer (NK) cells are CD2, and other members of the CD2 superfamily. CD2 can act as a co-stimulatory molecule on both T cells and NK cells.

In a preferred embodiment, the antigen binding domain B specifically binds to the antigen CD3. In a more preferred embodiment, the antigen binding domain B specifically binds to the α/β T cell receptor (TCR)/CD3 complex.

In some embodiments, the CDRH3 comprises an amino acid sequence of GSYYDYEGFVY [SEQ ID NO:50] or GSYYDYDGFVY [SEQ ID NO:58], and optionally, CDRH3 comprises up to three amino acid mutations, preferably amino acid substitutions.

In a preferred embodiment of an antigen binding protein comprising a binding domain B, _VH comprises: (a) a CDRH1 comprising an amino acid sequence of _X1YVMH [SEQ ID NO:111], wherein _X1 is S, R, K, or H, (b) a CDRH2 comprising _an amino _acid sequence _{of YINPX1X2DVTKYX3X4KFX5G} [SEQ ID NO: ₁₁₂ ], wherein _X1 is Y, R, K, or H, preferably Y or R; _X2 is N, R, or K, preferably N or R; _X3 is A or N; _X4 is E or Q, preferably E; and _X5 is Q or K, and (c) a CDRH3 comprising an amino acid sequence of GSYYDYEGFVY [SEQ ID NO:50] or GSYYDYDGFVY [SEQ ID NO:58], and V _L comprises: (a) a CDRL1 comprising an amino acid sequence of SATSSVXYMH [SEQ ID NO: 113], wherein X is S, R, or K, (b) a CDRL2 comprising an amino acid sequence of DTSKLAX [SEQ ID NO: 114], wherein X is S, R, or K, and (c) a CDRL3 comprising an amino acid sequence of QQWSSNPLT [SEQ ID NO: 53]; and optionally, wherein the CDRH1, the CDRH2 and the CDRH3 and each of the CDRL1, the CDRL2 and the CDRL3 comprise up to three amino acid substitutions.

More preferably, the _VH comprises: (a) a CDRH1 comprising the amino acid sequence of SYVMH [SEQ ID NO:48], (b) a CDRH2 comprising the amino acid sequence of YINPRNDVTKYAEKFQG [SEQ ID NO:49] or YINPYNDVTKYAEKFQG [SEQ ID NO:57], and (c) a CDRH3 comprising the amino acid sequence of GSYYDYEGFVY [SEQ ID NO:50] or GSYYDYDGFVY [SEQ ID NO:58], and the _VL comprises: (a) a CDRL1 comprising the amino acid sequence of SATSSVSYMH [SEQ ID NO:51], (b) a CDRL2 comprising the amino acid sequence of DTSKLAS [SEQ ID NO:52], and (c) a CDRH3 comprising the amino acid sequence of QQWSSNPLT [SEQ ID NO:53]. NO:53]; and optionally, wherein the CDRH1, the CDRH2 and the CDRH3 and each of the CDRL1, the CDRL2 and the CDRL3 comprises at most three amino acid substitutions.

In some embodiments, the ABP comprises a variable domain _VH comprising CDRH1, CDRH2, and CDRH3 having SEQ ID NOs: 48, 57, 50, respectively, and a _VL comprising CDRL1, CDRL2, and CDRL3 having SEQ ID NOs: 51-53, respectively, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 form the antigen binding domain B.

In some embodiments, the ABP comprises a variable domain _VH comprising CDRH1, CDRH2, and CDRH3 having SEQ ID NOs: 48, 49, 58, respectively, and a _VL comprising CDRL1, CDRL2, and CDRL3 having SEQ ID NOs: 51-53, respectively, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 form the antigen binding domain B.

In some embodiments, the ABP comprises a variable domain _VH comprising CDRH1, CDRH2, and CDRH3 having SEQ ID NOs: 48, 57, 58, respectively, and a _VL comprising CDRL1, CDRL2, and CDRL3 having SEQ ID NOs: 51-53, respectively, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 form the antigen binding domain B.

In a preferred embodiment, the ABP comprises a variable domain _VH comprising CDRH1, CDRH2, and CDRH3 having SEQ ID NOs: 48, 49, 50, respectively, and a _VL comprising CDRL1, CDRL2, and CDRL3 having SEQ ID NOs: 51-53, respectively, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 form the antigen binding domain B.

In some embodiments of the antigen binding protein, the _VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 46, 54, 55, and 56, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 46, 54, 55, or 56, and preferably comprises CDRH1, CDRH2, and CDRH3 as disclosed above; and the _VL comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 47, and preferably comprises CDRL1, CDRL2, and CDRL3 as disclosed above; and optionally, wherein the CDRH1, CDRH2 and the CDRH3 and each of the CDRL1, the CDRL2 and the CDRL3 comprise at most three amino acid substitutions.

In a preferred embodiment, antigen binding site B comprises: (i) a _VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 54, 55, and 56 or antigen-binding fragment(s) thereof and (ii) a _VL comprising an amino acid sequence of SEQ ID NO: 47 or antigen-binding fragment(s) thereof.

In some embodiments, the ABP comprises a variable domain _VH comprising the amino acid sequence of SEQ ID NO:46 and a variable domain _VL comprising the amino acid sequence of SEQ ID NO:47.

In some embodiments, the ABP comprises a variable domain _VH comprising the amino acid sequence of SEQ ID NO:54 and a variable domain _VL comprising the amino acid sequence of SEQ ID NO:47.

In some embodiments, the ABP comprises a variable domain _VH comprising the amino acid sequence of SEQ ID NO:55 and a variable domain _VL comprising the amino acid sequence of SEQ ID NO:47.

In some embodiments, the ABP comprises a variable domain _VH comprising the amino acid sequence of SEQ ID NO:56 and a variable domain _VL comprising the amino acid sequence of SEQ ID NO:47.

In some embodiments, the ABP comprises a variable domain _VH comprising the amino acid sequence of SEQ ID NO:115 and a variable domain _VL comprising the amino acid sequence of SEQ ID NO:116.

In some embodiments, the ABP comprises a variable domain _VH comprising the amino acid sequence of SEQ ID NO:117 and a variable domain _VL comprising the amino acid sequence of SEQ ID NO:118.

In some embodiments, the ABP comprises a variable domain _VH comprising the amino acid sequence of SEQ ID NO:119 and a variable domain _VL comprising the amino acid sequence of SEQ ID NO:120.

In some embodiments, the ABP comprises a variable domain _VH comprising the amino acid sequence of SEQ ID NO:121 and a variable domain _VL comprising the amino acid sequence of SEQ ID NO:122.

In some embodiments, the ABP comprises a variable domain _VH comprising the amino acid sequence of SEQ ID NO:123 and a variable domain _VL comprising the amino acid sequence of SEQ ID NO:124.

In some embodiments, the ABP comprises a variable domain _VH comprising the amino acid sequence of SEQ ID NO:125 and a variable domain _VL comprising the amino acid sequence of SEQ ID NO:126.

In some embodiments, the ABP comprises a variable domain _VH comprising the amino acid sequence of SEQ ID NO:127 and a variable domain _VL comprising the amino acid sequence of SEQ ID NO:128.

In some embodiments, the ABP comprises a variable domain _VH comprising the amino acid sequence of SEQ ID NO:129 and a variable domain _VL comprising the amino acid sequence of SEQ ID NO:130.

In some embodiments, the ABP comprises a variable domain _VH comprising the amino acid sequence of SEQ ID NO:131 and a variable domain _VL comprising the amino acid sequence of SEQ ID NO:132.

Other recruiting bodies can also be used for the antigen-binding proteins of the present invention, as disclosed in, for example, WO2022/233957, Zhu et al. (J lmmunol, 1995, 155, 1903-1 0 1910), Shearman et al. (J lmmunol, 1991, 147, 4366-73), WO2021/023657, Liu et al. (mAbs, 2023 Structure-based engineering of a novel CD3ε-targeting antibody for reduced polyreactivity, mAbs, 15:1, 2189974).

In some embodiments, the antigen binding protein comprises: - _VA , which comprises: - CDRa1 comprising or consisting of: SEQ ID NO:2, - CDRa2 comprising or consisting of: SEQ ID NO:7, and - CDRa3 comprising or consisting of: SEQ ID NO:3, - _VB , which comprises: - CDRb1 comprising or consisting of: SEQ ID NO:5, - CDRb2 comprising or consisting of: SEQ ID NO:9, and/or - CDRb3 comprising or consisting of: SEQ ID NO:6. - _VH comprising: - CDRH1 comprising or consisting of SEQ ID NO:48, - CDRH2 comprising or consisting of SEQ ID NO:49, and - CDRH3 comprising or consisting of SEQ ID NO:50, - and _VL comprising: - CDRL1 comprising or consisting of SEQ ID NO:51, - CDRL2 comprising or consisting of SEQ ID NO:52, and - CDRL3 comprising or consisting of SEQ ID NO:53.

In some embodiments, the antigen binding protein comprises: - _VA comprising: - CDRa1 comprising or consisting of: SEQ ID NO:2, - CDRa2 comprising or consisting of: SEQ ID NO:8, - CDRa3 comprising or consisting of: SEQ ID NO:4, - _VB comprising: - CDRb1 comprising or consisting of: SEQ ID NO:5, - CDRb2 comprising or consisting of: SEQ ID NO:10, - CDRb3 comprising or consisting of: SEQ ID NO:6, - _VH comprising: - CDRH1 comprising or consisting of: SEQ ID NO:48, - CDRH2 comprising or consisting of: SEQ ID NO:49, - CDRH3 comprising or consisting of: SEQ ID NO:50, - and _VL comprising: - - CDRL1 comprising or consisting of SEQ ID NO:51, - CDRL2 comprising or consisting of SEQ ID NO:52, and - CDRL3 comprising or consisting of SEQ ID NO:53.

In some embodiments, the antigen binding protein comprises: - _VA comprising: - CDRa1 comprising or consisting of: SEQ ID NO:2, - CDRa2 comprising or consisting of: SEQ ID NO:7, - CDRa3 comprising or consisting of: SEQ ID NO:3, - _VB comprising: - CDRb1 comprising or consisting of: SEQ ID NO:5, - CDRb2 comprising or consisting of: SEQ ID NO:10, - CDRb3 comprising or consisting of: SEQ ID NO:6, - _VH comprising: - CDRH1 comprising or consisting of: SEQ ID NO:48, - CDRH2 comprising or consisting of: SEQ ID NO:49, - CDRH3 comprising or consisting of: SEQ ID NO:50, - and _VL comprising: - - CDRL1 comprising or consisting of SEQ ID NO:51, - CDRL2 comprising or consisting of SEQ ID NO:52, and - CDRL3 comprising or consisting of SEQ ID NO:53.

In these embodiments, the CDRa1, CDRa3, CDRb1, CDRb3, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3 sequences may comprise one, two or three amino acid mutations.

In some embodiments, the first polypeptide and the second polypeptide of the antigen-binding protein are covalently linked together, for example, directly or via a cleavable or non-cleavable linker. In some embodiments, the first polypeptide and the second polypeptide of the antigen-binding protein are non-covalently linked together.

In some embodiments, the binding domains A and/or B are in a form selected from the group consisting of an antibody, a single chain variable fragment (scFv), a disulfide-stabilized Fv (dsFv), Fab, Fab', F(ab')2, a nanobody, a DARPin, a knottin, a bifunctional antibody, or an oligomer thereof. A single chain variable fragment (scFv) may also include (one or more) single chain fragments of a TCR, such as a scTV.

In some embodiments, binding domains A and B are in the form of scFv. The antigen binding protein may also comprise binding domains A and B on two scFvs, for example on two polypeptide chains.

In some embodiments, the antigen binding protein comprises a single-chain TCR (scTCR) and/or a single-chain bispecific antibody.

In the following, further specific forms of antigen binding proteins are exemplified, in which antigen binding site A and antigen binding site B are in a specific configuration, for example on a specific polypeptide chain. Such forms/configurations are merely exemplary features, and the antigen binding sites (e.g., A and B) may, for example, be located on one, two, three, four or more polypeptide chains. For example, variable domains V1 to V4 are each present on one polypeptide chain, i.e., on four polypeptide chains; or V1 and V2 are present on one polypeptide chain and V3 and V4 are present on one polypeptide chain, for example, on two polypeptide chains; or V1, V2, and V3 are present on one polypeptide chain, and V4 is present on one polypeptide chain, for example, on two polypeptide chains.

As another example, as long as VA and VB can form antigen binding site A and VH and VL can form antigen binding site B, VB, VA, and VH can be on a polypeptide chain and VL can be on another polypeptide chain (or any other other configuration), or can be included in any other binding site in the antigen binding protein. Alternatively, VB, VA, and VH, and dimerization protein can be on a polypeptide chain and VL can be on another polypeptide chain, and another part of the dimerization protein can be on a third polypeptide chain. The examples provided herein illustrate other antigen binding proteins such as TCER types. Therefore, the further indicated formula does not have a limiting feature, but is merely an example.

The indicated formulas V1-V4 comprise the CDR regions of the invention provided herein. For example, Vα comprises the CDRs of the α variable domain provided herein, and Vβ comprises the CDRs of the β variable domain provided herein.

In some embodiments, the antigen binding protein comprises two antigen binding sites A and B, wherein the polypeptide chain comprises a structure represented by formula [I]: V1-L1-V2       [I] wherein V1 is the first variable domain; V2 is the second variable domain; L1 is a linker domain, which is present or absent; and wherein the other polypeptide chain comprises a structure represented by formula [II]: V3-L3-V4       [II] wherein V3 is the third variable domain; V4 is the fourth variable domain; L3 is a linker, which is present or absent; optionally, wherein V1 and V2 form the antigen binding site A and V3 and V4 form the antigen binding site B, or V1 and V2 form the antigen binding site B and V3 and V4 form the antigen binding site A, or V1 and V3 form the antigen binding site A and V2 and V4 form the antigen binding site B, or V1 and V3 form the antigen binding site B and V2 and V4 form the antigen binding site A, or V1 and V4 form the antigen binding site A and V2 and V3 form the antigen binding site B, or V1 and V4 form the antigen binding site B and V2 and V3 form the antigen binding site A, and Optionally, wherein the first polypeptide and the second polypeptide are covalently or non-covalently linked together.

As also disclosed above, V1 and V2 are not necessarily on the same polypeptide chain, or V2 and V3 are not necessarily on the same polypeptide chain. For example, in some embodiments, the antigen binding protein comprises two antigen binding sites A and B, wherein the polypeptide chain comprises a structure represented by formula [I]: V1-L1-V2 -L2-V3 [I] wherein V1 is the first variable domain; V2 is the second variable domain; V3 is the third variable domain; L1 and L2 are (one or more) linker domains, which are present or absent; and wherein the other polypeptide chain comprises a structure represented by formula [II]: V4       [II] wherein V4 is the fourth variable domain; optionally, wherein V1 and V2 form the antigen binding site A and V3 and V4 form the antigen binding site B, or V1 and V2 form the antigen binding site B and V3 and V4 form the antigen binding site A, or V1 and V3 form the antigen binding site A and V2 and V4 form the antigen binding site B, or V1 and V3 form the antigen binding site B and V2 and V4 form the antigen binding site A, or V1 and V4 form the antigen binding site A and V2 and V3 form the antigen binding site B, or V1 and V4 form the antigen binding site B and V2 and V3 form the antigen binding site A, and Optionally, wherein the first polypeptide and the second polypeptide are covalently or non-covalently linked together.

The antigen binding proteins or polypeptide chains of the antigen binding proteins may be further covalently or non-covalently linked to additional proteins/domains, such as constant domains of antibodies or TCRs, such as Fc domains, or albumin, dimerizing proteins or fragments thereof, or additional binding domains, for example to further improve stability or include additional binding sites.

In some embodiments, the antigen binding protein comprises a polypeptide chain that forms antigen binding sites A and B, wherein, for example, the polypeptide chain comprises a structure represented by any one of formulas [I] to [VIII]: V1-L1-V4-L2-V2-L3-V3-C   [I], V4-L1-V1-L2-V2-L3-V3-C   [II], V1-L1-V4-L2-V3-L3-V2-C   [III], V4-L1-V1-L2-V3-L3-V2-C   [IV], V2-L1-V3-L2-V1-L3-V4-C   [V], V3-L1-V2-L2-V1-L3-V4-C   [VI], V2-L1-V3-L2-V4-L3-V1-C   [VII], V3-L1-V2-L2-V4-L3-V1-C   [VIII], where V1 is the first variable domain; V2 is the second variable domain; V3 is the third variable domain; V4 is the fourth variable domain; L1, L2, and L3 are connectors; C is a constant domain, which may or may not exist.

In a preferred embodiment, V1 and V4 may form the antigen binding site A and V2 and V3 may form the antigen binding site B.

In some embodiments, the antigen binding protein comprises a polypeptide chain that forms antigen binding sites A and B, wherein the polypeptide chain comprises a structure represented by any one of formulas [I] to [VIII]: V1-L1-V2-L2-V3-L3-V4-C   [I], V1-L1-V3-L2-V2-L3-V4-C   [II], V2-L1-V1-L2-V4-L3-V3-C   [III], V2-L1-V4-L2-V1-L3-V3-C   [IV], V3-L1-V1-L2-V4-L3-V2-C   [V], V3-L1-V4-L2-V1-L3-V2-C   [VI], V4-L1-V2-L2-V3-L3-V1-C   [VII], V4-L1-V3-L2-V2-L3-V1-C   [VIII], where V1 is the first variable domain; V2 is the second variable domain; V3 is the third variable domain; V4 is the fourth variable domain; L1, L2, and L3 are connecting subdomains; C is a constant domain, which may or may not exist.

As indicated above, in a preferred embodiment, V1 and V4 may form the antigen binding site A and V2 and V3 may form the antigen binding site B.

In some embodiments, the antigen binding protein comprises two polypeptide chains forming antigen binding sites A and B, wherein one polypeptide chain comprises a structure represented by one of formulas [I] to [IV]: V2-L1-V3-L2-V1-C1 [I] V2-L1-V3-L2-V4-C1 [II] V3-L1-V2-L2-V1-C1 [III] V3-L1-V2-L2-V4-C1 [IV] wherein V1 is the first variable domain; V2 is the second variable domain; V3 is the third variable domain; V4 is the fourth variable domain; C1 and C2 are constant domains; L1 and L2 are linker domains; and wherein the other polypeptide chain comprises a structure represented by formulas [V] and [VI]: V4-C2       [V] V1-C2       [VI] wherein [I] or [III] is bound to [VI], and [II] or [IV] is bound to [VI].

In a preferred embodiment, V1 and V4 may form the antigen binding site A and V2 and V3 may form the antigen binding site B.

In some embodiments, the antigen binding protein comprises two polypeptide chains that together form antigen binding sites A and B, wherein the first polypeptide chain comprises a structure represented by formula [III]: V1-L1-V2-L2-C1 [III] wherein V1 is the first variable domain; V2 is the second variable domain; L1 and L2 are linkers; L2 may be present or absent; C1 may be a homeostatic domain or its dimerization portion (e.g., CL) or another dimerization protein or fragment thereof and may be present or absent, or specifically a light chain homeostatic domain or its dimerization portion and may be present or absent; and wherein the second polypeptide chain comprises a structure represented by formula [IV]: V3-L3-V4-L4-C2 [IV] wherein V3 is the third variable domain; V4 is the fourth variable domain; L3 and L4 are linkers; L4 may be present or absent; C2 may be a constant domain or a dimerization portion thereof (e.g., CH) or another dimerization protein or a fragment thereof and may be present or absent, or specifically a heavy chain constant domain 1 or a dimerization portion thereof and may be present or absent; and wherein V1 and V2 form the antigen binding site A and V3 and V4 form the antigen binding site B, or V1 and V2 form the antigen binding site B and V3 and V4 form the antigen binding site A, or V1 and V3 form the antigen binding site A and V2 and V4 form the antigen binding site B, or V1 and V3 form the antigen binding site B and V2 and V4 form the antigen binding site A, or V1 and V4 form the antigen binding site A and V2 and V3 form the antigen binding site B, or V1 and V4 form the antigen binding site B and V2 and V3 form the antigen binding site A, and Optionally, wherein the first polypeptide and the second polypeptide are covalently or non-covalently linked together.

In a specific aspect, the antigen binding comprises: - a first polypeptide of the formula _V1 - _L1 - _V2 - _L2 - _C1 - _L5 - _FC1 , wherein _L2 , _C1 , and _L5 may be absent, and - a second polypeptide of the formula _V3 - _L3 - _V4 - _L4 - _C2 - _L6 - _FC2 , wherein _L4 , _C2 , and _L6 may be absent, wherein L2, L4, L5, and L6 are linkers, C1 may be a CL light chain constant domain or a dimerization portion thereof, or a CH heavy chain constant domain or a dimerization portion thereof; and if C1 is CL, then C2 may be a CH heavy chain constant domain or a dimerization portion thereof, and if C1 is CH, then C2 may be CL or a dimerization portion thereof.

In some embodiments, the first polypeptide chain has a structure represented by formula [V]: V1-L1-V2-L2-C1-L5- _FC1 [V] and the second polypeptide chain has a structure represented by formula [VI]: V3-L3-V4-L4-C2-L6- _FC2 [VI] wherein V1-V4, L1-L4, C1, and C2 are as defined above, and wherein L2, C1, L5, L4, C2, and C6 may be present or absent, and _FC1 and _FC2 may be Fc domains, and wherein _FC1 and _FC2 are the same or different.

In an exemplary embodiment, the antigen-binding protein of the present invention has variable domain orientations of VA, VB, VL, and VH according to orientation D in FIG. 8 .

In a specific embodiment as provided herein above, _V1 comprises CDRa1, CDRa2, and CDRa3 as defined above, _V2 comprises CDRH1, CDRH2, and CDRH3 as defined above, _V3 comprises CDRL1, CDRL2, and CDRL3 as defined above, and _V4 comprises CDRb1, CDRb2, and/or CDRb3 as defined above. For example, the antigen binding protein may comprise V ₁ , comprising: CDRa1 comprising or consisting of: SEQ ID NO: 2, CDRa2 comprising or consisting of: SEQ ID NO: 7, and CDRa3 comprising or consisting of: SEQ ID NO: 3, V ₂ , comprising: CDRH1 comprising or consisting of: SEQ ID NO: 48, CDRH2 comprising or consisting of: SEQ ID NO: 49, and CDRH3 comprising or consisting of: SEQ ID NO: 50, V ₃ , comprising: CDRL1 comprising or consisting of: SEQ ID NO: 51 , CDRL2 comprising or consisting of: SEQ ID NO: 52, and CDRL3 comprising or consisting of: SEQ ID NO: 53, V ₄ , comprising: CDRb1 comprising or consisting of: SEQ ID NO: 5, A CDRb2 comprising or consisting of: SEQ ID NO: 9, and/or a CDRb3 comprising or consisting of: SEQ ID NO: 6.

In a specific aspect, the antigen-binding protein comprises: - a polypeptide of the formula V ₁ -L ₁ -V ₂ , and - another polypeptide of the formula V ₃ -L ₃ -V ₄ wherein V1 is the first variable domain; V2 is the second variable domain; V3 is the third variable domain; V4 is the fourth variable domain; and L1 and L3 are linkers; and wherein V1 is Vα, V2 is VH, V3 is VL, and V4 is Vβ; V1 is VL, V2 is Vα, V3 is Vβ, and V4 is VH; V1 is Vβ, V2 is VL, V3 is VH, and V4 is Vα; V1 is VH, V2 is Vα, V3 is Vβ, and V4 is VL; V1 is Vα, V2 is VL, V3 is VH, and V4 is Vβ; V1 is VL, V2 is Vβ, V3 is Vα, and V4 is VH; V1 is Vβ, V2 is VH, V3 is VL, and V4 is Vα; or V1 is VH, V2 is Vβ, V3 is Vα, and V4 is VL.

In a more preferred embodiment, V1 is Vα, V2 is VH, V3 is VL, and V4 is Vβ.

In an even more preferred aspect, the antigen binding is a TCER as exemplified in the embodiments, and comprises a polypeptide of the formula _V1 - _L1 - _V2 - _FC1 , and a polypeptide of the formula _V3 - _L3 - _V4 - _FC2 wherein V1 is the first variable domain; V2 is the second variable domain; V3 is the third variable domain; V4 is the fourth variable domain; and L1 and L3 are linkers; and wherein Fc1 and Fc2 are Fc domains, and wherein _Fc1 and _Fc2 are the same or preferably different, and wherein V1 and V4 form the antigen binding site A and V2 and V3 form the antigen binding site B, or V1 and V4 form the antigen binding site B and V2 and V3 form the antigen binding site A. In this preferred embodiment, V1 is Vα, V2 is VH, V3 is VL, and V4 is Vβ; V1 is VL, V2 is Vα, V3 is Vβ, and V4 is VH; V1 is Vβ, V2 is VL, V3 is VH, and V4 is Vα; V1 is VH, V2 is Vα, V3 is Vβ, and V4 is VL; V1 is Vα, V2 is VL, V3 is VH, and V4 is Vβ; V1 is VL, V2 is Vβ, V3 is Vα, and V4 is VH; V1 is Vβ, V2 is VH, V3 is VL, and V4 is Vα; or V1 is VH, V2 is Vβ, V3 is Vα, and V4 is VL.

In a more preferred embodiment, V1 is Vα, V2 is VH, V3 is VL, and V4 is Vβ.

In other exemplary embodiments, the antigen-binding protein comprises: - a polypeptide of formula _V1 - _L1 - _V2 , and - another polypeptide of formula _V3 - _L3 - _V4 , and wherein _V1 comprises CDRa1, CDRa2, and CDRa3 as defined above, _V2 comprises CDRH1, CDRH2, and CDRH3 as defined above, _V3 comprises CDRL1, CDRL2, and CDRL3 as defined above, and _V4 comprises CDRb1, CDRb2, and/or CDRb3 as defined above. For example, the antigen binding protein may comprise V ₁ , comprising: CDRa1 comprising or consisting of: SEQ ID NO: 2, CDRa2 comprising or consisting of: SEQ ID NO: 7, and CDRa3 comprising or consisting of: SEQ ID NO: 3, V ₂ , comprising: CDRH1 comprising or consisting of: SEQ ID NO: 48, CDRH2 comprising or consisting of: SEQ ID NO: 49, and CDRH3 comprising or consisting of: SEQ ID NO: 50, V ₃ , comprising: CDRL1 comprising or consisting of: SEQ ID NO: 51 , CDRL2 comprising or consisting of: SEQ ID NO: 52, and CDRL3 comprising or consisting of: SEQ ID NO: 53, V ₄ , comprising: CDRb1 comprising or consisting of: SEQ ID NO: 5, A CDRb2 comprising or consisting of: SEQ ID NO: 9, and/or a CDRb3 comprising or consisting of: SEQ ID NO: 6.

In a preferred aspect of the present invention, F _C1 and/or F _C2 comprise or consist of the amino acid sequence of SEQ ID NO:24 [Fc knob] and/or SEQ ID NO:25 [Fc hole]; and/or L ₁ and L ₃ comprise or consist of the amino acid sequence of SEQ ID NO:26 GGGSGGGG.

In these embodiments, optionally, it may further comprise the following: _L1 and _L3 comprise or consist of the amino acid sequence GGGSGGGG of SEQ ID NO:26, and _L2 , _L4 , _L5 , and _L6 are absent, and optionally, wherein F _C1 comprises or consists of the amino acid sequence SEQ ID NO:25 [Fc hole], and F _C2 comprises or consists of the amino acid sequence SEQ ID NO:24 [Fc knob], or F _C1 comprises or consists of the amino acid sequence SEQ ID NO:24 [Fc knob], and F _C2 comprises or consists of the amino acid sequence SEQ ID NO:25 [Fc hole].

Preferably, the antigen binding proteins of the present invention have variable domain orientations of Vα, Vβ, VL, and VH according to orientation D in FIG8 .

In a preferred embodiment, the antigen binding protein of the present invention comprises the following CDR amino acid sequence: CDRa1 comprising or consisting of: SEQ ID NO:2, CDRa2 comprising or consisting of: SEQ ID NO:7, CDRa3 comprising or consisting of: SEQ ID NO:3, CDRb1 comprising or consisting of: SEQ ID NO:5, CDRb2 comprising or consisting of: SEQ ID NO:9, CDRb3 comprising or consisting of: SEQ ID NO:6, CDRH1 comprising or consisting of: SEQ ID NO:48, CDRH2 comprising or consisting of: SEQ ID NO:49, CDRH3 comprising or consisting of: SEQ ID NO:50, CDRL1 comprising or consisting of: SEQ ID NO:51, CDRL2 comprising or consisting of: SEQ ID NO:52, and CDRL3 comprising or consisting of: SEQ ID NO:53; Optionally, wherein the CDRa1, CDRa3, CDRb1, CDRb3, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3 sequences may comprise one, two or three amino acid mutations.

In another preferred embodiment, the antigen binding protein of the present invention comprises the following CDR amino acid sequence: CDRa1 comprising or consisting of: SEQ ID NO:2, CDRa2 comprising or consisting of: SEQ ID NO:8, CDRa3 comprising or consisting of: SEQ ID NO:4, CDRb1 comprising or consisting of: SEQ ID NO:5, CDRb2 comprising or consisting of: SEQ ID NO:10, CDRb3 comprising or consisting of: SEQ ID NO:6, CDRH1 comprising or consisting of: SEQ ID NO:48, CDRH2 comprising or consisting of: SEQ ID NO:49, CDRH3 comprising or consisting of: SEQ ID NO:50, CDRL1 comprising or consisting of: SEQ ID NO:51, CDRL2 comprising or consisting of: SEQ ID NO:52, and CDRL3 comprising or consisting of: SEQ ID NO:53; Optionally, wherein the CDRa1, CDRa3, CDRb1, CDRb3, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3 sequences may comprise one, two or three amino acid mutations.

In a further preferred embodiment, the antigen binding protein of the present invention comprises the following CDR amino acid sequence: CDRa1 comprising or consisting of: SEQ ID NO:2, CDRa2 comprising or consisting of: SEQ ID NO:7, CDRa3 comprising or consisting of: SEQ ID NO:3, CDRb1 comprising or consisting of: SEQ ID NO:5, CDRb2 comprising or consisting of: SEQ ID NO:10, CDRb3 comprising or consisting of: SEQ ID NO:6, CDRH1 comprising or consisting of: SEQ ID NO:48, CDRH2 comprising or consisting of: SEQ ID NO:49, CDRH3 comprising or consisting of: SEQ ID NO:50, CDRL1 comprising or consisting of: SEQ ID NO:51, CDRL2 comprising or consisting of: SEQ ID NO:52, and CDRL3 comprising or consisting of: SEQ ID NO:53; Optionally, wherein the CDRa1, CDRa3, CDRb1, CDRb3, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and/or CDRL3 sequences may comprise one, two or three amino acid mutations.

In the following, the formats of the sequences provided are explained by way of example. It is well known to those skilled in the art that the binding domains can also be included in other formats.

In a preferred embodiment, the antigen-binding protein of the present invention comprises: - a first polypeptide having an amino acid sequence (SEQ ID NO: 27) of formula _V1 - _L1 - _V2 - _L2 - _C1 - _L5 - _FC1 [V] : EDVEQSSFLSVREGDSAVINCTYTDASSTY FYWYKQEPGAGLQLLTY IYSNMDM KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLL FGDVTQLVVKP GGGSGGGG EVQLVQSGAEVKKPGASVKVSCKASGYKFT SYVMHWVRQAPGQGLEWMG YINPRNDVTKYAEKFQG RVTLTSDTSTSTAYMELSSLRSEDTAVYYCAR GSYYDYEGFVY WGQGTLVTVSS EPKSS DKTHTCPPCPAP PVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASI EKTISKAKGQPREPQVTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP wherein L2 _{, C1} , _L5 are absent and it comprises _V1 (mature TCR-derived α variable domain) having sequence SEQ ID NO: 11 (comprising the CDRa1, CDRa2, CDRa3 having SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 3, respectively; in bold), _L1 having sequence SEQ ID NO: 26 (TCER linker; underlined), _V2 having the sequence of SEQ ID NO:46 (BMhanced T cell recruiter _VH , which contains the CDRH1, the CDRH2, the CDRH3 having SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively; bold), and _Fc1 having the sequence of SEQ ID NO:25 (fc hole; italic and underlined); and a second polypeptide having the amino acid sequence (SEQ ID NO:34) of the formula _V3 - _L3 - _V4 - _L4 - _C2 - _L6 - _FC2 [VI] : QIQMTQSPSSLSASVGDRVTITC SATSSVSYMH WYQQKPGKAPKRWIY DTSKLAS GVPSRFSGSGSGTDYTLTISSLQPEDAATYYC QQWSSNPLT FGGGTKVEIK GGGSGGGG AVISQKPSRVIVQRGTSVTIQCQVD RPVTM MYWYRQQPGQSLTLIAT AYDEGSA TYESGFDIDKFPISRPNLTFSTLTTVSNVEPEDSSIYLC SVLGAYGYT FGSGTRLTVV EPKS S DKTHTCPPCPAP PVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA S IEKTISKAKGQPREPQVYTLPP C RDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP where L ₄ , C ₂ , and L ₆ do not exist and they contain the sequence SEQ ID NO:47 _V3 (BMhanced T cell recruiter VL, which contains the CDRL1, the CDRL2, the CDRL3 having SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; bold), _L3 having the sequence of SEQ ID NO:26 (TCER linker; underlined), _V4 having the sequence of SEQ ID NO:15 (TCR derived β variable domain) (containing the CDRb1, the CDRb2, the CDRb3 having SEQ ID NO:5, SEQ ID NO:9, and SEQ ID NO:6, respectively; bold), and _Fc2 having the sequence of SEQ ID NO:24 (fc knob; italic and underlined), wherein _Fc1 and Fc2 are The _C2 sequence includes: an amino acid substitution of cysteine to serine (S indicated in bold) because the light chain does not need to be linked via this cysteine; amino acid substitutions in the IgG1 hinge region for analogy to the IgG2 hinge to eliminate _Fc -γ receptor interaction (PVA in positions 233 to 235, and 331S, and G236 removal indicated in bold), an amino acid substitution of N297Q for removal of the N-glycosylation site within the _Fc portion to eliminate _Fc -γ receptor interaction (Q indicated in bold), and additionally, hole mutations in the case of _Fc1 (Y349C, T366S, L368A, and Y407V indicated in bold) and knob mutations in the case of _Fc2 (S354C and T366W indicated in bold). In addition, the C-terminal glycine-lysine (G446, K447) residues of the human IgG1 heavy chain can be deleted to reduce C-terminal heterogeneity.

In another preferred embodiment, the antigen-binding protein of the present invention comprises: - a first polypeptide having an amino acid sequence (SEQ ID NO: 28) of formula _V1 - _L1 - _V2 - _L2 - _C1 - _L5 - _FC1 [V] : EDVEQSSFLSVREGDSAVINCTYTDASSTY FYWYKQEPGAGLQLLTY IFSNMDM KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLM FGDVTQLVVKP GGGSGGGG EVQLVQSGAEVKKPGASVKVSCKASGYKFT SYVMHWVRQAPGQGLEWMG YINPRNDVTKYAEKFQG RVTLTSDTSTSTAYMELSSLRSEDTAVYYCAR GSYYDYEGFVY WGQGTLVTVSS EPKSS DKTHTCPPCPAP PVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASI EKTISKAKGQPREPQVTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP wherein L2 _{, C1} , _L5 are absent and it comprises _V1 (mature TCR-derived α variable domain) having sequence SEQ ID NO:12 (comprising the CDRa1, CDRa2, CDRa3 having SEQ ID NO:2, SEQ ID NO:8, and SEQ ID NO:4, respectively; in bold), _L1 having sequence SEQ ID NO:26 (TCER linker; underlined), _V2 having the sequence of SEQ ID NO:46 (BMhanced T cell recruiter _VH , which contains the CDRH1, the CDRH2, the CDRH3 having SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively; bold), and _Fc1 having the sequence of SEQ ID NO:25 (fc hole; italicized and underlined); and a second polypeptide having the amino acid sequence (SEQ ID NO:35) of formula _V3 - _L3 - _V4 - _L4 - _C2 - _L6 - _FC2 [VI] : QIQMTQSPSSLSASVGDRVTITC SATSSVSYMH WYQQKPGKAPKRWIY DTSKLAS GVPSRFSGSGSGTDYTLTISSLQPEDAATYYC QQWSSNPLT FGGGTKVEIK GGGSGGGG AVISQKPSRYIVQRGTSVTIQCQVD RPVTM MYWYRQQPGQSLTLIAT AYDVGSA TYESGFVIDKFPISRPNLTSTLTVSSNVEPEDSSIYLC SVLGAYGYT FGSGTRLTVV EPKS S DKTHTCPPCPAP PVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA S IEKTISKAKGQPREPQVYTLPP C RDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP where L ₄ , C ₂ , and L ₆ do not exist and they contain the sequence SEQ ID NO:47 _V3 (BMhanced T cell recruiter VL, which contains the CDRL1, the CDRL2, the CDRL3 having SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; bold), _L3 having the sequence of SEQ ID NO:26 (TCER linker; underlined), _V4 having the sequence of SEQ ID NO:16 (TCR derived β variable domain) (containing the CDRb1, the CDRb2, the CDRb3 having SEQ ID NO:5, SEQ ID NO:10, and SEQ ID NO:6; bold), and Fc2 having the sequence of SEQ ID NO: ₂₄ (fc knob; italic and underlined), wherein _Fc1 and Fc2 are The _C2 sequence includes: an amino acid substitution of cysteine to serine (S indicated in bold) because the light chain does not need to be linked via this cysteine; amino acid substitutions in the IgG1 hinge region for analogy to the IgG2 hinge to eliminate _Fc -γ receptor interaction (PVA in positions 233 to 235, and 331S, and G236 removal indicated in bold), an amino acid substitution of N297Q for removal of the N-glycosylation site within the _Fc portion to eliminate _Fc -γ receptor interaction (Q indicated in bold), and additionally, hole mutations in the case of _Fc1 (Y349C, T366S, L368A, and Y407V indicated in bold) and knob mutations in the case of _Fc2 (S354C and T366W indicated in bold). In addition, the C-terminal glycine-lysine (G446, K447) residues of the human IgG1 heavy chain can be deleted to reduce C-terminal heterogeneity.

In another preferred embodiment, the antigen-binding protein of the present invention comprises: - a first polypeptide having an amino acid sequence (SEQ ID NO: 29) of formula _V1 - _L1 - _V2 - _L2 - _C1 - _L5 - _FC1 [V] : EDVEQSSFLSVREGDSAVINCTYTDASSTY FYWYKQEPGAGLQLLTY IYSNMDM KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLL FGDGTQLVVKP GGGSGGGG EVQLVQSGAEVKKPGASVKVSCKASGYKFT SYVMHWVRQAPGQGLEWMG YINPRNDVTKYAEKFQG RVTLTSDTSTSTAYMELSSLRSEDTAVYYCAR GSYYDYEGFVY WGQGTLVTVSS EPKSS DKTHTCPPCPAP PVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASI EKTISKAKGQPREPQVTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP wherein L2 _{, C1} , _L5 are absent and it comprises _V1 (mature TCR-derived α variable domain) having sequence SEQ ID NO:13 (comprising the CDRa1, CDRa2, CDRa3 having SEQ ID NO:2, SEQ ID NO:7, and SEQ ID NO:3, respectively; in bold), _L1 having sequence SEQ ID NO:26 (TCER linker; underlined), _V2 (BMhanced T cell recruiter _VH having the sequence of SEQ ID NO:46, which contains the CDRH1, the CDRH2, the CDRH3 having the sequences of SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively; in bold), and _Fc1 (fc hole; italicized and underlined) having the sequence of SEQ ID NO:25; and a second polypeptide having the amino acid sequence (SEQ ID NO:36) of the formula _V3 - _L3 - _V4 - _L4 - _C2 - _L6 - _FC2 [VI] : QIQMTQSPSSLSASVGDRVTITC SATSSVSYMH WYQQKPGKAPKRWIY DTSKLAS GVPSRFSGSGSGTDYTLTISSLQPEDAATYYC QQWSSNPLT FGGGTKVEIK GGGSGGGG AVISQKPSRYIVQRGTSVTIQCQVD RPVTM MYWYRQQPGQSLTLIAT AYDVGSA TYESGFDIDKFPISRPNLTFSTLTVSNMEPEDSSIYLC SVLGAYGYT FGSGTRLTVV EPKS S DKTHTCPPCPAP PVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY Q STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA S IEKTISKAKGQPREPQVYTLPP C RDELTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP where L ₄ , C ₂ , and L ₆ do not exist and they contain the sequence SEQ ID NO:47 _V3 (BMhanced T cell recruiter VL, which contains the CDRL1, the CDRL2, the CDRL3 having SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, respectively; bold), _L3 having the sequence of SEQ ID NO:26 (TCER linker; underlined), _V4 having the sequence of SEQ ID NO:17 (TCR derived β variable domain) (containing the CDRb1, the CDRb2, the CDRb3 having SEQ ID NO:5, SEQ ID NO:10, and SEQ ID NO:6, respectively; bold), and _Fc2 having the sequence of SEQ ID NO:24 (fc knob; italic and underlined), wherein _Fc1 and Fc2 are The _C2 sequence includes: an amino acid substitution of cysteine to serine (S indicated in bold) because the light chain does not need to be linked via this cysteine; amino acid substitutions in the IgG1 hinge region for analogy to the IgG2 hinge to eliminate _Fc -γ receptor interaction (PVA in positions 233 to 235, and 331S, and G236 removal indicated in bold), an amino acid substitution of N297Q for removal of the N-glycosylation site within the _Fc portion to eliminate _Fc -γ receptor interaction (Q indicated in bold), and additionally, hole mutations in the case of _Fc1 (Y349C, T366S, L368A, and Y407V indicated in bold) and knob mutations in the case of _Fc2 (S354C and T366W indicated in bold). In addition, the C-terminal glycine-lysine (G446, K447) residues of the human IgG1 heavy chain can be deleted to reduce C-terminal heterogeneity.

In some embodiments, the amino acid sequences of binding domains A and B are chimeric, humanized or human.

In some embodiments, the (one or more) N-terminal or C-terminal amino acid of the polypeptide chain of the antigen binding protein comprises (one or more) modifications, such as post-translational modifications. Preferably, the (one or more) N-terminal amino acid of the polypeptide chain of the antigen binding protein comprises a modification.

In one embodiment, the (one or more) N-terminal or C-terminal amino acid of the polypeptide chain of the antigen binding protein comprises (one or more) modifications, such as naturally occurring modifications in aqueous solution, preferably a pyroglutamine modification.

In one embodiment, the (one or more) N-terminal amino acids of the polypeptide chain of the antigen binding protein, for example in VL, comprise a modification, preferably a pyroglutamine modification.

In some embodiments, the soluble antigen binding protein according to the present invention is capable of activating CD4+T cells, preferably CD4+CD8-T cells and/or CD8+T cells, preferably CD8+CD4-T cells.

In some embodiments, the antigen binding protein binds to the MAGEB2 antigenic peptide having SEQ ID NO: 1 with a _KD of less than 50 nM, preferably less than 20 nM, more preferably less than 5 nM as measured by biointerferometry.

In one embodiment, the antigen binding protein of the present invention specifically binds to a MAGEB2 antigen peptide comprising the amino acid sequence of SEQ ID NO: 1 and an HLA molecule, preferably HLA-A*02, or consisting thereof, with a KD of ≤100 μM, ≤50 μM, ≤30 μM, ≤25 μM, ≤1 μM, ≤500 nM, ≤100 nM, ≤50 nM, ≤10 nM, preferably ≤5 nM, such as 50 pM to 100 μM, 50 pM to 10 μM, 50 pM to 1 μM, more specifically 50 pM to 500 nM, 50 pM to 100 nM, 50 pM to 50 nM, 50 pM to 10 nM, 50 pM to 5 nM. " _{KD "} and "affinity" are as defined herein above in the "Definitions" section.

Therefore, in one example, the antigen binding protein of the present invention is, for example, expressed as a soluble TCER® described herein above, and its binding affinity to the HLA-A*02/MAGEB2 antigen peptide complex is analyzed. General conditions for bio-laminar interferometry are provided above. In general, measurements are performed using the settings recommended by the manufacturer, for example, on an Octet RED384 system. In brief, binding kinetics are generally measured at 30° C. and, for example, at a shaking speed of 1000 rpm using PBS, 0.05% Tween-20, 0.1% BSA as a buffer. Antigen binding proteins can be loaded onto biosensors such as FAB2G or AHC, or analyzed in solution.

In some embodiments, the antigen binding protein is capable of killing tumor cells expressing MAGEB2 in an in vitro cytotoxicity assay, wherein the number of MAGEB2 copies per cell of the tumor cells expressing MAGEB2 is less than 200, preferably less than 100, more preferably less than 50. In a preferred embodiment, the number of MAGEB2 copies per cell of the tumor cells expressing MAGEB2 is determined by AbsQuant® (e.g., as disclosed in PCT/EP2015/079873).

In some embodiments, the antigen binding protein is capable of killing tumor cells expressing MAGEB2 in an in vitro cytotoxicity assay, wherein the number of MAGEB2 copies per cell of the tumor cells expressing MAGEB2 is less than 200, and the EC50 is less than 200 pM, preferably less than 100 pM, and more preferably less than 30 pM. Nucleic Acids, Vectors, Host Cells

The polypeptides of the antigen binding proteins of the present invention can be encoded by nucleic acids and expressed in vivo, in vitro, or in vitro. Thus, in a second aspect, the present invention provides one or more nucleic acids that encode the antigen binding proteins of the first aspect of the present invention. For example, one, two, three, or four or more nucleic acids can encode any antigen binding protein as defined herein.

The nucleic acid molecules disclosed herein can be obtained using standard molecular biology techniques, including but not limited to RNA amplification and reverse transcription methods. Once a DNA fragment encoding, for example, a variable chain is obtained, such DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert a variable region gene into a full-length chain gene. In such manipulations, the DNA fragment encoding the variant is operably linked to another DNA molecule, or to a fragment encoding another protein such as a constant region or a flexible linker. As used in this context, the term "operably linked" is intended to mean that two DNA fragments are linked in a functional manner, for example, so that the amino acid sequence encoded by the two DNA fragments remains in frame, or so that the protein is expressed under the control of a desired promoter. The isolated DNA encoding the variable region, such as the variable α region and/or the variable β region, can be converted to a full-length chain gene by operably linking the DNA encoding the variable to another DNA molecule encoding the constant region. The sequences of human constant region genes, such as TCR or antibodies, are known in the art, and DNA fragments covering these regions can be obtained by standard PCR amplification.

Generally, the nucleic acid can be a DNA or RNA molecule contained in a suitable vector.

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

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

A variety of expression vectors can be used to express polynucleotides encoding antigen binding proteins or functional fragments thereof. Both viral-based expression vectors and non-viral expression vectors can be used to produce the antigen binding proteins or functional fragments thereof described herein in mammalian host cells. Non-viral vectors and systems include a variety of plastids, plastids, cosmids, episomes, artificial chromosomes, phages or viral vectors.

The vectors may contain regulatory elements such as promoters, enhancers, terminators and the like to cause or direct the expression of the polypeptide after administration to a subject. Examples of promoters and enhancers for expression vectors in animal cells include the early promoter and enhancer of SV40 (Mizukami T. et al. 1987), the LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), the promoter of antibody heavy chain (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983), and the like.

For example, non-viral vectors for expressing the polynucleotides and polypeptides described herein in mammalian (e.g., human or non-human) cells include all suitable vectors known in the art for expressing proteins. Other examples of plasmids include replicating plasmids or integrating plasmids comprising a replication origin, such as pUC, pcDNA, pBR, and the like.

The first polypeptide and the second polypeptide described herein may be present in the same vector or in separate vectors.

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

The nucleic acids of the present invention can be used to produce the recombinant antigen-binding proteins of the present invention in a suitable expression system.

Common expression systems include Escherichia coli host cells and plasmid vectors, insect host cells and bacilli virus 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 Escherichia coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, HEK cells, etc.), and primary or established mammalian cell cultures (e.g., derived from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2/0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells in which the dihydrofolate reductase gene is defective (Urlaub G et al; 1980), rat YB2/3HL.P2.G11.16Ag.20 cells (ATCC CRL1662), and the like. In some embodiments, YB2/0 cells may be preferred because the ADCC activity of chimeric antibodies or humanized antibodies is enhanced when they are expressed in these cells.

According to the above, in one embodiment, the present invention relates to a host cell comprising an antigen binding protein of the present invention as defined herein above, or a nucleic acid encoding an antigen binding protein of the present invention, or a vector encoding an antigen binding protein of the present invention, wherein the host cell may be 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.

Specifically, in order to express some of the antigen-binding proteins of the present invention, specifically antigen-binding proteins comprising two unlinked polypeptides, the expression vector may be a type in which the gene encoding the antibody heavy chain and the gene encoding the antibody light chain are present on separate vectors or a type in which both genes are present on the same vector (tandem type). With regard to the ease of construction of the antigen-binding protein expression vector, the ease of introduction into animal cells, and the balance between the expression levels of the antibody H and L chains in animal cells, a tandem humanized antibody expression vector is preferred (Shitara K et al. J Immunol Methods. 1994 Jan. 3; 167(1-2):271-8). Examples of tandem humanized antibody expression vectors include pKANTEX93 (WO97/10354), pEE18, and the like.

In one embodiment, the recombinant host cells can be used to produce at least one antigen binding protein of the present invention.

For the purpose of producing recombinant antigen binding proteins such as TCRs, polypeptides, or proteins, the host cell is preferably a mammalian cell. In a preferred embodiment, the host cell is a cell for recombinant expression such as a Chinese hamster ovary (CHO) cell.

The present invention further provides a composition comprising or consisting of: an antigen-binding protein of the first aspect of the present invention, one or more nucleic acids of the second aspect of the present invention, or one or more vectors of the third aspect of the present invention. Method for producing antigen-binding protein

The recombinant host cells of the fourth aspect can be used to produce at least one antigen binding protein of the first aspect of the present invention. Therefore, in a fifth aspect, the present invention provides a method for producing any of the antigen binding proteins of the first aspect of the present invention, comprising culturing the host cells of the fourth aspect of the present invention under suitable conditions, and optionally, isolating the antigen binding protein produced by the host cells.

Therefore, the antigen-binding protein of the present invention can be produced by the method of the fifth aspect.

In one embodiment, the method comprises the steps of: (a) providing a host cell according to the fourth aspect of the invention, (b) providing a vector of the third aspect comprising a coding sequence encoding an antigen-binding protein of the first aspect, (c) introducing the vector into the host cell, and (d) expressing the vector by the host cell.

In one embodiment, the method further comprises isolating and purifying the antigen binding protein from the host cell.

The antigen-binding proteins of the present invention can be produced by any technique known in the art, such as but not limited to any chemical, biological, genetic or enzymatic techniques alone or in combination.

The antigen-binding proteins of the present invention are suitably separated and purified from the culture medium by antibody purification procedures such as protein A-agarose, hydroxapatite chromatography, gel electrophoresis, chromatography, or affinity chromatography.

In one embodiment, recovering the expressed antigen-binding protein or polypeptide herein refers to performing protein A chromatography, Kappa select chromatography and/or size exclusion chromatography, preferably protein A chromatography and/or size exclusion chromatography, more preferably protein A chromatography and size exclusion chromatography.

Knowing the amino acid sequence of the desired sequence, one of ordinary skill in the art can produce the antigen-binding proteins of the present invention by standard techniques for producing polypeptides. For example, the antigen-binding proteins of the present invention can be synthesized using well-known solid phase methods, specifically using commercially available peptide synthesis equipment (e.g., manufactured by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, the antibodies and antigen-binding proteins of the present invention can be produced by recombinant DNA and gene transfection techniques well known in the art (see Morrison SL. et al . (1984) and patent documents US5,202,238 and US5,204,244). For example, after incorporating a DNA sequence encoding a desired (poly)peptide into expression vectors and introducing these vectors into appropriate eukaryotic or prokaryotic hosts expressing the desired polypeptides, fragments can be obtained as DNA expression products and can then be isolated from these hosts using well-known techniques.

Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, e.g., 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.

In one example, the vector used to express the recombinant antigen binding protein of the present invention is designed as a monocistronic, for example, controlled by a HCMV-derived promoter element pUC19 derivative. Plasmid DNA can be amplified, for example, in E. coli according to standard culture methods and subsequently purified using a commercially available kit (Macherey & Nagel). Purified plasmid DNA can be used to temporarily transfect, for example, CHO-S cells according to the manufacturer's instructions (ExpiCHO™ system; Thermo Fisher Scientific). Transfected CHO cells can be cultured, for example, at 32° C. to 37° C., for example, for 6-14 days, and receive one or two feedings of ExpiCHO™ feed solution.

The conditioned cell supernatant can be clarified by filtering (0.22 μm), for example, using, for example, Sartoclear ^Dynamics® laboratory filter aid (Sartorius). The bispecific antigen-binding protein can be purified using, for example, an Äkta Pure 25 L FPLC system (GE Lifesciences) configured to perform affinity chromatography and size exclusion chromatography in concert. Affinity chromatography can be performed, for example, on a protein A or L column (GE Lifesciences) following standard affinity chromatography procedures. For example, size exclusion chromatography can be performed directly after elution (pH 2.8) using, for example, a Superdex 200 pg 16/600 column (GE Lifesciences) autoaffinity column to obtain highly pure monomeric protein following standard procedures. Protein concentration can be determined, for example, on a NanoDrop system (Thermo Scientific) using an extinction coefficient calculated based on the predicted protein sequence. If necessary, the concentration can be adjusted by using a Vivaspin device (Sartorius). Finally, the purified molecule can be stored, for example, in phosphate-buffered saline at a concentration of about 1 mg/mL and a temperature of 2-8°C.

The quality of the purified bispecific antigen binding protein can be determined by HPLC-SEC on a MabPac SEC-1 column (5 µm, 7.8 x 300 mm) run, for example, in 50 mM sodium phosphate pH 6.8 containing 300 mM NaCl in a Vanquish UHPLC system. Pharmaceutical compositions

In a sixth aspect, the present invention provides a pharmaceutical composition comprising the antigen binding protein of the first aspect of the present invention, one or more nucleic acids of the second aspect of the present invention, or one or more vectors of the third aspect of the present invention, and optionally, a pharmaceutically acceptable carrier.

The antigen binding proteins of the present invention have been shown to be able to achieve cytotoxicity against cells presenting the MAGEB2 antigenic peptide. Since this peptide is specifically presented by tumor cells, the antigen binding proteins of the present invention can be used to destroy tumor cells in patients. An immune response in a patient can be induced by directly administering the described antigen binding proteins to the patient, ideally in combination with an agent that enhances immunogenicity (i.e., an adjuvant). Because the peptide GVYDGEEHSV (SEQ ID NO: 1) is not presented or is over-presented in significant copy numbers on normal tissues, it is expected that the immune response resulting from such therapeutic vaccination will be highly specific to tumor cells, thereby preventing the risk of unwanted autoimmune responses against normal tissue cells in the patient.

The present invention also relates to a pharmaceutical composition of the present invention for use as a medicament (see the section "Treatment methods and uses").

The pharmaceutical compositions may comprise a therapeutically effective amount of the antigen-binding protein of the present invention, or a mixture of the antigen-binding protein of the present invention further comprising a therapeutic agent and a pharmaceutically or physiologically acceptable formulation agent selected to be suitable for the mode of administration.

In some embodiments, the antigen binding proteins of the invention will be supplied as part of a sterile pharmaceutical composition, which will generally include at least one pharmaceutically acceptable carrier.

Examples of pharmaceutically acceptable carriers or diluents that can be used in the present invention include stabilizers such as SPGA, carbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose, polydextrose); proteins such as albumin or casein; protein-containing reagents such as bovine serum or skim milk; and buffers (e.g., phosphate buffer).

The form, route of administration, dosage and regimen of the pharmaceutical composition will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc. This pharmaceutical composition may be in any suitable form (depending on the desired method of administering it to the patient). It may be provided in unit dosage form, will usually be provided in a sealed container, and may be provided as part of a kit. Such kits will usually (although not necessarily) include instructions for use. It may include a plurality of such unit dosage forms.

Preferably, the pharmaceutical composition is administered by injection, such as intravenous injection. When the pharmaceutical composition comprises host cells expressing the antigen binding protein of the present invention, preferably TCR, the pharmaceutically acceptable carrier for the injectable cells may include any isotonic carrier such as physiological saline (about 0.90% w/v NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0 g NaCl/liter water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMALYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

Empirical considerations such as biological half-life will generally aid in dosage determination. The frequency of administration can be determined and adjusted during the course of therapy and is based on reducing cancer cell numbers, maintaining cancer cell reduction, reducing cancer cell proliferation, or killing cancer cells. Alternatively, sustained continuous release formulations of antigen binding proteins may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

In one embodiment, the dosage of the antigen binding protein can be determined empirically in an individual to whom one or more administrations have been given. Increasing doses of the antigen binding protein are given to the individual. In order to assess the efficacy of the antigen binding protein, indicators of the state of the cancer cells can be tracked. Such indicators include direct measurements of cancer cell proliferation and cell death by FACS, other imaging techniques; improved health assessed by such measurements, or improved quality of life, or prolonged survival as measured by recognized tests. It will be apparent to one of ordinary skill in the art that dosage will vary depending on the individual, the stage of the disease, and the past and concurrent treatments used.

The dosage for administration can be adapted according to various parameters and in particular according to the mode of administration used, the pathology involved or, alternatively, the desired duration of treatment.

The pharmaceutical compositions of the present invention (including antigen binding proteins, vectors, and/or nucleic acids) can be provided in a substantially pure form, for example, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.

In some embodiments, the antigen binding protein is dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. In some embodiments, when the antigen binding protein is dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium, certain amino acid residues may undergo post-translational modification. In some embodiments, the post-translational modification is an N-terminal modification. In some embodiments, the post-translational N-terminal modification is a cyclization of one or more glutamine (Q) residues of the antigen binding protein. In some embodiments, the post-translational N-terminal modification is a cyclization of one or more glutamine (E) residues of the antigen binding protein. In some embodiments, the post-translational N-terminal modification produces a pyroglutamine residue. Treatment Methods and Uses

In a seventh aspect, the invention provides an antigen binding protein of the first aspect of the invention, one or more nucleic acids of the second aspect of the invention, one or more vectors of the third aspect of the invention, or a pharmaceutical composition of the sixth aspect of the invention for treating a proliferative disease, wherein preferably, the proliferative disease is cancer.

The antigen binding proteins of the invention are particularly useful in immunotherapy for the treatment of proliferative diseases.

In a preferred embodiment, the antigen binding protein of the first aspect of the present invention, one or more nucleic acids of the second aspect of the present invention, one or more vectors of the third aspect of the present invention, or the pharmaceutical composition of the sixth aspect of the present invention is used to treat cancer expressing MAGEB2.

In a similar aspect, the present invention also relates to a method for treating a proliferative disease, wherein preferably, the proliferative disease is cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding protein of the first aspect of the present invention, one or more nucleic acids of the second aspect of the present invention, one or more vectors of the third aspect of the present invention, or a pharmaceutical composition of the sixth aspect of the present invention. In one embodiment, the cancer cells present GVYDGEEHSV (SEQ ID NO: 1) complexed with MHC molecules on the cell surface.

In a preferred embodiment of the method of treatment, the cancer is a cancer expressing MAGEB2.

In some embodiments of the medical uses and treatment methods, the cancer expressing MAGEB2 is a solid cancer.

Prior art such as WO2016/102272 shows beneficial treatment of specific cancers/tumors.

Therefore and in further embodiments, the medical use and the method of treatment may be selected from the group consisting of liver cancer, lung cancer, chronic lymphocytic leukemia (CLL), colorectal cancer (CRC), gallbladder cancer (GBC), glioblastoma (GBM), gastric cancer (GC), hepatocellular carcinoma (HCC), head and neck cancer, head and neck squamous cell carcinoma (HNSCC), melanoma (MEL), non Hodgkin lymphoma (NHL), non-small cell lung cancer adenocarcinoma (NSCLCadeno), non-small cell lung cancer (NSCLC), squamous cell non-small cell lung cancer (NSCLCsquam), ovarian cancer (OC), esophageal cancer (OSCAR), renal cell carcinoma (RCC), small cell lung cancer (SCLC), bladder cancer (UBC), and uterine and endometrial cancer (UEC).

In certain embodiments, the medical use and treatment method can be selected from the group consisting of hepatocellular carcinoma (HCC), melanoma, non-small cell lung cancer (NSCLC), and non-Hodgkin's lymphoma (NHL), and UEC (uterine, endometrial cancer).

In a preferred embodiment, the antigen binding protein is or comprises TCR or (one or more) functional fragments thereof.

In some embodiments, the antigen binding protein is conjugated to a therapeutic agent.

Preferably, the therapeutically active agent is selected from the group consisting of radionuclides, chemotherapeutic agents, and toxins.

In one embodiment, the treatment further comprises administering at least one chemotherapeutic agent to a subject in need of treatment.

In one embodiment, the treatment further comprises administering radiation therapy to the subject in need of treatment.

In a related aspect, the present invention relates to a method of inducing an immune response in a patient suffering from a proliferative disease, a cancer that specifically presents MAGEB2 in complex with an MHC protein, comprising administering to the patient an antigen binding protein of the present disclosure, wherein the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), melanoma, non-small cell lung cancer (NSCLC), non-Hodgkin lymphoma (NHL), and UEC (uterine, endometrial cancer). In one embodiment, the immune response mentioned in the method is a cytotoxic T cell response.

In yet another aspect, the invention relates to the use of an antigen binding protein, nucleic acid(s), vector(s), or pharmaceutical composition according to the invention for the manufacture of a medicament for treating a proliferative disease in a subject.

In yet another aspect, the present invention relates to the use of an antigen binding protein, (one or more) nucleic acid, (one or more) vector, or pharmaceutical composition according to the present invention for treating a disease in a subject.

In this context, guidance for cancer therapy is provided by Cancer, Principles and Practice of Oncology, 4th Edition, DeVita et al, Eds. J. B. Lippincott Co., Philadelphia, Pa. (1993). As is known in the art, the appropriate treatment is selected based on the specific type of cancer and other factors such as the patient's general condition. In treating cancer patients, the antigen binding proteins of the present invention can be used alone or can be added to a treatment regimen using other anti-tumor agents.

Thus, in some embodiments, the antigen binding proteins may be administered simultaneously with, prior to, or after a variety of drugs and treatments widely used in cancer treatment, such as chemotherapeutics, non-chemotherapeutic agents, antineoplastic agents, and/or radiation.

In one embodiment, the efficacy of treatment with an antigen binding protein of the invention is determined in vivo, for example, in a mouse cancer model and by measuring, for example, changes in tumor volume between treated and control groups.

The antigen binding protein of the present invention, the nucleic acid of the present invention, the vector of the present invention, or the pharmaceutical composition of the present invention can be administered by any feasible method.

In one aspect, a TCR-induced immune response or T cell response may refer to the proliferation and activation of effector functions induced in vitro, in vitro, or in vivo by a peptide such as GVYDGEEHSV (SEQ ID NO: 1). For MHC class I-restricted cytotoxic T cells, for example, the effector function may be the lysis of target cells pulsed with peptides, pulsed with peptide promotors, or presented by natural peptides, the secretion of cytokines, preferably interferon-γ, TNF-α, or IL-2, the secretion of effector molecules such as granzymes or perforins induced by peptides, or degranulation.

For the purposes of the present invention, the amount or dosage of the antigen binding protein of the first aspect of the present invention, the (one or more) nucleic acid of the second aspect of the present invention, the (one or more) vector of the third aspect of the present invention, or the pharmaceutical composition of the fifth aspect of the present invention administered may be sufficient to achieve, for example, a therapeutic or preventive response in a subject or animal within a reasonable time period. For example, the dosage of the antigen binding protein, (one or more) nucleic acid, (one or more) vector, or pharmaceutical composition according to the present invention should be sufficient to bind to a cancer antigen, or detect, treat, or prevent cancer within a time period of about 2 hours or longer, such as 12 to 24 hours or longer from the time of administration. In certain embodiments, the time period may be even longer. The dosage will be determined based on the efficacy of the antigen binding protein, nucleic acid(s), vector(s), or pharmaceutical composition of the present invention, the condition of the animal (e.g., human), and the weight of the animal (e.g., human) to be treated. Kit

In an eighth aspect, the present invention also provides a kit comprising the antigen-binding protein of the first aspect of the present invention, one or more nucleic acids of the second aspect of the present invention, or one or more vectors of the third aspect of the present invention.

In one embodiment, the kit comprises a) at least one antigen binding protein of the present invention as defined herein above in the "Antigen Binding Protein" section, (one or more) nucleic acids encoding the antigen binding protein, or (one or more) vectors comprising the nucleic acid(s), b) optionally, packaging material, and c) optionally, a label or drug instruction sheet contained in the packaging material indicating that the antigen binding protein is effective for a method of treating cancer or is effective for treating cancer.

In a preferred embodiment, the kit comprises (one or more) nucleic acids encoding the antigen binding protein of the present invention, or (one or more) vectors comprising the nucleic acid(s).

The kits of the present disclosure may further include any other reagents that can be used for transfection/transduction to introduce nucleic acid(s) or expression vector(s) into cells.

The components of the kits may be present in separate containers, or multiple components may be present in a single container. Suitable containers include a single tube, one or more wells of a plate (e.g., a 96-well plate, a 384-well plate, etc.), or the like.

In a related embodiment, at least one antigen-binding protein of the invention is contained in a single-chamber and/or multi-chamber loading syringe (eg, a liquid syringe and a lyophilizer syringe).

In one embodiment, the invention encompasses a kit for producing a single dosage administration unit.

Therefore, in one embodiment, the at least one antigen-binding protein of the present invention as mentioned in a) of the kit of the present invention is a dried antigen-binding protein of the present invention contained in a first container. Subsequently, the kit further contains a second container with an aqueous formulation.

Thus, in one embodiment, the kit comprises a) a first container comprising at least one dried antigen binding protein of the invention as defined herein above in the "Antigen Binding Protein" section, b) a second container comprising an aqueous formulation; c) optionally, a packaging material, and d) optionally, a label or package insert contained in the packaging material indicating that the antigen binding protein is effective for a method of treating cancer or is effective for treating cancer.

The aqueous formulation is typically an aqueous solution comprising a pharmaceutically acceptable carrier as defined herein above.

The definitions of the words or elements in the drawings described herein are intended to include not only the combinations of elements described literally, but also all equivalent structures, materials or actions for performing substantially the same function in substantially the same manner to obtain substantially the same results. In this sense, it is therefore expected that any of the described elements and their various embodiments may be replaced with two or more elements, or a single element may be replaced with two or more elements within the scope of the patent application.

Variations of the claimed subject matter that are now known or later conceived as apparent to one of ordinary skill in the art are expressly contemplated as equivalents and various embodiments thereof within the intended scope. Therefore, obvious substitutions now or later known to one of ordinary skill in the art are defined as being within the scope of the defined elements. Therefore, the present disclosure is intended to be understood to include what is specifically illustrated and described herein, what is conceptually equivalent, what is obviously substituted, and what incorporates the basic concept. Items

The present invention can also be characterized by the following items. 1. An antigen binding protein that specifically binds to a MAGEB2 antigen peptide complexed with a major histocompatibility complex (MHC) protein, wherein the MAGEB2 antigen peptide comprises or consists of the amino acid sequence GVYDGEEHSV (SEQ ID NO: 1), wherein the antigen binding protein comprises complementary determining regions (CDRs) CDRa1, CDRa2, and CDRa3 and CDRb1, CDRb2, and CDRb3, wherein CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 form an antigen binding domain A; preferably, wherein the variable domain V _A comprises or consists of the amino acid sequence according to SEQ ID NO: 152 or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 152, preferably, wherein the variable domain V _B comprises or consists of: an amino acid sequence according to SEQ ID NO:154 or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:154, preferably, wherein CDRa1 comprises the amino acid sequence of SEQ ID NO:2, CDRa3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3 and 4, CDRb1 comprises the amino acid sequence of SEQ ID NO:5, and CDRb3 comprises the amino acid sequence of SEQ ID NO:6; wherein the CDRa1, CDRa3, CDRb1 and/or CDRb3 sequences may comprise one, two or three amino acid mutations. 2. The antigen binding protein of item 1, wherein CDRa2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 8, and CDRb2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 10; wherein the CDRa2 and/or CDRb2 sequences may comprise one, two or three amino acid mutations. 3. An antigen binding protein that specifically binds to a MAGEB2 antigen peptide complexed with a major histocompatibility complex (MHC) protein, wherein the MAGEB2 antigen peptide comprises or consists of the amino acid sequence GVYDGEEHSV (SEQ ID NO: 1), wherein the antigen binding protein comprises: a first polypeptide comprising a variable domain _VA comprising complementary determining regions (CDRs) CDRa1, CDRa2, and CDRa3 and a second polypeptide comprising a variable domain _VB comprising CDRb1, CDRb2, and CDRb3, wherein CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 form an antigen binding domain A; preferably, wherein the variable domain _VA comprises or consists of the amino acid sequence according to SEQ ID NO: 152 or the amino acid sequence according to SEQ ID NO: 2 NO:152 has an amino acid sequence with at least 85%, 90%, 95%, 98%, or 99% identity, particularly preferably, wherein the _VA comprises the amino acid sequence according to SEQ ID NO:153 or an amino acid sequence with at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:153, preferably, wherein the variable domain _VB comprises or consists of the amino acid sequence according to SEQ ID NO:154 or an amino acid sequence with at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:154, particularly preferably, wherein the _VB comprises the amino acid sequence according to SEQ ID NO:155 or an amino acid sequence with at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:155, preferably, wherein (1) CDRa1 comprises SEQ ID NO:2, CDRa3 comprises SEQ ID NO:3, CDRb1 comprises SEQ ID NO:5, and CDRb3 comprises SEQ ID NO:6; or (2) CDRa1 comprises SEQ ID NO:2, CDRa3 comprises SEQ ID NO:4, CDRb1 comprises SEQ ID NO:5, and CDRb3 comprises SEQ ID NO:6; wherein the CDRa1, CDRa3, CDRb1 and/or CDRb3 sequences may comprise one, two or three amino acid mutations. 4. The antigen binding protein of item 3, wherein (1) CDRa1 comprises SEQ ID NO:2, CDRa2 comprises SEQ ID NO:7, CDRa3 comprises SEQ ID NO:3, CDRb1 comprises SEQ ID NO:5, CDRb2 comprises SEQ ID NO:9, and CDRb3 comprises SEQ ID NO:6; (2) CDRa1 comprises SEQ ID NO:2, CDRa2 comprises SEQ ID NO:8, CDRa3 comprises SEQ ID NO:4, CDRb1 comprises SEQ ID NO:5, CDRb2 comprises SEQ ID NO:10, and CDRb3 comprises SEQ ID NO:6; (3) CDRa1 comprises SEQ ID NO:2, CDRa2 comprises SEQ ID NO:7, CDRa3 comprises SEQ ID NO:3, CDRb1 comprises SEQ ID NO:5, CDRb2 comprises SEQ ID NO:10, and CDRb3 comprises SEQ ID NO:6; or (4) CDRa1 comprises SEQ ID NO:2, CDRa2 comprises SEQ ID NO:8, CDRa3 comprises SEQ ID NO:4, CDRb1 comprises SEQ ID NO:5, CDRb2 comprises SEQ ID NO:9, and CDRb3 comprises SEQ ID NO:6; wherein the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may comprise one, two or three amino acid mutations. 5. The antigen binding protein of any one of items 1 to 4, wherein the V _A comprises an amino acid sequence according to SEQ ID NO: 153, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 153, or wherein the V _A comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, and 14, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 11 and comprising the CDRa1, the CDRa2, and the CDRa3 having SEQ ID NOs: 2, 7, and 3, respectively, an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 12 and comprising the CDRa1, the CDRa2, and the CDRa3 having SEQ ID NOs: 2, 8, and 4, respectively, NO:13 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequence of said CDRa1, said CDRa2, and said CDRa3 of SEQ ID NOs: 2, 7, and 3, respectively, or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:14 and comprises the amino acid sequence of said CDRa1, said CDRa2, and said CDRa3 of SEQ ID NOs: 2, 8, and 4, respectively; and wherein said _VB comprises the amino acid sequence according to SEQ ID NO:155, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:155, or wherein said VB comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, and 23, or has at least 85%, 90%, 95%, 98%, or 99 _% identity to SEQ ID NO:14 NO:15 has at least 85%, 90%, 95%, 98%, or 99% identity and includes the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively, and SEQ ID NO:16 has at least 85%, 90%, 95%, 98%, or 99% identity and includes the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively, and SEQ ID NO:17 has at least 85%, 90%, 95%, 98%, or 99% identity and includes the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively, and SEQ ID NO:18 has at least 85%, 90%, 95%, 98%, or 99% identity and includes the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively. NO:5, 10, and 6, the amino acid sequence of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:19 and comprising the amino acid sequence of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively, having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:20 and comprising the amino acid sequence of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively, having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:21 and comprising the amino acid sequence of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively, NO:22 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively, or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:23 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively, wherein optionally, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may comprise one, two or three amino acid mutations, preferably amino acid substitutions. 6. The antigen-binding protein of any one of items 1 to 5, wherein the _VA comprises the amino acid sequence of SEQ ID NO: 11 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 11 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs: 2, 7, and 3, respectively, and wherein the _VB comprises the amino acid sequence of SEQ ID NO: 15 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 15 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs: 5, 9, and 6, respectively; wherein the _VA comprises the amino acid sequence of SEQ ID NO: 12 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 12 and comprises the amino acid sequences of SEQ ID NO: 13, respectively. NO:2, 8, and 4, and wherein the _{VB comprises the amino acid sequence of SEQ ID NO:16 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:16 and comprises the amino acid sequences of the CDRb1} , the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; wherein the _VA comprises the amino acid sequence of SEQ ID NO:13 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:13 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and wherein the _VB comprises the amino acid sequence of SEQ ID NO:17 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:17 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6, respectively; NO:5, 10, and 6 of the said CDRb1, the said CDRb2, and the said CDRb3; wherein the _VA comprises the amino acid sequence of SEQ ID NO:14 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:14 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively, and wherein the VB comprises the amino acid sequence of SEQ ID NO:18 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:18 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6 of the said CDRb1, the said CDRb2, and the said CDRb3; wherein the _{VA comprises the amino acid sequence of SEQ ID NO:13 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:14 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively, and wherein the VB} comprises the amino acid sequence of SEQ ID NO:18 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:18 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6 of the said CDRb1, the said CDRb2, and the said CDRb3; wherein the _VA comprises the amino acid sequence of SEQ ID NO:13 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:13 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 7, and 3, respectively, and wherein the _VB comprises the amino acid sequence of SEQ ID NO:19 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:19 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; wherein the _VA comprises the amino acid sequence of SEQ ID NO:11 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:11 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 7, and 3, respectively, and wherein the _VB comprises the amino acid sequence of SEQ ID NO:19 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: NO:19 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; wherein the _VA comprises the amino acid sequence of SEQ ID NO:14 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:14 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 8, and 4, respectively, and wherein the _VB comprises the amino acid sequence of SEQ ID NO:20 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:20 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; wherein the VA comprises the amino acid sequence of SEQ ID NO:14 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:14 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID _NOs :2, 8, and 4, respectively. NO:13 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:13 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 7, and 3, respectively, and wherein the _{VB comprises the amino acid sequence of SEQ ID NO:21 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:21 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3} of SEQ ID NOs:5, 10, and 6, respectively; wherein the _{VA comprises the amino acid sequence of SEQ ID NO:11 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:11 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 7, and 3, respectively, and wherein the VB} comprises the amino acid sequence of SEQ ID NO:21 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:21 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the _CDRb3 of SEQ ID NOs:5, 10, and 6, respectively. NO:21 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:21 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; wherein the _VA comprises the amino acid sequence of SEQ ID NO:12 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:12 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:2, 8, and 4, respectively, and wherein the _VB comprises the amino acid sequence of SEQ ID NO:18 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:18 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; wherein the V wherein said _{V A} comprises the amino acid sequence of SEQ ID NO: 13 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 13 and comprises the amino acid sequences of said CDRa1, said CDRa2, and said CDRa3 of SEQ ID NOs: 2, 7, and 3, respectively, and wherein said V _B comprises the amino acid sequence of SEQ ID NO: 22 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 22 and comprises the amino acid sequences of said CDRb1, said CDRb2, and said CDRb3 of SEQ ID NOs: 5, 9, and 6, respectively; or wherein said V _A comprises the amino acid sequence of SEQ ID NO: 14 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 14 and comprises the amino acid sequences of said CDRa1, said CDRa2, and said CDRa3 of SEQ ID NOs: 2, 8, and 4, respectively, and wherein said V 7. The antigen binding protein of any one of items 1 to 6, wherein _{the VA} comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, and 14; and wherein the _VB comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: ₁₅ , 16, 17, 18, 19, 20, 21, 22, and 23. 8. The antigen-binding protein of any one of items 1 to 7, wherein the V _A comprises the amino acid sequence of SEQ ID NO: 11 and the Vb comprises the amino acid sequence of SEQ ID NO: 15; wherein the V _A comprises the amino acid sequence of SEQ ID NO: 12 and the Vb comprises the amino acid sequence of SEQ ID NO: 16; wherein the V _A comprises the amino acid sequence of SEQ ID NO: 13 and the Vb comprises the amino acid sequence of SEQ ID NO: 17; wherein the V _A comprises the amino acid sequence of SEQ ID NO: 14 and the Vb comprises the amino acid sequence of SEQ ID NO: 18; wherein the V _A comprises the amino acid sequence of SEQ ID NO: 13 and the Vb comprises the amino acid sequence of SEQ ID NO: 19; wherein the V _A comprises the amino acid sequence of SEQ ID NO: 11 and the Vb comprises the amino acid sequence of SEQ ID NO: 19; wherein the V _A comprises the amino acid sequence of SEQ ID NO: 14 and the Vb comprises the amino acid sequence of SEQ ID NO: NO:20; wherein the _VA comprises the amino acid sequence of SEQ ID NO:13 and the Vb comprises the amino acid sequence of SEQ ID NO:21; wherein the _VA comprises the amino acid sequence of SEQ ID NO:11 and the Vb comprises the amino acid sequence of SEQ ID NO:21; wherein the _VA comprises the amino acid sequence of SEQ ID NO:12 and the Vb comprises the amino acid sequence of SEQ ID NO:18; wherein the _VA comprises the amino acid sequence of SEQ ID NO:13 and the Vb comprises the amino acid sequence of SEQ ID NO:22; or wherein the _VA comprises the amino acid sequence of SEQ ID NO:14 and the Vb comprises the amino acid sequence of SEQ ID NO:23; optionally, wherein the framework region(s) comprises at least one amino acid substitution selected from the group consisting of: - an amino acid substitution at position 19 in FR1-a, wherein the amino acid substitution is S19A, S19I, S19L, or S19V, preferably S19A or S19V, more preferably S19A, - an amino acid substitution at position 39 in FR2-a, wherein the amino acid substitution is L39F, L39I, or L39V, most preferably L39F, - an amino acid substitution at position 13 in FR1-b, wherein the amino acid substitution is C13V, C13T, C13S, or C13A, preferably C13V, - Amino acid substitutions at positions 94 and/or 95 in FR3-b, wherein the amino acid substitution at position 94 is M94V and/or the amino acid substitution at position 95 is S95E or S95D, preferably S95E, respectively; and wherein the positions of the substitutions are given according to the IMGT nomenclature. 9. An antigen-binding protein as described in any one of items 1 to 8, further comprising: a constitutive domain, wherein the constitutive domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 and 25 or an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NOs: 24 and 25; and/or a linker, wherein the linker comprises an amino acid sequence of SEQ ID NO: 26 or an amino acid sequence that is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 26. 10. The antigen binding protein of any one of items 1 to 9, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:27 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 7, and 3, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:28 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:28 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 8, and 4, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:29 and comprises the amino acid sequences of SEQ ID NOs: NO:2, 7, and 3 of the CDRa1, the CDRa2, and the CDRa3; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:30 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:31 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:31 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:32 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:32 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively. NO:2, 8, and 4 of the said CDRa1, the said CDRa2, and the said CDRa3; or wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:33 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:33 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:34 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:34 and comprises the amino acid sequences of SEQ ID NO:5, 9, and 6, respectively; the second polypeptide comprises the amino acid sequence of SEQ ID NO:35 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:35 and comprises the amino acid sequences of SEQ ID NO:5, 9, and 6, respectively. NO:5, 10, and 6 of the CDRb1, the CDRb2, and the CDRb3 amino acid sequence; the second polypeptide comprises the amino acid sequence of SEQ ID NO:36 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:36 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6, respectively; the second polypeptide comprises the amino acid sequence of SEQ ID NO:37 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:37 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6, respectively; the second polypeptide comprises the amino acid sequence of SEQ ID NO:38 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:39 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6, respectively; NO:38 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; the second polypeptide comprises the amino acid sequence of SEQ ID NO:39 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:39 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; the second polypeptide comprises the amino acid sequence of SEQ ID NO:40 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:40 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; the second polypeptide comprises the amino acid sequence of SEQ ID NO:40 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:40 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; NO:41 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:41 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; the second polypeptide comprises the amino acid sequence of SEQ ID NO:42 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:42 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; the second polypeptide comprises the amino acid sequence of SEQ ID NO:43 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:43 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; The second polypeptide comprises the amino acid sequence of SEQ ID NO:44, or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:44 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; or the second polypeptide comprises the amino acid sequence of SEQ ID NO:45, or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:45 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; wherein optionally, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may comprise one, two or three amino acid mutations, preferably amino acid substitutions. 11. The antigen-binding protein of any one of items 1 to 10, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 27 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 27 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs: 2, 7, and 3, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 34 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 34 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs: 5, 9, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 28 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 28 and comprises the amino acid sequences of SEQ ID NOs: NO:2, 8, and 4 of the CDRa1, the CDRa2, and the CDRa3; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:35 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:35 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:29 and comprises the amino acid sequences of SEQ ID NO:2, 7, and 3, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:36 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:36 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6, respectively. NO:5, 10, and 6 of the said CDRb1, the said CDRb2, and the said CDRb3 amino acid sequence; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:30 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:37 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:37 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:29 and comprises the amino acid sequences of SEQ ID NO:30, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:37 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:37 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6, respectively; NO:29 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs: 2, 7, and 3, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:38 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:38 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs: 5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:27 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs: 2, 7, and 3, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:38 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: NO:38 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs: 5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:30 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs: 2, 8, and 4, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:39 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:39 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs: 5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:29 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 7, and 3, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:40 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:40 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:27 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 7, and 3, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:40 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:40 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively. NO:40 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:40 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:28 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:28 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:2, 8, and 4, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:37 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:37 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:29 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 7, and 3, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:41 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:41 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:30 and comprises the amino acid sequences of SEQ ID NO:41, NO:2, 8, and 4 of the said CDRa1, the said CDRa2, and the said CDRa3; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:42 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:42 and comprises the amino acid sequences of the said CDRb1, the said CDRb2, and the said CDRb3 of SEQ ID NO:5, 9, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:31 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:31 and comprises the amino acid sequences of the said CDRa1, the said CDRa2, and the said CDRa3 of SEQ ID NO:2, 8, and 4, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:43 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:43 and comprises the amino acid sequences of SEQ ID NO:5, 9, and 6, respectively. NO:5, 9, and 6 of the said CDRb1, the said CDRb2, and the said CDRb3; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:32 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:32 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively; and the second polypeptide comprises the amino acid sequence of SEQ ID NO:44 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:44 and comprises the amino acid sequences of SEQ ID NO:5, 9, and 6 of the said CDRb1, the said CDRb2, and the said CDRb3; or wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:33 or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:33 and comprises the amino acid sequences of SEQ ID NO:2, 8, and 4, respectively; The second polypeptide comprises the amino acid sequence of SEQ ID NO:45 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:45 and comprises the amino acid sequences of the CDRb1, CDRb2, and CDRb3 of SEQ ID NO:5, 9, and 6, respectively; wherein optionally, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may comprise one, two or three amino acid mutations, preferably amino acid substitutions. 12. The antigen-binding protein of any one of items 1 to 11, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:34; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:28 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:35; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:36; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:37; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:38; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:38; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:37 NO:39; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:40; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:40; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:28 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:37; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:41; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:42; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:31 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:43; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:32 and the second polypeptide comprises the amino acid sequence of SEQ ID NO: NO:44; or wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:33 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:45. 13. The antigen binding protein of any one of items 1 to 12, wherein the antigen binding domain A is derived from TCR, or is TCR or its (one or more) fragments. 14. The antigen binding protein of item 13, wherein the TCR is selected from the group consisting of: α/β TCR, γ/δ TCR, a functional fragment of TCR, and a fusion protein or chimeric protein comprising (one or more) functional fragments of TCR. 15. The antigen binding protein of item 13 or item 14, wherein VA and _VB are TCR _α and TCR β domains, respectively. 16. The antigen binding protein of any one of items 1 to 15, wherein the MHC protein is an HLA protein, preferably HLA-A, more preferably HLA-A*02. 17. The antigen binding protein of any one of items 1 to 16, wherein the antigen binding protein does not significantly bind to at least 1, at least 2, at least 3, at least 4, at least 5, or all similar peptides selected from the group consisting of SEQ ID NOs: 59 to 67, 106, and 133 to 151. 18. The antigen binding protein of any one of items 1 to 17, wherein the antigen binding protein is a soluble protein. 19. The antigen binding protein of any one of items 1 to 18, wherein the antigen binding protein is a multispecific antigen binding protein, particularly a bispecific antigen binding protein. 20. The antigen binding protein of any one of items 1 to 19, wherein the antigen binding protein comprises an additional antigen binding domain B. 21. The antigen binding protein of any one of items 1 to 19, wherein the antigen binding protein further comprises: a variable domain VH comprising CDRH1, CDRH2, and CDRH3 and a VL comprising CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 form an antigen binding domain B. 22. The antigen binding protein of item 20 or item 21, wherein the antigen binding domain B specifically binds to immune cells, preferably T cells or natural killer (NK) cells. 23. The antigen-binding protein of item 22, wherein the antigen-binding domain B specifically binds to an antigen selected from the group consisting of: CD2; CD3, specifically CD3γ, CD3δ and/or CD3ε; CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD4 4. CD45RA, CD49, CD55, CD56, CD61, CD64, CD68, CD69, CD89, CD90, CD94, CD95, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Ly-6.2C, Mel14, Nkp46, NKG2D, GITR, _FcεRI , TCRα/β, TCRγ/δ, HLA-DR, and 4-1BB, or a combination thereof. 24. The antigen binding protein of item 22 or item 23, wherein the antigen binding domain B specifically binds to the α/β T cell receptor (TCR)/CD3 complex. 25. The antigen binding protein of any one of items 21 to 24, wherein the CDRH3 comprises the amino acid sequence of SEQ ID NO:50 [GSYYDYEGFVY] or SEQ ID NO:58 [GSYYDYDGFVY], and optionally, wherein the CDRH3 comprises up to three amino acid mutations, preferably amino acid substitutions. 26. The antigen binding protein of any one of items 21 to 25, wherein the VH comprises: (a) a CDRH1 comprising the amino acid sequence of _X1YVMH [SEQ ID NO:111], wherein _X1 is S, R, K, or H; (b) a CDRH2 comprising _the amino _acid sequence _{of YINPX1X2DVTKYX3X4KFX5G} [SEQ ID NO: ₁₁₂ ], wherein _X1 is Y, R, K, or H, preferably Y or R; _X2 is N, R, or K, preferably N or R; _X3 is A or N; _X4 is E or Q, preferably E; and _X5 is Q or K, and (c) a CDRH2 comprising GSYYDYEGFVY [SEQ ID NO:50] or GSYYDYDGFVY [SEQ ID NO:51]. NO:58], and wherein the VL comprises: (a) a CDRL1 comprising an amino acid sequence of SATSSVXYMH [SEQ ID NO:113], wherein X is S, R, or K, (b) a CDRL2 comprising an amino acid sequence of DTSKLAX [SEQ ID NO:114], wherein X is S, R, or K, and (c) a CDRL3 comprising an amino acid sequence of QQWSSNPLT [SEQ ID NO:53]; and optionally, wherein the CDRH1, the CDRH2, and the CDRH3 and each of the CDRL1, the CDRL2, and the CDRL3 comprise up to three amino acid substitutions. 27. The antigen binding protein of any one of items 21 to 26, wherein the VH comprises: (a) CDRH1 comprising the amino acid sequence of SYVMH [SEQ ID NO:48], (b) CDRH2 comprising the amino acid sequence of YINPRNDVTKYAEKFQG [SEQ ID NO:49] or YINPYNDVTKYAEKFQG [SEQ ID NO:57], and (c) CDRH3 comprising the amino acid sequence of GSYYDYEGFVY [SEQ ID NO:50] or GSYYDYDGFVY [SEQ ID NO:58], and wherein the VL comprises: (a) CDRL1 comprising the amino acid sequence of SATSSVSYMH [SEQ ID NO:51], (b) CDRL2 comprising the amino acid sequence of DTSKLAS [SEQ ID NO:52], and (c) CDRH3 comprising the amino acid sequence of QQWSSNPLT [SEQ ID NO:53]. NO:53]; and optionally, wherein said CDRH1, said CDRH2, and said CDRH3 and each of said CDRL1, said CDRL2, and said CDRL3 comprises at most three amino acid substitutions. 28. The antigen binding protein of any one of items 21 to 27, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 46, 54, 55, and 56 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 46, 54, 55, or 56 and preferably comprises the amino acid sequence of CDRH1, CDRH2, and CDRH3 according to any one of the preceding items; and wherein the VL comprises the amino acid sequence of SEQ ID NO: 47 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 47 and preferably comprises the amino acid sequence of CDRL1, CDRL2, and CDRL3 according to any one of the preceding items; and optionally, wherein each of said CDRH1, said CDRH2, and said CDRH3 and said CDRL1, said CDRL2, and said CDRL3 comprises at most three amino acid substitutions. 29. The antigen binding protein of any one of items 1 to 28, wherein said antigen binding site B comprises: (i) _VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO:46, 54, 55, and 56 or an antigen binding fragment thereof, and (ii) _VL comprising an amino acid sequence of SEQ ID NO:47 or an antigen binding fragment thereof. 30. The antigen binding protein of any one of items 1 to 29, wherein said first polypeptide and said second polypeptide are covalently or non-covalently linked together. 31. The antigen binding protein of any one of items 1 to 30, wherein the binding domain A and/or the binding domain B is in the form of an antibody, a single chain variable fragment (scFv), a disulfide-stabilized Fv (dsFv), Fab, Fab', F(ab')2, a nanobody, a DARPin, a knottin, a bifunctional antibody, or an oligomer thereof. 32. The antigen binding protein of any one of items 21 to 31, wherein the binding domain A and the binding domain B are in the form of scFv. 33. The antigen binding protein of any one of items 21 to 31, wherein the antigen binding protein comprises a single chain TCR (scTCR) and/or a single chain bispecific antibody. 34. The antigen binding protein of any one of items 21 to 33, wherein the antigen binding protein comprises two antigen binding sites A and B, wherein the first polypeptide chain comprises a structure represented by the following formula: V1-L1-V2 [I] wherein V1 is the first variable domain; V2 is the second variable domain; L1 is a linker domain; and wherein the second polypeptide chain comprises a structure represented by the following formula: V3-L3-V4 [II] wherein V3 is the third variable domain; V4 is the fourth variable domain; L3 is a linker; optionally, wherein: V1 and V2 form the antigen binding site A and V3 and V4 form the antigen binding site B, or V1 and V2 form the antigen binding site B and V3 and V4 form the antigen binding site A, or V1 and V3 form the antigen binding site A and V2 and V4 form the antigen binding site B, or V1 and V3 form the antigen binding site B and V2 and V4 form the antigen binding site A, or V1 and V4 form the antigen binding site A and V2 and V3 form the antigen binding site B, or V1 and V4 form the antigen binding site B and V2 and V3 form the antigen binding site A, and optionally, wherein the first polypeptide and the second polypeptide are covalently or non-covalently linked together. 35. An antigen binding protein according to any one of items 21 to 34, wherein the antigen binding protein comprises two antigen binding sites A and B, wherein the first polypeptide chain comprises a structure represented by the following formula: V1-L1-V2-L2-C1 [III] wherein V1 is the first variable domain; V2 is the second variable domain; L1 and L2 are linkers; L2 may be present or absent; C1 is a homeostatic domain or a dimerization portion thereof and is present or absent, preferably, wherein C1 is a CL light chain homeostatic domain or a dimerization portion thereof; and wherein the second polypeptide chain comprises a structure represented by the following formula: V3-L3-V4-L4-C2 [IV] wherein V3 is the third variable domain; V4 is the fourth variable domain; L3 and L4 are linkers; L4 may be present or absent; C2 is a constant domain or a dimerization portion thereof and is present or absent, preferably, wherein C2 is a CH heavy chain constant domain or a dimerization portion thereof; and wherein preferably V1 and V2 form the antigen binding site A and V3 and V4 form the antigen binding site B, or V1 and V2 form the antigen binding site B and V3 and V4 form the antigen binding site A, or V1 and V3 form the antigen binding site A and V2 and V4 form the antigen binding site B, or V1 and V3 form the antigen binding site B and V2 and V4 form the antigen binding site A, or V1 and V4 form the antigen binding site A and V2 and V3 form the antigen binding site B, or V1 and V4 form the antigen binding site B and V2 and V3 form the antigen binding site A, and optionally, wherein the first polypeptide and the second polypeptide are covalently or non-covalently linked together. 36. An antigen-binding protein as described in item 35, wherein the first polypeptide chain has a structure represented by formula [V]: V1-L1-V2-L2-C1-L5-FC1 [V] and the second polypeptide chain has a structure represented by formula [VI]: V3-L3-V4-L4-C2-L6-FC2 [VI] wherein V1 to V4, L1 to L4, C1, and C2 are as defined in item 35, and wherein L5 and L6 are linkers and are present or absent, and FC1 and FC2 are Fc domains, and wherein FC1 and FC2 are the same or different. 37. The antigen binding protein of item 36, wherein F _C1 and/or F _C2 comprise or consist of the amino acid sequence of SEQ ID NO:24 [Fc knob] and/or SEQ ID NO:25 [Fc hole], and/or wherein L ₁ and L ₃ comprise or consist of the amino acid sequence GGGSGGGG [SEQ ID NO:26]. 38. The antigen binding protein of item 36, wherein _V1 comprises CDRa1, CDRa2, and CDRa3 as defined in the preceding item, _V2 comprises CDRH1, CDRH2, and CDRH3 as defined in the preceding item, _V3 comprises CDRL1, CDRL2, and CDRL3 as defined in the preceding item, _V4 comprises CDRb1, CDRb2, and/or CDRb3 as defined in the preceding item; optionally, wherein _L1 and _L3 comprise or consist of the amino acid sequence GGGSGGGG of SEQ ID NO:26, and wherein _L2 , _L4 , _L5 , and _L6 are absent and optionally, wherein _Fc1 comprises or consists of the amino acid sequence SEQ ID NO:25 [Fc hole], and _Fc2 comprises or consists of the amino acid sequence SEQ ID NO:24 [Fc knob], or wherein F _C1 comprises or consists of the amino acid sequence SEQ ID NO:24 [Fc knob], and F _C2 comprises or consists of the amino acid sequence SEQ ID NO:25 [Fc hole]. 39. The antigen binding protein of any one of items 1 to 38, wherein the amino acid sequences of the binding domain A and the binding domain B are chimeric, humanized or human. 40. The antigen binding protein of any one of items 1 to 39, wherein the (one or more) N-terminal or C-terminal amino acids of the polypeptide chains of the antigen binding protein comprise (one or more) modifications. 41. The antigen binding protein of any one of items 1 to 39, wherein the (one or more) N-terminal amino acids of the polypeptide chains of the antigen binding protein comprise modifications. 42. The antigen binding protein of any one of items 18 to 41, wherein the antigen binding protein is capable of activating: - CD4+ T cells, preferably CD4+ CD8- T cells, and/or - CD8+ T cells, preferably CD8+ CD4- T cells. 43. The antigen binding protein of any one of items 18 to 42, wherein the antigen binding protein binds to the MAGEB2 antigen peptide having SEQ ID NO: 1 with a KD of less than 50 nM, preferably less than 20 nM, more preferably less than 5 nM as measured by biointerference. 44. The antigen binding protein of any one of items 18 to 43, wherein in an in vitro cytotoxicity assay, the antigen binding protein is capable of killing tumor cells expressing MAGEB2, wherein the number of copies of MAGEB2 per cell of the tumor cells expressing MAGEB2 is less than 200. 45. The antigen binding protein of item 44, wherein the number of copies of MAGEB2 per cell of the tumor cells expressing MAGEB2 is less than 100, preferably less than 50. 46. One or more nucleic acids encoding the antigen binding protein of any one of items 1 to 45. 47. One or more vectors comprising one or more nucleic acids of item 46. 48. A host cell comprising one or more nucleic acids of item 46 or (one or more) vectors of item 47. 49. A host cell of item 48, wherein the host cell is a cell for recombinant expression such as a Chinese Hamster Ovary (CHO) cell or a yeast cell. 50. A method for producing an antigen binding protein of any one of items 1 to 45, comprising culturing a host cell of item 48 or item 49 under suitable conditions, and optionally isolating the antigen binding protein produced by the host cells. 51. An antigen binding protein produced by the method of item 50. 52. A pharmaceutical composition comprising an antigen binding protein as in any one of items 1 to 45 or 51, a nucleic acid (or more) as in item 46, or a vector (or more) as in item 47, and optionally, a pharmaceutically acceptable carrier. 53. A pharmaceutical composition as in item 52, wherein the antigen binding protein is dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. 54. An antigen binding protein as in any one of items 1 to 45 or 51, a nucleic acid (or more) as in item 46, a vector (or more) as in item 47, or a pharmaceutical composition as in item 52 or 53, for use in the treatment of cancer. 55. An antigen binding protein according to any one of items 1 to 45 or 51, a nucleic acid (or more) according to item 46, a vector (or more) according to item 47, or a pharmaceutical composition according to item 52 or 53, for use in treating a cancer expressing MAGEB2. 56. A method for treating a cancer expressing MAGEB2, comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding protein according to any one of items 1 to 45 or 51, a nucleic acid (or more) according to item 46, a vector (or more) according to item 47, or a pharmaceutical composition according to item 52 or 53. 57. The antigen binding protein of item 54 or item 55 or the treatment method of item 56, wherein the cancer expressing MAGEB2 is selected from the group consisting of hepatocellular carcinoma (HCC), melanoma, non-small cell lung cancer (NSCLC), and non-Hodgkin's lymphoma (NHL), and uterine and endometrial carcinoma (UEC). 58. The antigen binding protein, (one or more) nucleic acid, (one or more) vector, or pharmaceutical composition of item 54, 55 or 57, or the treatment method of item 56 or item 57, wherein the antigen binding protein is conjugated with a therapeutic agent. 59. The antigen binding protein, nucleic acid(s), vector(s), or pharmaceutical composition of item 58, or the method of treatment of item 58, wherein the therapeutic agent is selected from the group consisting of radionuclides, chemotherapeutic agents, and toxins. 60. The antigen binding protein, nucleic acid(s), vector(s), or pharmaceutical composition of any one of items 54, 55, or 57 to 59, or the method of treatment of any one of items 56 to 59, wherein the treatment further comprises administering at least one chemotherapeutic agent to the subject in need of treatment. 61. An antigen binding protein, nucleic acid(s), vector(s), or pharmaceutical composition as in any one of items 54, 55, or 57 to 60, or a method of treatment as in any one of items 56 to 60, wherein the treatment further comprises administering radiation therapy to the subject in need of treatment. 62. A kit comprising an antigen binding protein as in any one of items 1 to 45 or 51, a nucleic acid(s), vector(s), and/or a vector(s) as in item 47. 63. A composition comprising or consisting of an antigen binding protein as in any one of items 1 to 45 or 51, a nucleic acid(s), vector(s), and/or a vector(s) as in item 47. 64. A method of treating cancer in a subject in need thereof, comprising administering to the subject an antigen binding protein of any one of items 1 to 45 or 51 and/or one or more nucleic acids of item 46, wherein cells of the cancer present GVYDGEEHSV (SEQ ID NO: 1) in complex with MHC molecules on the cell surface. 65. An antigen binding protein comprising: a first polypeptide comprising a variable domain _VA comprising complementary determining regions (CDRs) CDRa1, CDRa2, and CDRa3 and a second polypeptide comprising a variable domain _VB comprising CDRb1, CDRb2, and CDRb3, wherein CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 form an antigen binding domain A; wherein (1) CDRa1 comprises SEQ ID NO:2, CDRa3 comprises SEQ ID NO:3, CDRb1 comprises SEQ ID NO:5, and CDRb3 comprises SEQ ID NO:6; or (2) CDRa1 comprises SEQ ID NO:2, CDRa3 comprises SEQ ID NO:4, CDRb1 comprises SEQ ID NO:5, and CDRb3 comprises SEQ ID NO:6; wherein the CDRa1, CDRa3, CDRb1 and/or CDRb3 sequences may comprise one, two or three amino acid mutations. 66. The antigen binding protein of item 65, wherein the one, two or three amino acid mutations are conservative substitutions. 67. The antigen binding protein of item 65, comprising: a first polypeptide comprising a variable domain _VA comprising complementary determining regions (CDRs) CDRa1, CDRa2, and CDRa3 and a second polypeptide comprising a variable domain _VB comprising CDRb1, CDRb2, and CDRb3, wherein CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 form an antigen binding domain A; wherein (1) CDRa1 comprises SEQ ID NO:2, CDRa3 comprises SEQ ID NO:3, CDRb1 comprises SEQ ID NO:5, and CDRb3 comprises SEQ ID NO:6; or (2) CDRa1 comprises SEQ ID NO:2, CDRa3 comprises SEQ ID NO:4, CDRb1 comprises SEQ ID NO:5, and CDRb3 comprises SEQ ID NO:6. Examples Example 1: Generation of stable scTCR

For the present invention, TCR R76P1E1 (SEQ ID NOs: 76 and 77) was converted into a single-chain TCR construct (scTCR R76P1E1, SEQ ID NO: 89) using variable α (SEQ ID NO: 78) and β (SEQ ID NO: 79) domains and a glycine-serine linker sequence (SEQ ID NO: 85). For TCR maturation via yeast surface display, DNA of the corresponding sequence was synthesized along with a pCT302-based yeast display vector and transformed into brewer's yeast EBY100 (MATa AGA1::GAL1¬AGA1::URA3 ura3¬52 trp1 leu2¬delta200 his3¬delta200 pep4::HIS3 prbd1.6R can1 GAL) (ATCC ^® MYA¬ 4941™) (Boder and Wittrup, Methods Enzymol. 2000;328:430-44). The fusion protein (SEQ ID NO: 88) produced after homologous recombination in yeast contains a leader peptide at the N-terminus of the Aga2p protein (SEQ ID NO: 84) (Boder and Wittrup, Nat Biotechnol. 1997 Jun; 15(6): 553-7), the protein of interest, i.e., scTCR R76P1E1 (SEQ ID NO: 89) or its variants, and additional peptide markers (FLAG and Myc (SEQ ID NOs: 86 and 87)) used to determine the expression level of the fusion protein. Libraries of scTCR variants were generated by PCR using degenerate primers, and transformation of yeast cells was performed as described in WO2018/091396 and up to 10 ⁹ yeast strains/library were generated.

A selection method for yeast strains carrying mutant scTCR variants with improved binding to MAGEB2-001 in the presence of HLA-A*02 was performed using prior art methods. Functional binding via HLA-A*02/MAGEB2-001 tetramer staining was used to select the most promising candidates. As a starting point for scTCR transformation, the mutant scTCR R76P1E1_bC13V (SEQ ID NO:90) was selected. scTCR transformation by yeast surface display revealed six framework mutations and two single-site CDR mutations (SEQ ID NO:4 and 92), leading to the further stabilized scTCR R76P1E1C (SEQ ID NO:91) displaying expression and HLA-A*02/MAGEB2-001 tetramer binding (Figure 1). Example 2: Affinity maturation of stabilized scTCR improves binding to HLA-A*02/MAGEB2-001 and provides highly specific binding

To generate scTCR molecules with higher binding affinity for HLA-A*02/MAGEB2-001, all CDRs were matured individually using a cysteine-patch version of the previously identified stabilized scTCR R76P1E1C (SEQ ID NO: 91) (scTCR R76P1E1S (SEQ ID NO: 93)). CDR residues were randomized by using degenerate DNA oligo primers, and the resulting DNA library was transformed as described in Example 1.

To select affinity-enhanced and specific R76P1E1S scTCR variants, each round of selection used decreasing concentrations of HLA-A*02/MAGEB2-001 tetramers or monomers. After four rounds of selection, individual scTCR clones were isolated and sequenced, generating a large number of affinity-matured CDR sequences. As shown, HLA-A*02/MAGEB2-001 tetramers or monomers can show strong improvement in binding to scTCRs with mature CDRb1 and CDRb2 (SEQ ID NOs: 5 and 107-110, Table 2). The selectivity of HLA-A*02/MAGEB2-001 binding was maintained during maturation, as confirmed by low binding of scTCR to a mixture of HLA-A*02 tetramers containing peptides with high sequence similarity to the MAGEB2-001 peptide (SEQ ID NO: 1) (analog peptides or SimPeps). While all selected CDRb2 scTCR mature variants showed extensive staining with HLA-A*02/MAGEB2-001 monomers, the immature stabilized scTCR R76P1E1S used as a reference showed no staining (Figure 2B and Table 2). Selected CDRb1 variants showed improved binding to HLA-A*02/MAGEB2-001 tetramers compared to the reference. Furthermore, no binding was detected for the mature scTCR to a mixture of similar peptides applied in the high-affinity format of the HLA-A*02 tetramer at 25 nM concentration, confirming the ability of the scTCR mature variants to bind the MAGEB2-001 target peptide in a highly specific manner. Table 2: Binding data for yeast carrying scTCRs with mutant CDR2s [1] CDRb1 Yeast cells positively stained with CDRb2 Yeast cells positively stained with sequence SEQ ID NO HLA-A*02/MAGEB2-001 , tetramer HLA- A*02/SimPep , tetramer mixture sequence SEQ ID NO HLA-A*02/MAGEB2-001 , monomer HLA-A*02/SimPep , tetramer mixture, RPVTM 5 23.0% 0.0% AYDNNRT 107 4.4% 0.0% SQVTM* 82 3.6% 0.0% AFDDNTT 108 3.0% 0.0% YDDGSA 109 2.7% 0.0% AYSDGTA 110 4.1% 0.0% ANQGSEA* 83 0.0% 0.0%

To further improve the affinity of the scTCR strains, the mature CDRs identified in the CDR library described above were systematically combined in a DNA library and transformed into the brewing yeast EBY100 as described in Example 1. This library was selected using HLA-A*02/MAGEB2-001 monomers, and scTCRs from individual yeast strains were sequenced and analyzed for binding to the HLA-A*02 monomer containing the MAGEB2-001 target peptide (SEQ ID NO: 1) or a peptide derived from a group of 9 peptides sharing sequence similarity with MAGEB2-001 (SEQ ID NOs: 59-67) (analog peptides). All selected high affinity scTCR variants CL-48730, CL-48731, and CL-48732 (individually, SEQ ID NOs: 94-96) strongly bound to HLA-A*02/MAGEB2001 monomers. In the case of HLA-A*02 tetramers applied at a concentration of 10 nM, none of the scTCR variants showed binding above background levels to any of the analogous peptides (SEQ ID NOs: 59-67) ( FIG. 3B ). The data presented confirm the high binding specificity of the scTCR variants with combined CDR mutations.

These selected high affinity scTCRs from yeast surface displayed were further examined with respect to functional epitopes, termed binding motifs, on target peptides in the context of HLA-A*02 presentation. This was addressed by single substitutions at positions 1, 3, 4, 5, 6, 7, 8, and 9 of the MAGEB2-001 target peptide (SEQ ID NO: 68-75) in the context of HLA-A*02 and evaluation of the binding of scTCR-bearing yeast cells to the respective MAGEB2-001 peptide variants. Yeast cells carrying high affinity scTCRs were stained with HLA-A*02 monomers with MAGEB2-001 or respective substituted peptides at four concentrations (31.6 nM, 10 nM, 3.2 nM, 1 nM) and revealed a broad binding motif for all scTCR variants with strong recognition of positions 3 to 8 ( FIG. 4 ). Example 3: Biooptimization of scTCRs yields low nanomolar to sub-nanomolar binding EC ₅₀ values

To further optimize the scTCR, CL-48730 (SEQ ID NO: 94) was chosen as a starting point. Two possible isomerization sites were repaired, one in CDRa1 and the other in CDRb2, generating CL-21753 (SEQ ID NO: 97). Subsequently, two yeast surface display libraries were generated as described in Example 1, one based on error-prone PCR and the other with defined amino acid substitutions for further optimization of binding properties.

After selection for decreasing concentrations of HLA-A*02/MAGEB2-001 monomers and sequential counterselection for HLA-A*02/peptide-like tetramers, eight clones were further investigated and compared to the parental scTCR CL-21753. All of these clones showed strong improvement in HLA-A*02/MAGEB2-001 binding compared to their parental clone CL-21753 (Figure 5), yielding low nanomolar to sub-nanomolar _EC50 values (Table 3). Table 3: Binding data of yeast carrying scTCR molecules and respective variable chain sequences. [2] Molecule name SEQ ID NO FRa mutation (compared to parental TCR ) CDRa1 CDRa2 CDRa3 FRb mutation (compared to parental TCR ) CDRb1 CDRb2 CDRb3 EC 50 of HLA-A*02/MAGEB2-001 binding _[ nM] Peptide-like binding CL-21753 97 L9S, S19A, L39F DASSTY IFSNMDM AVKDNARLM C13V, T17N, L19V RPVTM AYDEGSA SVLGAYGYT nd 0 CL-25821 102 L9S, S19A, L39F, G121V DASSTY IFSNMDM AVKDNARLM D11V, C13V, T17N, L19V, F40Y, M94V RPVTM AYDEGSA SVLGAYGYT 0.7 0 CL-25720 101 L9S, S19A, L39F DASSTY IFSNMDM AVKDNARLM D11Y, C13V, T17N, L19V, L50P RPVTM YDV SVLGAYGYT 0.4 0 CL-25693 100 L9S, S19A, L39F DASSTY IFSNMDM AVKDNARLM C13V, T17N, L19V, F40Y, V72D, L103F RPVTM AYDEGSA SVLGAYGYT 1.0 0 CL-25688 98 L9S, S19A, L39F DDSSTY IFSNMDM AVKDNARLM C13V, T17N, L19V, L50P RPVTM AYDEGSA CVLGAYGYT 2.0 0 CL-25687 99 L9S, S19A, L39F DASSTY IYS NMDM AVKDNARLL C13V, T17N, L19V, V72D RPVTM AYDEGSA SVLGAYGYT 0.5 0 CL-24597 103 L9S, S19A, L39F DSSSTY IFSNMDM AVKDNARLM C13V, L19V, M94L, S95E RPVTM AYDEGSA SVLGAYGYT 51 0 CL-24505 104 L9S, S19A, L39F DASSTY IFSNMDM AVKDNARLM C13V, L19V, S95E RPVTM AYDEGSA SVLGAYGYT 35 0 CL-24478 105 L9S, S19A, L39F DASSTY IFSNMDM AVKDNARLM C13V, L19V, N93S, S95E RPVTM AYDEGSA SVLGAYGYT 50 0 Example 4: Bispecific variants can be produced in the form of soluble molecules and show good stability

TCR variants containing different mutations were included into an exemplary bispecific TCR format represented as a TCER molecule containing a humanized recruitment domain "BMhanced" (VH and VL: SEQ ID NO: 46 and 47, respectively) and a human IgG1 Fc domain (cf. WO2019/012138). DNA encoding the polypeptide chain was synthesized and cloned into a monocistronic, pUC19 derivative controlled by a HCMV-derived promoter element. Plasmid DNA was amplified in E. coli according to standard culture methods and subsequently purified using a commercially available kit (Macherey & Nagel). Chemically mediated transient transfection of CHO-S cells was performed using purified plasmid DNA. Transfected CHO cells were cultured at 32°C to 37°C for 10-12 days and received one to three feedings of Cellboost 7a and 7b (GE Healthcare™) solutions.

Conditioned cell supernatants were clarified by filtration (0.22 µm) using Sartoclear Dynamics® laboratory filter aid (Sartorius). Bispecific antigen binding proteins were purified using an Äkta Pure 25 L FPLC system (GE Lifesciences) equipped to coordinate affinity chromatography and size exclusion chromatography. Affinity chromatography was performed on MAbSelect SuRE or Protein L columns (GE Lifesciences) following standard affinity chromatography procedures. Size exclusion chromatography was performed directly after elution (pH 2.8) using a Superdex 200 pg 26/600 column (GE Lifesciences) autoaffinity column to obtain highly pure monomeric protein following standard procedures. Protein concentration was determined on a NanoDrop system (Thermo Scientific) using an extinction coefficient calculated based on the predicted protein sequence. If necessary, the concentration was adjusted by using a Vivaspin device (Sartorius). Finally, the purified molecules were stored in phosphate-buffered saline at a concentration of approximately 1 mg/mL at 2-8°C.

The quality of the purified bispecific antigen binding protein was determined by HPLC-SEC on a MabPac SEC-1 column (5 µm, 4 × 300 mm) or XBridge Premier SEC (dimensions 4.6 × 150 mm, particle size 2.5 µm) running in 50 mM sodium phosphate pH 6.8 containing 300 mM NaCl in a Vanquish uHPLC system. The detector wavelength was set to 214 nm.

Starting with the goal of comparing the orientation of different variable domain orders, TCER molecules (TPP-4017 to TPP-4020) with the same variable domain sequence were successfully generated and provided acceptable thermal stress stability (Table 4). TPP-4020 was selected as the base molecule for further engineering.

All additional variants showed yields exceeding 10 mg/L. In addition, all subsequent molecules showed a significant increase in productivity compared to the precursor molecule TPP-4020. Molecular thermal stress testing at 40°C revealed that all produced bispecific molecules showing less than 13% high-molecular-weight species (HMWS) formation had very good to acceptable stability after 14 days of thermal stress. Furthermore, all subsequent molecules exhibited significantly higher monomer content after thermal stress compared to the precursor molecule TPP-4020, indicating improved stability of the generated variants. Table 4 : Productivity and quality of purified TCER molecules TCER variants Yield [mg/L] Monomer after acid elution from capture column [%] Final unit [%] Monomers after 14 days at 40 ° C [%] TPP-4017 15 65 97.12 84.86 TPP-4018 12 65 91.88 47.87 TPP-4019 5 41 95.78 66.55 TPP-4020 11 59 97.59 87.36 TPP-4606 40 75 98.38 91.76 TPP-4784 35 78 98.55 94.16 TPP-4787 46 83 98.38 95.97 TPP-4788 31 82 98.49 92.48 TPP-4794 31 87 98.49 93.98 TPP-4798 28 64 96.42 91.85 TPP-4799 83 83 98.49 96.75 TPP-4805 43 85 98.52 94.58 TPP-4810 26 77 97.93 95.20 TPP-4812 36 85 98.32 94.75 TPP-4813 59 85 98.25 93.48 Example 5 : Bispecific TCR variants showing strong binding to HLA-A*02/MAGEB2-001

The binding affinity of mature R76P1E1S TCR variants expressed as soluble bispecific TCR molecules (e.g., TCER molecules) to HLA-A*02/MAGBE2-001 monomers was analyzed by bio-interferometry in various orientations. Measurements were performed on an Octet RED384 or HTX system using the manufacturer's recommended settings. Briefly, binding kinetics were measured at 30°C and 1000 rpm shaking using PBS, 0.05% Tween, 0.1% BSA as buffer. HLA-A*02/MAGEB2-001 was immobilized on the HIS1K sensor (concentration of 10 µg/ml for 120 s) before analyzing serial dilutions of the bispecific variants. Binding of bispecific TCR variants to HLA-A*02/MAGEB2-001 monomers was demonstrated for all orientations with KD values ranging from 1.61 nM to 13.7 nM (Table 5). The orientation of the TCER molecule did not affect the binding properties, as exemplified by TPP-4017 to TPP-4020 with comparable affinities of 9.4 nM to 13.7 nM. Table 5: Binding affinity analysis of different bispecific variants to HLA-A*02/MAGEB2-001 monomers by bio-interferometry. Bispecific variant KD [M] Bispecific variant KD [M] TPP-4017 1.29E-08 TPP-4794 3.20E-09 TPP-4018 1.05E-08 TPP-4798 1.61E-09 TPP-4019 9.40E-09 TPP-4799 2.72E-09 TPP-4020 1.37E-08 TPP-4805 2.78E-09 TPP-4606 3.54E-09 TPP-4810 3.08E-09 TPP-4784 2.95E-09 TPP-4812 2.96E-09 TPP-4787 2.43E-09 TPP-4813 2.47E-09 TPP-4788 2.54E-09 Example 6: Bispecific TCR molecules show high selectivity

In order to identify possible off-target peptides, exemplary bispecific TCR molecules (e.g., TCER molecules) incorporating TCR variants of R76P1E1S were analyzed for binding to MAGEB2-001 and possible off-target peptides. XPRESIDENT® MS technology was used to search for peptides in human proteomes that showed high sequence similarity to the target peptide and that have been confirmed as naturally occurring HLA ligands on normal tissues, as disclosed in, for example, PCT/EP03/050383. The most relevant possible off-target peptides of 28 were identified and screened for bispecific TCR binding using SP-01-0010 as a negative control. MAGEB2-001 and off-target peptide binding were measured by biointerferometry, and off-target binding signals of 20% or less compared to the MAGEB2 binding signal were considered irrelevant. Measurements were performed on the Octet RED384 or HTX system using the manufacturer's recommended settings. Briefly, binding kinetics were measured at 30°C and 1000 rpm shaking speed using PBS, 0.05% Tween, 0.1% BSA as buffer. HLA-A*02/MAGEB2-001 was immobilized on the HIS1K sensor (concentration of 10 µg/ml for 120 s) prior to analysis of TCR variants.

No relative response signal greater than 20% compared to MAGEB2-001 was observed for any of the 29 analyzed peptides that displayed high selectivity for the bispecific TCR molecule - see Table 6. Table 6: MAGEB2-001 and off-target peptide recognition ^as measured by biointerferometry. TPP-4787 TPP-4794 TPP-4799 TPP-4810 TPP-4812 TPP-4813 SP-02-1621 0 1 0 1 1 1 SP-02-1622 1 2 1 3 2 1 SP-02-1623 1 0 1 2 1 1 SP-02-1624 0 0 0 0 0 0 SP-02-1625 0 0 0 0 0 0 SP-02-1626 0 0 0 0 0 0 SP-02-1627 0 0 0 0 0 0 SP-02-1628 0 0 0 0 0 0 SP-02-1629 0 0 0 0 0 0 SP-01-0010 0 0 0 0 0 0 SP-02-1631 11 15 11 13 14 18 SP-02-1635 10 11 8 11 11 15 SP-02-1640 9 9 8 10 10 12 SP-02-1641 12 15 10 15 15 18 SP-02-1642 13 16 12 15 13 19 SP-02-1643 7 10 8 7 10 11 SP-02-1644 15 16 13 17 17 19 SP-02-1647 13 15 13 15 15 20 SP-02-1648 14 16 14 15 15 19 SP-02-1650 13 15 12 15 17 18 SP-02-1651 13 16 17 16 16 20 SP-02-1652 11 11 9 9 14 14 SP-02-1653 12 13 10 13 15 14 SP-02-1655 15 14 11 14 15 15 SP-02-1656 13 13 10 13 12 18 SP-02-1664 10 14 10 13 15 17 SP-02-1673 9 12 10 12 13 15 SP-02-1722 12 19 12 19 14 17 SP-02-1724 8 15 18 17 15 17 ³ Analysis of the binding of bispecific TCR molecules to MAGEB2-001 and 28 potential off-target peptides and SP-01-0010 as a negative control. The response signal of the off-target peptides is depicted as % of the response signal as measured against the MAGEB2-001 target peptide. Example 7: Bispecific TCR molecules effectively mediate T cell-dependent tumor cell lysis

The activity of bispecific TCR molecules (e.g., TCER molecules) targeting MAGEB2-001 with respect to inducing tumor cell lysis was assessed by evaluating human PBMC-mediated lysis of human cancer cell lines SKMEL-5, RPMI7951, and SCC25 presenting different copy numbers of the MAGEB2-001 peptide on the tumor cell surface in the context of HLA-A*02 (SKMEL-5 - 194, RPMI7951 - 23, and SCC25 - 16 copies of MAGEB2-001 per cell, as determined by quantitative M/S analysis), as determined by LDH release assay. The bispecific TCR molecular construct targeting MAGEB2-001 induced concentration-dependent lysis of MAGEB2-001-positive tumor cell lines (Figure 6). These results demonstrate that MAGEB2-001-directed bispecific TCR molecules can effectively mediate T cell-dependent tumor cell lysis even on tumor cells expressing as few as 20 copies of MAGEB2-001 per tumor cell. Example 8: Bispecific TCR molecules show a beneficial safety profile

All bispecific TCR molecules induced dose-dependent lysis of the target-positive cell line SKMEL5 (194 cpc), while no cytotoxicity was observed against any of the primary or iPSC-derived cell types tested at bispecific doses up to 100 nM (Figure 7). Example 9: In vivo efficacy in NOG mice bearing tumor xenografts

Pharmacodynamic studies were performed in the hyperimmune-deficient NOG mouse strain to test the ability of TCER molecules to recruit human immune T cell activity by specific binding to T cell antigens and by specific binding to human tumor-specific HLA-peptide complexes on human cancer cells (Figure 9). Human peripheral blood mononuclear (PBMC) cell xenografts were injected intravenously into the NOG mouse strain loaded with subcutaneous injections of the human tumor cell line BCPAP (including 450 to 500 copies of the MAGEB2-001 peptide in the case of HLA-A*02). When individual BCPAP tumors reached >120 mm3 in ^volume as calculated based on caliper measurements, human PBMCs (1× ¹⁰⁷ cells/mouse) were transplanted within 24 hours. Treatment was initiated within one hour after transplantation of human blood cells. Six female mice per group (randomized stratified by tumor size) received intravenous bolus injections (5 mL/kg body weight, once weekly for three doses) via the tail vein. The injection dose of TCER molecules was 0.25 mg/kg body weight per injection (Group 2), and PBS was used in the vehicle control group (Group 1). As shown in Figure 9 for individual mice, treatment with TCER molecules induced a significant anti-tumor response in all mice in Group 2. Treatment with TCER TPP-4799, which targets the HLA-A*02/MAGEB2-001 peptide complex, strongly inhibited tumor growth, as indicated by a decrease in individual tumor volume compared to the increase observed in control Group 1. Starting on Day 18, treatment with TCER TPP-4799 induced complete remission of BCPAP tumors in three of six mice, with the remaining tumor volume ranging between 9 mm ³ and 14 mm ³ on Day 21, compared to a range of 742 mm ³ and 1286 mm ³ in vehicle-treated mice in Group 1. Sequence Listing 7: Exemplary amino acid sequences mentioned herein Serial number Description /TCR Description / Link Description / Area / Region sequence 1 MAGEB2 target peptide GVYDGEEHSV 2 TPP-4799, TPP-4787, TPP-4810, TPP-4794, TPP-4812, TPP-4813, TPP-4606, TPP- 4784, TPP-4788, TPP-4798, TPP-4805, TPP-4020, TPP-4017, TPP-4018, TPP-4019 α CDR1 DASSTY 3 TPP-4799, TPP-4810, TPP-4812, TPP-4813, TPP-4784, TPP-4788, TPP-4805 α CDR3 AVKDNARLL 4 TPP-4787, TPP-4794, TPP-4606, TPP-4798, TPP-4020, TPP-4017, TPP-4018, TPP-4019 α CDR3 AVKDNARLM 5 TPP-4799, TPP-4787, TPP-4810, TPP-4794, TPP-4812, TPP-4813, TPP-4606, TPP-4784 , TPP-4788, TPP-4798, TPP-4805, TPP-4020, TPP-4017, TPP-4018, TPP-4019, CL-48553 β CDR1 RPVTM 6 TPP-4799, TPP-4787, TPP-4810, TPP-4794, TPP-4812, TPP-4813, TPP-4606, TPP- 4784, TPP-4788, TPP-4798, TPP-4805, TPP-4020, TPP-4017, TPP-4018, TPP-4019 β CDR3 SVLGAYGYT 7 TPP-4799, TPP-4810, TPP-4812, TPP-4813, TPP-4784, TPP-4788, TPP-4805 α CDR2 IYS NMDM 8 TPP-4787, TPP-4794, TPP-4606, TPP-4798, TPP-4020, TPP-4017, TPP-4018, TPP-4019 α CDR2 IFSNMDM 9 TPP-4799, TPP-4805, TPP-4020, TPP-4017, TPP-4018, TPP-4019 β CDR2 AYDEGSA 10 TPP-4787, TPP-4810, TPP-4794, TPP-4812, TPP-4813, TPP-4606, TPP-4784, TPP-4788, TPP-4798 β CDR2 YDV 11 TPP-4799, TPP-4813, TPP-4788 α Variable Domain E DVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIYSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLLFGDVTQLVVKP 12 TPP-4787, TPP-4798 α Variable Domain E DVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDVTQLVVKP 13 TPP-4810, TPP-4812, TPP-4784, TPP-4805 α Variable Domain E DVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIYSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLLFGDGTQLVVKP 14 TPP-4794, TPP-4606, TPP-4020, TPP-4017, TPP-4018, TPP-4019 α Variable Domain E DVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKP 15 TPP-4799 β Variable Domain AVISQKPSRVIVQRGTSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDEGSATYESGFDIDKFPISRPNLTFSTLTVSNVEPEDSSIYLCSVLGAYGYTFGSGTRLTVV 16 TPP-4787 β Variable Domain AVISQKPSRYIVQRGTSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDVGSATYESGFVIDKFPISRPNLTFSTLTVSNVEPEDSSIYLCSVLGAYGYTFGSGTRLTVV 17 TPP-4810 β Variable Domain AVISQKPSRYIVQRGTSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDVGSATYESGFDIDKFPISRPNLTFSTLTVSNMEPEDSSIYLCSVLGAYGYTFGSGTRLTVV 18 TPP-4794, TPP-4798 β Variable Domain AVISQKPSRVIVQRGTSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDVGSATYESGFVIDKFPISRPNLTFSTLTVSNVEPEDSSIYLCSVLGAYGYTFGSGTRLTVV 19 TPP-4812, TPP-4813 β Variable Domain AVISQKPSRYIVQRGTSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDVGSATYESGFDIDKFPISRPNLTFSTLTVSNVEPEDSSIYLCSVLGAYGYTFGSGTRLTVV 20 TPP-4606 β Variable Domain AVISQKPSRYIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSPTLIATAYDVGSATYESGFVIDKFPISRPNLTFSTLTVSNMEPEDSSIYLCSVLGAYGYTFGSGTRLTVV twenty one TPP-4784, TPP-4788 β Variable Domain AVISQKPSRYIVQRGTSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDVGSATYESGFDIDKFPISRPNLTFSTLTVSNMEPEDSSIYLCSVLGAYGYTFGSGTRLTVV twenty two TPP-4805 β Variable Domain AVISQKPSRDIVQRGTSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDEGSATYESGFDIDKFPISRPNLTFSTLTVSNMEPEDSSIYLCSVLGAYGYTFGSGTRLTVV twenty three TPP-4020, TPP-4017, TPP-4018, TPP-4019 β Variable Domain AVISQKPSRDIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDEGSATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVV twenty four TPP-4799, TPP-4787, TPP-4810, TPP-4794, TPP-4812, TPP-4813, TPP-4606, TPP- 4784, TPP-4788, TPP-4798, TPP-4805, TPP-4020, TPP-4017, TPP-4018, TPP-4019 fc pestle EPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA SIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 25 TPP-4799, TPP-4787, TPP-4810, TPP-4794, TPP-4812, TPP-4813, TPP-4606, TPP- 4784, TPP-4788, TPP-4798, TPP-4805, TPP-4020, TPP-4017, TPP-4018, TPP-4019 fc mortar EPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA SIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 26 TPP-4799, TPP-4787, TPP-4810, TPP-4794, TPP-4812, TPP-4813, TPP-4606, TPP- 4784, TPP-4788, TPP-4798, TPP-4805, TPP-4020, TPP-4017, TPP-4018, TPP-4019 TCER connector GGGSGGGG 27 TPP-4799, TPP-4813, TPP-4788 First polypeptide E DVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIYSNDMMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLLFGDVTQLVVKPGGGSGGGGE VQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWMGYINPRNDVTKYAEKFQGRVTLTSDTSSTAYMELSSLRSEDTAVYYCARGSYYDYEGFVYWGQGTLVT VSSEPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 28 TPP-4787, TPP-4798 First polypeptide E DVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIFSNDMMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDVTQLVVKPGGGSGGGGE VQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWMGYINPRNDVTKYAEKFQGRVTLTSDTSSTAYMELSSLRSEDTAVYYCARGSYYDYEGFVYWGQGTLVT VSSEPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 29 TPP-4810, TPP-4812, TPP-4784, TPP-4805 First polypeptide E DVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIYSNDMMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLLFGDGTQLVVKPGGGSGGGGE VQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWMGYINPRNDVTKYAEKFQGRVTLTSDTSSTAYMELSSLRSEDTAVYYCARGSYYDYEGFVYWGQGTLVT VSSEPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 30 TPP-4794, TPP-4606, TPP-4020 First polypeptide E DVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIFSNDMMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGSGGGGE VQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWMGYINPRNDVTKYAEKFQGRVTLTSDTSSTAYMELSSLRSEDTAVYYCARGSYYDYEGFVYWGQGTLVT VSSEPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 31 TPP-4017 First polypeptide E DVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIFSNDMMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGSG GGGQIQMTQSPSSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIKEPKS SDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASI EKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 32 TPP-4018 First polypeptide Q IQMTQSPSSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIKGGGSGGG GEDVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPEPKS SDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASI EKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 33 TPP-4019 First polypeptide E VQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWMGYINPRNDVTKYAEKFQGRVTLTSDTSSTAYMELSSLRSEDTAVYYCARGSYYDYEGFVYWGQGTLVT VSSGGGSGGGGEDVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLV VKPEPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP ASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 34 TPP-4799 The second polypeptide Q IQMTQSPSSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIKGGGSGGGG AVISQKPSRVIVQRGTSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDEGSATYESGFDIDKFPISRPNLTFSTLTVSNVEPEDSSIYLCSVLGAYGYTFGSGTRLTVVEP KSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 35 TPP-4787 The second polypeptide Q IQMTQSPSSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIKGGGSGGGG AVISQKPSRYIVQRGTSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDVGSATYESGFVIDKFPISRPNLTFSTLTVSNVEPEDSSIYLCSVLGAYGYTFGSGTRLTVVEP KSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 36 TPP-4810 The second polypeptide Q IQMTQSPSSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIKGGGSGGGG AVISQKPSRYIVQRGTSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDVGSATYESGFDIDKFPISRPNLTFSTLTVSNMEPEDSSIYLCSVLGAYGYTFGSGTRLTVVEP KSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 37 TPP-4794, TPP-4798 The second polypeptide Q IQMTQSPSSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIKGGGSGGGG AVISQKPSRVIVQRGTSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDVGSATYESGFVIDKFPISRPNLTFSTLTVSNVEPEDSSIYLCSVLGAYGYTFGSGTRLTVVEP KSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 38 TPP-4812, TPP-4813 The second polypeptide Q IQMTQSPSSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIKGGGSGGGG AVISQKPSRYIVQRGTSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDVGSATYESGFDIDKFPISRPNLTFSTLTVSNVEPEDSSIYLCSVLGAYGYTFGSGTRLTVVEP KSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 39 TPP-4606 The second polypeptide Q IQMTQSPSSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIKGGGSGGGG AVISQKPSRYIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSPTLIATAYDVGSATYESGFVIDKFPISRPNLTFSTLTVSNMEPEDSSIYLCSVLGAYGYTFGSGTRLTVVEP KSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 40 TPP-4784, TPP-4788 The second polypeptide Q IQMTQSPSSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIKGGGSGGGG AVISQKPSRYIVQRGTSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDVGSATYESGFDIDKFPISRPNLTFSTLTVSNMEPEDSSIYLCSVLGAYGYTFGSGTRLTVVEP KSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 41 TPP-4805 The second polypeptide Q IQMTQSPSSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIKGGGSGGGG AVISQKPSRDIVQRGTSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDEGSATYESGFDIDKFPISRPNLTFSTLTVSNMEPEDSSIYLCSVLGAYGYTFGSGTRLTVVEP KSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 42 TPP-4020 The second polypeptide Q IQMTQSPSSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIKGGGSGGGG AVISQKPSRDIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDEGSATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVVEP KSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 43 TPP-4017 The second polypeptide E VQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWMGYINPRNDVTKYAEKFQGRVTLTSDTSSTAYMELSSLRSEDTAVYYCARGSYYDYEGFVYWGQGTLVT VSSGGGSGGGGAVISQKPSRDIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDEGSATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGT RLTVVEPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 44 TPP-4018 The second polypeptide AVISQKPSRDIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDEGSATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVVGGGSGG GGEVQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWMGYINPRNDVTKYAEKFQGRVTLTSDTSTSTAYMELSSLRSEDTAVYYCARGSYYDYEGFVYWGQGTL VTVSSEPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PASIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 45 TPP-4019 The second polypeptide AVISQKPSRDIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDEGSATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVVGG GSGGGGQIQMTQSPSSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIKEP KSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAS IEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 46 BMhanced VH T cell recruiting VH E VQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWMGYINPRNDVTKYAEKFQGRVTLTSDTSTSTAYMELSSLRSEDTAVYYCARGSYYDYEGFVYWGQGTLVTVSSEPKSS 47 BMhanced VL T cell recruiting body VL Q IQMTQSPSSSLSASVGDRVTITCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTLTISSLQPEDAATYYCQQWSSNPLTFGGGTKVEIK 48 BMhanced HCDR1 Recruiting HCDR1 (Kabat) SYVMH 49 BMhanced HCDR2 Recruiting HCDR2 (Kabat) YINPRNDVTKYAEKFQG 50 BMhanced HCDR3 Recruiting HCDR3 (Kabat) GSYYDYEGFVY 51 BMhanced LCDR1 Recruiting factor LCDR1 (Kabat) SATSSVSYMH 52 BMhanced LCDR2 Recruiting factor LCDR2 (Kabat) DTSKLAS 53 BMhanced LCDR3 Recruiting factor LCDR3 (Kabat) QQWSSNPLT 54 BMA031-V10 VH VH for alternative BM recruitment E VQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWMGYINPYNDVTKYAEKFQGRVTLTSDTSSTAYMELSSLRSEDTAVHYCARGSYYDYDGFVYWGQGTLVTVSS 55 BMA031-V36 VH VH for alternative BM recruitment E VQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWMGYINPYNDVTKYAEKFQGRVTLTSDTSSTAYMELSSLRSEDTAVHYCARGSYYDYEGFVYWGQGTLVTVSS 56 BMA031D01VH VH for alternative BM recruitment E VQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWMGYINPRNDVTKYAEKFQGRVTLTSDTSSTAYMELSSLRSEDTAVHYCARGSYYDYEGFVYWGQGTLVTVSS 57 BMA HCDR2 variants YINPYNDVTKYAEKFQG 58 BMA HCDR3 variants GSYYDYDGFVY 59 SP-02-1621 TPP-142 Seq-SimPep FIVDGVEVSV 60 SP-02-1622 TPP-143 Seq-SimPep FVYDEPGHAV 61 SP-02-1623 TPP-144 Seq-SimPep GLSDGEWQLV 62 SP-02-1624 TPP-145 Seq-SimPep GLYDGPVHEV 63 SP-02-1625 TPP-146 Seq-SimPep GVIDGHIYAV 64 SP-02-1626 TPP-147 Seq-SimPep GVMAGDIYSV 65 SP-02-1627 TPP-148 Seq-SimPep GVYGGSVHEA 66 SP-02-1628 TPP-149 Seq-SimPep TVYGGYLCSV 67 SP-02-1629 TPP-150 Seq-SimPep YTYDEAIHSV 68 MAGEB2-001_A1 TPP-151 ScanPep AVYDGEEHSV 69 MAGEB2-001_A3 TPP-153 ScanPep GVADGEEHSV 70 MAGEB2-001_A4 TPP-154 ScanPep GVYAGEEHSV 71 MAGEB2-001_A5 TPP-155 ScanPep GVYDAEEHSV 72 MAGEB2-001_A6 TPP-156 ScanPep GVYDGAEHSV 73 MAGEB2-001_A7 TPP-157 ScanPep GVYDGEAHSV 74 MAGEB2-001_A8 TPP-158 ScanPep GVYDGEEASV 75 MAGEB2-001_A9 TPP-159 ScanPep GVYDGEEHAV 76 R76P1E1 α chain CL-6120 Parental TCR α chain (TRAV5*01_TRAJ31*01_TRAC) E DVEQSLFLSVREGDSSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNDMMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEKDNARLMFGDGTQLVVKPNIQNPDPAVYQLRDSKS SDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 77 R76P1E1 β chain CL-6120 Parental TCR β chain (TRBV29-1*01_TRBJ1-2*01_TRBC1*01) AVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLA TGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF 78 R76P1E1 Vα CL-6120 Parental TCR Vα E DVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNDMMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEKDNARLMFGDGTQLVVKP 79 R76P1E1 Vβ CL-6120 Parental TCR Vβ AVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVV 80 R76P1E1 CDRa1 CL-6120 R76P1E1 CDRa1 DSSSTY 81 R76P1E1 CDRa3 CL-6120 R76P1E1 CDRa3 AEKDNARLM 82 R76P1E1 CDRb1 CL-6120 R76P1E1 CDRb1 SQVTM 83 R76P1E1 CDRb2 CL-6120 R76P1E1 CDRb2 ANQGSEA 84 Aga2p+leader sequence MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVF 85 Show connector GGGGSGGGGSGGGGSGGGGSGGGGS 86 FLAG logo DYKDDDDK 87 Myc logo EQKLISEEDL 88 Fusion protein scTCR R76P1E1 MQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSLSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSPINTQYVFGGGGSDYKDDDDKGGGASEDVEQSLFLSVREGDSAVINCTYTDSSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQ TGDSAIYFCAEKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGGSGGGGSGGGGSGAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVVAAAGGSGGEQKLISEEDL 89 scTCR R76P1E1 CL-19871 scTv without Aga2p, or preceding or succeeding connectors/logos EDVEQSLFLSVREGDSSVINCTYTDSSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSGAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVV 90 scTCR R76P1E1_bC13V CL-19872 Parental strain for scTv transformation (aS19A+bC13V) EDVEQSLFLSVREGDSAVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSGAVISQKPSRDIVQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVV 91 R76P1E1C CL-48396 Stabilized clones from scTv transformation with free Cys EDVEQSSFLSVREGDSAVINCTYTDSSSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSGAVISQKPSRDIVQRGNSVTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCCVLGAYGYTFGSGTRLTVV 92 R76P1E1C CDRb3 CL-48396 CVLGAYGYT 93 scTCR R76P1E1S CL-48541 The Cys-free form of R76P1E1C was used as the basis for affinity maturation EDVEQSSFLSVREGDSAVINCTYTDSSSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSGAVISQKPSRDIVQRGNSVTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVV 94 CL-48730 CL-48730 Selection from CDR library EDVEQSSFLSVREGDSAVINCTYTDSSSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSGAVISQKPSRDIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDDGSATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVV 95 CL-48731 CL-48731 Selection from CDR library EDVEQSSFLSVREGDSAVINCTYTDSSSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSGAVISQKPSRDIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYSDGTATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVV 96 CL-48732 CL-48732 Selection from CDR library EDVEQSSFLSVREGDSAVINCTYTDSSSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSGAVISQKPSRDIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDDNTTTYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVV 97 CL-21753 CL-21753 SeqLiab-complemented clones for bio-optimization (less than 1 G in the linker), clones for TCER-targeted runs EDVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSAVISQKPSRDIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDEGSATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVV 98 CL-25688 CL-25688 From biologically optimized strains EDVEQSSFLSVREGDSAVINCTYTDDSSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSAVISQKPSRDIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSPTLIATAYDEGSATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCCVLGAYGYTFGSGTRLTVV 99 CL-25687 CL-25687 Two mutations in the linker from the bio-optimized clones EDVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIYSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLLFGDGTQLVVKPGGGGSRGGGSGGGG SGGGGSGGGDSAVISQKPSRDIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDEGSATYESGFDIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVV 100 CL-25693 CL-25693 From biologically optimized strains EDVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSAVISQKPSRDIVQRGNSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDEGSATYESGFDIDKFPISRPNLTFSTLTVSNMSPEDSSIYFCSVLGAYGYTFGSGTRLTVV 101 CL-25720 CL-25720 From biologically optimized strains EDVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSAVISQKPSRYIVQRGNSVTIQCQVDRPVTMMFWYRQQPGQSPTLIATAYDVGSATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVLGAYGYTFGSGTRLTVV 102 CL-25821 CL-25821 From biologically optimized strains EDVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDVTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSAVISQKPSRVIVQRGNSVTIQCQVDRPVTMMYWYRQQPGQSLTLIATAYDEGSATYESGFVIDKFPISRPNLTFSTLTVSNVSPEDSSIYLCSVLGAYGYTFGSGTRLTVV 103 CL-24597 CL-24597 From biologically optimized strains EDVEQSSFLSVREGDSAVINCTYTDSSSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSAVISQKPSRDIVQRGTSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDEGSATYESGFVIDKFPISRPNLTFSTLTVSNLEPEDSSIYLCSVLGAYGYTFGSGTRLTVV 104 CL-24505 CL-24505 From biologically optimized strains EDVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSAVISQKPSRDIVQRGTSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDEGSATYESGFVIDKFPISRPNLTFSTLTVSNMEPEDSSIYLCSVLGAYGYTFGSGTRLTVV 105 CL-24478 CL-24478 From biologically optimized strains EDVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLMFGDGTQLVVKPGGGGSGGGGSGGGG SGGGGSGGGGSAVISQKPSRDIVQRGTSVTIQCQVDRPVTMMFWYRQQPGQSLTLIATAYDEGSATYESGFVIDKFPISRPNLTFSTLTVSSMEPEDSSIYLCSVLGAYGYTFGSGTRLTVV 106 SP-02-1640 Seq-SimPep RLYDGLFKVI 107 CL-48641 β CDR2 AYDNNRT 108 CL-48644 β CDR2 AFDDNTT 109 CL-48650 β CDR2 YDDGSA 110 CL-48715 β CDR2 AYSDGTA 111 CommonBMhanced CDRH1 _X1YVMH , where _X1 is S, R, K, or H 112 CommonBMhanced CDRH2 YINPX ₁ X ₂ DVTKYX ₃ X ₄ KFX ₅ G, wherein X ₁ is Y, R, K, or H, preferably Y or R; X ₂ is N, R, or K, preferably N or R; X ₃ is A or N; X ₄ is E or Q, preferably E; and X ₅ is Q or K 113 CommonBMhanced CDRL1 SATSSVXYMH, where X is S, R, or K 114 CommonBMhanced CDRL2 DTSKLAX, where X is S, R, or K 115 20G6-Alternative T cell recruiter (anti-CD3) VH QVQLVESGGGVVQPGRSLRLSCAASGFTFTKAWMHWVRQAPGKQLEWVAQIKDKSNSYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCRGVYYALSPFDYWGQGTLVTVSS 116 20G6-Alternative T cell recruiter (anti-CD3) V L DIVMTQTPLSLSVTPGQPASISCKSSQSLVHNNANTYLSWYLQKPGQSPQSLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQYPFTFGSGTKVEIK 117 2B4-Alternative T cell recruiter (anti-CD3) VH E VQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS 118 2B4-Alternative T cell recruiter (anti-CD3) V L DIVMTQSPDSLAVSLGERATINCKSSQSLFNVRSRKNYLAWYQQKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYDLFTFGSGTKLEIK 119 38E4v-Alternative T cell recruiter (anti-CD3) VH E VQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRQAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSS 120 38E4v-Alternative T cell recruiter (anti-CD3) V L DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWTSTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSFILRTFGQGTKVEIK 121 CE115-Alternative T cell recruiter (anti-CD3) VH Q VQLVESGGGVVQPGGSLRLSCAASGFTFSNAWMHWVRQAPGKGLEWVAQIKDKSQNYATYVAESVKGRFTISRADSKNSIYLQMNSLKTEDTAVYYCRYVHYAAGYGVDIWGQGTTVTVSS 122 CE115-Alternative T cell recruiter (anti-CD3) V L DIVMTQSPLSLPVTPGEPASISCRSSQPLVHSNRNTYLHWYQQKPGQAPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQVPYTFGQGTKLEIK 123 CRIS7-Alternative T Cell Recruiter (Anti-CD3) VH Q VQLVQSGPEVKKPGSSVKVSCKASGYTFSRSTMHWVRQAPGQGLEWIGYINPSSAYTNYNQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARPQVHYDYNGFPYWGQGTLVTVSS 124 CRIS7-Alternative T Cell Recruiter (Anti-CD3) V L DIQMTQSPSTLSASSVGDRVTMTCSASSSVSYMNWYQQKPGKAPKRWIYDSSKLASGVPSRFSGSGSGTDYTLTISSLQPDDFATYYCQQWSRNPPTFGGGTKVEIK 125 F2B-Alternative T cell recruiter (anti-CD3) VH E VQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDSRGYGDYRLGGAYWGQGTLVTVSS 126 F2B-Alternative T cell recruiter (anti-CD3) V L E IVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPWTFGQGTKVEIK 127 H2M2691N - Alternative T cell recruiter (anti-CD3) VH E VQLVESGGGLVQPGRSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDNSGYGHYYYGMDVWGQGTTVTVAS 128 H2M2691N - Alternative T cell recruiter (anti-CD3) V L E IVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHYINWPLTFGGGTKVEIK 129 MT301-Alternative T cell recruiter (anti-CD3) VH Q VQLVQSGAEVKKPGASVKVSCKASGYTFTKYVIHWVRQAPGQGLEWMGYINPYNDVSKYNEKFRGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARNKDYYPMDYWGQGTTVTVSS 130 MT301-Alternative T cell recruiter (anti-CD3) V L E IVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIK 131 Alternative T cell recruiter (anti-CD3) VH Q VQLVQSGGGVVQPGRSLRLSCVASGFTFSSYGMHWVRQAPGKGLEWVAAIWYNARKQDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRGTGYNWFDPWGQGTLVTVSS 132 Alternative T cell recruiter (anti-CD3) V L DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK 133 SP-01-0010 Seq-SimPep YLLPAIVHI 134 SP-02-1631 Seq-SimPep GMYDGPVYEV 135 SP-02-1635 Seq-SimPep FIYDNTEFTL 136 SP-02-1641 Seq-SimPep NQWDGTQHGV 137 SP-02-1642 Seq-SimPep SQYSGQLHEV 138 SP-02-1643 Seq-SimPep VLYEGPVRGL 139 SP-02-1644 Seq-SimPep GLYEGEFTLA 140 SP-02-1647 Seq-SimPep GMYDGPVFDL 141 SP-02-1648 Seq-SimPep GLYDGPVCEV 142 SP-02-1650 Seq-SimPep GLYEGLDWLA 143 SP-02-1651 Seq-SimPep GLYEGLDWLS 144 SP-02-1652 Seq-SimPep FLHDGQEYTL 145 SP-02-1653 Seq-SimPep ALYPGTDYTV 146 SP-02-1655 Seq-SimPep SLYPFQIHSI 147 SP-02-1656 Seq-SimPep RMYPFQIHSI 148 SP-02-1664 Seq-SimPep LLYDGKLSSA 149 SP-02-1673 Seq-SimPep ALYPGTNYTV 150 SP-02-1722 Seq-SimPep ALENSEEHSA 151 SP-02-1724 Seq-SimPep QFYDGVVQ 152 Common V _A E DVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIXSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLXFGDXTQLVVKP 153 Common V _A E DVEQSSFLSVREGDSAVINCTYTDASSTYFYWYKQEPGAGLQLLTYIX ₁ SNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVKDNARLX ₂ FGDX ₃ TQLVVKP, where X ₁ is Y or F; X ₂ is L or M; X ₃ is V or G 154 Common _VB AVISQKPSRXIVQRGXSVTIQCQVDRPVTMMXWYRQQPGQSXTLIATAYDXGSATYESGFXIDKFPISRPNLTFSTLTVSNXXPEDSSIYLCSVLGAYGYTFGSGTRLTVV 155 Common _VB AVISQKPSRX ₁ IVQRGX ₂ SVTIQCQVDRPVTMMX ₃ WYRQQPGQSX ₄ TLIATAYDX ₅ GSATYESGFX ₆ IDKFPISRPNLTFSTLTVSNX ₇ X ₈ PEDSSIYLCSVLGAYGYTFGSGTRLTVV, where _X1 is V or Y or D; _X2 is T or N; _X3 is Y or F; _X4 is L or P; _X5 is E or V; _X6 is D or V; _X7 is V or M; _X8 is E or S

[ Figure 1 ] Target tetramer titration of stabilized scTCRs. HLA-A*02/MAGEB2-001 tetramers were titrated on selected strain R76P1E1C from the scTCR stabilized library. [Figure 2] Target binding and specificity analysis on affinity matured scTCRs Selected yeast strains with scTCRs from the CDRb1 and CDRb2 libraries were analyzed for target and analog peptide binding. The parental library strain R76P1E1S was included in the analysis. CDRb1 strains were stained for HLA-A*02/MAGEB2-001 tetramers ( Figure 2A ), while CDRb2 strains were stained for HLA-A*02/MAGEB2-001 monomers ( Figure 2B ). Specificity was treated using a mixture of 9 similar peptides with HLA-A*02 at a concentration of 25 nM each as peptide-HLA tetramers ( Figure 2C ). [Figure 3] Target binding and specificity analysis on scTCR after CDR assembly The binding of scTCR variants to the target HLA-A*02/MAGEB2-001 on yeast cells was studied as a titration series in monomer format ( Figure 3A ), while the 9 similar peptides were treated individually with HLA-A*02 as peptide-HLA tetramers at a concentration of 25 nM each ( Figure 3B ). [Figure 4] scTCR motif determination The binding of MAGEB2-001 peptide or its single amino acid substitutions to yeast cells displaying scTCR in the context of HLA-A*02 was studied. Yeast cells carrying high affinity scTCRs were stained with four concentrations of HLA-A*02 monomers with MAGEB2-001 or substituted peptides (31.6 nM, 10 nM, 3.2 nM, 1 nM, respectively), as indicated by four bars for each peptide. [Figure 5] Target titration after bio-optimization HLA-A*02/MAGEB2-001 monomers were titrated on yeast strains selected from the bio-optimization library and compared to their parental molecule CL-21753. The closed-sign strains were derived from a library based on error-prone PCR, and the open-sign strains were derived from a library with targeted amino acid substitutions. [FIG. 6] Results of LDH release assay using bispecific TCR/mAb bifunctional antibodies. LDH release assay using bispecific TCR/mAb bifunctional antibody constructs targeting the tumor-associated peptide MAGEB2-001 (SEQ ID NO. 1) presented on HLA-A*02 was performed (with TPP-4784, TPP-4787, TPP-4810 and TPP-4812 (each in FIG. 6A ); with TPP-4788, TPP-4805 and TPP-4813 (each in FIG. 6B ); and with TPP-4794, TPP-4798, TPP-4799 and TPP-4606 (each in FIG. 6C )). PBMCs isolated from healthy donors were co-cultured with cancer cell lines SKMEL-5, RPMI7951, and SCC25 (194 copies per cell for SKMEL-5, 23 copies per cell for RPMI7951, and 16 copies per cell for SCC25, as determined by M/S analysis) displaying different amounts of MAGB2-001:HLA-A*02 complexes on the cell surface in the presence of increasing concentrations of TCR/mAb bifunctional antibody molecules at an effector:target ratio of 10:1. After 48 hours of co-culture, target cell lysis was quantified using an LDH release assay according to the manufacturer's instructions (Promega). [FIG. 7] Safety of bispecific molecules The reactivity of several bispecific molecules targeting MAGEB2-001 was tested against the target-positive cell line SKMEL5 (194 cpc) and several primary cells and iPSC-derived cell types in cytotoxicity assays using PBMCs from healthy donors as effector cells ( FIG. 7A to 7C ). [FIG. 8] Different orientations of variable domains in bispecific molecules Arranging the variable domains VA, VB, VL, and VH in different positions in the first polypeptide and/or the second polypeptide of a bispecific molecule as disclosed herein results in different "orientations" A to D. In each case, VA and VB are capable of forming a first binding site (e.g., binding domain A), and VL and VH are capable of forming a second binding site (e.g., binding domain B). L represents a linker, C represents a constant domain, and KiH represents "knob-into-hole." Exemplary markers for V1 to V4 are indicated. [ FIG. 9 ] In vivo efficacy in NOG mice bearing tumor xenografts TCER-mediated in vivo efficacy in human PBMC-implanted immunodeficient female NOG mice against tumor xenografts of BCPAP cells, which are tumor cell lines with detectable HLA-A*02/MAGEB2-001 presentation. Group 1 represents a vehicle-treated control group, and Group 2 represents mice treated three times weekly with TCER TPP-4799 [0.25 mg/kg body weight]. Each symbol connected by a line represents a single animal, and the treatment days are indicated by arrows.

TW202506719A_113127984_SEQL.xml

Claims (15)

An antigen binding protein specifically binds to a MAGEB2 antigen peptide complexed with a major histocompatibility complex (MHC) protein, wherein the MAGEB2 antigen peptide comprises or consists of the following amino acid sequence GVYDGEEHSV (SEQ ID NO: 1), wherein the antigen binding protein comprises: a first polypeptide comprising a variable domain _VA comprising complementary determining regions (CDRs) CDRa1, CDRa2, and CDRa3 and a second polypeptide comprising a variable domain _VB comprising CDRb1, CDRb2, and CDRb3, wherein CDRa1, CDRa2, CDRa3, CDRb1, CDRb2, and CDRb3 form an antigen binding domain A; wherein the variable domain _VA comprises or consists of the following amino acid sequence according to SEQ ID NO: 152 or a polypeptide having a sequence similar to SEQ ID NO: 153; NO:152 has an amino acid sequence with at least 85%, 90%, 95%, 98%, or 99% identity, wherein the variable domain _VB comprises or consists of: an amino acid sequence according to SEQ ID NO:154 or an amino acid sequence with at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:154, preferably, wherein (1) CDRa1 comprises SEQ ID NO:2, CDRa3 comprises SEQ ID NO:3, CDRb1 comprises SEQ ID NO:5, and CDRb3 comprises SEQ ID NO:6; or (2) CDRa1 comprises SEQ ID NO:2, CDRa3 comprises SEQ ID NO:4, CDRb1 comprises SEQ ID NO:5, and CDRb3 comprises SEQ ID NO:6; wherein the CDRa1, CDRa3, CDRb1 and/or CDRb3 sequences may comprise one, two or three amino acid mutations. The antigen binding protein of claim 1, wherein (1) CDRa1 comprises SEQ ID NO:2, CDRa2 comprises SEQ ID NO:7, CDRa3 comprises SEQ ID NO:3, CDRb1 comprises SEQ ID NO:5, CDRb2 comprises SEQ ID NO:9, and CDRb3 comprises SEQ ID NO:6; (2) CDRa1 comprises SEQ ID NO:2, CDRa2 comprises SEQ ID NO:8, CDRa3 comprises SEQ ID NO:4, CDRb1 comprises SEQ ID NO:5, CDRb2 comprises SEQ ID NO:10, and CDRb3 comprises SEQ ID NO:6; (3) CDRa1 comprises SEQ ID NO:2, CDRa2 comprises SEQ ID NO:7, CDRa3 comprises SEQ ID NO:3, CDRb1 comprises SEQ ID NO:5, CDRb2 comprises SEQ ID NO:10, and CDRb3 comprises SEQ ID NO:6; or (4) CDRa1 comprises SEQ ID NO:2, CDRa2 comprises SEQ ID NO:8, CDRa3 comprises SEQ ID NO:4, CDRb1 comprises SEQ ID NO:5, CDRb2 comprises SEQ ID NO:9, and CDRb3 comprises SEQ ID NO:6; wherein the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may comprise one, two or three amino acid mutations. The antigen binding protein of claim 1 or claim 2, wherein the V _A comprises an amino acid sequence according to SEQ ID NO: 153 or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 153, or wherein the V _A comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, and 14, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 11 and comprising the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 having SEQ ID NOs: 2, 7, and 3, respectively, an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 12 and comprising the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 having SEQ ID NOs: 2, 8, and 4, respectively, NO:13 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequence of said CDRa1, said CDRa2, and said CDRa3 of SEQ ID NOs: 2, 7, and 3, respectively, or has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:14 and comprises the amino acid sequence of said CDRa1, said CDRa2, and said CDRa3 of SEQ ID NOs: 2, 8, and 4, respectively; and wherein said _VB comprises the amino acid sequence according to SEQ ID NO:155, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:155, or wherein said VB comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:15, 16, 17, 18, 19, 20, 21, 22, and 23, or has at least 85%, 90%, 95%, 98%, or 99 _% identity to SEQ ID NO:14 NO:15 has at least 85%, 90%, 95%, 98%, or 99% identity and includes the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively, and SEQ ID NO:16 has at least 85%, 90%, 95%, 98%, or 99% identity and includes the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively, and SEQ ID NO:17 has at least 85%, 90%, 95%, 98%, or 99% identity and includes the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively, and SEQ ID NO:18 has at least 85%, 90%, 95%, 98%, or 99% identity and includes the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 10, and 6, respectively. NO:5, 10, and 6, the amino acid sequence of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:19 and comprising the amino acid sequence of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively, having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:20 and comprising the amino acid sequence of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively, having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:21 and comprising the amino acid sequence of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively, NO:22 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively, or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:23 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively, wherein optionally, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may comprise one, two or three amino acid mutations, preferably amino acid substitutions. The antigen-binding protein of claim 1 or claim 2, wherein the V _A comprises the amino acid sequence of SEQ ID NO:11 and the Vb comprises the amino acid sequence of SEQ ID NO:15; wherein the V _A comprises the amino acid sequence of SEQ ID NO:12 and the Vb comprises the amino acid sequence of SEQ ID NO:16; wherein the V _A comprises the amino acid sequence of SEQ ID NO:13 and the Vb comprises the amino acid sequence of SEQ ID NO:17; wherein the V _A comprises the amino acid sequence of SEQ ID NO:14 and the Vb comprises the amino acid sequence of SEQ ID NO:18; wherein the V _A comprises the amino acid sequence of SEQ ID NO:13 and the Vb comprises the amino acid sequence of SEQ ID NO:19; wherein the V _A comprises the amino acid sequence of SEQ ID NO:11 and the Vb comprises the amino acid sequence of SEQ ID NO:19; wherein the V _A comprises the amino acid sequence of SEQ ID NO:14 and the Vb comprises the amino acid sequence of SEQ ID NO:19 NO:20; wherein the V _A comprises the amino acid sequence of SEQ ID NO:13 and the Vb comprises the amino acid sequence of SEQ ID NO:21; wherein the V _A comprises the amino acid sequence of SEQ ID NO:11 and the Vb comprises the amino acid sequence of SEQ ID NO:21; wherein the V _A comprises the amino acid sequence of SEQ ID NO:12 and the Vb comprises the amino acid sequence of SEQ ID NO:18; wherein the V _A comprises the amino acid sequence of SEQ ID NO:13 and the Vb comprises the amino acid sequence of SEQ ID NO:22; or wherein the V _A comprises the amino acid sequence of SEQ ID NO:14 and the Vb comprises the amino acid sequence of SEQ ID NO:23. The antigen binding protein of claim 1 or claim 2, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:27 and comprises the amino acid sequences of CDRa1, CDRa2, and CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:34 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:34 and comprises the amino acid sequences of CDRb1, CDRb2, and CDRb3 of SEQ ID NO:5, 9, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:28 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:28 and comprises the amino acid sequences of SEQ ID NO: NO:2, 8, and 4 of the CDRa1, the CDRa2, and the CDRa3, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:35 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:35 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:29 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:36 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:37. NO:36 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:30 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 8, and 4, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:37 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:37 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; wherein the first polypeptide comprises SEQ ID NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:29 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:38 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:38 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:27 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and The second polypeptide comprises the amino acid sequence of SEQ ID NO:38 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:38 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:30 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 8, and 4, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:39 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:39 and comprises the amino acid sequences of SEQ ID NO:5, 10, and 6, respectively. NO:5, 10, and 6 of the CDRb1, the CDRb2, and the CDRb3 amino acid sequence; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:29 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:40 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:40 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:27 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:27 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively. NO:27 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:40 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:40 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:28 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:28 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 8, and 4, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:40 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:40 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; NO:37 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:37 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 10, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:29 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:29 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 7, and 3, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:41 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:41 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 9, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:30 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:30 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 8, and 4, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:42 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:42 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 9, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:31 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:31 and comprises the amino acid sequences of SEQ ID NO:5, 9, and 6, respectively. NO:2, 8, and 4 of the CDRa1, the CDRa2, and the CDRa3 amino acid sequence, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:43 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:43 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NO:5, 9, and 6, respectively; wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:32 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:32 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NO:2, 8, and 4, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:44 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:44. NO:44 has at least 85%, 90%, 95%, 98%, or 99% identity and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; or wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:33 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:33 and comprises the amino acid sequences of the CDRa1, the CDRa2, and the CDRa3 of SEQ ID NOs:2, 8, and 4, respectively, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:45 or has at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO:45 and comprises the amino acid sequences of the CDRb1, the CDRb2, and the CDRb3 of SEQ ID NOs:5, 9, and 6, respectively; Optionally, the CDRa1, CDRa2, CDRa3, CDRb1, CDRb2 and/or CDRb3 sequences may contain one, two or three amino acid mutations, preferably amino acid substitutions. An antigen binding protein as claimed in claim 1 or claim 2, wherein the antigen binding protein further comprises: a variable domain VH comprising CDRH1, CDRH2, and CDRH3 and a VL comprising CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 form an antigen binding domain B. The antigen-binding protein of claim 6, wherein the antigen-binding domain B specifically binds to immune cells, preferably T cells or natural killer (NK) cells. The antigen binding protein of claim 6, wherein the VH comprises: (a) CDRH1 comprising the amino acid sequence of SYVMH [SEQ ID NO:48], (b) CDRH2 comprising the amino acid sequence of YINPRNDVTKYAEKFQG [SEQ ID NO:49] or YINPYNDVTKYAEKFQG [SEQ ID NO:57], and (c) CDRH3 comprising the amino acid sequence of GSYYDYEGFVY [SEQ ID NO:50] or GSYYDYDGFVY [SEQ ID NO:58], and wherein the VL comprises: (a) CDRL1 comprising the amino acid sequence of SATSSVSYMH [SEQ ID NO:51], (b) CDRL2 comprising the amino acid sequence of DTSKLAS [SEQ ID NO:52], and (c) CDRH3 comprising the amino acid sequence of QQWSSNPLT [SEQ ID NO:53]. NO:53]; and optionally, wherein the CDRH1, the CDRH2, and the CDRH3 and each of the CDRL1, the CDRL2, and the CDRL3 comprises at most three amino acid substitutions. An antigen binding protein as claimed in claim 6, wherein the antigen binding protein comprises two antigen binding sites A and B, wherein the first polypeptide chain comprises a structure represented by the following formula: V1-L1-V2 [I] wherein V1 is the first variable domain; V2 is the second variable domain; L1 is the linker domain; and wherein the second polypeptide chain comprises a structure represented by the following formula: V3-L3-V4 [II] wherein V3 is the third variable domain; V4 is the fourth variable domain; L3 is the linker; optionally, wherein: V1 and V2 form the antigen binding site A and V3 and V4 form the antigen binding site B, or V1 and V2 form the antigen binding site B and V3 and V4 form the antigen binding site A, or V1 and V3 form the antigen binding site A and V2 and V4 form the antigen binding site B, or V1 and V3 form the antigen binding site B and V2 and V4 form the antigen binding site A, or V1 and V4 form the antigen binding site A and V2 and V3 form the antigen binding site B, or V1 and V4 form the antigen binding site B and V2 and V3 form the antigen binding site A, and Optionally, wherein the first polypeptide and the second polypeptide are covalently or non-covalently linked together. An antigen-binding protein as claimed in claim 1 or claim 2, wherein the (one or more) N-terminal or C-terminal amino acids of the polypeptide chains of the antigen-binding protein comprise (one or more) modifications. One or more nucleic acids encoding the antigen binding protein of any one of claims 1 to 10. One or more vectors, which (etc.) contain one or more nucleic acids as claimed in claim 11. A host cell comprising one or more nucleic acids of claim 11 or (one or more) vectors of claim 12. A pharmaceutical composition comprising an antigen-binding protein as described in any one of claims 1 to 10, (one or more) nucleic acids as described in claim 11, or (one or more) vectors as described in claim 12, and optionally, a pharmaceutically acceptable carrier. A use of an antigen binding protein as claimed in any one of claims 1 to 10, a nucleic acid(s) as claimed in claim 11, a vector(s) as claimed in claim 12, or a pharmaceutical composition as claimed in claim 14 for the preparation of a medicament for treating cancer. [1] Stabilized scTCRs containing unmodified mature CDR1β and CDR2β were stained with 10 nM HLA-A*02/MAGEB2-001 tetramer or 100 nM HLA-A*02/MAGEB2-001 monomer, and contrasted with a mixture of HLA-A*02 tetramers containing peptides with high sequence similarity to MAGEB2-001 (SEQ ID NO:1) (analog peptides or SimPeps, SEQ ID NO:59-67), each tetramer was applied at a concentration of 25 nM. *: corresponding CDRs from R76P1E1S scTCR (SEQ ID NO:93). [2] For yeast cells carrying scTCR, binding to HLA-A*02/MAGEB2-001 monomers is presented as EC ₅₀ values, and binding to 9 similar peptides (SEQ ID NO:59-67) in the presence of 10 nM HLA-A*02 tetramer is presented as % positive cells. FR: framework region; nd: not determined