Modified bispecific anti-CD3 antibodies

Abstract

The present invention relates to bispecific antigen-binding proteins for MHC-presented target antigens (TA). The present invention particularly provides bispecific antigen-binding proteins comprising at least two antigen-binding sites (A and B), wherein antigen-binding site A binds to CD3 and antigen-binding site B binds to a target antigen (TA) peptide / MHC complex. In particular, the bispecific antigen-binding proteins of the present invention comprise 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 can be used to diagnose, treat, and prevent TA-related diseases, such as cancerous diseases expressing tumor-associated antigens (TAAs). Also provided are 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.

Description

[0001] The present invention relates to bispecific antigen-binding proteins that present a target antigen (TA) against major histocompatibility (MHC). The present invention particularly provides bispecific antigen-binding proteins comprising at least two antigen-binding sites (A and B), wherein antigen-binding site A binds to CD3 and antigen-binding site B binds to a target antigen (TA) peptide / MHC complex. In particular, the bispecific antigen-binding proteins of the present invention 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 can be used to diagnose, treat, and prevent TA-related diseases, such as cancerous diseases expressing tumor-associated antigens (TAAs). Also provided are 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. Background of the Invention

[0003] Specific peptides are presented on the surface of these cells by the major histocompatibility (MHC) complex. Therefore, TCR-based molecules have attracted great attention for the development of disease or tumor-specific immunotherapies. Although progress has been made in the development of molecular targeted drugs for cancer treatment, there is still a need in the art to develop new anti-cancer drugs that specifically target molecules that are highly specific for cancer cells but not for normal cells. Similarly, targeting molecules that are highly specific for diseased cells but not for normal cells are also very important for the development of drugs targeting infectious diseases (e.g., HIV).

[0004] In the context of the present invention, the source protein of the target antigen (TA) peptide 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 in the form of a peptide-MHC (pMHC) complex (TA peptide / MHC). The TA-induced recognition pattern allows the immune system to distinguish diseased cells (e.g., transformed tumor cells in the case of TAA antigen peptides) from surrounding normal tissue cells and trigger an immune cascade response against these cells.

[0005] To develop such TA-targeted drugs, TCRs that specifically target TAs have been identified, and the Vα and Vβ domains have been used to engineer novel TA-targeted molecules, particularly TAA-targeted molecules.

[0006] Regarding molecules targeting TAAs, it is important to note that natural T cell receptors (TCRs) that specifically bind to MHC-presented cancer antigens typically have lower affinity (KD = 1-300 μM) than T cell receptors (TCRs) that specifically bind to MHC-presented viral antigens. This phenomenon appears to be partially explained by the negative selection (tolerance induction) of T cells developing in the thymus on self-peptide MHC ligands, thereby eliminating T cells with excessively high affinity for these self-peptide MHCs. This low affinity is a possible explanation for tumor immune escape (Aleksic et al. 2012, Eur J Immunol. 2012 Dec; 42(12): 3174-9). Therefore, there seems to be a need to design TCR variants with higher binding affinity to cancer antigens for use as antigen recognition constructs in adoptive cell therapy (ACT) or as recognition modules for soluble approaches, i.e., using bispecific molecules (Hickman et al. 2016, J Biomol Screen. 2016 Sep; 21(8): 769-85).

[0007] However, simply increasing TCR affinity may also increase the risk of side effects. As mentioned above, naturally, high-affinity TCRs against tumor-associated antigens (self-proteins) are eliminated through thymic selection, thereby preventing cross-reactivity with self-peptides present on normal tissues. Therefore, simply increasing the affinity of a TCR for a target sequence may also increase affinity for similar non-cancer-specific peptides, thereby increasing the risk of cross-reactivity and adverse cytotoxic effects on normal tissues. This is not merely a theoretical risk, as it has been painfully discovered with engineered TCRs targeting melanoma-associated antigen A3 (MAGE-A3). In particular, previously published results showed that two patients developed fatal toxicities after receiving T cells engineered to express a TCR targeting MAGE-A3 that cross-reacted with a peptide from the muscle protein titin, a cross-reactivity not predicted in preclinical studies (Linette GP et al. Blood 2013; 122: 863–71, Cameron BJ, et al. Sci. Transl. Med. 2013; 5: 197–103). These patients suggest that TCR-engineered T cells can have severe and unpredictable off-target and organ-specific toxicities.

[0008] Therefore, despite advances in TCR technology, there remains a need for additional therapeutics, particularly cancer therapeutics, particularly therapeutics that can effectively target and kill diseased cells (particularly cancer cells) while maintaining a high safety profile.

[0009] As mentioned above, natural TCRs typically have very low affinity for their target TAA / MHC complexes, and low affinity can prevent cross-reactivity and recognition of self-peptides present on normal tissues. However, increasing the affinity for their target TAA / MHC complexes is desirable for the development of effective anticancer drugs.

[0010] To address this issue, the inventors of the present invention combined affinity-matured TCR variable domains that bind to the relevant TA / MHC with variable light and heavy chain domains that target CD3 (with lower affinity than existing anti-CD3 heavy and light chain variable domains) in a single molecule. The resulting molecules have the advantage of being able to recognize diseased cells (e.g., cancer cells) even when TA (e.g., TAA) is present only in small amounts on the surface of diseased cells, while maintaining a high safety profile. In particular, due to the very low affinity of the CD3-binding domain, the resulting molecules mimic the natural T cell / molecule (TCR) / TA relationship because, in the case of 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 occurs in natural TCR-expressing T cells bound to TA / MHC.

[0011] One of the technical advantages of using a low-affinity CD3 binding domain is that the resulting bispecific antigen-binding protein is specific for the relevant TA due to the use of a high-affinity TCR variable domain, while also having a high safety profile, i.e., a safety window, according to which a dose approximately 1000 times greater than that used to treat TA-presenting cells (e.g., cancer cells) would be required to kill cells in normal or healthy tissue (e.g., normal tissue cells expressing off-target peptides). Therefore, the combination of a high-affinity antigen-binding protein and a low-affinity CD3 binding domain can result in specific binding to the target peptide with reduced or no cross-recognition of off-target peptides on healthy tissue (e.g., off-target peptides), thereby providing a surprisingly large safety window.

[0012] Therefore, the bispecific antigen-binding proteins of the present invention that combine a low-affinity CD3 binding domain with an affinity-matured TA / MHC binding domain have the following advantages: the resulting bispecific antigen-binding proteins can effectively target diseased cells (rather than healthy cells) and also have a favorable or even improved safety profile. Advantageously, the stability and / or solubility of the resulting bispecific antigen molecules are further improved compared to bispecific molecules known in the art that use anti-CD3 domains, thereby providing promising bispecific molecules suitable for medical use.

[0013] In summary, the CD3 binding domain of the present invention, when used in a bispecific format in combination with a TCR or its MHC-peptide complex binding fragment or antigen binding protein, has the following advantages over the prior art, inter alia: (i) reducing the 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) improving the safety profile of the TCR or its MHC-peptide complex binding fragment or antigen binding protein; (iii) reducing off-target and off-tumor cytotoxic effects of the TCR or its MHC-peptide complex binding fragment or antigen binding protein; and (iv) providing a TCR or its MHC-peptide complex binding fragment or antigen binding protein with improved specificity, selectivity and safety.

[0014] definition

[0015] In this article, the term " Antigen binding proteins ” refers to a polypeptide or binding protein capable of binding to at least one antigen.

[0016] The term " antigen " refers to a molecule or a portion of a molecule or complex that is capable of binding to at least one antigen binding site, such as that present in a conventional antibody, a conventional TCR, and / or a bispecific antigen binding protein of the invention.

[0017] The present invention Bispecific antigen binding proteins "It has at least two valencies and binding specificities for at least two different antigens, antigen binding site A binds to CD3 and antigen binding site B binds to target antigen (TA) peptide / MHC complex. In the context of the present invention, the antigen binding site A with specificity for CD3 is derived from a new humanized version of the mouse monoclonal antibody UCHT1, more specifically, from an improved humanized version of the mouse monoclonal antibody UCHT1, and preferably, the antigen binding site B is derived from TCR. The "bispecific antigen binding protein" of the present invention is also referred to herein as the "antigen binding protein of the present invention", which comprises at least 6 CDRs as defined in the context of the present invention, more preferably, the antigen binding protein comprises V derived from the improved humanized UCHT1 antibody. L and V H domains, especially V L and V H Domain variants. The antigen binding site B in the context of the present invention binds to a target antigen (TA) peptide / MHC complex, in particular to a tumor-associated antigen (TAA) peptide / MHC complex, and can be derived from an antibody or TCR, preferably from a TCR. Therefore, 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, wherein the antigen binding site B preferably comprises at least one variable α domain (vα ) and at least one variable beta domain (v β ).

[0018] Such bispecific antigen binding proteins may also be referred to herein as "bispecific molecules."

[0019] As used in the context of the present invention, “ Target antigen (TA) peptide ” refers to a peptide isolated and identified from infectious or tumor material, such as material isolated from tuberculosis patients or from Epstein-Barr virus infected patients or cancer patients. The source protein of the TA peptide undergoes antigen processing in infected cells or tumor cells, so that it can be recognized by the host's immune effector cells (such as T cells or NKT cells) through MHC molecules and the ten (especially TA peptide / MHC complex) presented on the cell surface by the cell. The TA peptide in the context of the present invention comprises or consists of 10, 12 or 14 (such as 8 to 14, 8 to 12, for example, 9 to 11) amino acids. In the context of the present invention, when referring to a specific TA peptide, it is referred to as TA-C. Examples of TA antigenic peptides (for example, TA-C peptides) are viral antigenic peptides, bacterial antigenic peptides or tumor-associated antigen (TAA) antigenic peptides, preferably TAA antigenic peptides. Therefore, in one embodiment, the TA antigenic peptide (especially TA-C) is a viral peptide, a bacterial peptide or a tumor-associated antigen (TAA) antigenic peptide, preferably a TAA antigenic peptide.

[0020] In the context of the present invention, Viral antigen peptides " is an antigenic peptide that is presented by MHC molecules on the surface of diseased cells and is of viral origin, that is, the cells are usually infected with the virus. Such viral antigenic peptides have been found in the context of infection with, 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. Therefore, the 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).

[0021] Viral antigenic peptides that can be used with the methods and embodiments described herein include, for example, those described in the following table. In one aspect, the viral antigenic peptides that can be used with the methods and embodiments described herein include at least one viral antigenic peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 146 to SEQ ID NO: 148, as shown in Table 1 below.

[0022] Table 1: List of viral antigenic peptides

[0023] SEQ ID NO: peptides Virus MHC 146 SLYNTVATL HIV HLA-A*02:01 147 GILGFVFTL Influenza A HLA-A*02:01 148 NLVPMVATV HCMV HLA-A*02:01

[0024] In the context of the present invention, Bacterial antigens "Peptides" are antigenic peptides that are presented by MHC molecules on the surface of diseased cells and are of bacterial origin, i.e., cells that are normally infected with said bacteria. Such bacterial antigenic peptides have been found, for example, in the context of infections with Mycobacterium tuberculosis. Thus, bacterial antigenic peptides in the context of the present invention may be Mycobacterium tuberculosis antigenic peptides.

[0025] “ Tumor-associated antigen (TAA) peptides " Also referred to herein as a "TAA peptide" refers to a peptide isolated and identified from tumor material, and undergoes antigen processing in tumor cells, thereby being recognized by the host's immune effector cells. The TAA peptide comprises or consists of 10, 12 or 14 (such as 8 to 14, 8 to 12, for example, 9 to 11) amino acids. The TAA peptide in the context of the present invention may be, for example, a cancer / testis (CT) antigen peptide. Examples of cancer / testis (CT) antigen peptides are the MAGE-A antigen peptide of the amino acid sequence SEQ ID NO: 10 and the PRAME antigen peptide of the amino acid sequence SEQ ID NO: 9. The TAA peptide in the context of the present invention comprises a T cell epitope and may also be referred to as a TAA peptide in general. When referred to as a specific TAA peptide, it may also be referred to as a TAA peptide C in the context of the present invention.

[0026] In one aspect, tumor associated antigen (TAA) peptides that can be used with the methods and embodiments described herein include, for example, U.S. Patent Publication No. 20160187351, U.S. Patent Publication No. 20170165335, U.S. Patent Publication No. 20170035807, U.S. Patent Publication No. 20160280759, U.S. Patent Publication No. 20160287687, U.S. Patent Publication No. 20160346371, U.S. Patent Publication No. 20160368965, U.S. Patent Publication No. 20170022251, U.S. Patent Publication No. 20170002055, U.S. Patent Publication No. 20170029486, U.S. Patent Publication No. 20170037 089, U.S. Patent Publication No. 20170136108, U.S. Patent Publication No. 20170101473, U.S. Patent Publication No. 20170096461, U.S. Patent Publication No. 20170165337, U.S. Patent Publication No. 20170189505, U.S. Patent Publication No. 20170173132, U.S. Patent Publication No. 20170296640, U.S. Patent Publication No. 20170253633, U.S. Patent Publication No. 20170260249, U.S. Patent Publication No. 20180051080, and U.S. Patent Publication No. 20180164315, the disclosures of these patents and the sequence listing described herein are incorporated herein by reference in their entirety.

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

[0028] Table 2: TAA List

[0029]

[0030]

[0031]

[0032] Furthermore, the TA antigen peptide in the context of the present invention is a specific ligand for MHC class I molecules or MHC class II molecules (preferably MHC class I molecules).

[0033] In the context of the present invention, TAA antigen peptide C is preferably selected from the group of TAA antigen 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 a PRAME antigen peptide comprising or consisting of the amino acid sequence "SLLQHLIGL" of SEQ ID NO: 9, or a MAGE-A antigen peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 10, more preferably SEQ ID NO: 10, wherein the MHC is preferably HLA-A*02.

[0034] “ PRAME "or" Antigens preferentially expressed in melanomaPRAME is an antigen first identified as overexpressed in melanoma (Ikeda et al Immunity. 1997 Feb;6(2):199-208); also known as CT130, MAPE, OIP-4, Uniprot accession number P78395 (available January 11, 2019). This protein acts 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. Testicular cancer antigens are attractive targets for immunotherapeutic intervention because these antigens have limited or no expression in normal adult tissues. PRAME is expressed in many 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 Treat. 2008 May;109(2):359-65). Res.2006Nov15;66(22):10639-42; Ercolak et al Breast Cancer Res Treat.2008May;109(2):359-65; Matsushita et al Leuk Lymphoma.2003Mar;44(3):439-44; Mitsuhashi et al Int.JHematol.2014;100(1):88-95;Proto-Sequeire et al Leuk Res.2006Nov;30(11):1333-9;Szczepanski et al Oral Oncol.2013Feb;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 treating cancers, including but not limited to lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), liver cancer, head and neck cancer, skin cancer, renal cell carcinoma, brain cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, leukemia, breast cancer, Merkel cell carcinoma, melanoma, ovarian cancer, bladder cancer, uterine cancer, gallbladder and bile duct cancer, and esophageal cancer.

[0035] In the context of the present invention, PRAME-derived peptides" 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, which can be searched using Uniprot accession number P78395 (available on January 11, 2019). The 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 protein or a tumor-specific protein and is presented on the cell surface through molecules of the major histocompatibility complex (MHC). More specifically, the PRAME-004-derived peptide is presented on the cell surface in complex with HLA-A*02 (Med. 2001 Jan 1; 193(1): 73-88). In the context of the present invention, "PRAME-derived peptide" or "PRAME-004" are used interchangeably to refer to a peptide comprising the amino acid sequence SLLQHLIGL (SEQ ID NO: 9). NO:9) or a PRAME-derived peptide consisting thereof.

[0036] “ MAGE-A "or" melanoma-associated antigen A MAGE-A subfamily proteins were the first tumor-associated antigens identified at the molecular level (van der Bruggen P, et al. Science. 1991;254:1643–47). MAGE-A is a subfamily of 12 genes (MAGE-A1 to MAGE-A12) located in the q28 region of chromosome X. Members of the MAGE-A subfamily are typically expressed only in the testis or placenta, and their restricted expression suggests that they may play a role in germ cell development. MAGE-A proteins have also been detected in the central nervous system, as well as in the early development of the spinal cord and brainstem, indicating that MAGE-A proteins may also be involved in neuronal development. Members of this family encode proteins with 50% to 80% sequence identity, and all MAGE proteins share a common MAGE homology domain (MHD), a highly conserved domain consisting of approximately 170 amino acids. The biological functions and potential regulatory mechanisms of MAGE-A protein expression in cancer are currently incompletely understood.

[0037] “ MAGE-A4 "or" melanoma-associated antigen 4The protein is a member of the MAGE-A gene family and has Uniprot accession number P43358 with SEQ ID NO: 111 (available on July 8, 2019). The location of MAGE-A4 is 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 and 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 gonocytes but is expressed in prespermatogonia and mature germ cells (Mitchell et al., 2014, Mod. Pathol. 27, 1255-1266). The expression of MAGE-A4 protein and mRNA is associated with the development and prognosis of various cancers.

[0038] “ MAGE-A8 "or" melanoma-associated antigen 8 The protein is a member of the MAGE-A superfamily and has Uniprot accession number P43361 (available on July 8, 2019) with SEQ ID NO: 112.

[0039] As determined by protein sequence alignment using the BLASTP 2.9.0 algorithm, MAGE-A4 "and" MAGE- A8 " proteins have 72% sequence identity (Stephen F et al. (1997) Nucleic Acids Res. 25: 3389-3402). In addition, both "MAGE-A4" and "MAGE-A8" contain the MAG-003 peptide, ie, KVLEHVVRV (SEQ ID NO: 10).

[0040] The term " Epitope ” encompasses the terms “structural epitope” and “functional epitope.” Structural Table Bit "" refers to the amino acids of the antigen that are covered by the antigen binding protein when the antigen binding protein binds to the antigen (e.g., peptide-MHC complex). Generally, the antigen is considered to be covered by any atom of the amino acid residue of the antigen binding protein. All amino acids within are covered. The structural epitope of an antigen can be determined by methods known in the art (including X-ray crystallography or NMR analysis). The structural epitope of an antibody typically contains 20 to 30 amino acids. The structural epitope of a TCR typically contains 20 to 30 amino acids. Functional epitope"" is a subset of amino acids that form a structural epitope, comprising amino acids of the antigen that are essential for forming the interface of the antigen-binding protein of the present invention (either directly through the formation of non-covalent interactions, such as H-bonds, salt bridges, aromatic stacking, or hydrophobic interactions, or indirectly through stabilization of the bound conformation of the antigen), as determined, for example, by mutational scanning. Typically, a functional epitope of an antigen that binds to an antibody comprises 4 to 6 amino acids. Typically, a functional epitope of a peptide-MHC complex comprises 2 to 6 amino acids of the peptide and 2 to 7 amino acids of the MHC molecule. Since MHC I presenting peptides are typically 8 to 10 amino acids in length, only a subset of amino acids of each given peptide is part of a 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 present invention, comprises or consists of amino acids of the antigen required for forming the binding interface. Thus, a functional epitope comprises at least 3 amino acids, and preferably at least 4 amino acids, of the MAGE-A antigenic peptide of SEQ ID NO: 10.

[0041] In the context of the present invention, CD3 "An antigen expressed on T cells as part of a multimolecular T cell receptor complex composed of at least three different chains: CD3ε, CD3δ, and CD3γ. The sequence identity and / or similarity between CD3δ and CD3γ and human CD3ε is low (less than 20% similarity and identity)." 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". Aggregation of CD3 on T cells, for example using immobilized anti-CD3 antibodies, can lead to T cell activation, similar to T cell receptor binding, but independent of the typical specificity of their clone." CD3ε ” contains three domains—an intracellular domain, a transmembrane domain, and an extracellular domain.

[0042] In the context of the present invention, UCHT1The monoclonal antibody specifically binds to a complex of human CD3δ chain and CD3ε chain (referred to herein as CD3ε / δ complex, which is a 36 kDa subunit of the CD3 / T cell antigen receptor complex). The mouse 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). The humanization of UCHT1 is described, for example, by Shalaby et al. (J. Exp. Med. (1992); 175(1): 217–225), which was then further modified to obtain humanized UCHT1 variant 9, designated hUCHT1 (V9), as 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 NO:39 or consisting of the amino acid sequence thereof). However, prior art humanized UCHT variants have low solubility, making them difficult to use in a molecular environment with soluble molecules. Furthermore, those prior art variants have a high affinity for CD3, which, as found in the context of the present invention, may be a disadvantage.

[0043] In the context of the present invention, BMA031 " indicates a monoclonal antibody (mAb) WT31 specific for human α / β TCR. Various humanized variants have been disclosed in the art, including, for example, the α / β TCR-specific humanized antibody BMA031, described in Shearman et al. (J Immunol, 1991, 147, 4366-73). The humanized antibody described by Sherman et al. (J Immunol, 1991, 147, 4366-73) comprises a VL domain comprising, or consisting of, the amino acid sequence of SEQ ID NO: 40 and a VH domain comprising, or consisting of, the amino acid sequence of SEQ ID NO: 41.

[0044] In the context of the present invention, major histocompatibility complex"(MHC) is a group of cell surface proteins that are essential for the adaptive immune system to recognize foreign molecules in vertebrates, which in turn determines tissue compatibility. The main function of MHC molecules is to bind to antigens derived from pathogens and display them on the cell surface for recognition by appropriate T cells. Human MHC is also known as the HLA (human leukocyte antigen) complex (usually just HLA). The MHC gene family is divided into three subgroups: class I, class II and class III. The complex of peptide and MHC class I is recognized by CD8-positive T cells carrying the appropriate T cell receptor (TCR), while the complex of peptide and MHC class I is recognized by CD8-positive T cells carrying the appropriate T cell receptor (TCR), while the complex of peptide and MHC class II is recognized by CD8-positive T cells carrying the appropriate T cell receptor (TCR). The complex of class II molecules is recognized by CD4-positive helper T cells carrying the appropriate TCR. Because both CD8-dependent and CD4-dependent responses contribute jointly and synergistically to anti-tumor effects, identifying and characterizing tumor-associated antigens and corresponding T cell receptors is important in the development of cancer immunotherapies, such as vaccines and cell therapies. The HLA-A gene is located on the short arm of chromosome 6 and encodes the larger α-chain component of HLA-A. Variation in the HLA-A α-chain is key to HLA function. This variation contributes to genetic diversity within the human population. Because each HLA has a different affinity for peptides of certain structures, a greater variety of HLAs means a greater variety of antigens "presented" on the cell surface. Each person can express up to two types of HLA-A, one of which comes from their parents. Some people will inherit the same HLA-A from both parents, which reduces their individual HLA diversity; however, most people inherit two different copies of HLA-A. All HLA groups have the same pattern. In other words, each person can only express one or two of the 2432 known HLA-A alleles. In the context of the present invention, MHC The class I HLA protein may be an HLA-A, HLA-B or HLA-C protein, preferably an HLA-A protein, more preferably HLA-A*02.

[0045] “ HLA-A*02 ” indicates a specific HLA allele, where the letter A represents the gene and the suffix “*02” indicates the A2 serotype.

[0046] In an MHC class I-dependent immune response, peptides must not only bind to certain MHC class I molecules expressed by tumor cells, but they must then be recognized by T cells bearing specific T cell receptors (TCRs).

[0047] In the context of the present invention, TCR" is a heterodimeric cell surface protein of the immunoglobulin superfamily that is associated with an invariant protein of the CD3 complex involved in mediating signal transduction. TCRs exist in αβ and γδ forms, which are structurally similar but have very different anatomical locations and, most likely, very different functions. The extracellular portion of the naturally occurring heterodimeric αβTCR and γδTCR each contains two polypeptides, each with a membrane-proximal constant domain and a membrane-distal variable domain. Each constant and variable domain includes an intrachain disulfide bond. The variable domain contains highly polymorphic loops similar to the complementarity determining regions (CDRs) of antibodies.

[0048] In this article, the term " TCR ” refers to TCRs and fragments thereof, as well as single-chain TCRs and fragments thereof, in particular single-domain TCRs and variable α and β domains of chimeric, humanized, bispecific or multispecific TCRs.

[0049] “ TCR fragment "Comprising a portion of an intact or natural TCR, in particular the antigen binding region or variable region of an intact or natural TCR. Examples of TCR fragments include fragments of α, β, δ, γ chains, such as Vα-Ca or Vβ-Cβ or portions thereof, such fragments may further comprise corresponding hinge regions or single variable domains, such as Vα, Vβ, Vδ, Vγ, single-chain VαVβ fragments or bispecific and multispecific TCRs formed by TCR fragments. Compared to the naturally occurring full-length TCR, the TCR fragment performs the same function, i.e., the fragment selectively and specifically binds to its target peptide.

[0050] In this article, Single-chain TCR (scTCR) " refers to a protein in which the variable domains of a TCR, e.g., Vα and Vβ or Vδ and Vγ, are located on one polypeptide. Typically, the variable domains are separated by a linker, wherein the linker typically comprises 5 to 20, e.g., 5 to 15, amino acids.

[0051] In the example " Natural TCR "Native" as used in the context of the present invention refers to wild-type TCR.

[0052] Natural α-β heterodimeric TCRs have an α chain and a β chain. Each α chain includes variable, joining, and constant regions. The β chain typically also includes a short diversity region between the variable and joining regions, but this diversity region is often considered part of the joining region. The constant regions, or C regions, of the TCR α and β chains are termed 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 the β variable domain) includes three complementarity determining regions (CDRs) embedded within framework sequences, one of which is a highly variable region termed CDR3. The α variable domain CDRs are referred to herein as CDRa1, CDRa2, and CDRa3, while 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 framework, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. Vα types are referred to in IMGT nomenclature by unique TRAV numbers, and Vβ types are referred to in IMGT nomenclature by unique TRBV numbers (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1):42-54; Scavner and Lefranc, (2000), Exp Clin Immunogenet 17(2):83-96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). For more information on immunoglobulin antibodies and TCR genes, see the International Immunogenetic Information System. , Lefranc MP et al. (Nucleic Acids Res. 2015 Jan; 43 (Database issue): D413-22; and http: / / www.imgt.org / ). Therefore, a conventional TCR antigen-binding site typically includes six CDRs, comprising a CDR set from the α and β chain variable regions, wherein the CDR1 and CDR3 sequences are involved in the recognition and binding of peptide antigens bound by HLA proteins, while the CDR2 sequence is involved in the recognition and binding of HLA proteins.

[0053] Similar to antibodies, TCR framework region"(FR) refers to the amino acid sequence inserted between the CDRs, i.e., to those portions of the variable regions of the TCR α and β chains that are conserved to some extent between different TCRs in a single species. The α and β chains of each TCR each have four FRs, referred to herein as FR1-a, FR2-a, FR3-a, FR4-a and FR1-b, FR2-b, FR3-b, FR4-b, respectively. Thus, the α chain variable domain can be referred to as (FR1-a)-(CDRa1)-(FR2-a)-(CDRa2)-(FR3-a)-(CDRa3)-(FR4-a), and the β chain variable domain can be referred to as (FR1-b)-(CDRb1)-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)-(FR4-b).

[0054] In the context of the present invention, the CDR / FR definitions in the α or β chain or the γ or δ chain are determined based on the IMGT definitions (Lefranc et al. Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org). Thus, according to the IMGT definitions, the CDR / FR amino acid positions associated with a TCR or TCR-derived domain are indicated. In one embodiment, the IMGT positions of the CDR / FR amino acid positions of the first variable domain are similar to the IMGT numbering of TRAV5 and / or the IMGT positions of the CDR / FR amino acid positions of the second variable domain are similar to the IMGT numbering of TRBV12-4, for example, for the antigen binding site B variable domain of the MAGE-A antigenic peptide of SEQ ID NO:10.

[0055] With respect to γ / δ TCRs, the term "TCRγ variable domain" as used herein refers to the concatenation of the TCRγV (TRGV) region without the leader region (L) and the TCRγJ (TRGJ) region; the term TCRγ constant domain refers to the extracellular TRGC region or a C-terminal truncated TRGC sequence. Similarly, the term "TCRδ variable domain" refers to the concatenation of the TCRδV (TRDV) region without the leader region (L) and the TCRδD / J (TRDD / TRDJ) region; the term TCRδ constant domain refers to the extracellular TRDC region or a C-terminal truncated TRDC sequence.

[0056] exist" Antibody ” (also known as “ immunoglobulinsIn an antibody, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, namely, λ (l) and κ (k). There are five major types (or isotypes) of heavy chains, which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains different sequence domains. The light chain includes two domains or regions, namely, the variable domain (V L ) and constant domain (C L The heavy chain consists of four domains, namely, one variable domain (VH) and three constant domains (C H1 、C H2 and C H3 , collectively referred to as C H ). Light chain variable region (V L ) and heavy chain variable region (V H ) determines the binding recognition and specificity to the antigen. L ) and heavy chain constant region domain (C H ) possess important biological properties, such as antibody chain association, secretion, transplacental migration, complement fixation, and binding to Fc receptors (FcRs). The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment and is composed of the variable portion of one light chain and one heavy chain. The specificity of an antibody depends on the structural complementarity between the antibody combining site (equivalent to the antibody binding site) and the antigenic determinant. The antibody combining site is composed primarily of residues from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable regions or framework regions (FRs) can affect the overall domain structure and thus the binding site. The complementarity determining regions or CDRs refer to amino acid sequences that together determine the binding affinity and specificity of the native Fv region of the natural immunoglobulin binding site. Immunoglobulin light and heavy chains each have three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, a conventional antibody antigen-binding site includes six CDRs, comprising a set of CDRs from each of the heavy and light chain V regions.

[0057] In the context of the present invention, an antibody or immunoglobulin is IgM, IgD, IgG, IgA or IgE.

[0058] “ Antibody framework region"(FR) refers to the amino acid sequence inserted between CDRs, that is, those parts of the immunoglobulin light chain and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. Each immunoglobulin light chain and heavy chain each has four FRs, referred to as FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively. Therefore, the light chain variable domain can be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable domain can be referred to as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H).

[0059] In the context of the present invention, the CDR / FR definitions 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) are based on Kabat (Kabat EA, Te, Wu T, Foeller C, Perry HM, Gottesman KS. (1992) Sequences of Proteins of Immunological Interest.). However, the CDR / FR amino acid positions of an immunoglobulin light or heavy chain (particularly a UCHT1 variant in the context of the present invention) are numbered ordinal. Thus, for example, the range for CDRH1 according to Kabat is amino acid positions 31 to 35, the range for CDRH2 according to Kabat is amino acid positions 50 to 66, and the range for CDRH3 according to Kabat is amino acid positions 99 to 111. Thus, for example, the range for CDRL1 according to Kabat is amino acid positions 24 to 34, and the range for CDRL2 according to Kabat is amino acid positions 50 to 56.

[0060] The “ Human frame area " is a framework region that is substantially identical (about 85% or higher, particularly 90%, 95%, 97%, 99% or 100% identical) to the framework region of a natural antigen-binding protein (e.g., a natural human antibody or human TCR).

[0061] the term" Antibody ” refers to antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular single domain antibodies and variable heavy chains of chimeric, humanized, bispecific or multispecific antibodies.

[0062] The “ Conventional antibodies " is an antibody having the same structural domain as an antibody isolated from nature and comprising antibody-derived CDRs and framework regions. Similarly, the " Conventional TCR " is a TCR that contains the same structural domains as a natural TCR and includes TCR-derived CDRs and framework regions.

[0063] the term" Humanized antibodies " refers to an antibody of fully or partially non-human origin that has been modified to replace certain amino acids, especially amino acids in the framework regions of the heavy and light chains of non-human monoclonal antibodies, so as to avoid or minimize the immune response in the human body. The constant domains of humanized antibodies in most cases mainly refer to human C H and C L domain.

[0064] Many methods for humanizing antibody sequences are known in the art; for example, see the review Almagro & Fransson (2008) Front Biosci. 13: 1619-1633. A commonly used method is CDR transplantation or antibody remodeling, which involves transplanting the CDR sequences of a donor antibody (usually a mouse antibody) into a human antibody framework with different specificity. Since CDR transplantation can reduce the binding specificity and affinity of the CDR-transplanted non-human antibody, thereby reducing its biological activity, back mutations can be introduced at selected positions of the CDR-transplanted antibody to maintain the binding specificity and affinity of the parent antibody. Possible back mutation positions can be identified using existing information in the literature and antibody databases. Another humanization technique for CDR transplantation and back mutation is surface remodeling, in which non-surface exposed residues of non-human origin are retained, while surface residues are changed to human residues. Another alternative technique is called "guided selection" (Jespers et al. (1994) Biotechnology 12, 899), which can be used to derive fully human antibodies that retain the epitope and binding characteristics of the parent antibody from mouse antibodies, etc. Another approach to humanization is so-called 4D humanization. For example, the 4D humanization approach is described in patent application US20110027266 A1 (WO2009032661 A1), which is incorporated herein by reference in its entirety. In the context of the present invention, the monoclonal mouse antibody UCHT1 was humanized as described in detail in Example 1 herein. For chimeric antibodies, humanization typically involves modifying the framework regions of the variable region sequences.

[0065] In the context of the present invention, cursor area " refers to the murine residues within the framework regions that have been shown to affect the conformation of the CDR loops and the affinity of the antibody. These residues are also referred to as " Vernier residues ", the β-pleated sheet framework region located immediately below the CDRs and not involved in direct interactions with the antigen, i.e., these residues are retained in "humanized" antibodies (Foote & Winter, 1992).

[0066] Although it may be desirable to alter individual CDR amino acid residues in certain circumstances, for example, to remove glycosylation sites, deamidation sites, isomerization sites, or undesirable cysteine ​​residues, amino acid residues that are part of the CDR are generally not altered in conjunction with humanization. N-linked glycosylation occurs by attaching the oligosaccharide chain to an asparagine residue of the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X can be any amino acid except Pro. Removal of N-glycosylation sites can be achieved by mutating the Asn or Ser / Thr residues to different residues, particularly through conservative substitutions. Deamidation of asparagine and glutamine residues may depend on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation (primarily when present in the Asn-Gly sequence) and less so when present in other dipeptide sequences such as Asn-Ala. When such a deamidation site (particularly Asn-Gly) exists in a CDR sequence, it may be desirable to remove the site, typically by conservatively substituting one of the involved residues. Substitution within the CDR sequence to remove one of the involved residues is also contemplated by the present invention.

[0067] In the context of the present invention, Antibody fragments " comprises a portion of an intact antibody, in particular the antigen binding or variable region of an intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed by antibody fragments. An antibody fragment may also be a single domain antibody, for example, a heavy chain antibody or VHH.

[0068] the term" Fab "" refers to an antibody fragment with a molecular weight of approximately 50,000 daltons and antigen-binding activity, wherein the N-terminal half of the H chain and the entire L chain are bound together by a disulfide bond in the fragment obtained by treating IgG with a protease (e.g., papain).

[0069] The term " form ” refers to a bispecific antigen-binding protein that comprises a specific number and type of domains and their spatial organization present in the bispecific antigen-binding protein.

[0070] Many different formats are described in the art, for example, bispecific formats, generally in the context of antibodies, including non-limiting examples such as diabodies, crossover dual variable domains (CODVs) and / or dual variable domain (DVD) proteins. An overview of these different bispecific antibodies and methods for their preparation is disclosed, for example, in Brinkmann U. and Kontermann REMAbs. 2017 Feb-Mar; 9(2): 182–212. More specifically, DVD formats are disclosed, for example, in the following scientific articles (Wu C et al. Nat Biotechnol 2007; 25: 1290-7; PMID: 17934452; Wu C. et al. MAbs 2009; 1: 339-47; Lacy SE et al. MAbs 2015; 7: 605-19; PMID: 25764208; Craig RB et al. PLoS One 2012; 7: e46778; PMID: 23056448; Piccione EC et al. MAbs 2015). CODV is disclosed, for example, in Onuoha SC et al. Arthritis Rheumatol. 2015 Oct; 67(10): 2661-72 or, for example, in WO 2012 / 135345, WO 2016 / 116626. For example, bispecific diabodies are described 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.

[0071] “Used in the context of antibodies Double Antibody” usually refers to a bivalent molecule composed of two chains, each containing the VH and VL domains from the same or different antibodies. The two chains usually have the configurations VHA-VLB and VHB-VLA (A and B represent the two different specificities) or VLA-VHB and VLB-VHA.

[0072] In the context of the present invention, the “ Diabody (Db) or" Diabody format ” refers to a bivalent molecule consisting of two polypeptide chains, each of which contains Db1 and L Db2 ), wherein two domains are defined in the context of the present invention as a first domain and a second domain (V1 and V2), while the other two domains may be TCR-derived or antibody-derived variable domains (V A 、V B ). The V1 and V2 domains are located on two different polypeptides. A and V B The domains are located on two different polypeptides, and these domains dimerize in a head-to-tail orientation. Thus, the orientation may be 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. In order to make the domains dimerize head to tail, the linker (i.e. L Db1 and L Db1 ) may be the same or different and are short linkers. Short linkers are linkers that are typically 2 to 12, 3 to 13 amino acids in length, such as 3, 4, 5, 6, 7, 8, or 9 amino acids in length, for example, 4 or 5 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” of SEQ ID NO: 114, “GGGGS” of SEQ ID NO: 115, or “GGGSGGGG” of SEQ ID NO: 118.

[0073] “ Dual variable domain immunoglobulin (DVD-Ig TM )The description of the "DVD-Ig" format was first seen in Wu C. et al. 2007 (Nat Biotechnol. 2007 Nov; 25(11): 1290-7). In this format, the target binding variable domain of a second monoclonal antibody (B) is usually fused to a conventional antibody (A) (comprising VLA and VHA domains), wherein the light chain of the conventional antibody (A) thus comprises an additional light chain variable domain (VLB), and the heavy chain of the conventional antibody (A) comprises an additional heavy chain variable domain (VHB). Therefore, the DVD-Ig described in the art TM Usually composed of two polypeptide chains - a heavy chain (containing VHB-L-VHA-CH1-CH2-CH3) and a light chain (containing V LB -LV LA -C L ). Therefore, V LA / V HA and V LA / V LA Domain pairs are paired in parallel.

[0074] In the context of the present invention, " Dual variable domain Ig format " refers to a protein comprising two polypeptide chains, each polypeptide chain comprising two variable domains connected by a linker (L1, L3), wherein two of the domains are defined in the context of the present invention as the first domain and the second domain (V1 and V2), and the other two domains are antibody-derived heavy and light chain variable domains (V HA and V HB In the context of the present invention, in the form of DVD-Ig, for example, the polypeptide chain contains the organization 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 length of the linker L1 and L3 is preferably between 5 and 20 amino acid residues, such as 5 to 15 amino acid residues, and / or the linker L2 and L4 may or may not be present.

[0075] The “ Crossover dual variable domain-Ig-like protein " represents a form in which two V H and two V L domain to allow for variable V H -V LThe domains are connected in a cross-pairing manner, and the domains (from N-terminus to C-terminus) are connected in a V HA -V HB and V LB -V LA In order of V HB -V HA and V LA -V LB Arranged in order.

[0076] In the context of the present invention, " Crossover dual variable domain-Ig-like protein " refers to a protein comprising two polypeptide chains, each polypeptide chain comprising two variable domains connected by a linker (L1, L2, L3 and L4), wherein two of the domains are defined in the context of the present invention as the first domain and the second domain (V1 and V2), and the other two domains are antibody-derived heavy and light chain variable domains (V HA 、V HB In the context of the present invention, in the form of CDVD-Ig, for example, the polypeptide chain contains the organization 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 L In this CDVD format, the linkers (L1 to L4) are generally of varying lengths, including all-glycine linkers and the linkers described in the linker section below. For example, L1 is 3 to 12 amino acid residues long, L2 is 3 to 14 amino acid residues long, L3 is 1 to 8 amino acid residues long, L4 is 1 to 3 amino acid residues long, or

[0077] The length of L1 is 5 to 10 amino acid residues, the length of L2 is 5 to 8 amino acid residues, the length of L3 is 1 to 5 amino acid residues, and the length of L4 is 1 to 2 amino acid residues, or the length of L1 is 7 amino acid residues, the length of L2 is 5 amino acid residues, the length of L3 is 1 amino acid residue, and the length of L4 is 2 amino acid residues.

[0078] In this article, At least one " refers to one or more of the specified objects, such as 1, 2, 3, 4, 5 or 6 or more specified objects. For example, at least one binding site herein refers to 1, 2, 3, 4, 5 or 6 or more binding sites.

[0079] “ At least 85% identical to the reference sequence ” refers to sequences that have 85% or higher sequence identity, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity over their entire length to the reference sequence.

[0080] In the context of this application, the global pairwise alignment method is used to calculate the " Percent identity " (i.e., comparing the full length of the two sequences). Methods for comparing the identity of two or more sequences are well known in the art. For example, the "Needle" program can be used, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48: 443-453) to find the best alignment (including gaps) of two sequences (taking into account their full length). For example, the Needle program can be downloaded from the World Wide Web and is further described in the following publication (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277). According to the present invention, the method for calculating the percentage of identity between two polypeptides is: EMBOSS: Needle (Global) program, "Gap Open" parameter equal to 10.0, "Gap Extend" parameter equal to 0.5, matrix Blosum62.

[0081] 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, relative 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 (different from the reference sequence) derived from another species.

[0082] "Amino acid substitutions" can be conservative or non-conservative. Preferably, the substitution is a conservative substitution, in which one amino acid is replaced by another amino acid having similar structural and / or chemical properties.

[0083] In one embodiment, conservative substitutions may include substitutions described by Dayhoff in "The Atlas of Protein Sequence and Structure. Vol. 5", Natl. Biomedical Research, the contents of which are incorporated herein by reference in their entirety. For example, in one aspect, amino acids belonging to one of the following groups can be exchanged with each other, thereby constituting a conservative exchange: 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 can be selected from the following substitutions: T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G and / or T→S.

[0084] In another embodiment, a conservative amino acid substitution may include replacing one amino acid with another amino acid of the same class, for example, (1) nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp; (2) uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) acidic: Asp, Glu; and (4) basic: Lys, Arg, His. Other conservative amino acid substitutions may also be made as follows: (1) aromatic: Phe, Tyr, His; (2) proton donors: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptors: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see U.S. Patent No. 10,106,805, the contents of which are incorporated herein by reference in their entirety).

[0085] In another embodiment, conservative substitutions can be made according to Table 3. Methods for predicting protein modification tolerance can be found in, for example, Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the contents of which are incorporated by reference in their entirety.

[0086] Table 3: Conservative amino acid substitutions

[0087]

[0088] In another embodiment, conservative substitutions may be those shown under the heading "Conservative Substitutions" in Table 3. If such substitutions result in a change in biological activity, then major changes, designated "Representative Substitutions" in Table 4, may be introduced and the products screened, if desired.

[0089] Table 4: Amino acid substitutions

[0090]

[0091] In some embodiments, the bispecific antigen-binding protein may include a variant antigen-binding protein, wherein the variant bispecific antigen-binding protein includes a first polypeptide chain (e.g., an α chain) and a second polypeptide chain (e.g., a β chain) that comprises up to 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions compared to the bispecific antigen-binding protein from which the variant is derived, preferably in the CDR regions of the first variable domain (e.g., a Vα domain) and the second variable domain (e.g., a Vβ domain). 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 CDR region of the bispecific antigen-binding protein or in all CDR regions of the first and / or second variable domains. The substitution may be in the CDRs of the first and / or second variable domains.

[0092] In one embodiment, the variant is a functional variant.

[0093] As used herein, the term “ Functional variants " refers to bispecific antigen-binding proteins that have substantial or significant sequence identity or similarity to a parent bispecific antigen-binding protein, for example, those containing conservative amino acid substitutions, wherein the functional variant retains the biological activity of the parent bispecific antigen-binding protein. In one aspect, for example, functional variants include those variants of the bispecific antigen-binding proteins described herein (then, the bispecific antigen-binding proteins described herein themselves are referred to as parent antigen-binding proteins) that retain target cell recognition ability similar to, the same as, or greater than that of the parent bispecific antigen-binding protein. Compared to the parent bispecific antigen-binding protein, for example, a functional variant can contain an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the parent bispecific antigen-binding protein.

[0094] For example, a functional variant may comprise the amino acid sequence of a parent bispecific antigen-binding protein with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant may comprise the amino acid sequence of a parent bispecific antigen-binding protein with at least one non-conservative amino acid substitution. In this case, the non-conservative amino acid substitution preferably 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, thereby increasing the biological activity of the functional variant relative to the parent bispecific antigen-binding protein.

[0095] The modified TCRs, polypeptides and antigen-binding proteins (including functional parts, fragments and functional variants) of the present invention may have any length, that is, may contain any number of amino acids, provided that the modified TCR, polypeptide or protein (or its functional part or functional variant) retains its biological activity, such as: the ability to specifically bind to an antigen, detect diseased cells in a host, or treat or prevent a disease in a host.

[0096] The bispecific antigen-binding proteins of the present invention (including functional portions, fragments, and functional variants) may comprise synthetic amino acids that replace one or more natural amino acids. Such synthetic amino acids are known in the art and may include, for example, aminocyclohexanecarboxylic acid, norleucine, α-aminodecanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline- 2-Carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 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.

[0097] In one embodiment, the bispecific antigen-binding proteins of the invention (including functional portions and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bonds), or converted into acid addition salts and / or optionally dimerized, multimerized, or conjugated.

[0098] The bispecific antigen binding proteins of the present disclosure may be synthetic, recombinant, isolated and / or purified.

[0099] This article's covalent bond ” refers to, for example, a disulfide bond or a peptide bond or a covalent bond connected by a linker or linker sequence such as a polypeptide linker.

[0100] As used herein, the term “ Linker" refers to one or more amino acid residues that are inserted between two domains to provide sufficient mobility for the domains, e.g., in a single-chain construct, between the first and second variable domains of a bispecific antigen-binding protein of the invention and optionally between the variable domains of the light and heavy chain variable domains, to fold correctly to form an antigen-binding site, or, in the case of a bispecific antigen-binding protein, to form an antigen-binding site and at least one other antigen-binding site in a cross-pairing format (in a CODV format or certain diabody formats) or parallel pairing format (e.g., in a DVD format) of the bispecific antigen-binding protein of the invention.

[0101] In some embodiments, the linker consists of zero amino acids, meaning that the linker is absent. At the amino acid sequence level, linkers are inserted at transitions between variable domains or between variable and constant domains, respectively. Since the approximate sizes of immunoglobulin domains and TCR domains are well known, transitions between domains can be determined. As is known to those skilled in the art, the precise location of domain transitions can be determined by locating peptide stretches that do not form secondary structural elements (e.g., β-sheets or α-helices) (e.g., as demonstrated by experimental data), or can be identified or hypothesized using modeling or secondary structure prediction techniques. The term linker used in the context of the present invention refers to, but is not limited to, linkers designated L1, L2, L3, L4, L5, and L6.

[0102] Unless otherwise specified in the respective context, the length of the linker (e.g., L1, L2, L3, L4, L5, and L6) can be at least 1 to 30 amino acids. In some embodiments, the length of the linker (e.g., L1, L2, L3, L4, L5, and L6) can be 2-25, 2-20, or 3-18 amino acids. In some embodiments, the linker (e.g., L1, L2, L3, L4, L5, and L6) can be a peptide having a length of no more than 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids. In other embodiments, the length of the linker (e.g., L1, L2, L3, L4, L5, and L6) can be 5-25, 5-15, 4-11, 10-20, or 20-30 amino acids. In other embodiments, the length of the linker (e.g., 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 specific embodiments, the length of the linker (e.g., L1, L2, L3, L4, L5, and L6) may be less than 24, less than 20, less than 16, less than 12, less than 10 (e.g., 5 to 24, 10 to 24, or 5-10) amino acid residues. In some embodiments, the length of the linker is equal to one or more amino acid residues, for example, greater than 1, greater than 2, greater than 5, greater than 10, greater than 20 amino acid residues, greater than 22 amino acid residues.Exemplary linkers (e.g., L1, L2, L3, L4, L5, and L6) comprise or consist of an amino acid sequence 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), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 122), NO: 122), GGSGGGGSGGGGSGGGGSGG (SEQ ID NO: 33), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 123), GGSGGGGSGGGGSGGGGSGGGGSGG (SEQ ID NO: 66), GSADDAKKDAAKKDGKS (SEQ ID NO: 97), GGQGSGGTGSGGQGSGGTGSGGQGS (SEQ ID NO: 122) NO:143), TVLSSAS (SEQ ID NO:124), GGGGSGT (SEQ ID NO:183) and GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:125), in particular GGGSGGGG (SEQ ID NO:118), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 125) and GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 123).

[0103] The term " Fc domain " encompasses native Fc domains as well as Fc domain variants and sequences as further defined below. As with Fc variants and native Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form, whether enzymatically cleaved from intact antibodies or generated by other means.

[0104] As used herein, the term “ native Fc" refers to a molecule comprising the sequence of a non-antibody binding fragment produced by antibody enzymatic cleavage or other means, whether in monomeric or multimeric form, and may include a hinge region. The original immunoglobulin source of natural Fc is particularly human, and may be any immunoglobulin, preferably IgG1 or IgG2, and most preferably IgG1. Natural Fc molecules are composed of monomeric polypeptides, which can be associated into dimers or multimers through covalent bonds (i.e., disulfide bonds) and non-covalent bonds. The number of intermolecular disulfide bonds between monomeric subunits of natural Fc molecules is 1-4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of natural Fc is a disulfide-bonded dimer produced by papain cleavage of IgG. The term "natural Fc" used herein is a general term for monomeric, dimeric, and multimeric forms. An example of a natural Fc amino acid sequence is SEQ ID The amino acid sequence of NO:126 is the native Fc amino acid sequence of IGHG1*01.

[0105] “ hinge "or" Hinge area "or" Hinge domain "Usually refers to the H1 The flexible portion of the heavy chain between the C-terminal domain and the CH2 domain. It is approximately 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 herein are shown below:

[0106] IgG1:E216PKSCDKTHTCPPCPAPELLG(SEQ ID No.127)

[0107] IgG2:E216RKCCVECPPCPAPPVAGP(SEQ ID No.128)

[0108] IgG3:ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPE216PKSCDTPPPCPRCPAPELLG(SEQ IDNo.129)

[0109] IgG4:E216SKYGPPCPSCPAPEFLG (SEQ ID No. 130).

[0110] In the context of the present invention it refers to the amino acid positions in the Fc domain, these amino acid positions or residues being indicated according to the EU numbering system as described in, for example, Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969).

[0111] As used herein, the term “ Fc variants " refers to a molecule or sequence that has been modified from a native Fc but still contains a binding site for the salvage receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with salvage receptors are known in the art. Thus, the term "Fc variant" can include molecules or sequences that have been humanized from a non-human native Fc. In addition, a native Fc contains regions that can be removed because these regions provide structural features or biological activities that are not required for the bispecific antigen binding proteins of the present invention. Thus, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues or in which one or more Fc sites or residues have been modified, which affects or involves: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity after expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0112] Thus, in one embodiment, the Fc domain (eg, Fc1 and / or Fc2) comprises a hinge domain.

[0113] In one embodiment, the Fc domain is a human IgG Fc domain, preferably derived from human IgG1, IgG2, IgG3 or IgG4, preferably IgG1 or IgG2, more preferably IgG1.

[0114] In some embodiments, in particular, when the bispecific antigen binding protein comprises two Fc domains (ie, In the embodiment of the present invention, for example, Fc1 and Fc2), the two Fc domains may have the same immunoglobulin isotype or isotype subclass or different immunoglobulin isotypes or isotype subclasses, and are preferably the same. Therefore, in some embodiments, Fc1 and Fc2 are IgG1 subclass, IgG2 subclass, IgG3 subclass, or IgG4 subclass, preferably IgG1 subclass or IgG2 subclass, and more preferably IgG1 subclass.

[0115] In some embodiments, the Fc domain is a variant Fc domain, thus comprising one or more amino acid substitutions described below.

[0116] In some embodiments, the Fc domain comprises or further comprises "RF" and / or "knob-in-hole" mutations, preferably " pestle-mortar structure ”.

[0117] “ RF mutation "" generally refers to an amino acid substitution in the CH3 domain of an Fc domain that replaces the amino acid HY with an amino acid RF, such as the amino acid substitutions H435R and Y436F in the CH3 domain, which are described in Jendeberg, L. et al. (1997, J. Immunological Meth., 201: 25-34) as being advantageous for purification purposes because they abolish binding to protein A. In the case where the bispecific antigen-binding protein comprises two Fc domains, the RF mutation may occur in one or both Fc domains, preferably in one Fc domain.

[0118] “ pestle-mortar structure ” (also known as “pestoise-and-mortar” technology) refers to the H3 -C H3 The amino acid substitutions T366S, L368A, and Y407V (hole) and T366W (knob) at the interface promote heteromultimer formation. These knob-to-hole mutations can be further stabilized by introducing additional cysteine ​​amino acid substitutions Y349C and S354C. The "knob-to-hole" technology and stabilizing cysteine ​​amino acid substitutions are described in US Patents 5,731,168 and 8,216,805.

[0119] In the context of the present invention, the "knob" mutation and the cysteine ​​amino acid substitution S354C are present in, for example, an Fc domain comprising or consisting of the amino acid sequence of SEQ ID NO: 131, and the "hole" mutation and the cysteine ​​amino acid substitution Y349C1 are present in an Fc domain comprising or consisting of the amino acid sequence of SEQ ID NO: 132.

[0120] In some embodiments, the Fc domain of one polypeptide (e.g., Fc1) comprises the amino acid substitution T366W (knob) in its CH3 domain, and the Fc domain of the other polypeptide (e.g., Fc2) comprises the amino acid substitutions T366S, L368A, and Y407V (hole) in its CH3 domain, or vice versa.

[0121] In some embodiments, the Fc domain of one polypeptide (e.g., Fc1) comprises or further comprises the amino acid substitution S354C in its CH3 domain, while the Fc domain of the other polypeptide (e.g., Fc2) comprises or further comprises the amino acid substitution Y349C in its CH3 domain, or vice versa.

[0122] Thus, in some embodiments, the Fc domain of one polypeptide (e.g., Fc1) comprises amino acid substitutions S354C and T366W (knob) in its CH3 domain, while the Fc domain of the other polypeptide (e.g., Fc2) comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (hole) in its CH3 domain, or vice versa.

[0123] As described by Wei et al., the set of amino acid substitutions can be further expanded by incorporating the amino acid substitution K409A on one polypeptide and F405K on another polypeptide (Structural basis of a novel heterodimericFc for bispecific antibody production, Oncotarget. 2017). Thus, in some embodiments, the Fc domain (e.g., Fc1) of one polypeptide is located at its C H3 The Fc domain of the polypeptide comprises or further comprises the amino acid substitution K409A, and the Fc domain of another polypeptide (eg, Fc2) is in its C H3 The domain comprises or further comprises the amino acid substitution F405K, or vice versa.

[0124] In some cases, artificially introduced cysteine ​​bridges can improve the stability of bispecific antigen-binding proteins, ideally without interfering with the binding properties of the bispecific antigen-binding protein. Such cysteine ​​bridges can further improve heterodimerization.

[0125] Further amino acid substitutions (eg charge pair substitutions) have been described in the art, for example in EP 2 970 484, to improve the heterodimerization of the resulting protein.

[0126] Thus, in some embodiments, the Fc domain of one polypeptide (e.g., Fc1) comprises or further comprises a charged pair substituent E356K, E356R, D356R, or D356K and D399K or D399R, and the Fc domain of the other polypeptide (e.g., Fc2) comprises or further comprises a charged pair substituent R409D, R409E, K409E, or K409D and N392D, N392E, K392E, or K392D, or vice versa.

[0127] In another embodiment, the Fc domains on one or both (preferably both polypeptide chains) may contain one or more alterations that inhibit Fcγ receptor (FcγR) binding. Such alterations may include L234A, L235A.

[0128] By hinge, C H2 and C H3When incorporating Fc portions consisting of a bispecific binding domain or a portion thereof into antigen-binding proteins, more specifically into bispecific antigen-binding proteins, the problem of nonspecific immobilization of these molecules induced by Fc:Fc-gamma receptor (FcgR) interactions arises. FcgR is composed of different cell surface molecules (FcgRI, FcgRIIa, FcgRIIb, FcgRIII) that have different affinities for epitopes displayed by the Fc portion of IgG molecules. This nonspecific (i.e., not induced by either of the two binding domains of the bispecific molecule) immobilization is disadvantageous due to i) the impact on the pharmacokinetics of the molecule and ii) off-target activation of immune effector cells. Therefore, various Fc variants and mutations that ablate FcgR binding have been identified. In this context, Morgan et al. 1995, Immunology (The N-terminal end of the CH2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, FcγRI and FcγRIII binding) discloses that residues 233-236 of human IgG1 are exchanged with the corresponding sequence from human IgG2 (i.e., residues 233P, 234V and 235A, wherein the amino acid at position 236 is absent), resulting in abrogation of FcgRI binding, abrogation of C1q binding and reduced FcgRIII binding. EP1075496 discloses antibodies and other Fc-containing molecules with Fc region variants (e.g., one or more of 233P, 234V, 235A, and no residue or G at position 236, as well as 327G, 330S and 331S), wherein the recombinant antibodies are able to bind to target molecules without inducing significant complement-dependent lysis or cell-mediated target destruction.

[0129] Therefore, in some embodiments, the Fc region comprises or further comprises one or more amino acids or deletions selected from the group consisting of 233P, 234V, 235A, 236 (no residue) or G, 327G, 330S, 331S, preferably, the Fc region comprises or further comprises amino acids 233P, 234V, 235A, 236 (no residue) or G and one or more amino acids selected from the group consisting of 327G, 330S, 331S, most preferably, the Fc region comprises or further comprises amino acids 233P, 234V, 235A, 236 (no residue) and 331S.

[0130] In another embodiment, the Fc domain comprises or further comprises the amino acid substitution N297Q, N297G or N297A, preferably N297Q.

[0131] Amino acid substituents N297Q "," N297G "or" N297A " refers to an amino acid substitution at position 297 that eliminates the native N-glycosylation site within the Fc domain. This amino acid substitution can further prevent Fc-γ-receptor interaction and reduce the variability of the final protein product (i.e., the bispecific antigen-binding protein of the present invention) caused by sugar residues, as described, for example, in Tao, MH and Morrison, SL (J Immunol. 1989 Oct 15; 143(8): 2595-601.).

[0132] In a further embodiment, in particular in the absence of a light chain, the Fc domain comprises or further comprises the amino acid substitution C220S. The amino acid substitution "C220S" may be deleted to form C H1 -C L disulfide bonds of cysteine.

[0133] In some embodiments, the Fc domain comprises or further comprises at least two additional cysteine ​​residues, e.g., S354C and Y349C or L242C and K334C, wherein S354C is located in the Fc domain of one polypeptide (e.g., Fc1) and Y349C is located in the Fc domain of another polypeptide (e.g., Fc2), to form a heterodimer and / or wherein L242C and K334C are located in the same Fc domain of Fc1 or Fc2 of one or both polypeptides to form an intradomain C-C bridge.

[0134] When referring to a polypeptide (i.e., a bispecific antigen binding protein of the invention) or nucleotide sequence, " Purified "and" Separated " refers to the presence of the indicated molecule in the substantial absence of other biomacromolecules of the same type. The term "purified" as used herein specifically refers to the presence of at least 75%, 85%, 95% or 98% by weight of the biomacromolecule of the same type.

[0135] Encoding a specific polypeptide Separated "Nucleic acid molecule" refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide of interest; however, the molecule may include some additional bases or moieties that do not adversely affect the basic properties of the composition.

[0136] “ domain " can be any region of a protein, generally defined by sequence homology and often associated with a specific structural or functional entity.

[0137] “ Reorganization A molecule is one that is prepared, expressed, produced, or isolated by recombinant means.

[0138] the term" Gene " refers to a DNA sequence that encodes or corresponds to a specific amino acid sequence that comprises all or part of one or more proteins or enzymes and may or may not include regulatory DNA sequences, such as promoter sequences, which determine the conditions for, for example, gene expression. Certain 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 act as regulators of structural genes or regulators of DNA transcription. In particular, the term gene may be applied to genomic sequences that encode proteins, i.e., sequences that include regulatory factors, promoters, introns, and exon sequences.

[0139] “ Affinity "Affinity is theoretically defined as the equilibrium binding between a bispecific antigen binding protein and an antigen, and in the context of the present invention, is defined as the equilibrium binding between a bispecific antigen binding protein and its antigen, TA / MHC or TA-C / MHC or CD3. For example, affinity can be expressed as half maximal effective concentration (EC50, sometimes also referred to as half maximal binding concentration (EC50)) or equilibrium dissociation constant (K D )express.

[0140] “ K D " is the equilibrium dissociation constant between the bispecific antigen-binding protein and its antigen, i.e., the ratio of koff / kon. KD is inversely proportional to affinity. K 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 values ​​can be experimentally assessed using a variety of known methods, such as measuring association and dissociation rates using surface plasmon resonance (SPR) or biolayer interferometry (BLI), as described in more detail in the "Biblispecific Antigen Binding Proteins" section below.

[0141] “ half maximum effective concentration ”, also known as “EC 50 ”, usually refers to the concentration of a molecule that induces a response intermediate between baseline and maximum after a specified exposure time. EC 50 Inversely proportional to affinity, EC 50 The lower the value, the higher the affinity of the molecule. In one embodiment, "EC 50 " refers to a concentration of a bispecific antigen-binding protein of the invention that induces a response intermediate between the baseline and the maximum value after a specified exposure time, more specifically, refers to a concentration of a bispecific antigen-binding protein of the invention that induces a response intermediate between the baseline and the maximum value after a specified exposure time. EC 50The value can be experimentally assessed by various known methods, for example, using an IFN-γ release assay or an LDH release assay, as described in more detail in the experimental sections of Examples 2 and 5. 50 The values ​​are preferably determined by LDH release assay and thus refer to the induction of cytotoxicity.

[0142] In this article, diagnostic agents "refers to a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule, or any other label known in the art that provides a signal (directly or indirectly).

[0143] Known in the art Fluorescent molecules "Includes fluorescein isothiocyanate (FITC), phycoerythrin (PE), fluorophores for blue lasers (e.g., PerCP, PE-Cy7, PE-Cy5, FL3 and APC or Cy5, FL4), fluorophores for red, violet or ultraviolet lasers (e.g., Pacific Blue, Pacific Orange).

[0144] “ radioactive molecules "Includes but is not limited to radioactive atoms used in scintigraphy, such as: I 123 , I 124 、In 111 、Re 186 、Re 188 、Tc 99 The bispecific antigen-binding proteins of the present invention may further comprise a spin label 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.

[0145] Such diagnostic agents may be directly coupled (ie, physically linked) to the bispecific antigen binding protein or may be indirectly linked.

[0146] In this article, therapeutic agents " refers to drugs that have therapeutic effects. In one embodiment, such therapeutic agents can be growth inhibitory agents, such as cytotoxic agents or radioactive isotopes.

[0147] "Can be used indiscriminately" Growth inhibitors "or" Antiproliferative agents ” refers to a compound or composition that inhibits the growth of cells (especially tumor cells) in vitro or in vivo.

[0148] As used herein, the term “ Cytotoxic agents ” refers to substances that inhibit or interfere with cell function and / or cause cell destruction. The term “ Cytotoxic agents" is intended to include chemotherapeutic agents, enzymes, antibiotics, and toxins (e.g., small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof), as well as various anti-tumor or anti-cancer agents disclosed below. In some embodiments, the cytotoxic agent is paclitaxel, vinca, a taxane, maytansinoids or maytansinoid analogs (e.g., DM1 or DM4), a small molecule drug, tometomycin or a pyrrolobenzodiazepine derivative, a cryptophycin derivative, a leptomycin derivative, an auristatin or a dolastatin analog, a prodrug, a topoisomerase II inhibitor, a DNA alkylating agent, an anti-tubulin agent, CC-1065, or a CC-1065 analog.

[0149] the term" radioactive isotopes "Intended to include radioisotopes suitable for the treatment of cancer, such as At 211 、Bi 212 、Er 169 , I 131 , I 125 、Y 90 、In 111 、P 32 、Re 186 、Re 188 、Sm 153 、Sr 89 and radioisotopes of Lu. Such radioisotopes typically emit primarily beta radiation. In one embodiment, the radioisotope is an alpha-emitting isotope, more specifically thorium-227, which emits alpha radiation.

[0150] This article's PK modification part" refers to a moiety that modifies the pharmacokinetics (PK) of the bispecific antigen binding protein of the invention. Thus, the moiety specifically modifies the in vivo half-life and distribution of the bispecific antigen binding protein of the invention. In a preferred embodiment, the PK modifying moiety increases the half-life of the bispecific antigen binding protein. Examples of PK modifying moieties include, but are not limited to, PEG (Dozier et al., (2015) Int J Mol Sci. Oct 28; 16(10):25831-64 and Jevsevar et al., (2010) Biotechnol J. Jan; 5(1):113-28), PASylation (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), Fc portion of antibodies and / or unstructured polypeptides (Schellenberger et al., (2009) Nat Biotechnol. Dec; 27(12):1186-90).

[0151] Bispecific antigen binding proteins

[0152] The inventors of the present invention humanized the mouse monoclonal anti-CD3 antibody UCHT1 as described in Example 1 to obtain the humanized monoclonal antibody UCHT1(V17). Compared to the known humanized monoclonal antibody UCHT1(V9), the obtained humanized monoclonal antibody UCHT1(V17) has increased stability and / or solubility.

[0153] The inventors then used proof-of-principle experiments to demonstrate in Example 2 that when the variable domains of a T cell-recruiting antibody with moderate affinity for its target (e.g., BMA31 targeting TCRαβ) are used in combination with mature TCR variable domains, the safety window of the resulting bispecific antigen-binding protein is much wider than that of an antigen-binding protein using a high-affinity anti-CD3 antibody (e.g., UCHT1 (V17)) and the same TCR variable domains.

[0154] These new antigen-binding proteins, especially Antigen binding proteins in molecular form show high cytotoxicity to tumor cells. The molecular form of the new antigen binding protein has a half-maximal effective concentration (EC) for NCI-H1755, Hs695T cells and U2OS 50) is 1 pM to 100 pM, more particularly, 1 pM to 20 pM for cells, and therefore, compared with the EC of normal tissue cells 50 In contrast, the antigen binding proteins of the present invention have an effect on the EC 50 It is reduced by more than 1000 times (e.g. tumor cell line Hs695T compared with primary cells), showing higher safety.

[0155] Therefore, the inventors generated intermediate-affinity variants (V20, V21, V23, V17opt, V20opt, V21opt, and V23opt) of the high-affinity anti-CD3 antibody UCHT1 (V17), thereby creating a variety of T cell-recruiting variable domains suitable for use in combination with TCR variable domains to obtain bispecific antibodies with a favorable safety window.

[0156] Therefore, the present invention relates to a bispecific antigen-binding protein comprising at least two antigen-binding sites (A and B), wherein the antigen-binding site A binds to CD3, preferably to the CD3ε / δ complex, and wherein the antigen-binding site B binds to the target antigen (TA) peptide / MHC complex, preferably to the TAA antigen peptide / MHC complex, wherein the antigen-binding site A comprises a heavy chain variable domain (V H ) and light chain variable domain (V L ),and

[0157] a) wherein the VL comprises three complementarity determining regions (CDRs) CDRL1, CDRL2 and CDRL3, wherein

[0158] -CDRL1 comprises or consists of the amino acid sequence "RASQDIRNYLN" of SEQ ID NO: 1,

[0159] -CDRL2 comprises or consists of the amino acid sequence "YTSRLHS" of SEQ ID NO: 2, and

[0160] -CDRL3 comprises or consists of the amino acid sequence "QQGQTLPWT" of SEQ ID NO: 3, and

[0161] b) wherein the VH comprises three complementarity determining regions (CDRs) CDRH1, CDRH2 and CDRH3, wherein

[0162] -CDRH1 comprises or consists of the amino acid sequence "X1YTMN" of SEQ ID NO: 4, wherein X1 is G or E, preferably G,

[0163] -CDRH2 comprises or consists of the amino acid sequence of "LINPX2X3GVX4TYAQKX5QX6" SEQ ID NO: 5, wherein X2 is any amino acid, preferably Q, Y or E, more preferably Q or Y (e.g., Q), X3 is any amino acid, preferably R, K or E, more preferably R or K (e.g., 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, and

[0164] -CDRH3 comprises or consists of the amino acid sequence "SGYYGX7SWYFDV" of SEQ ID NO: 6, wherein X7 is any amino acid, preferably E or D, more preferably D,

[0165] 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.

[0166] In one embodiment, the VH comprises a CDRH1 according to SEQ ID NO:4, a CDRH2 according to SEQ ID NO:5 and a CDRH3 according to 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.

[0167] In a particular embodiment, the present invention relates to a bispecific antigen-binding protein comprising at least two antigen-binding sites (A and B), wherein the antigen-binding site A binds to CD3, preferably to the CD3ε / δ complex, wherein the antigen-binding site B binds to the target antigen (TA) peptide / MHC complex, preferably to the TAA antigen peptide / MHC complex, wherein the antigen-binding site A comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), and

[0168] a) wherein the VL comprises three complementarity determining regions (CDRs) CDRL1, CDRL2 and CDRL3, wherein

[0169] -CDRL1 comprises or consists of the amino acid sequence "RASQDIRNYLN" of SEQ ID NO: 1,

[0170] -CDRL2 comprises or consists of the amino acid sequence "YTSRLHS" of SEQ ID NO: 2, and

[0171] -CDRL3 comprises or consists of the amino acid sequence "QQGQTLPWT" of SEQ ID NO: 3, and

[0172] b) wherein the VH comprises three complementarity determining regions (CDRs) CDRH1, CDRH2 and CDRH3, wherein

[0173] - CDRH1 comprises 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 an amino acid sequence that optionally differs from SEQ ID NO: 133 or 134 (the difference being at least one amino acid substituent, preferably one or two amino acid substituents, or only one amino acid substituent), or consists thereof, wherein the amino acid sequence that preferably differs from SEQ ID NO: 133 or 134 comprises 31G or 31E,

[0174] - CDRH2 comprises or consists of an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 135 to 142, or an amino acid sequence that optionally differs from SEQ ID NOs: 133 or 134 by at least one amino acid substituent, preferably one, two, three or four amino acid substituents, preferably one or two amino acid substituents, or only one amino acid substituent, wherein preferably the amino acid sequence that differs from SEQ ID NOs: 133 or 134 comprises amino acid 61A and optionally at least one of amino acids 54Q, 54E or 54Y, 55R or 55E, 58S or 58T, 64F or 64V, 65Q, 66D or 66G, preferably 66D, and

[0175] -CDRH3 comprises or consists of the amino acid sequence of SEQ ID NO: 8 or 144, or optionally an amino acid sequence that differs from SEQ ID NO: 8 or 144 (the difference being at least one amino acid substituent, preferably one, two, three or four amino acid substituents, preferably one or two amino acid substituents, or only one amino acid substituent), wherein the amino acid sequence that differs from SEQ ID NO: 8 or 134 preferably comprises amino acid 104E

[0176] The present invention further relates 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, such variants may comprise at least one, for example four, three, two or one, preferably one, two or three amino acid substituents, wherein the preferred number of amino acid substituents preferably depends on the length of each CDR.

[0177] In some embodiments, CDRL1 comprises or consists of an amino acid sequence that differs from the CDRL1 amino acid sequence disclosed herein in that the difference is: at least one amino acid substituent, preferably one, two, three or four amino acid substituents, preferably one, two or three amino acid substituents, preferably one or two amino acid substituents (e.g., one amino acid substituent), wherein the amino acid substituents are preferably at positions 27, 28, 30 and 31.

[0178] In some embodiments, CDRL2 comprises or consists of an amino acid sequence that differs from the CDRL2 amino acid sequence disclosed herein, wherein the difference is: at least one amino acid substituent, preferably one, two or three amino acid substituents, preferably one or two amino acid substituents (e.g., one amino acid substituent), wherein the amino acid substituents are preferably at positions 51, 52 and 53.

[0179] In some embodiments, CDRL3 comprises or consists of an amino acid sequence that differs from the CDRL3 amino acid sequence disclosed herein, wherein the difference is that: there is at least one amino acid substituent, preferably one, two, three or four amino acid substituents, preferably one or two amino acid substituents (e.g., one amino acid substituent), wherein the amino acid substituent is preferably at any of amino acid positions 93, 94 and 95.

[0180] In a preferred embodiment, the mutations may occur in the CDRs of the heavy chain variable domain of antigen binding site A.

[0181] Thus, in some embodiments, CDRH1 comprises or consists of an amino acid sequence that is different from the CDRH1 amino acid sequence disclosed herein, wherein the difference is that there is at least one amino acid substituent, preferably one or two or three amino acid substituents, preferably one amino acid substituent, wherein the amino acid substituent is preferably at any of amino acid positions 31 to 35.

[0182] In some embodiments, the CDRH2 comprises or consists of an amino acid sequence that differs from the CDRH2 amino acid sequence disclosed herein in that it differs by at least one amino acid substituent, preferably one, two, three or four amino acid substituents, preferably one, two or three amino acid substituents, preferably one or two amino acid substituents (e.g., one amino acid substituent), wherein the amino acid substituent is preferably at amino acid positions 54, 55, and any of 57 to 59.

[0183] In some embodiments, the CDRH3 comprises or consists of an amino acid sequence that differs from the CDRH3 amino acid sequence disclosed herein, wherein the difference is at least one amino acid substituent, preferably one, two, three or four amino acid substituents, preferably one or two amino acid substituents (e.g., one amino acid substituent), wherein the amino acid substituent is preferably at any of amino acid positions 105, 107 and 110.

[0184] In a preferred embodiment, the light chain variable domain and the heavy chain variable domain further comprise light chain and heavy chain framework regions.

[0185] In one embodiment, the light chain variable domain further comprises one or more framework regions, preferably FR1-L, FR2-L, FR3-L and FR4-L selected from the group consisting of FR1-L, FR2-L, FR3-L and FR4-L, wherein

[0186] -FR1-L comprises or consists of the amino acid sequence "DIQMTQSPSSLSASVGDRVTITC" of SEQ ID NO: 11 or an amino acid sequence having at least 85% identity thereto, wherein the amino acid sequence having at least 85% identity thereto preferably comprises amino acids 6Q and / or 23C,

[0187] -FR2-L comprises the amino acid sequence of "WYQQKPGKAPKLLIY" of SEQ ID NO: 12 or "WYQQKPGKAVKLLIY" of SEQ ID NO: 13 (preferably SEQ ID NO: 12), or an amino acid sequence having at least 85% identity with SEQ ID NO: 12 or 13, or consists thereof, wherein the amino acid sequence having at least 85% identity with SEQ ID NO: 12 or 13 preferably comprises amino acids 35W, 36Y, 38Q, 44P, 46L and / or 49Y,

[0188] -FR3-L comprises or consists of the amino acid sequence "GVPSRFSGSGSGTDYTLTISSLQPEDIATYFC" of SEQ ID NO: 14, or an amino acid sequence having at least 85% identity thereto, wherein the amino acid sequence having at least 85% identity thereto preferably comprises amino acids 57G, 59P, 62F, 64G, 66G, 71Y, 82D, 86Y, 87F, 88C,

[0189] - FR4-L comprises or consists of the amino acid sequence "FGQGTKVEIKR" of SEQ ID NO: 15 or an amino acid sequence having at least 85% identity to SEQ ID NO: 15, wherein the amino acid sequence having at least 85% identity to SEQ ID NO: 15 preferably comprises amino acids 98F and / or 101G,

[0190] wherein VH further comprises one or more framework regions selected from the group consisting of FR1-H, FR2-H, FR3-H and FR4-H, and wherein

[0191] -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 amino acids 6Q, 14P, 22C, 24A, 26G, 27Y, 28S, 29F and / or 30T and optionally comprises at least one of the amino acid substitutions Q5V, P9A, L11V, V12K, M18V and / or I20V,

[0192] -FR2-H comprises or consists of the amino acid sequence "WVRQAPGQGLEWMG" of SEQ ID NO: 17, or an amino acid sequence having at least 85% identity thereto, wherein the amino acid sequence having at least 85% identity thereto preferably comprises 36W, 37V, 39Q, 45L, 46E and / or 47W and optionally comprises at least one of the amino acid substitutions K38R, S40A, H41P, K43Q, N44G,

[0193] - FR3-H comprises or consists of the amino acid sequence "RVTLTVDKSTSTAYMELSSLRSEDTAVYYCAR" of SEQ ID NO: 18, or an amino acid sequence having at least 85% identity thereto, wherein the amino acid sequence having at least 85% identity thereto preferably comprises 70L, 72V, 79A, 90D, 94Y, 95Y, 96C, 97A and / or 98R and optionally comprises at least one of the amino acid substitutions K67R, A68V, K74T, S76T, L84S, T87R and / or S91T, and

[0194] -FR4-H comprises or consists of the "WGQGTLVTVSS" amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 85% identity to SEQ ID NO: 19, wherein the amino acid sequence having at least 85% identity to SEQ ID NO: 19 preferably comprises 112W, 113G, 115G and optionally comprises at least one of the amino acid substitutions A114Q and / or T117L.

[0195] 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 or FR4-L, preferably FR1-L, FR2-L, FR3-L and FR4-L, wherein

[0196] -FR1-L comprises or consists of the amino acid sequence "DIQMTQSPSSLSASVGDRVTITC" of SEQ ID NO: 11 or an amino acid sequence having at least 85% identity thereto, wherein the amino acid sequence having at least 85% identity thereto preferably comprises amino acids 6Q and / or 23C,

[0197] -FR2-L comprises or consists of the amino acid sequence "WYQQKPGKAPKLLIY" of SEQ ID NO: 12 or "WYQQKPGKAVKLLIY" of SEQ ID NO: 13, preferably SEQ ID NO: 12 or an amino acid sequence having at least 85% identity with SEQ ID NO: 12 or 13, wherein the amino acid sequence having at least 85% identity with SEQ ID NO: 12 or 13 preferably comprises amino acids 35W, 36Y, 38Q, 44P, 46L and / or 49Y,

[0198] -FR3-L comprises or consists of the amino acid sequence "GVPSRFSGSGSGTDYTLTISSLQPEDIATYFC" of SEQ ID NO: 14, or an amino acid sequence having at least 85% identity thereto, wherein the amino acid sequence having at least 85% identity thereto preferably comprises amino acids 57G, 59P, 62F, 64G, 66G, 71Y, 82D, 86Y, 87F, 88C,

[0199] - FR4-L comprises or consists of the amino acid sequence "FGQGTKVEIK" of SEQ ID NO: 285 or an amino acid sequence having at least 85% identity to SEQ ID NO: 15, wherein the amino acid sequence having at least 85% identity to SEQ ID NO: 15 preferably comprises amino acids 98F and / or 101G,

[0200] wherein VH further comprises one or more framework regions selected from the group consisting of FR1-H, FR2-H, FR3-H and FR4-H, and wherein

[0201] -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 amino acids 6Q, 14P, 22C, 24A, 26G, 27Y, 28S, 29F and / or 30T and optionally comprises at least one of the amino acid substitutions Q5V, P9A, L11V, V12K, M18V and / or I20V,

[0202] -FR2-H comprises or consists of the amino acid sequence "WVRQAPGQGLEWMG" of SEQ ID NO: 17, or an amino acid sequence having at least 85% identity thereto, wherein the amino acid sequence having at least 85% identity thereto preferably comprises 36W, 37V, 39Q, 45L, 46E and / or 47W and optionally comprises at least one of the amino acid substitutions K38R, S40A, H41P, K43Q, N44G,

[0203] - FR3-H comprises or consists of the amino acid sequence "RVTLTVDKSTSTAYMELSSLRSEDTAVYYCAR" of SEQ ID NO: 18, or an amino acid sequence having at least 85% identity thereto, wherein the amino acid sequence having at least 85% identity thereto preferably comprises 70L, 72V, 79A, 90D, 94Y, 95Y, 96C, 97A and / or 98R and optionally comprises at least one of the amino acid substitutions K67R, A68V, K74T, S76T, L84S, T87R and / or S91T, and

[0204] -FR4-H comprises or consists of the "WGQGTLVTVSS" amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 85% identity to SEQ ID NO: 19, wherein the amino acid sequence having at least 85% identity to SEQ ID NO: 19 preferably comprises 112W, 113G, 115G and optionally comprises at least one of the amino acid substitutions A114Q and / or T117L.

[0205] 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 or FR4-L, preferably FR1-L, FR2-L, FR3-L and FR4-L, wherein

[0206] -FR1-L comprises or consists of the amino acid sequence "DIQMTQSPSSLSASVGDRVTITC" of SEQ ID NO: 11 or an amino acid sequence having at least 90% identity thereto, wherein the amino acid sequence having at least 90% identity thereto preferably comprises amino acids 6Q and / or 23C,

[0207] -FR2-L comprises or consists of the amino acid sequence "WYQQKPGKAPKLLIY" of SEQ ID NO: 12 or "WYQQKPGKAVKLLIY" of SEQ ID NO: 13, preferably 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 amino acids 35W, 36Y, 38Q, 44P, 46L and / or 49Y,

[0208] - 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 amino acids 57G, 59P, 62F, 64G, 66G, 71Y, 82D, 86Y, 87F, 88C,

[0209] - 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,

[0210] wherein VH further comprises one or more framework regions selected from the group consisting of FR1-H, FR2-H, FR3-H and FR4-H, and wherein

[0211] -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 amino acids 6Q, 14P, 22C, 24A, 26G, 27Y, 28S, 29F and / or 30T and optionally comprises at least one of the amino acid substitutions Q5V, P9A, L11V, V12K, M18V and / or I20V,

[0212] -FR2-H comprises or consists of the amino acid sequence "WVRQAPGQGLEWMG" of SEQ ID NO: 17, or an amino acid sequence having at least 90% identity thereto, wherein the amino acid sequence having at least 90% identity thereto preferably comprises 36W, 37V, 39Q, 45L, 46E and / or 47W and optionally comprises at least one of the amino acid substitutions K38R, S40A, H41P, K43Q, N44G,

[0213] - 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 comprises at least one of the amino acid substitutions K67R, A68V, K74T, S76T, L84S, T87R and / or S91T, and

[0214] -FR4-H comprises or consists of the "WGQGTLVTVSS" amino acid sequence 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 comprises at least one of the amino acid substitutions A114Q and / or T117L.

[0215] 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 or FR4-L, preferably FR1-L, FR2-L, FR3-L and FR4-L, wherein

[0216] -FR1-L comprises or consists of the amino acid sequence "DIQMTQSPSSLSASVGDRVTITC" of SEQ ID NO: 11 or an amino acid sequence having at least 95% identity thereto, wherein the amino acid sequence having at least 95% identity thereto preferably comprises amino acids 6Q and / or 23C,

[0217] -FR2-L comprises or consists of the amino acid sequence "WYQQKPGKAPKLLIY" of SEQ ID NO: 12 or "WYQQKPGKAVKLLIY" of SEQ ID NO: 13, preferably 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 amino acids 35W, 36Y, 38Q, 44P, 46L and / or 49Y,

[0218] - 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 amino acids 57G, 59P, 62F, 64G, 66G, 71Y, 82D, 86Y, 87F, 88C,

[0219] - FR4-L comprises or consists of the amino acid sequence "FGQGTKVEIK" of SEQ ID NO: 285 or an amino acid sequence having at least 95% identity to SEQ ID NO: 15, wherein the amino acid sequence having at least 95% identity to SEQ ID NO: 15 preferably comprises amino acids 98F and / or 101G,

[0220] Among them, V H further comprising one or more framework regions selected from the group consisting of FR1-H, FR2-H, FR3-H, and FR4-H, and wherein

[0221] -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 amino acids 6Q, 14P, 22C, 24A, 26G, 27Y, 28S, 29F and / or 30T and optionally comprises at least one of the amino acid substitutions Q5V, P9A, L11V, V12K, M18V and / or I20V,

[0222] -FR2-H comprises or consists of the amino acid sequence "WVRQAPGQGLEWMG" of SEQ ID NO: 17, or an amino acid sequence having at least 95% identity thereto, wherein the amino acid sequence having at least 95% identity thereto preferably comprises 36W, 37V, 39Q, 45L, 46E and / or 47W and optionally comprises at least one of the amino acid substitutions K38R, S40A, H41P, K43Q, N44G,

[0223] - 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 comprises at least one of the amino acid substitutions K67R, A68V, K74T, S76T, L84S, T87R and / or S91T, and

[0224] -FR4-H comprises or consists of the "WGQGTLVTVSS" amino acid sequence 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 comprises at least one of the amino acid substitutions A114Q and / or T117L.

[0225] The inventors determined that amino acids 35W, 36Y, 46L, and 49Y of FR2-L and amino acids 64G, 71Y of FR3-L are located in the Vernier region and are preferably not substituted.

[0226] The inventors determined that 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 are located in the Vernier region and are preferably not substituted.

[0227] Variants of the antigen binding proteins described herein are contemplated and specifically referred to using the term " At least 85% identical to the 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 suitably differ from the reference sequences SEQ ID NO: 11 to SEQ ID NO: 19 by at least one amino acid substitution, in particular at least one conservative amino acid substitution and / or canonical residue substitution. 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 SEQ ID NO: 11 to SEQ ID NO: 19 only by conservative amino acid substitutions.

[0228] The amino acid sequences and corresponding DNA sequences of the bispecific antigen-binding proteins of the present invention can be modified and altered individually and still produce functional antigen-binding proteins or polypeptides with the desired properties. Modifications can be made in the heavy and light chain variable domains of antigen-binding site A or in the α and β or γ and δ variable domains of antigen-binding site B, in particular in the framework regions or in each or all CDRs comprised by the heavy and light chain variable domains of antigen-binding site A or in the framework regions or in each or all CDRs comprised by the α and β or γ and δ variable domains.

[0229] The bispecific antigen binding protein may comprise a light chain variable region comprising FR2-L, 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 (located in the human germline sequence Vk1-018) has the advantage of deimmunizing the humanized variable domain because a proline is commonly found at this position in the 10 most similar human germlines.

[0230] This article's Deimmunization "Reducing immunogenicity" refers to reducing immunogenicity, i.e., the ability to induce an immune response in a subject. This is accomplished by replacing amino acids with those most commonly found in the human germline, thereby rendering them incapable of being recognized as foreign by the immune system.

[0231] Thus, in one embodiment, in the context of a bispecific antigen binding protein of the invention, antigen binding site A comprises a heavy chain variable domain (V H ) and light chain variable domain (V L ),

[0232] wherein V L comprising or consisting of the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 145, wherein the amino acid sequence having at least 85% identity 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, and

[0233] wherein V H comprising or consisting of an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NO: 149 to SEQ ID NO: 160, or an amino acid sequence having at least 85% identity with an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NO: 149 to SEQ ID NO: 160, and

[0234] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0235] Among them, preferably, the amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 150 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 139, and CDRH3 of SEQ ID NO: 8, and

[0236] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and CDRH3 of SEQ ID NO: 8, and

[0237] Among them, preferably, the amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 152 comprises the amino acid sequence of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 140, and CDRH3 of SEQ ID NO: 8, and

[0238] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0239] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0240] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0241] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0242] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0243] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0244] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0245] Among them, preferably, the amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 160 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138, and CDRH3 of SEQ ID NO: 144.

[0246] Thus, in one embodiment, in the context of a bispecific antigen binding protein of the invention, antigen binding site A comprises a heavy chain variable domain (V H ) and light chain variable domain (VL),

[0247] wherein V L comprising or consisting of the amino acid sequence of SEQ ID NO: 286 or an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 286, wherein the amino acid sequence having at least 85% identity 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, and

[0248] wherein V H comprising or consisting of an amino acid sequence selected from the group of amino acid sequences selected from the group of amino acid sequences of SEQ ID NO: 149 to SEQ ID NO: 160, or an amino acid sequence having at least 85% identity with an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NO: 149 to SEQ ID NO: 160, and wherein, preferably, the amino acid sequence having at least 85% identity with 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, and

[0249] Among them, preferably, the amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 150 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 139, and CDRH3 of SEQ ID NO: 8, and

[0250] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and CDRH3 of SEQ ID NO: 8, and

[0251] Among them, preferably, the amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 152 comprises the amino acid sequence of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 140, and CDRH3 of SEQ ID NO: 8, and

[0252] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0253] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0254] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0255] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0256] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0257] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0258] Among them, preferably, the amino acid sequence having at least 85% identity with 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, and

[0259] Among them, preferably, the amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 160 comprises the amino acid sequences of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138, and CDRH3 of SEQ ID NO: 144.

[0260] In a preferred embodiment, the VL 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 the 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, and

[0261] wherein V H comprising or consisting 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 the amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 156 preferably 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, or

[0262] wherein V H comprising or consisting 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 the amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 149 preferably comprises the amino acid sequence of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 138 and CDRH3 of SEQ ID NO: 8, or

[0263] wherein V H A method comprising or consisting 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 the amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 151 preferably comprises the amino acid sequence of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138 and CDRH3 of SEQ ID NO: 8.

[0264] In a preferred embodiment, the V Lcomprising or consisting of the amino acid sequence of SEQ ID NO: 286 or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 286, wherein the amino acid sequence having at least 90% identity 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, and

[0265] wherein V H comprising or consisting 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 the amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 156 preferably 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, or

[0266] wherein V H comprising or consisting 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 the amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 149 preferably comprises the amino acid sequence of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 138 and CDRH3 of SEQ ID NO: 8, or

[0267] wherein V H A method comprising or consisting of the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 151, wherein the amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 151 preferably comprises the amino acid sequence of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138 and CDRH3 of SEQ ID NO: 8.

[0268] In a preferred embodiment, the V Lcomprising or consisting of the amino acid sequence of SEQ ID NO: 286 or an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 286, wherein the amino acid sequence having at least 85% identity 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, and

[0269] wherein V H comprising or consisting 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 the amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 156 preferably 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, or

[0270] wherein V H comprising or consisting 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 the amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 149 preferably comprises the amino acid sequence of CDRH1 of SEQ ID NO: 133, CDRH2 of SEQ ID NO: 138 and CDRH3 of SEQ ID NO: 8, or

[0271] wherein V H A method comprising or consisting 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 the amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 151 preferably comprises the amino acid sequence of CDRH1 of SEQ ID NO: 134, CDRH2 of SEQ ID NO: 138 and CDRH3 of SEQ ID NO: 8.

[0272] In some embodiments, the antigen binding site B of the bispecific antigen-binding protein of the present invention comprises or consists of an antibody or fragment thereof, or an α chain variable domain (vα) and a β chain variable domain (vβ) or a γ chain variable domain (vγ) or a δ chain variable domain (vδ). Antibodies and fragments thereof are defined in the "Definitions" section above.

[0273] In some embodiments, the antigen binding site B of the bispecific antigen-binding protein of the present invention comprises an α chain variable domain (vα) and a β chain variable domain (vβ) or a γ chain variable domain (vγ) or a δ chain variable domain (vδ), preferably a vα and vβ domain. Detailed descriptions of the α chain variable domain (vα) and the β chain variable domain (vβ) or the γ chain variable domain (vγ) or the δ chain variable domain (vδ) that may be used in the context of the present invention and that bind to a specific TA are described in WO2018172533, WO2018033291, WO2017158103, WO2018104438, WO2018104478, WO2019002444, and WO2017158116.

[0274] Therefore, in one embodiment, the vα and vβ or vγ and vδ comprise or consist of the amino acid sequences disclosed in WO2018172533, WO2018033291, WO2017158103, WO2018104438, WO2018104478, WO2019002444, WO2017158116, and the vα and vβ or vγ and vδ cited in the prior art are combined with the TA peptide (particularly the TAA peptide) disclosed in the same patent application.

[0275] In one embodiment, the bispecific antigen binding protein of the invention comprises vα and vβ domains or vγ and vδ domains, wherein

[0276] i) vα or vγ comprises or consists of an amino acid sequence selected from the group consisting of:

[0277] "EDVEQSLFLSVREGDSVVINCTYTDSSSTYLYWYKQEPGKGLQLLTYIYSSQDSKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP" SEQ ID NO: 20,

[0278] "EDVEQSLFLSVREGDSVVINCTYTDSSSTYLYWYKQEPGKGLQLLTYIYSSQDQKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP" SEQ ID NO: 21,

[0279] “EDVEQSLFLSVREGDSVVINCTYTESSSTYLYWYKQEPGKGLQLLTYIYSSQDQKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP” SEQ ID NO: 22, or an amino acid sequence having at least 85% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21 and 22, and wherein the amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 20 preferably comprises the amino acid sequence of CDRa1 of SEQ ID NO: 23, CDRa2 of SEQ ID NO: 24 and CDRa3 of SEQ ID NO: 25, wherein the amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 21 preferably comprises the amino acid sequence of CDRa1 of SEQ ID NO: 23, CDRa2 of SEQ ID NO: 26 and CDRa3 of SEQ ID NO: 25, wherein the amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: The amino acid sequence of SEQ ID NO:22 having at least 85% identity preferably comprises 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 acids of the first variable domain preferably comprise amino acids 19V and / or 48K, and

[0280] vβ or vδ contains

[0281] "DAGVIQSPRHEVTEMGQEVTLRCKPIPGHDYLFWYRQTMMRGLELLFYFCYGTPCDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRADTGELFFGEGSRLTVL" the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 85% identity with the amino acid sequence consisting of SEQ ID NO: 30, or consisting thereof, wherein preferably the amino acid sequence having at least 85% identity with 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, and CDRb3 of SEQ ID NO: 35, respectively, and optionally comprises amino acids 54F and / or 66C, or

[0282] (ii) vα or vγ comprises or consists of the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 48, wherein preferably the amino acid sequence that 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, and

[0283] vβ or vδ comprises or consists of the amino acid sequence of SEQ ID NO:44, or an amino acid sequence that is at least 85% identical to SEQ ID NO:44, wherein preferably the amino acid sequence that 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.

[0284] 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 IMGT numbering. In a preferred embodiment, the amino acid at position 44 is substituted with another suitable amino acid to improve pairing. In a specific embodiment, preferably an embodiment in which the antigen binding protein is a TCR, the amino acid substitution improves, for example, the pairing of chains (i.e., the pairing of α and β chains or the pairing of γ and δ). In a preferred embodiment, one or both amino acids present at position 44 of the first variable domain (v144) and at position 44 of the second variable domain (v244) are substituted with a residue selected from v144D / v244R, v1 44R / v244D, v1 The amino acid pair v144 / v244 is composed of the amino acid pair group consisting of v144E / v244K, v144K / v244E, v144D / v244K, v144K / v244D, v144R / v244E; v144E / v244R, v144L / v244W, v144W / v244L, v144V / v244W, and v144W / v244V.

[0285] Thus, in another embodiment, the antigen binding protein may further comprise one of the preferred substitution pairs (v144 / v244) 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; v 1W44E / v2Q44K;v1W44K / v2Q44E;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;v1K44E / v2Q44R;v1K44R / v2Q44E;v1K44L / v2Q44W;v1K44W / v2Q44L;v1K44V / v2Q44W; and v1K44W / v2Q44V;v1E44D / v2Q44R;v1E44R / v2Q44 D; v1E44 / v2Q44K; v1E44K / v2Q44E; v1E44D / v2Q44K; v1E44K / v2Q44D; v1E44 / v2Q44R; v1E44R / v2Q44E; v1E44L / v2Q44W; v1E44W / v2Q44L; v1E44V / v2Q44W ; and v1E44W / v2Q44V; 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 / v2R44 K; v1W44K / v2R44D; v1W44E / v2R44; v1W44R / v2R44E; v1W44L / v2R44W; v1W44 / v2R44L; v1W44V / v2R44W; and v1W44 / v2R44V; v1H44D / v2R44; v1H44R / v2R44D; v1H44E / v2R44K; 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;v1 K44L / v2R44W;v1K44W / v2R44L;v1K44V / v2R44W; and v1K44W / v2R44V;v1E44D / v2R44;v1E44R / v2R44D;v1E44 / v2R44K;v1E44K / v2R44E;v1E44D / v2R44K;v 1E44K / v2R44D;v1E44R / v2R44E;v1E44L / v2R44W;v1E44W / v2R44L;v1E44V / v2R44W; and v1E44W / v2R44V;v1Q44D / v2K44R;v1Q44R / v2K44D;v1Q44E / v244K; v1Q44K / 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; v1H44E / v244K; v1H44K / v2K44E; v1H44D / v244K; v1H44K / v2K44D; v1H44E / v2K4 4R;v1H44R / v2K44E;v1H44L / v2K44W;v1H44W / v2K44L;v1H44V / v2K44W; and v1H44W / v2K44V;v1K44D / v2K44R;v1K44R / v2K44D;v1K44E / v244K;v1K44 / v2K44E;v1K44D / v244K;v1K44 / v2K44D;v1K44E / v2K44R;v1K44R / v2K44E;v1K44L / v2K44W;v1K44W / v2K44L;v1K44V / v2K44W; and v1K44W / v2K44V;v1E44D / v2K44R;v1E44R / v2K44D;v1 E44 / v244K;v1E44K / v2K44E;v1E44D / v244K;v1E44K / v2K44D;v1E44 / v2K44R;v1E44R / v2K44E;v1E44L / v2K44W;v1E44W / v2K44L;v1E44V / v2K44W; and v1E44W / v2Q44V. ;

[0286] In the above content, for example, "v1Q44R / v2Q44D" means that in the first variable domain, Q44 is substituted by R, and in the second variable domain, Q44 is substituted by D. Other substitutions and descriptions can be found in U.S. Patent Application No. 2018-0162922.

[0287] 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.

[0288] Definitions of bispecific antibodies and TCRs and their respective fragments are given above in the "Definitions" section.

[0289] In one embodiment, the antigen binding protein is of human origin, which is understood to be produced from a human antigenic locus and therefore comprises human sequences, particularly human TCR or antibody sequences.

[0290] In one embodiment, the light chain variable domain and the heavy chain variable domain are linked together and / or the VA and VB or VA and v domains are linked together, preferably by a covalent bond.

[0291] In one embodiment, the bispecific antigen binding protein comprises at least two polypeptides.

[0292] 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.

[0293] In the same embodiment, the α chain variable domain (vα) and the β chain variable domain (vβ) or the γ chain variable domain (vγ) or the δ chain variable domain (vδ), preferably the να and νβ domains, are located on the same or different polypeptides.

[0294] In a preferred embodiment, the antigen binding protein is a soluble protein.

[0295] "Covalent bond," "linker sequence," or "polypeptide linker" are defined above under "Linker."

[0296] In one embodiment, the bispecific antigen binding protein of the invention further comprises one or more of the following:

[0297] (i) diagnostic agents;

[0298] (ii) a therapeutic agent; or

[0299] (iii) PK modification moiety.

[0300] "Diagnostic agent," "therapeutic agent," and "PK modifying moiety" are defined above in the Definitions section.

[0301] In some embodiments, the antigen binding protein of the present invention is covalently attached to at least one growth inhibitory agent directly or via a cleavable or non-cleavable linker.Antigen binding proteins attached to such at least one growth inhibitory agent may also be referred to as conjugates.

[0302] The preparation of such conjugates (e.g., immunoconjugates) is described in application 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 can be transferred by a person skilled in the art to the preparation of the antigen-binding proteins of the present invention, to which at least one growth inhibitor of this type is attached.

[0303] In the context of at least one growth inhibitor attached Linker ” refers to a chemical moiety comprising a covalent bond or chain of atoms that covalently attaches the polypeptide to the drug moiety.

[0304] Conjugates can be prepared by in vitro methods. In order to link the drug or prodrug to the antibody, a linking group is used. Suitable linking groups are well known in the art and include disulfide bonds, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. The conjugation of the antigen-binding protein of the present invention to the cytotoxic agent or growth inhibitory agent can be carried out using a variety of bifunctional protein coupling agents, including but not limited to N-succinimidyl pyridinyl dithiobutyrate (SPDB), butyric acid 4-[(5-nitro-2-pyridinyl) dithio]-2,5-dioxo-1-pyrrolidinyl ester (nitro SPDB), 4-(pyridin-2-yldisulfanyl)-2-sulfobutyric acid (sulfo-SPDB), N-succinimidyl (2-pyridinyldithio) propionate (SPDP), succinimidyl (N-maleimide) Examples of the immunotoxin include iminomethyl)cyclohexane-1-carboxylate (SMCC), iminothiane (IT), bifunctional derivatives of iminoesters (e.g., dimethyl hexamethylenediamine HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazido compounds (e.g., bis-(p-azidobenzyl)-hexanediamine), bisdiazonium derivatives (e.g., bis-(p-diazoniumbenzyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described by Vitetta et al. (1987). Carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies (WO 94 / 11026).

[0305] The linker can be a "cleavable linker" that facilitates release of the cytotoxic agent or growth inhibitor in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, an esterase-labile linker, a photolabile linker, or a disulfide-containing linker can be used (see, e.g., U.S. Patent No. 5,208,020). The linker can also be a "non-cleavable linker" (e.g., an SMCC linker), which may improve tolerability in some cases.

[0306] Alternatively, fusion proteins comprising a bispecific antigen-binding protein of the invention and a cytotoxic or growth inhibitory polypeptide can be prepared by recombinant techniques or peptide synthesis. The length of DNA may include corresponding regions encoding the two parts of the conjugate, the two parts being adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

[0307] The antigen binding proteins of the present invention can also be used in enzyme-mediated prodrug therapy by conjugating the polypeptide to a prodrug-activating enzyme that converts a prodrug (e.g., a peptide-based chemotherapeutic agent, see WO 81 / 01145 ) into an active anticancer drug (see, e.g., WO 88 / 07378 and U.S. Pat. No. 4,975,278 ).

[0308] In one embodiment, the antigen binding protein of the present invention further comprises one or more of the following: an enzyme, a cytokine (eg, human IL-2, IL-7, or IL-15), a nanocarrier, or a nucleic acid.

[0309] Various bispecific formats are described in the art and are described above under "Formats" in the "Definitions" section. Techniques for preparing proteins in various formats are also disclosed in the art (referenced in the corresponding sections), so that one skilled in the art can readily use the variable domains defined in the context of the present invention in various formats, particularly those disclosed herein. Antigen binding proteins (particularly soluble bispecific binding proteins, such as: Those skilled in the art will appreciate that when the antigen binding protein comprises two polypeptides, the light and heavy chain variable domains can be, for example, in a parallel orientation, and the α and β variable domains can be in a parallel orientation, as in a DVD format, or the light and heavy chain variable domains can be, for example, in a staggered orientation, and the α and β variable domains can be in a staggered orientation, as in a CODV format.

[0310] Therefore, the present invention further relates to a bispecific antigen-binding protein comprising two polypeptide chains forming two antigen-binding sites (A and B), wherein the first polypeptide chain has a structure represented by the following formula:

[0311] V3–L1-V4-L2–C L [I]

[0312] Wherein, V3 is the third variable domain; V4 is the fourth variable domain; L1 and L2 are linkers; L2 may or may not exist; C L is a light chain constant domain or a portion thereof and may or may not be present;

[0313] The second polypeptide chain has the structure represented by the following formula:

[0314] V5-L3-V6-L4–C H1 [II]

[0315] Wherein, V5 is the fifth variable domain; V6 is the sixth variable domain; L3 and L4 are linkers; L4 may or may not exist; C H1is heavy chain constant domain 1 or a portion thereof and is present or absent; wherein

[0316] V3 is a Vα or Vγ variable domain and V5 is a Vβ or Vδ variable domain as defined above, and 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,

[0317] V3 is a Vβ or Vδ variable domain and V5 is a Vα or Vγ variable domain as defined above, and 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,

[0318] V3 is a Vα or Vγ variable domain and V6 is a Vβ or Vδ variable domain as defined above, and 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

[0319] V3 is a Vβ or Vδ variable domain and V6 is a Vα or Vγ variable domain as defined above, and 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,

[0320] V4 is a Vα or Vγ variable domain as defined above and V5 is a Vβ or Vδ variable domain, and V3 is a light chain variable domain and V6 is a heavy chain variable domain, or V3 is a heavy chain variable domain and V6 is a light chain variable domain, or V4 is a Vβ or Vδ variable domain as defined above and V5 is a Vα or Vγ variable domain, and 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,

[0321] In which, the light chain variable domain and the heavy chain variable domain together form an antigen binding site A, and the Vα and Vβ or Vγ and Vδ variable domains form an antigen binding site B, wherein the Vα or Vγ variable domain is preferably Vα, and Vβ or Vδ is preferably Vβ. The definitions of linkers L1, L2, L3, and L4 are as described above in the "Definitions" section. However, in some embodiments, some linker lengths may be preferred for specific forms. However, knowledge about linker lengths and their amino acid sequences is common knowledge in the art, and linkers and linkers and amino acid sequences for different forms are part of the prior art and are disclosed in the disclosures cited above.

[0322] In a preferred embodiment, V3 is Vα as defined above and V6 is Vβ as defined above, and V4 is a light chain variable domain as defined in the context of the present invention and V5 is a heavy chain variable domain as defined in the context of the present invention or

[0323] V3 is Va and V6 is Vβ as defined above, and V4 is a heavy chain variable domain and V5 is a light chain variable domain as defined in the context of the present invention.

[0324] In one embodiment, the polypeptide of formula [I] further comprises a linker (L5) and an Fc domain or a portion thereof at the C-terminus of the polypeptide of formula [I], and / or wherein the polypeptide of formula [II] further comprises a linker (L6) and an Fc domain or a portion thereof at the C-terminus of the polypeptide of formula [II].

[0325] The definition of Fc domain is given in the "Definitions" section above.

[0326] In one embodiment, the antigen binding protein comprises two polypeptide chains forming two antigen binding sites (A and B),

[0327] One of the polypeptide chains has a structure represented by formula [III]:

[0328] V3-L1-V4-L2-C L -L5-F c1 [III]

[0329] And a polypeptide chain has a structure represented by formula [IV]:

[0330] V5-L3-V6-L4-C H1 -L6-F c2 [IV]

[0331] Among them, V3, L1, V4, L2, C L 、V5、L3、V6、L4、C H1 As defined above, and wherein L5 and L6 are linkers that are present or absent, and wherein Fc1 and Fc2 are Fc domains, and wherein Fc1 and Fc2 are identical or different, preferably different. The definition of Fc domain is as described above in the "Definitions" section.

[0332] In one embodiment, Fc1 comprises or consists of the amino acid sequence of SEQ ID NO: 132 (hole), and Fc2 comprises or consists of the amino acid sequence of SEQ ID NO: 131 (knob), or vice versa.

[0333] More preferably, when V4 or V3 is a heavy chain variable domain, Fc1 comprises or consists of the amino acid sequence of SEQ ID NO: 132, and therefore, when V5 or V6 is a light chain variable domain, Fc2 comprises or consists of the amino acid sequence of SEQ ID NO: 131, or when V4 or V3 is a light chain variable domain, Fc1 comprises or consists of the amino acid sequence of SEQ ID NO: 131, and therefore, when V5 or V6 is a heavy chain variable domain, Fc2 comprises or consists of the amino acid sequence of SEQ ID NO: 132.

[0334] As can be seen from the examples, the inventors of the present invention have demonstrated in principle that low affinity recruits (antigen binding site A) can be combined with mature TCR variable domains. The forms are used in combination (Example 2).

[0335] Therefore, in a preferred embodiment, the antigen binding protein comprises two polypeptide chains forming two antigen binding sites (A and B), wherein one polypeptide chain has a structure represented by formula [III]:

[0336] V3-L1-V4-L2-C L -L5-F c1 [III]

[0337] And a polypeptide chain has a structure represented by formula [IV]:

[0338] V5-L3-V6-L4-CH1-L6-F c2 [IV]

[0339] Among them, L2, C L , L5 and L4, C H1 , L6 does not exist, and

[0340] Wherein V3, L1, V4, V5, L3, V6 are as defined above,

[0341] Preferably, V3 is a Vα or Vγ domain and V6 is a Vβ or Vδ domain as defined in the context of the present invention, and 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

[0342] Preferably, V3 is a Vα or Vγ variable domain and V6 is a Vβ or Vδ domain as defined in the context of the present invention, and V4 is a heavy chain variable domain and V5 is a light chain variable domain as defined in the context of the present invention, and

[0343] Preferably, L1 and L3 comprise or consist of the amino acid sequence "GGGSGGGG" (SEQ ID NO: 118), and

[0344] Preferably, Fc1 comprises or consists of the amino acid sequence of SEQ ID NO: 132, and Fc2 comprises or consists of the amino acid sequence of SEQ ID NO: 131, or vice versa.

[0345] More preferably, when V4 is a heavy chain variable domain, Fc1 comprises or consists of the amino acid sequence of SEQ ID NO: 132, and therefore, when V5 is a light chain variable domain, Fc2 comprises or consists of the amino acid sequence of SEQ ID NO: 131, or

[0346] More preferably, when V4 is a light chain variable domain, Fc1 comprises or consists of the amino acid sequence of SEQ ID NO: 131, and accordingly, when V5 is a heavy chain variable domain, Fc2 comprises or consists of the amino acid sequence of SEQ ID NO: 132, and

[0347] The light chain variable domain and the heavy chain variable domain together form an antigen binding site A that binds to CD3.

[0348] And wherein the Vα and Vβ or Vγ and Vδ variable domains form an antigen binding site B, which specifically binds to the TA antigen peptide / MHC complex (preferably the TAA antigen peptide / MHC complex) defined in the context of the present invention.

[0349] The antigen binding protein of this embodiment may also be referred to as

[0350] A bispecific T cell receptor (TCR) is a soluble antigen binding protein comprising two antigen binding domains - a heavy and light chain variable domain that binds CD3 as defined in the context of the present invention and a Vα and Vβ or a Vγ and Vδ domain as defined in the context of the present invention.

[0351] In one embodiment, the present invention relates to an antigen binding protein comprising the formula V3-L1-V4-L2-C L -L5-Fc1[III] (comprising or consisting of the amino acid sequence of SEQ ID NOs: 165 to 167), and a first polypeptide of the formula V5-L3-V6-L4-C H1 - a second polypeptide of L6-Fc2[IV] (which comprises or consists of the amino acid sequence of SEQ ID NO: 163 or 164).

[0352] It may also be desirable to modify the antigen-binding proteins of the invention with respect to effector functions, thereby enhancing or reducing the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antigen-binding protein. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antigen-binding protein, also referred to herein as Fc-variants in the context of the antigen-binding proteins of the invention. Alternatively or additionally, cysteine ​​residues may be introduced into the Fc region, thereby forming interchain disulfide bonds in this region. The resulting homodimeric antigen-binding protein may have improved or reduced internalization capacity and / or increased complement-mediated cell killing and / or antibody-dependent cellular cytotoxicity (ADCC) (Caron PC. et al. 1992; and Shopes B. 1992).

[0353] Another type of amino acid modification of the antigen-binding proteins of the present invention can be used to alter the native glycosylation pattern of the antigen-binding protein by deleting one or more carbohydrate moieties present in the antigen-binding protein and / or adding one or more glycosylation sites not present in the antigen-binding protein. The presence of either of the tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) creates a potential glycosylation site. Addition or deletion of glycosylation sites in an antigen-binding protein can be conveniently accomplished by altering the amino acid sequence to include one or more of these tripeptide sequences (for N-linked glycosylation sites).

[0354] Another type of modification involves removing sequences identified by computational or experimental methods that may result in degradation products or heterogeneity in the antigen-binding protein preparation. For example, the deamination of asparagine and glutamine residues may depend on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamination (primarily when present in Asn-Gly sequences) and less so in other dipeptide sequences (e.g., Asn-Ala). When such a deamination site (particularly Asn-Gly) is present in the antigen-binding proteins of the invention, it may be desirable to remove the site, typically by conservatively replacing one of the involved residues. Such substitutions within the sequence to remove one or more of the involved residues are also contemplated by the present invention.

[0355] Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antigen-binding protein. These procedures have the advantage that they do not require the production of the antigen-binding protein in a host cell capable of glycosylation for N- or O-linked glycosylation. Depending on the coupling method used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as cysteine ​​groups, (d) free hydroxyl groups, such as serine, threonine, or hydroxyproline groups, (e) aromatic residues, such as phenylalanine, tyrosine, or tryptophan residues, or (f) the amide group of glutamine. Such methods are described, for example, in WO 87 / 05330.

[0356] Removal of any carbohydrate moieties present on the antigen-binding protein can be achieved chemically or enzymatically. Chemical deglycosylation requires exposure of the antigen-binding protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars other than the linking sugar (N-acetylglucosamine or N-acetylglucosamine), while leaving the antigen-binding protein intact. Chemical deglycosylation is described in Sojahr H. et al. (1987) and Edge, AS. et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved using a variety of endo- and exo-glycosidases, as described in Thotakura, NR. et al. (1987).

[0357] Another type of covalent modification of the antigen binding protein involves linking the antigen binding protein to one of a variety of nonprotein polymers (e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes) by the methods described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.

[0358] The present invention also includes particles displaying antigen-binding proteins of the present invention, and particles comprising particle libraries. Such particles include, but are not limited to, phage, yeast, ribosomes, or mammalian cells. Methods for producing such particles and libraries are known in the art (e.g., see WO 2004 / 044004; WO 01 / 48145, Chervin et al. (2008) J. Immuno. Methods 339.2:175-184).

[0359] As disclosed herein, the antigen-binding proteins of the present invention bind to CD3 at antigen-binding site A and to the target antigen (TA) peptide / MHC complex at antigen-binding site B. CD3 molecules are typically present on the surface of CD3-presenting cells (e.g., effector cells, preferably T cells). Target antigen (TA) peptide / MHC complexes are typically present on the surface of target antigen (TA) peptide / MHC complex-presenting cells (e.g., diseased cells, such as cancer cells). Binding of the antigen-binding protein to CD3 and the target antigen (TA) peptide / MHC complex brings the effector and target cells into proximity, and the binding of the bispecific antigen-binding protein can elicit an immune response upon binding. Thus, the antigen-binding proteins of the present invention can induce an immune response in effector cells.

[0360] Thus, in one embodiment, the antigen binding proteins of the invention can induce an immune response in CD3 presenting cells (e.g., effector cells, such as T cells or NK cells), preferably wherein the immune response is characterized by increased levels of interferon (IFN) gamma. Thus, in one embodiment, the immune response can be characterized by ECs, preferably using an IFN-gamma release assay. 50 Value to describe.

[0361] In the context of the present invention, the bispecific antigen binding protein of the invention is K D (A) binds to CD3, antigen binding site B is K D (C) binds to the TA antigen peptide C (TA-C) / MHC complex (preferably the TAA antigen peptide C (TAA-C) / MHC complex), and K D (A) / K D The ratio of (C) 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, for example: between 1 and 150, 4 to 140, 6 to 100, 8 to 100, 10 to 100, preferably between 10 and 100.

[0362] the term" Affinity "and" K D " is defined in the "Definitions" section above. Measuring affinity (such as K D ) methods are known to those skilled in the art, including, for example, surface plasmon resonance and biofilm interferometry. As is known to those skilled in the art, the experimental conditions used in those experiments (e.g., the buffer used, protein concentration, or temperature) may affect the results.

[0363] Therefore, in one embodiment, the bispecific antigen binding protein of the present invention is expressed as a soluble The binding affinity of the HLA-A*02 / MAG-003 complex to the monomer is analyzed. Typically, the measurement is performed on an Octet RED384 system, for example, using the manufacturer's recommended settings. Briefly, the binding kinetics are typically measured at 30°C and, for example, 1000 rpm using, for example, PBS, 0.05% Tween-20, and 0.1% BSA as a buffer. ), the peptide HLA-A:02 complex is loaded onto a biosensor (e.g., HIS1K) prior to being loaded onto a biosensor. The same antigen binding protein is then typically further analyzed for its binding affinity to CD3. Thus, CD3 binding is measured as described above.

[0364] In some embodiments, the antigen binding site A is ≥3 nM, ≥5 nM, ≥8 nM, ≥10 nM, ≥12 nM, ≥14 nM, ≥16 nM, ≥18 nM, ≥20 nM, ≥25 nM, ≥30 nM, ≥35 nM, ≥40 nM, ≥45 nM and ≤1000 nM, ≤800 nM, ≤600 nM, ≤500 nM, ≤400 nM (e.g., 3 nM to 1000 nM, 3 nM to 600 nM, The invention further comprises binding to CD3 with a KD(A) of 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, preferably 5 nM to 100 nM), which is preferably determined using surface plasmon resonance (SPR) or bio-layer interferometry (BLI), preferably bio-layer interferometry (BLI).

[0365] In some embodiments, antigen binding site B binds to TA antigen peptide C (TA-C) / MHC complex (preferably TAA antigen peptide C (TAA-C) / MHC complex) with a KD(C) of ≤100 μM, ≤1 μM, ≤100 nM, ≤50 nM, ≤10 nM, 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), and the KD(C) is determined using surface plasmon resonance (SPR) or biofilm interferometry (BLI), preferably biofilm interferometry (BLI).

[0366] In one embodiment, the antigen binding protein binds to a MAGE-A antigen peptide, preferably HLA-A*02, comprising or consisting of the amino acid sequence “KVLEHVVRV” of SEQ ID NO: 10, with a KD of ≤100 nM, ≤50 nM, ≤10 nM, ≤1 nM, for example, 10 pM to 100 nM, 10 pM to 50 nM, 10 pM to 10 nM, particularly 50 pM to 100 nM, 100 pM to 50 nM, 100 pM to 10 nM, 500 pM to 10 nM, preferably 500 pM to 10 nM, wherein the KD is ≤100 nM, ≤50 nM, ≤10 nM, ≤10 nM, ≤10 nM, for example, 10 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. D Surface plasmon resonance (SPR) or biolayer interferometry (BLI) is used for determination, preferably biolayer interferometry (BLI).

[0367] In one embodiment, the antigen binding protein binds to a PRAME peptide / MHC complex comprising or consisting of the amino acid sequence SEQ ID NO: 9, D is ≤100 nM, ≤50 nM, ≤10 nM, ≤1 nM, ≤1 nM, e.g., 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, 100 pM to 50 nM, wherein K D Preferably, the measurement is performed using surface plasmon resonance (SPR) or biolayer interferometry (BLI), preferably biolayer interferometry (BLI).

[0368] In one embodiment, the EC50 of the bispecific antigen binding protein for TA-C / MHC presenting cells (preferably TAA-C / MHC presenting cells) is greater than the EC50 for normal tissue cells. 50 The value is ≥5 times, ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥500 times, ≥1000 times lower, among which EC 50 Preferably, it is determined with respect to induced cytotoxicity.

[0369] In this article, TA-C / MHC presenting cells "or" TA presenting cells" refers to a cell that presents a TA antigen peptide (e.g., a specific TA-C antigen peptide) complexed with an MHC protein on its surface, wherein the copy number of the TA peptide / MHC complex on the cell surface can generally be determined using 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 embodiments, the TA antigen peptide is a viral or bacterial peptide, in which case the TA / MHC presenting cell is typically a diseased cell or an infected cell, wherein the diseased cell is infected with the corresponding virus or bacteria. In some embodiments, the TA antigen peptide is a TAA antigen peptide, and in such embodiments, the TAA / MHC presenting cell is typically a cancer cell.

[0370] In one embodiment, the TA / MHC presenting cell, preferably a TAA / MHC presenting cell, for example, the TA / MHC copy number or TAA / MHC copy number of the TAA / MHC presenting cancer cell is greater than 50, greater than 100, greater than 150, greater than 200, greater than 300, greater than 600, greater than 800, greater than 1000, greater than 1500, greater than 2000, respectively, preferably a TAA / MHC copy number of 50 to 5000.

[0371] In this article, Copy number " refers to the number of TA antigen peptide / MHC complexes present on the cell surface of a cell (e.g., a TA / MHC presenting cell, such as a TAA / MHC presenting cell, such as a cancer cell or a normal healthy cell).

[0372] Such copy number depends on, for example, the specific TA and cell type and can be detected using methods known to those skilled in the art (e.g., FACS analysis, mass spectrometry (MS) and RNA sequencing, preferably mass spectrometry (MS) and RNA sequencing).

[0373] “ healthy cells ” can also be called “ Normal cells " " herein refers to cells without cancer cells, preferably healthy cells herein refer to tissue cells surrounding TA-presenting cells. Preferably, when TA is a viral or bacterial antigen peptide, the healthy cells are preferably not diseased, that is, not infected by the corresponding virus or bacteria. However, in some cases, healthy cells may also express and present TA peptide / MHC complexes on their surface, such as TAA peptide / MHC complexes, such as TAA-C / MHC complexes. Generally, as those skilled in the art will appreciate, the number (copy number) of TA peptide / MHC complexes presented in healthy cells in the context of the present invention is less than that in TA-presenting cells (e.g., cancer cells).

[0374] Therefore, in one embodiment, the TA / MHC copy number, preferably the TAA / MHC copy number (e.g., TAA-C / MHC copy number) of healthy cells is less than 5000, less than 1000, less than 500, less than 100, less than 50, less than 20, less than 10, preferably the TAA / MHC copy number is less than 10, preferably the TAA / MHC copy number is between 0 and 10 (e.g., 0 to 5).

[0375] Healthy cells are preferably selected from the group consisting of astrocytes, GABA neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, coronary artery endothelial cells, skin 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).

[0376] In one embodiment, the EC50 of the antigen binding protein for TA / MHC complex presenting cells (such as TA-C / MHC complex presenting cells), such as MAGE-A / MHC complex presenting cells, is greater than the EC50 for healthy cells. 50 The value is ≥1000, ≥15000, ≥9000 times lower, and the healthy cells are preferably selected from the group consisting of astrocytes, GABA neurons, cardiomyocytes, cardiac microvascular endothelial cells, chondrocytes, coronary artery endothelial cells, skin 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).

[0377] The bispecific antigen binding proteins of the present invention have a high safety profile.

[0378] In this article, Safety features " refers to the ability to distinguish tumor cells from normal healthy tissue cells or similar peptide-presenting cells. In the art, the safety window is used to describe the safety characteristics.

[0379] In this article, Safety window "or" therapeutic window ” refers to the ratio of the half-maximal concentration of the compound required to induce 100% cytotoxicity in tumor cell lines to the half-maximal concentration of the compound required to induce 100% cytotoxicity in normal tissue cells. If the EC 50 The EC determined for example on primary cells is 1 pM. 50 If the value is 1000pM, the safety window is 1000, because the EC50 1000-fold smaller than the EC50 against primary cells.

[0380] In one embodiment, the bispecific antigen binding protein of the present invention is directed to the EC of a TA / MHC complex presenting cell, preferably a TAA / MHC complex presenting cell (such as a TAA-C / MHC complex presenting cell). 50 ≥100-fold, ≥500-fold, ≥1000-fold, ≥2000-fold, ≥3000-fold, ≥4000-fold, ≥5000-fold, ≥6000-fold lower than the EC50 for normal tissue cells, for example, the EC50 for TA / MHC complex presenting cells, preferably TAA / MHC complex presenting cells (such as TAA-C / MHC complex presenting cells) 50 EC targeting normal tissue cells 50 The value is 500 to 12,000 times lower, preferably 1000 to 10,000 times lower.

[0381] When EC cannot be calculated 50 In the case of The safety window is determined by the ratio of the "lowest observed effect level" (LOEL) of a molecule on target cells and normal tissue cells.

[0382] This article's LOEL ” is defined as the first time a response exceeds a critical value The critical value is defined as the background value (no addition of The quantification of the quantification of the molecule was performed by adding three times the standard deviation of all assay wells.

[0383] In one embodiment, the LOEL of the bispecific antigen-binding protein of the present invention against TA / MHC complex-presenting cells, preferably TAA / MHC complex-presenting cells (such as TAA-C / MHC complex-presenting cells), is ≥100-fold, ≥500-fold, ≥1000-fold, ≥2000-fold, ≥3000-fold, ≥4000-fold, ≥5000-fold, or ≥6000-fold lower than the LOEL against normal cells. For example, the LOEL against TA / MHC complex-presenting cells, preferably TAA / MHC complex-presenting cells (such as TAA-C / MHC complex-presenting cells), is 500 to 12,000-fold lower than the LOEL against normal cells, preferably 1,000 to 10,000-fold lower.

[0384] In one embodiment, the bispecific antigen binding protein of the present invention is directed to the EC of a TA / MHC complex presenting cell, preferably a TAA / MHC complex presenting cell (such as a TAA-C / MHC complex presenting cell). 50 ECs of similar peptide / MHC presenting cells50 The values ​​were ≥5 times, ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥500 times, ≥1000 times, ≥2,000 times, ≥3,000 times, ≥4,000 times, ≥5,000 times, ≥6,000 times lower, among which EC 50 Preferably, it is determined based on the induction of cytotoxicity, such as ECs of TA / MHC complex presenting cells. 50 , preferably ECs presenting TAA / MHC complexes 50 ECs of similar peptide / MHC presenting cells 50 The value is 500 to 12,000 times lower, preferably 1,000 to 12,000 times lower.

[0385] In the context of the present invention, “ Peptide-like ” can also be called “ Off-target ", relates to peptides generally comprising 8 to 16 amino acids in length. Similar peptides in the context of the present invention are generally presented by MHC. Furthermore, similar 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 antigen peptide, and in a more preferred embodiment in the context of the present invention, these peptides comprise epitopes compared to TA antigen peptide epitopes, wherein at least one amino acid (e.g. at least 1, 2 or 3, preferably 1, 2 or 3, more preferably 1) of said epitopes is substituted compared to the TA antigen peptide epitopes. Due to this sequence similarity, similar peptides may be incorporated into the bispecific antigen binding proteins of the present invention. Binding, in this case, for example, if the similar peptide is presented by the MHC protein and is therefore bound by the bispecific antigen binding protein, the ability of a given bispecific antigen binding protein to bind to the similar peptide will not result in the desired effector cell response, but may result in adverse reactions. Such adverse reactions may be "out-of-tumor" side effects, such as the cross-reaction of specific TCRs that cross-react with normal tissue peptides reported on page 7 of "Cytotherapy" published by Lowdell et al. on December 4, 2018. Similar peptides in the context of the present invention can be selected from, for example, a database of HLA-A class 1 binding peptides presented by normal tissue (XPRESI / DENT database), such as HLA-A*02 binding peptides in the case of MAGE-A, based on, for example, a high degree of sequence similarity (similarity BLAST search) to TA antigen peptides (e.g., MAG-003). Due to these adverse reactions, the bispecific antigen binding proteins of the present invention are therefore modified to avoid cytotoxicity to normal tissue cells that present similar peptides.

[0386] This article's Peptide-like / MHC-presenting cells ” denotes cells that present similar peptide / MHC complexes on their cell surface.

[0387] In one embodiment, the similar peptide / MHC copy number of the similar peptide / MHC presenting cell is greater than 50, greater than 100, greater than 150, greater than 200, greater than 300, greater than 600, greater than 800, greater than 1000, greater than 1500, greater than 2000, preferably the TAA / MHC copy number is 50 to 5000.

[0388] In one embodiment, the similar peptide / MHC presenting cell is a MAGE-A / MHC complex presenting cell, and the copy number of the MAGE-A / MHC complex is greater than 50, greater than 80, greater than 100, greater than 120, greater than 150, greater than 300, greater than 400, greater than 600, greater than 800, greater than 1000, greater than 1500, or greater than 2000, preferably the MAGE-A / MHC copy number is 50 to 2000, e.g., 80 to 2000, e.g., 100 to 2000, e.g., 120 to 2000.

[0389] 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.

[0390] Thus, in one embodiment, the bispecific antigen binding protein of the invention does not bind or does not significantly bind to at least one analogous peptide, such as at least two, at least three, at least four, at least five, such as one, two, three, four, five, preferably at least three, or three or all analogous peptides selected from the group 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, with the proviso that the analogous peptide forms a complex with an MHC protein, preferably with HLA-A*02.

[0391] In the context of similar peptides and in the context of bispecific antigen binding proteins herein, “ Not significant Combined with " is characterized by a low binding signal and / or K D For example, in the context of the present invention, the binding response of the bispecific antigen binding protein to at least one similar peptide / MHC complex is less than 50%, less than 45%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3% of the binding response of the same bispecific antigen binding protein to the MAGE-A antigen peptide / MHC complex, under the same experimental conditions and at the same concentration of the bispecific antigen binding protein, and / or, for example, in the context of the present invention, the bispecific antigen binding protein binds to at least one similar peptide / MHC complex with a decreased affinity compared to its affinity for a specific antigen (i.e., a MAGE-A antigen peptide / MHC complex described herein), wherein the corresponding K for each similar peptide is less than 50%, less than 45%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3% of the binding response of the same bispecific antigen binding protein to the MAGE-A antigen peptide / MHC complex. D 5, 7, 10, 15, 20, 30, 40, 50, 100-fold, preferably 20 to 100-fold, more preferably 30 to 100-fold, such as 40 to 100-fold, usually 40 to 50-fold. For example, when the bispecific antigen binding protein has a K of 1 nM D Binds to the MAG-003 / MHC complex, and the bispecific antigen binding protein binds with a K of 100 nM D When bound to, for example, a RABGAP1L-001 / MHC complex, the K of the bispecific antigen binding protein bound to RABGAP1L-001 / MHC is D A 100-fold increase results in a 100-fold decrease in affinity. In these examples, the binding response, dissociation constant, and binding affinity are preferably measured using biofilm interferometry techniques such as those described in Examples 4 and 5.

[0392] From the examples, especially Examples 1 and Figure 8 It can be further seen that the bispecific antigen binding proteins of the present invention are not only soluble, but can also be transiently expressed in CHO cells at levels >10 mg / L (cell culture), >20 mg / L or even 40 mg / L. Thus, in one embodiment, the bispecific antigen binding proteins of the present invention can be expressed in high yields in host cells, wherein preferably the host cells are CHO cells, and wherein the yield is preferably greater than 10, greater than 15, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45 mg / L (cell culture), for example, 5 to 50, 5 to 45, 5 to 40, 5 to 35, 10 to 35, 10 to 30, 10 to 25, 10 to 20 mg / L (cell culture). In one embodiment, the antigen binding protein is expressed in transiently transfected CHO-S cells, wherein the cells are cultured in a total volume of 320 mL of GE Healthcare™ medium at T=0 and a density of 4x10 6 / mL, 37° C. One day later, feed solution (CellBoost 7a and b) was added and the temperature was lowered to 32° C. After a total of 12 days in culture and 3 feedings, the cells and thus the antigen binding protein were harvested.

[0393] As can be further seen in the Examples, the inventors demonstrated that the stability of the bispecific antigen-binding proteins of the present invention is comparable to or even higher than a reference protein, for example, an antigen-binding protein comprising a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 39 and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, or a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 137 and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 145, preferably a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 137 and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 145.

[0394] Thus, in one embodiment, the stability of the antigen-binding proteins of the present invention is comparable or improved, optionally compared to a reference protein. In the context of the present invention, comparable or improved stability refers to, for example, comparable or increased physical stability when exposed to thermal stress. Thus, the newly developed antigen-binding proteins of the present invention tolerate stress conditions, in particular thermal stress, comparable or better than a reference antigen-binding protein, wherein the antigen-binding proteins are preferably in the same form.

[0395] The term " stability" refers to physical stability and can be assessed qualitatively and / or quantitatively using various analytical techniques described in the art, for review see, e.g., 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 involve assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and / or by visual inspection). To measure stability, samples comprising an antigen binding protein of the invention can be tested in stability studies wherein the sample is exposed to stress conditions for a selected period of time and then the chemical and physical stability is analyzed quantitatively and, optionally, qualitatively, using appropriate analytical techniques.

[0396] In one embodiment, the antigen binding proteins of the invention are physically stable, for example, when exposed to stress conditions for a period of time, for example, after exposure to a temperature of 40°C for 14 days.

[0397] In the context of the present invention, " Physical stability "Substantially" means that the antigen binding protein shows no signs of aggregation, precipitation and / or denaturation.

[0398] Examples of methods for assessing physical stability are: size exclusion chromatography (SEC), dynamic light scattering (DLS), light obscuration (LO), and color and clarity.

[0399] “ No signs of gathering ” means, for example, that after a sample comprising the antigen binding protein is exposed to stress conditions (e.g., a temperature of 40° C.) in a buffer (e.g., PBS) for 14 days, the monomer content is 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%, or greater than 99%, for example, the monomer content is 94% to 99%, 95% to 99%, 96% to 99%, or 97% to 99% when measured by SEC (e.g., SEC-HPLC) in a buffer (e.g., PBS).

[0400] Thus, in one embodiment, the antigen binding proteins of the invention have the same or lower aggregation potential, for example compared to a reference protein.

[0401] When using size exclusion chromatography (SEC), depending on the column used, the operating pressure and the buffer flow rate, a difference in monomer content of 1%, 2%, 3%, 4%, preferably 1% or 2%, more preferably 2% is considered to be a significant difference in the context of the present invention under the conditions tested.

[0402] This means that when the monomer content of the reference antigen binding protein is 96% and the monomer content of the antigen binding protein of the invention is 98%, the monomer content of the antigen binding protein of the invention is significantly different and, therefore, is significantly increased compared to the reference antigen binding protein when measured under the same conditions.

[0403] Nucleic acids, vectors and recombinant host cells

[0404] Another object of the present invention relates to an isolated nucleic acid sequence comprising or consisting of a sequence encoding the bispecific antigen binding protein of the present invention as defined above.

[0405] Typically, the nucleic acid is a DNA or RNA molecule, which can be contained in any suitable vector (eg, a plasmid, a cosmid, an episome, an artificial chromosome, a phage, or a viral vector).

[0406] the term" carrier "," cloning vector "and" expression vector " refers to a vector that can introduce DNA or RNA sequences (eg, foreign genes) into host cells to transform the host and promote the expression (eg, transcription and translation) of the introduced sequences.

[0407] Therefore, another object of the present invention relates to a vector comprising the nucleic acid according to the invention.

[0408] Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., to cause or direct the expression of the polypeptide after administration to a subject. Examples of promoters and enhancers used in animal cell expression vectors include the early promoter and enhancer of SV40 (Mizukami T. et al. 1987), the LTR promoter and enhancer of Moloney murine leukemia virus (Kuwana Y et al. 1987), the promoter of immunoglobulin H chain (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983), etc.

[0409] Any animal cell expression vector can be used as long as it can insert and express the gene encoding the human antibody C region. Examples of suitable vectors include (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 pSG1βd2-4- (Miyaji H et al. 1990). Other examples of plasmids include replicating plasmids or integrating plasmids containing an origin of replication, such as pUC, pcDNA, and pBR.

[0410] Other examples of viral vectors include adenovirus, retrovirus, herpes virus, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with a helper plasmid or virus. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, and the like. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, US Pat. No. 5,882,877, US Pat. No. 6,013,516, US Pat. No. 4,861,719, US Pat. No. 5,278,056, and WO 94 / 19478.

[0411] the term" viral vectors " refers to a nucleic acid vector construct that includes at least one element of viral origin, has the ability to be packaged into viral vector particles, and encodes at least one exogenous nucleic acid. The vectors and / or particles can be used for the purpose of transferring any nucleic acid into cells in vitro or in vivo. Various forms of viral vectors are known in the art. The term "virion" refers to a single infectious viral particle. "Viral vector," "viral vector particle," and "viral particle" also refer to the complete viral particle with its DNA or RNA core and protein coat, as the virus exists outside the cell. For example, the viral vector can be selected from adenovirus, poxvirus, alphavirus, coronavirus, flavivirus, rhabdovirus, retrovirus, lentivirus, herpesvirus, paramyxovirus, or picornavirus.

[0412] Virus may refer to naturally occurring viruses as well as artificial viruses. According to some embodiments of the present invention, the virus may be an enveloped virus or a non-enveloped virus. Parvoviruses (e.g., AAV) are examples of non-enveloped viruses. In a preferred embodiment, the virus may be an enveloped virus. In a preferred embodiment, the virus may be a retrovirus, particularly a lentivirus. Viral envelope proteins that can promote viral infection of eukaryotic cells may include HIV-1 derived lentiviral vectors (LVs), which are pseudotyped with the envelope glycoprotein (GP) of vesicular stomatitis virus (VSV-G), modified feline endogenous retrovirus (RD114TR), and modified gibbon ape leukemia virus (GALVTR). These envelope proteins can effectively promote the entry of other viruses, such as parvoviruses, including adeno-associated viruses (AAV), thereby demonstrating their wide efficiency. For example, other viral envelope proteins can be used, including Moloney murine leukemia virus (MLV) 4070 env (as described in Merten et al., J. Virol. 79:834-840, 2005; incorporated herein by reference), RD114 env, the chimeric envelope protein RD114pro, or RDpro (an RD114-HIV chimera constructed by replacing the R peptide cleavage sequence of RD114 with the HIV-1 matrix / capsid (MA / CA) cleavage sequence, as described in Bell et al. Experimental Biology and Medicine 2010;235:1269-1276; incorporated herein by reference), baculovirus GP64 env (as described in Wang et al., J. Virol. 81:10869-10878, 2007; incorporated herein by reference), or GALV env (as described in Merten et al., J. Virol. 81:10869-10878, 2007; incorporated herein by reference), or GALV env (as described in Merten et al., J. Virol. 81:10869-10878, 2007; incorporated herein by reference). al., J. Virol. 79:834-840, 2005; the contents of which are incorporated herein by reference) or derivatives thereof.

[0413] Another object of the present invention relates to a host cell transformed, transduced or transfected with the nucleic acid and / or the vector according to the invention.

[0414] the term" conversion Originally, it referred to the natural process of gene transfer into a host cell, which involves the cell taking up genetic material (e.g., nucleic acids such as DNA or RNA) through the cell membrane, resulting in the host cell expressing the introduced gene or sequence to produce a desired substance, usually a protein or enzyme encoded by the introduced gene or sequence. There are two types of transformation, known as natural transformation and artificial or induced transformation. Artificial or induced transformation methods are performed under laboratory conditions.

[0415] A host cell that accepts and expresses exogenous nucleic acid (e.g., DNA or RNA) through a transformation process has been " conversion ”.

[0416] the term" Transfection " refers to a method of gene transfer that involves creating pores in the cell membrane of a host cell, enabling the host cell to receive foreign genetic material. Typically, transfection refers to the transformation of eukaryotic cells (e.g., insect or mammalian cells). Chemically mediated transfection involves the use of, for example, calcium phosphate or cationic polymers or liposomes. Non-chemically mediated transfection methods are typically electroporation, sonoporation, transfection by penetration, optical transfection, or hydrodynamic delivery. Particle-based transfection uses gene gun technology, where nanoparticles are used to transfer nucleic acids into the host cell, or by another method called magnetofection. Nucleofection and the use of heat shock are additional evolved methods for successful transfection. A host cell that receives foreign nucleic acid via a transfection method has been "transfected."

[0417] the term" transduction " is generally understood to involve the transfer of foreign nucleic acid (e.g., DNA or RNA) into cells via viruses or viral vectors. Host cells that receive and express foreign nucleic acid (e.g., DNA or RNA) via viruses or viral vectors have been " transduction ”.

[0418] In some embodiments, cells can be transduced using the methods described in US20190216852, the contents of which are incorporated herein by reference in their entirety.

[0419] The nucleic acids of the present invention can be used to produce the recombinant antigen-binding proteins of the present invention in a suitable expression system.

[0420] the term" Expression system ” means a host cell and a compatible vector under appropriate conditions, e.g., for the expression of a protein encoded by foreign DNA carried by the vector and introduced into the host cell.

[0421] Common expression systems include: Escherichia coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include: Escherichia coli, Kluyveromyces or Saccharomyces cerevisiae, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., cultures formed from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neural cells, adipocytes, etc.). Examples also include: mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells (in which the dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") is defective, Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells"), etc. In some embodiments, YB2 / 0 cells may be preferred because when chimeric or humanized antibodies are expressed in these cells, their ADCC activity is enhanced.

[0422] The present invention also relates to host cells comprising bispecific antigen recognition constructs according to the present invention. Specifically, the host cells of the present invention comprise nucleic acids or vectors as described above. The host cells can be eukaryotic cells, such as plants, animals, fungi or algae, or prokaryotic cells, such as bacteria or protozoa. The host cells can be cultured cells or primary cells, i.e., directly isolated from an organism (e.g., a human). The host cells can be adherent cells or suspension cells (i.e., cells grown in suspension). For the purpose of producing bispecific antigen-binding proteins (e.g., bispecific TCRs, polypeptides or proteins), the host cells are preferably mammalian cells.

[0423] In a particular embodiment, the host cell is a stem cell, preferably a mesenchymal stem cell.

[0424] In accordance with the above, in one embodiment, the present invention relates to a host cell comprising a bispecific antigen-binding protein of the invention or a nucleic acid or vector of the invention as defined above, wherein the host cell is preferably a) a mesenchymal stem cell, or b) a cell for recombinant expression, such as a Chinese Hamster Ovary (CHO) cell.

[0425] In particular, for expressing some bispecific antigen-binding proteins of the present invention, the expression vector can be of a type in which the gene encoding the first polypeptide (e.g., antibody heavy chain or α chain) and the gene encoding the second polypeptide (e.g., antibody light chain or β chain) are present on separate vectors, or of a type in which both genes are present on the same vector (tandem type). Tandem humanized antibody expression vectors are preferred from the perspectives of ease of constructing bispecific antigen-binding protein expression vectors, ease of introduction into animal cells, and balance between antibody H and L chain expression levels in animal cells (Shitara K et al. J Immunol Methods. 1994 Jan. 3; 167(1-2): 271-8). Examples of tandem humanized antibody expression vectors include pKANTEX93 (WO 97 / 10354), pEE18, and the like.

[0426] In one embodiment, such recombinant host cells can be used to produce at least one antigen binding protein of the invention.

[0427] Methods for producing bispecific antigen-binding proteins of the invention

[0428] The present invention also relates to a method for producing an antigen binding protein as defined above, comprising

[0429] a. Propose a suitable host cell,

[0430] b. Propose a gene construct comprising a coding sequence encoding a bispecific antigen-binding protein of the present invention,

[0431] c. introducing the gene construct into (preferably in vitro or ex vivo) the appropriate host cell, and

[0432] d. expressing the gene construct by the suitable host cell, and optionally

[0433] e. selecting cells that express and / or secrete the antibody.

[0434] The bispecific antigen-binding proteins of the present invention are defined in the corresponding sections.

[0435] This article's Gene constructs " denotes a nucleic acid that can express the coding region in a host, and thus refers to a nucleic acid (such as the vector described above) or RNA.

[0436] In a specific embodiment, the method may further comprise the step of presenting the bispecific antigen recognition construct on the cell surface of the suitable host cell.

[0437] In other preferred embodiments, the gene construct in b) comprises a nucleic acid encoding a bispecific antigen-binding protein of the invention. Such nucleic acids are defined in the "Nucleic Acids, Vectors, and Recombinant Host Cells" section above.

[0438] In a related embodiment, the genetic construct is an expression construct comprising a promoter sequence operably linked to the coding sequence.

[0439] In a related embodiment, the gene construct is introduced into a suitable host using transformation, transduction or transfection methods. The definitions of transformation, transduction or transfection are given above in the relevant sections.

[0440] Ideally, the transduction system used to introduce the gene construct into the appropriate host cell is a retroviral or lentiviral vector system as described above in the nucleic acid, vector and recombinant host cell sections. Such systems are well known to those skilled in the art.

[0441] 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 T cells.

[0442] The bispecific antigen-binding proteins of the present invention can be produced by any technique known in the art, such as, but not limited to, any chemical, biological, genetic, or enzymatic technique, which can be used alone or in combination.

[0443] Standard techniques for producing polypeptides (e.g., antibodies or fragments thereof and TCRs or fragments thereof) are known in the art, and those skilled in the art can use these techniques to produce the bispecific antigen-binding proteins of the present invention. For example, well-known solid-phase methods can be used for synthesis, in particular using commercially available peptide synthesis equipment (e.g., peptide synthesis equipment manufactured by Applied Biosystems in Foster City, California) and following the manufacturer's instructions. Alternatively, the bispecific antigen-binding proteins of the present invention (e.g., 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 for expressing the desired polypeptide, the fragment can be obtained as a DNA expression product, which can then be isolated using well-known techniques.

[0444] In one embodiment, that is, In the case of bispecific molecules, DNA sequences encoding various combinations of VH and VL, variable α (Vα) and variable β (Vβ), and sequences encoding linkers can be obtained, for example, by gene synthesis. The resulting DNA sequences can be cloned in frame into the hinge region, C region, and C region encoding sequences derived from human IgG4 [Accession No.: K01316] and IgG1 [Accession No.: P01857], respectively. H2 and C H3 The knob-hole mutation can be incorporated into the C H3 domains with and without additional interchain disulfide stabilization; removal of C H2 or introducing an N-glycosylation site in the VL or VH fragments (e.g., N297Q mutation); or introducing an Fc silencing mutation; or introducing additional disulfide bond stabilization into the VL and VH fragments, respectively, according to the method described by Reiter et al. (Stabilization of the FvFragments in Recombinant Immunotoxins by Disulfide Bonds Engineered into Conserved Framework Regions. Biochemistry, 1994, 33, 5451–5459).

[0445] The bispecific antigen-binding proteins of the invention can be suitably separated from the culture medium by immunoglobulin purification methods (eg, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography).

[0446] In one embodiment, recovering the expressed bispecific antigen-binding protein or polypeptide herein refers to performing protein A chromatography, kappa-selective chromatography and / or size exclusion chromatography, preferably protein A chromatography and / or size exclusion chromatography, more preferably protein A chromatography and size exclusion chromatography.

[0447] Methods for producing the bispecific antigen-binding proteins of the present invention involve recombinant DNA, and gene transfection techniques are well known in the art (see Morrison SL. et al. (1984) and patent documents US Pat. No. 5,202,238 and US Pat. No. 5,204,244).

[0448] 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 produce the bispecific antigen-binding proteins of the present invention.

[0449] In one embodiment, the vector for expressing the recombinant antigen-binding protein of the present invention is designed as a monocistronic vector, for example, controlled by a pUC19-derivative promoter element derived from HCMV. For example, plasmid DNA is amplified in E. coli according to standard culture methods and then purified using a commercially available kit (Macherey & Nagel). For example, according to the manufacturer's instructions (ExpiCHO TM Purified plasmid DNA was used for transient transfection of CHO-S cells using an electroporation system (MaxCyte STX). For example, transfected CHO cells were cultured at 32°C to 37°C for 6-14 days and subjected to one or two ExpiCHO TM Feed or Cellboost 7a and 7b (GE Healthcare TM ) solution feed.

[0450] For example, using Sartoclear The conditioned cell supernatant was clarified by filtration (0.22 μm) using Lab Filter Aid (Sartorius). The bispecific antigen-binding protein is purified using a Pure 25L FPLC system (GE Lifesciences) for affinity and size exclusion chromatography. For example, affinity chromatography is performed on a protein A or L column (GE Lifesciences) according to a standard affinity chromatography protocol. For example, size exclusion chromatography is performed directly after elution from the affinity column (pH 2.8) to obtain a highly pure monomeric protein using a Superdex 200pg 16 / 600 column (GE Lifesciences) according to a standard protocol. For example, protein concentration is determined on a NanoDrop system (Thermo Scientific) using an extinction coefficient calculated based on the predicted protein sequence. If necessary, a Vivaspin device (Sartorius) is used to adjust the concentration. Finally, the purified molecule is stored in, for example, phosphate-buffered saline at a temperature of 2-8°C at a concentration of approximately 1 mg / mL.

[0451] The quality of the purified bispecific antigen binding protein is determined, for example, by HPLC-SEC on a MabPac SEC-1 column (5 μm, 4×300 mm) run in a Vanquish uHPLC system, for example, in 50 mM sodium phosphate pH 6.8 containing 300 mM NaCl.

[0452] Pharmaceutical composition

[0453] The present invention also relates to a pharmaceutical composition comprising the bispecific antigen-binding protein of the present invention, the nucleic acid of the present invention, the vector of the present invention or the host cell of the present invention and a pharmaceutically acceptable carrier.

[0454] The present invention also relates to a bispecific antigen binding protein according to the present invention for use as a medicament. The present invention also relates to a pharmaceutical composition according to the present invention for use as a medicament.

[0455] The present invention also relates to the use of the bispecific antigen binding protein according to the present invention and / or the pharmaceutical composition according to the present invention in the preparation of a medicament.

[0456] The term " Pharmaceutical composition "or" Therapeutic compositions ” refers to a compound or composition that is capable of inducing a desired therapeutic effect when properly administered to a subject.

[0457] In some embodiments, a subject may also be referred to as a patient.

[0458] Such therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of a bispecific antigen binding protein of the invention, or further comprising a therapeutic agent, mixed with a pharmaceutically or physiologically acceptable formulation selected to be suitable for the mode of administration.

[0459] The bispecific antigen binding proteins of the present invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier.

[0460] “ Pharmaceutical "or" medicinal " refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when properly administered to mammals, especially humans. Pharmaceutical carriers or excipients refer to any type of non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0461] “ Pharmaceutical carriers ” can also be called “ Pharmaceutical diluents "or" pharmaceutical solvents ", may include solvents, fillers, stabilizers, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents and absorption delaying agents, etc., which are physiologically compatible. Therefore, in one embodiment, the carrier is an aqueous carrier.

[0462] On the other hand, aqueous carriers can improve properties when combined with the bispecific antigen binding proteins described herein, such as improved solubility, therapeutic efficacy, and / or improved immunotherapy.

[0463] The dosage form, route of administration, dosage and dosage regimen of the pharmaceutical composition will naturally depend on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, the desired duration of treatment, etc. The pharmaceutical composition may be in any suitable dosage form (depending on the desired method of administration for the patient). It may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such kits typically (but not necessarily) include instructions for use. It may include a plurality of such unit dosage forms.

[0464] Empirical considerations (e.g., biological half-life) will generally contribute to determining the dosage. The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of cancer cells, maintaining a reduction in cancer cells, reducing cancer cell proliferation, or killing cancer cells. Alternatively, sustained-release formulations of bispecific antigen-binding proteins may be suitable. Various formulations and devices for achieving sustained release are known in the art.

[0465] In one embodiment, the dosage of the antigen-binding protein can be determined empirically in individuals who have received one or more doses. The dose of the antigen-binding protein is gradually increased in the individual. To assess the efficacy of the antigen-binding protein, markers of cancer cell status can be tracked. These include direct measurement of cancer cell proliferation and cell death using FACS or other imaging techniques; assessment of health improvements using such measurements; or measurement of improved quality of life or prolonged survival using recognized tests. It will be apparent to those skilled in the art that the dosage will vary depending on the individual, the stage of the disease, and prior and concomitant treatments being used.

[0466] In particular, the pharmaceutical composition comprises a solvent which is pharmaceutically acceptable for injectable formulations. In particular, these may be isotonic sterile saline solutions (mono- or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts) or dry, in particular lyophilized, compositions which, as appropriate, can be prepared into injectable solutions by adding sterile water or physiological saline.

[0467] To prepare a pharmaceutical composition, an effective amount of the bispecific antigen-binding protein of the present invention can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0468] Pharmaceutical dosage forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or propylene glycol in water; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the dosage form must be sterile and fluid enough to allow easy syringability. It must be stable under the conditions of manufacture and storage and must be preserved against contamination by microorganisms (e.g., bacteria and fungi).

[0469] Solutions of the active compound as a free base or a pharmaceutically acceptable salt can be prepared in water suitably mixed with a surfactant (e.g., hydroxypropylcellulose). Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof, and oils. Under conventional storage and use conditions, these preparations contain a preservative to prevent microbial growth.

[0470] The antigen binding proteins of the present invention can be formulated into compositions in neutral or salt form using pharmaceutically acceptable salts.

[0471] Sterile injectable solutions are prepared by incorporating the desired amount of the active compound into an appropriate solvent containing the various other ingredients as described above, as needed, followed by filter sterilization. Dispersions are typically prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the other desired ingredients as described above. Preferred methods for preparing sterile powders for the preparation of sterile injectable solutions are vacuum drying and freeze-drying techniques, which produce a powder of the active ingredient plus any other desired ingredients from a previously sterile-filtered solution.

[0472] Preparation of more or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as a solvent is envisioned to allow for extremely rapid penetration, thereby delivering high concentrations of active agent to small tumor areas.

[0473] After formulation, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulation is readily administered in a variety of dosage forms, such as the injectable solution type described above, but drug release capsules and the like may also be employed.

[0474] Treatment methods and uses

[0475] The inventors have shown in Example 2 of the in vitro experiments section that MAG-003 targeting antigen binding protein is combined with BMA031 (V36) or UCHT1 (V17) as a recruiting agent, in particular with The cytotoxic activity of these molecules against different MAG-003-positive cancer cell lines in the form of cytotoxicity assays was also demonstrated by the inventors, as the cytotoxic activity was highly specific and limited to TAA-positive cells (e.g., MAG-003-positive cells), as the bispecific antigen-binding proteins induced only a small amount of lysis in cell lines expressing HLA-A*02 but not presenting TAA peptides (e.g., MAG-003). Thus, these examples demonstrate the technical advantages of combining low-affinity binding domains for CD3 (disclosed in the context of the present invention) with high-affinity TCR variable domains. In these examples, such TCR variable domains specifically bind to the TAA MAGE-A, but those skilled in the art will appreciate that the advantages of the exemplary bispecific antigen-binding proteins in the context of TAAs as targets can also be transferred to bispecific antigen-binding proteins targeting another TA (e.g., a viral or bacterial antigenic peptide, rather than a TAA). Those skilled in the art will understand that, in several embodiments, once the antigen binding protein is administered to a subject, it binds to target cells (e.g., TA / MHC complex presenting cells) and recruits endogenous effector cells, binds to them through CD3, and activates them, thereby positioning those effector cells near target cells (particularly target cancer cells) to achieve killing of the target cells, particularly achieving anti-cancer activity.

[0476] In one aspect, binding of the antigen binding protein to CD3 of target and effector cells elicits an immune response, which may include proliferation and initiation of effector functions in vitro or in vivo. For MHC class I-restricted cytotoxic T cells, for example, effector functions may include lysis of peptide-pulsed, peptide precursor-pulsed, or naturally peptide-presenting target cells, secretion of cytokines, preferably peptide-induced interferon-γ, TNF-α, or IL-2, secretion of effector molecules, such as peptide-induced granzymes or perforins, or degranulation.

[0477] Therefore, the bispecific antigen binding proteins of the present invention, in particular The molecules can be used to treat a wide range of diseases, including, for example, various forms of cancer and / or infectious diseases. The bispecific antigen binding proteins of the present invention can be used for therapeutic purposes in humans and / or non-human mammals (particularly humans).

[0478] In one embodiment, the bispecific antigen-binding proteins of the present invention can bind to diseased cells and slow the growth of and / or kill diseased cells that present TA peptide / MHC complexes on their cell surfaces. In a preferred embodiment, the bispecific antigen-binding proteins of the present invention can bind to tumor cells and slow the growth of and / or kill tumor cells that present TAA peptide / MHC complexes on their cell surfaces. It will be appreciated that the bispecific antigen-binding proteins are administered at a concentration that promotes binding under physiological (e.g., in vivo) conditions.

[0479] Therefore, in one embodiment, the bispecific antigen binding proteins of the present invention can be used for immunotherapy against tumor cells in different tissues (e.g., colon, lung, breast, prostate, ovary, pancreas, kidney, etc.). In another embodiment, the antigen binding proteins of the present invention can bind to tumor cells and reduce the growth of tumor cells and / or kill tumor cells.

[0480] Therefore, the present invention relates to a method for treating or preventing a proliferative disease or disorder, comprising administering to a subject in need thereof an effective therapeutic amount of a bispecific antigen binding protein, nucleic acid or vector, host cell or pharmaceutical composition according to the present invention, as defined above in the sections "Bispecific antigen binding protein", "nucleic acid" or "pharmaceutical composition".

[0481] In a specific embodiment, the invention relates to a method of treating a subject with a disease, said method comprising administering to said subject a bispecific antigen binding protein of the invention.

[0482] In another embodiment, the invention relates to a method of eliciting an immune response in a diseased subject, said method comprising administering to said subject a composition comprising an antigen recognition construct of the invention, optionally expressed in a host cell.

[0483] In one embodiment, the present invention relates to the use of the bispecific antigen binding protein, nucleic acid or vector, host cell or pharmaceutical composition according to the present invention in treating or preventing a disease in a subject.

[0484] In one embodiment, the immune response mentioned in the method is a cytotoxic T cell response. The cytotoxic T cell response is induced by binding of the antigen binding protein to CD3 on the effector cell and to the TA antigen peptide / MHC complex, thereby bringing the effector cell and target cell into proximity with each other.

[0485] The present invention also relates to the bispecific antigen binding protein of the present invention, the nucleic acid of the present invention, the vector of the present invention, the host cell of the present invention or the pharmaceutical composition of the present invention for use in diagnosing, preventing and / or treating a disease.

[0486] The present invention also relates to the use of the bispecific antigen-binding protein of the present invention, the nucleic acid of the present invention, the vector of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention in the manufacture of a medicament for diagnosing, preventing and / or treating a disease.

[0487] the term" Subjects "or" individual " can be used interchangeably, for example, can be a human or a non-human mammal, preferably a human.

[0488] In the context of the present invention, the term " treat " refers to therapeutic use (i.e., for a subject suffering from a particular disease), meaning reversing, alleviating, or inhibiting the progression of one or more symptoms of such disease or condition. Thus, treatment refers not only to treatment that results in a complete cure of the disease, but also to treatment that slows the progression of the disease and / or prolongs the survival of the subject.

[0489] “ prevention " is intended to refer to prophylactic use (i.e., use in a subject susceptible to a particular disease).

[0490] the term" Need treatment " refers to a subject already suffering from a disorder as well as a subject in whom prevention of the disorder is desired. Thus, in one embodiment, the subject is a patient.

[0491] In one embodiment, " disease "or" disease In one embodiment, this includes chronic and acute conditions or diseases, including those pathological conditions that predispose the subject to the condition. In particular, the disease involved in the context of the present invention may be a proliferative disease or a disease caused by a virus or bacteria. Diseases caused by viruses or bacteria may also be referred to as viral infections or bacterial infections. In the context of the present invention, the virus causing the disease 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, bacteria causing the disease include, for example, Mycobacterium tuberculosis. It will be understood by those skilled in the art that when the bispecific antigen binding protein targets a viral antigenic peptide (e.g., HIV), the bispecific antigen binding protein is used to treat HIV. Thus, the bispecific antigen binding protein targeting the viral or bacterial antigenic peptide TA-C may be used to treat the virus or bacteria from which the antigenic viral or bacterial antigenic peptide is derived.

[0492] “ Proliferative diseases ” (e.g., cancer) involves the unregulated and / or inappropriate proliferation of cells.

[0493] In one embodiment, the proliferative disorder or disease is, for example, a neoplastic disease characterized by expression of a TA, more particularly a TAA, by the cancer or tumor cells of said neoplastic disease.

[0494] Therefore, particularly preferred cancers are TA-positive cancers, in particular TAA-positive cancers.

[0495] In another embodiment, the proliferative disorder or disease is, for example, a neoplastic disease characterized by expression of MAGEA4 and / or MAGEA8 by cancer or tumor cells of said neoplastic disease.

[0496] Therefore, particularly preferred cancers are MAGEA4 and / or MAGEA8 positive cancers.

[0497] In another embodiment, the proliferative disorder or disease is, for example, a neoplastic disease characterized by expression of PRAME by cancer or tumor cells of said neoplastic disease.

[0498] Therefore, particularly preferred cancers are PRAME positive cancers.

[0499] In the context of the present invention, a cancer is considered "cancer-associated" if, according to the NCI guidelines, the relevant TAA peptide (e.g., one of the TAA peptides defined in the "Definitions" section above, such as MAG-003 peptide or PRAME-004) is presented in >98% of all cancers. TAA Positive ",like," MAGEA4 and / or MAGEA8 positive "or" PRAME positive In all other indications noted herein, a biopsy can be performed as this is standard practice in the treatment of these cancers and can be performed according to and related methods for identifying peptides (according to WO 03 / 100432; WO 2005 / 076009; WO 2011 / 128448; WO 2016 / 107740, US Pat. No. 7,811,828, US Pat. No. 9,791,444, and US Pat. No. 2016 / 0187351, the contents of each of which are incorporated herein by reference in their entirety). In one embodiment, for example, cancer can be more easily detected (i.e., diagnosed) using the bispecific antigen-binding proteins of the present invention. Methods for identifying antigen-expressing cancers using antigen-binding proteins are known to those skilled in the art.

[0500] In one embodiment, the cancer is selected from the list consisting of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), liver cancer, head and neck cancer, skin cancer, renal cell carcinoma, brain cancer, stomach 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.

[0501] Cancer, Principles and Practice of Oncology, 4th Edition, DeVita et al., Eds. JB Lippincott Co., Philadelphia, Pa. (1993) provides guidance for cancer treatment. The appropriate treatment method is selected based on the specific type of cancer and other factors recognized in the relevant field (e.g., the patient's general condition). The bispecific antigen-binding proteins of the present invention can be used alone or in combination with other anticancer drugs commonly used to treat cancer patients.

[0502] Thus, in some embodiments, the bispecific antigen binding proteins of the present invention can be administered simultaneously with, before, or after a variety of drugs and treatments widely used in cancer treatment (e.g., chemotherapeutic agents, non-chemotherapeutic agents, anti-tumor agents and / or radiation therapy, preferably chemotherapeutic agents).

[0503] In a further embodiment, the bispecific antigen binding protein can also be used to treat infectious diseases, e.g., infectious viral or bacterial diseases, wherein the viral disease is 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), and wherein the bacterial disease is, for example, tuberculosis.

[0504] The antigen-binding protein or pharmaceutical composition thereof of the present invention can be administered alone, or can be administered simultaneously with, before, or after administration of other therapeutic drugs for treating such infectious diseases.

[0505] In this article, diagnosis ” refers to medical diagnosis and means determining which disease or condition explains a patient’s signs and symptoms.

[0506] “ Effective therapeutic dose"A bispecific antigen binding protein or pharmaceutical composition thereof is intended to be a sufficient amount of the bispecific antigen binding protein to treat the proliferative disease at a reasonable benefit / risk ratio applicable to any medical treatment. However, it will be understood that the total daily or monthly usage of the antigen binding protein, nucleic acid or vector, host cell or pharmaceutical composition of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific effective therapeutic dosage level for any particular patient will depend on a variety of factors, including: the condition or disease being treated and the severity of the condition; the activity of the specific bispecific antigen binding protein used; the specific composition used, the patient's age, weight, general health, sex and diet; the time of administration, route of administration and excretion rate of the specific polypeptide used; the duration of treatment; drugs used in combination or concomitantly with the specific polypeptide used; and factors well known in the medical field. For example, it is well known to those skilled in the art to start the dose of the compound at a dose level lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0507] In one embodiment, the therapeutic efficacy of a bispecific antigen binding protein of the invention is determined in vivo, for example, in a mouse model of cancer, by measuring, for example, changes in tumor volume between treated and control groups.

[0508] The pharmaceutical compositions, vectors, nucleic acids and cells of the invention can be provided in a substantially pure form, e.g., wherein 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 a bispecific antigen binding protein of the same type is present.

[0509] The bispecific antigen-binding protein of the present invention, the nucleic acid of the present invention, the vector of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention can be administered by any feasible method.

[0510] The present invention also provides a method for killing target cells in a patient, comprising administering an antigen binding protein to the patient. In the context of this method, the antigen binding protein, after administration to the subject, binds to target cells and effector cells expressing CD3, preferably triggering an immune response.

[0511] In a particular approach, the host cell can be a stem cell (e.g., a mesenchymal stem cell) and engineered to express a bispecific antigen binding protein of the invention. In this embodiment, the bispecific antigen binding protein is a Therefore, the host cells of the present invention, preferably the stem cells defined above, can be used as active ingredients in therapeutic compositions. Therefore, the present invention also provides a method for killing target cells in a patient, the method comprising administering to the patient an effective amount of the host cells defined above, preferably mesenchymal stem cells.

[0512] For the purposes of the methods of the present invention, wherein a host cell or cell population is administered to a subject, the host cell can be allogeneic (derived from another subject) or autologous to the subject. Preferably, the cell is autologous to the subject. If the host cell is an allogeneic cell, i.e., derived from another subject, the other subject is healthy.

[0513] "Healthy" means that the subject is in generally good condition, preferably has a competent immune system, and more preferably is free of any easily testable or detectable disease.

[0514] Thus, the host cell is transformed, transduced or transfected with the nucleic acid and / or vector according to the invention as defined above in the section "Nucleic Acids, Vectors and Recombinant Host Cells".

[0515] When a host cell is transformed, transduced, or transfected to express a bispecific antigen-binding protein of the present invention, it is preferred that the cell contain an expression vector capable of expressing the antigen-binding protein. After a host cell expresses a bispecific antigen-binding protein of the present invention, the host cell can be referred to as a primed host cell.

[0516] In one aspect, a TCR-induced immune response or T cell response can refer to the induction of proliferation and activation of effector functions by binding a bispecific antigen to a T cell and a TA antigen peptide / MHC complex (e.g., a TA-C / MHC complex) in vitro or in vivo. For MHC class I-restricted cytotoxic T cells, for example, effector functions may include lysis of peptide-pulsed, peptide precursor-pulsed, or naturally peptide-presenting target cells, secretion of cytokines, preferably peptide-induced interferon-γ, TNF-α, or IL-2, secretion of effector molecules, such as peptide-induced granzymes or perforins, or degranulation.

[0517] Therefore, another aspect of the present invention provides an activated host cell prepared by the aforementioned method of the present invention.

[0518] The activated host cells produced by the above method can selectively recognize target cells.

[0519] In this article, target cells ” refers to the above “ Bispecific antigen binding proteins TA-C / MHC presenting cells or TA presenting cells as defined in the ” section.

[0520] In a preferred embodiment, ie when the TA / MHC complex is a TAA / MHC complex, the target cell is a cancer cell, wherein the cancer is as defined above.

[0521] According to the present invention, the in vivo target cells of CD3-positive effector cells can be tumor cells (sometimes expressing MHC class II antigens) and / or stromal cells surrounding the tumor (tumor cells) (sometimes also expressing MHC class II antigens; (Dengjel, J. et al., Clin Cancer Res 12 (2006): 4163-4170).

[0522] Kit

[0523] Finally, the present invention also provides a kit comprising at least one bispecific antigen binding protein of the present invention.

[0524] In one embodiment, the kit comprises

[0525] a) at least one bispecific antigen-binding protein of the invention as defined above in the section “Bispeci?c Antigen-Binding Proteins”,

[0526] b) optionally packaging material, and

[0527] c) optionally, a label or leaflet contained in the packaging material, indicating that the bispecific antigen binding protein is effective in treating a disease, preferably cancer, or can be used to treat a disease, preferably cancer.

[0528] In a related embodiment, at least one antigen binding protein of the invention is contained in a single-chamber and / or multi-chamber pre-filled syringe (eg, liquid syringes and lyophilized syringes).

[0529] In one embodiment, the invention includes a kit for producing single-dosage administration units.

[0530] Therefore, in one embodiment, the at least one bispecific antigen-binding protein of the invention as mentioned in a) of the kit of parts of the invention is a dried bispecific antigen-binding protein of the invention contained in a first container. The kit further comprises a second container having an aqueous formulation.

[0531] Thus, in one embodiment, the kit comprises

[0532] a) a first container comprising at least one dried bispecific antigen-binding protein of the invention as defined above in the section "Antigen-binding proteins",

[0533] b) a second container comprising an aqueous formulation;

[0534] c) optionally packaging material, and

[0535] d) optionally, a label or leaflet contained in the packaging material, indicating that the bispecific antigen binding protein is effective in treating a disease, preferably cancer, or can be used to treat a disease, preferably cancer.

[0536] Aqueous formulations generally refer to aqueous solutions containing a pharmaceutically acceptable carrier as defined above in the "Pharmaceutical Compositions" section.

[0537] In a related embodiment, "first container" and "second container" refer to the chambers of a multi-chamber pre-filled syringe (eg, a lyophilizing syringe).

[0538] Cancer in the context of the present invention is defined above.

[0539] The invention also relates to the items and aspects cited below.

[0540] In another aspect, the present invention relates to a bispecific antigen binding protein comprising at least two antigen binding sites (D and B), wherein the antigen binding site D binds to TCRα / β, wherein the antigen binding site B binds to the target antigen (TA) peptide / MHC complex, wherein the antigen binding site D comprises a heavy chain variable domain (V H ) and light chain variable domain (V L ), wherein the VL comprises or consists of the amino acid sequence of SEQ ID NO:42, and wherein the VH comprises or consists of the amino acid sequence of SEQ ID NO:43.

[0541] In a related item of the above aspect, the antigen binding site D of the bispecific antigen binding protein of the above aspect binds to TCRα / β with KD(D), and the antigen binding site B binds to the target antigen peptide C (TA-C) / MHC complex with KD(C), wherein K D (D) / K D The ratio of (C) 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, for example: between 1 and 150, 4 to 140, 6 to 100, 8 to 100, 10 to 100, preferably between 10 and 100.

[0542] In another related item of said aspect, the antigen binding site D is ≥3 nM, ≥5 nM, ≥8 nM, ≥10 nM, ≥12 nM, ≥14 nM, ≥16 nM, ≥18 nM, ≥20 nM, ≥25 nM, ≥30 nM, ≥35 nM, ≥40 nM, ≥45 nM and preferably ≤1000 nM, ≤800 nM, ≤600 nM, ≤500 nM, ≤4 00nM (e.g., 3nM to 1000nM, 3nM to 600nM, 5nM to 600nM, 10nM to 600nM, 12nM to 600nM, 14nM to 600nM, 16nM to 600nM, 18nM to 600nM, 20nM to 600nM, preferably 5nM to 100nM) for binding to TCRα / β. D (D) is preferably measured using surface plasmon resonance (SPR) or biolayer interferometry (BLI), preferably biolayer interferometry (BLI).

[0543] In another related item of that aspect, the antigen binding site B binds to the target antigen peptide C (TA-C) / MHC complex with a KD(C) of ≤100 μM, ≤1 μM, ≤100 nM, ≤50 nM, ≤10 nM, e.g., 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), wherein the KD(C) D (C) Measured using surface plasmon resonance (SPR) or biolayer interferometry (BLI), preferably biolayer interferometry (BLI).

[0544] In another related item of the above aspect, the antigen binding protein is resistant to EC of TA-C / MHC presenting cells. 50 EC compared to normal tissue cells 50 The value is ≥100, ≥500, or ≥1000 times lower.

[0545] In another related item of the aspect, the TA antigen peptide C is a viral peptide, a bacterial peptide or a tumor-associated antigen (TAA) peptide, preferably a tumor-associated antigen (TAA) peptide.

[0546] In another related item of the above aspect, the EC50 of the antigen binding protein for TA-C / MHC complex presenting cells is higher than the EC50 for normal tissue cells. 50 The value is ≥5 times, ≥10 times, ≥20 times, ≥50 times, ≥100 times, ≥500 times, ≥1000 times lower.

[0547] In another related item of the aspect, the TA antigen peptide C is a tumor-associated antigen (TAA) peptide C, wherein the TAA-C is selected from the group of TAA antigen peptides comprising or consisting of the amino acid sequences of SEQ ID NOs: 162 to 317, SEQ ID NOs: 9 and 10, such as, a PRAME antigen peptide comprising or consisting of the amino acid sequence "SLLQHLIGL" of SEQ ID NO: 9, or a MAGE-A antigen peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 10, wherein the MHC is preferably HLA-A*02.

[0548] In another related item of the aspect, the TA antigen peptide C is a MAGE-A antigen peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO: 10, wherein 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.

[0549] In another related item of this aspect, 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.

[0550] In another related item of the aspect, the antigen binding site B comprises an antibody or fragment thereof or an α chain variable domain (vα) and a β chain variable domain (vβ) or a γ chain variable domain (vγ) or a δ chain variable domain (vδ), preferably an α chain variable domain (vα) and a β chain variable domain (vβ) or a γ chain variable domain (vγ) and a δ chain variable domain (vδ), preferably vα and vβ.

[0551] In another related aspect,

[0552] i) vα comprises or consists of an amino acid sequence selected from the group consisting of:

[0553] "EDVEQSLFLSVREGDSVVINCTYTDSSSTYLYWYKQEPGKGLQLLTYIYSSQDSKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP" SEQ ID NO: 20,

[0554] "EDVEQSLFLSVREGDSVVINCTYTDSSSTYLYWYKQEPGKGLQLLTYIYSSQDQKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP" SEQ ID NO: 21,

[0555] “EDVEQSLFLSVREGDSVVINCTYTESSSTYLYWYKQEPGKGLQLLTYIYSSQDQKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEMTSESKIIFGSGTRLSIRP” SEQ ID NO: 22, or an amino acid sequence having at least 85% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21 and 22, and wherein the amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 20 preferably comprises the amino acid sequence of CDRa1 of SEQ ID NO: 23, CDRa2 of SEQ ID NO: 24 and CDRa3 of SEQ ID NO: 25, wherein the amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 21 preferably comprises the amino acid sequence of CDRa1 of SEQ ID NO: 23, CDRa2 of SEQ ID NO: 26 and CDRa3 of SEQ ID NO: 25, wherein the amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: The amino acid sequence of SEQ ID NO:22 having at least 85% identity preferably comprises 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 acids of the first variable domain preferably comprise amino acids 19V and / or 48K, and

[0556] vβ contains

[0557] "DAGVIQSPRHEVTEMGQEVTLRCKPIPGHDYLFWYRQTMMRGLELLFYFCYGTPCDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRADTGELFFGEGSRLTVL" the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 85% identity thereto or consisting of the same, wherein preferably the amino acid sequence having at least 85% identity thereto preferably comprises the amino acid sequence of CDRb1 of SEQ ID NO: 31, CDRb2 of SEQ ID NO: 34, and CDRb3 of SEQ ID NO: 35, respectively, and optionally comprises amino acids 54F and / or 66C, or

[0558] (ii) vα or vγ comprises or consists of the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 48, wherein preferably the amino acid sequence that 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, and

[0559] vβ or vδ comprises or consists of the amino acid sequence of SEQ ID NO:44, or an amino acid sequence that is at least 85% identical to SEQ ID NO:44, wherein preferably the amino acid sequence that 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.

[0560] In one of said aspects, the antigen binding protein comprises the formula V3-L1-V4-L2-C L -L5-Fc1[III] (comprising or consisting of the amino acid sequence of SEQ ID NO: 282 or 284), and a first polypeptide of the formula V5-L3-V6-L4-C H1 - a second polypeptide of L6-Fc2[IV] (which comprises or consists of the amino acid sequence of SEQ ID NO: 283).

[0561] In another related item of that aspect, the bispecific antigen binding protein further comprises one or more of the following:

[0562] (i) diagnostic agents;

[0563] (ii) a therapeutic agent; or

[0564] (iii) Pharmacokinetic (PK) modification part.

[0565] In another related item of said aspect, the isolated nucleic acid comprises a sequence encoding the bispecific antigen binding protein as defined in the above aspects and items, or the nucleic acid vector comprises said nucleic acid.

[0566] In another related item of the aspect, a recombinant host cell comprises the bispecific antigen binding protein defined in the above aspects and items, or the nucleic acid or vector defined in the above items, wherein the 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.

[0567] Another related item of the aspect relates to a pharmaceutical composition comprising the bispecific antigen binding protein defined in the above aspects and items, the nucleic acid or vector defined in the above aspects and items, or the host cell defined in the above aspects and items, and a pharmaceutically acceptable carrier, diluent stabilizer and / or excipient.

[0568] Another related item of said aspects relates to a method for producing a bispecific antigen binding protein as defined in the above aspects and items, comprising

[0569] a. Propose a suitable host cell,

[0570] b. Proposing a gene construct comprising a coding sequence encoding a bispecific antigen-binding protein as defined in the above aspects and items,

[0571] c. introducing the gene construct into the appropriate host cell, and

[0572] d. expressing the gene construct by the suitable host cell.

[0573] In another related item, the method defined in the above aspects and items further comprises isolating and purifying the bispecific antigen binding protein from a suitable host cell.

[0574] Another related item of said aspect relates to the use of 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 in medicine.

[0575] In another related item, the use of the bispecific antigen-binding protein defined in the above aspects and items, the nucleic acid or vector defined in the above aspects and items, the host cell defined in the above aspects and items, or the pharmaceutical composition defined in the above aspects and items in the diagnosis, prevention and / or treatment of diseases (such as viral or bacterial infections or proliferative diseases, preferably cancer, more preferably TAA / MHC-positive cancer).

[0576] The definitions and embodiments used in this patent application apply to the above-mentioned aspects and items herein with appropriate modifications.

[0577] Throughout the present application, the term "and / or" is a grammatical conjunction and should be interpreted as encompassing the possibility that one or more of the circumstances to which it is connected may occur. For example, the phrase "such native sequence proteins can be prepared using standard recombinant and / or synthetic methods" means that the native sequence proteins can be prepared using standard recombinant and synthetic methods, or that the native sequence proteins can be prepared using standard recombinant methods or that the native sequence proteins can be prepared using synthetic methods.

[0578] Furthermore, throughout the present application, the term “ Include " should be interpreted as including all the features specifically mentioned as well as optional, additional, and unspecified features. As used herein, the use of the term "comprising" also discloses embodiments in which there are no features other than those specifically mentioned (i.e., " Composed of ”).

[0579] In addition, the indefinite article "a" or " one A plurality of measures is not excluded. 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.

[0580] The present invention will now be described in more detail with reference to the following drawings and examples. All documents and patent files cited herein are incorporated herein by reference in their entirety. Although the present invention has been described and illustrated in detail in the foregoing description, the embodiments are intended to be illustrative or exemplary rather than restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0581] Figure 1 The alignment of the UCHT1 VL domain with the defined human acceptor framework is shown. The CDRs identified in the figure are defined according to the Cothia definition.

[0582] Figure 2 The alignment of the UCHT1 VH domain with the determined human acceptor framework is shown. The CDRs identified in the figure are defined according to the Cothia definition.

[0583] Figure 3 Demonstrated specificity of PRAME-004 as measured by flow cytometry Concentration-dependent binding of the molecules to Jurkat cells.

[0584] Figure 4 Representative LDH release assay results using PBMCs from healthy HLA-A*02 positive donors are shown. MAG-003 specificity using UCHT1 (V17) or BMA031 (V36) was tested on MAG-003 positive and MAG-003 negative as well as off-target positive tumor cell lines. Molecules. Cell lines tested (from left to right): H695T, A375, T98G, BV173. Error bars represent the standard deviation of three replicates.

[0585] Figure 5 The specificity of MAG-003 based on UCHT1 (V17) was shown. Results of LDH release assays on incubated healthy cells. Each cytotoxicity graph shows LDH release by primary healthy cell types (open circles) relative to control tumor cell line Hs695T (filled circles) in the same culture medium composition after co-incubation of PBMC with increasing concentrations of TCER molecules. A safety window calculated based on the EC50 value is also depicted.

[0586] Figure 6 The specificity of MAG-003 based on BMA031 (V36) was shown. Results of LDH release assay on incubated healthy cells. Each cytotoxicity graph shows LDH release of primary healthy cell types (open circles) relative to control tumor cell line Hs695T (filled circles) after incubation of PBMC with increasing concentrations of TCER molecules in the same medium composition. 50 value or safety window calculated based on LOEL.

[0587] Figure 7Visualizations of introduced point mutations are 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 nearby. Panel B shows the wt Tyr and the substituted Gln at position 54 of the UCHT1 heavy chain. Substitution of the aromatic side chain with a less polar amino acid eliminates hydrophobic interactions with the nonpolar stem of Asp at position 48 of CD3ε. Panel C shows the replacement of the same Tyr at position 54 of the UCHT1 heavy chain with Glu and the negatively charged side chains of CD3ε (48D, 49E, 50D, 51D) located nearby, which may lead to electrostatic repulsion of the newly introduced Glu on UCHT1. Panel D shows the wt Lys at position 55 of the UCHT1 heavy chain forming a hydrogen bond (dashed line) with the Ser backbone at position 56 of CD3ε (left). Substitution of the Lys with Arg eliminates polar interactions and introduces additional bulk, resulting in a strained rotamer of the Arg side chain (right). Figure E shows the same lysine at position 55 of the UCHT1 heavy chain replaced by Glu; this mutation also eliminates the H-bond formed between Lys and Ser at position 56 of CD3ε. In addition, it introduces a negatively charged side chain near the negatively charged patch on the CD3ε surface formed by Asp 48, Glu 49, Asp 50, and Asp 51.

[0588] Figure 8 Shown are humanized UCHT1 variants based on Summary of the yield and stability characteristics of the molecule. (na) Not applicable, (nd) Not done.

[0589] Figure 9A Shows the biolayer interferometry measurements Binding curves of antigen binding proteins (comprising different UCHT1 variants) for MAG-003 complexed with HLA-A*02. The concentration of the molecule is expressed in nM.

[0590] Figure 9B Shows the biolayer interferometry measurements Binding curves of antigen binding proteins (including different UCHT1 variants) for CD3δε-Fc. The concentration of the molecule is expressed in nM.

[0591] Figure 10 Results from two independent LDH release assays using PBMCs from two healthy HLA-A*02 positive donors (HBC-982 and HBC-720) are shown. MAG-003 specificity using various affinity UCHT1 variants was tested on MAG-003 positive tumor cell lines. Numerator. Error bars represent the standard deviation of three replicates. Example

[0592] Example 1: Humanization of mouse monoclonal antibody UCHT1

[0593] Humanization of the mouse monoclonal antibody UCHT1 was performed using CDR grafting according to published methods. The VH and VL CDRs were determined according to the Cothia definition. A sequence alignment was generated to compare the UCHT1 variable domain with human germlines. Based on overall sequence identity, matching interface positions, and class-similar CDR canonical positions, a germline was identified as the most promising acceptor framework for each light and heavy chain: VK1-018 for the light chain and VH-1-46 for the heavy chain. The FR4 of the J segment gene was compared with the parental sequence, and J segments JK1 and JH4 were selected for the light and heavy chains, respectively.

[0594] A list of all positions containing different residues between the parental and acceptor frameworks was generated. All positions were analyzed and considered in isolation and with other possible substitutions. Positions were classified as neutral, critical, or contributing, and suggestions were made for which residues could be substituted and evaluated in humanized variants. TM (Lonza) screened potential humanized variant sequences. For each epitope or epitope cluster, substitutions that could eliminate the epitope or further reduce the predicted immunogenicity were analyzed. Potential sites of post-translational modification within the CDRs were further identified and corresponding remedial measures were proposed. Overall, this resulted in the production of four different VH domains and five different 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 based on expression titer, aggregation level and EC binding to Jurkat cells. 50 Based on these results, humanized UCHT1 (V17) defined by SEQ ID No: 137 and SEQ ID No: 145 was selected to determine the inventors' molecular.

[0595] PRAME-004 (SEQ ID No: 9) targeting dUTP was constructed using the recruitment domain of humanized UCHT1 (V17) (obtaining molecules comprising SEQ ID No: 171 and SEQ ID No. 170) or humanized BMA031 (V10) (obtaining SEQ ID No: 168 and SEQ ID No: 169), respectively. The vector used to express the recombinant protein was designed as a monocistronic vector controlled by a pUC19 derivative of the 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). The expression of the recombinant protein was performed according to the manufacturer's instructions (ExpiCHO TM system; Thermo Fisher Scientific) and the purified plasmid DNA was used for transient transfection of CHO-S cells. The transfected CHO cells were cultured at 32°C to 37°C for 6-14 days and received one or two ExpiCHO TM Feed solution.

[0596] Conditioned cell supernatants were collected by centrifugation (4000×g; 30 min) and clarified by filtration (0.22 μm). The bispecific molecules were purified using a Pure 25L FPLC system (GE Lifesciences) for online affinity and size exclusion chromatography. Affinity chromatography was performed on a Protein A column (GE Lifesciences) according to a standard affinity chromatography protocol. Size exclusion chromatography was performed directly after elution from the affinity column (pH 2.8) to obtain highly pure monomeric protein using a Superdex 200pg 16 / 600 column (GE Lifesciences) according to a standard protocol. Protein concentration was determined on a NanoDrop system (Thermo Scientific) using an extinction coefficient calculated based on the predicted protein sequence. Concentration (if necessary) and buffer exchange were performed using a Vivaspin device (Sartorius). Finally, the purified molecules were stored in phosphate-buffered saline at a concentration of approximately 1 mg / mL at 2-8°C.

[0597] These The binding affinity of the molecules to effector cells was assessed by flow cytometry. Therefore, Jurkat cells (CD3+ and TCRab+) were incubated with increasing concentrations of After washing, the cell-bound The molecules are dyed. Cells were analyzed on the iQue cell screening instrument. Figure 3 Results from one of four independent experiments are shown, demonstrating that PRAME-004 is specific The molecules bind to Jurkat cells in a concentration-dependent manner. The EC50 of the molecule binding is about 2-3nM, and the BMA031-based The binding of the molecules to Jurkat cells was at least 50-100 times weaker.

[0598] Example 2: Demonstration of the principle of cytotoxicity using recruits of different affinities

[0599] The method for producing an antigen binding protein targeting peptide MAG-003 (SEQ ID No: 10) is as follows: The engineered variable domains of the T cell receptor (SEQ ID No: 20 and SEQ ID No: 30) were combined with the variable domains of UCHT1 (V17) (SEQ ID No: 137 and SEQ ID No: 145) or BMA031 (V36) (SEQ ID No: 42 and SEQ ID No: 43), respectively. The vector of the molecule is designed as a monocistronic vector controlled by a pUC19 derivative, a promoter element derived from HCMV. Plasmid DNA was amplified in E. coli according to standard culture methods and subsequently purified using a commercially available kit (Macherey & Nagel). The purified plasmid DNA was used to transiently transfect CHO-S cells using an electroporation system (MaxCyte STX). The transfected CHO cells were cultured at 32°C to 37°C for 10-12 days and received one to three injections of Cellboost 7a and 7b (GE Healthcare). TM ) solution feed.

[0600] Using Sartoclear The conditioned cell supernatant was clarified by filtration (0.22 μm) using Lab Filter Aid (Sartorius). The bispecific antigen-binding protein was purified using a Pure 25L FPLC system (GE Lifesciences) for online affinity and size exclusion chromatography. Affinity chromatography was performed on a MAbSelect SuRE or Protein L column (GE Lifesciences) according to a standard affinity chromatography protocol. Size exclusion chromatography was performed directly after elution from the affinity column (pH 2.8) to obtain highly pure monomeric protein using a Superdex 200pg26 / 600 column (GE Lifesciences) according to a standard protocol. Protein concentration was determined on a NanoDrop system (ThermoScientific) using an extinction coefficient calculated based on the predicted protein sequence. Concentrations were adjusted using a Vivaspin device (Sartorius) if necessary. Finally, the purified molecules were stored in phosphate-buffered saline at a concentration of approximately 1 mg / mL at 2-8°C.

[0601] The cytotoxic activity of the bispecific molecules against MAG-positive and MAG-negative tumor cell lines was analyzed by LDH-release assay. Tumor cell lines expressing varying amounts of HLA-A*02 / MAG-003 on their cell surface were incubated with PBMCs isolated from healthy donors (HLA-A*02+) in the presence of increasing concentrations of the molecule. After 48 hours, lysis of the target cell lines was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (PROMEGA).

[0602] Exemplary results of such an assay are shown in the accompanying Figure (Example 2). Table 5 summarizes the EC values ​​obtained. 50 value.

[0603] Table 5: Comparison of EC obtained from killing assays with different recruiting antibodies 50 Value summary.

[0604]

[0605] These results suggest that BMA031 (V36)-based molecules are more effective than UCHT1 (V17)-based molecules. The potency of the molecule was reduced (23-fold against target-high expressing cell lines and 96-fold against target-low expressing cell lines). 50 The safety window can be calculated as described in the “Definition” section above. In short, the safety window is defined as the EC value for killing off-target expressing cells. 50 ECs that kill target-expressing (TAA) cells 50 This means that based on UCHT1 (V17) The safety window is about 49 times, and the BMA031 (V36) The safety window was increased by approximately 312-fold (comparing off-target expressing tumor cell lines with target (TAA) high expressing tumor cell lines).

[0606] These findings suggest that, in general, the use of low-affinity recruitment domains may improve discrimination between targets and off-targets, thereby increasing the safety window. To further test this hypothesis, the specificity of MAG-003 was evaluated. To this end, 11 different primary healthy tissue cells (HLA-A*02+) were co-cultured with PBMC effector cells from healthy HLA-A*02+ donors at an E:T ratio of 10:1 and LDH was measured in increasing concentrations. Cells were co-incubated in a 50% mixture of primary tissue cell-specific medium and optimal T cell medium. To determine the safety window, primary cells and the corresponding medium composition of 100% optimal T cell medium were cultured. The molecules were co-incubated with the MAG-003-positive tumor cell line Hs695T under the same conditions to eliminate bias caused by different culture media. After 48 hours of co-culture, the supernatant was collected and analyzed for cell lysis by measuring LDH release using the LDH-Glo™ kit (Promega).

[0607] exist Figure 5 and Figure 6 In the figures, each cytotoxicity graph shows LDH release from primary healthy cell types (open circles) relative to the control tumor cell line Hs695T (filled circles) after incubation of PBMCs with increasing concentrations of TCER molecules in the same medium composition. Figure 5 Summary of MAG-003 specificity based on UCHT1(V17) Results for the molecule. Strong reactivity was detected for all cell types tested, except for nasal epithelial cells and PBMCs, and respective EC50 values ​​could be determined. Based on the EC50 values, the safety window was calculated as described in the definition section above. The x-fold safety window is indicated in the figure. For the UCHT1 (V17)-based The most critical safety windows were determined for the following cells: astrocytes (48-fold), dermal microvascular endothelial cells (94-fold), and mesenchymal stem cells (170-fold).

[0608] Using BMA031(V36) When the molecule was expressed, all responses to healthy primary cells were too low to calculate an EC50. Instead, we defined a safety window based on the lowest observed effect level (LOEL), which was determined as the first response exceeding the cutoff value. Concentration. The critical value is defined as

[0609] ([standard deviation of all triplicate values ​​x 3] + [no TCER control])

[0610] (The no TCER control is represented by a dotted line in each cytotoxicity graph) and used as the threshold for determining the LOEL and safety window between healthy tissue cells and tumor control cell lines. All confirmed safety windows are greater than 1000 times. Figure 5 and Figure 6 It can be clearly concluded from the comparison that MAG-003 specificity In the context of this molecule, the safety window can be significantly expanded by utilizing the low-affinity recruiter BMA031(V36).

[0611] Example 3: Generation of affinity-reduced humanized UCHT1 variants

[0612] To obtain a low-affinity variant of the CD3-specific humanized antibody UCHT1 (V17), a structure-guided design was performed. Based on the solved structure of UCHT1 in complex with its target CD3δ / ε (PDB ID: 1xiw), point mutations were introduced into the antibody that were hypothesized to reduce affinity without destabilizing the protein itself.

[0613] To achieve this goal, positions were selected primarily in the CDRs; from the solved structures it could be inferred that the interface between the two proteins is primarily formed between CD3ε and the antibody heavy chain, and therefore only mutations in positions in the heavy chain were considered.

[0614] For clarification, positions on CD3ε are numbered sequentially according to the PDB entry ID 1xiw, chain ID: A.

[0615] G31E will introduce a negative charge on the antibody surface facing the negatively charged surface patch formed by CD3ε48D, CD3ε49E, CD3ε50D and CD3ε51D, which may cause electrostatic repulsion and thus reduce affinity.

[0616] Y54Q alters the shape complementarity of the binding surface and abolishes the hydrophobic interaction between the Y54 aromatic ring and the nonpolar stem of CD3ε48D.

[0617] Y54E alters the shape complementarity of the binding surface, abolishes the hydrophobic interaction between the Y54 aromatic ring and the nonpolar stem of CD3ε48D, and additionally introduces a negative charge facing the negatively charged patch formed by CD3ε48D, CD3ε49E, CD3ε50D, and CD3ε51D.

[0618] K55R introduces a larger side chain with similar physicochemical properties, thereby eliminating the H-bond formed between the Nζ of 55K and the CD3ε56S backbone. The increased side chain size may also lead to slight changes in binding geometry.

[0619] K55E replaces the positive charge with a negative charge, thereby eliminating the H-bond formed between the Nζ of 55K and the CD3ε36S backbone. In addition, the introduction of a negative charge can lead to electrostatic repulsion with the negatively charged patch formed by CD3ε57D, CD3ε58E, and CD3ε59D.

[0620] Based on these findings, sequences encoding UCHT1(V20) to UCHT1(V27) were generated and are summarized in Table 6.

[0621] In an attempt to further optimize the humanized UCHT1 sequence, a potential post-translational modification site within CDR-H3 (Asp-isomerization, 106D107S) was eliminated by introducing 106E. This modification was introduced into UCHT1(V17), UCHT1(20), UCHT1(V21), and UCHT1(V23), generating the variants UCHT1(V17opt), UCHT1(V20opt), UCHT1(V21opt), and UCHT1(V23opt), respectively.

[0622] Table 6: Sequence combinations to generate humanized UCHT1 variants.

[0623]

[0624]

[0625] Using the humanized UCHT1 variants described 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) can be generated, prepared and purified as described above, respectively. molecular.

[0626] Example 4: Affinity determination of designed UCHT1 variants

[0627] In order to determine affinity using biofilm interferometry, the molecule CD3δε-Fc was generated. Thus, the extracellular domains of human CD3δ and CD3ε were fused to the N-terminus of the Fc domain, as The same constructs were used (including the knob-to-hole mutation and an additional C-terminal His-tag), resulting in SEQ ID No: 161 and SEQ ID No: 162, respectively.

[0628] CD3δε-Fc molecules were expressed in ExpiCHO cells and purified using protein A affinity chromatography followed by size exclusion chromatography as described above.

[0629] For bispecifics comprising different UCHT1 variants (as shown in Table 6) Antigen binding protein, using biofilm interferometry to identify MAGE-A antigen peptide (SEQ ID NO: 10) complexed with HLA-A*02 ( Figure 9A , Table 7) and for CD3δε-Fc ( Figure 9B The binding affinity characteristics of the α-binding peptides (A, B, C, and D) were analyzed. The measurements were performed on an OctetRED384 system using the manufacturer's recommended settings. Briefly, binding kinetics were measured at 30°C and 1000 rpm using PBS, 0.05% Tween-20, and 0.1% BSA as buffer. Before the molecule was added, peptide-HLA-A*02 complex or CD3δε-Fc was loaded onto the biosensor (HIS1K). The molecules all showed similar binding to HLA-A*02 / MAG-003, K D The CD3δε affinity encompasses a window greater than 200-fold, with a K D The values ​​ranged from 3 to 750 nM.

[0630] Table 7: Comprising different UCHT1 variants according to Table 6 Molecular affinity analysis. K D Biofilm interferometry

[0631]

[0632]

[0633] Example 5: Low-affinity humanized UCHT1 variants have reduced potency

[0634] As described above in Example 2, MAG-003 specific The cytotoxic potency of the molecules was assessed using an LDH release assay. Representative assay results are shown in Figure 2. Figure 10 As expected, the high affinity recruitment domain of UCHT1(V17) was detected. Compared to the newly designed variants (UCHT1 (V17opt), UCHT1 (V20), UCHT1 (V21), UCHT1 (V23)), the efficacy was reduced. Ranking the molecules also closely reflects the affinity of each elicitor variant (highest potency: UCHT1(V17) < UCHT1(V21) < UCHT1(V20) < UCHT1(V17opt) < UCHT1V23). These effects on potency can only be attributed to the elicitor domain, since the TCR domains have comparable affinity for MAG-003 complexed with HLA-A*02 (Figure 9). Sequence Listing <110> Immatics Biotechnologies GmbH <120> Modified Bispecific Anti-CD3 Antibody <130> 1017-23 <150> DE 10 2019 121 022.4 <151> 2019-08-02 <150> US 62 / 882,364 <151> 2019-08-02 <160> 286 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL5_CDRL1 <400> 1 Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn 1 5 10<​​​​​​​​​​​​​​​​​​​​​​​​​​​​<223> VL5_CDRL3 <400> 3 Gln Gln Gly Gln Thr Leu Pro Trp Thr 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial sequence <220> <223> CDRH1_Consensus <220> <221> MISC_FEATURE <222> (1)..(1) <223> Wherein, Xaa is any amino acid, preferably G or E, more preferably G <400> 4 Xaa Tyr Thr Met Asn 1 5 <210> 5 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDRH2_Consensus <220> <221> MISC_FEATURE <222> (5)..(5) <223> Wherein, Xaa is any amino acid, preferably Q, Y or E, more preferably Q <220> <221> MISC_FEATURE <222> (6) <223> Wherein, Xaa is any amino acid, preferably R, K or E, more preferably Preferably R or K, for example K <220> <221> MISC_FEATURE <222> (9)..(9) <223> Wherein, Xaa is any amino acid, preferably S or T, more preferably Preferably S <220> <221> MISC_FEATURE <222> (15)..(15) <223> Wherein, Xaa is any amino acid, preferably F or V, more preferably F <220> <221> MISC_FEATURE <222> (17) <223> Wherein, Xaa is any amino acid, preferably G or D, more preferably D <400> 5 Leu Ile Asn Pro Xaa Xaa Gly Val Xaa Thr Tyr Ala Gln Lys Xaa Gln 1 5 10 15 Xaa <210> 6 <211> 12 <212> PRT <213> Artificial sequence <220> <223> CDRH3_Consensus <220> <221> MISC_FEATURE <222> (6) <223> Wherein, Xaa is any amino acid, preferably D or E, more preferably D <400> 6 Ser Gly Tyr Tyr Gly Xaa Ser Trp Tyr Phe Asp Val 1 5 10 <210> 7 <211> 17 <212> PRT <213> Artificial sequence <220> <223> CDRH2_ VH2,8,16,19,20 <400> 7 Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Tyr Ala Gln Lys Phe Gln 1 5 10 15 Asp <210> 8 <211> 13 <212> PRT <213> Artificial sequence <220> <223> CDRH3_VH2-15,19,21 <400> 8 Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val 1 5 10 <210> 9 <211> 9 <212> PRT <213> Artificial sequence <220> <223> PRAME-004 <400> 9 Ser Leu Leu Gln His Leu Ile Gly Leu 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial sequence <220> <223> MAG-003 <400> 10 Lys Val Leu Glu His Val Val Arg Val 1 5 <210> 11 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> FR1-a (L) <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 <210> 12 <211> 15 <212> PRT <213> Artificial sequence <220> <223> FR2-a (L) <400> 12 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 13 <211> 15 <212> PRT <213> Artificial sequence <220> <223> FR2-a (L) <400> 13 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile Tyr 1 5 10 15 <210> 14 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR3-a (L) <400> 14 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys 20 25 30 <210> 15 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FR4-a (L) <400> 15 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 <210> 16 <211> 30 <212> PRT <213> Artificial sequence [[ID=第十七条 <223> FR1-b (H) <400> 16 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr 20 25 30 <210> 17 <211> 14 <212> PRT <213> Artificial sequence <220> <223> FR2-b (H) <400> 17 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 <210> 18 <211> 32 <212> PRT <213> Artificial sequence <220> <223> FR3-b (H) <400> 18 Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 ​Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 19 <211> 11 <212> PRT <213> artificial sequence <220> <223> FR3-b (H) <400> 19 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 20 <211> 109 <212> PRT <213> artificial sequence <220> <223> Va_114_iso0 <400> 20 Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp Ser 1 5 10 15 Val Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu Tyr 20 25 30 Trp Tyr Lys Gln Glu Pro Gly Lys Gly Leu Gln Leu Leu Thr Tyr Ile 35 40 45 Tyr Ser Ser Gln Asp Ser Lys Gln Asp Gln Arg Leu Thr Val Leu Leu 50 55 60 Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr Gln Thr 65 70 75 80 Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Met Thr Ser Glu Ser Lys 85 90 95 Ile Ile Phe Gly Ser Gly Thr Arg Leu Ser Ile Arg Pro 100 105 <210> 21 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Va_114_iso1 <400> 21 Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp Ser 1 5 10 15 Val Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu Tyr 20 25 30 Trp Tyr Lys Gln Glu Pro Gly Lys Gly Leu Gln Leu Leu Thr Tyr Ile 35 40 45 Tyr Ser Ser Gln Asp Gln Lys Gln Asp Gln Arg Leu Thr Val Leu Leu 50 55 60<\(0002045\)>Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr Gln Thr 65 70 75 80 Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Met Thr Ser Glu Ser Lys 85 90 95 Ile Ile Phe Gly Ser Gly Thr Arg Leu Ser Ile Arg Pro 100 105 <210> 22 <211> 109 <212> PRT <213> artificial sequence <220> <223> Va_114_iso2 <400> 22 Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp Ser 1 5 10 15 Val Val Ile Asn Cys Thr Tyr Thr Glu Ser Ser Ser Thr Tyr Leu Tyr 20 25 30 Trp Tyr Lys Gln Glu Pro Gly Lys Gly Leu Gln Leu Leu Thr Tyr Ile 35 40 45 Tyr Ser Ser Gln Asp Gln Lys Gln Asp Gln Arg Leu Thr Val Leu Leu 50 55 60 Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr Gln Thr 65 70 75 80 Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Met Thr Ser Glu Ser Lys 85 90 95 Ile Ile Phe Gly Ser Gly Thr Arg Leu Ser Ile Arg Pro 100 105 <210> 23 <211> 6 <212> PRT <213> artificial sequence <220> <223> Va_114_iso0 / 1_CDRa1 <400> 23 Asp Ser Ser Ser Thr Tyr 1 5 <210> twenty four <211> 7 <212> PRT <213> Artificial sequence <220> <223> Va_114_iso0_CDRa2 <400> twenty four Ile Tyr Ser Ser Gln Asp Ser 1 5 <210> 25 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Va_114_CDRa3 <400> 25 Cys Ala Glu Met Thr Ser Glu Ser Lys Ile Ile Phe 1 5 10 <210> 26 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Va_114_iso1 / 2_CDRa2 <400> 26 Ile Tyr Ser Ser Gln Asp Gln 1 5 <210> 27 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Va_114_iso2_CDRa1 <400> 27 Glu Ser Ser Ser Thr Tyr 1 5 <210> 28 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 28 Gly Gly Ser Gly Gly 1 5 <210> 29 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 29 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 1 5 10 <210> 30 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Vb_114 <400> 30 Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly 1 5 10 15 Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Pro Gly His Asp Tyr Leu 20 25 30 Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Phe Tyr 35 40 45 Phe Cys Tyr Gly Thr Pro Cys Asp Asp Ser Gly Met Pro Glu Asp Arg 50 55 60 Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln 65 70 75 80 Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Arg Ala 85 90 95 Asp Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu 100 105 110 <210> 31 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Vb_114_CDRb1 (rather PGHDY) <400> 31 Gly His Asp Tyr 1 <210> 32 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 32 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 1 5 10 15 <210> 33 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 33 Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly 20 <210> 34 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Vb_114_CDRb2 <400> 34 Phe Cys Tyr Gly Thr Pro 1 5 <210> 35 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Vb_114_CDRb3 <400> 35 Cys Ala Ser Arg Ala Asp Thr Gly Glu Leu Phe Phe 1 5 10 <210> 36 <211> 108 <212> PRT <213> Artificial sequence <220> <223> VL mUCHT1 <400> 36 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 ​​​​​ 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 37 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> VH_mUCHT1 <400> 37 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met 35 40 45 Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 38 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL_V9_Shalaby <400> 38 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 39 <211> 122 <212> PRT <213> Artificial sequence <220> <223> VH_V9_Shalaby <400> 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr[[ID=3i]] 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 40 <211> 106 <212> PRT <213> Artificial sequenceIt should be noted that there seems to be a misspelling in the original text where in line 31 the tag is " " but the text refers to "3i" which might be an error. This has been left as is in the translation. <220> <223> VL_BMA_Shearman <400> 40 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Met Thr Cys Ser Ala Thr Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ile Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 41 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH_BMA_Shearman <400> 41 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser ​​Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val His Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Asp Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 42 <211> 106 <212> PRT <213> artificial sequence <220> <223> VL_BMA(36) <400> 42 Gln Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Thr Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 43 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> VH_BMA(36) <400> 43 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Val Thr Lys Tyr Ala Glu Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ser Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val His Tyr Cys 85 90 95 Ala Arg Gly Ser Tyr Tyr Asp Tyr Glu Gly Phe Val Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 44 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> HiAff1_Vb <400> 44 Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys Thr Arg Gly 1 5 10 15 Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Pro Gly His Arg Ala Val 20 25 30 Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe Leu Phe Glu 35 40 45 Tyr Val His Gly Glu Glu Arg Asn Lys Gly Asn Phe Pro Gly Arg Phe 50 55 60 Ser Gly Arg Gln Phe Ser Asn Ser Ser Ser Glu Met Asn Ile Ser Asn 65 70 75 80 Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser Pro Trp 85 90 95 Asp Ser Pro Asn Val Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val 100 105 110 Thr Glu Asp Leu Lys Asn 115 <210> 45 <211> 5 <212> PRT <213> Artificial sequence <220> <223> HiAff1_CDRb1 <400> 45 Pro Gly His Arg Ala 1 5 <210> 46 <211> 6 <212> PRT <213> Artificial sequence <220> <223> HiAff1_CDRb2 <400> 46 Tyr Val His Gly Glu Glu 1 5 <210> 47 <211> 14 <212> PRT <213> Artificial sequence <220> <223> HiAff1_CDRb3 <400> 47 Cys Ala Ser Ser Pro Trp Asp Ser Pro Asn Val Gln Tyr Phe 1 5 10 <210> 48 <211> 117 <212> PRT <213> artificial sequence <220> <223> HiAff1_Va <400> 48 Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser 20 25 30 Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 35 40 45 Ser Ile Tyr Gln Glu Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 50 55 60 Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Ala Val Ile Asp Asn Asp 85 90 95 Gln Gly Gly Ile Leu Thr Phe Gly Thr Gly Thr Arg Leu Thr Ile Ile 100 105 110 Pro Asn Ile Gln Asn 115 <210> 49 <211> 6 <212> PRT <213> artificial sequence <220> <223> HiAff1_CDRa1 <400> 49 Asp Arg Gly Ser Gln Ser 1 5 <210> 50 <211> 6 <212> PRT <213> Artificial sequence <220> <223> HiAff1_CDRa2 <400> 50 Ile Tyr Gln Glu Gly Asp 1 5 <210> 51 <211> 15 <212> PRT <213> Artificial sequence <220> <223> HiAff1_CDRa3 <400> 51 Cys Ala Ala Val Ile Asp Asn Asp Gln Gly Gly Ile Leu Thr Phe 1 5 10 15 <210> 52 <211> 10 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 52 Tyr Leu Tyr Asp Ser Glu Thr Lys Asn Ala 1 5 10 <210> 53 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 53 His Leu Met Asp Gln Pro Leu Ser Val 1 5 <210> 54 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 54 Gly Leu Leu Lys Lys Ile Asn Ser Val 1 5 <210> 55 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 55 Phe Leu Val Asp Gly Ser Ser Ala Leu 1 5 <210> 56 <211> 10 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 56 Phe Leu Phe Asp Gly Ser Ala Asn Leu Val 1 5 10 <210> 57 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 57 Phe Leu Tyr Lys Ile Ile Asp Glu Leu 1 5 <210> 58 <211> 11 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 58 Phe Ile Leu Asp Ser Ala Glu Thr Thr Thr Leu 1 5 10 <210> 59 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 59 Ser Val Asp Val Ser Pro Pro Lys Val 1 5 <210> 60 <211> 8 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 60 Val Ala Asp Lys Ile His Ser Val 1 5 <210> 61 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 61 Ile Val Asp Asp Leu Thr Ile Asn Leu 1 5 <210> 62 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 62 Gly Leu Leu Glu Glu Leu Val Thr Val 1 5 <210> 63 <211> 10 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 63 Thr Leu Asp Gly Ala Ala Val Asn Gln Val 1 5 10 <210> 64 <211> 10 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 64 Ser Val Leu Glu Lys Glu Ile Tyr Ser Ile 1 5 10 <210> 65 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 65 Leu Leu Asp Pro Lys Thr Ile Phe Leu 1 5 <210> 66 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 66 Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 <210> 67 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 67 Tyr Leu Met Asp Asp Phe Ser Ser Leu 1 5 <210> 68 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 68 Lys Val Trp Ser Asp Val Thr Pro Leu 1 5 <210> 69 <211> 11 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 69 Leu Leu Trp Gly His Pro Arg Val Ala Leu Ala 1 5 10 <210> 70 <211> 11 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 70 Lys Ile Trp Glu Glu Leu Ser Val Leu Glu Val 1 5 10 <210> 71 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 71 Leu Leu Ile Pro Phe Thr Ile Phe Met 1 5 <210> 72 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 72 Phe Leu Ile Glu Asn Leu Leu Ala Ala 1 5 <210> 73 <211> 11 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 73 Leu Leu Trp Gly His Pro Arg Val Ala Leu Ala 1 5 10 <210> 74 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 74 Phe Leu Leu Glu Arg Glu Gln Leu Leu 1 5 <210> 75 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 75 Ser Leu Ala Glu Thr Ile Phe Ile Val 1 5 <210> 76 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 76 Thr Leu Leu Glu Gly Ile Ser Arg Ala 1 5 <210> 77 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 77 Ile Leu Gln Asp Gly Gln Phe Leu Val 1 5 <210> 78 <211> 10 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 78 Val Ile Phe Glu Gly Glu Pro Met Tyr Leu 1 5 10 <210> 79 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 79 Ser Leu Phe Glu Ser Leu Glu Tyr Leu 1 5 <210> 80 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 80 Ser Leu Leu Asn Gln Pro Lys Ala Val 1 5 <210> 81 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 81 Gly Leu Ala Glu Phe Gln Glu Asn Val 1 5 <210> 82 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 82 Lys Leu Leu Ala Val Ile His Glu Leu 1 5 <210> 83 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 83 Thr Leu His Asp Gln Val His Leu Leu 1 5 <210> 84 <211> 11 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 84 Thr Leu Tyr Asn Pro Glu Arg Thr Ile Thr Val 1 5 10 <210> 85 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 85 Lys Leu Gln Glu Lys Ile Gln Glu Leu 1 5 <210> 86 <211> 10 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 86 Ser Val Leu Glu Lys Glu Ile Tyr Ser Ile 1 5 10 <210> 87 <211> 11 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 87 Arg Val Ile Asp Asp Ser Leu Val Val Gly Val 1 5 10 <210> 88 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 88 Val Leu Phe Gly Glu Leu Pro Ala Leu 1 5 <210> 89 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 89 Gly Leu Val Asp Ile Met Val His Leu 1 5 <210> 90 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 90 Phe Leu Asn Ala Ile Glu Thr Ala Leu 1 5 <210> 91 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 91 Ala Leu Leu Gln Ala Leu Met Glu Leu 1 5 <210> 92 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 92 Ala Leu Ser Ser Ser Gln Ala Glu Val 1 5 <210> 93 <211> 11 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 93 Ser Leu Ile Thr Gly Gln Asp Leu Leu Ser Val 1 5 10 <210> 94 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 94 Gln Leu Ile Glu Lys Asn Trp Leu Leu 1 5 <210> 95 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 95 Leu Leu Asp Pro Lys Thr Ile Phe Leu 1 5 <210> 96 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 96 Arg Leu His Asp Glu Asn Ile Leu Leu 1 5 <210> 97 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 97 Gly Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Gly Lys 1 5 10 15 Ser <210> 98 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 98 Gly Leu Pro Ser Ala Thr Thr Thr Val 1 5 <210> 99 <211> 11 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 99 Gly Leu Leu Pro Ser Ala Glu Ser Ile Lys Leu 1 5 10 <210> 100 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 100 Lys Thr Ala Ser Ile Asn Gln Asn Val 1 5 <210> 101 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 101 Tyr Leu Met Asp Asp Phe Ser Ser Leu 1 5 <210> 102 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 102 Leu Met Tyr Pro Tyr Ile Tyr His Val 1 5 <210> 103 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 103 Lys Val Trp Ser Asp Val Thr Pro Leu 1 5 <210> 104 <211> 11 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 104 Leu Leu Trp Gly His Pro Arg Val Ala Leu Ala 1 5 10 <210> 105 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 105 Val Leu Asp Gly Lys Val Ala Val Val 1 5 <210> 106 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 106 Gly Leu Leu Gly Lys Val Thr Ser Val 1 5 <210> 107 <211> 9 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 107 Lys Met Ile Ser Ala Ile Pro Thr Leu 1 5 <210> 108 <211> 11 <212> PRT <213> Artificial sequence <220> <223> TAA <400> 108 Gly Leu Leu Glu Thr Thr Gly Leu Leu Ala Thr 1 5 10 <210> 109 <211> 9 <212> PRT <213> artificial sequence <220> <223> AAT <400> 109 Thr Leu Asn Thr Leu Asp Ile Asn Leu 1 5 <210> 110 <211> 9 <212> PRT <213> artificial sequence <220> <223> AAT <400> 110 Val Ile Ile Lys Gly Leu Glu Glu Ile 1 5 <210> 111 <211> 317 <212> PRT <213> artificial sequence <220> <223> MAGE-A4_P43358 <400> 111 Met Ser Ser Glu Gln Lys Ser Gln His Cys Lys Pro Glu Glu Gly Val 1 5 10 15 Glu Ala Gln Glu Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Thr 20 25 30 Thr Glu Glu Gln Glu Ala Ala Val Ser Ser Ser Ser Pro Leu Val Pro 35 40 45 Gly Thr Leu Glu Glu Val Pro Ala Ala Glu Ser Ala Gly Pro Pro Gln 50 55 60 Ser Pro Gln Gly Ala Ser Ala Leu Pro Thr Thr Ile Ser Phe Thr Cys 65 70 75 80 Trp Arg Gln Pro Asn Glu Gly Ser Ser Ser Gln Glu Glu Glu Gly Pro 85 90 95 Ser Thr Ser Pro Asp Ala Glu Ser Leu Phe Arg Glu Ala Leu Ser Asn 100 105 110 Lys Val Asp Glu Leu Ala His Phe Leu Leu Arg Lys Tyr Arg Ala Lys 115 120 125 Glu Leu Val Thr Lys Ala Glu Met Leu Glu Arg Val Ile Lys Asn Tyr 130 135 140 Lys Arg Cys Phe Pro Val Ile Phe Gly Lys Ala Ser Glu Ser Leu Lys 145 150 155 160 Met Ile Phe Gly Ile Asp Val Lys Glu Val Asp Pro Ala Ser Asn Thr 165 170 175 Tyr Thr Leu Val Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly 180 185 190 Asn Asn Gln Ile Phe Pro Lys Thr Gly Leu Leu Ile Ile Val Leu Gly 195 200 205 Thr Ile Ala Met Glu Gly Asp Ser Ala Ser Glu Glu Glu Ile Trp Glu 210 215 220 Glu Leu Gly Val Met Gly Val Tyr Asp Gly Arg Glu His Thr Val Tyr 225 230 235 240 Gly Glu Pro Arg Lys Leu Leu Thr Gln Asp Trp Val Gln Glu Asn Tyr 245 250 255 Leu Glu Tyr Arg Gln Val Pro Gly Ser Asn Pro Ala Arg Tyr Glu Phe 260 265 270 Leu Trp Gly Pro Arg Ala Leu Ala Glu Thr Ser Tyr Val Lys Val Leu 275 280 285 Glu His Val Val Arg Val Asn Ala Arg Val Arg Ile Ala Tyr Pro Ser 290 295 300 Leu Arg Glu Ala Ala Leu Leu Glu Glu Glu Glu Gly Val 305 310 315 <210> 112 <211> 318 <212> PRT <213> Synthetic sequence <220> <223> MAGE - A8_P43361 <400> 112 Met Leu Leu Gly Gln Lys Ser Gln Arg Tyr Lys Ala Glu Glu Gly Leu 1 5 10 15 Gln Ala Gln Gly Glu Ala Pro Gly Leu Met Asp Val Gln Ile Pro Thr 20 25 30 Ala Glu Glu Gln Lys Ala Ala Ser Ser Ser Ser Thr Leu Ile Met Gly 35 40 45 Thr Leu Glu Glu Val Thr Asp Ser Gly Ser Pro Ser Pro Pro Gln Ser 50 55 60 Pro Glu Gly Ala Ser Ser Ser Leu Thr Val Thr Asp Ser Thr Leu Trp 65 70 75 80 Ser Gln Ser Asp Glu Gly Ser Ser Ser Asn Glu Glu Glu Gly Pro Ser 85 90 95 Thr Ser Pro Asp Pro Ala His Leu Glu Ser Leu Phe Arg Glu Ala Leu 100 105 110 Asp Glu Lys Val Ala Glu Leu Val Arg Phe Leu Leu Arg Lys Tyr Gln 115 120 125 Ile Lys Glu Pro Val Thr Lys Ala Glu Met Leu Glu Ser Val Ile Lys 130 135 140 Asn Tyr Lys Asn His Phe Pro Asp Ile Phe Ser Lys Ala Ser Glu Cys 145 150 155 160 Met Gln Val Ile Phe Gly Ile Asp Val Lys Glu Val Asp Pro Ala Gly 165 170 175 His Ser Tyr Ile Leu Val Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu 180 185 190 Leu Gly Asp Asp Gln Ser Thr Pro Lys Thr Gly Leu Leu Ile Ile Val 195 200 205 Leu Gly Met Ile Leu Met Glu Gly Ser Arg Ala Pro Glu Glu Ala Ile 210 215 220 Trp Glu Ala Leu Ser Val Met Gly Leu Tyr Asp Gly Arg Glu His Ser 225 230 235 240 Val Tyr Trp Lys Leu Arg Lys Leu Leu Thr Gln Glu Trp Val Gln Glu 245 250 255 Asn Tyr Leu Glu Tyr Arg Gln Ala Pro Gly Ser Asp Pro Val Arg Tyr 260 265 270 Glu Phe Leu Trp Gly Pro Arg Ala Leu Ala Glu Thr Ser Tyr Val Lys 275 280 285 Val Leu Glu His Val Val Arg Val Asn Ala Arg Val Arg Ile Ser Tyr 290 295 300 Pro Ser Leu His Glu Glu Ala Leu Gly Glu Glu Lys Gly Val 305 310 315 <210> 113 <211> 5 <212> PRT<(...)><213> Artificial sequence <...

Claims

1. A bispecific antigen-binding protein comprising at least two antigen-binding sites B and D, wherein: Antigen binding site D binds to T cell receptor (TCR) α / β, and wherein antigen binding site B binds to target antigen (TA) peptide / MHC complex, and wherein antigen binding site D comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), and wherein (i) the VL consists of the amino acid sequence of SEQ ID NO: 42, and (ii) the VH consists of the amino acid sequence of SEQ ID NO:

43.

2. The bispecific antigen-binding protein according to claim 1, wherein The antigen binding protein binds to the EC of TA-C / MHC presenting cells 50 EC compared to normal tissue cells 50 The value is ≥100 times, ≥500 times, or ≥1000 times lower.

3. The bispecific antigen-binding protein of claim 1, wherein the antigen-binding site B binds to a target antigen (TA) peptide C / MHC complex, and the TA peptide C is a viral peptide, a bacterial peptide, or a tumor-associated antigen (TAA) peptide.

4. The bispecific antigen-binding protein of claim 3, wherein the TA peptide C is a tumor-associated antigen (TAA) peptide C (TAA-C), and wherein the TAA-C is selected from the group of TAA antigen peptides comprising or consisting of the amino acid sequences of SEQ ID NOs: 52 to 65, 67 to 96, 98, SEQ ID NOs: 172 to 182, 184 to 268, SEQ ID NOs: 9 and 10.

5. The bispecific antigen-binding protein according to claim 4, wherein The TA peptide C is a MAGE-A antigen peptide comprising or consisting of the amino acid sequence "KVLEHVVRV" of SEQ ID NO:

10.

6. The bispecific antigen-binding protein of claim 1, wherein the bispecific antigen-binding protein is a bispecific T cell receptor (TCR) or a fragment thereof or a bispecific single-chain TCR (scTCR).

7. The bispecific antigen-binding protein according to any one of claims 1 to 6, further comprising one or more of the following: (i) diagnostic agents; (ii) a therapeutic agent; or (iii) Pharmacokinetic (PK) modification part.

8. One or more isolated nucleic acids or a nucleic acid vector comprising a sequence encoding the bispecific antigen-binding protein according to any one of claims 1 to 7.

9. A recombinant host cell comprising the bispecific antigen binding protein according to any one of claims 1 to 7 or one or more isolated nucleic acids or vectors according to claim 8, wherein the host cell is a cell for recombinant expression.

10. The recombinant host cell of claim 9, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

11. A pharmaceutical composition comprising the bispecific antigen binding protein according to any one of claims 1 to 7, one or more isolated nucleic acids or vectors according to claim 8 or the host cell according to claim 9 or 10, and a pharmaceutically acceptable carrier, diluent, stabilizer and / or excipient.

12. A method for producing a bispecific antigen-binding protein according to any one of claims 1 to 7, comprising a) providing a suitable host cell, b) providing a gene construct comprising a coding sequence encoding the bispecific antigen-binding protein according to any one of claims 1 to 7, c) introducing the gene construct into the appropriate host cell, and d) expressing the bispecific antigen binding protein by the suitable host cell.

13. The method of claim 12, further comprising isolating and purifying the bispecific antigen binding protein from a suitable host cell.

14. Use of the bispecific antigen binding protein according to any one of claims 1 to 7, the one or more isolated nucleic acids or vectors according to claim 8, the host cell according to claim 9 or 10, or the pharmaceutical composition according to claim 11 for the preparation of a medicament for treating melanoma.

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