Modified bispecific anti CD3 antibodies

Abstract

To provide additional therapeutics, in particular cancer therapeutics, in particular bispecific antigen-binding proteins that efficiently target and kill diseased cells, in particular cancer cells, while a high safety profile is maintained.SOLUTION: The present invention relates to bispecific antigen-binding proteins directed against MHC-presented target antigens (TAs). In particular, the present invention provides bispecific antigen-binding proteins comprising at least two antigen-binding sites (A and B), in which an antigen-binding site A binds to CD3 and an antigen-binding site B binds to a target antigenic (TA) peptide / MHC complex. The bispecific antigen-binding proteins of the present invention comprise, in particular, the CDRs of the VL and VH domains of novel, engineered anti-CD3 antibodies with reduced affinity. The bispecific antigen-binding proteins of the present invention are useful for the diagnosis, treatment, and prevention of TA-related diseases, such as tumor-associated antigens (TAAs) that manifest in cancerous diseases.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to bispecific antigen-binding proteins directed against major histocompatibility complex (MHC)-presented target antigens (TA). The present invention particularly provides bispecific antigen-binding proteins comprising at least two antigen-binding sites (A and B), in which antigen-binding site A binds to CD3 and antigen-binding site B binds to a target antigenic (TA) peptide / MHC complex. The bispecific antigen-binding proteins of the present invention particularly comprise the complementarity-determining regions (CDRs) of the VL and VH domains of novel engineered anti-CD3 antibodies with reduced affinity. The bispecific antigen-binding proteins of the present invention are useful for the diagnosis, treatment, and prevention of TA-related diseases, such as tumor-associated antigens (TAAs) that manifest as cancerous diseases. Furthermore, nucleic acids encoding the bispecific antigen-binding proteins of the present invention, vectors comprising these nucleic acids, recombinant cells expressing the antigen-binding proteins, and pharmaceutical compositions comprising the bispecific antigen-binding proteins of the present invention are provided. [Background technology]

[0002] T cells recognize (virus) infected cells and tumor cells by detecting the presence of disease-specific peptides presented on the cell surface by the major histocompatibility complex (MHC) with clone-specific T cell receptors (TCRs).Therefore, TCR-based molecules have attracted great interest in the development of disease- or tumor-specific immunotherapy.While progress has been made in the development of molecular targeting drugs for cancer treatment, there is still a need in the art to develop new anticancer drugs that specifically target molecules that are highly specific to cancer cells but not to normal cells.Similarly, targeting molecules that are highly specific to diseased cells but not to normal cells are also very important for the development of drugs targeting infectious diseases such as HIV.

[0003] In the context of the present invention, the protein from which the target antigenic (TA) peptide is derived is degraded by the proteasome into short peptides, transported to the endoplasmic reticulum, packaged into the groove of newly synthesized MHC molecules, and delivered to the cell membrane as a peptide-MHC (pMHC) complex (TA peptide / MHC). The recognition pattern induced by the TA enables the immune system to distinguish diseased cells, such as transformed tumor cells in the case of TAA antigenic peptides, from surrounding normal tissue cells and to trigger an immune cascade against them.

[0004] To develop such TA-targeting drugs, TCRs that specifically target TAs have been identified, and V has been developed to engineer novel TA-targeting molecules, particularly TAAA-targeting molecules. α and v β The domain was used.

[0005] Regarding TAA targeting molecules, natural T cell receptors (TCRs) that specifically bind to MHC-presented cancer antigens often have lower affinity (K ) than TCRs that specifically bind to MHC-presented viral antigens. D It should be noted that the affinity of TCRs is typically between 1 and 300 μM. Part of the explanation for this phenomenon appears to be that T cells developing in the thymus are negatively selected on self-peptide MHC ligands (tolerance induction), resulting in the elimination of T cells with too high affinity for such self-peptide MHC. This low affinity may be one possible explanation for tumor immune escape (Non-Patent Document 1). Therefore, it seems desirable to design TCR variants that bind with higher affinity to cancer antigens for use as antigen recognition constructs in adoptive cell therapy (ACT) or as recognition modules in soluble approaches, i.e., using bispecific molecules (Non-Patent Document 2).

[0006] However, simply increasing TCR affinity can also increase the risk of side effects. As mentioned above, in nature, high-affinity TCRs against tumor-associated antigens (self-proteins) are eliminated by thymic selection to avoid recognition of self-peptides present on normal tissues through cross-reactivity. Therefore, simply increasing TCR affinity for its target sequence may also increase affinity for similar non-cancer-specific peptides, thus increasing the risk of cross-reactivity and undesirable cytotoxic effects on normal tissues. This is not just a theoretical risk; it has been painfully observed with engineered TCRs targeting melanoma-associated antigen A3 (MAGE-A3). In particular, previously published results have shown fatal toxicity in two patients infused with engineered T cells expressing TCRs targeting MAGE-A3, which cross-react with peptides from the muscle protein titin, despite preclinical studies predicting no cross-reactivity (Non-Patent Document 3). These patients demonstrated that TCR-engineered T cells can have severe and unpredictable off-target and organ-specific toxicities. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Aleksic et al.2012,Eur J Immunol.2012 Dec;42(12):3174-9 [Non-patent document 2] Hickman et al.2016,J Biomol Screen.2016 Sep;21(8):769-85 [Non-patent document 3] Linette GP et al.Blood 2013;122:863-71,Cameron BJ,et al.Sci.Transl.Med.2013;5:197-103 Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, despite advances in TCR technology, there remains a need for additional therapeutic agents, particularly cancer therapeutic agents, that efficiently target and kill diseased cells, particularly cancer cells, while maintaining a high safety profile.

[0009] As shown herein above, natural TCRs often have fairly low affinity for their target TAA / MHC complexes, which avoids the recognition of self-peptides present on normal tissues through cross-reactivity. However, it is preferable to increase the affinity for the target TAA / MHC complex to create an efficient anti-cancer drug. [Means for solving the problem]

[0010] To address this issue, the present inventors have combined in one molecule an affinity-matured TCR variable domain that binds to a TA / MHC of interest with variable light and heavy chain domains that target CD3 with lower affinity than prior art anti-CD3 heavy and light chain variable domains. The resulting molecule has the advantage of recognizing diseased cells, such as cancer cells, even when TAs, such as TAAs, are present at the cell surface in low abundance, while maintaining a high safety profile. In particular, thanks to the significantly lower affinity of the CD3-binding domain, the resulting molecule mimics the natural T cell / molecule (TCR) / TA relationship because, in the bispecific antigen-binding proteins of the present invention, low-affinity binding occurs at the interface between the T cell and the CD3-binding domain of the bispecific antigen-binding protein, rather than at the interface between the TCR and the TA / MHC complex, as is the case for natural TCRs expressing T cells that bind to the TA / MHC.

[0011] Thus, one technically advantageous effect of using a low-affinity CD3-binding domain is that the resulting bispecific antigen-binding protein is specific for the TA of interest while having a high safety profile due to the use of a high-affinity TCR variable domain, i.e., a safety margin in which killing cells of normal or healthy tissue, such as cells of normal tissue expressing the off-target peptide, requires about 1000 times or more the dose used to treat TA-presenting cells, such as cancer cells. Thus, the combination of a high-affinity antigen-binding protein with a low-affinity CD3-binding domain results in specific binding to the target peptide with reduced or no cross-recognition of off-target peptides, for example, on healthy tissue, such as off-target peptides, thus providing a surprisingly large safety margin.

[0012] Thus, bispecific antigen-binding proteins of the invention that combine a low-affinity CD3-binding domain with an affinity-matured TA / MHC-binding domain have the advantage that the resulting bispecific antigen-binding protein efficiently targets diseased cells but not healthy cells and also has a favorable or even improved safety profile. Advantageously, the resulting bispecific antigen molecule further has increased stability and / or increased solubility compared to bispecific molecules using anti-CD3 domains known in the art, providing promising bispecific molecules suitable for medical applications.

[0013] In summary, the CD3 binding domains of the present invention, when used in combination with TCRs or MHC-peptide complex binding fragments thereof or antigen binding proteins, e.g., in a bispecific format, offer, inter alia, the following advantages to the art: (i) reduced cross-reactivity of a given TCR or antigen binding protein with similar peptides on healthy tissues, while maintaining high tumor selectivity and / or specificity; (ii) an improved safety profile of the TCRs or MHC-peptide complex binding fragments thereof, or antigen binding proteins; (iii) reduced off-target and extra-tumor cytotoxicity of the TCRs or MHC-peptide complex binding fragments thereof, or antigen binding proteins; and (iv) improved specific, selective, and safe provision of TCRs or MHC-peptide complex binding fragments thereof, or antigen binding proteins.

[0014] definition The term "antigen-binding protein" as used herein refers to a polypeptide or binding protein that is capable of binding to at least one antigen.

[0015] The term "antigen" as used herein refers to a molecule, or part of a molecule or complex, capable of being bound by at least one antigen-binding site, wherein said one antigen-binding site is present, for example, in a conventional antibody, a conventional TCR, and / or a bispecific antigen-binding protein of the invention.

[0016] A "bispecific antigen-binding protein" of the present invention has at least two valencies and binding specificities for at least two different antigens, with antigen-binding site A binding to CD3 and antigen-binding site B binding to a target antigen (TA) peptide / MHC complex.

[0017] In the context of the present invention, it is preferred that antigen-binding site A specific for CD3 is derived from a novel humanized version of the murine monoclonal antibody UCHT1, more particularly from an improved humanized version of the murine monoclonal antibody UCHT1, and antigen-binding site B is derived from a TCR.

[0018] The "bispecific antigen-binding proteins" of the invention are also referred to herein as "antigen-binding proteins of the invention", which comprise at least six CDRs as defined in the context of the present invention, more preferably antigen-binding protein V derived from an improved humanized UCHT1 antibody. L and V H Domain, especially V L and V H Antigen-binding site B in the context of the present invention binds to a target antigen (TA) peptide / MHC complex, in particular a tumor-associated antigen (TAA) peptide / MHC complex, and may be derived from an antibody or a TCR, preferably a TCR. Thus, in a preferred embodiment, the antigen-binding protein of the present invention comprises a bispecific antigen-binding site B that binds to a target antigen (TA) peptide / MHC complex, in which said antigen-binding site B preferably comprises at least one variable alpha domain (v alpha ) derived from a TCR. α ) and at least one variable β domain (v β )

[0019] Bispecific antigen-binding proteins may also be referred to herein as "bispecific molecules."

[0020] As used in the context of the present invention, a "target antigenic (TA) peptide" refers to a peptide that has been isolated and identified from infected or neoplastic material, such as material isolated from an individual with tuberculosis, an Epstein-Barr virus infection, or cancer. The protein from which the TA peptide is derived undergoes antigen processing in infected or tumor cells and is then presented on the cell surface by MHC molecules and cells, particularly TA peptide / MHC complexes, and is then recognized by host immune effector cells, such as T cells or NKT cells. A TA peptide in the context of the present invention comprises or consists of 10, 12, or 14 amino acids, e.g., 8-14, 8-12, or 9-11. When a specific TA peptide is referred to in the context of the present invention, it is referred to as a TA-C. Examples of TA antigenic peptides, such as TA-C peptides, are viral antigenic peptides, bacterial antigenic peptides, or tumor-associated antigen (TAA) antigenic peptides, preferably TAA antigenic peptides. Thus, in one embodiment, the TA antigenic peptide, particularly TA-C, is a viral peptide, a bacterial peptide, or a tumor-associated antigen (TAA) antigenic peptide, preferably a TAA antigenic peptide.

[0021] A "viral antigenic peptide" in the context of the present invention refers to an antigenic peptide of viral origin that is presented by MHC molecules on the surface of diseased cells, i.e., cells typically infected with said viruses. Such viral antigenic peptides have been found in association with infections from, for example, human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), cytomegalovirus (CMV), human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), and influenza virus. Thus, a viral antigenic peptide in the context of the present invention may be an antigenic peptide selected from the group consisting of HIV antigenic peptides, HCMV antigenic peptides, CMV antigenic peptides, HPV antigenic peptides, HBV antigenic peptides, HCV antigenic peptides, EBV antigenic peptides, and influenza antigenic peptides, preferably HIV, HBV, influenza, and HCMV antigenic peptides.

[0022] Viral antigenic peptides that can be used in the methods and embodiments described herein include, for example, the viral antigenic peptides set forth in the table below. Viral antigenic peptides that can be used in the methods and embodiments described herein include at least one viral antigenic peptide set forth in Table 1 below, comprising or consisting of an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 146 to 148.

[0023] Table 1: List of viral antigenic peptides [Table 1]

[0024] A "bacterial antigenic peptide" in the context of the present invention is an antigenic peptide of bacterial origin that is presented by MHC molecules on the surface of diseased cells, i.e., cells typically infected with said bacteria. Such bacterial antigenic peptides are found, for example, in the context of infections from Mycobacterium tuberculosis. Thus, a bacterial antigenic peptide in the context of the present invention may be a Mycobacterium tuberculosis antigenic peptide.

[0025] A "tumor-associated antigen (TAA) peptide," also referred to herein as a "TAA peptide," refers to a peptide isolated and identified from tumor material, undergoes antigen processing in tumor cells, and can therefore be recognized by host immune effector cells. A TAA peptide comprises or consists of 10, 12, or 14 amino acids, e.g., 8-14, 8-12, or 9-11 amino acids. A TAA peptide in the context of the present invention can be, for example, a cancer / testis (CT) antigenic peptide. Examples of cancer / testis (CT) antigenic peptides are the MAGE-A antigenic peptide of the amino acid sequence of SEQ ID NO: 10 and the PRAME antigenic peptide of the amino acid sequence of SEQ ID NO: 9. A TAA peptide in the context of the present invention comprises a T-cell epitope and may also be referred to in a general context as a TAA peptide, or, when referring to one specific TAA peptide, as a TAA peptide C in the context of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] In one aspect, tumor associated antigens, TAA peptides that can be used in the methods and embodiments described herein include, for example, those described in U.S. Patent Application Publication No. 20160187351, U.S. Patent Application Publication No. 20170165335, U.S. Patent Application Publication No. 20170035807, U.S. Patent Application Publication No. 20160280759, U.S. Patent Application Publication No. 20160287687, U.S. Patent Application Publication No. 20160346371, U.S. Patent Application Publication No. 20160368965, U.S. Patent Application Publication No. 20170022251, U.S. Patent Application Publication No. 20170002055, U.S. Patent Application Publication No. 20170029486, U.S. Patent Application Publication No. 20170037089, U.S. Patent Application Publication No. 20170 and US Patent Publication Nos. 20170173132, 20170296640, 20170253633, 20170260249, 20180051080, and 20180164315, the contents of each of which publications and the sequence listings set forth therein are incorporated herein by reference in their entirety.

[0027] In one aspect, a bispecific antigen-binding protein described herein, and particularly antigen-binding site B in the context of the present invention, selectively recognizes cells presenting a TAA peptide described in one or more of the above patents and publications. In another aspect, a TAA that can be used in the methods and embodiments described herein includes at least one TAA consisting of an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 52-65, 67-96, 98-110, SEQ ID NOs: 172-182, 184-268, SEQ ID NOs: 9 and 10, preferably SEQ ID NOs: 9 and 10. In one aspect, a bispecific antigen-binding protein, and particularly antigen-binding site B of a bispecific antigen-binding protein, selectively recognizes cells presenting a TAA peptide / MHC complex, wherein the TAA peptide comprises or consists of the amino acid sequence of SEQ ID NOs: 52-65, 67-96, 98, SEQ ID NOs: 172-182, 184-268, SEQ ID NOs: 9 and 10, or any of the amino acid sequences described in the patents or patent applications described herein, preferably SEQ ID NOs: 9 and 10.

[0028] Table 2: List of TAAs [Table 2-1] [Table 2-2]

[0029] Furthermore, a TAA antigenic peptide in the context of the present invention is a specific ligand for an MHC class I molecule or an MHC class II molecule, preferably an MHC class I molecule.

[0030] The TAA antigenic peptide C in the context of the invention is preferably selected from the group of TAA antigenic peptides consisting of the amino acid sequences of SEQ ID NOs: 52 to 65, 67 to 96, 98 to 110, SEQ ID NOs: 172 to 182, 184 to 268, SEQ ID NO: 9 and SEQ ID NO: 10, preferably the PRAME antigenic peptide comprising or consisting of the amino acid sequence "SLLQHLIGL" of SEQ ID NO: 9, or the MAGE-A antigenic peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 10, more preferably the MHC is preferably HLA-A. * The number is 02.

[0031] "PRAME," or "preferentially expressed antigen in melanoma," was first identified as an antigen overexpressed in melanoma (Ikeda et al. Immunity. 1997 Feb;6(2):199-208); it is also known as CT130, MAPE, and OIP-4 and has Uniprot accession number P78395 (available as of January 11, 2019). This protein functions as a repressor of retinoic acid receptor signaling (Epping et al., Cell. 2005 Sep 23;122(6):835-47). PRAME belongs to a family of germline-encoded antigens known as cancer-testis antigens. Cancer-testis antigens are attractive targets for immunotherapeutic intervention because they typically have restricted or no expression in normal adult tissues. PRAME is expressed in several solid tumors, as well as in leukemias and lymphomas (Doolan et al Breast Cancer Res Treat. 2008 May;109(2):359-65; Epping et al Cancer Res. 2006 Nov 15;66(22):10639-42; Ercolak et al Breast Cancer Res Treat. 2008 May;109(2):359-65; Matsushita et al Leuk Lymphoma. 2003 Mar;44(3):439-44; Mitsuhashi et al Int. J Hematol. 2014;100(1):88-95; Proto-Sequeire et al Leuk Res. 2006 Nov;30(11):1333-9; Szczepanski et al Oral Oncol. 2013 Feb;49(2):144-51; Van Baren et al Br J Haematol.1998 Sep;102(5):1376-9).The PRAME targeted therapy of the present invention may be particularly suitable for the treatment of cancers including, but not limited to, lung cancer, such as non-small cell lung cancer and small cell lung cancer, liver cancer, head and neck cancer, skin cancer, renal cell cancer, brain cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, leukemia, breast cancer, Merkel cell carcinoma, melanoma, ovarian cancer, bladder cancer, uterine cancer, gallbladder and bile duct cancer, and esophageal cancer.

[0032] A "PRAME-derived peptide" in the context of the present invention comprises or consists of the amino acid sequence SLLQHLIGL (SEQ ID NO: 9), which corresponds to amino acids 425-433 of the full-length PRAME protein of the amino acid sequence of SEQ ID NO: 7, accessible under Uniprot accession number P78395 (available as of January 11, 2019). A PRAME-derived peptide comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 9) is also referred to herein as PRAME-004. The PRAME-004 peptide is a peptide epitope derived from a tumor-associated or tumor-specific protein and is presented on the cell surface by molecules of the major histocompatibility complex (MHC). More specifically, the PRAME-004-derived peptide binds to HLA-A * It is presented on the cell surface in a complex with .02 (Med. 2001 Jan 1;193(1):73-88). In the context of the present invention, "PRAME-derived peptide" or "PRAME-004" are used synonymously and thus refer to a PRAM-derived peptide comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 9).

[0033] The "MAGE-A" or "melanoma-associated antigen A" subfamily proteins were the first tumor-associated antigens identified at the molecular level (van der Bruggen P, et al. Science. 1991;254:1643-47). MAGE-A is a subfamily of 12 genes (MAGE-A1-A12) located in the q28 region of the X chromosome. Members of the MAGE-A subfamily proteins are normally expressed only in the testis or placenta, and their restricted expression suggests that they may function in germ cell development. MAGE-A proteins have also been detected in the early development of the central nervous system, spinal cord, and brainstem, suggesting that MAGE-A proteins may also be involved in neurogenesis. Members of this family encode proteins with 50-80% sequence identity to each other, and all MAGE proteins share a common MAGE homology domain (MHD), a highly conserved domain consisting of approximately 170 amino acids. The biological functions of MAGE-A protein expression in cancer and the underlying regulatory mechanisms are still not fully understood.

[0034] The "MAGE-A4" or "melanoma-associated antigen 4" protein is a member of the MAGE-A gene family and has Uniprot accession number P43358 with SEQ ID NO: 111 (available as of July 8, 2019). MAGE-A4 localization has been described as cytoplasmic. However, MAGE-A4 staining has also been detected in the nucleus, with differential distribution between the nucleus and cytoplasm in well-differentiated versus poorly differentiated cancers (Sarcevic B et al., 2003, Oncology 64, 443-449). MAGE-A4 is used as a male germ cell marker. It is not expressed in spermatogonia, but is expressed in prespermatogonia and mature germ cells (Mitchell et al., 2014, Mod. Pathol. 27, 1255-1266). MAGE-A4 protein and mRNA expression have been associated with the development and prognosis of various cancers.

[0035] "MAGE-A8" or "melanoma associated antigen 8" protein is a member of the MAGE-A superfamily and has Uniprot accession number P43361 with SEQ ID NO: 112 (available as of July 2019).

[0036] The "MAGE-A4" and "MAGE-A8" proteins share 72% sequence identity as determined by protein sequence alignment using the BLASTP 2.9.0 algorithm (Stephen F et al. (1997) Nucleic Acids Res. 25:3389-3402). Furthermore, both "MAGE-A4" and "MAGE-A8" comprise the MAG-003 peptide, i.e., KVLEHVVRV (SEQ ID NO: 10).

[0037] The term "epitope" as used in the context of the present invention comprises the terms "structural epitope" and "functional epitope." A "structural epitope" is an amino acid of an antigen, such as a peptide-MHC complex, that is covered by an antigen-binding protein when bound to the antigen. Typically, all amino acids of an antigen that are within 5 Å of any atom of an amino acid of the antigen-binding protein are considered to be covered. The structural epitope of an antigen may be determined by methods known in the art, including X-ray crystallography or NMR analysis. The structural epitope of an antibody typically comprises 20 to 30 amino acids. The structural epitope of a TCR typically comprises 20 to 30 amino acids. A "functional epitope" is a subset of amino acids that form a structural epitope, comprising amino acids of an antigen that are important for forming an interface with an antigen-binding protein of the invention, either by directly forming non-covalent bonds such as H-bonds, salt bridges, aromatic stacking, or hydrophobic interactions, or by indirectly stabilizing the antigen's bound conformation, as determined, for example, by mutational scanning. Typically, a functional epitope of an antigen to which an antibody binds comprises 4-6 amino acids. Typically, a functional epitope of a peptide-MHC complex comprises 2-6 amino acids of the peptide and 2-7 amino acids of the MHC molecule. Because MHCCI-presented peptides are typically 8-10 amino acids long, only a subset of the amino acids of each given peptide are part of the functional epitope of the peptide-MHC complex. In the context of the present invention, an epitope, in particular a functional epitope bound by a bispecific antigen binding protein of the invention, comprises or consists of a functional epitope comprising amino acids of the antigen required for the formation of the binding interface, and thus at least three, preferably at least four amino acids of the MAGE-A antigenic peptide of SEQ ID NO: 10.

[0038] In the context of the present invention, "CD3" refers to an antigen expressed on T cells as part of a multimolecular T cell receptor complex and consisting of at least three different chains: CD3ε, CD3δ, and CD3γ. CD3δ and CD3γ have low sequence identity and / or similarity with human CD3ε (similarities and identities are less than 20%). The "CD3ε / δ-complex" refers to the complex formed by CD3ε and CDR3δ. CD3ε also forms a complex with CDR3γ, the so-called "CD3ε / γ complex." For example, clustering of CD3 on T cells by immobilized anti-CD3 antibodies results in T cell activation similar to T cell receptor engagement, but independent of the typical specificity of the clone. "CD3ε" comprises three domains: an intracellular domain, a transmembrane domain, and an extracellular domain.

[0039] The "UCHT1" monoclonal antibody in the context of the present invention specifically binds to the complex of human CD3δ chain and CD3ε chain, a 36 kDa subunit of the CD3 / T cell antigen receptor complex, referred to herein as the CD3ε / δ-complex. The murine monoclonal antibody UCHT-1 comprises a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 36 and a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 37. Humanization of UCHT1 has been described, for example, by Shalaby et al. (J. Exp. Med. (1992); 175(1):217-225), which was then further modified to result in the humanized variant 9 of UCHT1, referred to as hUCHT1(V9), described by Zhu et al. (J. Immunol., 1995, 155, 1903-1910). hUCHT1(V9) comprises a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 38 and a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 39. However, prior art variants of humanized UCHT have low solubility and are difficult to use in the molecular context of soluble molecules. Furthermore, these prior art variants have high affinity for CD3, which may be disadvantageous as discovered in the context of the present invention.

[0040] In the context of the present invention, "BMA031" refers to the monoclonal antibody (mAb) WT31 specific for human α / β TCR. Different humanized variants have been disclosed in the art, including the α / β TCR-specific humanized antibody BMA031 described by Shearman et al. (J Immunol, 1991, 147, 4366-73). The humanized antibody described by Sherman et al. (J Immunol, 1991, 147, 4366-73) comprises or consists of the amino acid sequence of SEQ ID NO: 40. L domain and a V domain comprising or consisting of the amino acid sequence of SEQ ID NO: 41. H and a domain.

[0041] In the context of this invention, the "major histocompatibility complex" (MHC) is a set of cell surface proteins essential for the adaptive immune system to recognize foreign molecules in vertebrates, which in turn determines histocompatibility. The primary function of MHC molecules is to bind to antigens derived from pathogens and present them on the cell surface for recognition by appropriate T cells. Human MHC is also referred to as the HLA (human leukocyte antigen) complex (often simply HLA). The MHC gene family is divided into three subgroups: class I, class II, and class III. Peptide-MHC class I complexes are recognized by CD8+ T cells bearing the appropriate T cell receptor (TCR), while peptide-MHC class II molecule complexes are recognized by CD4+ helper T cells bearing the appropriate TCR. Because both CD8- and CD4-dependent responses synergistically contribute to antitumor effects, the identification and characterization of tumor-associated antigens and corresponding T cell receptors are important for the development of cancer immunotherapies, such as vaccines and cell therapies. The HLA-A gene, located on the short arm of chromosome 6, encodes the larger α chain of HLA-A. Diversity in the HLA-A α chain is key to HLA function. This diversity promotes genetic diversity within a population. Because each HLA has a different affinity for peptides with specific structures, the more HLA variants there are, the greater the variety of antigens "presented" on cell surfaces. Each individual can express up to two HLA-A types, one from each parent. Some individuals inherit identical HLA-A alleles from both parents, reducing individual HLA diversity; however, the majority of individuals inherit two distinct copies of HLA-A. This same pattern continues across all HLA groups. In other words, each individual expresses only one or two of the 2,432 known HLA-A alleles.

[0042] An MHC class I HLA protein in the context of the present invention is an HLA-A, HLA-B or HLA-C protein, preferably an HLA-A protein, more preferably an HLA-A * It could also be 02.

[0043] "HLA-A* 02" represents a specific HLA allele, in which the letter A represents the gene and the suffix " * "02" indicates the A2 serotype.

[0044] In an MHC class I-dependent immune response, peptides must not only be able to bind to specific MHC class I molecules expressed by tumor cells, but they must also be subsequently recognized by T cells bearing specific T cell receptors (TCRs).

[0045] A "TCR," in the context of this invention, is a heterodimeric cell surface protein of the immunoglobulin superfamily, which is associated with the invariant protein of the CD3 complex, which is involved in mediating signal transduction. TCRs exist in αβ and γδ forms, which are structurally similar but have quite different anatomical locations and likely functions. The extracellular portions of naturally occurring heterodimeric αβ and γδ TCRs each contain two polypeptides, each of which has a membrane-proximal constant domain and a membrane-distal variable domain. Each of the constant and variable domains contains intrachain disulfide bonds. The variable domains contain highly polymorphic loops similar to the complementarity-determining regions (CDRs) of antibodies.

[0046] The term "TCR" herein refers to TCRs and fragments thereof, as well as single chain TCRs and fragments thereof, in particular the variable α and β domains of single domain TCRs, and chimeric, humanized, bispecific or multispecific TCRs.

[0047] A "fragment of a TCR" comprises a portion of an intact or naturally occurring TCR, in particular the antigen binding or variable region of an intact or naturally occurring TCR. Examples of TCR fragments include the V α -C a or V β -C β Such fragments include fragments or portions of the α, β, δ, and γ chains, such as the corresponding hinge region; or V α , V β , V δ , Vγ single-chain VαVβ fragments; bispecific and multispecific TCRs formed from TCR fragments. TCR fragments perform the same function as naturally occurring full-length TCRs, i.e., they selectively and specifically bind to their target peptides.

[0048] "Single chain TCR (scTCR)" refers herein to a protein, in which V α and V β , or V δ and V γ The variable domains of a TCR, such as the TCR V1, V2, V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, V13, V14, V15, V16, V17, V18, V19, V20, V21, V22, V23, V24, V25, V26, V27, V28, V29, V30, V31, V32, V33, V34, V35, V36, V37, V38, V39, V40, V41, V42, V43, V44, V45, V46, V47, V48, V49, V49, V50, V51, V52, V53, V54, V55, V56, V57, V58, V59, V60, V61, V62, V63, V64, V65, V66, V67, V68, V69, V69, V70, V71, V72, V73, V74, V75, V76, V77, V78, V79, V79, V79, V80, V81, V82, V83, V84, V85, V86, V87, V88, V89, V90, V91, V92, V93, V94, V95, V96, V97, V98, V99, V99, V91, V91, V91, V92, V93, V94, V95, V96, V97, V98, V99, V99, V91, V99, V91, V91, V91, V92, V93, V94, V95, V96, V9

[0049] For example, "native" as used in the phrase "native TCR" refers to a wild-type TCR. A native α-β heterodimeric TCR has an α chain and a β chain. Each α chain comprises a variable region, a joining region, and a constant region; the β chain usually also contains a short diversity region between the variable region and the joining region, although this diversity region is often considered part of the joining region. The constant regions or C regions of the TCR α chain and β chain are referred to as TRAC and TRBC, respectively (Lefranc, (2001), Curr Protoc Immunol Appendix 1:Appendix 10). Each variable region, referred to herein as the α variable domain and β variable domain, comprises three "complementarity-determining regions" (CDRs) embedded in framework sequences, one of which is a hypervariable region designated CDR3. The α variable domain CDRs are referred to herein as CDRa1, CDRa2, and CDRa3, and the β variable domain CDRs are referred to herein as CDRb1, CDRb2, and CDRb3. There are several types of α chain variable (Vα) regions and several types of β chain variable (Vβ) regions, distinguished by their frameworks, CDR1 and CDR2 sequences, and by partially defined CDR3 sequences. Vα types are referred to by unique TRAV numbers in the IMGT nomenclature, and Vβ types are referred to by unique TRBV numbers in the IMGT nomenclature (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1):42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2):83-96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). For more information about immunoglobulin genes, see the international ImMunoGeneTics information system®, Lefranc M-Pbet et al (Nucleic Acids Res. 2015 Jan;43(Database Publication):D413-22; and http: / / www.imgt.org / ).Thus, a conventional TCR antigen-binding site typically contains six CDRs, comprising a set of CDRs from each of the α and β chain variable regions, in which the CDR1 and CDR3 sequences are involved in recognizing and binding to peptide antigens that bind to HLA proteins, and the CDR2 sequence is involved in recognizing and binding to HLA proteins.

[0050] As with antibodies, "TCR framework region" (FR) refers to the amino acid sequences interposed between the CDRs, i.e., portions of the TCR α and β chain variable regions that are conserved to some extent among different TCRs within a single species. The α and β chains of a TCR each have four FRs, referred to herein as FR1-a, FR2-a, FR3-a, and FR4-a, and FR1-b, FR2-b, FR3-b, and FR4-b, respectively. Thus, the α chain variable domain may be referred to as (FR1-a)-(CDRa1)-(FR2-a)-(CDRa2)-(FR3-a)-(CDRa3)-(FR4-a), and the β chain variable domain may be referred to as (FR1-b)-(CDRb1)-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)-(FR4-b).

[0051] In the context of the present invention, the CDR / FR definitions in the α or β chain or the γ or δ chain are to be determined based on the IMGT definition (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org). Therefore, the CDR / FR amino acid positions when related to a TCR or TCR-derived domain are indicated according to the above-mentioned IMGT definition. In one example, the IMGT positions of the CDR / FR amino acid positions of the first variable domain are indicated by analogy with the IMGT numbering of TRAV5, and / or the IMGT positions of the CDR / FR amino acid positions of the second variable domain are indicated by analogy with the IMGT numbering of TRBV12-4, such as in the variable domain of antigen-binding site B directed against the MAGE-A antigenic peptide of SEQ ID NO: 10.

[0052] With respect to gamma / delta TCRs, the term "TCRgamma variable domain" as used herein refers to the junction of the TCRgamma V (TRGV) region without the leader region (L) and the TCRgamma J (TRGJ) region, and the term TCRgamma constant domain refers to the extracellular TRGC region or a C-terminally truncated TRGC sequence. Similarly, the term "TCRdelta variable domain" refers to the junction of the TCRdelta V (TRDV) region without the leader region (L) and the TCRdelta D / J (TRDD / TRDJ) region, and the term "TCRdelta constant domain" refers to the extracellular TRDC region or a C-terminally truncated TRDC sequence.

[0053] In "antibodies," also called "immunoglobulins," two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (l) and kappa (k). There are five major heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. Light chains contain a variable domain (V L ) and constant domain (C L The heavy chain contains two domains or regions: a variable domain (V H ) and three constant domains (collectively C H It is called C H1 , C H2 , and C H3 ) and four domains. L ) and heavy chain (V H The variable regions of both the light chain (C) and the heavy chain (C) determine antigen binding recognition and specificity. L ) and heavy chain (C H The constant region domains of the antibody are responsible for antibody chain binding, secretion, placental transport, complement fixation, and F c Receptor (F cThe Fv fragment is the N-terminal portion of an immunoglobulin's Fab fragment and consists of one light chain variable portion and one heavy chain variable portion. Antibody specificity resides in the structural complementarity between the antibody combining site (synonymous with antibody combining site) and an antigenic determinant. The antibody combining site is primarily composed of residues from the hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) influence the overall domain structure and thus the binding site. Complementarity-determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, and CDR3-H, respectively. Thus, a conventional antibody antigen-binding site contains six CDRs comprising a set of CDRs from each of the heavy and light chain V regions.

[0054] In the context of the present invention, an antibody or immunoglobulin is IgM, IgD, IgG, IgA or IgE.

[0055] "Antibody framework region" (FR) refers to the amino acid sequences inserted between the CDRs, i.e., the portions of the immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins of a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Thus, the light chain variable domain may also be designated as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable domain may also be designated as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H).

[0056] In the context of the present invention, the definition of CDR / FR in an immunoglobulin light or heavy chain, particularly an immunoglobulin light or heavy chain of an anti-CD3 antibody variant in the context of the present invention, is determined based on Kabat (Kabat EA, Te, Wu T, Foeller C, Perry HM, Gottesman KS. (1992) Sequences of Proteins of Immunological Interest.). However, the position numbering of CDR / FR amino acids in an immunoglobulin light or heavy chain, particularly an UCHT1 variant in the context of the present invention, is ordinal. Thus, for example, CDRH1 determined according to Kabat ranges from amino acid positions 31 to 35, CDRH2 determined according to Kabat ranges from amino acid positions 50 to 66, and CDRH3 determined according to Kabat ranges from amino acid positions 99 to 111. Thus, for example, CDRL1 determined according to Kabat ranges from amino acid positions 24 to 34, and CDRL2 determined according to Kabat ranges from amino acid positions 50 to 56.

[0057] As used herein, a "human framework region" is a framework region that is substantially identical (about 85% or more, particularly 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody or antigen-binding protein, such as a human TCR.

[0058] The term "antibody" refers to antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular the variable heavy chains of single domain antibodies, and chimeric, humanized, bispecific or multispecific antibodies.

[0059] A "conventional antibody" herein is an antibody that has the same domains as an antibody isolated from nature and comprises CDRs and framework regions derived from the antibody. Similarly, a "conventional TCR" referred to herein is a TCR that comprises the same domains as a naturally occurring TCR and CDRs and framework regions derived from a TCR.

[0060] The term "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and that has been modified to replace certain amino acids, particularly in the framework regions of the heavy and light chains of a non-human monoclonal antibody, in order to avoid or minimize a human immune response. The constant domains of a humanized antibody are primarily composed of human C H and C L It is a domain.

[0061] Many methods for humanizing antibody sequences are known in the art; see, for example, the review by Almagro & Fransson (2008) Front Biosci. 13:1619-1633. One commonly used method is CDR grafting, or antibody reshaping, which involves grafting the CDR sequences of a donor antibody, usually a mouse antibody, onto the framework scaffold of a human antibody of different specificity. Because CDR grafting can sometimes reduce the binding specificity and affinity, and therefore the biological activity, of the CDR-grafted non-human antibody, back mutations may be introduced at selected positions of the CDR-grafted antibody to retain the binding specificity and affinity of the parent antibody. Identification of possible back mutation positions can be performed using information available in the literature and antibody databases. An alternative humanization technique to CDR grafting and back mutation is resurfacing, in which non-surface-exposed residues of the non-human origin are retained while surface residues are modified to human residues. Another alternative technique is known as "guided selection" (Jespers et al. (1994) Biotechnology 12, 899), which can be used to derive fully human antibodies from, for example, murine antibodies that retain the epitope and binding properties of the parent antibody. A further method of humanization is so-called 4D humanization. 4D humanization protocols are described, for example, in U.S. Patent Application Publication No. 20110027266A1 (WO 2009032661A1), which is incorporated herein by reference in its entirety. The monoclonal murine antibody UCHT1 in the context of the present invention has been humanized as described in detail in Example 1 herein. For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences.

[0062] "Vernier zone" in the context of the present invention refers to murine residues in the framework region that have been demonstrated to influence the conformation of the CDR loops and the affinity of the antibody. These residues, also called "Vernier residues," are located in the beta-sheet framework region immediately below the CDRs and are not involved in direct interactions with the antigen, i.e., these residues are retained in "humanized" antibodies.

[0063] Although amino acid residues that are part of CDRs are typically not modified in connection with humanization, in certain instances, it may be desirable to modify individual CDR amino acid residues, for example, to remove glycosylation sites, deamidation sites, isomerization sites, or unwanted cysteine ​​residues. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X can be any amino acid except Pro. Removal of N-glycosylation sites may be achieved by mutating either the Asn or Ser / Thr residue to a different residue, particularly through conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation when present primarily in the sequence Asn-Gly, and less so in other dipeptide sequences such as Asn-Ala. Therefore, where such a deamidation site, particularly Asn-Gly, is present in a CDR sequence, it may be desirable to eliminate the site by removing one of the involved residues, typically by conservative substitution. Substitutions in the CDR sequence to remove one of the involved residues are also intended to be encompassed by the present invention.

[0064] An "antibody fragment" in the context of the present invention comprises a portion of an intact antibody, in particular the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, and bispecific and multispecific antibodies formed from antibody fragments. An antibody fragment may also be a heavy chain antibody or a single domain antibody such as a VHH.

[0065] The term "Fab" refers to an antibody fragment having a molecular weight of approximately 50,000 daltons and antigen-binding activity, in which approximately the N-terminal half of the heavy chain and the entire light chain are linked together via disulfide bonds, for example, a fragment obtained by treating IgG with a protease such as papain.

[0066] The term "morphology" in the context of the present invention refers to a bispecific antigen-binding protein that comprises a particular number and type of domains present in the bispecific antigen-binding protein and its spatial organization.

[0067] Many different forms, such as bispecific forms, have been described in the art in the context of antibodies, and such forms typically include diabodies, crossover dual variable domain (CODV) and / or dual variable domain (DVD) proteins. An overview of these different bispecific antibodies and methods for producing them is disclosed, for example, in Brinkmann U. and Kontermann REMAbs. 2017 Feb-Mar;9(2):182-212. More specifically, DVD forms are disclosed, for example, in the following scientific papers (Wu C et al. Nat Biotechnol 2007;25:1290-7; PMID:17934452; Wu C. et al. MAbs 2009;1:339-47; Lacy SE et al. MAbs 2015;7:605-19; PMID:25764208; Craig RB et al. PLoS One 2012;7:e46778; PMID:23056448; Piccione EC et al. MAbs 2015). CODV is disclosed, for example, in Onuoha SC et al. Arthritis Rheumatol. 2015 Oct;67(10):2661-72, or, for example, in WO 2012 / 135345 and WO 2016 / 116626. Bispecific diabodies are described, for example, in Holliger P et al. Protein Eng 1996;9:299-305; PMID:8736497; Atwell JL et al. Mol Immunol 1996;33:1301-12; PMID:9171890; Kontermann RE, Nat Biotechnol 1997;15:629-31; PMID:9219263; Kontermann RE et al. Immunotechnology 1997;3:137-44; PMID:9237098; Cochlovius B et al. Cancer Res 2000;60:4336-41; PMID:10969772; and DeNardo DG et al. Cancer Biother Radiopharm 2001;16:525-35;PMID:11789029.

[0068] A "bispecific antibody" as used in the context of antibodies refers to a bivalent molecule composed of two chains, each comprising a VH and a VL domain, typically from the same or different antibodies. The two chains typically have the configurations VHA-VLB and VHB-VLA (A and B representing two different specificities), or VLA-VHB and VLB-VHA.

[0069] A "diabody (Db)" or "diabody format" in the context of the present invention is defined herein as a bispecific antibody, each of which is formed by linking a linker (L Db1 and L Db2 ), in which two of the domains are the first and second domains (V1 and V2) as defined in the context of the present invention, and the other two domains are TCR- or antibody-derived variable domains (V A , V B The V1V2 domains may be located on two different polypeptides, and the V A V B The domains are located on two different polypeptides, and the domains dimerize in a head-to-tail orientation. Thus, the orientation is V1-L. Db1 -V A and V B -L Db2 -V2, V2-L Db1 -V A and V B -L Db2 -V1, V1-L Db1 -V B and V A -L Db2 -V2, or V2-L Db1 -V B and V A -L Db2 -V1. To allow the domains to dimerize in a head-to-tail orientation, a linker, i.e., L Db1 and L Db1can be the same or different and are short linkers. Short linkers are typically between 2 and 12 amino acids in length, 3 and 13 amino acids in length, e.g., 4, 5, 6, 7, 8, 9 amino acids in length (Brinkmann U. and Kontermann RE (MAbs. 2017 Feb-Mar;9(2):182-212)), or 8 amino acids in length, such as "GGGS" in SEQ ID NO: 114, "GGGGS" in SEQ ID NO: 115, or "GGGSGGGG" in SEQ ID NO: 118.

[0070] The "dual variable domain immunoglobulin (DVD-Ig™)" format was first described in 2007 by Wu C. et al. (Nat Biotechnol. 2007 Nov;25(11):1290-7). In this format, the target-binding variable domain of a second monoclonal antibody (B) is typically a target-binding variable domain of a conventional antibody (A) (domain V). LA and V HA ) in which the light chain of the conventional antibody (A) is thus fused to an additional light chain variable domain (V LB ), and the heavy chain of the conventional antibody (A) comprises an additional heavy chain variable domain (V HB DVD-Ig™ as described in the art therefore typically comprises V HB -LV HA -C H1 -C H2 -C H3 and one heavy chain comprising V LB -LV LA -C L It is composed of two polypeptide chains with one light chain comprising domain pair V LA / V HA and V LA / V LA is therefore involuted in parallel.

[0071] A "dual variable domain Ig form" in the context of the present invention refers to a protein comprising two polypeptide chains each comprising two variable domains, linked by a linker (L1, L3), in which two of the domains are the first and second domains (V1 and V2) as defined in the context of the present invention, and the other two domains are the heavy and light chain variable domains (V HA and V HB In the context of the present invention, in the DVD-Ig form, the polypeptide chains are, for example, V1-L1-V HA -L2-C H1 -C H2 -C H3 and V2-L3-V LA -L4-C L , or V2-L1-V HA -L2-C H1 -C H2 -C H3 and V1-L3-V LA -L4-C L The connecting linkers L1 and L3 are preferably between 5 and 20 amino acid residues in length, such as 5 to 15 amino acid residues, and / or the connecting linkers L2 and L4 may or may not be present.

[0072] A "crossover dual variable domain Ig-like protein" as described in the art in the context of antibodies is a protein that contains two V H and two V's L The domain is variable V H -V L are linked in a manner that allows crossover pairing of the domains, HA -V HB and V LB -V LA order, or V HB -V HA and V LA -V LB This corresponds to a form in which the amino acids are arranged in either order (from the N- to C-terminus).

[0073] In the context of the present invention, a "crossover dual variable domain Ig-like protein" refers to a protein comprising two polypeptide chains, each comprising two variable domains connected by a linker (L1, L2, L3 and L4), in which two domains are the first and second domains (V1 and V2) as defined in the context of the present invention, and the other two domains are heavy and light chain variable domains (V) derived from an antibody. HA , V HB In the context of the present invention, in the CDVD-Ig form, the polypeptide chain is, for example, V1-L1-V HA -L2-C H1 -C H2 -C H3 and V LA -L3-V2-L4-C L , V2-L1-V HA -L2-C H1 -C H2 -C H3 and V LA -L3-V1-L4-C L、 V HA -L1-V1-L2-C H1 -C H2 -C H3 and V2-L3-V LA -L DVD3 -C L , or V HA -L1-V2-L2-C H1 -C H2 -C H3 and V1-L3-V LA -L4-C LIn this CDVD configuration, the linkers (L1-L4) are typically of different lengths, including all-glycine linkers and those described below in the linker section of this specification. For example, L1 is 3-12 amino acid residues long, L2 is 3-14 amino acid residues long, L3 is 1-8 amino acid residues long, and L4 is 1-3 amino acid residues long; or L1 is 5-10 amino acid residues long, L2 is 5-8 amino acid residues long, L3 is 1-5 amino acid residues long, and L4 is 1-2 amino acid residues long; or L1 is 7 amino acid residues long, L2 is 5 amino acid residues long, L3 is 1 amino acid residue long, and L4 is 2 amino acid residues long.

[0074] "At least one" herein refers to one or more specific entities, such as 1, 2, 3, 4, 5, or 6 or more specific entities. For example, at least one binding site herein refers to 1, 2, 3, 4, 5, or 6 or more binding sites.

[0075] A sequence that is "at least 85% identical to a reference sequence" is a sequence that has 85% or more, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity over its entire length with the entire length of the reference sequence.

[0076] In the context of this application, "percent identity" is calculated using a global pairwise alignment (i.e., two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. For example, the "needle" program may be used, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimal alignment (including gaps) of two sequences considering their entire length. The Needle program is available, for example, on the World Wide Web and is further described in the following publications: EMBOSS: The European Molecular Biology Open Software Suite (2000); Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277). The percentage of identity between two polypeptides according to the invention is calculated using the EMBOSS:needle (global) program with a "Gap Open" parameter equal to 10.0, a "Gap Extend" parameter equal to 0.5, and a Blosum62 matrix.

[0077] A protein consisting of an amino acid sequence that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical" to a reference sequence may contain amino acid mutations, such as deletions, insertions, and / or substitutions, compared to the reference sequence. In the case of substitutions, a protein consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence may correspond to a homologous sequence derived from a different species than the reference sequence.

[0078] "Amino acid substitutions" may be conservative or non-conservative. Preferably, the substitutions are conservative, in which one amino acid is replaced by another amino acid having similar structural and / or chemical properties.

[0079] In one embodiment, conservative substitutions may include those described by Dayhoff in "The Atlas of Protein Sequence and Structure. Vol. 5," Natl. Biomedical Research, the entire contents of which are incorporated by reference. For example, in one embodiment, amino acids belonging to one of the following groups may be exchanged for one another, and thus constitute conservative exchanges: Group 1: alanine (A), proline (P), glycine (G), asparagine (N), serine (S), threonine (T); Group 2: cysteine ​​(C), serine (S), tyrosine (Y), threonine (T); Group 3: valine (V), isoleucine (I), leucine (L), methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K), arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H); and Group 6: aspartic acid (D), glutamic acid (E). In one aspect, conservative amino acid substitutions may be selected from T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G, and / or T→S.

[0080] In further embodiments, conservative amino acid substitutions may include the substitution of an amino acid with another amino acid of the same class, such as, for example, (1) non-polar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp; (2) uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) acidic: Asp, Glu; and (4) basic: Lys, Arg, His. Other conservative amino acid substitutions may also be made, such as: (1) aromatic: Phe, Tyr, His; (2) proton donor: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, e.g., U.S. Pat. No. 10,106,805, the entire contents of which are incorporated by reference).

[0081] In another embodiment, conservative substitutions may be made according to Table 3. Methods for predicting tolerance to protein modifications can be found, for example, in Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the entire contents of which are incorporated by reference.

[0082] Table 3: Conservative amino acid substitutions [Table 3]

[0083] In another embodiment, conservative substitutions may be those shown in Table 3 under the heading of "conservative substitutions." If such substitutions result in altered biological activity, more substantial changes, designated "exemplary substitutions" in Table 4, may be introduced and the products screened as appropriate.

[0084] Table 4: Amino acid substitutions [Table 4]

[0085] In some embodiments, the bispecific antigen-binding protein may comprise a mutant antigen-binding protein, wherein said mutant bispecific antigen-binding protein comprises a first polypeptide chain (such as an α chain) and a second polypeptide chain (such as a β chain) that comprise up to 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions, preferably in the CDR regions of the first variable domain (such as the Vα domain) and the second variable domain (such as the Vβ domain), compared to the bispecific antigen-binding protein from which the variant is derived. In this regard, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions in each of the CDR regions of the bispecific antigen-binding protein, or in all of the CDR regions of the first and / or second variable domains. The substitutions may be in the CDRs of either the first and / or second variable domain.

[0086] In one embodiment, the variant is a functional variant.

[0087] The term "functional variant", as used herein, refers to a bispecific antigen-binding protein that has substantial or significant sequence identity or similarity to a parent bispecific antigen-binding protein, such as a bispecific antigen-binding protein containing conservative amino acid substitutions, wherein said functional variant retains the biological activity of the parent bispecific antigen-binding protein. In one aspect, functional variants include, for example, variants of a bispecific antigen-binding protein described herein (the bispecific antigen-binding protein itself described herein is referred to as the parent antigen-binding protein) that retain the ability to recognize target cells to about the same extent, the same extent, or a greater extent than the parent bispecific antigen-binding protein. With respect to the parent bispecific antigen-binding protein, a functional variant may, for example, have an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence to the parent bispecific antigen-binding protein.

[0088] A functional variant may, for example, comprise the amino acid sequence of the parent bispecific antigen-binding protein containing at least one conservative amino acid substitution. Alternatively, or in addition, a functional variant may comprise the amino acid sequence of the parent bispecific antigen-binding protein with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent bispecific antigen-binding protein.

[0089] The modified TCRs, polypeptides, and antigen binding proteins (including functional portions, fragments, and functional variants) of the present invention can be of any length, i.e., comprise any number of amino acids, provided that the modified TCR, polypeptide, or protein (or functional portion or functional variant thereof) retains their biological activity, such as, for example, the ability to specifically bind to an antigen, the ability to detect diseased cells in a host, or the ability to treat or prevent disease in a host.

[0090] Bispecific antigen-binding proteins of the invention (including functional parts, fragments, and functional variants) may comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2- Carboxylic acids, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine may be mentioned.

[0091] In one embodiment, the bispecific antigen-binding proteins of the invention (including functional portions and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts, and / or optionally dimerized or polymerized, or conjugated.

[0092] The bispecific antigen-binding proteins of the present disclosure may be synthetic, recombinant, isolated, and / or purified.

[0093] As used herein, "covalent bond" refers to a disulfide bridge or a peptide bond, or a covalent bond, for example, via a linker or linker sequence, such as a polypeptide linker.

[0094] The term "linker" as used herein refers to one or more amino acid residues that are inserted between two domains, such as between the first and second variable domains, and optionally between the light chain variable domain and the heavy chain variable domain, of a bispecific antigen-binding protein of the invention in a single chain construct, to provide sufficient flexibility for the domains to fold correctly to form the antigen-binding site, or, in the case of a bispecific antigen-binding protein, to form the antigen-binding site and at least one further antigen-binding site, either in crossover pairing (in CODV format, or in some bispecific antibody formats) or parallel pairing configuration (e.g. in DVD format) of the bispecific antigen-binding protein of the invention.

[0095] In some embodiments, the linker consists of 0 amino acids, i.e., there is no linker. The linker is inserted at the transition between variable domains or between variable and constant domains at the amino acid sequence level, respectively. Because the approximate sizes of immunoglobulin domains and TCR domains are well understood, the transition between domains can be identified. Those skilled in the art know that the precise location of the domain transition can be determined by identifying peptide stretches that do not form secondary structure elements such as β-sheets or α-helices, as demonstrated by experimental data or as identified or assumed using modeling or secondary structure prediction techniques. The term linker as used in the context of the present invention refers to, but is not limited to, linkers designated L1, L2, L3, L4, L5, and L6.

[0096] Unless otherwise specified in the respective context, L1, L2, L3, L4, L5, and L 6、Linkers such as L1, L2, L3, L4, L5, and L6 can be at least 1-30 amino acids in length. In some embodiments, linkers such as L1, L2, L3, L4, L5, and L6 can be 2-25, 2-20, or 3-18 amino acids in length. In some embodiments, linkers such as L1, L2, L3, L5, and L6 can be 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids in length or less. In other embodiments, linkers such as L1, L2, L3, L4, L5, and L6 can be 5-25, 5-15, 4-11, 10-20, or 20-30 amino acids in length. In other embodiments, linkers such as L1, L2, L3, L4, L5, and L6 can be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In certain embodiments, linkers such as L1, L2, L3, L4, L5, and L6 can be less than 24, less than 20, less than 16, less than 12, or less than 10 amino acid residues in length, e.g., 5-24, 10-24, or 5-10 amino acid residues in length. In some embodiments, the linkers are one 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, or greater than 22 amino acid residues in length.Exemplary linkers such as L1, L2, L3, L4, L5, L6 are selected from the group consisting of TVAAP (SEQ ID NO:113), GGGS (SEQ ID NO:114), GGSGG (SEQ ID NO:28), GGGGS (SEQ ID NO:115), TVLRT (SEQ ID NO:116), TVSSAS (SEQ ID NO:117), GGGSGGGG (SEQ ID NO:118), GGGGSGGGGS (SEQ ID NO:119), GGGGSAAA (SEQ ID NO:120), GGSGGGGSGG (SEQ ID NO:29), GGSGGGGSGGGGSGG (SEQ ID NO:32), GGGGSGGGGSGGGGS (SEQ ID NO:121), GGGGSGGGGSGGGGSGGGGSGGGGSGS (SEQ ID NO:122), GGSGGGGSGGGGSGGGGSGG (SEQ ID NO:33), GGGGSGGGGSG or consisting of an amino acid sequence selected from the group consisting of the amino acid sequences: GGGSGGGGSGGGGS (SEQ ID NO: 123), GGSGGGGSGGGGSGGGGSGGGGSGG (SEQ ID NO: 66), GSADDAKKDAAKKDGKS (SEQ ID NO: 97), GGQGSGGTGSGGQGSGGTGSGGQGS (SEQ ID NO: 143), TVLSSAS (SEQ ID NO: 124), GGGGSGT (SEQ ID NO: 183), and GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 125), in particular the amino acid sequences: GGGSGGGG (SEQ ID NO: 118), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 125), and GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 123).

[0097] As used in the context of the present invention, c The term "domain" refers to a natural F domain, as further defined below. c Domain and F c Includes variants and sequences of domains. c Mutant and natural F c As with the numerator, "F c The term "domain" includes molecules in either monomeric or multimeric form, whether resolved from whole antibodies or generated by other means.

[0098] "Natural F cThe term "antigen-binding fragment" as used herein refers to a molecule comprising the sequence of a non-antigen-binding fragment obtained from the digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and which may contain a hinge region. c The original immunoglobulin source of the Fc is particularly of human origin and can be any immunoglobulin, preferably IgG1 or IgG2, most preferably IgG1. Native Fc molecules are composed of monomeric polypeptides that can be bound into dimeric or multimeric forms by covalent (i.e., disulfide) and non-covalent bonds. c The number of intermolecular disulfide bonds between the monomeric subunits of the molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). c An example of a natural F is the disulfide-linked dimer obtained from papain digestion of IgG. c The term " is used herein generically to monomeric, dimeric, and multimeric forms. c An example of an amino acid sequence is IGHG1 * 01 Natural F c The amino acid sequence is the amino acid sequence of sequence number 126.

[0099] "Hinge" or "hinge region" or "hinge domain" typically refers to a C H1 Domain and C H2 It refers to the flexible part of the heavy chain located between the domains. It is about 25 amino acids long and is divided into the "upper hinge," "middle hinge" or "core hinge," and "lower hinge." The "hinge subdomain" refers to the upper hinge, middle (or core) hinge, or lower hinge. The amino acid sequences of the hinges of IgG1, IgG2, IgG3, and IgG4 molecules are shown below: IgG1:E 216 PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 127) IgG2:E 216 RKCCVECPPCPAPPVAGP (SEQ ID NO: 128) IgG3:ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPE 216 PKSCDTPPPCPRCPAPELLG (SEQ ID NO: 129) IgG4:E 216 SKYGPPCPSCPAPEFLG (SEQ ID NO: 130).

[0100] In the context of the present invention, this refers to amino acid positions in the Fc domain, and 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).

[0101] "F c The term "variant" as used herein refers to a naturally occurring F c Although modified from F, the salvage receptor c Rn (neonatal F c refers to a molecule or sequence that still comprises a binding site for a target molecule (receptor). c Mutants and their interactions with salvage receptors are known in the art. c The term "mutant" refers to a non-human naturally occurring F c Furthermore, the natural F c comprises regions that can be removed because they provide structural features or biological activity that are not required for the bispecific antigen binding proteins of the invention. c The term "variant" refers to one or more naturally occurring F c or a molecule or sequence lacking one or more F c Sites or residues may be modified to prevent (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity upon expression in the selected host cell, (4) glycosylation, (5) interaction with complement, or (6) F other than the salvage receptor. creceptor binding, or (7) affecting or involved in antibody-dependent cellular cytotoxicity (ADCC).

[0102] Thus, in one embodiment, F c1 and / or F c2 The Fc domain, such as, comprises a hinge domain.

[0103] In one embodiment, the Fc domain is a human IgGFc domain, preferably from human IgG1, IgG2, IgG3 or IgG4, preferably IgG1 or IgG2, more preferably IgG1.

[0104] In some embodiments, in particular, the bispecific antigen binding protein comprises two Fc domains, i.e., in the TCER® form (F c1 and F c2 In some embodiments, the two Fc domains may be of the same immunoglobulin isotype or isotype subclass, or of different immunoglobulin isotypes or isotype subclasses, preferably the same. c1 and F c2 is the IgG1 subclass, or the IgG2 subclass, or the IgG3 subclass, or the IgG4 subclass, preferably the IgG1 subclass or the IgG2 subclass, more preferably the IgG1 subclass.

[0105] In some embodiments, the Fc domain is a variant Fc domain and thus comprises one or more amino acid substitutions as described herein below.

[0106] In some embodiments, F c The domain comprises or further comprises a "RF" and / or "knob-into-hole" mutation, preferably a "knob-into-hole".

[0107] "RF mutation" refers to a C mutation as described by Jendeberg, L. et al. (1997, J. Immunological Meth., 201:25-34). H3 amino acid substitutions H435R and Y436F in the F domain c Domain C H3 It generally refers to the amino acid substitution of amino acids HY in the Fc domain with RF, which is said to be advantageous for purification purposes as it abolishes binding to Protein A. When the bispecific antigen-binding protein comprises two Fc domains, the RF mutation may be in one or both, preferably in one of the Fc domains.

[0108] "Knob-in-hole" technology, also known as "knob-in-hole," is a technology that H3 -C H3 These "knob-into-hole" mutations, T366S, L368A, and Y407V (hole) and T366W (knob), promote heteromultimer formation at the interface. These "knob-into-hole" mutations can be further stabilized by the introduction of additional cysteine ​​amino acid substitutions, Y349C and S354C. The "knob-into-hole" technology, along with stabilizing cysteine ​​amino acid substitutions, is described in U.S. Pat. Nos. 5,731,168 and 8,216,805.

[0109] In the context of the present invention, a "knob" mutation is present in an Fc domain comprising, for example, the amino acid sequence of SEQ ID NO: 131 together with the cysteine ​​amino acid substitution S354C, and a "hole" mutation is present in an Fc domain comprising, for example, the amino acid sequence of SEQ ID NO: 132 together with the cysteine ​​amino acid substitution Y349C.

[0110] In some embodiments, for example, F c1 One Fc domain of a polypeptide such as H3 domain, comprising the amino acid substitution T366W (knob), e.g., F c2 The Fc domain of other polypeptides such asH3 It comprises the amino acid substitutions T366S, L368A, and Y407V (hole) in the domain, and vice versa.

[0111] In some embodiments, for example, F c1 One Fc domain of a polypeptide such as H3 The F domain may comprise or further comprise the amino acid substitution S354C, e.g., c2 F of other polypeptides such as c The domain is that C H3 It may comprise or further comprise the amino acid substitution Y349C in the domain, or vice versa.

[0112] Thus, in some embodiments, for example, F c1 One Fc domain of a polypeptide such as H3 domain, comprising the amino acid substitutions S354C and T366W (knob), e.g., F c2 The Fc domain of other polypeptides such as H3 It comprises the amino acid substitutions Y349C, T349S, L368A, and Y407V (hole) in the domain, and vice versa.

[0113] This series of amino acid substitutions was reported by Wei et al. (Structural basis of a novel heterodimeric F c This can be further expanded by including the amino acid substitution K409A in one polypeptide and F405K in the other polypeptide, as described by Oncotarget et al. (2017) for bispecific antibody production. Thus, in some embodiments, e.g., F c1 One F of a polypeptide such as c The domain is that C H3 and / or further comprising the amino acid substitution K409A in the F domain, e.g., c2 F of other polypeptides such as cThe domain is that C H3 It may comprise or further comprise the amino acid substitution F405K in the domain, or vice versa.

[0114] In some cases, artificially introduced cysteine ​​bridges may improve the stability of the bispecific antigen-binding protein, optimally without interfering with the binding properties of the bispecific antigen-binding protein. Such cysteine ​​bridges may further improve heterodimerization.

[0115] Further amino acid substitutions, such as charge pair substitutions, to improve heterodimerization of the resulting protein have been described in the art, for example in EP 2970484.

[0116] Thus, in one embodiment, for example, F c1 One Fc domain of a polypeptide such as, for example, comprises or further comprises the charge pair substitutions E356K, E356R, D356R, or D356K and D399K or D399R, e.g., F c2 F of other polypeptides such as c The domain comprises or further comprises the charge pair substitutions R409D, R409E, K409E, or K409D and N392D, N392E, K392E, or K392D, or vice versa.

[0117] In a further embodiment, F on one or both, preferably both, of the polypeptide chains. c The domain is F c It may comprise one or more modifications that inhibit FcyR binding. Such modifications may include L234A, L235A.

[0118] hinge, H2 , and C H3The inclusion of an Fc portion, or a portion thereof, consisting of domains in antigen-binding proteins, and more specifically in bispecific antigen-binding proteins, has led to the problem of nonspecific immobilization of these molecules induced by Fc:Fc-γ receptor (FcgR) interactions. FcgR is composed of different cell surface molecules (FcgRI, FcgRIIa, FcgRIIb, and FcgRIII) that bind with different affinities to epitopes presented by the Fc portion of IgG molecules. Such nonspecific immobilization (i.e., not induced by either of the two binding domains of the bispecific molecule) is undesirable due to i) its impact on the pharmacokinetics of the molecule and ii) off-target activation of immune effector cells. Therefore, various Fc variants and mutations have been identified to eliminate FcgR binding. In this context, Morgan et al. (1995), Immunology (The N-terminal end of the C H2 The chimeric human IgG1 anti-HLA-DR domain is necessary for C1q, FcyRI, and FcyRIII binding discloses the replacement of residues 233-236 of human IgG1 with the corresponding sequence from human IgG2, i.e., residues 233P, 234V, and 235A, in which the absence of an amino acid at position 236 abolishes FcgRI binding, abolishes C1q binding, and reduces FcgRIII binding. EP 1075496 discloses antibodies and other Fc-containing molecules with variations in the Fc region (one or more of 233P, 234V, 235A, no G at position 236, 327G, 330S, and 331S), in which the recombinant antibodies are able to bind to target molecules without inducing significant complement-dependent lysis or cell-mediated destruction of the target.

[0119] Thus, in some embodiments, the Fc region comprises or further comprises one or more or deletions of amino acids selected from the group consisting of 233P, 234V, 235A, 236 (no residues) or G; and 327G, 330S, 331S, preferably the Fc region comprises or further comprises amino acids 233P, 234V, 235A, 236 (no residues) or G; and one or more amino acids selected from the group consisting of 327G, 330S, 331S, most preferably the Fc region comprises or further comprises amino acids 233P, 234V, 235A, 236 (no residues) and 331S.

[0120] In a further embodiment, the Fc domain comprises or further comprises the amino acid substitution N297Q, N297G or N297A, preferably N297Q.

[0121] The amino acid substitution "N297Q," "N297G," or "N297A" refers to an amino acid substitution at position 297 that suppresses the native N-glycosylation site within the Fc domain. This amino acid substitution further prevents Fc-γ-receptor interactions and reduces the variability of the final protein product, i.e., the bispecific antigen-binding protein of the invention, due to sugar residues as described, for example, in Tao, MH and Morrison, SL (J Immunol. 1989 Oct 15;143(8):2595-601).

[0122] In a further embodiment, particularly in the absence of a light chain, the Fc domain comprises or further comprises the amino acid substitution C220S. H1 The cysteines that form the CL disulfide bridges are deleted.

[0123] In some embodiments, F c The domain comprises or further comprises at least two additional cysteine ​​residues, such as, for example, S354C and Y349C or L242C and K334C, wherein S354C is a cysteine ​​residue that is present in the F c1and Y349C is in the Fc domain of one polypeptide such as c2 and / or in which L242C and K334C are present in the Fc domain of one or both Fc domains of the polypeptides. c1 or F c2 are located in the same Fc domain and form an intradomain CC bridge.

[0124] "Purified" and "isolated," when referring to a polypeptide (i.e., a bispecific antigen-binding protein of the invention) or nucleotide sequence, mean that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" particularly means that at least 75%, 85%, 95%, or 98%, by weight, of the same type of biological macromolecules is present.

[0125] An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may contain some additional bases or moieties that do not adversely affect the basic characteristics of the composition.

[0126] A "domain" can be any region of a protein, generally defined on the basis of sequence homology, and often associated with a particular structural or functional entity.

[0127] A "recombinant" molecule is one prepared, expressed, produced, or isolated by recombinant means.

[0128] The term "gene" refers to a DNA sequence that encodes or corresponds to a specific sequence of amino acids comprising all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as, for example, promoter sequences, which determine the conditions under which the gene is expressed. Some genes that are not structural genes may be transcribed from DNA into RNA but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or regulators of DNA transcription. In particular, the term gene may refer to a genomic sequence that encodes a protein, i.e., a sequence comprising regulatory elements, promoter, intron, and exon sequences.

[0129] "Affinity" is theoretically defined by the equilibrium binding between the bispecific antigen-binding protein and the antigen, and in the context of the present invention is defined by the equilibrium binding between the bispecific antigen-binding protein and its antigen TA / MHC, or TA-C / MHC or CD3. Affinity can be measured, for example, by the half-maximal effective concentration (EC 50 )(half-maximal binding concentration (EC 50 ) or equilibrium dissociation constant (K D ) may also be expressed as

[0130] "K D ” is the equilibrium dissociation constant between the antigen-binding protein and its antigen, k off / k on is the ratio of K D and affinity are inversely related. D The value is related to the concentration of the bispecific antigen-binding protein, K D The lower the value, the higher the affinity of the bispecific antigen-binding protein. D The value can be experimentally assessed by various known methods, such as measuring the association and dissociation rates using surface plasmon resonance (SPR) or biolayer interferometry (BLI), as described in detail herein below in the section "Bispecific Antigen Binding Proteins."

[0131] "EC 50The "half-maximal effective concentration," also referred to as the "EC" (Effective Concentration of a Single Substance), usually refers to the concentration of a molecule that elicits a response halfway between the baseline and maximum after a specified exposure time. 50 and affinity are inversely proportional, and EC 50 The lower the value, the higher the affinity of the molecule. 50 " refers to the concentration of a bispecific antigen-binding protein of the invention that elicits a response halfway between baseline and maximum after a specified exposure time, and more specifically refers to the concentration of a bispecific antigen-binding protein of the invention that elicits a response halfway between baseline and maximum after a specified exposure time. EC 50 The EC value can be experimentally assessed by a variety of known methods, for example, using an IFN-γ release assay or an LDH release assay, as described in more detail in the experimental section of Examples 2 and 5. 50 The values ​​are preferably determined by an LDH release assay and therefore refer to the cytotoxicity induced.

[0132] As used herein, a "diagnostic agent" refers to a detectable molecule or substance that provides a signal (directly or indirectly), such as a fluorescent molecule, a radioactive molecule, or any other label known in the art.

[0133] "Fluorescent molecules" known in the art include fluorescein isothiocyanate (FITC), phycoerythrin (PE), fluorophores for use with blue lasers (e.g., PerCP, PE-Cy7, PE-Cy5, FL3 and APC or Cy5, FL4), fluorophores for use with red, violet, or UV lasers (e.g., Pacific Blue, Pacific Orange).

[0134] As a "radioactive molecule," I 123 , I 124 , In 111 ,Re 186 ,Re 188 , Tc 99Bispecific binding proteins of the invention may also comprise spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0135] Such diagnostic agents may be directly bound (ie, physically linked) or indirectly bound to the bispecific antigen-binding protein.

[0136] "Therapeutic agent" herein refers to an agent that has a therapeutic effect. In one embodiment, such a therapeutic agent may be a growth inhibitory agent, such as a cytotoxic agent or a radioisotope.

[0137] "Growth inhibitory agent" or "anti-proliferative agent," which can be used indiscriminately, refers to a compound or composition that inhibits the growth of cells, particularly tumor cells, either in vitro or in vivo.

[0138] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term "cytotoxic agent" is intended to include chemotherapeutic agents, enzymes, antibiotics, and toxins, such as small molecule or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof), as well as various antitumor or anticancer agents disclosed below. In some embodiments, the cytotoxic agent is a taxoid, vinca, taxane, maytansinoid or maytansinoid analogue such as DM1 or DM4, a small drug, tomaymycin or a pyrrolobenzodiazepine derivative, a cryptophycin derivative, a leptomycin derivative, an auristatin or dolastatin analogue, a prodrug, a topoisomerase II inhibitor, a DNA alkylating agent, an antitubulin agent, CC-1065 or a CC-1065 analogue.

[0139] The term "radioisotope" means At211 , Bi 212 , Er 169 , I 131 , I 125 , Y 90 , In 111 , P 32 ,Re 186 ,Re 188 , Sm 153 , Sr 89 The term "radioisotopes" is intended to include radioisotopes suitable for cancer therapy, such as thorium-227, thorium-227, and thorium-227, which emit alpha radiation. Such radioisotopes generally emit primarily beta radiation. In one embodiment, the radioisotope is an alpha-radioisotope, more precisely, thorium-227, which emits alpha radiation.

[0140] A "PK-modifying moiety" herein refers to a moiety that modifies the pharmacokinetics (PK) of a bispecific antigen-binding protein of the invention. Thus, the moiety, in particular, alters the in vivo half-life and distribution of the bispecific antigen-binding protein of the invention. In a preferred embodiment, the PK-modifying moiety extends the half-life of the bispecific antigen-binding protein. Examples of PK modifying moieties include PEG (Dozier et al., (2015) Int J Mol Sci. Oct 28;16(10):25831-64, and Jevsevar et al., (2010) Biotechnol J. Jan;5(1):113-28), PAS (Schlapschy et al., (2013) Protein Eng Des Sel. Aug;26(8):489-501), albumin (Dennis et al., (2002) J Biol Chem. Sep 20;277(38):35035-43), F of antibodies and / or unstructured polypeptides. c Examples of such fragments include, but are not limited to, the fragments (Schellenberger et al., (2009) Nat Biotechnol. Dec;27(12):1186-90).

[0141] Bispecific antigen-binding proteins The present inventors have humanized the murine monoclonal anti-CD3 antibody UCHT1 to obtain the humanized monoclonal antibody UCHT1(V17), as disclosed in Example 1. The resulting humanized monoclonal antibody UCHT1(V17) has improved stability and / or improved solubility compared to UCHT1(V9) known in the art.

[0142] Next, the inventors demonstrated in Example 2 in a proof-of-principle experiment that when using the variable domain of a T cell-engaging antibody with intermediate affinity for its target (such as BMA31 targeting TCRαβ) in combination with a mature TCR variable domain, the resulting bispecific antigen-binding protein has a much wider margin of safety than an antigen-binding protein that uses a high-affinity anti-CD3 antibody (such as UCHT1(V17)) in association with the same TCR variable domain.

[0143] Furthermore, these novel antigen-binding proteins, particularly in the form of TCER® molecules, exhibit high cytotoxicity against tumor cells. For example, the novel antigen-binding proteins in the form of TCER® molecules exhibited half-maximal effective concentrations (EC ) of 1000-12000 for NCI-H1755, Hs695T cells, and U2OS. 50 ) is in the range of 1 pM to 100 pM, more specifically, between 1 pM and 20 pM, for cells, and therefore the EC 50 EC obtained from normal tissue cells, such as the tumor cell line Hs695T versus primary cells. 50 It is over 1000 times lower than the conventional method, and its high safety has been demonstrated.

[0144] Therefore, we have generated intermediate affinity variants (V20, V21, V23, V17opt, V20opt, V21opt, and V23opt) of the high affinity anti-CD3 antibody UCHT1 (V17), and thus a range of T cell-engaging variable domains suitable for use in combination with TCR variable domains to obtain bispecific antibodies with beneficial safety margins.

[0145] Thus, the present invention refers to a bispecific antigen-binding protein comprising at least two antigen-binding sites (A and B), in which antigen-binding site A binds to CD3, preferably to the tCD3ε / δ-complex, in which antigen-binding site B binds to a target antigenic (TA) peptide / MHC complex, preferably to a TAA antigenic peptide / MHC complex, and in which antigen-binding site A binds to a heavy chain variable domain (V H ) and the light chain variable domain (V L ) a) wherein V L comprises three complementarity determining regions (CDRs), CDRL1, CDRL2, and CDRL3, wherein: CDRL1 comprises or consists of the amino acid sequence "RASQDIRNYLN" of SEQ ID NO: 1, CDRL2 comprises or consists of the amino acid sequence "YTSRLHS" of SEQ ID NO: 2, CDRL3 comprises or consists of the amino acid sequence "QQGQTLPWT" of SEQ ID NO: 3, b) wherein V H comprises three complementarity determining regions (CDRs), CDRH1, CDRH2, and CDRH3, wherein: CDRH1 comprises or consists of the amino acid sequence "X1YTMN" of SEQ ID NO: 4, wherein X1 is G or E, preferably G; -CDRH2 comprises or consists of the amino acid sequence of SEQ ID NO: 5 "LINPX2X3GVX4TYAQKX5QX6", wherein X2 is any amino acid, preferably Q, Y or E, more preferably Q or Y, such as Q; X3 is any amino acid, preferably R, K or E, more preferably R or K, such as K; X4 is any amino acid, preferably S or T, more preferably S; X5 is any amino acid, preferably F or V, more preferably F; X6 is any amino acid, preferably G or D, more preferably D; -CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO: 6, "SGYYGX7SWYFD," wherein X7 is any amino acid, preferably E or D, more preferably D.

[0146] In one embodiment, when CDRH2 comprises or consists of the amino acid sequence "LINPYKGVSTYAQKFQD" of SEQ ID NO: 7 and CDRH3 comprises or consists of the amino acid sequence "SGYYGDSDWYFDV" of SEQ ID NO: 8, X1 of CDRH1 is E.

[0147] In one embodiment, the V H comprises a CDRH1 set forth in SEQ ID NO:4, a CDRH2 set forth in SEQ ID NO:5, and a CDRH3 set forth in SEQ ID NO:7, with the proviso that CDRH1 does not comprise or consist of SEQ ID NO:133, CDRH2 does not comprise or consist of SEQ ID NO:7, and CDRH3 does not comprise or consist of SEQ ID NO:8.

[0148] In one particular embodiment, the present invention refers to a bispecific antigen-binding protein comprising at least two antigen-binding sites (A and B), in which antigen-binding site A binds to CD3, preferably to the tCD3ε / δ-complex, and in which antigen-binding site B binds to a target antigenic (TA) peptide / MHC complex, preferably to a TAA antigenic peptide / MHC complex, and in which antigen-binding site A binds to a heavy chain variable domain (V H ) and the light chain variable domain (V L ) a) wherein V L comprises three complementarity determining regions (CDRs), CDRL1, CDRL2, and CDRL3, wherein: CDRL1 comprises or consists of the amino acid sequence "RASQDIRNYLN" of SEQ ID NO: 1, CDRL2 comprises or consists of the amino acid sequence "YTSRLHS" of SEQ ID NO: 2, CDRL3 comprises or consists of the amino acid sequence "QQGQTLPWT" of SEQ ID NO: 3, b) wherein V H comprises three complementarity determining regions (CDRs), CDRH1, CDRH2, and CDRH3, wherein: - CDRH1 comprises or consists of the amino acid sequence "GYTMN" of SEQ ID NO: 133 or "EYTMN" of SEQ ID NO: 134, preferably GYTMN of SEQ ID NO: 133, or optionally an amino acid sequence which differs from SEQ ID NO: 133 or 134 by at least one amino acid substitution, preferably by one or two amino acid substitutions, or by only one amino acid substitution, wherein preferably the amino acid sequence which differs from SEQ ID NO: 133 or 134 comprises 31G or 31E, - CDRH2 comprises or consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 135 to 142, or amino acid sequences which optionally differ from SEQ ID NOs: 133 or 134 by at least one amino acid substitution, preferably 1, 2, 3 or 4 amino acid substitutions, preferably 1 or 2 amino acid substitutions, or only 1 amino acid substitution, wherein preferably the amino acid sequence which differs from SEQ ID NO: 133 or 134 comprises amino acid 61A and, optionally, at least one of the amino acids 54Q, 54E or 54Y, 55R or 55E, 58S or 58T, 64F or 64V, 65Q, 66D or 66G, preferably 66D; -CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO: 8 or 144, or optionally an amino acid sequence which differs from SEQ ID NO: 8 or 144 by at least one amino acid substitution, preferably by 1, 2, 3 or 4 amino acid substitutions, preferably by 1 or 2 amino acid substitutions, or by only 1 amino acid substitution, wherein preferably the amino acid sequence which differs from SEQ ID NO: 8 or 134 comprises amino acid 104E.

[0149] The present invention further refers to antigen binding proteins comprising variants of the CDR amino acid sequences disclosed in the context of the present invention, typically variants of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and / or CDRH3, wherein such variants may comprise at least one, such as 4, 3, 2 or 1, preferably 1, 2 or 3 amino acid substitutions, wherein the preferred number of amino acid substitutions preferably depends on the length of the respective CDR.

[0150] In some embodiments, CDRL1 comprises or consists of an amino acid sequence that differs from the CDRL1 amino acid sequence disclosed herein by at least one amino acid substitution, preferably by one, two, three or four amino acid substitutions, preferably by one, two or three amino acid substitutions, preferably by one or two amino acid substitutions, such as one amino acid substitution, wherein said amino acid substitutions are preferably at positions 27, 28, 30, and 31.

[0151] In some embodiments, CDRL2 comprises or consists of an amino acid sequence that differs from a CDRL2 amino acid sequence disclosed herein by at least one amino acid substitution, preferably by one, two or three amino acid substitutions, preferably by one or two amino acid substitutions, such as one amino acid substitution, wherein said amino acid substitutions are preferably at positions 51, 52, and 53.

[0152] In some embodiments, the CDRL3 comprises or consists of an amino acid sequence that differs from a CDRL3 amino acid sequence disclosed herein by at least one amino acid substitution, preferably by one, two, three or four amino acid substitutions, preferably by one or two amino acid substitutions, such as one amino acid substitution, wherein the amino acid substitution is preferably at any of amino acid positions 93, 94, and 95.

[0153] In a preferred embodiment, mutations may occur in the CDRs of the heavy chain variable domain of antigen binding site A.

[0154] Thus, in some embodiments, CDRH1 comprises or consists of an amino acid sequence that differs from a CDRH1 amino acid sequence disclosed herein by at least one amino acid substitution, preferably by one or two or three amino acid substitutions, preferably by one amino acid substitution, wherein preferably said amino acid substitution is at any of amino acid positions 31-35.

[0155] In some embodiments, CDRH2 comprises or consists of an amino acid sequence that differs from a CDRH2 amino acid sequence disclosed herein by at least one amino acid substitution, preferably by one, two, three, or four amino acid substitutions, preferably by one or two amino acid substitutions, such as one, two, or three amino acid substitutions, preferably one amino acid substitution, wherein the amino acid substitutions are preferably at any of amino acid positions 54, 55, and 57-59.

[0156] In some embodiments, the CDRH3 comprises or consists of an amino acid sequence that differs from a CDRH3 amino acid sequence disclosed herein by at least one amino acid substitution, preferably by one, two, three, or four amino acid substitutions, preferably by one or two amino acid substitutions, such as one amino acid substitution, wherein the amino acid substitution is preferably at any of amino acid positions 105, 107, and 110.

[0157] In preferred embodiments, the light chain variable domain and the heavy chain variable domain further comprise light chain and heavy chain framework regions.

[0158] In one embodiment, the light chain variable domain further comprises one or more framework regions selected from the group consisting of FR1-L, FR2-L, FR3-L, and FR4-L, preferably FR1-L, FR2-L, FR3-L, and FR4-L, wherein: -FR1-L comprises or consists of the amino acid sequence "DIQMTQSPSSLSASVGDRVTITC" of SEQ ID NO: 11, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 11, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 11 preferably comprises the amino acids 6Q and / or 23C, -FR2-L comprises or consists of "WYQQKPGKAPKLLIY" of SEQ ID NO: 12, or "WYQQKPGKAVKLLI" of SEQ ID NO: 13, preferably the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 12 or 13, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 12 or 13 preferably comprises the amino acids 35W, 36Y, 38Q, 44P, 46L and / or 49Y, -FR3-L comprises or consists of the amino acid sequence "GVPSRFSGSGSGTDYTLTISSLQPEDIATYFC" of SEQ ID NO: 14, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 14, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 14 preferably comprises the amino acids 57G, 59P, 62F, 64G, 66G, 71Y, 82D, 86Y, 87F, 88C; -FR4-L comprises or consists of the amino acid sequence "FGQGTKVEIKR" of SEQ ID NO: 15, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 15, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 15 preferably comprises amino acids 98F and / or 101G; wherein VH further comprises one or more framework regions selected from the group consisting of FR1-H, FR2-H, FR3-H, and FR4-H, wherein: -FR1-H comprises or consists of the amino acid sequence "EVQLVQSGAEVKKPGASVKVSCKASGYSFT" of SEQ ID NO: 16, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 16, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 16 preferably comprises the amino acids 6Q, 14P, 22C, 24A, 26G, 27Y, 28S, 29F and / or 30T, and optionally at least one of the amino acid substitutions Q5V, P9A, L11V, V12K, M18V and / or I20V; -FR2-H comprises or consists of the amino acid sequence "WVRQAPGQGLEWMG" of SEQ ID NO: 17, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 17, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 17 preferably comprises 36W, 37V, 39Q, 45L, 46E and / or 47W, and optionally at least one of the amino acid substitutions K38R, S40A, H41P, K43Q, N44G; -FR3-H comprises or consists of the amino acid sequence "RVTLTVDKSTSTAYMELSSLRSEDTAVYYCAR" of SEQ ID NO: 18, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 18, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 18 preferably comprises 70L, 72V, 79A, 90D, 94Y, 95Y, 96C, 97A, and / or 98R, and optionally at least one of the amino acid substitutions K67R, A68V, K74T, S76T, L84S, T87R and / or S91T; -FR4-H comprises or consists of the amino acid sequence "WGQGTLVTVSS" of SEQ ID NO: 19, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 19, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 19 preferably comprises 112W, 113G, 115G, and optionally at least one of the amino acid substitutions A114Q and / or T117L.

[0159] In another embodiment, the light chain variable domain further comprises one or more framework regions selected from the group consisting of FR1-L, FR2-L, FR3-L, and FR4-L, preferably FR1-L, FR2-L, FR3-L, and FR4-L, wherein: -FR1-L comprises or consists of the amino acid sequence "DIQMTQSPSSLSASVGDRVTITC" of SEQ ID NO: 11, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 11, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 11 preferably comprises the amino acids 6Q and / or 23C, -FR2-L comprises or consists of "WYQQKPGKAPKLLIY" of SEQ ID NO: 12, or "WYQQKPGKAVKLLI" of SEQ ID NO: 13, preferably the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 12 or 13, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 12 or 13 preferably comprises the amino acids 35W, 36Y, 38Q, 44P, 46L and / or 49Y, -FR3-L comprises or consists of the amino acid sequence "GVPSRFSGSGSGTDYTLTISSLQPEDIATYFC" of SEQ ID NO: 14, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 14, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 14 preferably comprises the amino acids 57G, 59P, 62F, 64G, 66G, 71Y, 82D, 86Y, 87F, 88C; -FR4-L comprises or consists of the amino acid sequence "FGQGTKVEIK" of SEQ ID NO: 285, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 15, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 15 preferably comprises amino acids 98F and / or 101G; wherein VH further comprises one or more framework regions selected from the group consisting of FR1-H, FR2-H, FR3-H, and FR4-H, wherein: -FR1-H comprises or consists of the amino acid sequence "EVQLVQSGAEVKKPGASVKVSCKASGYSFT" of SEQ ID NO: 16, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 16, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 16 preferably comprises the amino acids 6Q, 14P, 22C, 24A, 26G, 27Y, 28S, 29F and / or 30T, and optionally at least one of the amino acid substitutions Q5V, P9A, L11V, V12K, M18V and / or I20V; -FR2-H comprises or consists of the amino acid sequence "WVRQAPGQGLEWMG" of SEQ ID NO: 17, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 17, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 17 preferably comprises 36W, 37V, 39Q, 45L, 46E and / or 47W, and optionally at least one of the amino acid substitutions K38R, S40A, H41P, K43Q, N44G; -FR3-H comprises or consists of the amino acid sequence "RVTLTVDKSTSTAYMELSSLRSEDTAVYYCAR" of SEQ ID NO: 18, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 18, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 18 preferably comprises 70L, 72V, 79A, 90D, 94Y, 95Y, 96C, 97A, and / or 98R, and optionally at least one of the amino acid substitutions K67R, A68V, K74T, S76T, L84S, T87R and / or S91T; -FR4-H comprises or consists of the amino acid sequence "WGQGTLVTVSS" of SEQ ID NO: 19, or an amino acid sequence that is at least 85% identical to SEQ ID NO: 19, wherein the amino acid sequence that is at least 85% identical to SEQ ID NO: 19 preferably comprises 112W, 113G, 115G, and optionally at least one of the amino acid substitutions A114Q and / or T117L.

[0160] In another embodiment, the light chain variable domain further comprises one or more framework regions selected from the group consisting of FR1-L, FR2-L, FR3-L, and FR4-L, preferably FR1-L, FR2-L, FR3-L, and FR4-L, wherein: -FR1-L comprises or consists of the amino acid sequence "DIQMTQSPSSLSASVGDRVTITC" of SEQ ID NO: 11, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 11, wherein the amino acid sequence that is at least 90% identical to SEQ ID NO: 11 preferably comprises the amino acids 6Q and / or 23C, -FR2-L comprises or consists of "WYQQKPGKAPKLLIY" of SEQ ID NO: 12, or "WYQQKPGKAVKLLI" of SEQ ID NO: 13, preferably the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 12 or 13, wherein the amino acid sequence that is at least 90% identical to SEQ ID NO: 12 or 13 preferably comprises the amino acids 35W, 36Y, 38Q, 44P, 46L and / or 49Y, -FR3-L comprises or consists of the amino acid sequence "GVPSRFSGSGSGTDYTLTISSLQPEDIATYFC" of SEQ ID NO: 14, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 14, wherein the amino acid sequence that is at least 90% identical to SEQ ID NO: 14 preferably comprises the amino acids 57G, 59P, 62F, 64G, 66G, 71Y, 82D, 86Y, 87F, 88C; -FR4-L comprises or consists of the amino acid sequence "FGQGTKVEIK" of SEQ ID NO: 285, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 15, wherein the amino acid sequence that is at least 90% identical to SEQ ID NO: 15 preferably comprises amino acids 98F and / or 101G; wherein VH further comprises one or more framework regions selected from the group consisting of FR1-H, FR2-H, FR3-H, and FR4-H, wherein: -FR1-H comprises or consists of the amino acid sequence "EVQLVQSGAEVKKPGASVKVSCKASGYSFT" of SEQ ID NO: 16, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 16, wherein the amino acid sequence that is at least 90% identical to SEQ ID NO: 16 preferably comprises the amino acids 6Q, 14P, 22C, 24A, 26G, 27Y, 28S, 29F and / or 30T, and optionally at least one of the amino acid substitutions Q5V, P9A, L11V, V12K, M18V and / or I20V; -FR2-H comprises or consists of the amino acid sequence "WVRQAPGQGLEWMG" of SEQ ID NO: 17, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 17, wherein the amino acid sequence that is at least 90% identical to SEQ ID NO: 17 preferably comprises 36W, 37V, 39Q, 45L, 46E and / or 47W, and optionally at least one of the amino acid substitutions K38R, S40A, H41P, K43Q, N44G; -FR3-H comprises or consists of the amino acid sequence "RVTLTVDKSTSTAYMELSSLRSEDTAVYYCAR" of SEQ ID NO: 18, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 18, wherein the amino acid sequence that is at least 90% identical to SEQ ID NO: 18 preferably comprises 70L, 72V, 79A, 90D, 94Y, 95Y, 96C, 97A, and / or 98R, and optionally at least one of the amino acid substitutions K67R, A68V, K74T, S76T, L84S, T87R and / or S91T; -FR4-H comprises or consists of the amino acid sequence "WGQGTLVTVSS" of SEQ ID NO: 19, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, wherein the amino acid sequence that is at least 90% identical to SEQ ID NO: 19 preferably comprises 112W, 113G, 115G, and optionally at least one of the amino acid substitutions A114Q and / or T117L.

[0161] In another embodiment, the light chain variable domain further comprises one or more framework regions selected from the group consisting of FR1-L, FR2-L, FR3-L, and FR4-L, preferably FR1-L, FR2-L, FR3-L, and FR4-L, wherein: -FR1-L comprises or consists of the amino acid sequence "DIQMTQSPSSLSASVGDRVTITC" of SEQ ID NO: 11, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 11, wherein the amino acid sequence that is at least 95% identical to SEQ ID NO: 11 preferably comprises the amino acids 6Q and / or 23C, -FR2-L comprises or consists of "WYQQKPGKAPKLLIY" of SEQ ID NO: 12, or "WYQQKPGKAVKLLI" of SEQ ID NO: 13, preferably the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 12 or 13, wherein the amino acid sequence that is at least 95% identical to SEQ ID NO: 12 or 13 preferably comprises the amino acids 35W, 36Y, 38Q, 44P, 46L and / or 49Y, -FR3-L comprises or consists of the amino acid sequence "GVPSRFSGSGSGTDYTLTISSLQPEDIATYFC" of SEQ ID NO: 14, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 14, wherein the amino acid sequence that is at least 95% identical to SEQ ID NO: 14 preferably comprises the amino acids 57G, 59P, 62F, 64G, 66G, 71Y, 82D, 86Y, 87F, 88C; -FR4-L comprises or consists of the amino acid sequence "FGQGTKVEIK" of SEQ ID NO: 285, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 15, wherein the amino acid sequence that is at least 95% identical to SEQ ID NO: 15 preferably comprises amino acids 98F and / or 101G; wherein VH further comprises one or more framework regions selected from the group consisting of FR1-H, FR2-H, FR3-H, and FR4-H, wherein: -FR1-H comprises or consists of the amino acid sequence "EVQLVQSGAEVKKPGASVKVSCKASGYSFT" of SEQ ID NO: 16, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 16, wherein the amino acid sequence that is at least 95% identical to SEQ ID NO: 16 preferably comprises the amino acids 6Q, 14P, 22C, 24A, 26G, 27Y, 28S, 29F and / or 30T, and optionally at least one of the amino acid substitutions Q5V, P9A, L11V, V12K, M18V and / or I20V; -FR2-H comprises or consists of the amino acid sequence "WVRQAPGQGLEWMG" of SEQ ID NO: 17, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 17, wherein the amino acid sequence that is at least 95% identical to SEQ ID NO: 17 preferably comprises 36W, 37V, 39Q, 45L, 46E and / or 47W, and optionally at least one of the amino acid substitutions K38R, S40A, H41P, K43Q, N44G; -FR3-H comprises or consists of the amino acid sequence "RVTLTVDKSTSTAYMELSSLRSEDTAVYYCAR" of SEQ ID NO: 18, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 18, wherein the amino acid sequence that is at least 95% identical to SEQ ID NO: 18 preferably comprises 70L, 72V, 79A, 90D, 94Y, 95Y, 96C, 97A, and / or 98R, and optionally at least one of the amino acid substitutions K67R, A68V, K74T, S76T, L84S, T87R and / or S91T; -FR4-H comprises or consists of the amino acid sequence "WGQGTLVTVSS" of SEQ ID NO: 19, or an amino acid sequence that is at least 95% identical to SEQ ID NO: 19, wherein the amino acid sequence that is at least 95% identical to SEQ ID NO: 19 preferably comprises 112W, 113G, 115G, and optionally at least one of the amino acid substitutions A114Q and / or T117L.

[0162] Amino acids 35W, 36Y, 46L, and 49Y of FR2-L and amino acids 64G, 71Y of FR3-L have been identified by the present inventors as being located in the Vernier zone and are preferably not substituted.

[0163] Amino acids 27Y, 28S, 29F, and 30T of FR-1H, amino acid 47W of FR2-H, amino acids 70L, 72V, 79A, 97A, and 98R of FR3-H, and 112W of FR4-H have been identified by the inventors as being located in the Vernier zone and are preferably not substituted.

[0164] Variants of the antigen-binding proteins described herein are contemplated and are expressly referred to herein using the phrase "at least 85% identical to a reference sequence," as defined in the "Definitions" section above. For example, the sequences FR1-L, FR2-L, FR3-L, and FR4-L, and FR1-H, FR2-H, FR3-H, and FR4-H may, where appropriate, differ from the reference sequences of SEQ ID NOs: 11 to 19 by at least one amino acid substitution, particularly at least one conservative amino acid substitution and / or substitution with a standard residue. In particular, the sequences FR1-L, FR2-L, FR3-L, and FR4-L, and FR1-H, FR2-H, FR3-H, and FR4-H of the light and heavy chain variable domains may differ from the reference sequences of SEQ ID NOs: 11 to 19 only by conservative amino acid substitutions.

[0165] Modifications and changes may be made to the amino acid sequences of the bispecific antigen-binding proteins of the invention, and the corresponding DNA sequences, respectively, which still result in a functional antigen-binding protein or polypeptide with the desired properties. Modifications may be made in the heavy and light chain variable domains of antigen-binding site A, or the α and β or γ and δ variable domains of antigen-binding site B, particularly in the framework regions, or in each CDR, or in all CDRs within the heavy and light chain variable domains of antigen-binding site A, or in the framework regions, or in each CDR, or in all CDRs within the α and β or γ and δ variable domains.

[0166] The bispecific antigen binding protein may comprise a light chain variable region comprising: wherein the amino acid sequence of FR2-L is at least 85% identical to SEQ ID NO: 12 or 13 and comprises amino acid 44P. This amino acid 44P (found in the human germline sequence Vk1-018) has the advantage of deimmunizing the humanized variable domain, as proline is common at this position in the 10 most similar human germlines.

[0167] "Deimmunization" herein refers to reducing immunogenicity, i.e., the ability to induce an immune response in a subject. This is achieved by replacing amino acids with those that are most common in the human germline and therefore are not recognized as foreign by the immune system.

[0168] Thus, in one embodiment, the antigen-binding site A in the bispecific antigen-binding protein of the invention comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ) Among them, the above V L comprises or consists of the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 145, wherein said amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 145 preferably comprises the amino acid sequence of CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3; Among them, the above V Hcomprises or consists of an amino acid sequence selected from the group of amino acid sequences selected from the amino acid sequences of SEQ ID NOs: 149 to 160, or an amino acid sequence which is at least 85% identical to an amino acid sequence selected from the group of amino acid sequences consisting of the amino acid sequences of SEQ ID NOs: 149 to 160, wherein preferably, the amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 149 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 138, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 151 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 151 comprises the amino acid sequence of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138, or CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 152 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 140, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 153 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 141, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 154 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 142, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 155 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 142, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 156 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 144; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 157 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 138, and CDRH3 of SEQ ID NO: 144; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 158 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 159 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 144; Among them, preferably, the amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 160 comprises the amino acid sequence of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138, and CDRH3 of SEQ ID NO: 144.

[0169] Thus, in one embodiment, the antigen-binding site A in the bispecific antigen-binding protein of the invention comprises a heavy chain variable domain (V H ) and a light chain variable domain (V L ) Among them, the above V L comprises or consists of the amino acid sequence of SEQ ID NO: 286 or an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 286, wherein said amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 286 preferably comprises the amino acid sequence of CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3; Among them, the above V Hcomprises or consists of an amino acid sequence selected from the group of amino acid sequences selected from the amino acid sequences of SEQ ID NOs: 149 to 160, or an amino acid sequence which is at least 85% identical to an amino acid sequence selected from the group of amino acid sequences consisting of the amino acid sequences of SEQ ID NOs: 149 to 160, wherein preferably, the amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 149 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 138, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 151 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 151 comprises the amino acid sequence of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138, or CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 152 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 140, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 153 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 141, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 154 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 142, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 155 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 142, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 156 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 144; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 157 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 138, and CDRH3 of SEQ ID NO: 144; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 158 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8; wherein, preferably, the amino acid sequence at least 85% identical to the amino acid sequence of SEQ ID NO: 159 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 144; Among them, preferably, the amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 160 comprises the amino acid sequence of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138, and CDRH3 of SEQ ID NO: 144.

[0170] In a preferred embodiment, the V L comprises or consists of the amino acid sequence of SEQ ID NO: 286 or an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 286, wherein said amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 286 preferably comprises the amino acid sequence of CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3; Among them, the above V Hcomprises or consists of the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 156, wherein preferably, said amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 156 comprises the amino acid sequence of SEQ ID NO: 156 and comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 144; Among them, the above V H comprises or consists of the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 149, wherein preferably, said amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 149 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 138, and CDRH3 of SEQ ID NO: 8; Among them, the above V H comprises or consists of the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 151, wherein preferably, said amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 151 comprises the amino acid sequence of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138, CDRH3 of SEQ ID NO: 8.

[0171] In a preferred embodiment, the V L comprises or consists of the amino acid sequence of SEQ ID NO: 286 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 286, wherein said amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 286 preferably comprises the amino acid sequence of CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3; Among them, the above V Hcomprises or consists of the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 156, wherein preferably, said amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 156 comprises the amino acid sequence of SEQ ID NO: 156 and comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 144; Among them, the above V H comprises or consists of the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 149, wherein preferably, said amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 149 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 138, and CDRH3 of SEQ ID NO: 8; Among them, the above V H comprises or consists of the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 151, wherein preferably said amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 151 comprises the amino acid sequence of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138, CDRH3 of SEQ ID NO: 8.

[0172] In a preferred embodiment, the V L comprises or consists of the amino acid sequence of SEQ ID NO: 286 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 286, wherein said amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 286 preferably comprises the amino acid sequence of CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3; Among them, the above V Hcomprises or consists of the amino acid sequence of SEQ ID NO: 156 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156, wherein preferably, said amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156 comprises the amino acid sequence of SEQ ID NO: 156 and comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 144; Among them, the above V H comprises or consists of the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149, wherein preferably, said amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 138, and CDRH3 of SEQ ID NO: 8; Among them, the above V H comprises or consists of the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 151, wherein preferably, said amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151 comprises the amino acid sequence of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138, CDRH3 of SEQ ID NO: 8.

[0173] In some embodiments, antigen-binding site B of the bispecific antigen-binding protein of the invention comprises an antibody or fragment thereof, or an alpha chain variable domain (v α ) and the β chain variable domain (v β ) or γ chain variable domain (v γ ) or δ chain variable domain (v δ Antibodies and fragments thereof are as defined herein above in the "Definitions" section.

[0174] In some embodiments, antigen-binding site B of the bispecific antigen-binding protein of the invention comprises an alpha chain variable domain (v α ) and the β chain variable domain (v β) or γ chain variable domain (v γ ) or δ chain variable domain (v δ ), preferably v α and v β The alpha chain variable domains (v) may be used in the context of the present invention in the context of the particular TA to which they bind. α ) and the β chain variable domain (v β ) or γ chain variable domain (v γ ) or δ chain variable domain (v δ ) are described in detail, for example, in International Publication No. 2018172533, International Publication No. 2018033291, International Publication No. 2017158103, International Publication No. 2018104438, International Publication No. 2018104478, International Publication No. 2019002444, and International Publication No. 2017158116.

[0175] Thus, in one embodiment, v α and v β or v γ and v δ comprises or consists of the amino acid sequence disclosed in WO 2018172533, WO 2018033291, WO 2017158103, WO 2018104438, WO 2018104478, WO 2019002444, WO 2017158116, and is described in the cited prior art. α and v β or v γ and v δ binds to the TA peptides, particularly the TAA peptides, disclosed in the same patent application cited.

[0176] In one embodiment, the bispecific antigen binding protein of the invention comprises α and v β domain or v γ and v δdomains, in which: i)v α or v γ is an amino acid sequence selected from the group consisting of "EDVEQSLFLSVREGDSVVINCTYTDSSSTYLYWYKQEPGKGLQLLTYIYSSQDSKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP" SEQ ID NO: 20, "EDVEQSLFLSVREGDSVVINCTYTDSSSTYLYWYKQEPGKGLQLLTYIYSSQDQKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP" SEQ ID NO: 21, and "EDVEQSLFLSVREGDSVVINCTYTESSSTYLYWYKQEPGKGLQLLTYIYSSQDQKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP" SEQ ID NO: 22, or an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, and 22. and wherein the amino acid sequence is at least 85% identical to the amino acid sequence of SEQ ID NO: 20, preferably comprising the amino acid sequence of CDRa1 of SEQ ID NO: 23, CDRa2 of SEQ ID NO: 24, and CDRa3 of SEQ ID NO: 25; and wherein the amino acid sequence is at least 85% identical to the amino acid sequence of SEQ ID NO: 21, preferably comprising the amino acid sequence of CDRa1 of SEQ ID NO: 23, CDRa2 of SEQ ID NO: 26, and CDRa3 of SEQ ID NO: 25; and wherein the amino acid sequence is at least 85% identical to the amino acid sequence of SEQ ID NO: 22, preferably comprising the amino acid sequence of CDRa1 of SEQ ID NO: 27, CDRa2 of SEQ ID NO: 26, and CDRa3 of SEQ ID NO: 25; and wherein the amino acid sequence of said first variable domain preferably comprises amino acids 19V and / or 48K; v β or v δcomprises or consists of the amino acid sequence 'DAGVIQSPRHEVTEMGQEVTLRCKPIPGHDYLFWYRQTMMRGLELLFYFCYGTPCDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRADTGELFFGEGSRLTVL' SEQ ID NO: 30, or an amino acid sequence which is at least 85% identical to an amino acid sequence consisting of SEQ ID NO: 30, wherein preferably the amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 30 preferably comprises the amino acid sequence of CDRb1 of SEQ ID NO: 31, CDRb2 of SEQ ID NO: 34, CDRb3 of SEQ ID NO: 35, respectively, and optionally comprising amino acids 54F and / or 66C; or (ii)v α or v γ comprises or consists of the amino acid sequence of SEQ ID NO: 48 or an amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 48, wherein preferably the amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 48 comprises the amino acid sequence of CDRa1 of SEQ ID NO: 49, CDRa2 of SEQ ID NO: 50, and CDRa3 of SEQ ID NO: 51; v β or v δ comprises or consists of the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence which is at least 85% identical to SEQ ID NO: 44, wherein preferably said amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 44 comprises the amino acid sequence of CDRb1 of SEQ ID NO: 45, CDRb2 of SEQ ID NO: 46, and CDRb3 of SEQ ID NO: 47.

[0177] In one embodiment, the first variable domain and the second variable domain as defined herein in the context of the antigen binding protein of the invention may comprise an amino acid substitution at position 44 according to the IMGT numbering. In a preferred embodiment, said amino acid at position 44 is substituted with another suitable amino acid to improve pairing. In a particular embodiment, preferably wherein said antigen binding protein is a TCR, said amino acid substitution improves, for example, chain pairing (i.e., α and β chain pairing or γ and δ chain pairing). In a preferred embodiment, one or both of the amino acids present at position 44 in the first variable domain (v144) and the amino acid present at position 44 in the second variable domain (v244) are v144D / v244R. 、v1 44R / v244D 、v1 The amino acid pair v144 / v244 is substituted with a pair of amino acids selected from the group consisting of: v144E / v244K, v144K / v244E, v144D / v244K, v144K / v244D, v144R / v244E; v144E / v244R, v144L / v244W, v144W / v244L, v144V / v244W, v144W / v244V.

[0178] Thus, in a further embodiment, the antigen binding protein is selected from the group consisting of: v1Q44D / v2Q44R; v1Q44R / v2Q44D; v1Q44E / v2Q44K; v1Q44K / v2Q44E; v1Q44D / v2Q44K; v1Q44K / v2Q44D; v1Q44E / v2Q44R; v1Q44R / v2Q44E; v1Q44L / v2Q44W; v1Q44W / v2Q44L; v1Q44V / v2Q44W; and v1Q44W / v2Q44V; v1W44D / v2Q44R; v1W44R / v2Q44D; v1W44E / v2Q44K; v1W44K / v2 Q44E;v1W44D / v2Q44K;v1W44K / v2Q44D;v1W44E / v2Q44R;v1W44R / v2Q44E;v1W44L / v2Q44W;v1W44 / v2Q44L;v1W44V / v2Q44W;and v1W44 / v2Q44V;v1H44D / v2Q44R;v1H44R / v2Q44D;v1H44E / v2Q44K;v1H44K / v2Q44E;v1H44D / v2Q44K ;v1H44K / v2Q44D;v1H44E / v2Q44R;v1H44R / v2Q44E;v1H44L / v2Q44W;v1H44W / v2Q44L;v1H44V / v2Q44W;and v1H44W / v2Q44V;v1K44D / v2Q44R;v1K44R / v2Q44D;v1K44E / v2Q44K;v1K44 / v2Q44E;v1K44D / v2Q44K;v1K44 / v2Q44D;v1K4 4E / v2Q44R;v1K44R / v2Q44E;v1K44L / v2Q44W;v1K44W / v2Q44L;v1K44V / v2Q 44W; and v1K44W / v2Q44V;v1E44D / v2Q44R;v1E44R / v2Q44D;v1E44 / v2Q44K;v 1E44K / v2Q44E; v1E44D / v2Q44K; v1E44K / v2Q44D; v1E44 / v2Q44R; v1E44R / v2Q44E; v1E44L / v2Q44W; v1E44W / v2Q44L; v1E44V / v2Q44W; and v1E44W / v2Q44 V;v1Q44D / v2R44;v1Q44R / v2R44D;v1Q44E / v2R44K;v1Q44K / v2R44E;v1Q44D / v2R44K;v1Q44K / v2R44D;v1Q44E / v2R44;v1Q44R / v2R44E;v1Q44L / v2R44W;v1Q44W / v2R44L; v1Q44V / v2R44W; and v1Q44W / v2R44V; v1W44D / v2R44; v1W44R / v2R44D; v1W44E / v2R44K; v1W44K / v2R44E; v1W44D / v2R44K; v1W44K / v2R44D; v1W44E / v2R44; v1W44R / v2R44E; v1W44L / v2R44W; v1W44 / v2R44L; v1W44V / v2R44W; and v1W44 / v2R44V; v1H44D / v2R44; v1H44R / v2R44D; v1H44E / v2R 44K;v1H44K / v2R44E;v1H44D / v2R44K;v1H44K / v2R44D;v1H44E / v2R44;v1H44R / v2R44E;v1H44L / v2R44W;v1H44W / v2R44L;v1H44V / v2R44W;and v1H44W / v2R44V;v1K44D / v2R44;v1K44R / v2R44D;v1K44E / v2R44K;v1K44 / v2R44E; v1K44D / v2R44K;v1K44 / v2R44D;v1K44E / v2R44;v1K44R / v2R44E;v1K44L / v2 R44W;v1K44W / v2R44L;v1K44V / v2R44W;and v1K44W / v2R44V;v1E44D / v2R44;v1E44R / v2R44D;v1E44 / v2R44K;v1E44K / v2R44E;v1E44D / v2R44K;v1E44K / v2R44D;v1E44R / v2R44E;v1E44L / v2R44W;v1E44W / v2R44L;v1E44V / v2R44W;and v1E44W / v2R44V;v1Q44D / v2K44R;v1Q44R / v2K44D;v1Q44E / v244K;v1Q 44K / v2K44E; v1Q44D / v244K; v1Q44K / v2K44D; v1Q44E / v2K44R; v1Q44R / v2K44E; v1Q44L / v2K44W; v1Q44W / v2K44L; v1Q44V / v2K44W; and v1Q44W / v2K44V ;v1W44D / v2K44R;v1W44R / v2K44D;v1W44E / v244K;v1W44K / v2K44E;v1W44D / v244K;v1W44K / v2K44D;v1W44E / v2K44R;v1W44R / v2K44E;v1W44L / v2K44W;v1W44 / v2K44L;v1W44V / v2K44W;and v1W44 / v2K44V;v1H44D / v2K44R;v1H44R / v2K44D;v1 H44E / v244K;v1H44K / v2K44E;v1H44D / v244K;v1H44K / v2K44D;v1H44E / v2K44R;v1H44R / v 2K44E;v1H44L / v2K44W;v1H44W / v2K44L;v1H44V / v2K44W;and v1H44W / v2K44V;v1K44D / v2K44R;v1K44R / v2K44D;v1K44E / v244K;v1K44 / v2K44E;v1K44D / v244K;v1K44 / v2K44D;v1K 44E / v2K44R;v1K44R / v2K44E;v1K44L / v2K44W;v1K44W / v2K44L;v1K44V / v2K44W;and v1K 44W / v2K44V;v1E44D / v2K44R;v1E44R / v2K44D;v1E44 / v244K;v1E44K / v2K44E;v1E44D / v2 v1E44K / v2K44D; v1E44 / v2K44R; v1E44R / v2K44E; v1E44L / v2K44W; v1E44W / v2K44L; v1E44V / v2K44W; and v1E44W / v2Q44V.

[0179] In the above, for example, "v1Q44R / v2Q44D" is intended to mean that in the first variable domain, Q44 is substituted with R, and in the second variable domain, Q44 is substituted with D. Additional substitutions and explanations are described in U.S. Patent Application No. 2018-0162922.

[0180] In one embodiment, the bispecific antigen-binding protein is a bispecific antibody or fragment thereof, a bispecific T-cell receptor (TCR) or fragment thereof, or a bispecific single-chain TCR (scTCR) or a bispecific single-chain antibody.

[0181] Bispecific antibodies, TCRs and respective fragments are defined herein above in the "Definitions" section.

[0182] In one embodiment, the antigen binding protein is of human origin, which is understood to be generated from human antigen loci and therefore comprise human sequences, in particular human TCR or antibody sequences.

[0183] In one embodiment, the light chain variable domain and the heavy chain variable domain are linked together and / or α and v β or v γ and v δ The domains are preferably linked together via covalent bonds.

[0184] In one embodiment, the bispecific antigen binding protein comprises at least two polypeptides.

[0185] In a related embodiment, the light chain variable domain and the heavy chain variable domain are located on the same or different polypeptides, preferably on different polypeptides.

[0186] In the same embodiment, the α chain variable domain (v α ) and the β chain variable domain (v β ) or γ chain variable domain (v γ ) and the δ chain variable domain (v δ ), preferably v α and v β are located on the same or different polypeptides.

[0187] In a preferred embodiment, the antigen binding protein is a soluble protein.

[0188] A "covalent bond," "linker sequence," or "polypeptide linker" is defined herein above in the "Definitions" section under "Linker."

[0189] In one embodiment, the bispecific antigen-binding protein of the invention further comprises one or more of the following: (i) Diagnostic agents; (ii) a therapeutic drug; or (iii) PK modification moiety.

[0190] "Diagnostic agent," "therapeutic agent," and "PK modifying moiety" are defined herein above in the definitions section.

[0191] In some embodiments, the antigen binding proteins of the present invention are covalently linked, either directly or via a cleavable or non-cleavable linker, to at least one growth inhibitory agent. Such antigen binding proteins having at least one growth inhibitory agent attached may also be referred to as conjugates.

[0192] The preparation of such conjugates, e.g., immunoconjugates, is described in WO 2004 / 091668, or Hudecz, F., Methods Mol. Biol. 298:209-223 (2005), and Kirin et al., Inorg Chem. 44(15):5405-5415 (2005), and may be transferred by one skilled in the art to the preparation of such antigen-binding proteins of the invention having at least one growth inhibitory agent attached.

[0193] "Linker" in the context of attaching at least one growth inhibitory agent means a chemical moiety comprising a covalent bond or chain of atoms, which covalently attaches the polypeptide to the drug moiety.

[0194] Conjugates may also be prepared by in vitro methods. Drugs or prodrugs are attached to antibodies using linking groups. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. Conjugation of antigen-binding proteins of the invention to cytotoxic or growth inhibitory agents can be achieved using N-succinimidyl pyridyldithiobutyrate (SPDB), butanoic acid 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl ester (nitro-SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sulfo-SPDB), N-succinimidyl (2-pyridyldithio)propionate (SPDP), succinimidyl (N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT); or the following bifunctional derivatives: imidoesters. This may be accomplished using a variety of bifunctional protein-binding agents, including, but not limited to, esters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al. (1987). Carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for attaching radionucleotides to antibodies (WO 94 / 11026).

[0195] The linker may be a "cleavable linker" that facilitates release of the cytotoxic or growth inhibitory agent inside the cell. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase-labile linker, a photolabile linker, or a disulfide-containing linker (see, e.g., U.S. Pat. No. 5,208,020) may be used. The linker may also be a "non-cleavable linker" (e.g., an SMCC linker), which may provide better resistance in some cases.

[0196] Alternatively, fusion proteins comprising a bispecific antigen-binding protein of the invention and a cytotoxic or growth inhibitory polypeptide may be produced by recombinant techniques or peptide synthesis. The length of DNA may include regions encoding the two parts of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate.

[0197] The antigen binding proteins of the invention may also be used in dependent enzyme-mediated prodrug therapy by conjugating the polypeptide to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81 / 01145) into an active anti-cancer drug (see, e.g., WO 88 / 07378 and U.S. Pat. No. 4,975,278).

[0198] In one embodiment, the antigen binding protein of the invention further comprises one or more of an enzyme, a cytokine (such as human IL-2, IL-7 or IL-15), a nanocarrier, or a nucleic acid.

[0199] Different bispecific formats have been described in the art and are set forth herein above in the "Forms" section under "Definitions." Techniques for producing different forms of proteins are also disclosed in the art cited in the corresponding sections, and therefore, those skilled in the art can easily use the variable domains defined in the context of the present invention in different formats, particularly those disclosed herein. The production of antigen-binding proteins, particularly soluble bispecific binding proteins such as TCER®, is also disclosed in the Examples section herein. Those skilled in the art will understand that when an antigen-binding protein is composed of two polypeptides, the light and heavy chain variable domains may be in a parallel orientation, as in the DVD format, and the α and β variable domains may be in a parallel orientation, or the light and heavy chain variable domains may be in a crossed orientation, as in the CODV format, and the α and β variable domains may be in a crossed orientation.

[0200] Thus, the present invention further refers to an antigen binding protein comprising two polypeptide chains that form two antigen binding sites (A and B), in which the first polypeptide chain has the formula: V3-L1-V4-L2-C L [I] (wherein V3 is a third variable domain; V4 is a fourth variable domain; L1 and L2 are linkers; L2 may be present or absent; C L a light chain constant domain or part thereof, which may or may not be present) and having a structure represented by wherein the second polypeptide chain has the formula: V5-L3-V6-L4-C H1 [II] (wherein V5 is the fifth variable domain; V6 is the sixth variable domain; L3 and L4 are linkers; L4 may be present or absent; C H1 is heavy chain constant domain 1 or a portion thereof, present or absent; wherein V3 is V, as defined herein above; α or Vγ V5 is a variable domain, β or V δ V4 is a light chain variable domain and V6 is a heavy chain variable domain, or V4 is a heavy chain variable domain and V6 is a light chain variable domain, or As defined herein above, V3 is V β or V δ V5 is a variable domain, α or V γ V4 is a light chain variable domain and V6 is a heavy chain variable domain, or V4 is a heavy chain variable domain and V6 is a light chain variable domain, or As defined herein above, V3 is V α or V γ variable domain, V6 is V β or V δ V4 is a light chain variable domain and V5 is a heavy chain variable domain, or V4 is a heavy chain variable domain and V5 is a light chain variable domain, or As defined herein above, V3 is V β or V δ variable domain, V6 is V α or V γ V4 is a light chain variable domain and V5 is a heavy chain variable domain, or V4 is a heavy chain variable domain and V5 is a light chain variable domain; As defined herein above, V4 is V α or V γ V5 is a variable domain, β or V δ V3 is a light chain variable domain and V6 is a heavy chain variable domain, or V3 is a heavy chain variable domain and V6 is a light chain variable domain, or As defined herein above, V4 is V β or V δ V5 is a variable domain, α or V γV3 is a light chain variable domain and V6 is a heavy chain variable domain, or V3 is a heavy chain variable domain and V6 is a light chain variable domain; In it, the light chain variable domain and the heavy chain variable domain together form an antigen-binding site A, in which V α and V β , or V γ and V δ The variable domains form the antigen-binding site B, in which V α or V γ The variable domain is preferably V α And V β or V δ is preferably V β is) It has a structure represented by:

[0201] The linkers L1, L2, L3, and L4 are defined herein above in the "Definitions" section. However, in some embodiments, some linker lengths may be preferred for specific forms. However, knowledge of linker lengths and their amino acid sequences is within the general knowledge of the art, and different forms of linkers, as well as linker and amino acid sequences, are part of the state of the art and are disclosed in the disclosures cited herein above.

[0202] In a preferred embodiment, V3 is V as defined herein above. α where V6 is V as defined herein above. β wherein V4 is a light chain variable domain as defined in the context of the present invention, V3 is V as defined herein above α where V6 is V as defined herein above. β wherein V4 is a heavy chain variable domain as defined in the context of the present invention and V5 is a light chain variable domain as defined in the context of the present invention.

[0203] In one embodiment, the polypeptide of formula [I] comprises at the C-terminus a polypeptide of formula [I], a linker (L5), and F cdomain or a portion thereof, and / or wherein the polypeptide of formula [II] comprises at the C-terminus a polypeptide of formula [II], a linker (L6), and F c It further comprises a domain or part thereof.

[0204] F c Domains are as defined herein above in the "Definitions" section.

[0205] In one embodiment, the antigen binding protein comprises two polypeptide chains that form two antigen binding sites (A and B), In the present invention, one polypeptide chain has the formula [III]: V3-L1-V4-L2-C L -L5-F c1 [III] and having a structure represented by One polypeptide chain has the formula [IV]: V5-L3-V6-L4-C H1 -L6-F c2 [IV] (In the formula, V3, L1, V4, L2, C L , V5, L3, V6, L4, C H1 is as defined herein above, wherein L5 and L6 are linkers that are present or absent, and wherein F c1、 and F c2 is F c domain, in which F c1 and F c2 are the same or different, preferably different) and F c Domains are as defined herein above in the "Definitions" section.

[0206] In one embodiment, F c1 comprises or consists of the amino acid sequence of SEQ ID NO: 132 (hole), and F c2 comprises or consists of the amino acid sequence of SEQ ID NO: 131 (Knob), or vice versa; More preferably, when V4 or V3 is a heavy chain variable domain, F c1 comprises or consists of the amino acid sequence of SEQ ID NO: 132, and accordingly, when V5 or V6 is a light chain variable domain, F c2 comprises or consists of the amino acid sequence of SEQ ID NO: 131, or When V4 or V3 is a light chain variable domain, F c1 comprises or consists of the amino acid sequence of SEQ ID NO: 131, and accordingly, when V5 or V6 is a heavy chain variable domain, F c2 comprises or consists of the amino acid sequence of SEQ ID NO: 132.

[0207] As can be seen from the examples, the inventors of the present invention have demonstrated, as proof of principle, the use of a low affinity recruiter (antigen binding site A) in combination with a mature TCR variable domain in TCER® form (Example 2).

[0208] Thus, in one preferred embodiment, the antigen-binding protein comprises two polypeptide chains that form two antigen-binding sites (A and B), in which one polypeptide chain has the formula [III]: V3-L1-V4-L2-C L -L5-F c1 [III] and having a structure represented by One polypeptide chain has the formula [IV]: V5-L3-V6-L4-CH1-L6-F c2 [IV] and having a structure represented by (In the formula, L2, C L , L5 and L4, C H1 , L6 does not exist, Among them, V 3、 L1, V4, V5, L3, V6 are as defined herein above; Preferably, as defined in the context of the present invention, V3 is V α or V γ domain, and V6 is Vβ or V δ domains, where V4 is a light chain variable domain and V5 is a heavy chain variable domain as defined in the context of the present invention, or Preferably, as defined in the context of the present invention, V3 is V α or V γ variable domain, V6 is V β or V δ domains, where V4 is a heavy chain variable domain and V5 is a light chain variable domain as defined in the context of the present invention; Preferably, L1 and L3 comprise or consist of the amino acid sequence "GGGSGGGG" of (SEQ ID NO: 118), Preferably F c1 comprises or consists of the amino acid sequence of SEQ ID NO: 132, F c2 comprises or consists of the amino acid sequence of SEQ ID NO: 131, or vice versa; More preferably, when V4 is a heavy chain variable domain, F c1 comprises or consists of the amino acid sequence of SEQ ID NO: 132, and accordingly, when V5 is a light chain variable domain, F c2 comprises or consists of the amino acid sequence of SEQ ID NO: 131, or More preferably, when V4 is a light chain variable domain, F c1 comprises or consists of the amino acid sequence of SEQ ID NO: 131, and accordingly, when V5 is a heavy chain variable domain, F c2 comprises or consists of the amino acid sequence of SEQ ID NO: 132, The light chain variable domain and the heavy chain variable domain together form one antigen-binding site A that binds to CD3, Among them, V α and V β or V γ and V δ The variable domains form one antigen binding site B, which specifically binds to a TA antigenic peptide / MHC complex, preferably a TAA antigenic peptide / MHC complex, as defined in the context of the present invention.

[0209] The antigen binding protein of this embodiment may also be referred to as TCER®.

[0210] A "TCER®" is a bispecific T cell receptor (TCR) that contains two antigen-binding domains, a heavy and light chain variable domain that binds to CD3 as defined in the context of the present invention, and a V α and V β or V γ and V δ It is a soluble antigen-binding protein comprising a domain.

[0211] In one embodiment, the present invention provides a method for the production of a medicament for the treatment of pulmonary arthritis, comprising or consisting of the amino acid sequence of SEQ ID NOs: 165-167, of the formula: V3-L1-V4-L2-C L -L5-F c1 [III] and a first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 163 or 164. H1 -L6-F c2 [IV] and a second polypeptide of [IV].

[0212] It may also be desirable to modify the antigen binding proteins of the invention with respect to effector function, for example to enhance or decrease the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antigen binding protein. This is referred to herein in the context of the antigen binding proteins of the invention as F c F of antigen-binding proteins, also called mutant forms c Alternatively, or in addition, this may be achieved by introducing one or more amino acid substitutions into the region. cCysteine ​​residues may be introduced into the region to allow interchain disulfide bond formation in this region, and the homodimeric antigen-binding protein thus generated may have improved or reduced internalization capability and / or increased complement-mediated cell killing and / or antibody-dependent cellular cytotoxicity (ADCC) (Caron PC, et al. 1992; and Shopes B, 1992).

[0213] Another type of amino acid modification of the antigen-binding proteins of the present invention may be useful to alter the native glycosylation pattern of the antigen-binding protein, namely by deleting one or more carbohydrate moieties found in the antigen-binding protein and / or adding one or more glycosylation sites that are not present in the antigen-binding protein. The presence of either the tripeptide sequences asparagine-X-serine, and asparagine-X-threonine (where X is any amino acid except proline) creates a potential glycosylation site. Addition or deletion of glycosylation sites to the antigen-binding protein is conveniently achieved by altering the amino acid sequence (in the case of N-linked glycosylation sites) so that it contains one or more of the above tripeptide sequences.

[0214] Another type of modification involves the removal of sequences identified in silico or experimentally as potentially contributing to degradation products or heterogeneity of antigen-binding protein preparations. For example, deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation when present primarily in Asn-Gly sequences, and less so in other dipeptide sequences such as Asn-Ala. Therefore, when such deamidation sites, particularly Asn-Gly, are present in the antigen-binding proteins of the present invention, it may be desirable to remove one of the involved residues, typically by conservative substitution. Such substitutions in the sequence to remove one or more involved residues are also intended to be encompassed by the present invention.

[0215] Another type of covalent modification involves chemically or enzymatically conjugating glycosides to antigen-binding proteins. These procedures are advantageous in that they do not require production of the antigen-binding protein in host cells with glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling mode used, sugars may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. For example, such methods are described in WO 87 / 05330.

[0216] Removal of any carbohydrate moieties present on the antigen-binding protein may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposing the antigen-binding protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the antigen-binding protein intact. Chemical deglycosylation is described by Sojahr H. et al. (1987); and Edge, A.S. et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by using various endo- and exoglycosidases, as described by Thotakura, N.R. et al. (1987).

[0217] Another type of covalent modification of an antigen-binding protein comprises coupling the antigen-binding protein to one of a variety of non-proteinaceous polymers, such as, for example, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner described in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.

[0218] The present invention also includes particles that display the antigen-binding proteins of the present invention, and including said particles in a particle library. Such particles include, but are not limited to, phage, yeast, ribosomes, or mammalian cells. Methods for generating such particles and libraries are known in the art (see, for example, WO 2004 / 044004; WO 01 / 48145; Chervin et al. (2008) J. Immuno. Methods 339.2:175-184).

[0219] As disclosed herein, the antigen binding proteins of the present invention bind to CD3 at antigen-binding site A and to a target antigen (TA) peptide / MHC complex at antigen-binding site B. CD3 molecules are typically present on the surface of effector cells, preferably CD3-presenting cells such as T cells. The target antigenic (TA) peptide / MHC complex is typically present on the surface of a target antigenic (TA) peptide / MHC complex-presenting cell, such as a diseased cell such as a cancer cell. Binding of the antigen binding protein to CD3 and the target antigen (TA) peptide / MHC complex brings the effector cell and target cell into close proximity with each other; thus, binding of the bispecific antigen binding protein may elicit an immune response upon binding. Thus, the antigen binding proteins of the present invention induce an immune response in the effector cell.

[0220] Thus, in one embodiment, the antigen binding proteins of the present invention preferably induce an immune response in CD3 presenting cells, such as effector cells, such as T cells or NK cells, in which the immune response is characterized by increased levels of interferon (IFN)γ. Thus, in one example, the immune response is preferably determined by an EC 50 It may be characterized by its value.

[0221] In the context of the present invention, the bispecific antigen-binding protein of the invention is D (A) binds to CD3, and the antigen-binding site B is K D (C) binds to a TA antigenic peptide C (TA-C) / MHC complex, preferably a TAA antigenic peptide C (TAA-C) / MHC complex, and K D (A) / K D (C) The ratio is greater than 1, greater than 4, greater than 6, greater than 8, greater than 10, greater than 15, greater than 20, greater than 25, greater than 30, greater than 40, greater than 50, between 1 and 150, between 4 and 140, between 6 and 100, between 8 and 100, between 10 and 100, preferably between 10 and 100.

[0222] "Affinity" and "K D The term " is defined herein above in the "Definitions" section. D Methods for measuring affinity, such as surface plasmon resonance and biolayer interferometry, are known to those skilled in the art. As known to those skilled in the art, the experimental conditions used in these experiments, such as the buffer used, protein concentration, or temperature, may affect the results.

[0223] Thus, in one example, the bispecific antigen binding protein of the invention is expressed as a soluble TCER®, e.g., as described herein above, and binds to the complex HLA-A *The peptide-HLA-A:02 complexes were analyzed for their binding affinity to the 02 / MAG-003 monomer. Typically, measurements are performed, for example, on an Octet RED384 system, typically using the settings recommended by the manufacturer. Briefly, binding kinetics were typically measured at 30°C, for example, at a shaking speed of 1000 rpm, using, for example, PBS, 0.05% Tween-20, 0.1% BSA as a buffer. The peptide-HLA-A:02 complexes were loaded onto a biosensor such as HIS1K before analyzing the antigen-binding protein, particularly TCER®. The same antigen-binding protein is then typically further analyzed for binding affinity to CD3. Therefore, measurements were performed as described herein above for CD3 binding.

[0224] In some embodiments, the antigen-binding site A has an affinity of 3 nM or more, 5 nM or more, 8 nM or more, 10 nM or more, 12 nM or more, 14 nM or more, 16 nM or more, 18 nM or more, 20 nM or more, 25 nM or more, 30 nM or more, 35 nM or more, 40 nM or more, preferably as measured using surface plasmon resonance (SPR) or biolayer interferometry (BLI), preferably biolayer interferometry (BLI). K is 45 nM or more, 1000 nM or less, 800 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 3 nM to 1000 nM, 3 nM to 600 nM, 5 nM to 600 nM, 10 nM to 600 nM, 12 nM to 600 nM, 14 nM to 600 nM, 16 nM to 600 nM, 18 nM to 600 nM, 20 nM to 600 nM, and the like, preferably 5 nM to 100 nM. D (A) binds to CD3.

[0225] In some embodiments, antigen-binding site B has a K of 100 μM or less, 1 μM or less, 100 nM or less, 50 nM or less, 10 nM or less, for example, 0.01 nM to 150 nM, 0.05 nM to 150 nM, 0.1 nM to 150 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.5 nM to 10 nM, 0.5 nM to 5 nM, 0.1 nM to 5 nM, preferably 0.5 nM to 5 nM, as measured using surface plasmon resonance (SPR) or biolayer interferometry (BLI), preferably biolayer interferometry (BLI). D In (A), it binds to a TA antigenic peptide C (TA-C) / MHC complex, preferably a TAA antigenic peptide C (TAA-C) / MHC complex.

[0226] In one example, the antigen-binding protein has a K of 100 nM or less, 50 nM or less, 10 nM or less, 1 nM or less, 1 nM or less, for example 10 pM to 100 nM, 10 pM to 50 nM, 10 pM to 10 nM, particularly 50 pM to 100 nM, 100 pM to 50 nM, 100 pM to 10 nM, 500 pM to 10 nM, preferably 500 pM to 10 nM, as measured using surface plasmon resonance (SPR) or biolayer interferometry (BLI), preferably biolayer interferometry (BLI). D a MAGE-A antigenic peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 10, preferably HLA-A * Combine with 02.

[0227] In one example, the antigen-binding protein has a K of 100 nM or less, 50 nM or less, 10 nM or less, 1 nM or less, 1 nM or less, for example 10 pM to 100 nM, 10 pM to 150 nM, 10 pM to 100 nM, particularly 50 pM to 100 nM, 100 pM to 100 nM, or 100 pM to 50 nM, as measured using surface plasmon resonance (SPR) or biolayer interferometry (BLI), preferably biolayer interferometry (BLI). D and binds to a PRAME peptide comprising or consisting of the amino acid sequence / MHC complex of SEQ ID NO:9.

[0228] In one embodiment, the bispecific antigen binding protein binds to ECs against normal tissue cells. 50 EC2 for TA-C / MHC-presenting cells, preferably TAA-C / MHC-presenting cells, that is 5-fold or more, 10-fold or more, 20-fold or more, 50-fold or more, 100-fold or more, 500-fold or more, or 1000-fold or more lower than the value 50 Among them, EC 50 is preferably determined in terms of induced cytotoxicity.

[0229] As used herein, "TA-C / MHC-presenting cell" or "TA-presenting cell" refers to a cell that presents a TA antigenic peptide, such as a specific TA-C antigenic peptide in a complex with an MHC protein, on its surface, in which the copy number of the TA peptide / MHC complex present on the cell surface can typically be determined by methods known to those skilled in the art. In one embodiment, the TA / MHC-presenting cell is a TA-C / MHC-presenting cell, preferably a TAA / MHC-presenting cell. In some examples, the TA antigenic peptide is a viral or bacterial peptide, and in such examples, the TA / MHC-presenting cell is typically a diseased or infected cell, in which the diseased cell is infected with the respective virus or bacterium. In some examples, the TA antigenic peptide is a TAA antigenic peptide, and in such examples, the TAA / MHC-presenting cell is typically a cancer cell.

[0230] In one embodiment, the TA / MHC-presenting cells, preferably TAA / MHC-presenting cells, for example TAA / MHC-presenting cancer cells, have a TA / MHC copy number or TAA / MHC copy number of more than 50, more than 100, more than 150, more than 200, more than 300, more than 600, more than 800, more than 1000, more than 1500, or more than 2000, preferably a TAA / MHC copy number of 50 to 5000.

[0231] "Copy number" herein refers to the number of TA antigenic peptide / MHC complexes present on the cell surface of a cell, such as a TAA / MHC-presenting cell, e.g., a cancer cell or a TA / MHC-presenting cell, such as a normal-shaped cell.

[0232] Such copy number depends, for example, on the particular TA and cell type and can be detected by methods known to those skilled in the art, such as FACS analysis, mass spectrometry (MS) and RNA sequencing, preferably mass spectrometry (MS) and RNA sequencing.

[0233] "Healthy cells," also sometimes referred to as "normal cells," herein refer to cells that are not cancer cells, and preferably, healthy cells herein refer to cells in the tissue surrounding TA-presenting cells. Preferably, when the TA is a viral or bacterial antigenic peptide, the healthy cells are preferably disease-free, i.e., not suffering from bacterial or viral infection with the respective virus or bacterium. However, in some cases, healthy cells may also express and present TA peptide / MHC complexes, e.g., TAA peptide / MHC complexes, such as TAA-C / MHC complexes, on their surface. Typically, in healthy cells in the context of the present invention, TA peptide / MHC complexes are present in lower amounts (copy numbers) than in TA-presenting cells, such as cancer cells, as will be understood by those skilled in the art.

[0234] Thus, in one embodiment, healthy cells have a TAA / MHC copy number, such as a TAA-C / MHC copy number, preferably a TAA / MHC copy number of less than 5000, less than 1000, less than 500, less than 100, less than 50, less than 20, less than 10, preferably less than 10, a TAA / MHC copy number between 0 and 10, such as 0-5.

[0235] The healthy cells are preferably selected from the group consisting of astrocytes, GABA neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, coronary artery endothelial cells, dermal microvascular endothelial cells, mesenchymal stem cells, nasal epithelial cells, peripheral blood mononuclear cells, and pulmonary artery smooth muscle cells, preferably GABA neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, coronary artery endothelial cells, nasal epithelial cells, peripheral blood mononuclear cells, and pulmonary artery smooth muscle cells.

[0236] In one embodiment, the antigen binding protein is selected from the group consisting of, for example, healthy cells, preferably astrocytes, GABA neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, coronary artery endothelial cells, dermal microvascular endothelial cells, mesenchymal stem cells, nasal epithelial cells, peripheral blood mononuclear cells, pulmonary artery smooth muscle cells, preferably GABA neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, coronary artery endothelial cells, nasal epithelial cells, peripheral blood mononuclear cells, pulmonary artery smooth muscle cells. 50 EC values ​​for TA / MHC complex-presenting cells, such as TA-C / MHC complex-presenting cells, including MAGE-A / MHC complex-presenting cells, that are 1,000 times or more lower, 15,000 times or more lower, or 9,000 times or more lower than the values ​​for TA / MHC complex-presenting cells, such as MAGE-A / MHC complex-presenting cells. 50 It has.

[0237] The bispecific antigen-binding proteins of the present invention have a high safety profile.

[0238] "Safety profile" herein refers to the ability to distinguish tumor cells from normal tissue cells or similar peptide-presenting cells. In the art, safety profiles are described using a safety margin.

[0239] As used herein, the term "safety margin" or "therapeutic margin" refers to the coefficient of the half-maximal concentration of a compound required to induce 100% cytotoxicity in a tumor cell line compared to the half-maximal concentration of a compound required to induce 100% cytotoxicity in normal tissue cells. The EC 50 is 1 pM, e.g., the EC determined for primary cells 50 EC50 values ​​of 1000 pM indicate a significant difference in the EC50 values ​​for tumor cell lines. 50 is EC versus primary cells 50 Since the value is 1000 times smaller than the reference value, the margin of safety is 1000.

[0240] In one embodiment, the bispecific antigen binding protein of the invention inhibits ECs against normal tissue cells. 50EC2 for TAA / MHC complex-presenting cells, preferably TAA-C / MHC complex-presenting cells, that is 500 to 12,000 times, preferably 1,000 to 10,000 times lower than the EC2 value. 50 EC against normal tissue cells, etc. 50 EC2 for TA / MHC complex-presenting cells, preferably TAA / MHC complex-presenting cells such as TAA-C / MHC complex-presenting cells, that is 100-fold or more, 500-fold or more, 1000-fold or more, 2000-fold or more, 3000-fold or more, 4000-fold or more, 5000-fold or more, or 6000-fold or more lower than the value 50 It has.

[0241] EC 50 If the margin of safety cannot be calculated, the margin of safety can also be determined by calculating the ratio of the "Lowest Observed Level" (LOEL) of the TCER® molecule to on-target cells and normal tissue cells, respectively.

[0242] The "LOEL" is defined herein as the first TCER® concentration associated with a response above the cutoff value, which was defined as the sum of the assay background (co-culture of healthy tissue cells with TCER® molecules without the addition of PBMCs) and three times the standard deviation within all assay wells.

[0243] In one embodiment, the bispecific antigen-binding protein of the invention has an LOEL for TA / MHC complex-presenting cells, preferably TAA / MHC complex-presenting cells such as TAA-C / MHC complex-presenting cells, that is at least 100-fold, at least 500-fold, at least 1000-fold, at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold, at least 6000-fold lower than the LOEL value for normal cells, such as an LOEL for TA / MHC complex-presenting cells, preferably TAA / MHC complex-presenting cells such as TAA-C / MHC complex-presenting cells, that is 500- to 12,000-fold, preferably 1,000- to 10,000-fold lower than the LOEL for normal cells.

[0244] In one embodiment, the bispecific antigen binding protein of the invention binds to ECs against similar peptide / MHC presenting cells. 50 EC2 for TA / MHC complex-presenting cells, preferably TAA / MHC complex-presenting cells, or TAA-C / MHC complex-presenting cells that is 5 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, 500 times or more, 1000 times or more, 2,000 times or more, 3,000 times or more, 4,000 times or more, 5,000 times or more, or 6,000 times or more lower than the value 50 Among them, EC 50 Preferably, ECs against similar peptide / MHC presenting cells 50 EC2 for TA / MHC complex-presenting cells, preferably TAA / MHC complex-presenting cells, that is 500 to 12,000, preferably 1,000 to 12,000 lower than the value 50 The cytotoxicity is determined by comparing it with the cytotoxicity induced by the cytotoxicity.

[0245] "Similar peptides" in the context of the present invention may also be referred to as "off-target" peptides and typically relate to peptides comprising 8 to 16 amino acids in length. Analogous peptides in the context of the present invention are typically MHC-presented. Furthermore, analogous peptides in the context of the present invention comprise or consist of an amino acid sequence similar to the amino acid sequence of a TA antigenic peptide, and in one preferred example in the context of the present invention, comprise an epitope, compared to the epitope of the TA antigenic peptide, in which at least one, two, or three, preferably one, two, or three, more preferably one, amino acid of said epitope has been substituted compared to the epitope of the TA antigenic peptide. Due to this sequence similarity, for example, if the analogous peptide is presented by an MHC protein, in this scenario the analogous peptide may be bound by the bispecific antigen-binding protein of the present invention, and thus the ability of a given bispecific antigen-binding protein to bind to the analogous peptide may not result in the desired effector cell response and may even result in an adverse reaction. Such adverse reactions may be "extra-tumor" side effects, such as cross-reactivity of specific TCRs that cross-react with peptides in normal tissues, as reported by Lowdell et al., Cytotherapy, published December 4, 2018, p. 7. Similar peptides in the context of the present invention may be selected based on high sequence similarity (similarity BLAST search) with TA antigenic peptides such as MAG-003, for example, HLA-A in the case of MAGE-A. * The peptides may be selected from a database of normal tissue-presented HLA class 1-binding peptides (XPRESIDENT database), such as 02-binding peptides. Therefore, because of these adverse reactions, the bispecific antigen-binding proteins of the invention are engineered to avoid cytotoxicity against cells of normal tissues that present similar peptides.

[0246] As used herein, the term "similar peptide / MHC-presenting cells" refers to cells that present similar peptide / MHC complexes on their cell surface.

[0247] In one embodiment, the analogous peptide / MHC presenting cells have a TAA / MHC copy number of more than 50, more than 100, more than 150, more than 200, more than 300, more than 600, more than 800, more than 1000, more than 1500, or more than 2000, preferably 50 to 5000.

[0248] In one embodiment, the analogous peptide / MHC presenting cells are MAGE-A / MHC complex presenting cells and have a MAGE-A / MHC complex copy number of more than 50, more than 80, more than 100, more than 120, more than 150, more than 300, more than 400, more than 600, more than 800, more than 1000, more than 1500, or more than 2000, preferably a MAGE-A / MHC copy number of 50 to 2000, such as 80 to 2000, such as 100 to 2000, for example 120 to 2000.

[0249] In one embodiment, the TA antigenic peptide is a Mage-A antigenic peptide, and the similar peptide is selected from the list consisting of the following peptides: RABGAP1L-001 consisting of the amino acid sequence of SEQ ID NO: 269, AXIN1-001 consisting of the amino acid sequence of SEQ ID NO: 270, ANO5-001 consisting of the amino acid sequence of SEQ ID NO: 271, TPX2-001 consisting of the amino acid sequence of SEQ ID NO: 272, SYNE3-001 consisting of the amino acid sequence of SEQ ID NO: 273, MIA3-001 consisting of the amino acid sequence of SEQ ID NO: 274, HERC4-001 consisting of the amino acid sequence of SEQ ID NO: 275, PSME2-001 consisting of the amino acid sequence of SEQ ID NO: 276, HEATR5A-001 consisting of the amino acid sequence of SEQ ID NO: 277, CNOT1-003 consisting of the amino acid sequence of SEQ ID NO: 278, TEP1-003 consisting of the amino acid sequence of SEQ ID NO: 279, ZFC-001 consisting of the amino acid sequence of SEQ ID NO: 281, and PITPNM3-001 consisting of the amino acid sequence of SEQ ID NO: 280.

[0250] Thus, in one embodiment the bispecific antigen binding protein of the invention is preferably HLA-A when said analogous peptide is in complex with an MHC protein. *When in a complex with .02, it does not bind or does not significantly bind to at least one analog peptide, such as at least two, at least three, at least four, at least five, 1, 2, 3, 4, 5, etc., preferably at least three or all analog peptides selected from the group of peptides consisting of RABGAP1L-001, AXIN1-001, ANO5-001, TPX2-001, SYNE3-001, MIA3-001, HERC4-001, PSME2-001, HEATR5A-001, CNOT1-003, TEP1-003, PITPNM3-001, ZFC-001, preferably HEATR5A-001, HERC4-001, and ZFC-001.

[0251] "Does not significantly bind" in the context of similar peptides, and in the context of the bispecific antigen-binding proteins herein, refers to a lower binding signal and / or a higher K D For example, in the context of the present invention, a bispecific antigen binding protein has a binding response to at least one similar peptide / MHC complex that is less than 50%, less than 45%, less than 40%, less than 30%, less than 20%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3% of the response of the same bispecific antigen binding protein binding to a MAGE-A antigenic peptide / MHC complex in the same experimental setting and at the same bispecific antigen binding protein concentration, and / or for example, in the context of the present invention, a bispecific antigen binding protein binds to at least one similar peptide / MHC complex with a reduced affinity compared to the affinity for the specific antigen, i.e., a MAGE-A antigenic peptide / MHC complex as described herein, in which the respective K D is increased by 5, 7, 10, 15, 20, 30, 40, 50, or 100 times, preferably 20 to 100 times, more preferably 30 to 100 times, 40 to 100 times, or typically 40 to 50 times. For example, when a bispecific antigen-binding protein binds to the MAG-003 / MHC complex at a K of 1 nM, D For example, the bispecific antigen-binding protein binds to the RABGAP1L-001 / MHC complex with a K of 100 nM.D Bispecific antigen-binding proteins exhibit a 100-fold increased K D and thus binds to RABGAP1L-001 / MHC with 100-fold reduced affinity. In these examples, the binding response, dissociation constant, and binding affinity are preferably measured using biolayer interferometry, e.g., as described in Examples 4 and 5.

[0252] As can be further seen from the Examples, particularly Example 1 and Figure 8, the bispecific antigen binding proteins of the invention are not only soluble but can also be expressed in CHO cells in transient expression at amounts of less than 10 mg / L (cell culture), less than 20 mg / L, or even up to 40 mg / L. Thus, in one embodiment, the bispecific antigen binding proteins of the invention can be expressed at high yields in host cells, preferably in amounts of more than 10, 15, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45 mg / L (cell culture), 5-50, 5-45, 5-40, 5-35, 10-35, 10-30, 10-25, 10-20 mg / L (cell culture), etc. In one example, the antigen binding protein is expressed in transiently transfected CHO-S cells, in which the cells are transfected at T=0 with 4x10 6 Cells were cultured at a density of 1000 / mL at 37°C in a total volume of 320 mL of GE Healthcare™ medium. After one day, feed solutions (CellBoost 7a and b) were added and the temperature was reduced to 32°C. Cells, and thus antigen-binding proteins, were harvested after a total culture time of 12 days and a total of three feedings.

[0253] As can be further seen from the examples, the inventors have demonstrated that the bispecific antigen-binding proteins of the invention have comparable or even improved stability compared to reference proteins, for example compared to an antigen-binding protein comprising a VH domain comprising, or alternatively consisting of, the amino acid sequence of SEQ ID NO: 39 and a VL domain comprising, or alternatively consisting of, the amino acid sequence of SEQ ID NO: 38, or a VH domain comprising, or alternatively consisting of, the amino acid sequence of SEQ ID NO: 137 and a VL domain comprising, or alternatively consisting of, the amino acid sequence of SEQ ID NO: 145, preferably a VH domain comprising, or alternatively consisting of the amino acid sequence of SEQ ID NO: 137 and a VL domain comprising, or alternatively consisting of the amino acid sequence of SEQ ID NO: 145.

[0254] Thus, in one embodiment, the antigen-binding proteins of the present invention optionally have comparable or improved stability compared to a reference protein. Comparable or improved stability in the context of the present invention refers to comparable or increased physical stability, for example, when exposed to temperature stress. Thus, the newly developed antigen-binding proteins of the present invention may withstand stress conditions, in particular temperature stress, comparable or better than the reference antigen-binding protein, wherein said antigen-binding protein is preferably in the same form.

[0255] The term "stability" in the context of the present invention refers to physical stability, which may be assessed qualitatively and / or quantitatively using various analytical techniques described in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991); and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). In the context of the present invention, these methods particularly refer to the assessment of aggregate formation (e.g., by using size exclusion chromatography, measuring turbidity, and / or visual inspection). To measure stability, samples comprising the antigen-binding protein of the present invention may be tested in stability tests, in which the sample is exposed to stress conditions for a selected period of time, followed by quantitative and optionally qualitative analysis of chemical and physical stability using appropriate analytical techniques.

[0256] In one embodiment, the antigen binding proteins of the invention are physically stable when exposed to stress conditions for a particular period of time, such as, for example, when exposed to a temperature of 40°C for, for example, 14 days.

[0257] "Physical stability", in the context of the present invention, refers to an antigen-binding protein that is substantially free from signs of aggregation, precipitation and / or denaturation.

[0258] Methods to access physical stability are, for example, size exclusion chromatography (SEC), dynamic light scattering (DLS), light obscuration (LO), color and clarity.

[0259] "No signs of aggregation" means, for example, that after exposure to stress conditions such as a temperature of 40°C for 14 days in a buffer such as PBS, a sample comprising the antigen binding protein has a monomer content of greater than 80%, greater than 86%, greater than 88%, greater than 90%, greater than 92%, greater than 94%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, such as a monomer content of 94%-99%, 95%-99%, 96%-99%, 97%-99% of the monomer content when measured by SEC, such as SEC-HPLC, in a buffer such as PBS.

[0260] Thus, in one embodiment, an antigen binding protein of the invention has the same or reduced aggregation, for example compared to a reference protein.

[0261] In size exclusion chromatography (SEC), depending on the column, operating pressure, and buffer velocity used, under the conditions tested, a difference of 1%, 2%, 3%, 4%, preferably 1 or 2%, more preferably 2% in monomer content is considered significant in the context of the present invention.

[0262] This means that if a reference antigen-binding protein has a monomer content of 96% and an antigen-binding protein of the invention has a monomer content of 98%, the monomer content of the antigen-binding protein of the invention is significantly different, and therefore significantly increased, compared to the reference antigen-binding protein when measured under the same conditions.

[0263] Nucleic acids, vectors, and recombinant host cells A further object of the present invention relates to an isolated nucleic acid sequence comprising or consisting of a sequence encoding a bispecific antigen-binding protein of the invention as defined herein above.

[0264] Typically, the nucleic acid is a DNA or RNA molecule that may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0265] "Vector," "cloning vector," and "expression vector" refer to vehicles by which DNA or RNA sequences (e.g., foreign genes) are introduced into a host cell, transforming the host and promoting expression (e.g., transcription or translation) of the introduced sequences.

[0266] Therefore, a further object of the present invention relates to a vector comprising a nucleic acid according to the invention.

[0267] Such vectors may comprise regulatory elements such as promoters, enhancers, terminators, etc., which may cause or direct the expression of the polypeptide when administered to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer (Mizukami T. et al. 1987), the Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), and promoters and enhancers of immunoglobulin H chains, etc. (Mason JO et al. 1985) and enhancers (Gillies SD et al. 1983).

[0268] Any expression vector for animal cells can be used as long as it can insert and express a gene encoding a human antibody C region. Examples of suitable vectors include pAGE107 (Miyaji H et al., 1990), pAGE103 (Mizukami T et al., 1987), pHSG274 (Brady G et al., 1984), pKCR (O'Hare K et al., 1981), and pSG1 β d2-4- (Miyaji H et al., 1990). Other examples of plasmids include autonomously replicating plasmids containing a replication origin, or integrative plasmids such as pUC, pcDNA, and pBR.

[0269] Other examples of viral vectors include adenovirus, retrovirus, herpes virus, and AAV vectors. Such recombinant viruses can be produced by transfection into packaging cells or by transient transfection with helper plasmids or viruses using techniques known in the art. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in International Publication No. 95 / 14785, International Publication No. 96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and International Publication No. 94 / 19478.

[0270] The term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin, has the ability to be packaged into a viral vector particle, and encodes at least an exogenous nucleic acid. The vector and / or particle can be used to transfer any nucleic acid into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. The term "virion" is used to refer to a single infectious viral particle. "Viral vector," "viral vector particle," and "viral particle" also refer to a complete viral particle with its DNA or RNA core and protein coat that is present outside a cell. For example, the viral vector may be selected from adenovirus, poxvirus, alphavirus, arenavirus, flavivirus, rhabdovirus, retrovirus, lentivirus, herpesvirus, paramyxovirus, or picornavirus.

[0271] Viruses may refer to naturally occurring viruses as well as artificial viruses. Viruses according to some embodiments of the present invention may be either enveloped or non-enveloped viruses. Parvoviruses (such as AAV) are examples of non-enveloped viruses. In preferred embodiments, the virus may be an enveloped virus. In preferred embodiments, the virus may be a retrovirus, particularly a lentivirus. Viral envelope proteins that can promote viral infection of eukaryotic cells may include vesicular stomatitis virus (VSV-G), modified feline endogenous retrovirus (RD114TR), and HIV-1-derived lentiviral vectors (LV) pseudotyped with envelope glycoproteins (GP) from modified gibbon ape leukemia virus (GALVTR). These envelope proteins can efficiently promote the entry of other viruses, such as parvoviruses, including adeno-associated virus (AAV), thereby demonstrating their broad efficacy. For example, Moloney murine leukemia virus (MLV) 4070 env (as described in Merten et al., J. Virol. 79:834-840, 2005, the contents of which are incorporated herein by reference), RD114 env, chimeric envelope proteins RD114 pro or RD pro (as described in Bell et al. Experimental Biology and Medicine 2010;235:1269-1276, the contents of which are incorporated herein by reference), RD114-HIV chimeras constructed by replacing the R peptide cleavage sequence of RD114 with the HIV-1 matrix / capsid (MA / CA) cleavage sequence, or baculovirus GP64 env (as described in Wang et al. J. Virol. 81:10869-10878, 2007, the contents of which are incorporated herein by reference), or GALV env (as described in Merten et al., J. Virol. 81:10869-10878, 2007, the contents of which are incorporated herein by reference). Other viral envelope proteins may also be used, including those described in (as described by) et al., J. Virol. 79:834-840, 2005) or derivatives thereof.

[0272] A further object of the present invention relates to host cells transformed, transduced or transfected with the nucleic acids and / or vectors according to the invention.

[0273] The term "transformation" originally referred to the naturally occurring process of gene transfer into a host cell, which involves the absorption of genetic material, e.g., nucleic acids such as DNA or RNA, by the cell through the cell membrane, resulting in the host cell expressing the introduced gene or sequence and producing a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. There are two types, termed natural transformation and artificial or induced transformation. Artificial or induced transformation methods are performed under laboratory conditions. A host cell that receives and expresses foreign nucleic acid, such as DNA or RNA, during the transformation process has been "transformed."

[0274] The term "transfection" refers to a mode of gene transfer involving the creation of pores in the cell membrane of a host cell, allowing the host cell to receive foreign genetic material. Typically, transfection refers to the transformation of eukaryotic cells, such as insect or mammalian cells. Chemical-mediated transfection involves the use of, for example, calcium phosphate or cationic polymers or liposomes. Non-chemically mediated transfection methods are typically electroporation, sonoporation, impalefection, phototransfection, or hydrodynamic delivery. Particle-based transfection uses gene gun technology, which uses nanoparticles to transfer nucleic acid into host cells, or another method called magnetofection. Nucleofection and the use of heat shock are other advanced methods for successful transfection. Host cells that receive foreign nucleic acid via transfection methods are "transfected."

[0275] The term "transduction" is generally understood to relate to the transfer of foreign nucleic acid, such as DNA or RNA, into a cell by a virus or viral vector. A host cell that receives and expresses foreign nucleic acid, such as DNA or RNA, by a virus or viral vector has been "transduced."

[0276] In some embodiments, the cells may be transduced using the methods described in US 20190216852, the entire contents of which are incorporated herein by reference.

[0277] The nucleic acids of the invention may be used to produce recombinant antigen binding proteins of the invention in a suitable expression system.

[0278] The term "expression system" means a host cell and a compatible vector under appropriate conditions for the expression of a protein encoded by, for example, foreign DNA carried by the vector and introduced into the host cell.

[0279] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other exemplary host cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, yeast of the genus Kluyveromyces or Saccharomyces, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., derived from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al; 1980), and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"). In some embodiments, YB2 / 0 cells may be preferred because the ADCC activity of chimeric or humanized antibodies is enhanced when expressed in these cells.

[0280] The present invention also relates to a host cell comprising a bispecific antigen recognition construct according to the present invention. Specifically, the host cell of the present invention comprises a nucleic acid or vector as described hereinabove. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or protozoan. The host cell can be a cultured cell or a primary cell, i.e., a cell directly isolated from an organism, such as a human. The host cell can be an adherent cell or a suspension cell, i.e., a cell that grows in suspension. For the purpose of producing a bispecific antigen-binding protein, such as a bispecific TCR, polypeptide, or protein, the host cell is preferably a mammalian cell.

[0281] In one particular embodiment, the host cells are stem cells, preferably mesenchymal stem cells.

[0282] In accordance with the above, in one embodiment the present invention refers to a host cell comprising the bispecific antigen binding protein of the invention, or the nucleic acid, or the vector of the invention as defined herein above, wherein said host cell is preferably a) a mesenchymal stem cell or b) a cell for recombinant expression such as a Chinese Hamster Ovary (CHO) cell.

[0283] In particular, for the expression of some bispecific antigen-binding proteins of the present invention, the expression vector may be of either type, including a type in which the gene encoding a first polypeptide, such as an antibody heavy chain or α chain, and a gene encoding a second polypeptide, such as an antibody light chain or β chain, are present on separate vectors, and a type in which both genes are present on the same vector (tandem type). Tandem-type humanized antibody expression vectors are preferred in terms of ease of construction of bispecific antigen-binding protein expression vectors, ease of introduction into animal cells, and balance between the expression levels of antibody H and L chains in animal cells (Shitara K et al. J Immunol Methods. 1994 Jan. 3;167(1-2):271-8). Examples of tandem-type humanized antibody expression vectors include pKANTEX93 (WO 97 / 10354 pamphlet) and pEE18.

[0284] In one embodiment, such recombinant host cells may be used for the production of at least one antigen binding protein of the invention.

[0285] Methods of Producing Bispecific Antigen-Binding Proteins of the Invention The present invention also provides a. providing a suitable host cell; b. providing a genetic construct comprising a coding sequence encoding a bispecific antigen binding protein of the invention; c. introducing said genetic construct into said suitable host cell, preferably ex vivo or in vitro; d. expressing said genetic construct by said suitable host cell; and optionally, e. Selecting cells that express and / or secrete the antibody; The present invention also relates to a method for producing an antigen binding protein as defined herein above, comprising:

[0286] The bispecific antigen-binding proteins of the present invention are as defined in the corresponding section herein above.

[0287] As used herein, "genetic construct" means a nucleic acid that allows for the expression of a coding region in a host, and thus refers to a nucleic acid such as a vector or RNA as described above.

[0288] In a specific embodiment, the method may further comprise the step of displaying said bispecific antigen recognition construct on the cell surface of said suitable host cell.

[0289] In another preferred embodiment, the genetic construct of b) comprises a nucleic acid encoding a bispecific antigen-binding protein of the invention, such nucleic acid being as defined herein above in the section "Nucleic Acids, Vectors and Recombinant Host Cells".

[0290] In a related embodiment, the genetic construct is an expression construct comprising a promoter sequence operably linked to said coding sequence.

[0291] In a related embodiment, the genetic construct is introduced into a suitable host using transformation, transduction or transfection, as defined herein above.

[0292] Desirably, the transduction system for introducing the genetic construct into said suitable host cells is a retroviral or lentiviral vector system as described herein above in the section on nucleic acids, vectors, and recombinant host cells. Such systems are well known to those skilled in the art.

[0293] In one embodiment, the method further comprises isolating and purifying the bispecific antigen-binding protein from the host cell, and optionally reconstituting the bispecific antigen-binding protein in a T cell.

[0294] The bispecific antigen binding proteins of the present invention may be produced by any technique known in the art, including but not limited to chemical, biological, genetic or enzymatic techniques, either alone or in combination.

[0295] Standard techniques for producing polypeptides such as antibodies or fragments thereof and TCRs or fragments thereof are known in the art, and these techniques can be used by those skilled in the art to produce the bispecific antigen-binding proteins of the present invention. For example, they can be synthesized using well-known solid-phase methods, particularly using commercially available peptide synthesizers (such as those manufactured by Applied Biosystems, Foster City, California) according to the manufacturer's instructions. Alternatively, antigen-binding proteins of the present invention, such as bispecific antibodies or fragments thereof and TCRs or fragments thereof, can be synthesized by recombinant DNA techniques well known in the art. For example, after incorporating a DNA sequence encoding the desired (poly)peptide into an expression vector and introducing such a vector into a suitable eukaryotic or prokaryotic host that expresses the desired polypeptide, the fragment can be obtained as a DNA expression product, from which the fragment can then be isolated using well-known techniques.

[0296] In one example, i.e., in the case of the TCER® bispecific molecule, V H and V LDNA sequences encoding various combinations of variable α (Vα) and variable β (Vβ), as well as sequences encoding linkers, may be obtained, for example, by gene synthesis. The resulting DNA sequences may contain, for example, hinge regions, C, D, E, F, G ... H2 , and C H3 The F domains may be cloned in frame into expression vectors encoding the F domains, respectively, and further manipulated. v Fragments in Recombinant Immunotoxins by Disulfide Bonds Engineered into Conserved Framework Regions. Biochemistry, 1994, 33, 5451-5459) were synthesized with or without additional interchain disulfide bond stabilization. H3 To incorporate "knob-into-hole" mutations into the C domain H2 to remove N-glycosylation sites (e.g., N297Q mutation); c -to introduce silencing mutations; or V L and V H Genetic engineering may be performed to introduce additional disulfide bond stabilization into each of the nucleotides.

[0297] The bispecific antigen-binding proteins of the invention are suitably separated from the culture medium by immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0298] In one embodiment, recovery of the expressed antigen binding protein or polypeptide herein refers to performing Protein A chromatography, Protein L chromatography, Kappa selection chromatography, and / or size exclusion chromatography, preferably Protein L chromatography and / or size exclusion chromatography, more preferably Protein L-chromatography and size exclusion chromatography.

[0299] Methods for producing the bispecific antigen-binding proteins of the present invention involve recombinant DNA and gene transfection techniques well known in the art (see Morrison SL. et al. (1984) and patent documents U.S. Pat. No. 5,202,238; and U.S. Pat. No. 5,204,244).

[0300] Furthermore, methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, for example, Riechmann L. et al. 1988; Neuberger MS. et al. 1985) and can be readily applied to the production of the bispecific antigen-binding proteins of the present invention.

[0301] In one example, a vector for expression of a recombinant antigen-binding protein of the present invention was designed as a monocistronic vector controlled by, for example, a pUC19 derivative, a promoter element derived from HCMV. Plasmid DNA was amplified in Escherichia coli (E. coli) using standard culture methods and subsequently purified using a commercially available kit (Macherey & Nagel). The purified plasmid DNA was used for transient transfection of CHO-S cells, for example, using the ExpiCHO™ system (Thermo Fisher Scientific) according to the manufacturer's instructions or an electroporation system (MaxCyte STX). The transfected CHO-cells were cultured, for example, at 32°C to 37°C for 6 to 14 days and fed once or twice with ExpiCHO™ Feed or Cellboost 7a and 7b (GE Healthcare™) solutions.

[0302] The adjusted cell supernatant was clarified by filtration (0.22 μm) using, for example, a Sartoclear Dynamics® Lab Filter Aid (Sartorius). The bispecific antigen-binding protein was purified using, for example, an Akta Pure 25 L FPLC system (GE Lifesciences) equipped to perform affinity and size-exclusion chromatography inline. Affinity chromatography was performed, for example, on a Protein A or L column (GE Lifesciences) according to standard affinity chromatography protocols. For example, size-exclusion chromatography was performed immediately after elution from the affinity column (pH 2.8) using a Superdex 200 pg 16 / 600 column (GE Lifesciences), to obtain highly pure monomeric protein according to standard protocols. Protein concentration was determined, for example, on a NanoDrop system (Thermo Scientific) using the extinction coefficient calculated according to the predicted protein sequence. If necessary, the concentration was adjusted using a Vivaspin device (Sartorius). Finally, the purified molecule was stored, for example, in phosphate buffered saline at a concentration of approximately 1 mg / mL at a temperature of 2-8°C.

[0303] The quality of the purified bispecific antigen-binding protein was determined by HPLC-SEC on, e.g., a MabPac SEC-1 column (5 μm, 4×300 mm) run in, e.g., a Vanquishu HPLC-System in 50 mM sodium phosphate, pH 6.8, containing 300 mM NaCl.

[0304] Pharmaceutical Composition The present invention further refers to a pharmaceutical composition comprising a bispecific antigen-binding protein of the invention, a nucleic acid of the invention, a vector of the invention, or a host cell of the invention, and a pharmaceutically acceptable carrier.

[0305] The present invention also relates to a bispecific antigen-binding protein according to the invention for use as a medicament. The present invention also relates to a pharmaceutical composition of the invention for use as a medicament.

[0306] The present invention also relates to the use of a bispecific antigen-binding protein according to the invention and / or a pharmaceutical composition of the invention in the manufacture of a medicament.

[0307] The term "pharmaceutical composition" or "therapeutic composition," as used herein, refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a subject.

[0308] In some embodiments, a subject may also be referred to as a patient.

[0309] Such therapeutic agents or pharmaceutical compositions may comprise a therapeutically effective amount of a bispecific antigen-binding protein of the invention in a mixture with a pharmaceutically or physiologically acceptable formulation selected for compatibility with the mode of administration, or may further comprise a therapeutic agent.

[0310] The bispecific antigen-binding proteins of the invention will normally be supplied as part of a sterile pharmaceutical composition, which will normally include a pharmaceutically acceptable carrier.

[0311] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecules and compositions that do not produce adverse, allergic or other untoward reactions, as appropriate, when administered to mammals, especially humans. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0312] A "pharmacologically acceptable carrier," which may also be referred to as a "pharmaceutically acceptable diluent" or a "pharmaceutically acceptable vehicle," may include physiologically compatible solvents, fillers, stabilizers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Thus, in one embodiment, the carrier is an aqueous carrier.

[0313] In another aspect, aqueous carriers can confer improved properties when combined with the bispecific antigen-binding proteins described herein, such as, for example, improved solubility, efficacy, and / or improved immunotherapy. The form, route of administration, dosage, and regimen of a pharmaceutical composition will necessarily depend on the condition being treated, the severity of the disease, the age, weight, and sex of the patient, the desired duration of treatment, etc. The pharmaceutical composition may be in any suitable form (depending on the desired method of administering it to a patient). It may be provided in unit dosage form, generally in a sealed container, and may be provided as part of a kit. Such a kit will usually (but not necessarily) include instructions for use. It may contain a plurality of said unit dosage forms.

[0314] Empirical considerations such as biological half-life generally contribute to determining the dosage. The frequency of administration may be determined and adjusted over the course of treatment and is based on reducing the number of cancer cells, maintaining a reduction in cancer cells, reducing the proliferation of cancer cells, or eliminating cancer cells. Alternatively, a sustained release formulation of the bispecific antigen-binding protein may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0315] In one embodiment, the dosage of the antigen-binding protein can be empirically determined in individuals who have received one or more doses. Individuals are given increasing doses of the antigen-binding protein. To evaluate the effectiveness of the antigen-binding protein, markers of cancer cell status can be monitored. These include direct measurement of cancer cell proliferation and cell death using FACS or other imaging techniques; and improved health as assessed by such measurements, or improved quality of life as measured by recognized tests or prolonged survival. Those skilled in the art will appreciate that dosages will vary depending on the individual, the stage of the disease, and previous and concurrent treatments being used.

[0316] In particular, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for injectable formulations, which may in particular be an isotonic, sterile, saline solution (such as mono- or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or a mixture of such salts), or a dried, in particular lyophilized, composition, which, upon addition of sterile water or saline, allows the constitution of an injectable solution.

[0317] To prepare a pharmaceutical composition, an effective amount of the bispecific antigen-binding protein of the invention may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0318] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to allow easy injection. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[0319] A solution of the active compound as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under normal storage and use conditions, these preparations contain a preservative to prevent the growth of microorganisms.

[0320] The antigen binding proteins of the present invention may be formulated into neutral or salt form compositions using pharmaceutically acceptable salts.

[0321] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in suitable solvent with various other ingredients as listed above, if necessary, and then by filtration sterilization.Generally, dispersion solution is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains basic dispersion medium and other necessary ingredients from those listed above.For the aseptic powder that is used to prepare sterile injectable solution, the preferred preparation method includes vacuum drying and freeze-drying technology, which produces powder of active ingredient and any additional desired ingredients from the solution that has been previously sterile-filtered.

[0322] The preparation of more concentrated or highly concentrated solutions for direct injection is also contemplated, in which the use of DMSO as a solvent is envisioned to result in very rapid penetration, delivering high concentrations of active agent to small tumor areas.

[0323] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above, although drug release capsules and the like can also be used.

[0324] Treatments and Uses The present inventors have demonstrated in vitro cytotoxic activity of MAG-003 target antigen binding proteins combined with either BMA031 (V36) or UCHT1 (V17) as recruiters, particularly in the TCER® form, against different MAG-003-positive cancer cell lines in Example 2 of the Experimental Section. Furthermore, the present inventors have demonstrated that MAG-003 target antigen binding proteins combined with either BMA031 (V36) or UCHT1 (V17) as recruiters, particularly in the TCER® form, demonstrate the cytotoxic activity of these molecules against different MAG-003-positive cancer cell lines in vitro. * In cell lines expressing CD3 but not displaying a TAA peptide such as MAG-003, only little lysis was induced by the dual antigen-binding protein, demonstrating that the cytotoxic activity is highly specific and restricted to TAA-positive cells, such as MAG-003-positive cells. Thus, the Examples demonstrate the technical advantages of combining a low-affinity binding domain directed against CD3 with a high-affinity TCR variable domain, as disclosed in the context of the present invention. While in the Examples, such TCR variable domains specifically bind to TAA A as a target, those skilled in the art will appreciate that the advantages observed for the exemplified bispecific antigen-binding proteins in the context of a TAA as a target are also transferable to bispecific antigen-binding proteins that target another TA, such as a viral or bacterial antigenic peptide, instead of a TAA. It will be appreciated by those skilled in the art that in some embodiments, antigen binding proteins, when administered to a subject, bind to target cells, such as TA / MHC complex-presenting cells, recruit endogenous effector cells, bind to them via CD3, and activate them, thus localizing those effector cells in the vicinity of target cells, particularly target cancer cells, to achieve killing of said target cells, particularly anti-cancer activity.

[0325] In one embodiment, the binding of the antigen-binding protein to the target cell and the binding of the effector cell to CD3 induces an immune response, and this response may refer to the proliferation and activation of effector functions in vitro or in vivo. For example, in the case of MHC class I-restricted cytotoxic T cells, the effector function may be the lysis of target cells that are peptide-pulsed, peptide precursor-pulsed, or naturally presenting the peptide; the secretion of cytokines, preferably interferon-γ, TNF-α, or IL-2, induced by the peptide; the secretion of effector molecules, such as granzymes or perforins, induced by the peptide; or degranulation.

[0326] Thus, the bispecific antigen-binding proteins of the invention, and in particular the TCER® molecules, may be used to treat a wide variety of conditions, including, for example, various forms of cancer and / or infectious disease conditions. The bispecific antigen-binding proteins of the invention may be used for therapeutic purposes in humans and / or non-human mammals, in particular humans.

[0327] In one embodiment, bispecific antigen-binding proteins of the invention may bind to diseased cells and reduce the proliferation and / or kill diseased cells that present TA peptide / MHC complexes on their cell surface. In a preferred embodiment, bispecific antigen-binding proteins of the invention may bind to tumor cells and reduce the proliferation and / or kill tumor cells that present TAA peptide / MHC complexes on their cell surface. It is understood that the bispecific antigen-binding protein is administered at a concentration that promotes binding under physiological (e.g., in vivo) conditions.

[0328] Thus, in one embodiment, the bispecific antigen binding proteins of the invention may be used in immunotherapy against tumor cells of different tissues such as colon, lung, breast, prostate, ovary, pancreas, kidney, etc. In another embodiment, the antigen binding proteins of the invention may bind to tumor cells, reduce their proliferation and / or kill them.

[0329] Accordingly, the present invention relates to a method of treating or preventing a proliferative disease or disorder comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific antigen-binding protein, nucleic acid or vector, host cell or pharmaceutical composition as defined herein above in the sections "Bispecific antigen-binding protein", "Nucleic acid" or "Pharmaceutical composition" in accordance with the present invention.

[0330] In certain embodiments, the present invention relates to a method of treating a subject having a disease comprising administering to the subject a bispecific antigen-binding protein of the invention.

[0331] In a further embodiment, the present invention refers to a method of eliciting an immune response in a subject with a disease, comprising administering to the subject a composition comprising an antigen recognition construct of the present invention, optionally expressed in a host cell.

[0332] In one embodiment the invention refers to the use of a bispecific antigen-binding protein, a nucleic acid or vector, a host cell or a pharmaceutical composition according to the invention for treating or preventing a disease in a subject.

[0333] In one embodiment, the immune response referred to in the method is a cytotoxic T cell response induced by binding of an antigen binding protein to CD3 present on an effector cell, by binding to a TA antigenic peptide / MHC complex, and thus by bringing the effector cell and target cell into close proximity.

[0334] The present invention further refers to a bispecific antigen-binding protein of the invention, a nucleic acid of the invention or a vector of the invention, a host cell of the invention or a pharmaceutical composition of the invention for use in the diagnosis, prevention and / or treatment of a disease.

[0335] The present invention further refers to the use of a bispecific antigen-binding protein of the invention, a nucleic acid of the invention or a vector of the invention, a host cell of the invention or a pharmaceutical composition of the invention for the manufacture of a medicament for the diagnosis, prevention and / or treatment of a disease.

[0336] The terms "subject" or "individual" are used interchangeably and may be, for example, a human or non-human mammal, preferably a human.

[0337] The term "treat" or "treatment" in the context of the present invention refers to therapeutic use (i.e., in a subject with a given disease) and means to reverse, alleviate, or inhibit the progression of one or more symptoms of such disorder or condition. Thus, treatment refers not only to treatment that results in a complete cure of the disease, but also to treatment that slows the progression of the disease and / or prolongs the survival of the subject.

[0338] By "preventing" is meant prophylactic use (ie, in a subject susceptible to a given disease).

[0339] The term "in need of treatment" refers to a subject already with the disorder, as well as a subject in which the disorder is to be prevented. Thus, in one embodiment, the subject is a patient.

[0340] In one embodiment, a "disease" or "disorder" is any medical condition that would benefit from treatment with an antigen-binding protein of the present invention. In one embodiment, this includes chronic and acute disorders or diseases, including pathological conditions that predispose the subject to the disorder in question. In particular, the disease referred to in the context of the present invention may be a proliferative disease, or a disease caused by a virus or bacteria. A "disease caused by a virus or bacteria" may also be referred to as a viral or bacterial infection. In the context of the present invention, the disease-causing virus may be selected from the group consisting of, for example, human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), cytomegalovirus (CMV), human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), influenza virus, preferably human immunodeficiency virus (HIV). In the context of the present invention, the disease-causing bacterium may be Mycobacterium tuberculosis, etc. It will be appreciated by those skilled in the art that where the bispecific antigen binding protein targets a viral antigenic peptide such as HIV, the bispecific antigen binding protein is for use in the treatment of HIV. Thus, a bispecific antigen binding protein targeting the viral or bacterial antigenic peptide TA-C is thus for use in the treatment of the virus or bacterium from which said antigenic viral or bacterial antigenic peptide is derived.

[0341] "Proliferative disorders," such as cancer, involve the uncontrolled and / or inappropriate growth of cells.

[0342] In one embodiment, the proliferative disorder or disease is a tumor disease characterized by the expression of TAs, more specifically TAAs, in, for example, cancer cells or tumor cells of the tumor disease.

[0343] Therefore, particularly preferred cancers are TA-positive cancers, especially TAA-positive cancers.

[0344] In a further embodiment, the proliferative disorder or disease is a tumor disease characterized by expression of MAGEA4 and / or MAGEA8, for example in cancer or tumor cells of said tumor disease.

[0345] Therefore, particularly preferred cancers are MAGEA4 and / or MAGEA8 positive cancers.

[0346] In a further embodiment, the proliferative disorder or disease is a tumor disease characterized by expression of PRAME, for example in cancer or tumor cells of said tumor disease.

[0347] Therefore, a particularly preferred cancer is a PRAME-positive cancer.

[0348] In the context of the present invention, a cancer is considered to be "TAA positive", such as "MAGEA4 and / or MAGEA8 positive" or "PRAME positive", if a relevant TAA peptide, such as one of the TAA peptides defined herein above in the "Definitions" section, e.g., MAG-003 peptide or PRAME-004, is present in more than 98% of all cancers according to NCI guidelines. For any other indications mentioned herein, a biopsy may be performed, as is the standard in the treatment of these cancers, and peptides may be identified according to XPRESIDENT® and related methods (per WO 03 / 100432; WO 2005 / 076009; WO 2011 / 128448; WO 2016 / 107740; U.S. Pat. No. 7,811,828; U.S. Pat. No. 9,791,444; and U.S. Pat. No. 2016 / 0187351, the contents of each of which are incorporated herein by reference in their entirety). In one embodiment, cancer is readily assayed (i.e., diagnosed), for example, by using the bispecific antigen binding proteins of the invention. Methods for identifying cancer-expressing antigens using antigen binding proteins are known to those of skill in the art.

[0349] In one embodiment, the cancer is selected from the list consisting of lung cancer, such as non-small cell lung cancer and small cell lung cancer, liver cancer, head and neck cancer, skin cancer, renal cell cancer, brain cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, leukemia, breast cancer, Merkel cell carcinoma, melanoma, ovarian cancer, bladder cancer, uterine cancer, gallbladder and bile duct cancer, osteosarcoma, and esophageal cancer.

[0350] Textbooks providing guidance in cancer treatment include Cancer, Principles and Practice of Oncology, 4th Edition, DeVita et al., Eds. J.B. Lippincott Co., Philadelphia, Pa. (1993). The appropriate treatment approach will depend on the particular type of cancer and other factors, such as the patient's general condition, as recognized in the relevant field. The bispecific antigen-binding proteins of the invention can be used by themselves or can be added to a treatment regimen using other anti-cancer agents typically used to treat cancer patients.

[0351] Thus, in some embodiments, the bispecific antigen-binding proteins of the invention may be administered simultaneously with, prior to, or following a variety of drugs and therapies commonly used in cancer treatment, such as, for example, chemotherapeutic agents, non-chemotherapeutic agents, anti-tumor agents, and / or radiation, preferably chemotherapeutic agents.

[0352] In a further embodiment, the bispecific antigen binding proteins may also be used to treat infectious diseases such as infectious viral or bacterial diseases, wherein the viral disease is, for example, selected from the group consisting of human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), cytomegalovirus (CMV), human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus infection (HPV), Epstein-Barr virus (EBV), influenza virus, preferably HIV, HBV, influenza, and HCMV, among others, and wherein the bacterial disease is, for example, tuberculosis.

[0353] The antigen binding protein of the present invention or pharmaceutical composition thereof may be administered by itself, or may be administered simultaneously with, before, or after the administration of other therapeutic agents used to treat such infections.

[0354] "Diagnosis" herein means a medical diagnosis and refers to determining what disease or condition explains a person's symptoms and signs.

[0355] A "therapeutically effective amount" of a bispecific antigen-binding protein or pharmaceutical composition thereof refers to a sufficient amount of bispecific antigen-binding protein to treat said proliferative disease at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily or monthly usage of the antigen-binding protein, nucleic acid or vector, host cell, or pharmaceutical composition of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective amount level for any particular patient will depend on a variety of factors, including the disorder or disease being treated and the severity of the disorder; the activity of the particular bispecific antigen-binding protein used; the particular composition used, the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the particular polypeptide used; the duration of treatment; drugs used in combination with or concomitantly with the particular polypeptide used; and similar factors well known in the medical arts. For example, it is well within the skill of one in the art to start administering a compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0356] In one embodiment, the efficacy of treatment with a bispecific antigen binding protein of the invention is assayed in vivo, e.g., in a mouse model of cancer, by measuring, e.g., the change in tumor volume between treatment and control groups.

[0357] For example, pharmaceutical compositions, vectors, nucleic acids, and cells of the invention may be provided, for example, in substantially pure form, in which at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% by weight of the same type of bispecific antigen-binding protein is present.

[0358] The bispecific antigen-binding protein of the invention, the nucleic acid of the invention or the vector of the invention, the host cell of the invention or the pharmaceutical composition of the invention may be administered by any practicable method.

[0359] The present invention also provides a method of killing target cells in a patient comprising administering to the patient an antigen binding protein, in the context of which the antigen binding protein, when administered to a subject, binds to target cells and CD3-expressing effector cells and preferably elicits an immune response.

[0360] In one particular approach, the host cells may be stem cells, such as mesenchymal stem cells, which are engineered to express the bispecific antigen-binding protein of the invention. In this example, the bispecific antigen-binding protein is TCER®, as described herein.

[0361] Therefore, the host cells of the present invention, preferably stem cells as defined above, may be used as an active ingredient in a therapeutic composition. Accordingly, the present invention also provides a method of killing target cells in a patient, the method comprising administering to the patient an effective number of host cells, preferably mesenchymal stem cells as defined above.

[0362] For purposes of the methods of the invention in which a host cell or population of cells is administered to a subject, the host cells can be allogeneic (from another subject) or autologous to the subject. Preferably, the cells are autologous to the subject.

[0363] If the host cells are allogeneic and therefore from another subject, said other subject is healthy.

[0364] By "healthy" it is meant that the subject is in generally good health, preferably has a competent immune system, and more preferably is free from any disease that can be easily tested for and detected.

[0365] Thus, host cells have been transformed, transduced or transfected with nucleic acids and / or vectors according to the invention, as described herein above in the section "Nucleic Acids, Vectors and Recombinant Host Cells."

[0366] Where a host cell is transformed, transduced or transfected to express an antigen binding protein of the invention, preferably the cell comprises an expression vector capable of expressing the bispecific antigen binding protein. Once the host cell expresses the bispecific antigen binding protein of the invention the host cell may be referred to as an activated host cell.

[0367] In one embodiment, a TCR-induced immune response or T cell response can refer to the proliferation and activation of effector functions induced by dual antigen binding to T cells and binding to a TA antigenic peptide / MHC complex, such as a TA-C / MHC complex, in vitro or in vivo. For example, in MHC class I-restricted cytotoxic T cells, the effector functions can be lysis of peptide-pulsed, peptide precursor-pulsed, or naturally peptide-presenting target cells; peptide-induced secretion of cytokines, preferably interferon-γ, TNF-α, or IL-2; peptide-induced secretion of effector molecules, such as granzymes or perforins; or degranulation.

[0368] Thus, a further aspect of the present invention provides activated host cells obtainable by the methods of the present invention described above.

[0369] The activated host cells produced by the above method may selectively recognize target cells.

[0370] "Target cells" herein refer to TA-C / MHC-presenting cells or TA-presenting cells as defined herein above in the section "Bispecific antigen-binding proteins."

[0371] In a preferred embodiment, ie, when the TA / MHC complex is a TAA / MHC complex, the target cell is a cancer cell, wherein cancer is as defined herein above.

[0372] In vivo, target cells for CD3-positive effector cells according to the present invention may be tumor cells (which sometimes express MHC class II) and / or stromal cells surrounding the tumor (tumor cells) (which sometimes also express MHC class II; (Dengjel, J. et al., Clin Cancer Res 12(2006):4163-4170).

[0373] kit Finally, the present invention also provides a kit comprising at least one bispecific antigen-binding protein of the present invention.

[0374] In one embodiment, the kit comprises: a) at least one bispecific antigen-binding protein of the invention as defined herein above in the section "Bispecific antigen-binding proteins"; b) optionally packaging materials, and c) optionally, a label or package insert contained within said packaging material indicating that said dual antigen binding protein is effective in treating or to be used for the treatment of a disease, preferably cancer; The compound comprises:

[0375] In a related embodiment, at least one antigen binding protein of the present invention is contained in single and / or multi-chambered pre-filled syringes (eg, liquid syringes and dissolution syringes).

[0376] In one embodiment, the invention encompasses a kit for producing a single-dose administration unit.

[0377] Thus, in one embodiment, the at least one bispecific antigen-binding protein of the invention referred to in a) of the kit of the invention is a dried bispecific antigen-binding protein of the invention contained in a first container, in turn the kit further comprises a second container with an aqueous formulation.

[0378] Therefore, the kit a) a first container comprising at least one dried bispecific antigen-binding protein of the invention as defined herein above in the section "Antigen-binding proteins", b) a second container comprising an aqueous formulation; c) optionally packaging materials, and d) optionally, a label or package insert contained within said packaging material indicating that said dual antigen binding protein is effective in treating or to be used for the treatment of a disease, preferably cancer. The compound comprises:

[0379] Aqueous formulations are typically aqueous solutions comprising a pharmaceutically acceptable carrier, as defined herein above under "Pharmaceutical Compositions."

[0380] In related embodiments, the terms "first container" and "second" container refer to the chambers of a multi-chambered pre-filled syringe (e.g., a dissolution syringe).

[0381] Cancer is as defined herein above in the context of the present invention.

[0382] The present invention further relates to the items and embodiments cited herein below.

[0383] In a further aspect, the present invention relates to a bispecific antigen-binding protein comprising at least two antigen-binding sites (D and B), in which antigen-binding site D binds to TCRα / β, in which antigen-binding site B binds to a target antigenic (TA) peptide / MHC complex, and in which antigen-binding site D binds to a heavy chain variable domain (V H) and the light chain variable domain (V L ), wherein said V L comprises or consists of the amino acid sequence of SEQ ID NO: 42, wherein said V H comprises or consists of the amino acid sequence of SEQ ID NO:43.

[0384] In one related aspect of the above embodiment, the antigen-binding site D of the bispecific antigen-binding protein of the above embodiment is K D (D) binds to TCRα / β, and antigen-binding site B binds to K D (C) binds to the target antigenic peptide C(TA-C) / MHC complex, in which K D (D) / K D (C) The ratio is greater than 1, greater than 4, greater than 6, greater than 8, greater than 10, greater than 15, greater than 20, greater than 25, greater than 30, greater than 40, greater than 50, between 1 and 150, between 4 and 140, between 6 and 100, between 8 and 100, between 10 and 100, preferably between 10 and 100.

[0385] In a further related aspect of one of the above embodiments, the antigen-binding site D has a denaturing activity of 3 nM or more, 5 nM or more, 8 nM or more, 10 nM or more, 12 nM or more, 14 nM or more, 16 nM or more, 18 nM or more, 20 nM or more, 25 nM or more, 30 nM or more, 35 nM or more, 40 nM or more, as measured by surface plasmon resonance (SPR) or biolayer interferometry (BLI), preferably biolayer interferometry (BLI). K is nM or more, 45 nM or more, preferably 1000 nM or less, 800 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 3 nM to 1000 nM, 3 nM to 600 nM, 5 nM to 600 nM, 10 nM to 600 nM, 12 nM to 600 nM, 14 nM to 600 nM, 16 nM to 60 nM, 18 nM to 600 nM, 20 nM to 600 nM, and the like, preferably 5 nM to 100 nM. D Binding to TCRα / β in (A).

[0386] In a further related item of one of the above embodiments, antigen-binding site B has a K of 100 μM or less, 1 μM or less, 100 nM or less, 50 nM or less, 10 nM or less, for example, 0.01 nM to 150 nM, 0.05 nM to 150 nM, 0.1 nM to 150 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.5 nM to 10 nM, 0.5 nM to 5 nM, preferably 0.5 nM to 5 nM, as measured using surface plasmon resonance (SPR) or biolayer interferometry (BLI), preferably biolayer interferometry (BLI). D In (C), the target antigenic peptide C (TA-C) binds to the MHC complex.

[0387] In a further related aspect of the above embodiment, the antigen binding protein is an EC 50 EC values ​​for TA-C / MHC-presenting cells that are 100 or more, 500 or more, or 1000 or more lower than the values 50 It has.

[0388] In a further related aspect of one of the above embodiments, the TA antigenic peptide C is a viral peptide, a bacterial peptide or a tumor-associated antigen (TAA) peptide, preferably a tumor-associated antigen (TAA) peptide.

[0389] In a further related aspect of the above embodiment, the antigen binding protein is an EC 50 EC values ​​for TA-C / MHC complex-presenting cells that are 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, or 1000-fold lower than the values 50 It has.

[0390] In a further related item of one of the above embodiments, the TA antigenic peptide C is a tumor-associated antigen (TAA) peptide C, wherein said TAA-C is a PRAME antigenic peptide comprising or consisting of the amino acid sequence "SLLQHLIGL" of SEQ ID NO: 9, or a MAGE-A antigenic peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 10 (wherein the MHC is preferably HLA-A* 02), and the like, are selected from the group of TAA antigenic peptides comprising or consisting of the amino acid sequences of SEQ ID NOs: 162 to 317, SEQ ID NOs: 9 and 10.

[0391] In a further related item of the above embodiment, TA antigenic peptide C is a MAGE-A antigenic peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 10, wherein the similar peptides are selected from the list consisting of RABGAP1L-001, AXIN1-001, ANO5-001, TPX2-001, SYNE3-001, MIA3-001, HERC4-001, PSME2-001, HEATR5A-001, CNOT1-003, TEP1-003, PITPNM3-001, ZFC-001, preferably HEATR5A-001, HERC4-001, and CNOT1-003.

[0392] In a further related aspect of one of the above embodiments, the bispecific antigen-binding protein is a bispecific antibody or fragment thereof, a bispecific T-cell receptor (TCR) or fragment thereof, or a bispecific single-chain TCR (scTCR) or a bispecific single-chain antibody.

[0393] In a further related aspect of the above embodiment, antigen-binding site B is an antibody or fragment thereof or an α chain variable domain (v α ) and the β chain variable domain (v β ) or γ chain variable domain (v γ ) or δ chain variable domain (v δ ), preferably an α chain variable domain (v α ) and the β chain variable domain (v β ) or γ chain variable domain (v γ ) and the δ chain variable domain (v δ ), preferably v α and v β The compound comprises:

[0394] In a further related aspect of the above embodiment, i)v αis an amino acid sequence selected from the group consisting of "EDVEQSLFLSVREGDSVVINCTYTDSSSTYLYWYKQEPGKGLQLLTYIYSSQDSKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP" SEQ ID NO: 20, "EDVEQSLFLSVREGDSVVINCTYTDSSSTYLYWYKQEPGKGLQLLTYIYSSQDQKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP" SEQ ID NO: 21, and "EDVEQSLFLSVREGDSVVINCTYTESSSTYLYWYKQEPGKGLQLLTYIYSSQDQKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP" SEQ ID NO: 22, or an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, and 22. and wherein the amino acid sequence is at least 85% identical to the amino acid sequence of SEQ ID NO: 20, preferably comprising the amino acid sequence of CDRa1 of SEQ ID NO: 23, CDRa2 of SEQ ID NO: 24, and CDRa3 of SEQ ID NO: 25; and wherein the amino acid sequence is at least 85% identical to the amino acid sequence of SEQ ID NO: 21, preferably comprising the amino acid sequence of CDRa1 of SEQ ID NO: 23, CDRa2 of SEQ ID NO: 26, and CDRa3 of SEQ ID NO: 25; and wherein the amino acid sequence is at least 85% identical to the amino acid sequence of SEQ ID NO: 22, preferably comprising the amino acid sequence of CDRa1 of SEQ ID NO: 27, CDRa2 of SEQ ID NO: 26, and CDRa3 of SEQ ID NO: 25; and wherein the amino acid sequence of said first variable domain preferably comprises amino acids 19V and / or 48K; v βcomprises or consists of the amino acid sequence "DAGVIQSPRHEVTEMGQEVTLRCKPIPGHDYLFWYRQTMMRGLELLFYFCYGTPCDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRADTGELFFGEGSRLTVL" SEQ ID NO: 30, or an amino acid sequence which is at least 85% identical to the amino acid sequence consisting of SEQ ID NO: 30, wherein preferably the amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 30 preferably comprises the amino acid sequence of CDRb1 of SEQ ID NO: 31, CDRb2 of SEQ ID NO: 34, CDRb3 of SEQ ID NO: 35, respectively, and optionally comprising amino acids 54F and / or 66C; or (ii)v α or v γ comprises or consists of the amino acid sequence of SEQ ID NO: 48 or an amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 48, wherein preferably the amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 48 comprises the amino acid sequence of CDRa1 of SEQ ID NO: 49, CDRa2 of SEQ ID NO: 50, and CDRa3 of SEQ ID NO: 51; v β or v δ comprises or consists of the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence which is at least 85% identical to SEQ ID NO: 44, wherein preferably said amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 44 comprises the amino acid sequence of CDRb1 of SEQ ID NO: 45, CDRb2 of SEQ ID NO: 46, and CDRb3 of SEQ ID NO: 47.

[0395] In one item of the above embodiment, the antigen binding protein has the formula V3-L1-V4-L2-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 282 or 284. L -L5-F c1 [III] and a first polypeptide of the formula V5-L3-V6-L4-C, comprising or consisting of the amino acid sequence of SEQ ID NO: 283. H1 -L6-F c2[IV] The second polypeptide.

[0396] In a further related aspect of one of the above embodiments, the bispecific antigen-binding protein comprises: (i) Diagnostic agents; (ii) a therapeutic drug; or (iii) a pharmacokinetic (PK)-modifying moiety and further comprising one or more of:

[0397] In a further related item of one of the above embodiments, the isolated nucleic acid comprises a sequence encoding a bispecific antigen-binding protein as defined in the above embodiment and item, or a nucleic acid vector comprises said nucleic acid.

[0398] In a further related item of one of the above embodiments, a recombinant host cell comprises a bispecific antigen-binding protein as defined in the above embodiments and items, or a nucleic acid or vector as defined in the above items, wherein said host cell is preferably a) a stem cell, preferably a mesenchymal stem cell, or b) a cell for recombinant expression such as a Chinese hamster ovary (CHO) cell.

[0399] A further related item of one of the above embodiments refers to a pharmaceutical composition comprising a bispecific antigen-binding protein as defined in the above embodiments and items, a nucleic acid or vector as defined in the above embodiments and items, or a host cell as defined in the above embodiments and items, and a pharmaceutically acceptable carrier, diluent, stabilizer and / or excipient.

[0400] A further related aspect of the above embodiment is a. providing a suitable host cell; b. Providing a genetic construct comprising a coding sequence encoding a bispecific antigen-binding protein as defined in the above aspects and clauses; c. introducing said genetic construct into said suitable host cell; d. expressing said genetic construct by said suitable host cell;

[0023] Reference is made to a method for producing a bispecific antigen-binding protein as defined in the above aspects and clauses, comprising:

[0401] In one further related item, the method defined in the above embodiment and item further comprises the isolation and purification of the bispecific antigen-binding protein from suitable host cells.

[0402] Another related item of the above aspect relates to a bispecific antigen-binding protein as defined in the above aspects and items, a nucleic acid or vector as defined in the above aspects and items, a host cell as defined in the above aspects and items, or a pharmaceutical composition as defined in the above aspects and items, for use in medicine.

[0403] In a further related item, a bispecific antigen binding as defined in the above aspects and items, a nucleic acid or vector as defined in the above aspects and items, a host cell as defined in the above aspects and items, or a pharmaceutical composition as defined in the above aspects and items for use in the diagnosis, prevention, and / or treatment of a disease such as a viral or bacterial infection or a proliferative disease, preferably cancer, more preferably a TAA / MHC positive cancer.

[0404] The definitions and embodiments used in this patent application apply mutatis mutandis to the above aspects and sections of this specification.

[0405] Throughout this application, the term "and / or" is a grammatical conjunction that should be interpreted to encompass one or more of the circumstances to which it is connected. For example, the phrase "such native sequence proteins can be prepared using standard recombinant and / or synthetic methods" indicates that the native sequence proteins can be prepared using standard recombinant and synthetic methods, that the native sequence proteins can be prepared using standard recombinant methods, or that the native sequence proteins can be prepared using synthetic methods.

[0406] Furthermore, throughout this application, the term "comprising" should be interpreted to encompass all specifically mentioned features as well as optional, additional, unspecified features. As used herein, use of the term "comprising" also discloses embodiments in which no features are present other than those specifically mentioned (i.e., "consisting of").

[0407] Furthermore, the indefinite articles "a" or "an" do not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0408] The present invention will now be described in more detail with reference to the following figures and examples. All documents and patents cited herein are incorporated herein by reference in their entirety. While the invention has been illustrated and described in detail in the foregoing description, the examples should be considered as illustrative or exemplary and not restrictive. [Brief explanation of the drawings]

[0409] [Figure 1]

[0023] Figure 1 shows the alignment of the UCHT1 VL domain with the identified human acceptor frameworks. The CDRs identified in the figure are identified according to the Cothia definition. [Figure 2]

[0023] Figure 1 shows the alignment of the UCHT1 VH domain with the identified human acceptor frameworks. The CDRs identified in the figure are identified according to the Cothia definition. [Figure 3] 1 shows the concentration-dependent binding of PRAME-004-specific TCER® molecules to Jurkat cells as measured by flow cytometry. [Figure 4]Representative LDH release assay results are shown using PBMCs from a healthy HLA-A*02-positive donor. MAG-003-specific TCER® molecules using UCHT1 (V17) or BMA031 (V36) were tested in MAG-003-positive and MAG-003-negative, as well as off-target-positive tumor cell lines. Tested cell lines (from left to right): H695T, A375, T98G, BV173. Error bars indicate standard deviation within triplicates. [Figure 5] Figure 1 shows the results of an LDH release assay on healthy cells incubated with the MAG-003-specific TCER® based on UCHT1 (V17). Each cytotoxicity plot shows the LDH release of a primary healthy cell type (open circles) compared to the control tumor cell line Hs695T (filled circles) in the same media combination after co-incubation with PBMCs and increasing concentrations of the TCER molecule. The calculated safety margin based on the EC50 value is also shown. [Figure 6] Figure 1 shows the results of an LDH release assay on healthy cells incubated with MAG-003-specific TCER® based on BMA031 (V36). Each cytotoxicity plot shows the LDH release of a primary healthy cell type (open circles) compared to the control tumor cell line Hs695T (filled circles) in the same media combination after co-incubation with PBMCs and increasing concentrations of TCER molecules. A calculated safety margin based on either the EC50 value or the LOEL is also shown. [Figure 7]Visualization of the introduced point mutations is shown. Panel A shows the newly introduced Asp side chain at position 31 of the UCHT1 heavy chain and the negatively charged side chain of CD3ε located in close spatial proximity. Panel B shows both the wild-type Tyr and the substituted Gln at position 54 of the UCHT1 heavy chain. Replacing the aromatic side chain with a smaller, polar amino acid eliminates the hydrophobic interaction with the apolar stem of Asp at position 48 of CD3ε. Panel C shows that replacing the same Tyr at position 54 of the UCHT1 heavy chain with the negatively charged side chain of CD3ε (48D, 49E, 50D, and 51D) in close spatial proximity to Glu likely causes electrostatic repulsion of the newly introduced Glu in UCHT1. Panel D shows that the wild-type Lys at position 55 of the UCHT1 heavy chain forms a hydrogen bond (dashed line) with the main chain of Ser at position 56 (left) of CD3ε. Substitution of this Lys with Arg removes polar interactions, introduces additional bulk, and distorts the Arg side chain into a rotamer (right). Panel E shows that the same Lys at position 55 of the UCHT1 heavy chain is replaced with Glu; this mutation also eliminates the hydrogen bond formed between Lys and Ser at position 56 of CD3ε. Furthermore, it introduces a negatively charged side chain in spatial proximity to the negatively charged patch on the surface of CD3ε formed by Asp48, Glu49, Asp50, and Asp51. [Figure 8] Figure 1 shows an overview of the production yield and stability properties of TCER® molecules based on humanized UCHT1 variants. (na) Not applicable, (nd) Not performed. [Figure 9] Figure 9A: Binding curves of TCER® antigen binding proteins comprising different UCHT1 variants to MAG-003 in complex with HLA-A*02, as measured by biolayer interferometry. Increasing concentrations of TCER® molecules in solution were applied and are shown in nM. Figure 9B: Binding curves of TCER® antigen binding proteins comprising different UCHT1 variants of CD3δε-Fc, as measured by biolayer interferometry. Increasing concentrations of TCER® molecules in solution were applied and are shown in nM. [Figure 10]The results of two independent LDH release assays using PBMCs from two healthy HLA-A*02-positive donors (HBC-982 and HBC-720) are shown. MAG-003-specific TCER® molecules were tested on MAG-003-positive tumor cell lines using UCHT1-mutants with various affinities. Error bars indicate standard deviation within triplicates. [Example]

[0410] Example 1: Humanization of mouse monoclonal Ab UCHT1 Humanization of the murine monoclonal antibody UCHT1 was performed by CDR-grafting according to published methods. Accordingly, the CDRs of the VH and VL were identified according to the Cothia definition. Sequence alignments were generated comparing the UCHT1 variable domain with the human germline. Based on overall sequence identity, matching interface positions, and similarly classified CDR canonical positions, the germline was identified as the most likely acceptor framework for each of the light and heavy chains: VK1-O18 for the light chain and VH-1-46 for the heavy chain. The J-segment genes were compared with the parental sequences on FR4, and J-segments JK1 and JH4 were selected for the light and heavy chains, respectively.

[0411] A list of all positions where residues differed between the parent and acceptor frameworks was generated. All positions were analyzed and considered both alone and in the context of other potential substitutions. Each position was ranked as neutral, critical, or contributing, and suggestions were made for which residues to replace and evaluate in the humanized variants. Potential humanized variant sequences were screened using Epibase™ (Lonza). Each epitope or cluster of epitopes was analyzed for substitutions that would either eliminate the epitope or further reduce predicted immunogenicity. In addition, potential sites of post-translational modification within the CDRs were identified, and suggestions for improvement were made for each. In total, this resulted in the generation of four distinct VH and five distinct VL domains. Thus, 17 humanized variants of UCHT1 were generated. All variants were expressed as Fab molecules in CHO cells. The expressed proteins were purified and analyzed for expression titers, aggregate levels, and EC2 values ​​for binding to Jurkat cells. 50 Based on these results, humanized UCHT1 (V17) as defined by SEQ ID NO: 137 and SEQ ID NO: 145 was selected for the establishment of our TCER® molecule.

[0412] The recruitment domain of humanized UCHT1 (V17) was utilized to construct PRAME-004 (SEQ ID NO: 9), resulting in molecules containing SEQ ID NO: 171 and SEQ ID NO: 170, or humanized BMA031 (V10), resulting in SEQ ID NO: 168 and SEQ ID NO: 169, respectively. The vector for recombinant protein expression was designed as a monocistronic vector controlled by a pUC19 derivative, an HCMV-derived promoter element. Plasmid DNA was amplified in Escherichia coli (E. coli) according to standard culture methods and subsequently purified using a commercially available kit (Macherey & Nagel). Purified plasmid DNA was used for transient transfection of CHO-S cells according to the manufacturer's instructions (ExpiCHO™ system; Thermo Fisher Scientific). Transfected CHO cells were cultured at 32°C to 37°C for 6 to 14 days and fed once or twice with ExpiCHO™ feed solution.

[0413] The adjusted cell supernatant was collected by centrifugation (4000 x g; 30 min) and clarified by filtration (0.22 μm). The bispecific antigen-binding protein was purified using an Äkta Pure 25 L FPLC system (GE Lifesciences) equipped to perform affinity and size-exclusion chromatography inline. Affinity chromatography was performed on a Protein A column (GE Lifesciences) according to standard affinity chromatography protocols. Size-exclusion chromatography was performed immediately after elution from the affinity column (pH 2.8) using a Superdex 200 pg 16 / 600 column (GE Lifesciences), yielding highly pure monomeric protein according to standard protocols. Protein concentration was determined using the extinction coefficient calculated according to the predicted protein sequence on a NanoDrop system (Thermo Scientific). Concentration, if necessary, was performed, and buffer exchange was performed using a Vivaspin device (Sartorius). Finally, the purified molecule was stored in phosphate-buffered saline at a concentration of approximately 1 mg / mL at a temperature of 2–8 °C.

[0414] The binding affinity of these TCER® molecules to effector cells was assessed by flow cytometry. Therefore, Jurkat cells (CD3+ and TCRab+) were incubated with increasing concentrations of TCER®. After washing, cells bound to the TCER® molecules were stained using a PE-labeled secondary reagent (#709-116-098, Jackson Immuno Research). The cells were finally analyzed using the Intellicyt® iQue Cell Screener. The results of one of four independent experiments are shown in Figure 3, demonstrating concentration-dependent binding of PRAME-004-specific TCER® molecules to Jurkat cells. UCHT1-based TCER® molecules exhibited an EC of approximately 2–3 nM. 50It is clear that while BMA031-based TCER® molecules exhibit at least 50-100 times weaker binding to Jurkat cells.

[0415] Example 2: Proof of principle cytotoxicity using recruiters with different affinities Antigen binding proteins targeting peptide MAG-003 were generated by combining engineered variable domains of the T cell receptor (SEQ ID NO:20 and SEQ ID NO:30) with the variable domains of either UCHT1 (V17) (SEQ ID NO:137 and SEQ ID NO:145) or BMA031 (V36) (SEQ ID NO:42 and SEQ ID NO:43), respectively, in a TCER® construct.

[0416] The vectors for expression of each TCER® molecule were designed as monocistronic vectors controlled by a pUC19 derivative, an 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). Purified plasmid DNA was used for transient transfection of CHO-S cells using an electroporation system (MaxCyte STX). Transfected CHO cells were cultured at 32°C to 37°C for 10 to 12 days and fed with Cellboost 7a and 7b (GE Healthcare™) solutions one to three times.

[0417] The conditioned cell supernatant was clarified by filtration (0.22 μm) using a Sartoclear Dynamics® Lab Filter Aid (Sartorius). The bispecific antigen-binding protein was purified using an Äkta Pure 25 L FPLC system (GE Lifesciences) equipped to perform affinity and size-exclusion chromatography inline. Affinity chromatography was performed on a MAbSelect SuRE or Protein L column (GE Lifesciences) according to standard affinity chromatography protocols. Size-exclusion chromatography was performed immediately after elution from the affinity column (pH 2.8) using a Superdex 200 pg 26 / 600 column (GE Lifesciences), yielding highly pure monomeric protein according to standard protocols. Protein concentration was determined using the extinction coefficient calculated according to the predicted protein sequence on a NanoDrop system (Thermo Scientific). If necessary, the concentration was adjusted using a Vivaspin device (Sartorius). Finally, the purified molecule was stored in phosphate-buffered saline at a concentration of approximately 1 mg / mL at a temperature of 2–8 °C.

[0418] The cytotoxic activity of the bispecific molecules against MAG-positive and MAG-negative tumor cell lines was analyzed by LDH release assay. Thus, increasing concentrations of TCER® molecules were used to induce tumor cell proliferation with varying amounts of HLA-A on the cell surface. * Tumor cell lines expressing 02 / MAG-003 were cultured in PBMCs (HLA-A) isolated from healthy donors. * After 48 hours, lysis of the target cell lines was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (PROMEGA).

[0419] Exemplary results of such an assay are shown in the figure (Example 2). The resulting EC 50 The values ​​are summarized in Table 5.

[0420] Table 5: EC of killing assay comparing different mobilized antibodies 50 Value Summary [Table 5]

[0421] These results demonstrate reduced potency of the BMA031 (V36)-based TCER® molecules compared to the UCHT1 (V17)-based molecules (23-fold and 96-fold on target high and low expressing cell lines, respectively). 50 Based on this value, the safety margin can be calculated as described in the definition section above. Briefly, the safety margin is the EC 50 This means that UCHT1 (V17)-based TCER® exhibits an approximately 49-fold safety margin, while BMA031 (V36)-based TCER® exhibits an approximately 312-fold increase in safety window (comparing off-target expressing tumor cell lines to target (TAA)-high expressing tumor cell lines).

[0422] These findings generally suggested that the use of low-affinity recruitment domains may improve discrimination between target and off-target targets, thereby increasing the safety margin. To further test this hypothesis, primary healthy tissue cells (HLA-A) were cultured. * The cytotoxic activity of the MAG-003-specific TCER® molecule against 11 different primary healthy tissue cells (HLA-A 02+) was evaluated. To this end, we used 11 different primary healthy tissue cells (HLA-A 02+) at an E:T ratio of 10:1. * 02+) and healthy HLA-A *LDH was measured at increasing TCER® concentrations during coculture with PBMC effector cells from a 02+ donor. Cells were co-incubated in a 50% mixture of primary tissue cell-specific medium and optimal T cell medium. To determine the safety margin, TCER® molecules were co-incubated with the MAG-003-positive tumor cell line Hs695T in the same setup, using the respective medium combinations of primary cells and 100% optimal T cell medium to eliminate bias caused by different media. After 48 hours of co-culture, supernatants were collected and cell lysis was analyzed by measuring LDH release using the LDH-Glo™ kit (Promega).

[0423] In Figures 5 and 6, each cytotoxicity plot shows LDH release of a primary healthy cell type (open circles) compared to the control tumor cell line Hs5T (closed circles) in the same media combination after co-incubation of PBMCs with increasing concentrations of TCER molecules. Figure 5 summarizes the results for the UCHT1 (V17)-based MAG-003-specific TCER® molecule. With the exception of nasal epithelial cells and PBMCs, strong reactivity was detectable in all cell types tested, with the respective EC 50 The value could be determined. 50 Based on the values, the safety margin was calculated as described above in the definitions section. The x-fold safety margin is shown in the figure. For UCHT1 (V17)-based TCER®, the most significant safety margins were determined for astrocytes (48-fold), dermal microvascular endothelial cells (94-fold), and mesenchymal stem cells (170-fold).

[0424] In the case of BMA031 (V36)-based TCER® molecules, all responses to healthy primary cells were too low and EC 50 Instead, we defined a margin of safety based on the lowest observed effect level (LOEL), determined as the first TCER® concentration with a response above the cutoff value.

[0425] ([standard deviation from all triplicates x 3] + [no TCER control]) The cutoff, defined as the no TCER control (no TCER control is shown as a dotted line in each cytotoxicity plot), was used as a threshold to determine the LOEL and safety margin between healthy tissue cells and tumor control cell lines. All determined safety margins for BMA031 (V36)-based TCER® were greater than 1000-fold.

[0426] From a comparison of Figures 5 and 6, it is clear that the safety margin is significantly expanded when using the low affinity recruiter BMA03(V36) in the context of the MAG-003-specific TCER1®(Reg) molecule.

[0427] Example 3: Generation of humanized UCHT1 mutants with reduced affinity To obtain a low-affinity variant of the CD3-specific humanized antibody UCHT1 (V17), we performed structure-guided design of mutants. Based on the solved structure of UCHT1 in complex with its target CD3δ / ε (PDB ID: 1xiw), we introduced point mutations into the antibody that are expected to reduce affinity without destabilizing the protein itself.

[0428] To achieve this goal, positions were selected primarily within the CDRs; as can be inferred from the solved structure, the interface between the two proteins is primarily formed between CD3ε and the antibody heavy chain, and therefore only positions within the heavy chain were considered for mutation.

[0429] For clarity, the CD3ε positions are numbered sequentially as in the PDB entry with ID 1xiw, chain ID:A.

[0430] G31E introduces a negative charge on the surface of the antibody facing the negatively charged surface patch formed by CD3ε48D, CD3ε49E, CD3ε50D, and CD3ε51D, likely causing electrostatic repulsion and reducing affinity.

[0431] Y54Q alters the shape complementarity of the binding surface, eliminating the hydrophobic interaction between the aromatic ring of Y54 and the apolar stem of CD3ε48D.

[0432] Y54E alters the shape complementarity of the binding surface, eliminating the hydrophobic interaction between the aromatic ring of Y54 and the apolar stem of CD3ε48D, and further introducing a negative charge facing the negatively charged patch formed by CD3ε48D, CD3ε49E, CD3ε50D, and CD3ε51D.

[0433] K55R introduces a bulkier side chain with similar physicochemical properties and eliminates the H-bond formed between Nζ of 55K and the backbone of CD3ε56S. The increase in side chain size may also induce a slight change in the binding geometry.

[0434] K55E replaces the positive charge with a negative one, eliminating the hydrogen bond formed between Nζ of 55K and the backbone of CD3ε36S. Furthermore, the introduction of the negative charge causes electrostatic repulsion from the negatively charged patches formed by CD3ε57D, CD3ε58E, and CD3ε59D.

[0435] Based on these findings, sequences encoding UCHT1(V20) to UCHT1(V27) were generated as summarized in Table 6.

[0436] In a further attempt to optimize the humanized UCHT1 sequence, a potential post-translational modification site (Asp-isomerization, 106D107S) within CDR-H3 was removed by introducing 106E. This modification was introduced into UCHT1(V17), UCHT1(20), UCHT1(V21), and UCHT1(V23), resulting in mutant UCHT1(V17opt), UCHT1(V20opt), UCHT1(V21opt), and UCHT1(V23opt), respectively.

[0437] Table 6: Sequence combinations resulting in humanized UCHT1 variants [Table 6]

[0438] TCER® molecules were generated, purified and purified as described above using the humanized UCHT1 variants listed in Table 6, PRAME-004 specific (Vα: SEQ ID NO: 48, Vβ: SEQ ID NO: 44) and MAG-003 specific (Vα: SEQ ID NO: 21, Vβ: SEQ ID NO: 30), respectively.

[0439] Example 4: Determining the affinity of designed UCHT1 variants For affinity determination using biolayer interferometry, the molecules CD3δε-Fc were generated by fusion to the N-terminus of the Fc domain utilized in the TCER® construct (containing knob-into-hole mutations and an additional C-terminal His-tag), resulting in SEQ ID NO: 161 and SEQ ID NO: 162, respectively.

[0440] CD3δε-Fc molecules were expressed in ExpiCHO cells and purified using protein A affinity chromatography followed by size exclusion chromatography as described above.

[0441] HLA-A using biolayer interferometry * Bispecific TCER® antigen-binding proteins (as shown in Table 6) comprising different UCHT1 variants were characterized for their binding affinity to the MAGE-A antigenic peptide (SEQ ID NO: 10) in complex with UCHT1 variants 02 (Figure 9A, Table 7) and CD3δε-Fc (Figure 9B, Table 7). Measurements were performed on an Octet RED384 system using the settings recommended by the manufacturer. Briefly, binding kinetics were measured at 30°C and a shaking speed of 1000 rpm using PBS, 0.05% Tween-20, 0.1% BSA as buffer. Peptide-HLA-A * The O2 complex or CD3δε-Fc was loaded onto a biosensor (HIS1K) before analyzing serial dilutions of the bispecific TCER® molecules. All TCER® molecules have a K of approximately 2 nM. D Values, HLA-A * CD3δε affinities ranged from 3 to 750 nM, with KD The values ​​covered a range of more than 200 times.

[0442] Table 7: Affinity analysis of TCER® molecules comprising different UCHT1 variants listed in Table 6. D Values ​​were measured by biolayer interferometry [Table 7]

[0443] Example 5: Reduced potency of low affinity humanized UCHT1 variants The cytotoxic potency of MAG-003-specific TCER® molecules was assessed in an LDH release assay as described above in Example 2. The results of a representative assay are shown in Figure 10. As expected, the newly designed variants (UCHT1(V17opt), UCHT1(V20), UCHT1(V21), UCHT1(V23)) showed reduced potency compared to TCER® containing the high-affinity recruitment domain of UCHT1(V17). Ranking the TCER® molecules according to their potency also closely reflects the affinity of each recruiter variant. (Highest potency: UCHT1(V17) <UCHT1(V21)<UCHT1(V20)<UCHT1(V17opt)<UCHT1V23)。HLA-A * These effects on potency can be attributed solely to the recruitment domain, as the affinity of the TCR domains for MAG-003 in complex with 02 is comparable (Figure 9).

Claims

1. A bispecific antigen-binding protein comprising at least two antigen-binding sites (A and B), wherein the antigen-binding site A binds to CD3 and the antigen-binding site B binds to a target antigenic (TA) peptide / MHC complex, and the antigen-binding site A binds to a heavy chain variable domain (V Ｈ ) and a light chain variable domain (V Ｌ ) and a) the V Ｌ comprises three complementarity determining regions (CDRs): CDRL1, CDRL2, and CDRL3, wherein: CDRL1 comprises or consists of the amino acid sequence "RASQDIRNYLN" of SEQ ID NO: 1, CDRL2 comprises or consists of the amino acid sequence "YTSRLHS" of SEQ ID NO: 2, CDRL3 comprises or consists of the amino acid sequence "QQGQTLPWT" of SEQ ID NO: 3, b) the V Ｈ comprises three complementarity determining regions (CDRs): CDRH1, CDRH2, and CDRH3, wherein: - CDRH1 has the amino acid sequence "X" of SEQ ID NO: 4 １ YTMN, wherein X １ is G or E, preferably G; CDRH2 has the amino acid sequence LINPX of SEQ ID NO: 5 ２ X ３ GVX ４ TYAQKX ５ QX ６ wherein X ２ is Q, Y or E, and X ３ is R, K or E, and X ４ is S or T, and X ５ is F or V, and X ６ is G or D, - CDRH3 has the amino acid sequence "SGYYGX" of SEQ ID NO: 6 ７ SWYFD" in which X ７ is E or D.

2. 2. The bispecific antigen-binding protein of claim 1, wherein the antigen-binding site A is Ｄ (A) binds to CD3, and the antigen-binding site B is K Ｄ (C) binds to the target antigenic peptide C (TA-C) / MHC complex, and the K Ｄ (A) / K Ｄ (C) Bispecific antigen-binding proteins with a ratio of greater than 1, greater than 4, greater than 6, greater than 8, greater than 10, greater than 15, greater than 20, greater than 25, greater than 30, greater than 40, greater than 50, between 1 and 150, between 4 and 140, between 6 and 100, between 8 and 100, between 10 and 100, etc., preferably between 10 and 100.

3. 3. A bispecific antigen-binding protein according to claim 1 or 2, wherein the antigen-binding site A has a denaturing activity of 3 nM or more, 5 nM or more, 8 nM or more, 10 nM or more, 12 nM or more, 14 nM or more, 16 nM or more, 18 nM or more, 20 nM or more, 25 nM or more, 30 nM or more, 3 nM or more, 4 nM or more, 5 nM or more, 6 nM or more, 7 nM or more, 8 nM or more, 9 ...10 nM or more, 12 nM or more, 14 nM or more, 16 nM or more, 18 nM or more, 20 nM or more, 25 nM K of 5 nM or more, 40 nM or more, 45 nM or more, 1000 nM or less, 800 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 3 nM to 1000 nM, 3 nM to 600 nM, 5 nM to 600 nM, 10 nM to 600 nM, 12 nM to 600 nM, 14 nM to 600 nM, 16 nM to 600 nM, 18 nM to 600 nM, 20 nM to 600 nM, etc., preferably 5 nM to 100 nM. Ｄ (A) A bispecific antigen-binding protein that binds to CD3.

4. 4. A bispecific antigen-binding protein according to any one of claims 1 to 3, wherein antigen-binding site B has a K of 100 μM or less, 1 μM or less, 100 nM or less, 50 nM or less, 10 nM or less, for example 0.01 nM to 150 nM, 0.05 nM to 150 nM, 0.1 nM to 150 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.5 nM to 10 nM, 0.5 nM to 5 nM, preferably 0.5 nM to 5 nM, as measured by surface plasmon resonance (SPR) or biolayer interferometry (BLI), preferably biolayer interferometry (BLI). Ｄ (C) A bispecific antigen-binding protein that binds to the target antigenic peptide C (TA-C) / MHC complex.

5. The antigen-binding protein binds to ECs against normal tissue cells. ５０ EC values ​​for TA-C / MHC-presenting cells that are 100 or more, 500 or more, or 1000 or more lower than the values ５０ 5. The bispecific antigen-binding protein of claim 1, wherein

6. 7. The bispecific antigen-binding protein of any one of claims 1 to 6, wherein said TA antigenic peptide C is a viral peptide, a bacterial peptide or a tumor-associated antigen (TAA) peptide, preferably a tumor-associated antigen (TAA) peptide.

7. The antigen-binding protein binds to ECs against normal tissue cells. ５０ EC for TA-C / MHC complex-presenting cells that is 5 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, 500 times or more, or 1000 times or more lower than the value ５０ 7. The bispecific antigen-binding protein of any one of claims 1 to 6, comprising:

8. 8. The bispecific antigen-binding protein according to any one of claims 1 to 7, wherein said TA antigenic peptide C is a tumor-associated antigen (TAA) peptide C, said TAA-C being selected from the group of TAA antigenic peptides comprising or consisting of the amino acid sequence of SEQ ID NOs: 52-65, 67-96, 98, SEQ ID NOs: 172-182, 184-268, SEQ ID NOs: 9 and 10, such as a PRAME antigenic peptide comprising or consisting of the amino acid sequence "SLLQHLIGL" of SEQ ID NO: 9, or a MAGE-A antigenic peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 10, and said MHC is preferably HLA-A. ＊ The number is 02.

9. 9. The bispecific antigen-binding protein of claim 8, wherein the TA antigenic peptide C is a MAGE-A antigenic peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 10, and the similar peptide is selected from the list consisting of RABGAP1L-001, AXIN1-001, ANO5-001, TPX2-001, SYNE3-001, MIA3-001, HERC4-001, PSME2-001, HEATR5A-001, CNOT1-003, TEP1-003, PITPNM3-001, ZFC-001, preferably HEATR5A-001, HERC4-001, and CNOT1-003.

10. 10. The bispecific antigen-binding protein of any one of claims 1 to 9, wherein the bispecific antigen-binding protein is a bispecific antibody or fragment thereof, a bispecific T-cell receptor (TCR) or fragment thereof, or a bispecific single chain TCR (scTCR) or a bispecific single chain antibody.

11. 11. The bispecific antigen-binding protein according to any one of claims 1 to 10, wherein the VL domain further comprises one or more framework regions selected from the group consisting of FR1-L, FR2-L, FR3-L, and FR4-L, wherein: - FR1-L comprises or consists of the amino acid sequence "DIQMTQSPSSLSASVGDRVTITC" of SEQ ID NO: 11 or an amino acid sequence which is at least 85% identical to SEQ ID NO: 11; - FR2-L comprises or consists of "WYQQKPGKAPKLLIY" of SEQ ID NO: 12 or "WYQQKPGKAVKLLI" of SEQ ID NO: 13, preferably the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence which is at least 85% identical to SEQ ID NO: 12 or 13, - FR3-L comprises or consists of the amino acid sequence "GVPSRFSGSGSGTDYTLTISSLQPEDIATYFC" of SEQ ID NO: 14, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 14; FR4-L comprises or consists of the amino acid sequence "FGQGTKVEIKR" of SEQ ID NO: 15 or an amino acid sequence which is at least 85% identical to SEQ ID NO: 15, wherein VH further comprises one or more framework regions selected from the group consisting of FR1-H, FR2-H, FR3-H, and FR4-H, wherein: - FR1-H comprises or consists of the amino acid sequence "EVQLVQSGAEVKKPGASVKVSCKASGYSFT" of SEQ ID NO: 16, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 16; - FR2-H comprises or consists of the amino acid sequence "WVRQAPGQGLEWMG" of SEQ ID NO: 17 or an amino acid sequence which is at least 85% identical to SEQ ID NO: 17; - FR3-H comprises or consists of the amino acid sequence "RVTLTVDKSTSTAYMELSSLRSEDTAVYYCAR" of SEQ ID NO: 18, or an amino acid sequence which is at least 85% identical to SEQ ID NO: 18, or - FR4-H comprises or consists of the amino acid sequence of "WGQGTLVTVSS" of SEQ ID NO: 19, or an amino acid sequence which is at least 85% identical to SEQ ID NO:

19.

12. The antigen-binding site B is an antibody or a fragment thereof or an α-chain variable domain (v α ) and a β chain variable domain (v β ) or a gamma chain variable domain (v γ ) or δ chain variable domain (v δ ), preferably an α chain variable domain (v α ) and a β chain variable domain (v β ) or a gamma chain variable domain (v γ ) and the δ chain variable domain (v δ ), preferably v α and v β 13. The bispecific antigen-binding protein of any one of claims 1 to 12, comprising:

13. 13. A bispecific antigen-binding protein according to any one of claims 1 to 12, comprising: i) v α "EDVEQSLFLSVREGDSVVINCTYTDSSSTYLYWYKQEPGKGLQLLTYIYSSQDSKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP" SEQ ID NO: 20, "EDVEQSLFLSVREGDSVVINCTYTDSSSTYLYWYKQEPGKGLQLLTYIYSSQDQKQDQRLTVLLNKKDKHLSLRIADTQT an amino acid sequence selected from the group consisting of "GDSAIYFCAEMTSESKIIFGSGTRLSIRP" SEQ ID NO: 21, "EDVEQSLFLSVREGDSVVINCTYTESSSTYLYWYKQEPGKGLQLLTYIYSSQDQKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP" SEQ ID NO: 22, or an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, and 22. Preferably, said amino acid sequence is at least 85% identical to the amino acid sequence of SEQ ID NO: 20, preferably comprising the amino acid sequences of CDRa1 of SEQ ID NO: 23, CDRa2 of SEQ ID NO: 24, and CDRa3 of SEQ ID NO: 25; preferably, said amino acid sequence is at least 85% identical to the amino acid sequence of SEQ ID NO: 21, preferably comprising the amino acid sequences of CDRa1 of SEQ ID NO: 23, CDRa2 of SEQ ID NO: 26, and CDRa3 of SEQ ID NO: 25; preferably, said amino acid sequence is at least 85% identical to the amino acid sequence of SEQ ID NO: 22, preferably comprising the amino acid sequences of CDRa1 of SEQ ID NO: 27, CDRa2 of SEQ ID NO: 26, and CDRa3 of SEQ ID NO: 25; and ... The above v β comprises or consists of the amino acid sequence "DAGVIQSPRHEVTEMGQEVTLRCKPIPGHDYLFWYRQTMMRGLELLFYFCYGTPCDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRADTGELFFGEGSRLTVL" SEQ ID NO: 30, or an amino acid sequence which is at least 85% identical to an amino acid sequence consisting of SEQ ID NO: 30, preferably said amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 30 preferably comprises the amino acid sequence of CDRb1 of SEQ ID NO: 31, CDRb2 of SEQ ID NO: 34, CDRb3 of SEQ ID NO: 35, respectively, and optionally comprising amino acids 54F and / or 66C; or (ii) the v α or v γ comprises or consists of the amino acid sequence of SEQ ID NO: 48 or an amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 48, preferably the amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 48 comprises the amino acid sequences of CDRa1 of SEQ ID NO: 49, CDRa2 of SEQ ID NO: 50, and CDRa3 of SEQ ID NO: 51; The above v β or v δ comprises or consists of the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence which is at least 85% identical to SEQ ID NO: 44, preferably wherein said amino acid sequence which is at least 85% identical to the amino acid sequence of SEQ ID NO: 44 comprises the amino acid sequences of CDRb1 of SEQ ID NO: 45, CDRb2 of SEQ ID NO: 46, and CDRb3 of SEQ ID NO:

47. Bispecific antigen-binding proteins.

14. (i) diagnostic agents; (ii) a therapeutic agent; or (iii) a pharmacokinetic (PK)-modifying moiety 14. The bispecific antigen-binding protein of any one of claims 1 to 13, further comprising one or more of:

15. 15. An isolated nucleic acid comprising a sequence encoding the bispecific antigen-binding protein of any one of claims 1 to 14, or a nucleic acid vector comprising said nucleic acid.

16. 16. A recombinant host cell comprising the bispecific antigen-binding protein of any one of claims 1 to 14, or the nucleic acid or vector of claim 15, which is preferably a) a stem cell, preferably a mesenchymal stem cell, or b) a cell for recombinant expression such as a Chinese Hamster Ovary (CHO) cell.

17. 17. A pharmaceutical composition comprising a bispecific antigen-binding protein according to any one of claims 1 to 14, or a nucleic acid or vector according to claim 15, or a host cell according to claim 16, and a pharmaceutically acceptable carrier, diluent, stabilizer and / or excipient.

18. a) providing a suitable host cell; b) providing a genetic construct comprising a coding sequence encoding a bispecific antigen-binding protein according to any one of claims 1 to 14; c) introducing said genetic construct into said suitable host cell; d) expressing said genetic construct by said suitable host cell; 15. A method for producing a bispecific antigen-binding protein according to any one of claims 1 to 14, comprising:

19. 20. The method of claim 18, further comprising the step of isolating and purifying said bispecific antigen-binding protein from said suitable host cell.

20. 18. A bispecific antigen-binding protein according to any one of claims 1 to 14, a nucleic acid or a vector according to claim 15, a host cell according to claim 16, or a pharmaceutical composition according to claim 17 for use in medicine.

21. 18. The bispecific antigen-binding protein of any one of claims 1 to 14, the nucleic acid or vector of claim 15, the host cell of claim 16 or the pharmaceutical composition of claim 17 for use in the diagnosis, prevention and / or treatment of a disease such as a viral or bacterial infection or a proliferative disease, preferably cancer, more preferably a TAA / MHC positive cancer.

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