Combination therapy with anti-EGFRvIII / anti-CD3 antibodies and tumor-targeted 4-1BB agonists

Abstract

This invention relates to a combination therapy of an anti-EGFRvIII / anti-CD3 bispecific antibody and a tumor-targeted 4-1BB(CD137) agonist.

Description

[Technical Field]

[0001] This invention relates to a combination therapy of an anti-EGFRvIII / anti-CD3 bispecific antibody and a tumor-targeted 4-1BB agonist. [Background technology]

[0002] Cancer is one of the leading causes of death worldwide. Despite advances in treatment options, the prognosis for patients with advanced cancer remains poor. Glioblastoma, in particular, has a poor prognosis. Only 41% of glioblastoma patients survive one year after initial diagnosis, and less than 7% survive five years. Therefore, there is a persistent and urgent medical need for optimal treatments to improve cancer patient survival without causing unacceptable toxicity. Recent results from clinical trials have shown that immunotherapy extends overall survival and provides a sustained response in cancer patients. Despite these promising results, current immuno-based therapies are only effective for a portion of patients, and combination strategies are needed to improve treatment efficacy.

[0003] One way to mobilize a patient's own immune system to fight cancer is through T-cell bispecific antibodies (TCBs). Anti-EGFRvIII / anti-CD3 bispecific antibodies are molecules that target epidermal growth factor receptor variant III (EGFRvIII), which is expressed on tumor cells, and the CD3 epsilon chain (CD3ε), which is present on T cells. Simultaneous binding activates T cells, leading to the release of cytokines and subsequent T-cell-mediated necrosis of tumor cells.

[0004] 4-1BB (CD137), a member of the TNF receptor superfamily, was first identified as an inducible molecule expressed by T cell activation (Kwon and Weissman, 1989, Proc Natl Acad Sci USA 86, 1963-1967). Subsequent studies have demonstrated that many other immune cells, including NK cells, B cells, NKT cells, monocytes, neutrophils, mast cells, dendritic cells (DCs), and non-hematopoietic cells such as endothelial and smooth muscle cells, also express 4-1BB (Vinay and Kwon, 2011, Cell Mol Immunol 8, 281-284). Expression of 4-1BB in different cell types is primarily inducible and driven by various stimulative signals, such as T cell receptor (TCR) or B cell receptor triggers, as well as signaling induced via receptors for co-stimulatory molecules or pro-inflammatory cytokines (Diehl et al., 2002, J Immunol 168, 3755-3762; Zhang et al., 2010, Clin Cancer Res 13, 2758-2767).

[0005] The ligand for 4-1BB (4-1BBL or CD137L) was identified in 1993 (Goodwin et al., 1993, Eur J Immunol 23, 2631-2641). Expression of 4-1BBL has been shown to be limited to specialized antigen-presenting cells (APCs) such as B cells, dendritic cells, and macrophages. Inducible expression of 4-1BBL is characteristic of T cells, including both αβ and γδ T cell subsets, as well as endothelial cells (Shao and Schwarz, 2011, J Leukoc Biol 89, 21-29).

[0006] Co-stimulation via the 4-1BB receptor (e.g., by 4-1BBL ligation) triggers multiple signaling cascades within T cells (CD4 + and CD8 +It activates both subsets and potently enhances T cell activation (Bartkowiak and Curran, 2015). It triggers TCR, and when used in combination with an agonist 4-1BB-specific antibody, it enhances T cell proliferation, stimulates lymphokine secretion, and reduces the sensitivity of T lymphocytes to activation-induced cell death (Snell et al., 2011, Immunol Rev 244, 197-217). This mechanism has been further advanced as the first proof of concept in cancer immunotherapy. In a preclinical model, administration of an agonist antibody against 4-1BB in tumor-bearing mice resulted in a potent antitumor effect (Melero et al., 1997, Nat Med 3, 682-685). Subsequent cumulative evidence has shown that 4-1BB is typically effective as an antitumor agent when administered in combination with other immunomodulatory compounds, chemotherapy agents, tumor-specific vaccines, or radiotherapy (Bartkowiak and Curran, 2015, Front Oncol 5, 117).

[0007] Because TNFR superfamily signaling requires cross-linking of trimerized ligands to bind to receptors, 4-1BB agonist antibodies must have wild-type Fc binding (Li and Ravetch, 2011, Science 333, 1030-1034). However, systemic administration of 4-1BB-specific agonist antibodies with functionally active Fc domains is associated with hepatotoxicity related to CD8 + This leads to T cell influx (Dubrot et al., 2010, Cancer Immunol Immunother 59, 1223-1233), which is reduced or significantly improved in mice in the absence of functional Fc receptors. In clinical trials, the Fc-competent 4-1BB agonist Ab (BMS-663513) (NCT00612664) caused grade 4 hepatitis, leading to the discontinuation of the trial (Simeone and Ascierto, 2012, J Immunotoxicol 9, 241-247). Therefore, there is a need for an effective and safer 4-1BB agonist.

[0008] Fusion proteins have been created consisting of a single extracellular domain of a 4-1BB ligand and a single-chain antibody fragment (Hornig et al., 2012, J Immunother 35, 418-429; Muller et al., 2008, J Immunother 31, 714-722) or a single 4-1BB ligand fused to the C-terminus of a heavy chain (Zhang et al., 2007, Clin Cancer Res 13, 2758-2767). PCT Publication No. 2010 / 010051 discloses the generation of a fusion protein consisting of three TNF ligand ectodomains that bind to each other and are fused to the antibody moiety. In this invention, an antigen-binding molecule consisting of a trimer, and therefore biologically active, 4-1BB ligand and an antigen-binding domain and an Fc-inactivating domain specific to the tumor-associated antigen FAP is shown to be particularly stable and robust (hereinafter referred to as FAP-4-1BBL). The FAP antigen-binding domain is replaced by FAP-targeted specific crosslinking, which replaces nonspecific FcγR-mediated crosslinking, particularly in the liver, causing Fc-mediated toxicity. Crosslinking by tumor (stromal) antigens allows for the administration of 4-1BB agonists.

[0009] Anti-EGFRvIII / anti-CD3 bispecific antibodies are described, for example, in PCT Publication No. 2020 / 127619. Tumor-targeted 4-1BB agonists are described, for example, in PCT Publication No. 2016 / 075278 or PCT Publication No. 2016 / 156291. [Overview of the project]

[0010] It has been found that combining a targeted 4-1BBL antigen-binding molecule with an anti-EGFRvIII / anti-CD3 bispecific antibody, i.e., an EGFRvIII TCB, achieves a better antitumor effect of 4-1BB agonism. The T cell bispecific antibody provides initial TCR activation signaling to T cells, and the combination with FAP-4-1BBL then results in a further boost to antitumor T cell immunity. Accordingly, we describe in this specification a novel combination therapy for tumors expressing EGFRvIII (EGFRvIII-positive cancer).

[0011] In one embodiment, the present invention provides an anti-EGFRvIII / anti-CD3 bispecific antibody combined with a tumor-targeted 4-1BB(CD137) agonist for use as an adjunctive therapy in the treatment of cancer. In a further embodiment, the present invention provides the use of an anti-EGFRvIII / anti-CD3 bispecific antibody combined with a tumor-targeted 4-1BB agonist in the manufacture of a pharmaceutical for treating cancer. In another embodiment, the present invention provides a method for treating cancer in an individual, comprising administering the individual an anti-EGFRvIII / anti-CD3 bispecific antibody in combination with a tumor-targeted 4-1BB agonist. In a further embodiment, the present invention provides a kit comprising a first pharmaceutical comprising an anti-EGFRvIII / anti-CD3 bispecific antibody and a second pharmaceutical comprising a tumor-targeted 4-1BB agonist, further optionally comprising a package insert containing instructions for administering the first pharmaceutical in combination with the second pharmaceutical for treating cancer in an individual.

[0012] In any one embodiment of the above aspects, an anti-EGFRvIII / anti-CD3 bispecific antibody combined with a tumor-targeted 4-1BB agonist for use as described in any one of the preceding claims, in a use, method or kit, the anti-EGFRvIII / anti-CD3 bispecific antibody (i) specifically binds to EGFRvIII and comprises a heavy chain variable region (VH) including heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a light chain complementarity-determining region (LCDR) of SEQ ID NO: 4. (ii) A first antigen-binding moiety comprising (ii) a light chain variable region (VL) including 1, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; and a second antigen-binding moiety comprising a heavy chain variable region (VH) that specifically binds to CD3 and includes a heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 9, HCDR2 of SEQ ID NO: 10, and HCDR3 of SEQ ID NO: 11, and a light chain variable region (VL) including a light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14.

[0013] In one embodiment, the anti-EGFRvIII / anti-CD3 bispecific antibody includes (i) a first antigen-binding moiety that specifically binds to EGFRvIII, comprising VH which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7 and VL which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8, and (ii) a second antigen-binding moiety that specifically binds to CD3, comprising VH which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 and VL which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16. In one embodiment, the first antigen-binding moiety and / or the second antigen-binding moiety of the anti-EGFRvIII / anti-CD3 bispecific antibody is a Fab molecule. In further embodiments, the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1, particularly the variable domains VL and VH of the Fab light chain and Fab heavy chain, are substituted for each other. In another embodiment, the first antigen-binding moiety is a Fab molecule in which, in the constant domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), in the constant domain CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering). In one embodiment, the first antigen-binding portion and the second antigen-binding portion are fused with each other, and optionally fused via a peptide linker.In further embodiments, the first and second antigen-binding moieties are each Fab molecules, and (i) the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain, or (ii) the first antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen-binding moiety at the C-terminus of the Fab heavy chain. In one embodiment, the anti-EGFRvIII / anti-CD3 bispecific antibody includes a third antigen-binding moiety. In another embodiment, the third antigen-binding moiety is identical to the first antigen-binding moiety. In one embodiment, the anti-EGFRvIII / anti-CD3 bispecific antibody includes an Fc domain composed of first and second subunits. In one embodiment, each of the first, second, and, if present, third antigen-binding moieties is a Fab molecule, and (i) the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain, and the first antigen-binding moiety is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, or (ii) the first antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen-binding moiety at the C-terminus of the Fab heavy chain, and the second antigen-binding moiety is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and, if present, the third antigen-binding moiety is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

[0014] In one embodiment, the Fc domain is an IgG Fc domain, particularly an IgG1 Fc domain. In another embodiment, the Fc domain is a human Fc domain. In a further embodiment, the Fc domain includes modifications that facilitate the association of the first and second subunits of the Fc domain, and / or the Fc domain includes one or more amino acid substitutions that reduce binding to the Fc receptor and / or effector function.

[0015] In one embodiment, the anti-EGFRvIII / anti-CD3 bispecific antibody against any embodiment includes the polypeptide sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0016] In one embodiment, the tumor-targeted 4-1BB agonist of any of the above embodiments includes an antigen-binding moiety that specifically binds to fibroblast-activating protein (FAP) or carcinoembryonic antigen (CEA). In one embodiment, the antigen-binding moiety that specifically binds to FAP includes a heavy chain variable region (VH) containing heavy chain CDR (HCDR)1 of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22, and HCDR3 of SEQ ID NO: 23, and a light chain variable region containing light chain CDR (LCDR)1 of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 26. In a further embodiment, the antigen-binding moiety that specifically binds to FAP includes VH which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27, and VL which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 28. In another embodiment, the antigen-binding moiety that specifically binds to FAP is a Fab molecule. In one embodiment, the tumor-targeted 4-1BB agonist comprises three ectodomains or fragments of 4-1BBL. In another embodiment, the three ectodomains of 4-1BBL comprise an amino acid sequence selected from the group consisting of SEQ ID NOs. 29, SEQ ID NOs. 30, SEQ ID NOs. 31, SEQ ID NOs. 32, SEQ ID NOs. 33, SEQ ID NOs. 34, SEQ ID NOs. 35, and SEQ ID NOs. 36, particularly an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NOs. 33. In yet another embodiment, the tumor-targeted 4-1BB agonist comprises first and second polypeptides linked to each other by disulfide bonds, the first polypeptide comprising two ectodomains or fragments of 4-1BBL linked to each other by a peptide linker, and the second polypeptide comprising one ectodomain or fragment of 4-1BBL.In one embodiment, the first polypeptide of the tumor-targeted 4-1BB agonist comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 37, 38, 39, 40, 41, 42, 43, and 44, and the second polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 29, 30, 31, 32, 33, 34, 35, and 36. In a further embodiment, the tumor-targeted 4-1BB agonist comprises an Fc domain composed of first and second subunits. In one embodiment, the Fc domain of a tumor-targeted 4-1BB agonist includes modifications that promote the association of the first and second subunits of the Fc domain, and / or the Fc domain includes one or more amino acid substitutions that reduce binding to the Fc receptor and / or effector function.In one embodiment, a tumor-targeted 4-1BB agonist is an antigen-binding molecule comprising: (i) first and second polypeptides, wherein the first polypeptide comprises two ectodomains or fragments of 4-1BBL linked together by a peptide linker, and the second polypeptide comprises one ectodomain or fragment of 4-1BBL; (ii) an antigen-binding moiety that specifically binds to FAP and is a Fab molecule; (iii) an Fc domain composed of first and second subunits; and (iv) a CL domain and a CH1 domain, wherein the antigen-binding molecule comprises (a) a first heavy chain fused at its C-terminus to the N-terminus of the CL domain (b) a first heavy chain comprising a lipeptide and a CL domain fused at its C-terminus to the N-terminus of one of the subunits of the Fc domain (e.g., the first subunit), and one of the subunits of the Fc domain (e.g., the first subunit); (b) a first light chain comprising a second polypeptide fused at its C-terminus to the N-terminus of the CH1 domain, and a CH1 domain; (c) a second heavy chain comprising a Fab molecule heavy chain fused at its C-terminus to the N-terminus of the other of the subunits of the Fc domain (e.g., the second subunit), and the other of the subunits of the Fc domain (e.g., the second subunit); and (d) a second light chain comprising a Fab molecule light chain.

[0017] In one embodiment, the tumor-targeted 4-1BB agonist of any of the above embodiments is an antigen-binding molecule comprising: a first heavy chain containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45; a first light chain containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46; a second heavy chain containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47; and a second light chain containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48. [Brief explanation of the drawing]

[0018] [Figure 1] Figures 1A-C: Tumor cell death by EGFRvIII TCB. The induction of tumor cell death at 24 hours (Figure 1A), 48 hours (Figure 1B), and 72 hours (Figure 1C) by EGFRvIII TCB alone or in combination with 1nM FAP-4-1BBL or 1nM DP47-4-1BBL was determined during co-culture of U87MG-huEGFRvIII with PBMCs isolated from healthy donors. [Figure 2] Figures 2-E: Cytokine release by EGFRvIII TCBs. The induction of IL2 (Figure 2A), TNFα (Figure 2B), IL10 (Figure 2C), granzyme B (Figure 2D), and IFNγ (Figure 2E) release by EGFRvIII TCBs alone or in combination with 1nM FAP-4-1BBL or 1nM DP47-4-1BBL was determined during co-culture of U87MG-huEGFRvIII cells with PBMCs isolated from healthy donors. Cytokine release was measured after 72 hours. [Figure 3] We present the results of efficacy experiments evaluating EGFRvIII-TCB as a monotherapy and in combination with the FAP-41BBL costimulatory molecule. Tumor growth inhibition in a subcutaneous xenograft model of glioblastoma was studied by subcutaneous injection of the U87-huEGFRvIII human glioblastoma cell line into humanized NSG mice. The amount of antibody injected per mouse (mg / kg) was 0.03 for the EGFRvIII-TCB construct and 1 for the FAP-41BBL molecule. Antibodies were administered intravenously once a week for four weeks. The EGFRvIII-TCB + FAP-41BBL combination mediated superior efficacy in tumor growth inhibition compared to EGFRvIII-TCB and FAP-41BBL as monotherapy. [Modes for carrying out the invention]

[0019] definition Unless otherwise defined below, terms are used herein in the manner commonly used in the art.

[0020] As used herein, the terms “first,” “second,” or “third,” if present, with respect to antigen-binding moieties, Fc domains, heavy chains, or light chains, are used for convenience to distinguish between parts of each type when more than one is present. The use of these terms is not intended to assign any particular order or orientation of the parts unless expressly stated otherwise.

[0021] As used herein, the term “antigen-binding molecule” broadly refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules include antibodies, immunoglobulins, and their derivatives (e.g., fragments).

[0022] The term "antibody" as used herein is used in its broadest sense and encompasses a wide range of antibody structures, including, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they exhibit the desired antigen-binding activity.

[0023] An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFab), single-domain antibodies, and multispecific antibodies formed from antibody fragments. For an overview of specific antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0024] The terms "full-length antibody," "intact antibody," and "whole antibody" are used herein as interchangeable to refer to antibodies having a structure substantially similar to that of a native antibody.

[0025] When used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies in the population are identical and / or bind to the same epitope, except for possible variant antibodies containing, for example, naturally occurring mutations or mutations arising during the manufacture of the monoclonal antibody preparation, such variants are generally present in small amounts. Typically, in contrast to polyclonal antibody preparations which contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring antibody production by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0026] An “isolated” antibody is one that has been separated from its natural environment. In some embodiments, antibodies are purified to a purity of 95% or greater than 99%, as determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC, affinity chromatography, size exclusion chromatography). For an overview of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007). In some embodiments, the antibodies provided by the present invention are isolated antibodies.

[0027] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, while the rest of the heavy chain and / or light chain originates from a different source or species.

[0028] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody substantially contains all of at least one, typically two, variable domains, in which all or substantially all of the CDRs correspond to the CDRs of a non-human antibody and all or substantially all of the FRs correspond to the FRs of a human antibody. Such variable domains are referred to herein as “humanized variable regions.” A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which CDR residues are derived) to restore or improve antibody specificity or affinity, for example. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0029] A "human antibody" is defined as an antibody produced by a human or human cell, or an antibody having an amino acid sequence corresponding to a non-human antibody that utilizes a sequence encoding a human antibody, such as a human antibody repertoire. This definition of a human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. In certain embodiments, a human antibody may originate from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In certain embodiments, a human antibody may originate from a hybridoma cell line. Antibodies or antibody fragments isolated from a human antibody library are also considered human antibodies or human antibody fragments in this specification.

[0030] The term "antigen-binding region" refers to a portion of an antibody that includes a region that binds to and is complementary to a part or all of an antigen. The antigen-binding region may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). In a preferred embodiment, the antigen-binding domain includes an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0031] A "variable region" or "variable domain" is a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and complementarity-determining regions (CDRs). For example, Kindt et al., Kuby Immunology, 6 th See ed., WH Freeman & Co., page 91 (2007). A single VH domain or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using the VH domain or VL domain of the antibody that binds to that antigen, and complementary libraries of VL domains or VH domains can be screened. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). As used herein with respect to variable region sequences, “Kabat numbering” refers to the numbering system defined by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0032] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "Kabat numbering" or "Kabat numbering." Specifically, the Kabat numbering system (see pp. 647–660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domain CL of kappa and lambda isotypes, and the Kabat EU index numbering system (see pp. 661–723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3). The Kabat EU index numbering system is further clarified herein by the terms "Kabat EU index numbering" or "Kabat EU index numbering."

[0033] As used herein, the terms “hypervariable region” or “HVR” mean each of the regions of the antibody variable domain, such as “complementarity-determining regions” (CDRs), that are hypervariable within the sequence and determine antigen-binding specificity. Generally, antibodies contain six CDRs: three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs as used herein include: (a) Hypervariable loops located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (h1), 53-55 (h2), and 96-101 (h3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)), and (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J.Mol.Biol.262:732-745 (1996)).

[0034] Unless otherwise specified, the designation of the CDR is determined according to Kabat et al. above. Those skilled in the art will understand that the designation of the CDR may also be determined according to Chothia, McCallum, or any other scientifically recognized nomenclature above.

[0035] The term "framework" or "FR" refers to variable domain residues other than the complementarity-determining region (CDR). The variable domain FR generally consists of the following four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences are usually represented in the following order in VH (or VL): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0036] Unless otherwise indicated, CDR residues and other residues within the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al. cited above.

[0037] For the purposes of this specification, “acceptor human framework” means a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived” from a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0038] The "Human Consensus Framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Typically, these sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

[0039] In this specification, the term “immunoglobulin molecule” refers to a protein having the structure of a naturally occurring antibody. For example, IgG class immunoglobulins are heterotetrameric glycoproteins with a weight of approximately 150,000 daltons, composed of two disulfide-linked light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy chain domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light chain domain or light chain variable region, followed by a constant light chain (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types called α(IgA), δ(IgD), ε(IgE), γ(IgG), or μ(IgM), some of which may be further divided into subtypes such as γ1(IgG1), γ2(IgG2), γ3(IgG3), γ4(IgG4), α1(IgA1), and α2(IgA2). The light chain of an immunoglobulin may be assigned to one of two types called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via an immunoglobulin hinge region.

[0040] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region present in the heavy chain of the antibody or immunoglobulin. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0041] A "Fab molecule" refers to a protein consisting of the VH domain and CH1 domain of the immunoglobulin heavy chain ("Fab heavy chain") and the VL domain and CL domain of the immunoglobulin light chain ("Fab light chain").

[0042] A “crossover” Fab molecule (also called a “Crossfab”) means a Fab molecule in which the variable or constant domains of the Fab heavy chain and light chain are exchanged (i.e., replaced by each other). That is, a crossover Fab molecule includes a peptide chain composed of a light chain variable domain VL and a heavy chain constant domain 1 CH1 (VL-CH1, from N-terminus to C-terminus), and a peptide chain composed of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL, from N-terminus to C-terminus). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and Fab heavy chain are exchanged, the peptide chain containing the heavy chain constant domain 1 CH1 is referred to herein as the “heavy chain” of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule in which the constant domains of the Fab light chain and Fab heavy chain are exchanged, the peptide chain containing the heavy chain variable domain VH is referred to herein as the “heavy chain” of the (crossover) Fab molecule.

[0043] In contrast, a “conventional” Fab molecule refers to a Fab molecule in its natural form, that is, one comprising a heavy chain (VH-CH1, N-terminus to C-terminus) composed of a heavy chain variable domain and a constant domain, and a light chain (VL-CL, N-terminus to C-terminus) composed of a light chain variable domain and a constant domain.

[0044] In this specification, the terms “Fc domain” or “Fc region” are used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more amino acids from the C-terminus of the heavy chain, particularly one or two amino acids. Thus, by expression of certain nucleic acid molecules encoding a full-length heavy chain, antibodies produced by host cells may contain the full-length heavy chain or cleaved variants of the full-length heavy chain. This is the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. The amino acid sequence of the heavy chain containing the Fc region (or a subunit of the Fc domain as defined herein) is shown herein without the C-terminal glycine-lysine dipeptide unless otherwise shown. In one embodiment, the heavy chain containing the Fc region (subunit) specified herein, as contained in the antibody according to the present invention, includes an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment, the heavy chain containing the Fc region (subunit) specified herein, as contained in the antibody according to the present invention, includes an additional C-terminal glycine residue (G446, numbered according to the Kabat EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above).As used herein, a “subunit” of an Fc domain refers to one of the two polypeptides that form a dimer Fc domain (i.e., a polypeptide containing the C-terminal constant region of an immunoglobulin heavy chain that is capable of stable self-assembly). For example, a subunit of an IgG Fc domain includes the IgG CH2 and IgG CH3 constant domains.

[0045] "Fused" means that the constituent elements (e.g., the Fab molecule and the Fc domain subunit) are linked directly by peptide bonds or via one or more peptide linkers.

[0046] The term "multispecificity" means that an antibody can specifically bind to at least two different antigenic determinants. A multispecific antibody may be, for example, a bispecific antibody. Typically, a bispecific antibody contains two antigen-binding sites, each specific to a different antigenic determinant. In certain embodiments, a multispecific (e.g., bispecific) antibody can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0047] As used herein, the term “valence” refers to the presence of a specific number of antigen-binding sites within an antigen-binding molecule. In this case, the term “monovalent binding to an antigen” refers to the presence of one (and not more than one) antigen-specific antigen-binding sites within the antigen-binding molecule.

[0048] An "antigen-binding site" refers to the part of an antigen-binding molecule that interacts with an antigen, i.e., one or more amino acid residues. For example, the antigen-binding site of an antibody contains amino acid residues from the complementarity-determining region (CDR). Native immunoglobulin molecules typically contain two antigen-binding sites, while Fab molecules typically have one antigen-binding site.

[0049] As used herein, the term “antigenic determinant” is synonymous with “antigen” and “epitope,” and refers to a site on a polypeptide macromolecule (e.g., a configuration consisting of a sequence of amino acids or different regions of non-contiguous amino acids) to which an antigen-binding moiety binds, forming an antigen-binding domain-antigen complex. Useful antigenic determinants may be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other affected cells, on the surface of immune cells, free in serum, and / or within the extracellular matrix (ECM). Proteins referred to herein as antigens (e.g., CD3, EGFRvIII, FAP, 4-1BB) may be any native form of a protein from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. In a preferred embodiment, the antigen is a human protein.

[0050] As used herein, “T cell activating antigen” refers to an antigenic determinant expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, that can induce T cell activation through interaction with an antigen-binding molecule. Specifically, the interaction of an antigen-binding molecule with a T cell activating antigen can induce T cell activation by triggering a cascade of signaling in the T cell receptor complex. In certain embodiments, the T cell activating antigen is CD3, particularly the epsilon subunit of CD3.

[0051] As used herein, “T cell activation” refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and are described herein.

[0052] Unless otherwise specified, “CD3” refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus macaques), and rodents (e.g., mice and rats). The term encompasses “full-length,” untreated CD3, and any form of CD3 resulting from intracellular processing. The term also encompasses naturally occurring variants of CD3, such as splice variants or allele variants. In one embodiment, CD3 refers to human CD3, particularly the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in SEQ ID NO 50 (without signal peptide). See also accession number P07766 (version 189) (www.uniprot.org) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. In another embodiment, CD3 is cynomolgus monkey (Macaca fascicularis) CD3, particularly cynomolgus monkey CD3ε. See also NCBI GenBank no. BAB71849.1. In certain embodiments, the antibody of the present invention binds to CD3 epitopes that are conserved among CD3 antigens from different species, particularly human and cynomolgus monkey CD3. In a preferred embodiment, the antibody binds to human CD3.

[0053] As used herein, “target cell antigen” refers to an antigenic determinant presented on the surface of target cells, such as cancer cells or cells in a tumor, such as tumor stromal cells (in which case, “tumor cell antigen”). Preferably, the target cell antigen is not and / or expressed on cells different from CD3. In one preferred embodiment, the target cell antigen is EGFRvIII, particularly human EGFRvIII. In another preferred embodiment, the target cell antigen is FAP. In one embodiment, the target cell antigen is CEA.

[0054] "EGFRvIII" represents epidermal growth factor receptor variant III, a variant of EGFR, formed by an in-frame deletion of exons 2-7, resulting in a 267-amino acid deletion with a glycine substitution at the junction. The sequence of human EGFRvIII is shown in SEQ ID NO: 51 (without signal peptide). The sequence of human EGFR is shown in SEQ ID NO: 52 (without signal peptide). See also UniProt entry number P00533 (version 258). As used herein, "EGFRvIII" refers to native EGFRvIII from any vertebrate, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term includes "full-length" unprocessed EGFRvIII (but not wild-type EGFR), as well as any form of EGFRvIII resulting from intracellular processing (e.g., EGFRvIII without signal peptide). In one embodiment, EGFRvIII is human EGFRvIII.

[0055] The term “fibroblast-activating protein (FAP),” also known as prolyl endopeptidase FAP or seplacase (EC3.4.21), means, unless otherwise specified, any native FAP derived from any vertebrate source, including primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and mammals such as rodents (e.g., mice and rats). The term encompasses not only “full-length,” untreated FAP, but also any form of FAP resulting from intracellular processing. The term also encompasses naturally occurring variants of FAP, such as splice variants or allele variants. In one embodiment, the antigen-binding molecule of the present invention can specifically bind to human, mouse, and / or cynomolgus monkey FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884 (version 149) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004451.2. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760. The amino acid sequence of mouse FAP is shown in UniProt accession number P97321 (version 126) or NCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to 761. Preferably, the anti-FAP binding molecule of the present invention binds to the extracellular domain of FAP. An exemplary anti-FAP binding molecule is described in International Patent Application No. WO2012 / 020006A2.

[0056] The term "carcinoembryonic antigen (CEA)," also known as carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5), means any native CEA derived from any vertebrate source, including primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and mammals such as rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human CEA is shown in UniProt accession number P06731 (version 151).

[0057] As used herein, the terms “4-1BB” or “CD137” refer to native 4-1BB derived from vertebrates, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses “full-length,” unprocessed 4-1BB and 4-1BB resulting from intracellular processing. The term also encompasses naturally occurring variants of 4-1BB, such as splice variants or allele variants. The amino acid sequence of an exemplary human 4-1BB is shown in Uniprot accession number Q07011; registration version 185. The amino acid sequence of an exemplary mouse 4-1BB is shown in Uniprot accession number P20334, and the amino acid sequence of an exemplary cynomolgus monkey 4-1BB (from Macaca mulatta) is shown in Uniprot accession number F6W5G6.

[0058] "4-1BBL," or "4-1BB ligand," or "CD137L," is a member of the co-stimulatory TNF ligand family that can co-stimulate T cell proliferation and cytokine production. Co-stimulatory TNF family ligands co-stimulate TCR signaling through interaction with their corresponding TNF receptors, and this interaction recruits TNFR-related factors (TRAFs), initiating a signaling cascade that leads to T cell activation. 4-1BBL is a type II transmembrane protein. Complete or full-length 4-1BBL having the amino acid sequence shown in UniProt accession number P41273 (registration version 153) has been described as forming trimers on the cell surface. Trimerization is enabled by a specific motif in the ectodomain of 4-1BBL, which is referred to herein as the "trimerization region." Amino acids 50-254 of the human 4-1BBL sequence (SEQ ID NO: 49) form the extracellular domain of 4-1BBL, but fragments of this sequence can also form trimers.

[0059] An "ectodomain" is a domain of a membrane protein that extends into the extracellular space (i.e., the space outside the target cell). The ectodomain is typically the portion that initiates contact with the protein surface and leads to signal transduction. Therefore, as defined herein, the ectodomain of 4-1BBL refers to the extracellular domain of 4-1BBL, but also includes shorter portions or fragments responsible for trimerization and binding to the corresponding receptor 4-1BB. Thus, the term "ectodomain or fragment of 4-1BBL" refers to the extracellular domain of 4-1BBL that forms the extracellular domain, or the portion that can bind to the receptor (receptor-binding domain). Exemplary ectodomains or fragments of 4-1BBL are shown in the amino acid sequences of SEQ ID NO: 32 (amino acids 52-254 of human 4-1BBL), SEQ ID NO: 29 (amino acids 71-254 of human 4-1BBL), SEQ ID NO: 31 (amino acids 80-254 of human 4-1BBL), SEQ ID NO: 30 (amino acids 85-254 of human 4-1BBL), SEQ ID NO: 33 (amino acids 71-248 of human 4-1BBL), SEQ ID NO: 34 (amino acids 85-248 of human 4-1BBL), SEQ ID NO: 35 (amino acids 80-248 of human 4-1BBL), and SEQ ID NO: 36 (amino acids 52-248 of human 4-1BBL). Other fragments of the trimerizable ectodomain are also included herein.

[0060] "Specifically binds" means that the binding is antigen - selective and can be distinguished from unwanted or non - specific interactions. The binding ability of an antigen - binding portion to a specific antigen determinant can be measured by enzyme - linked immunosorbent assay (ELISA) or other techniques well - known to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed with a BIAcore device) (Liljeblad et al., Glyco J 17, 323 - 329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217 - 229 (2002)). In one embodiment, the degree of binding of the antigen - binding portion to an irrelevant protein is less than about 10% of the binding of the antigen - binding portion to the antigen measured, for example, by SPR. In certain embodiments, the antigen - binding portion that binds to an antigen, or an antigen - binding molecule containing such an antigen - binding portion, has a dissociation constant (K -8 M) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM or ≤ 0.001 nM (e.g., 10 -8 M - 10 -13 M, e.g., 10 -9 M - 10 -13 M).

[0061] "Affinity" refers to the total strength of non - covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured by well - established methods known in the art, including those described herein. A preferred method for measuring affinity is surface plasmon resonance (SPR).

[0062] ​​"Reduced binding," for example, reduced binding to the Fc receptor, refers to a decrease in affinity for each interaction, as measured, for example, by SPR. For clarity, this term also includes a reduction in affinity to zero (or below the detection limit of the analytical method), i.e., complete termination of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for each interaction.

[0063] As used herein, “T cell activation” refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and are described herein.

[0064] "Modifications that promote the association of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the association of a peptide containing an Fc domain subunit with an identical polypeptide for homodimer formation. As used herein, association-promoting modifications preferably involve distinct modifications made to each of two Fc domain subunits that are desired to associate (i.e., the first and second subunits of the Fc domain), and these modifications are complementary to each other in order to promote the association of the two Fc domain subunits. For example, the association-promoting modifications may alter the structure or charge of one or both of the Fc domain subunits so that they perform a sterically or electrostatically desired association, respectively. Thus, (hetero)dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, which may not be identical in the sense that the further components fused to each subunit (e.g., antigen-binding domains) are not the same. In some embodiments, modifications that facilitate the association of the first and second subunits of the Fc domain include amino acid mutations, specifically amino acid substitutions, within the Fc domain. In preferred embodiments, modifications that facilitate the association of the first and second subunits of the Fc domain include distinct amino acid mutations, specifically amino acid substitutions, in each of the two subunits of the Fc domain.

[0065] The term "effector function" refers to the biological activity resulting from the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immunoconjugate-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0066] An "activated Fc receptor" is an Fc receptor that, following the binding of the antibody's Fc domain, triggers a signaling event that stimulates receptor-hosting cells to perform effector functions. Examples of human activated Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0067] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism in which immune effector cells lyse antibody-coated target cells. Target cells are cells to which antibodies or derivatives containing an Fc region specifically bind, typically via a protein moiety at the N-terminus of the Fc region. As used herein, the term “reduced ADCC” is defined as either a reduction in the number of target cells lysed at a given time by a given concentration of antibody in the culture medium surrounding the target cells via the ADCC mechanism as defined above, and / or an increase in the concentration of antibody in the culture medium surrounding the target cells required to achieve the lysis of a given number of target cells at a given time via the ADCC mechanism. ADCC reduction is compared to unmanipulated ADCC mediated by the same antibody produced by the same type of host cells using the same standard production, purification, formulation, and storage methods (known to those skilled in the art). For example, ADCC reduction mediated by an antibody containing an amino acid substitution in its Fc domain that reduces ADCC is compared to ADCC mediated by the same antibody that does not contain this amino acid substitution in its Fc domain. Appropriate assays for measuring ADCC are well known in the art (see, for example, PCT Publication No. 2006 / 082515 or PCT Publication No. 2012 / 130831).

[0068] As used herein, the terms “to manipulate,” “to be manipulated,” and “to manipulate” are considered to include any manipulation or post-translational modification of the peptide backbone of naturally occurring or recombinant polypeptides or fragments thereof. Manipulation includes modification of amino acid sequences, modification of glycosylation patterns, or modification of the side chain groups of individual amino acids, and combinations of these approaches.

[0069] As used herein, the term “amino acid mutation” encompasses amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be made to arrive at the final construct, insofar as the final construct has the desired characteristics, e.g., reduced binding to the Fc receptor or increased association with another peptide. Deletions and insertions of amino acid sequences include deletions and insertions of the amino-terminus and / or carboxy-terminus of an amino acid. The preferred amino acid mutation is amino acid substitution. For example, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid with different structural and / or chemical properties, are particularly preferred for the purpose of altering the binding properties of the Fc region. Amino acid substitutions include substitutions with non-natural amino acids or substitutions with natural amino acid derivatives of 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be induced using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods other than genetic manipulation to alter the side chain groups of amino acids, such as chemical modification, may also be useful. Various names can be used herein to describe the same amino acid mutation. For example, the substitution of proline to glycine at position 329 of the Fc domain is referred to as 329G, G329 329 It can be written as P329G or Pro329Gly.

[0070] The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum possible sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or FASTA program packages. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. Alternatively, the identity percentage value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code is filed in the user documentation of the US Copyright Office (Washington DC, 20559), registered under US Copyright Registration No. TXU510087, and listed in PCT Publication No. International 2001 / 007611.

[0071] Unless otherwise specified, for the purposes of this specification, amino acid sequence identity % values ​​are generated using the ggsearch program in FASTA package version 36.3.8c or later, along with the BLOSUM50 comparison matrix. The FASTA program package was created by WRPearson and DJLipman ("Improved Tools for Biological Sequence Analysis", PNAS 85(1988)2444-2448), WRPearson ("Effective protein sequence comparison", MethEnzymol.266(1996)227-258) and Pearson et al. (Genomics 46(1997)24-36) and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, you can use the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi to perform a global alignment (not just a local one) and compare sequences using the ggsearch(global protein:protein) program with default options (BLOSUM50;open:-10;ext:-2;Ktup=2). The amino acid identity percentage is shown in the output alignment header.

[0072] The term “polynucleotide” or “nucleic acid molecule” includes any compound and / or substance containing a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented 5' to 3'. In this specification, the term nucleic acid molecule includes deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or cyclic. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. In addition, nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides, including those containing derivatized sugar or phosphate backbone links or chemically modified residues, include modified nucleotide bases. Nucleic acid molecules also include DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the present invention in vitro and / or in vivo in a host or patient, for example. Such DNA (e.g., cDNA) vectors or RNA (e.g., mRNA) vectors may or may not be modified. For example, mRNA may be chemically modified to improve the stability of the RNA vector and / or the expression of the encoded molecule, allowing the mRNA to be injected into a target to generate antibodies in vivo (see, e.g., Stadler et al. (2017) Nature Medicine 23:815-817, or European Patent No. 2101823).

[0073] The terms “pharmaceutical composition” or “pharmaceutical preparation” refer to a preparation that is in a form that enables the biological activity of the active ingredient contained herein and does not contain any additional components that would be unacceptably toxic to the subject to which the composition is to be administered.

[0074] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition or preparation other than the active ingredient that is non-toxic to the target. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0075] As used herein, “treatment” (and its grammatical variations, e.g., “treat” or “treating”) refers to a clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and may be carried out for preventive purposes or during the course of a clinicopathological disease. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological outcomes of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the condition, and achieving remission or improving the prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of the disease or to slow the progression of the disease.

[0076] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human.

[0077] The “effective amount” of a drug, such as a pharmaceutical composition, refers to the amount that is effective in the dosage and duration required to achieve the desired therapeutic or preventive outcome.

[0078] The term “package insert” is used to refer to instructions that are typically included in the market packaging of therapeutic products and that contain information about indications, usage, dosage, administration, combination therapy, contraindications and / or warnings relating to the use of such therapeutic products. Bispecific antibodies that bind to EGFRvIII and CD3

[0079] The anti-EGFRvIII / anti-CD3 bispecific antibody (also referred to herein as "EGFRvIII TCB") used in the combination therapies described herein comprises at least two antigen-binding moieties capable of specifically binding to two different antigenic determinants (first and second antigens). The anti-EGFRvIII / anti-CD3 bispecific antibody comprises at least one antigen-binding moiety capable of specifically binding to EGFRvIII and one binding moiety capable of specifically binding to CD3. Suitable bispecific antigen-binding molecules that bind to EGFRvIII and CD3 for use in the present invention are described, for example, in PCT Publication No. 2020 / 127619.

[0080] First antigen-binding site A bispecific antigen-binding molecule comprises at least one antigen-binding moiety, particularly a Fab molecule, that binds to EGFRvIII (the first antigen). In certain embodiments, the bispecific antigen-binding molecule comprises two antigen-binding moieties (first and third), particularly a Fab molecule, that bind to EGFRvIII. In such particular embodiments, each of these antigen-binding moieties binds to the same antigenic determinant. In further particular embodiments, all of these antigen-binding moieties are identical, i.e., they contain the same amino acid sequence, including the same amino acid substitutions in the CH1 domain and CL domain as described herein (if present). In one embodiment, the bispecific antigen-binding molecule comprises two or fewer antigen-binding moieties, particularly a Fab molecule, that bind to EGFRvIII.

[0081] In one embodiment, the first (and, if present, third) antigen-binding region includes a heavy chain variable region (VH) containing the heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) containing the light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0082] In one embodiment, the first (and, if present, the third) antigen-binding portion is a humanized antibody (derived from). In one embodiment, the first (and, if present, the third) antigen-binding portion is a humanized antigen-binding portion (i.e., the antigen-binding portion of a humanized antibody). In one embodiment, the VH and / or VL of the first (and, if present, the third) antigen-binding portion are humanized variable regions.

[0083] In one embodiment, the VH and / or VL of the first (and, if present, third) antigen-binding moiety includes an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0084] In one embodiment, the first (and, if present, third) antigen-binding moiety VH includes one or more heavy chain framework sequences of SEQ ID NO: 7 (i.e., FR1, FR2, FR3, and / or FR4 sequences). In one embodiment, VH includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7. In one embodiment, VH includes an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 7. In one embodiment, VH includes an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 7. In certain embodiments, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody containing that sequence retains the ability to bind to EGFRvIII. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 7. In certain embodiments, substitutions, insertions, or deletions occur in the region outside the CDR (i.e., at the FR). In one embodiment, VH contains the amino acid sequence of SEQ ID NO: 7. Optionally, VH contains the amino acid sequence of SEQ ID NO: 7, including post-translational modifications of that sequence.

[0085] In one embodiment, the first (and, if present, third) antigen-binding moiety VL contains one or more light chain framework sequences of SEQ ID NO: 8 (i.e., FR1, FR2, FR3, and / or FR4 sequences). In one embodiment, the VL contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8. In one embodiment, the VL contains an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 8. In one embodiment, the VL contains an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody containing that sequence retains the ability to bind to EGFRvIII. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 8. In certain embodiments, substitutions, insertions, or deletions occur in the region outside the CDR (i.e., in the FR). In one embodiment, the VL contains the amino acid sequence of SEQ ID NO: 8. Optionally, the VL contains the amino acid sequence of SEQ ID NO: 8, including post-translational modifications of that sequence.

[0086] In one embodiment, the first (and, if present, third) antigen-binding moiety VH that specifically binds to EGFRvIII includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7, and the first (and, if present, third) antigen-binding moiety VL includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8. In one embodiment, VH includes the amino acid sequence of SEQ ID NO: 7, and VL includes the amino acid sequence of SEQ ID NO: 8.

[0087] In a further embodiment, the first (and third, if present) antigen-binding domain comprises a VH containing the sequence of SEQ ID NO: 7 and a VL containing the sequence of SEQ ID NO: 8.

[0088] In a further embodiment, the first (and third, if present) antigen-binding moiety includes the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 8.

[0089] In another embodiment, the first (and third, if present) antigen-binding moiety comprises VH containing the heavy chain CDR sequence of VH of SEQ ID NO: 7 and VL containing the light chain CDR sequence of VL of SEQ ID NO: 8.

[0090] In a further embodiment, the first (and, if present, third) antigen-binding moiety includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of VH in SEQ ID NO: 7 and the LCDR1, LCDR2, and LCDR3 amino acid sequences of VL in SEQ ID NO: 8.

[0091] In one embodiment, the first (and, if present, the third) antigen-binding portion is a Fab molecule.

[0092] Second antigen-binding site The bispecific antigen-binding molecule comprises at least one antigen-binding moiety, particularly a Fab molecule, that binds to CD3 (the second antigen). In a preferred embodiment, CD3 is human CD3 or cynomolgus monkey CD3, most particularly human CD3. In one embodiment, the first antigen-binding domain is cross-reactive (i.e., specifically binds) to both human and cynomolgus monkey CD3. In some embodiments, CD3 is the epsilon subunit of CD3 (CD3 epsilon).

[0093] In a preferred embodiment, the bispecific antibody comprises one or fewer antigen-binding domains that bind to CD3. In one embodiment, the bispecific antibody provides monovalent binding to CD3.

[0094] In one embodiment, the second antigen-binding region includes a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 9, the HCDR2 of SEQ ID NO: 10, and the HCDR3 of SEQ ID NO: 11, as well as a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 12, the LCDR2 of SEQ ID NO: 13, and the LCDR3 of SEQ ID NO: 14.

[0095] In one embodiment, the second antigen-binding portion is a humanized antibody (derived from a humanized antibody). In another embodiment, the second antigen-binding portion is a humanized antigen-binding portion (i.e., the antigen-binding portion of a humanized antibody). In another embodiment, the VH and / or VL of the second antigen-binding portion are humanized variable regions.

[0096] In one embodiment, the VH and / or VL of the second antigen-binding moiety include an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0097] In one embodiment, the VH of the second antigen-binding moiety includes one or more heavy chain framework sequences of SEQ ID NO: 15 (i.e., FR1, FR2, FR3, and / or FR4 sequences). In one embodiment, VH includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 15. In one embodiment, VH includes an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 88. In one embodiment, VH includes an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 15. In certain embodiments, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antibody containing that sequence retains the ability to bind to CD3. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 15. In certain embodiments, substitutions, insertions, or deletions occur in the region outside the CDR (i.e., in the FR). In one embodiment, VH contains the amino acid sequence of SEQ ID NO: 15. Optionally, VH contains the amino acid sequence of SEQ ID NO: 15, including post-translational modifications of that sequence.

[0098] In one embodiment, the VL of the second antigen-binding moiety includes one or more light chain framework sequences of SEQ ID NO: 16 (i.e., FR1, FR2, FR3, and / or FR4 sequences). In one embodiment, the VL includes an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 16. In one embodiment, the VL includes an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 16. In one embodiment, the VL includes an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 16. In certain embodiments, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity includes substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody containing that sequence retains the ability to bind to CD3. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 16. In certain embodiments, substitutions, insertions, or deletions occur in the region outside the CDR (i.e., in the FR). In one embodiment, the VL contains the amino acid sequence of SEQ ID NO: 16. Optionally, the VL contains the amino acid sequence of SEQ ID NO: 16, including post-translational modifications of that sequence.

[0099] In one embodiment, the second antigen-binding moiety VH, which specifically binds to CD3, comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 16, and the first (and, if present, third) antigen-binding moiety VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 16. In one embodiment, VH comprises the amino acid sequence of SEQ ID NO: 15, and VL comprises the amino acid sequence of SEQ ID NO: 16.

[0100] In a further embodiment, the second antigen-binding domain includes VH containing the sequence of SEQ ID NO: 15 and VL containing the sequence of SEQ ID NO: 16.

[0101] In a further embodiment, the second antigen-binding portion includes the VH sequence of SEQ ID NO: 15 and the VL sequence of SEQ ID NO: 16.

[0102] In another embodiment, the second antigen-binding portion includes VH, which contains the heavy chain CDR sequence of VH of SEQ ID NO: 15, and VL, which contains the light chain CDR sequence of VL of SEQ ID NO: 16.

[0103] In a further embodiment, the second antigen-binding moiety includes the HCDR1, HCDR2, and HCDR3 amino acid sequences of VH in SEQ ID NO: 15 and the LCDR1, LCDR2, and LCDR3 amino acid sequences of VL in SEQ ID NO: 16.

[0104] In one embodiment, the second antigen-binding moiety is a Fab molecule. In some embodiments, the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and Fab heavy chain, particularly the variable domains VL and VH, are replaced by each other (i.e., according to such embodiments, the second antigen-binding moiety is a crossover Fab molecule in which the variable or constant domains of the Fab light chain and Fab heavy chain are exchanged). In such one embodiment, the first (and third, if present) antigen-binding moiety is a conventional Fab molecule.

[0105] charge modification Anti-EGFRvIII / anti-CD3 bispecific antibodies used in the combination therapies described herein may contain amino acid substitutions in their Fab molecules, which are particularly effective in reducing mispairing with the heavy chain that does not match the light chain (Bence-Jones type byproducts), which can occur in the production of Fab-based multispecific antibodies with VH / VL exchange in one of the binding arms (two or more in the case of molecules containing two or more antigen-binding Fab molecules) (see International Publication No. 2015 / 150447 of PCT Publication No. 150447, in particular for examples, which are incorporated herein by reference in their entirety). The ratio of desirable multispecific antibodies to undesirable byproducts, particularly Bence-Jones type byproducts that occur in multispecific antibodies with VH / VL domain exchange in one binding arm, can be improved by introducing charged amino acids with opposite charges at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modification").

[0106] Therefore, in some embodiments where both the first and second (and, if present, the third) antigen-binding domains of a bispecific antibody are Fab molecules, and in one of those antigen-binding domains (particularly the second antigen-binding domain), the variable domains VL and VH of the Fab light chain and Fab heavy chain are more interchangeable with each other, i) In the constant domain CL of the first (and third, if present) antigen-binding domain, the amino acid at position 124 is substituted with a positively charged amino acid (numbered by Kabat), and in the constant domain CH1 of the first (and third, if present) antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is substituted with a negatively charged amino acid (numbered by the Kabat EU index), or ii) In the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is substituted with a positively charged amino acid (numbered by Kabat), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is substituted with a negatively charged amino acid (numbered by the Kabat EU index).

[0107] Bispecific antibodies do not contain both of the modifications described in i) and ii). The constant domains CL and CH1 of the antigen-binding domain with VH / VL exchange are not substituted for each other (i.e., not exchanged).

[0108] In more specific cases, i) In the constant domain CL of the first (and third, if present) antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat), and in the constant domain CH1 of the first (and third, if present) antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat EU index), or ii) In the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat EU index).

[0109] In one such embodiment, in the constant domain CL of the first (and third, if present) antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), and in the constant domain CH1 of the first (and third, if present) antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0110] In a further embodiment, in the constant domain CL of the first (and, if present, the third) antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat), and in the constant domain CH1 of the first (and, if present, the third) antigen-binding domain, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat EU index).

[0111] In a preferred embodiment, in the constant domain CL of the first (and third, if present) antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (Kabat numbering), in the constant domain CH1 of the first (and third, if present) antigen-binding domain, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0112] In a more preferred embodiment, in the constant domain CL of the first (and third, if present) antigen-binding domain, the amino acid at position 124 is substituted with lysine (K) (Kabat numbering), the amino acid at position 123 is substituted with lysine (K) (Kabat numbering), and in the constant domain CH1 of the first (and third, if present) antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (Kabat EU index numbering), and the amino acid at position 213 is substituted with glutamic acid (E) (Kabat EU index numbering).

[0113] In a more preferred embodiment, in the constant domain CL of the first (and third, if present) antigen-binding domain, the amino acid at position 124 is substituted with lysine (K) (Kabat numbering), the amino acid at position 123 is substituted with arginine (R) (Kabat numbering), and in the constant domain CH1 of the first (and third, if present) antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (Kabat EU index numbering), and the amino acid at position 213 is substituted with glutamic acid (E) (Kabat EU index numbering).

[0114] In a preferred embodiment, when the amino acid substitution according to the above embodiment is performed on the constant domain CL and constant domain CH1 of the first (and, if present, the third) antigen-binding domain, the constant domain CL of the first (and, if present, the third) antigen-binding domain is a kappa isotype.

[0115] Alternatively, the amino acid substitutions according to the above embodiments may be carried out in the constant domain CL and constant domain CH1 of the second antigen-binding domain instead of the constant domain CL and constant domain CH1 of the first (and, if present, third) antigen-binding domain. In a preferred embodiment, the constant domain CL of the second antigen-binding domain is a kappa isotype.

[0116] Therefore, in one embodiment, in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat EU index).

[0117] In a further embodiment, in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat EU index).

[0118] In another embodiment, in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat), the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (numbered by Kabat), in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat EU index), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered by Kabat EU index).

[0119] In one embodiment, in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is substituted with lysine (K) (Kabat numbering), the amino acid at position 123 is substituted with lysine (K) (Kabat numbering), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (Kabat EU index numbering), and the amino acid at position 213 is substituted with glutamic acid (E) (Kabat EU index numbering).

[0120] In another embodiment, in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is substituted with lysine (K) (Kabat numbering), the amino acid at position 123 is substituted with arginine (R) (Kabat numbering), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (Kabat EU index numbering), and the amino acid at position 213 is substituted with glutamic acid (E) (Kabat EU index numbering).

[0121] In a preferred embodiment, the bispecific antibody of the present invention is (a) A first and optionally third antigen-binding domain that binds to EGFRvIII (the first antigen-binding portion is a Fab molecule containing a heavy chain variable region (VH) including heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) including light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6), and (b) A second antigen-binding moiety that binds to CD3 (the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) containing heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 9, HCDR2 of SEQ ID NO: 10, and HCDR3 of SEQ ID NO: 11, and a light chain variable region (VL) containing light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, wherein the variable domains VL and VH of the Fab heavy chain are substituted for each other), In the first (and, if present, the third) constant domain CL of the antigen-binding moiety, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H) (in a preferred embodiment, independently substituted with lysine (K) or arginine (R)) (Kabat numbering), the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H) (in a preferred embodiment, independently substituted with lysine (K) or arginine (R)) (Kabat numbering), in the first (and, if present, the third) constant domain CH1 of the antigen-binding domain, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0122] Bispecific antibody format Techniques for producing multispecific antibodies include, but are not limited to, the recombination co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and the "knob-in-hole" operation (see, e.g., U.S. Patent No. 5,731,168 and Atwell et al., J.Mol.Biol.270:26 (1997)). Multispecific antibodies also involve the manipulation of electrostatic steering effects to produce antibody Fc heterodimer molecules (see, e.g., International Publication No. 2009 / 089004); crosslinking of two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); and the production of bispecific antibodies using leucine zippers (see, e.g., Kostelny et al.) They can be produced by the use of general light chain techniques to avoid light chain mispairing problems (e.g., see International Publication No. 2011 / 034605); the use of "diabody" techniques to produce bispecific antibody fragments (e.g., see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single-stranded Fv(sFv) dimers (e.g., see Gruber et al., J. Immunol., 152:5368 (1994)); as well as by the preparation of triplicate antibodies as described, for example, Tutt et al. J. Immunol. 147:60 (1991).

[0123] For example, this also includes manipulated antibodies having three or more antigen-binding sites, such as "octopus antibodies," or DVD-Ig (see, for example, International Publication 2001 / 77342 and International Publication 2008 / 024715). Other examples of multispecific antibodies having three or more antigen-binding sites can be found in International Publication 2010 / 115589, International Publication 2010 / 112193, International Publication 2010 / 136172, International Publication 2010 / 145792 and International Publication 2013 / 026831. Bispecific antibodies or their antigen-binding fragments also include "dual-acting FAb" or "DAF" containing antigen-binding sites that bind to EGFRvIII and CD3 (see, for example, U.S. Patent Application Publication 2008 / 0069820 and International Publication 2015 / 095539).

[0124] Multispecific antibodies can also be provided in an asymmetric form with domain crossovers in one or more binding arms of the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., International Publications 2009 / 080252 and 2015 / 150447), CH1 / CL domains (see, e.g., International Publication 2009 / 080253), or complete Fab arms (see, International Publications 2009 / 080251, 2016 / 016299, PNAS, Schaefer et al., 108(2011)1187-1191, and Klein et al., MAbs 8(2016)1010-20). Asymmetric Fab arms can also be manipulated by introducing charged or uncharged amino acid mutations into the domain interface to direct correct Fab pairing. See, e.g., International Publication 2016 / 172485.

[0125] Various further molecular formats of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67(2015) 95-106).

[0126] Examples of bispecific antibody formats that may be useful for this purpose include, but are not limited to, so-called "BiTE" (bispecific T cell engager) molecules in which two scFv molecules are fused by a flexible linker (see, e.g., International Publication Nos. 2004 / 106381, 2005 / 061547, 2007 / 042261, and 2008 / 119567, Nagorsen and Baeuerle, Exp Cell Res 317, 1255-1260 (2011)), diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies ("TandAb", Kipriyanov et al., J Mol Biol Examples include 293,41-56(1999)), “DART” (Dual Affinity Retargeting) molecules based on the diabody format but featuring a C-terminal disulfide crosslink for further stabilization (Johnson et al., J Mol Biol 399,436-449(2010)), and so-called triomabs (which are entire mouse / rat IgG hybrid molecules (seen in Seimetz et al., Cancer Treat Rev 36,458-467(2010))). Specific T cell bispecific antibody formats included herein are described in International Publication Nos. 2013 / 026833, 2013 / 026839, 2016 / 020309, and Bacac et al., Oncoimmunology 5(8)(2016)e1203498.

[0127] The components of the bispecific antigen-binding molecule may be fused with each other in various configurations.

[0128] In a preferred embodiment, the antigen-binding portion contained in the anti-EGFRvIII / anti-CD3 bispecific antibody is a Fab molecule. In such an embodiment, the first, second, third, and so on antigen-binding portions may be referred to herein as the first, second, third, and so on Fab molecules, respectively.

[0129] In one embodiment, the first antigen-binding moiety and the second antigen-binding moiety of a bispecific antibody are optionally fused to each other via a peptide linker. In a preferred embodiment, the first and second antigen-binding moieties are each a Fab molecule. In such an embodiment, the first antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain at the C-terminus of the Fab heavy chain. In another such embodiment, the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain. In embodiments where (i) the first antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the second antigen-binding moiety at the C-terminus of the Fab heavy chain, or (ii) the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain, the Fab light chain of the first antigen-binding domain and the Fab light chain of the second antigen-binding moiety may further be fused to each other via a peptide linker.

[0130] Bispecific antigen-binding molecules, which have a single antigen-binding moiety (such as a Fab molecule) that can specifically bind to target cell antigens such as EGFRvIII, are particularly useful when internalization of the target cell antigen is expected after binding of the high-affinity antigen-binding moiety. In such cases, the presence of more than one antigen-binding moiety specific to the target cell antigen may enhance the internalization of the target cell antigen, thereby reducing its availability.

[0131] However, in other cases, it is advantageous to have a bispecific antigen-binding molecule that contains two or more antigen-binding moieties (e.g., Fab molecules) that are specific to the target cell antigen, for example, to optimize targeting to the target site or to enable crosslinking of the target cell antigen.

[0132] Therefore, in certain embodiments, the bispecific antigen-binding molecule includes a third antigen-binding portion, the third antigen-binding portion being identical to the first antigen-binding portion.

[0133] In certain embodiments, the bispecific antigen-binding molecule includes an Fc domain composed of first and second subunits. The first and second subunits of the Fc domain are capable of stable association.

[0134] The bispecific antigen-binding molecule may have different configurations, i.e., the first, second (and optionally third) antigen-binding moieties may be fused to each other and to the Fc domain in different ways. The components may be fused to each other directly, or preferably via one or more suitable peptide linkers. If the fusion of the Fab molecule is to the N-terminus of a subunit of the Fc domain, it is typically via an immunoglobulin hinge region.

[0135] In some embodiments, the first and second antigen-binding moieties are each a Fab molecule, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In such embodiments, the first antigen-binding moiety may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the second antigen-binding moiety or to the N-terminus of the other subunit of the Fc domain. In certain such embodiments, the first antigen-binding moiety is a conventional Fab molecule, and the second antigen-binding moiety is a crossover Fab molecule as described in the specification (i.e., a Fab molecule in which the variable domains VH and VL or constant domains CL and CH1 of the Fab heavy and light chains are exchanged / replaced by each other). In other such embodiments, the first Fab molecule is a crossover Fab molecule, and the second Fab molecule is a conventional Fab molecule.

[0136] In one embodiment, the first antigen-binding moiety and the second antigen-binding moiety are each Fab molecules, the second antigen-binding moiety being fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the first antigen-binding moiety being fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety. In a specific embodiment, the bispecific antigen-binding molecule essentially consists of first and second Fab molecules, an Fc domain composed of first and second subunits, and optionally one or more peptide linkers, the first Fab molecule being fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule being fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Optionally, the Fab light chains of the first Fab molecule and the Fab light chains of the second Fab molecule may be additionally fused to each other.

[0137] In another embodiment, the first antigen-binding moiety and the second antigen-binding moiety are each a Fab molecule, and the first and second antigen-binding moieties are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. In a specific embodiment, the bispecific antigen-binding molecule essentially consists of the first and second Fab molecules, the Fc domain comprising the first and second subunits and optionally one or more peptide linkers, and the first and second Fab molecules each have the C-terminus of the Fab heavy chain fused at the N-terminus of one of the subunits of the Fc domain. The first and second Fab molecules may be fused to the Fc domain directly or via a peptide linker. In a particular embodiment, the first and second Fab molecules are each fused to the Fc domain by an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1Fc domain.

[0138] In some embodiments, the first antigen-binding moiety and the second antigen-binding moiety are each a Fab molecule, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In such embodiments, the second antigen-binding moiety may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety or (as described above) to the N-terminus of the other subunit of the Fc domain. In certain such embodiments, the first antigen-binding moiety is a conventional Fab molecule, and the second antigen-binding moiety is a crossover Fab molecule as described in the specification (i.e., a Fab molecule in which the variable domains VH and VL or constant domains CL and CH1 of the Fab heavy and light chains are exchanged / replaced by each other). In other such embodiments, the first Fab molecule is a crossover Fab molecule, and the second Fab molecule is a conventional Fab molecule.

[0139] In one embodiment, the first antigen-binding moiety and the second antigen-binding moiety are each Fab molecules, the first antigen-binding moiety being fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the second antigen-binding moiety being fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding moiety. In a specific embodiment, the bispecific antigen-binding molecule essentially consists of first and second Fab molecules, an Fc domain composed of first and second subunits, and optionally one or more peptide linkers, the second Fab molecule being fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule being fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Optionally, the Fab light chains of the first Fab molecule and the Fab light chains of the second Fab molecule may be additionally fused to each other.

[0140] In some embodiments, a third antigen-binding moiety, particularly a third Fab molecule, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In certain such embodiments, the first and third Fab molecules are each conventional Fab molecules, and the second Fab molecule is a crossover Fab molecule as described in the specification (i.e., a Fab molecule in which the variable domains VH and VL or constant domains CL and CH1 of the Fab heavy and light chains are exchanged / replaced by each other). In other such embodiments, the first and third Fab molecules are each crossover Fab molecules, and the second Fab molecule is a conventional Fab molecule.

[0141] In certain such embodiments, the second and third antigen-binding moieties are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one subunit of the Fc domain, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In specific embodiments, the bispecific antigen-binding molecule essentially consists of first, second and third Fab molecules, an Fc domain composed of first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. The second and third Fab molecules may be fused directly to the Fc domain or via a peptide linker. In certain embodiments, the second and third Fab molecules are fused to the Fc domain via an immunoglobulin hinge region. In specific embodiments, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1Fc domain. Optionally, the Fab light chains of the first Fab molecule and the Fab light chains of the second Fab molecule may be additionally fused to each other.

[0142] In another such embodiment, the first and third antigen-binding moieties are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one subunit of the Fc domain, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first antigen-binding moiety. In a specific embodiment, the bispecific antigen-binding molecule essentially consists of first, second and third Fab molecules, an Fc domain composed of first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. The first and third Fab molecules may be fused to the Fc domain directly or via a peptide linker. In certain embodiments, the first and third Fab molecules are fused to an Fc domain by an immunoglobulin hinge region. In specific embodiments, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1Fc domain. Optionally, the Fab light chains of the first Fab molecule and the Fab light chains of the second Fab molecule may be additionally fused to each other.

[0143] In a configuration of a bispecific antigen-binding molecule in which the C-terminus of the Fab heavy chain is fused to the N-terminus of each subunit of the Fc domain via an immunoglobulin hinge region, the two Fab molecules, the hinge region, and the Fc domain essentially form an immunoglobulin molecule. In certain embodiments, the immunoglobulin molecule is an IgG class immunoglobulin. In even more specific embodiments, the immunoglobulin is an IgG1 subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG4 subclass immunoglobulin. In yet another embodiment, the immunoglobulin is a human immunoglobulin. In yet another embodiment, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one embodiment, the immunoglobulin includes a human constant region, in particular a human Fc region.

[0144] In some bispecific antigen-binding molecules, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are optionally fused to each other via a peptide linker. Depending on the structures of the first and second Fab molecules, the Fab light chain of the first Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. The fusion of the Fab light chains of the first and second Fab molecules further reduces mispairing of incompatible Fab heavy and light chains and also reduces the number of plasmids required for the expression of some bispecific antigen-binding molecules.

[0145] The antigen-binding moiety may be directly attached to the Fc domain or fused to each other via a peptide linker containing one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n (SG4) n (G4S) n Or G4 (SG4) n A peptide linker is an example. "n" is generally an integer from 1 to 10, typically from 2 to 4. In one embodiment, the peptide linker has a length of at least 5 amino acids, in one embodiment it has a length of 5 to 100 amino acids, and in a further embodiment it has a length of 10 to 50 amino acids. In one embodiment, the peptide linker is (GxS) n or (GxS) n G mThe peptide linker is (G4S)2, where G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, m=0, 1, 2 or 3), or (x=4, n=2, 3, 4 or 5, m=0, 1, 2 or 3), where in one embodiment x=4, n=2 or 3, and in a further embodiment x=4, n=2. In one embodiment, the peptide linker is (G4S)2. The peptide linker particularly suitable for fusing the Fab light chains of the first and second Fab molecules to each other is (G4S)2. An exemplary peptide linker suitable for linking the Fab heavy chains of the first and second Fab fragments includes sequence (D)-(G4S)2 (SEQ ID NOs: 7 and 8). Another suitable such linker includes sequence (G4S)4. Furthermore, the linker may include (part of) an immunoglobulin hinge region. In particular, when a Fab molecule is fused to the N-terminus of an Fc domain subunit, it may be fused with or without an additional peptide linker via the immunoglobulin hinge region or a portion thereof.

[0146] In a particular embodiment, the bispecific antigen-binding molecule is such that the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region), thereby the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit of the polypeptide (VL (2) -CH1 (2) -CH2-CH3(-CH4)) and the Fab heavy chain of the first Fab molecule share a polypeptide (VH) with the Fc domain subunit and a carboxy-terminal peptide bond. (1) -CH1 (1) The bispecific antigen-binding molecule includes -CH2-CH3(-CH4)). In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1)) further includes. In certain embodiments, the polypeptides are covalently bonded, for example, by disulfide bonds.

[0147] In a particular embodiment, the bispecific antigen-binding molecule has a Fab heavy chain variable region of the second Fab molecule that shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the heavy chain constant region is replaced by the light chain constant region), thereby the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit of the polypeptide (VH (2) -CL (2) -CH2-CH3(-CH4)) and the Fab heavy chain of the first Fab molecule share a polypeptide (VH) with the Fc domain subunit and a carboxy-terminal peptide bond. (1) -CH1 (1) The bispecific antigen-binding molecule includes -CH2-CH3(-CH4)). In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule. (2) -CH1 (2) )) and the Fab light chain polypeptide (VL) of the first Fab molecule. (1) -CL (1) ) further includes. In certain embodiments, the polypeptides are covalently bonded, for example, by disulfide bonds.

[0148] In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region), thereby the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, thereby the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit. (2) -CH1(2) -VH (1) -CH1 (1) In other embodiments, the bispecific antigen-binding molecule contains a polypeptide (VH) such that the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, thereby the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region), thereby the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit. (1) -CH1 (1) -VL (2) -CH1 (2) Includes -CH2-CH3(-CH4)).

[0149] In some of these embodiments, the bispecific antigen-binding molecule is a crossover Fab light chain polypeptide (VH) of the second Fab molecule, in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) further includes. In some other embodiments of these embodiments, where appropriate, the bispecific antigen-binding molecule comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, and the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule. (2) -CL (2) -VL (1) -CL (1)), or a polypeptide (VL) in which the Fab light chain polypeptide of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, and the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (1) -CL (1) -VH (2) -CL (2) ) further includes.

[0150] The bispecific antigen-binding molecules according to these embodiments are (i) Fc domain subunit polypeptides (CH2-CH3(-CH4)), or (ii) polypeptides in which the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide (VL) of the third Fab molecule (3) -CL (3) ) may further include. In certain embodiments, the polypeptides are covalently bonded, for example, by disulfide bonds.

[0151] In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VH) in which the variable region of the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the constant region of the heavy chain is replaced by the constant region of the light chain), thereby the constant region of the Fab light chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, thereby the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit. (2) -CL (2) -VH (1) -CH1 (1)In other embodiments, the bispecific antigen-binding molecule contains a polypeptide (VH) such that the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, thereby the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the heavy chain constant region is replaced by the light chain constant region), thereby the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit. (1) -CH1 (1) -VH (2) -CL (2) Includes -CH2-CH3(-CH4)).

[0152] In some of these embodiments, the bispecific antigen-binding molecule is a crossover Fab light chain polypeptide (VL) of the second Fab molecule, in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule. (2) -CH1 (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) further includes. In some other embodiments of these embodiments, where appropriate, the bispecific antigen-binding molecule comprises a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule, and the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule. (2) -CH1 (2) -VL (1) -CL (1) ), or a polypeptide (VL) in which the Fab light chain polypeptide of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, and the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (1) -CL (1)-VL (2) -CH1 (2) ) further includes.

[0153] The bispecific antigen-binding molecules according to these embodiments are (i) Fc domain subunit polypeptides (CH2-CH3(-CH4)), or (ii) polypeptides in which the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide (VL) of the third Fab molecule (3) -CL (3) ) may further include. In certain embodiments, the polypeptides are covalently bonded, for example, by disulfide bonds.

[0154] In certain embodiments, the bispecific antigen-binding molecule does not contain an Fc domain. In certain such embodiments, the first and, if present, third Fab molecules are each conventional Fab molecules, and the second Fab molecule is a crossover Fab molecule as described in the specification (i.e., a Fab molecule in which the variable domains VH and VL or constant domains CL and CH1 of the Fab heavy and light chains are exchanged / replaced by each other). In other such embodiments, the first and, if present, third Fab molecules are each crossover Fab molecules, and the second Fab molecule is a conventional Fab molecule.

[0155] In one such embodiment, the bispecific antigen-binding molecule essentially consists of a first and a second antigen-binding moiety, and optionally one or more peptide linkers, where both the first and second antigen-binding moieties are Fab molecules, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding moiety.

[0156] In another such embodiment, the bispecific antigen-binding molecule essentially consists of a first and a second antigen-binding moiety, and optionally one or more peptide linkers, where both the first and second antigen-binding moieties are Fab molecules, and the second antigen-binding moiety is fused to the N-terminus of the Fab heavy chain of the first antigen-binding moiety at the C-terminus of the Fab heavy chain.

[0157] In some embodiments, a first Fab molecule is fused at the C-terminus of the Fab heavy chain of a second Fab molecule to the N-terminus of the Fab heavy chain of a second Fab molecule, and the bispecific antigen-binding molecule further comprises a third antigen-binding moiety, in particular a third Fab molecule, the third Fab molecule being fused at the C-terminus of the Fab heavy chain of a first Fab molecule to the N-terminus of the Fab heavy chain of a first Fab molecule. In certain such embodiments, the bispecific antigen-binding molecule essentially consists of first, second and third Fab molecules, and optionally one or more peptide linkers, the first Fab molecule being fused at the C-terminus of the Fab heavy chain of a second Fab molecule to the N-terminus of the Fab heavy chain of a second Fab molecule, and the third Fab molecule being fused at the C-terminus of the Fab heavy chain of a first Fab molecule to the N-terminus of the Fab heavy chain of a first Fab molecule.

[0158] In some embodiments, a second Fab molecule is fused to the N-terminus of the Fab heavy chain of a first Fab molecule at the C-terminus of the Fab heavy chain, and the bispecific antigen-binding molecule further comprises a third antigen-binding moiety, in particular a third Fab molecule, the third Fab molecule being fused to the C-terminus of the Fab heavy chain of a first Fab molecule at the N-terminus of the Fab heavy chain. In certain such embodiments, the bispecific antigen-binding molecule essentially consists of first, second, and third Fab molecules, and optionally one or more peptide linkers, the second Fab molecule being fused to the N-terminus of the Fab heavy chain of a first Fab molecule at the C-terminus of the Fab heavy chain, and the third Fab molecule being fused to the C-terminus of the Fab heavy chain of a first Fab molecule at the N-terminus of the Fab heavy chain.

[0159] In certain embodiments, the bispecific antigen-binding molecule is such that the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, whereby the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain with the heavy chain variable region replaced by the light chain variable region) polypeptide (VH (1) -CH1 (1) -VL (2) -CH1 (2) ). In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide (VH (2) -CL (2) ) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule.

[0160] In certain embodiments, the bispecific antigen-binding molecule is such that the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain with the heavy chain variable region replaced by the light chain variable region), whereby the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, polypeptide (VH (2) -CH1 (2) -VL (1) -CH1 (1) ). In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide (VH (2) -CL (2) ) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule, and the Fab light chain polypeptide (VL (1) -CL (1) ) of the first Fab molecule.

[0161] In a particular embodiment, the bispecific antigen-binding molecule is a polypeptide (VH) in which the variable region of the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the constant region of the heavy chain is replaced by the constant region of the light chain), thereby the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule. (2) -CL (2) -VH (1) -CH1 (1) ) includes. In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule. (2) -CH1 (2) )) and the Fab light chain polypeptide (VL) of the first Fab molecule. (1) -CL (1) ) and further include.

[0162] In a particular embodiment, the bispecific antigen-binding molecule according to the present invention has a Fab light chain variable region of a second Fab molecule that shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region), thereby the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, thus forming a polypeptide (VL (2) -CH1 (2) -VH (1) -CH1 (1) ) includes. In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) and further include.

[0163] In a particular embodiment, the bispecific antigen-binding molecule is such that the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, thereby the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, thereby the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region), and the polypeptide (VH (3) -CH1 (3) -VH (1) -CH1 (1) -VL (2) -CH1 (2) ) includes. In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) further includes. In some embodiments, the bispecific antigen-binding molecule is the Fab light chain polypeptide (VL) of the third Fab molecule. (3) -CL (3) ) further includes.

[0164] In a particular embodiment, bispecific antigen binding occurs when the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, thereby the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, thereby the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the heavy chain constant region is replaced by the light chain constant region), and polypeptide (VH) (3) -CH1 (3) -VH (1) -CH1 (1) -VH(2) -CL (2) ) includes. In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule. (2) -CH1 (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) further includes. In some embodiments, the bispecific antigen-binding molecule is the Fab light chain polypeptide (VL) of the third Fab molecule. (3) -CL (3) ) further includes.

[0165] In a particular embodiment, the bispecific antigen-binding molecule is a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region), thereby the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, thereby the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the third Fab molecule. (2) -CH1 (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) ) includes. In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) further includes. In some embodiments, the bispecific antigen-binding molecule is the Fab light chain polypeptide (VL) of the third Fab molecule. (3) -CL (3) ) further includes.

[0166] In a particular embodiment, the bispecific antigen-binding molecule is a polypeptide (VH) in which the variable region of the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the constant region of the Fab light chain of the second Fab molecule (i.e., the second Fab molecule contains a crossover Fab heavy chain, and the constant region of the heavy chain is replaced by the constant region of the light chain), thereby the constant region of the Fab light chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, thereby the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the third Fab molecule. (2) -CL (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) ) includes. In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule. (2) -CH1 (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) further includes. In some embodiments, the bispecific antigen-binding molecule is the Fab light chain polypeptide (VL) of the third Fab molecule. (3) -CL (3) ) further includes.

[0167] In a particular embodiment, the bispecific antigen-binding molecule is such that the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, thereby the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region), thereby the Fab heavy chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the third Fab molecule, thereby the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region), and polypeptide (VH (2) -CH1 (2) -VL (1) -CH1 (1) -VL (3) -CH1 (3) ) includes. In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide, and the Fab heavy chain variable region of the first Fab molecule is the Fab light chain constant region (VH) of the first Fab molecule. (1) -CL (1) ) and the Fab light chain polypeptide (VL) of the second Fab molecule (2) -CL (2) ) and share a carboxy-terminal peptide bond. In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule. (3) -CL (3) ) further includes.

[0168] In a particular embodiment, the bispecific antigen-binding molecule is such that the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, thereby sharing a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule contains a crossover Fab heavy chain, and the heavy chain constant region is replaced by the light chain constant region), thereby sharing a carboxy-terminal peptide bond with the Fab heavy chain variable region of the third Fab molecule, thereby sharing a carboxy-terminal peptide bond with the Fab heavy chain variable region of the third Fab molecule (i.e., the third Fab molecule contains a crossover Fab heavy chain, and the heavy chain constant region is replaced by the light chain constant region), and the polypeptide (VH (2) -CH1 (2) -VH (1) -CL (1) -VH (3) -CL (3) ) includes. In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide, and the Fab light chain variable region of the first Fab molecule is the Fab heavy chain constant region (VL) of the first Fab molecule. (1) -CH1 (1) ) and the Fab light chain polypeptide (VL) of the second Fab molecule (2) -CL (2) ) and share a carboxy-terminal peptide bond. In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VL) in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule. (3) -CH1 (3) ) further includes.

[0169] In a particular embodiment, the bispecific antigen-binding molecule is a polypeptide (VL) in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region), thereby the Fab heavy chain constant region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, thereby the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule contains a crossover Fab heavy chain, and the heavy chain variable region is replaced by the light chain variable region), thereby the Fab heavy chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule. (3) -CH1 (3) -VL (1) -CH1 (1) -VH (2) -CH1 (2) ) includes. In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide, and the Fab heavy chain variable region of the first Fab molecule is the Fab light chain constant region (VH) of the first Fab molecule. (1) -CL (1) ) and the Fab light chain polypeptide (VL) of the second Fab molecule (2) -CL (2) ) and share a carboxy-terminal peptide bond. In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule. (3) -CL (3) ) further includes.

[0170] In a particular embodiment, the bispecific antigen-binding molecule is a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (i.e., the third Fab molecule contains a crossover Fab heavy chain, and the heavy chain constant region is replaced by the light chain constant region), thereby the Fab heavy chain constant region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, thereby the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule contains a crossover Fab heavy chain, and the heavy chain constant region is replaced by the light chain constant region), thereby the Fab light chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule. (3) -CL (3) -VH (1) -CL (1) -VH (2) -CH1 (2) ) includes. In some embodiments, the bispecific antigen-binding molecule further comprises a polypeptide, and the Fab light chain variable region of the first Fab molecule is the Fab heavy chain constant region (VL) of the first Fab molecule. (1) -CH1 (1) ) and the Fab light chain polypeptide (VL) of the second Fab molecule (2) -CL (2) ) and share a carboxy-terminal peptide bond. In some embodiments, the bispecific antigen-binding molecule is a polypeptide (VL) in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule. (3) -CH1 (3) ) further includes.

[0171] In certain embodiments, the bispecific antigen-binding molecule used in the combination therapy described herein is a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is EGFRvIII, and the first antigen-binding moiety comprises a heavy chain variable region (VH) including heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a light chain including light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6 a) a Fab molecule comprising a variable region (VL) and a first antigen-binding portion, b) a second antigen-binding portion that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding portion is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and Fab heavy chain are replaced by each other, and the Fab molecule comprises a heavy chain variable region (VH) including the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 9, HCDR2 of SEQ ID NO: 10, and HCDR3 of SEQ ID NO: 11, and SEQ ID NO: (i) The first antigen-binding portion of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second antigen-binding portion of the Fab heavy chain, and the second antigen-binding portion of b) is fused at the C-terminus of the Fab heavy chain, and the second antigen-binding portion of b) is fused at the N-terminus of the Fab heavy chain. The antigen-binding portion of (i) and the third antigen-binding portion of (c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of (d), or (ii) the second antigen-binding portion of (b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first antigen-binding portion of (a), and the first antigen-binding portion of (a) and the third antigen-binding portion of (c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of (d).

[0172] In another embodiment, the present invention relates to a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is EGFRvIII, and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) including heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) including light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; and b) a second antigen-binding moiety that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and Fab heavy chain are interchangeable. The obtained Fab molecule is a bispecific antigen-binding molecule comprising a second antigen-binding moiety comprising a heavy chain variable region (VH) including heavy chain complementarity-determining region (HCDR)1 of SEQ ID NO: 9, HCDR2 of SEQ ID NO: 10, and HCDR3 of SEQ ID NO: 11, and a light chain variable region (VL) including light chain complementarity-determining region (LCDR)1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, and c) an Fc domain composed of first and second subunits, wherein (i) the first antigen-binding moiety of a) and the second antigen-binding moiety of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

[0173] In certain embodiments, particularly when the amino acid substitutions described herein are made in the first (and, if present, third) antigen-binding moiety / Fab molecule, the constant domain CL of the first (and, if present, third) Fab molecule of the bispecific antigen-binding molecule is a kappa isotype. In other embodiments of the bispecific antigen-binding molecule according to the present invention, particularly in embodiments where the amino acid substitutions described herein are made in the second antigen-binding moiety / Fab molecule, the constant domain CL of the second antigen-binding moiety / Fab molecule is a kappa isotype. In some embodiments, the constant domain CL of the first (and, if present, third) antigen-binding moiety / Fab molecule and the constant domain CL of the second antigen-binding moiety / Fab molecule are kappa isotypes.

[0174] In one embodiment, the bispecific antigen-binding molecule is: a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is EGFRvIII, and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) including heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) including light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; b) a second antigen-binding moiety that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted for each other, and the Fab molecule comprises a heavy chain variable region including heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 9, HCDR2 of SEQ ID NO: 10, and HCDR3 of SEQ ID NO: 11. a) (Numbered according to the EU Index), the amino acid at position 213 is substituted by glutamic acid (E) (Kabat(Numbered according to the EU index), (i) the first antigen-binding portion of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second antigen-binding portion of b) and the third antigen-binding portion of c) is each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of d), or (ii) the second antigen-binding portion of b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first antigen-binding portion of a) and the first antigen-binding portion of a) and the third antigen-binding portion of c) is each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of d).

[0175] In certain embodiments, the bispecific antigen-binding molecule is: a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is EGFRvIII, and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) including heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) including light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; b) a second antigen-binding moiety that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted for each other, and the Fab molecule comprises a heavy chain variable region (HCDR) including heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 9, HCDR2 of SEQ ID NO: 10, and HCDR3 of SEQ ID NO: 11. a) (Numbered according to the EU Index), the amino acid at position 213 is substituted by glutamic acid (E) (Kabat(Numbered according to the EU index), (i) the first antigen-binding portion of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second antigen-binding portion of b), and the second antigen-binding portion of b) and the third antigen-binding portion of c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of d), or (ii) the second antigen-binding portion of b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first antigen-binding portion of a), and the first antigen-binding portion of a) and the third antigen-binding portion of c) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of d).

[0176] In another embodiment, the bispecific antigen-binding molecule is: a) a first antigen-binding moiety that binds to a first antigen, wherein the first antigen is EGFRvIII, and the first antigen-binding moiety is a Fab molecule comprising a heavy chain variable region (VH) including heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3, and a light chain variable region (VL) including light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; b) a second antigen-binding moiety that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted for each other, and the Fab molecule comprises a heavy chain variable region including heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 9, HCDR2 of SEQ ID NO: 10, and HCDR3 of SEQ ID NO: 11 a) (Numbered according to the EU index), the amino acid at position 213 is substituted with glutamic acid (E) (numbered according to the Kabat EU index), and the first antigen-binding portion of a) and the second antigen-binding portion of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

[0177] According to any of the embodiments described above, the components of the bispecific antigen-binding molecule (e.g., Fab molecule, Fc domain) may be fused directly or via various linkers, particularly via peptide linkers containing one or more amino acids, typically about 2 to 20 amino acids, as described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S)n, (SG4)n, (G4S)n, or G4(SG4)n peptide linkers, where "n" is usually an integer from 1 to 10, typically from 2 to 4.

[0178] In a particular embodiment, the bispecific antigen-binding molecule comprises: a) first and third antigen-binding moieties that bind to a first antigen, wherein the first antigen is EGFRvIII, and the first and second antigen-binding moieties are (conventional) Fab molecules, each comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 8; and b) a second antigen-binding moiety that binds to a second antigen, wherein the second antigen is CD3, and the second antigen-binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted for each other, and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15. a) comprising a second antigen-binding region including a light chain variable region containing the amino acid sequence of SEQ ID NO: 16, and c) an Fc domain composed of first and second subunits, wherein in the constant domains CL of the first and third antigen-binding regions of a), the amino acid at position 124 is substituted with lysine (K) (Kabat numbering), the amino acid at position 123 is substituted with lysine (K) or arginine (R) (particularly arginine (R)) (Kabat numbering), and in the constant domains CH1 of the first and third antigen-binding regions of a), the amino acid at position 147 is substituted with glutamic acid (E) (Kabat numbering). (Numbered according to the EU index), the amino acid at position 213 is substituted with glutamic acid (E) (numbered according to the Kabat EU index), furthermore, the first antigen-binding portion of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second antigen-binding portion of b) of the Fab heavy chain, and the second antigen-binding portion of b) and the third antigen-binding portion of a) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

[0179] In one embodiment, in the first subunit of the Fc domain of the bispecific antigen-binding molecule, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), optionally the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbered according to the Kabat EU index).

[0180] In further embodiments, in the first subunit of the Fc domain of the bispecific antigen-binding molecule, the serine residue at position 354 is further replaced by a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C) (particularly when the serine residue at position 354 is replaced by a cysteine ​​residue), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is further replaced by a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index).

[0181] In a further embodiment, in each of the first and second subunits of the Fc domain of the bispecific antigen-binding molecule, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbered according to the Kabat EU index).

[0182] In a further embodiment, the Fc domain is a human IgG1 Fc domain.

[0183] In another specific embodiment, the bispecific antigen-binding molecule includes a polypeptide having an amino acid sequence identical to at least 95%, 96%, 97%, 98%, or 99% of the sequence of SEQ ID NO: 17; a polypeptide having an amino acid sequence identical to at least 95%, 96%, 97%, 98%, or 99% of the sequence of SEQ ID NO: 18; a polypeptide having an amino acid sequence identical to at least 95%, 96%, 97%, 98%, or 99% of the sequence of SEQ ID NO: 19; and a polypeptide having an amino acid sequence identical to at least 95%, 96%, 97%, 98%, or 99% of the sequence of SEQ ID NO: 20. In a further specific embodiment, the bispecific antigen-binding molecule includes a polypeptide having an amino acid sequence of SEQ ID NO: 17; a polypeptide having an amino acid sequence of SEQ ID NO: 18; a polypeptide having an amino acid sequence of SEQ ID NO: 19; and a polypeptide having an amino acid sequence of SEQ ID NO: 20.

[0184] Fc domain variant In certain embodiments, the bispecific antigen-binding molecule includes an Fc domain composed of a first and a second subunit.

[0185] The Fc domain of a bispecific antigen-binding molecule consists of a pair of polypeptide chains containing the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, with each subunit containing the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other. In one embodiment, the bispecific antigen-binding molecule of the present invention contains not more than one Fc domain.

[0186] In one embodiment, the Fc domain of the bispecific antigen-binding molecule is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain containing an amino acid substitution at position S228, in particular the amino acid substitution S228P (Kabat EU index numbering). This amino acid substitution reduces Fab arm exchange of the IgG4 antibody in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further specific embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG1 Fc domain. An exemplary sequence of the human IgG1 Fc region is shown in Sequence ID No. 53.

[0187] Fc domain modification that promotes heterodimerization Bispecific antigen-binding molecules contain different antigen-binding moieties that can be fused to one or the other of two subunits of the Fc domain, and therefore the two subunits of the Fc domain are typically contained in two non-identical polypeptide chains. Several combinations of two polypeptides are possible through recombinant co-expression of these polypeptides and subsequent dimerization. To increase the yield and purity of bispecific antigen-binding molecules in recombinant production, it is advantageous to introduce modifications to the Fc domain of the bispecific antigen-binding molecule that promote the association of the desired polypeptide.

[0188] Therefore, in certain embodiments, the Fc domain of a bispecific antigen-binding molecule includes modifications that facilitate the association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is located within the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is located within the CH3 domain of the Fc domain.

[0189] To enhance heterodimerization, several methods exist for modifying the CH3 domain of the Fc domain, which are well described, for example, in International Publications 96 / 27011, 98 / 050431, European Patent No. 1870459, 2007 / 110205, 2007 / 147901, 2009 / 089004, 2010 / 129304, 2011 / 90754, 2011 / 143545, 2012058768, 2013157954, and 2013096291. Typically, in all such methods, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both manipulated in a complementary manner so that each CH3 domain (or the heavy chain containing it) does not homodimerize with itself but heterodimerizes with other complementaryly manipulated CH3 domains (resulting in heterodimerization of the first and second CH3 domains, and no homodimer is formed between the two first CH3 domains or the two second CH3 domains). These different methods for improved heavy chain heterodimerization are envisioned as different alternatives to heavy-light chain modifications in bispecific antigen-binding molecules with reduced heavy / light chain mispairing and Bence Jones-type byproducts (e.g., exchange / substitution of VH and VL in one binding arm and introduction of substitution of charged amino acids with opposite charges at the CH1 / CL interface).

[0190] In a specific embodiment, the modification that facilitates the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which includes a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain.

[0191] The knob-into-hole technique is described, for example, in U.S. Patents 5,731,168, 7,695,936, Ridgway et al., Prot Eng 9,617-621 (1996), and Carter, J Immunol Meth 248,7-15 (2001). Generally, this method involves introducing a projection ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, so that the projection can be positioned within the cavity to promote heterodimerization and inhibit homodimerization. The projection is constructed by replacing a smaller amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the projection is created at the interface of the second polypeptide by replacing a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0192] Therefore, in certain embodiments, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen-binding molecule, an amino acid residue is replaced with an amino acid residue having a larger side-chain volume, thereby generating a repositionable protrusion within the CH3 domain of the first subunit within the cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side-chain volume, thereby generating a cavity within the CH3 domain of the second subunit, within which the protrusion in the CH3 domain of the first subunit is repositionable.

[0193] Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0194] Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0195] The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0196] In a specific embodiment, in the first subunit of the Fc domain ("knob" subunit) (the CH3 domain), the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain ("hole" subunit) (the CH3 domain), the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbered according to the Kabat EU index).

[0197] In further embodiments, in the first subunit of the Fc domain, the serine residue at position 354 is further replaced by a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C) (particularly when the serine residue at position 354 is replaced by a cysteine ​​residue), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is further replaced by a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index). The introduction of these two cysteine ​​residues creates a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0198] In certain embodiments, the first subunit of the Fc domain includes the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain includes the amino acid substitutions Y349C, T366S, L368A and Y407V (numbered according to the Kabat EU index).

[0199] In certain embodiments, the antigen-binding moiety that binds to a second antigen (e.g., an activated T cell antigen) is fused to a first subunit of the Fc domain (including the "knob" modification) (optionally via a first antigen-binding moiety that binds to EGFRvIII and / or a peptide linker). While we do not wish to be constrained by theory, the fusion of the antigen-binding moiety that binds to a second antigen (e.g., an activated T cell antigen) to the knob-containing subunit of the Fc domain further minimizes the generation of an antigen-binding molecule containing two antigen-binding moieties that bind to the activated T cell antigen (steric collision of two knob-containing polypeptides).

[0200] Other techniques for CH3 modification that enhance heterodimerization have been considered as alternatives to the present invention and are described, for example, in International Publication Nos. 96 / 27011, 98 / 050431, European Patent No. 1870459, International Publication Nos. 2007 / 110205, 2007 / 147901, 2009 / 089004, 2010 / 129304, 2011 / 90754, 2011 / 143545, 2012 / 058768, 2013 / 157954, and 2013 / 096291.

[0201] In one embodiment, the heterodimerization method described in European Patent No. 1870459 is used instead. This method is based on the introduction of charged amino acids with opposite charges to specific amino acid positions at the CH3 / CH3 domain interface between two subunits of the Fc domain. One preferred embodiment of the bispecific antigen-binding molecule of the present invention is amino acid mutation R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutation D399K; E357K in the other CH3 domain of the Fc domain (numbered according to the Kabat EU index).

[0202] In another embodiment, the bispecific antigen-binding molecule contains the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain, the amino acid mutations T366S, L368A, and Y407V in the CH3 domain of the second subunit of the Fc domain, further amino acid mutations R409D and K370E in the CH3 domain of the first subunit of the Fc domain, and amino acid mutations D399K and E357K in the CH3 domain of the second subunit of the Fc domain (numbered according to the Kabat EU index).

[0203] In another embodiment, the bispecific antigen-binding molecule contains amino acid mutations S354C and T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, and Y407V in the CH3 domain of the second subunit of the Fc domain, or the bispecific antigen-binding molecule contains amino acid mutations Y349C and T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, and Y407V in the CH3 domain of the second subunit of the Fc domain, and further contains amino acid mutations R409D and K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K and E357K in the CH3 domain of the second subunit of the Fc domain (all numbered according to the Kabat EU index).

[0204] In one embodiment, the heterodimerization method described in International Publication No. 2013 / 157953 is used instead. In one embodiment, the first CH3 domain contains the amino acid mutation T366K, and the second CH3 domain contains the amino acid mutation L351D (numbered according to the Kabat EU index). In a further embodiment, the first CH3 domain contains a further amino acid mutation L351K. In a further embodiment, the second CH3 domain further contains an amino acid mutation selected from Y349E, Y349D, and L368E (preferably L368E) (numbered according to the Kabat EU index).

[0205] In one embodiment, the heterodimerization method described in International Publication No. 2012 / 058768 is used instead. In one embodiment, the first CH3 domain contains amino acid mutations L351Y, Y407A, and the second CH3 domain contains amino acid mutations T366A, K409F. In further embodiments, the second CH3 domain contains further amino acid mutations at position T411, D399, S400, F405, N390 or K392, e.g., (a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, (b) D399R, D399W, D399Y or D (c) S400E, S400D, S400R or S400K, (d) F405I, F405M, F405T, F405S, F405V or F405W, (e) N390R, N390K or N390D, (f) K392V, K392M, K392R, K392L, K392F or K392E (numbered according to the Kabat EU index). In further embodiments, the first CH3 domain includes amino acid mutations L351Y, Y407A, and the second CH3 domain includes amino acid mutations T366V, K409F. In further embodiments, the first CH3 domain includes amino acid mutation Y407A, and the second CH3 domain includes amino acid mutations T366A, K409F. In further embodiments, the second CH3 domain further includes amino acid mutations K392E, T411E, D399R, and S400R (numbered according to the Kabat EU index).

[0206] In one embodiment, a heterodimerization technique described in International Publication No. 2011 / 143545 is used instead, having, for example, amino acid modifications at positions selected from the group consisting of 368 and 409 (numbered according to the Kabat EU index).

[0207] In one embodiment, a heterodimerization technique described in International Publication No. 2011 / 090762 is used instead, which also employs the technique of inserting a knob into the hole described above. In one embodiment, the first CH3 domain contains the amino acid mutation T366W, and the second CH3 domain contains the amino acid mutation Y407A. In another embodiment, the first CH3 domain contains the amino acid mutation T366Y, and the second CH3 domain contains the amino acid mutation Y407T (numbered according to the Kabat EU index).

[0208] In one embodiment, the bispecific antigen-binding molecule or its Fc domain is an IgG2 subclass, and the heterodimerization method described in International Publication No. 2010 / 129304 is used instead.

[0209] In alternative embodiments, modifications that facilitate the association of the first and second subunits of the Fc domain include modifications that mediate electrostatic maneuvering effects, as described, for example, in PCT International Publication 2009 / 089004. Generally, this method involves the substitution of one or more amino acid residues with charged amino acid residues at the interface of the two Fc domain subunits such that homodimerization is electrostatically undesirable, but heterodimerization is electrostatically desirable. In one such embodiment, the first CH3 domain includes an amino acid substitution with a negatively charged amino acid of K392 or N392 (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain includes an amino acid substitution with a positively charged amino acid of D399, E356, D356 or E357 (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K, more preferably D399K and E356K). In a further embodiment, the first CH3 domain further includes an amino acid substitution with a negatively charged amino acid of K409 or R409 (e.g., glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In further embodiments, the first CH3 domain further comprises, or is replaced by, an amino acid substitution with negatively charged amino acids at K439 and / or K370 (e.g., glutamic acid (E) or aspartic acid (D)) (all numbered according to the Kabat EU index).

[0210] In further embodiments, the heterodimerization method described in International Publication No. 2007 / 147901 is used instead. In one embodiment, the first CH3 domain contains amino acid mutations K253E, D282K, and K322D, and the second CH3 domain contains amino acid mutations D239K, E240K, and K292D (numbered according to the Kabat EU index).

[0211] In yet another embodiment, the heterodimerization method described in International Publication No. 2007 / 110205 may be used instead.

[0212] In one embodiment, the first subunit of the Fc domain includes amino acid substitutions K392D and K409D, and the second subunit of the Fc domain includes amino acid substitutions D356K and D399K (numbered according to the Kabat EU index).

[0213] Fc domain modification that reduces Fc receptor binding and / or effector function. The Fc domain confers desirable pharmacokinetic properties to bispecific antigen-binding molecules, including a long serum half-life that contributes to good accumulation in target tissues and a desirable tissue-to-blood distribution ratio. However, at the same time, it can lead to undesirable targeting of bispecific antigen-binding molecules to cells expressing the Fc receptor rather than the preferred antigen-holding cells. Furthermore, co-activation of the Fc receptor signaling pathway, combined with T cell activation properties (e.g., in embodiments of bispecific antigen-binding molecules where the second antigen-binding portion binds to activated T cell antigens) and the long half-life of the bispecific antigen-binding molecule, can result in cytokine release that leads to overactivation of cytokine receptors and severe side effects upon systemic administration. Activation of immune cells other than T cells (those possessing the Fc receptor), for example due to potential T cell destruction by NK cells, can even reduce the effectiveness of bispecific antigen-binding molecules (particularly those where the second antigen-binding portion binds to activated T cell antigens).

[0214] Therefore, in certain embodiments, the Fc domain of a bispecific antigen-binding molecule exhibits reduced binding affinity to the Fc receptor and / or reduced effector function compared to a native IgG1Fc domain. In one such embodiment, the Fc domain (or a bispecific antigen-binding molecule containing said Fc domain) exhibits a binding affinity of less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% compared to a native IgG1Fc domain (or a bispecific antigen-binding molecule containing a native IgG1Fc domain), and / or exhibits effector function of less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% compared to a native IgG1Fc domain (or a bispecific antigen-binding molecule containing a native IgG1Fc domain). In one embodiment, the Fc domain (or a bispecific antigen-binding molecule containing said Fc domain) does not substantially bind to the Fc receptor and / or induce effector function. In certain embodiments, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, and most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a specific embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the native IgG1Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or a bispecific antigen-binding molecule containing said Fc domain) exhibits a binding affinity to FcRn that is greater than about 70%, particularly greater than about 80%, and more specifically greater than about 90%, compared to the native IgG1Fc domain (or a bispecific antigen-binding molecule containing said IgG1Fc domain).

[0215] In certain embodiments, the Fc domain is engineered to have reduced binding affinity to the Fc receptor and / or reduced effector function compared to an unengineered Fc domain. In certain embodiments, the Fc domain of a bispecific antigen-binding molecule contains one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to the Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to the Fc receptor. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there are more than one amino acid mutations that reduce the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to the Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, a bispecific antigen-binding molecule containing an engineered Fc domain exhibits a binding affinity to the Fc receptor of less than 20%, particularly less than 10%, and more specifically less than 5%, compared to a bispecific antigen-binding molecule containing an unengineered Fc domain. In certain embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activated Fc receptor. In specific embodiments, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, and most specifically human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complementary components (specifically binding affinity to C1q) is also reduced. In one embodiment, binding affinity to the neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn (i.e., protection of the binding affinity of the Fc domain to the receptor) is achieved when the Fc domain (or a bispecific antigen-binding molecule containing the Fc domain) exhibits a binding affinity greater than approximately 70% of the binding affinity of the unmodified form of the Fc domain (or a bispecific antigen-binding molecule containing this unmodified form of Fc) to FcRn. The Fc domain, or the bispecific antigen-binding molecule of the present invention containing the Fc domain, may exhibit an affinity greater than approximately 80% and even greater than approximately 90% of such affinity. In certain embodiments, the Fc domain of the bispecific antigen-binding molecule is modified to have reduced effector function compared to the unmodified Fc domain. Reduced effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cell-mediated cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cell phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced cross-linking with target-binding antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment, reduced effector function is reduced ADCC. In one embodiment, reduced ADCC is less than 20% of ADCC induced by an unmanipulated Fc domain (or a bispecific antigen-binding molecule containing an unmanipulated Fc domain).

[0216] In one embodiment, the amino acid mutation that reduces the binding affinity and / or effector function of the Fc domain to the Fc receptor is an amino acid substitution. In one embodiment, the Fc domain contains an amino acid substitution at a position selected from the group E233, L234, L235, N297, P331, and P329 (numbered by the Kabat EU index). In a more specific embodiment, the Fc domain contains an amino acid substitution at a position selected from the group L234, L235, and P329 (numbered by the Kabat EU index). In some embodiments, the Fc domain contains amino acid substitutions L234A and L235A (numbered by the Kabat EU index). In one such embodiment, the Fc domain is an IgG1Fc domain, in particular a human IgG1Fc domain. In one embodiment, the Fc domain contains an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, in particular P329G (numbered by the Kabat EU index). In one embodiment, the Fc domain includes an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297, and P331 (numbered according to the Kabat EU index). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc domain includes amino acid substitutions at positions P329, L234, and L235 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc domain includes amino acid mutations L234A, L235A, and P329G ("P329G LALA", "PGLALA", or "LALAPG").In particular, in certain embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (numbering according to the Kabat EU index), i.e., in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to the Kabat EU index).

[0217] In such an embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The combination of amino acid substitutions "P329G LALA" almost completely abolishes Fcγ receptor (and similarly, complement) binding of the human IgG1 Fc domain, as described in PCT International Publication No. WO 2012 / 130831, which is hereby incorporated by reference in its entirety. WO 2012 / 130831 also describes methods for preparing such variant Fc domains and methods for determining their properties, such as Fc receptor binding or effector functions.

[0218] IgG4 antibodies exhibit a reduced binding affinity to Fc receptors and a reduced effector function compared to IgG1 antibodies. Thus, in some embodiments, the Fc domain of the bispecific antigen-binding molecule is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment, the IgG4 Fc domain contains an amino acid substitution at position S228, specifically including the amino acid substitution S228P (numbering according to the Kabat EU index). In order to further reduce the binding affinity to Fc receptors and / or its effector function, in one embodiment, the IgG4 Fc domain contains an amino acid substitution at position L235, specifically including the amino acid substitution L235E (numbering according to the Kabat EU index). In another embodiment, the IgG4 Fc domain contains an amino acid substitution at position P329, specifically including the amino acid substitution P329G (numbering according to the Kabat EU index). In certain embodiments, the IgG4 Fc domain contains amino acid substitutions at positions S228, L235, and P329, specifically including the amino acid substitutions S228P, L235E, and P329G (numbering according to the Kabat EU index). Such IgG4 Fc domain variants and their Fcγ receptor binding characteristics are described in International Publication No. WO 2012 / 130831, which is hereby incorporated by reference in its entirety.

[0219] In certain embodiments, an Fc domain that exhibits a reduced binding affinity to Fc receptors and / or a reduced effector function compared to the native IgG1 Fc domain is a human IgG1 Fc domain containing the amino acid substitutions L234A, L235A, and optionally P329G, or a human IgG4 Fc domain containing the amino acid substitutions S228P, L235E, and optionally P329G (numbering according to the Kabat EU index).

[0220] In certain embodiments, the N-glycosylation of the Fc domain is removed. In one such embodiment, the Fc domain includes an amino acid mutation at position N297, specifically an amino acid substitution replacing asparagine with alanine (N297A) or an amino acid substitution replacing aspartic acid with aspartic acid (N297D) (numbered according to the Kabat EU index).

[0221] In addition to the Fc domains described herein and in PCT International Publication No. 2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also include those having one or more substitutions at Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (numbered by the Kabat EU Index). Such Fc variants include the so-called "DANA" Fc variant, which has substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, as well as Fc variants having substitutions at amino acid positions 265, 269, 270, 297, and 327 (U.S. Patent No. 7,332,581).

[0222] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of coding DNA sequences, PCR, gene synthesis, etc. Correct nucleotide changes can be confirmed, for example, by sequencing.

[0223] Binding to the Fc receptor can be readily determined, for example, by ELISA, or by surface plasmon resonance (SPR) using Fc receptors obtained by recombinant expression with standard equipment such as a BIAcore instrument (GE Healthcare). Alternatively, the binding affinity of an Fc domain or a bispecific antigen-binding molecule containing an Fc domain to the Fc receptor may be evaluated using cell lines known to express a specific Fc receptor (e.g., human NK cells expressing the FcγIIIa receptor).

[0224] The effector function of an Fc domain, or a bispecific antigen-binding molecule containing an Fc domain, can be measured by methods known in the art. Examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362, Hellstrom et al., Proc Natl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,1499-1502 (1985), U.S. Patent No. 5,821,337, Bruggemann et al., J Exp Med 166,1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA) and the CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or further, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, for example, as disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0225] In some embodiments, the binding of the Fc domain to complementary components (specifically to C1q) is reduced. Therefore, in some embodiments where the Fc domain is manipulated to have reduced effector function, this reduced effector function includes reduced CDC. C1q binding assays may be performed to determine whether a bispecific antigen-binding molecule containing an Fc domain can bind to C1q and thus possesses CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and 2005 / 100402. A CDC assay may be performed to assess complement activation (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0226] The determination of fcrn binding and in vivo clearance / half-life can also be carried out using methods known in the art (see, for example, Petkova, Sbet al., Int'l. Immunol. 18(12):1759-1769 (2006); International Publication No. 2013 / 120929).

[0227] Tumor-targeted 4-1BB agonists In one embodiment, the tumor-targeted 4-1BB agonist used in the combination therapy described herein is an antigen-binding molecule comprising an antigen-binding moiety that specifically binds to a tumor antigen and an antigen-binding moiety that specifically binds to 4-1BB. In one embodiment, the tumor-targeted 4-1BB agonist used in the combination therapy described herein is an anti-FAP / anti-4-1BB bispecific antibody. Tumor-targeted 4-1BB agonists suitable for use in the present invention (also referred to herein as "FAP-4-1BBL") are described, for example, in International Publication No. 2016 / 075278 and International Publication No. 2016 / 156291.

[0228] Tumor antigen binding site Tumor-targeted 4-1BB agonists include at least one antigen-binding moiety, particularly a Fab molecule, that binds to tumor cell antigens, especially targets on cancer cells or in the stroma. Therefore, in one embodiment, the tumor-targeted 4-1BB agonist is an antigen-binding molecule. In one embodiment, the tumor-targeted 4-1BB agonist includes an antigen-binding moiety that specifically binds to fibroblast-activating protein (FAP). In one embodiment, the tumor-targeted 4-1BB agonist includes an antigen-binding moiety that specifically binds to carcinoembryonic antigen (CEA).

[0229] In one embodiment, the antigen-binding portion includes a heavy chain variable region (VH) comprising the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22, and HCDR3 of SEQ ID NO: 23, and a light chain variable region (VL) comprising the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 26.

[0230] In one embodiment, VH contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 27. In one embodiment, VH contains an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 27. In one embodiment, VH contains an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 27. In certain embodiments, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antibody containing that sequence retains the ability to bind to FAP. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 27. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., in the FR). In one embodiment, VH contains the amino acid sequence of SEQ ID NO: 27. Optionally, VH contains the amino acid sequence of SEQ ID NO: 27, including post-translational modifications of that sequence.

[0231] In one embodiment, VL contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 28. In one embodiment, VL contains an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 28. In one embodiment, VL contains an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 28. In certain embodiments, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but the antibody containing that sequence retains the ability to bind to FAP. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., in the FR). In one embodiment, VL contains the amino acid sequence of SEQ ID NO: 28. Optionally, VL includes the amino acid sequence of SEQ ID NO: 28, including post-translational modifications of that sequence.

[0232] In one embodiment, the antigen-binding portion VH, which specifically binds to FAP, includes an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 27, and the antigen-binding portion VL includes an amino acid sequence that is at least approximately 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 28. In one embodiment, VH includes the amino acid sequence of SEQ ID NO: 27, and VL includes the amino acid sequence of SEQ ID NO: 28.

[0233] In a further embodiment, the antigen-binding domain includes VH containing the sequence of SEQ ID NO: 27 and VL containing the sequence of SEQ ID NO: 28.

[0234] In a further embodiment, the antigen-binding portion includes the VH sequence of SEQ ID NO: 27 and the VL sequence of SEQ ID NO: 28.

[0235] In another embodiment, the antigen-binding portion includes VH, which contains the heavy chain CDR sequence of VH in SEQ ID NO: 27, and VL, which contains the light chain CDR sequence of VL in SEQ ID NO: 28.

[0236] In a further aspect, the antigen-binding portion comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of VH of SEQ ID NO: 27 and the LCDR1, LCDR2, and LCDR3 amino acid sequences of VL of SEQ ID NO: 28.

[0237] In one aspect, the antigen-binding portion that specifically binds to FAP is a Fab molecule.

[0238] 4-1BB binding portion The tumor-targeted 4-1BB agonist comprises at least one portion that specifically binds to 4-1BB. In particular, the tumor-targeted 4-1BB agonist used herein comprises three ectodomains of 4-1BBL or a fragment thereof.

[0239] In certain aspects, the tumor-targeted 4-1BB agonist comprises three ectodomains of 4-1BBL or a fragment thereof, and the ectodomain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, particularly an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 33. In one aspect, the ectodomain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, particularly the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 33. In one aspect, the ectodomain comprises the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36, particularly the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 33. In certain aspects, the tumor-targeted 4-1BB agonist comprises three ectodomains of 4-1BBL or a fragment thereof, and the ectodomain comprises the amino acid sequence of SEQ ID NO: 33.

[0240] In one embodiment, a tumor-targeted 4-1BB agonist comprises a first and a second polypeptide linked to each other by a disulfide bond, the first polypeptide comprising two ectodomains or fragments of 4-1BBL linked to each other by a peptide linker, and the second polypeptide comprising one ectodomain or fragment of 4-1BBL.

[0241] In one embodiment, the tumor-targeted 4-1BB agonist comprises a first polypeptide containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 37, 38, 39, 40, 40, 42, 43, and 44, and the second polypeptide contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 29, 30, 31, 32, 33, 34, 35, and 36. In a further embodiment, the first polypeptide is selected from the group consisting of SEQ ID NOs: 37, 38, 39, 40, 40, 42, 43, and 44, and the second polypeptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, and 36. In one embodiment, the tumor-targeted 4-1BB agonist comprises a first polypeptide containing the amino acid sequence of SEQ ID NO: 41 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 33.

[0242] In one embodiment, a tumor-targeted 4-1BB agonist comprises a first and a second polypeptide linked to each other by a disulfide bond, the first polypeptide comprising two ectodomains or fragments of 4-1BBL linked to each other by a peptide linker, and the second polypeptide comprising one ectodomain or fragment of 4-1BBL, with each polypeptide linked to a CH1 or CL domain.

[0243] Tumor-targeted 4-1BB agonist format The techniques for producing multispecific antibodies are also applicable to the tumor-targeted 4-1BB agonists described herein.

[0244] In one embodiment, the tumor-targeted 4-1BB agonist includes an Fc domain composed of first and second subunits capable of stable association. In another embodiment, the tumor-targeted 4-1BB agonist includes an IgG Fc domain, specifically an IgG1Fc domain or an IgG4Fc domain.

[0245] In one embodiment, a tumor-targeted 4-1BB agonist used in the combination therapy described herein comprises (a) a Fab fragment capable of specifically binding to a tumor-associated antigen, (b) a first and second polypeptide linked to each other by a disulfide bond (the antigen-binding molecule is characterized in that the first polypeptide comprises two ectodomains or two fragments of 4-1BBL linked to each other by a peptide linker, and the second polypeptide comprises only one ectodomain or a fragment of 4-1BBL), and (c) an Fc domain composed of first and second subunits capable of stable association (in one of the Fab fragments, either the variable domains VH and VL or the constant domains CH1 and CL are exchanged). Thus, a tumor-targeted 4-1BB agonist can be prepared according to Crossmab technology.

[0246] Multispecific antibodies with domain substitution / exchange in one binding arm (CrossMabVH-VL or CrossMabCH-CL) are described in International Publication No. 2009 / 080252 and Schaefer, W. et al., PNAS, 108(2011)11187-1191. These multispecific antibodies can significantly reduce byproducts resulting from mismatches between the light chain for the first antigen and the wrong heavy chain for the second antigen (compared to approaches without such domain exchange).

[0247] In one embodiment, a tumor-targeted 4-1BB agonist comprises (a) a first Fab fragment capable of specifically binding to a tumor-associated antigen; (b) first and second polypeptides linked to each other by disulfide bonds (the antigen-binding molecule is characterized in that the first polypeptide comprises two ectodomains or fragments of 4-1BBL linked to each other by a peptide linker, and the second polypeptide comprises only one ectodomain or fragment of 4-1BBL, each of which is linked to a CH1 or CL domain); and (c) an Fc domain composed of first and second subunits capable of stable association (the constant domains CL and CH1 adjacent to 4-1BBL are replaced by each other such that the CH1 domain is part of the light chain and the CL domain is part of the heavy chain).

[0248] In one embodiment, the tumor-targeted 4-1BB agonist used in the combination therapy described herein is (a) A first heavy chain comprising a first polypeptide fused at its C-terminus to the N-terminus of a CL domain, a CL domain fused at its C-terminus to the N-terminus of one of the subunits of the Fc domain (e.g., the first subunit), (b) A first light chain comprising a second polypeptide fused to the N-terminus of the CH1 domain at its C-terminus, and the CH1 domain, (c) A second heavy chain comprising a heavy chain of a Fab molecule that specifically binds to a tumor antigen, the other of the subunits of the Fc domain (e.g., the second subunit), and the other of the subunits of the Fc domain. (d) Contains a second light chain containing a Fab molecule that specifically binds to the tumor antigen.

[0249] In one embodiment, the tumor-targeted 4-1BB agonist used in the combination therapy described herein is (i) First and second polypeptides, wherein the first polypeptide comprises two ectodomains or fragments of 4-1BBL linked together by a peptide linker, and the second polypeptide comprises one ectodomain or fragment of 4-1BBL, (ii) An antigen-binding portion that specifically binds to FAP, and is a Fab molecule, (iii) Fc domain consisting of the first and second subunits, (iv) An antigen-binding molecule containing a CL domain and a CH1 domain, Antigen-binding molecules, (a) A first heavy chain comprising a first polypeptide fused at its C-terminus to the N-terminus of a CL domain, a CL domain fused at its C-terminus to the N-terminus of one of the subunits of the Fc domain (e.g., the first subunit), (b) A first light chain comprising a second polypeptide fused to the N-terminus of the CH1 domain at its C-terminus, and the CH1 domain, (c) A second heavy chain comprising the heavy chain of the Fab molecule, the other of the subunits of the Fc domain (e.g., the second subunit), and the other of the subunits of the Fc domain (e.g., the second subunit), and (d) A second light chain containing the light chain of the Fab molecule, It consists of.

[0250] In one embodiment, a tumor-targeted 4-1BB agonist used in the combination therapy described herein comprises a first heavy chain containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45; a first light chain containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46; a second heavy chain containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 47; and a second light chain containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48. In one embodiment, the tumor-targeted 4-1BB agonist used in the combination therapy described herein comprises a first heavy chain containing the amino acid sequence of SEQ ID NO: 45, a first light chain containing the amino acid sequence of SEQ ID NO: 46, a second heavy chain containing the amino acid sequence of SEQ ID NO: 47, and a second light chain containing the amino acid sequence of SEQ ID NO: 48. In another embodiment, the tumor-targeted 4-1BB agonist used in the combination therapy described herein comprises the polypeptide sequences of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48.

[0251] charge modification In another embodiment, to further improve correct pairing, a tumor-targeted 4-1BB agonist may include (a) a first Fab fragment capable of specifically binding to a tumor-associated antigen, (b) first and second polypeptides linked to each other by disulfide bonds (the antigen-binding molecule is characterized in that the first polypeptide contains two ectodomains or two fragments of 4-1BBL linked to each other by a peptide linker, and the second polypeptide contains only one ectodomain or fragment of 4-1BBL, each of which is linked to a CH1 or CL domain), and (c) an Fc domain composed of first and second subunits capable of stable association, and may contain different charged amino acid substitutions (so-called "charged residues"). These modifications are introduced into the cross- or non-cross-linked CH1 and CL domains. In a particular embodiment, the present invention relates to a bispecific antigen-binding molecule in which, in one of the CL domains, the amino acid at position 123 (EU numbering) is replaced with arginine (R), the amino acid at position 124 (EU numbering) is replaced with lysine (K), and in one of the CH1 domains, the amino acids at positions 147 (EU numbering) and 213 (EU numbering) are replaced with glutamic acid (E).

[0252] Preferably, in the CL domain adjacent to 4-1BBL, the amino acid at position 123 (EU numbering) is replaced by arginine (R), and the amino acid at position 124 (EU numbering) is replaced by lysine (K). In the CH1 domain adjacent to 4-1BBL, the amino acids at position 147 (EU numbering) and position 213 (EU numbering) are replaced by glutamic acid (E).

[0253] Fc domain variant The tumor-targeted 4-1BB agonists used in the combination therapies described herein may include any modifications to the Fc domain of an anti-EGFRvIII / anti-CD3 bispecific antibody as described above.

[0254] In one embodiment, the Fc domain of a tumor target 4-1BB agonist includes a modification that facilitates the association of the first and second subunits of the Fc domain. In a particular embodiment, the modification is a so-called "knob-into-hole" modification, which includes a "knob" modification of one of the two subunits of the Fc domain and a "hole" modification of the other of the two subunits of the Fc domain.

[0255] Accordingly, the present invention relates to an antigen-binding molecule comprising (a) at least one antigen-binding moiety capable of specifically binding to a tumor-associated antigen, (b) first and second polypeptides linked to each other by a disulfide bond (the antigen-binding molecule is characterized in that the first polypeptide comprises two ectodomains or two fragments of 4-1BBL linked to each other by a peptide linker, and the second polypeptide comprises only one ectodomain or fragment of 4-1BBL), and (c) an Fc domain composed of first and second subunits capable of stable association (characterized in that, according to the knob-into-hole method, the first subunit of the Fc domain comprises a knob and the second subunit of the Fc domain comprises a hole). Accordingly, in certain embodiments, in the CH3 domain of the first subunit of the Fc domain of the tumor-targeting 4-1BB agonist, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protrusion within the CH3 domain of the first subunit that can be positioned within the cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit that can accommodate the protrusion within the CH3 domain of the first subunit. In certain embodiments, the first subunit of the Fc domain includes amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain includes amino acid substitutions Y349C, T366S and Y407V (numbered according to the Kabat EU index).

[0256] In one embodiment, the Fc domain of a tumor-targeted 4-1BB agonist contains one or more substitutions that reduce binding to and / or effector function of the Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In particular, the Fc domain contains amino acid substitutions at positions E233, L234, L235, N297, P331, and P329 (EU numbered). Specifically, the Fc domain contains amino acid substitutions at positions 234 and 235 (EU numbered) and / or 329 (EU numbered) of the IgG heavy chain. More specifically, tumor-targeted 4-1BB agonists used in combination therapies described herein contain an Fc domain having amino acid substitutions L234A, L235A, and P329G ("P329G LALA", EU numbered) in the IgG heavy chain.

[0257] Compositions, formulations, and routes of administration In further embodiments, the present invention provides a pharmaceutical composition comprising any of the anti-EGFRvIII / anti-CD3 bispecific antibodies and / or tumor-targeted 4-1BB agonists provided herein for use in, for example, any of the following therapeutic methods. In one embodiment, the pharmaceutical composition comprises the anti-EGFRvIII / anti-CD3 bispecific antibody and / or tumor-targeted 4-1BB agonist provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises the anti-EGFRvIII / anti-CD3 bispecific antibody and / or tumor-targeted 4-1BB agonist provided herein and at least one additional therapeutic agent, for example, described below.

[0258] Furthermore, a method is provided for producing a bispecific antibody and / or tumor-targeted 4-1BB agonist in a form suitable for in vivo administration, comprising (a) obtaining a bispecific antibody and / or tumor-targeted 4-1BB agonist, and (b) formulating the bispecific antibody and / or tumor-targeted 4-1BB agonist with at least one pharmaceutically acceptable carrier, thereby formulating a preparation of the bispecific antibody and / or tumor-targeted 4-1BB agonist for in vivo administration.

[0259] The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a bispecific antibody and / or a tumor-targeted 4-1BB agonist dissolved or dispersed in a pharmaceutically acceptable carrier. The expression “pharmaceutically or pharmacologically acceptable” means molecular entities and compositions that are generally non-toxic to the recipient at the dose and concentration employed, i.e., that do not cause adverse reactions, allergic reactions, or other adverse reactions when administered to animals such as humans. The preparation of pharmaceutical compositions comprising a bispecific antibody and / or a tumor-targeted 4-1BB agonist and optionally additional active ingredients will be known to those skilled in the art in light of this disclosure, as shown in Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, incorporated herein by reference. Furthermore, for animal (e.g., human) administration, it will be understood that the preparation should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards or other corresponding national agencies. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial agents, antifungal agents), isotonic agents, absorption retarders, salts, preservatives, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such similar materials and combinations thereof, as is known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, pp. 1289–1329, incorporated herein by reference). Any conventional carrier is intended for use in therapeutic or pharmaceutical compositions unless it is incompatible with the active ingredient.

[0260] Anti-EGFRvIII / anti-CD3 bispecific antibodies and / or tumor-targeted 4-1BB agonists (and any additional therapeutic agents) may be administered by any preferred means, including parenteral administration, intrapulmonary administration, intranasal administration, and, if desired for topical treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be carried out by injection, such as intravenous or subcutaneous injection, via any appropriate route, depending in part whether the administration is short-term or long-term.

[0261] Parenteral compositions include those designed for administration by injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, anti-EGFRvIII / anti-CD3 bispecific antibodies and / or tumor-targeted 4-1BB agonists may be formulated in aqueous solution, preferably in a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or saline buffer. The solution may contain formulation agents such as suspension agents, stabilizers, and / or dispersants. Alternatively, anti-EGFRvIII / anti-CD3 bispecific antibodies and / or tumor-targeted 4-1BB agonists may be in powder form for preparation with a suitable vehicle, such as sterile pyrogen-free water, before use. Sterile injection solutions are prepared by incorporating the anti-EGFRvIII / anti-CD3 bispecific antibodies and / or tumor-targeted 4-1BB agonists in the required amounts in a suitable solvent, along with various other components as listed below, if necessary. Sterilization can be easily achieved, for example, by filtration with a sterile filtration membrane. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a base dispersion medium and / or other components. For sterile powders for preparing sterile injection solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or freeze-drying, where powders of the active ingredients and any additional desired components are obtained from an already sterile filtered liquid medium. The liquid medium should be adequately buffered if necessary, and the liquid diluent should be first isotonic with sufficient saline or glucose before injection. The composition must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. It will be recognized that endotoxin contamination should be kept to a safe level, for example, less than 0.5 ng / mg of protein.Suitable pharmaceutically acceptable carriers include, but are not limited to, buffers, e.g., phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens, e.g., methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than approximately 10 residues) polypeptides; proteins Examples of substances include: substances such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts such as sodium; metal complexes such as Zn-protein complexes; and / or nonionic surfactants such as polyethylene glycol (PEG). The aqueous injection suspension may also contain compounds that increase the viscosity of the suspension (e.g., sodium carboxymethylcellulose, sorbitol, dextran, etc.). Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to enable the preparation of a highly concentrated solution. Furthermore, the suspension of the active compound may also be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethylcrete or triglycerides, or liposomes.

[0262] The active ingredient may also be encapsulated within colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsifies, nanoparticles, and nanocapsules) or in macroemulsifies, for example, by coacervation technology or by interfacial polymerization, in the form of microcapsules such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may also be prepared. A suitable example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing a polypeptide, which may be in the form of a molded article such as a film or microcapsule. In certain embodiments, sustained absorption of the injectable composition may be achieved by using it in a composition containing an absorption-delaying agent, such as aluminum monostearate, gelatin, or a combination thereof.

[0263] Furthermore, the aforementioned compositions may be formulated as depot preparations. Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, anti-EGFRvIII / anti-CD3 bispecific antibodies and / or tumor-targeted 4-1BB agonists can be formulated as suitable polymers or hydrophobic materials (e.g., as emulsions in acceptable oils) or ion-exchange resins, or as somewhat poorly soluble derivatives, such as somewhat poorly soluble salts.

[0264] Pharmaceutical compositions can be manufactured by conventional mixing, dissolution, emulsification, encapsulation, containment, or lyophilization processes. Pharmaceutical compositions can also be formulated conventionally using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate the processing of proteins into pharmaceutically usable formulations. The appropriate formulation depends on the selected route of administration.

[0265] Anti-EGFRvIII / anti-CD3 bispecific antibodies and / or tumor-targeted 4-1BB agonists can be formulated into compositions in the form of free acids or bases, neutral or salts. Pharmaceutically acceptable salts are those that substantially retain the biological activity of the free acid or free base. These include acid addition salts, e.g., those formed from free amino groups of proteinaceous compositions, or those formed from inorganic acids, e.g., hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed from free carboxyl groups can be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide; or from organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than their corresponding free base forms.

[0266] Treatment method and composition The present invention includes a combination therapy comprising an anti-EGFRvIII / anti-CD3 bispecific antibody in combination with a tumor-targeted 4-1BB agonist.

[0267] The present invention includes a method for treating a patient in need of treatment, characterized by administering to the patient a therapeutically effective dose of a combination therapy of an anti-EGFRvIII / anti-CD3 bispecific antibody combined with a tumor-targeting 4-1BB agonist.

[0268] One preferred embodiment of the present invention is a combination therapy of an anti-EGFRvIII / anti-CD3 bispecific antibody and a tumor-targeted 4-1BB agonist for use in the treatment of tumor cancer.

[0269] One embodiment of the present invention is an anti-EGFRvIII / anti-CD3 bispecific antibody described herein for use in the treatment of cancer or tumors in combination with a tumor-targeted 4-1BB agonist described herein.

[0270] Another embodiment of the present invention is a tumor-targeted 4-1BB agonist described herein for use in the treatment of cancer or tumors in combination with an anti-EGFRvIII / anti-CD3 bispecific antibody described herein.

[0271] Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, hematological cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferation disorders that can be treated with the combination therapies of the present invention include, but are not limited to, tumors located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and genitourinary system. Precancerous symptoms or lesions and cancer metastases are also included. In certain embodiments, the cancer is selected from the group consisting of kidney cancer, bladder cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer, and prostate cancer. In one embodiment, the cancer is glioblastoma.

[0272] One embodiment of the present invention is an anti-EGFRvIII / anti-CD3 bispecific antibody as described herein, combined with a tumor-targeted 4-1BB agonist as described herein, for use in the treatment of any of the above-mentioned cancers or tumors.

[0273] One embodiment of the present invention is an anti-EGFRvIII / anti-CD3 bispecific antibody described herein, combined with a tumor-targeted 4-1BB agonist described herein, for use in the treatment of glioblastoma.

[0274] The present invention includes a method for treating a patient in need of treatment, characterized by administering to the patient a therapeutically effective combination dose of an anti-EGFRvIII / anti-CD3 bispecific antibody described herein and a tumor-targeted 4-1BB agonist described herein.

[0275] The present invention provides a method for treating cancer in an individual, comprising administering to the individual an anti-EGFRvIII / anti-CD3 bispecific antibody as described herein in combination with a tumor-targeted 4-1BB agonist as described herein.

[0276] The present invention includes a method for preventing or treating metastasis in a patient requiring prevention or treatment of metastasis, characterized by administering to the patient a therapeutically effective combination dose of an anti-EGFRvIII / anti-CD3 bispecific antibody and a tumor-targeted 4-1BB agonist as described herein.

[0277] The present invention comprises the use of a tumor-targeted 4-1BB agonist and an anti-EGFRvIII / anti-CD3 bispecific antibody according to the present invention for the described combination therapy.

[0278] In a preferred embodiment, the anti-EGFRvIII / anti-CD3 bispecific antibody used in the above combination therapy and medical use comprises the polypeptide sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In a further embodiment, the tumor-targeted 4-1BB agonist used in the above combination therapy and medical use comprises the polypeptide sequences of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48. In another embodiment, the anti-EGFRvIII / anti-CD3 bispecific antibody used in the above combination therapy and medical use comprises the polypeptide sequences of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, and the tumor-targeted 4-1BB agonist used in the above combination therapy and medical use comprises the polypeptide sequences of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48.

[0279] The present invention comprises an anti-EGFRvIII / anti-CD3 bispecific antibody as described herein, combined with a tumor-targeted 4-1BB agonist as described herein, for use in the manufacture of a pharmaceutical for the treatment of cancer.

[0280] In another embodiment, the present invention provides a composition, such as a pharmaceutical composition, comprising an anti-EGFRvIII / anti-CD3 bispecific antibody and a tumor target 4-1BB agonist as described herein, formulated with a pharmaceutically acceptable carrier.

[0281] As used herein, "pharmaceutically acceptable carriers" include any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic agents and absorption / reabsorption retarders. Preferably, the carrier is suitable for injection or infusion.

[0282] The compositions of the present invention can be administered by various methods known in the art. As will be understood by those skilled in the art, the route and mode of administration will vary depending on the desired outcome.

[0283] Examples of pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions. The use of such media and drugs for pharmaceutically active substances is known in the art. In addition to water, carriers may be, for example, isotonic buffered saline.

[0284] Regardless of the selected route of administration, the compounds of the present invention, and / or the pharmaceutical compositions of the present invention, which can be used in a preferred hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0285] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an effective amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. The selected dose level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention used, or its ester, salt, or amide; the mode of administration; the time of administration; the elimination rate of the particular compound used; other drugs, compounds, and / or materials used in combination with the particular composition used; the age, sex, weight, symptoms, overall health, and medical history of the patient being treated, as well as similar factors well known in the medical field.

[0286] The present invention comprises a tumor-targeted 4-1BB agonist as described herein, combined with an anti-EGFRvIII / anti-CD3 bispecific antibody as described herein, for use in the manufacture of a pharmaceutical for the treatment of cancer. In one embodiment, the anti-EGFRvIII / anti-CD3 bispecific antibody for use in the manufacture of a pharmaceutical for the treatment of cancer according to the present invention comprises the polypeptide sequences of SEQ ID NOs. 17, 18, 19, and 20. In one embodiment, the tumor-targeted 4-1BB agonist for use in the manufacture of a pharmaceutical for the treatment of cancer according to the present invention comprises the polypeptide sequences of SEQ ID NOs. 45, 46, 47, and 48. In one embodiment, the anti-EGFRvIII / anti-CD3 bispecific antibody for use in the manufacture of a pharmaceutical for the treatment of cancer according to the present invention comprises the polypeptide sequences of SEQ ID NOs. 17, 18, 19, and 20, and the tumor-targeted 4-1BB agonist for use in the manufacture of a pharmaceutical for the treatment of cancer according to the present invention comprises the polypeptide sequences of SEQ ID NOs. 45, 46, 47, and 48.

[0287] The present invention further provides the use of the anti-EGFRvIII / anti-CD3 bispecific antibody and the tumor-targeted 4-1BB agonist according to the present invention, as described herein, for the manufacture of a pharmaceutical product, preferably together with a pharmaceutically acceptable carrier, for the treatment of patients suffering from cancer.

[0288] In one embodiment, the present invention provides a kit intended for the treatment of a disease, comprising (a) an anti-EGFRvIII / anti-CD3 bispecific antibody as described herein and (b) a tumor-targeted 4-1BB agonist as described herein, in the same or separate containers, and optionally further comprising a package insert including printed instructions for the use of combination therapy as a method for treating the disease.

[0289] Furthermore, the kit may include (a) a first container containing a composition comprising the anti-EGFRvIII / anti-CD3 bispecific antibody described herein; (b) a second container containing a composition comprising the tumor-targeted 4-1BB agonist described herein; and optionally (c) a third container containing a composition comprising an additional cytotoxic agent or other therapeutic agent. The kit in this embodiment may further include a package insert indicating that the composition can be used to treat specific symptoms. Alternatively, or in addition thereto, the kit may further include a fourth container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. This may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0290] In another embodiment, the present invention provides a kit intended for the treatment of a disease, comprising (a) a container comprising the anti-EGFRvIII / anti-CD3 bispecific antibody described herein, and (b) a package insert containing instructions for the use of the anti-EGFRvIII / anti-CD3 bispecific antibody in combination therapy with a tumor-targeted 4-1BB agonist described herein as a method for treating the disease.

[0291] In another embodiment, the present invention provides a kit intended for the treatment of a disease, comprising (a) a container comprising a tumor-targeted 4-1BB agonist as described herein, and (b) a package insert containing instructions for the use of the tumor-targeted 4-1BB agonist in combination therapy with an anti-EGFRvIII / anti-CD3 bispecific antibody as described herein as a method for treating the disease.

[0292] In a further embodiment, the present invention provides a pharmaceutical product intended for the treatment of a disease, comprising an anti-EGFRvIII / anti-CD3 bispecific antibody as described herein, which is intended for use in combination therapy with a tumor-targeted 4-1BB agonist as described herein, and optionally includes a package insert containing printed instructions directing the use of combination therapy as a method for treating the disease.

[0293] The term “treatment method” or its equivalent, when applied to cancer, refers to a treatment or set of measures designed to reduce or eliminate the number of cancer cells in a patient, or to alleviate the symptoms of cancer. “Treatment method” of cancer or another proliferative disorder does not necessarily mean that cancer cells or other disorders are actually eliminated, that the number of cells or disorders is actually reduced, or that the symptoms of cancer or other disorders are actually alleviated. Often, treatment methods for cancer are performed even when the probability of success is low, but are still considered to induce a set of measures that are beneficial overall, given the patient’s medical history and estimated survival.

[0294] The terms “administered in combination with,” “co-administration,” “co-administering,” “combination therapy,” or “combination treatment” refer to the administration of the anti-EGFRvIII / anti-CD3 bispecific antibody and the tumor-targeted 4-1BB agonist described herein, for example, as separate formulations / uses (or as a single formulation / use). Co-administration may be simultaneous or sequential in either order, and it is desirable that there be a period in which both (or all) activators exert biological activity simultaneously. The activators may be co-administered simultaneously or sequentially (e.g., intravenously (iv)) by continuous administration, or by oral administration. When both therapeutic agents are co-administered sequentially, the doses may be administered on the same day in two separate doses, or one drug may be administered on day 1 and the second drug co-administered from day 2 to day 7, preferably from day 2 to day 4. Therefore, in one embodiment, the term “continuously” means within 7 days after administration of the first component, preferably within 4 days after administration of the first component, and the term “simultaneously” means administration at the same time. The term “co-administration” with respect to maintenance doses of anti-EGFRvIII / anti-CD3 bispecific antibodies and / or tumor-targeted 4-1BB agonists means that maintenance doses can be co-administered simultaneously, for example, weekly, if the treatment cycle is appropriate for all drugs.

[0295] It is clear that an antibody is administered to a patient in a “therapeutic effective dose” (or simply “effective dose”), which is the amount of each compound or combination that elicits a biological or medical response in a tissue, system, animal, or human being that is being sought by researchers, veterinarians, physicians, or other clinicians.

[0296] The dosage and timing of co-administration will depend on the type (species, sex, age, weight, etc.) and the symptoms of the patient being treated, as well as the severity of the disease or symptoms being treated. The anti-EGFRvIII / anti-CD3 bispecific antibody and / or tumor-targeted 4-1BB agonist should be administered to the patient appropriately, either once or over a series of treatments, for example, on the same day, on subsequent days, or at weekly intervals.

[0297] Those skilled in the art will readily recognize that, in many cases, combination therapies may not result in a cure and may only provide partial benefits. In some embodiments, any physiological change that provides some benefit may also be considered therapeutically beneficial. Therefore, in some embodiments, the amount of combination therapy that produces a physiological change may be considered an "effective dose" or "therapeutic effective dose." [Examples]

[0298] Example 1: Functional characterization of EGFRvIII-TCB in combination with FAP-4-1BBL Isolation of PBMCs Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation of blood obtained from buffy coats derived from healthy human donors. The buffy coats were diluted with sterile PBS and layered on a Histopaque gradient (Sigma, #H8889). After centrifugation (450 × g, 30 min, room temperature), the plasma above the interface containing the PBMCs was discarded, and the PBMCs were subsequently transferred to a new Falcon tube filled with 50 ml of PBS. The mixture was centrifugated (400 × g, 10 min, room temperature), the supernatant was discarded, and the PBMC pellet was washed twice with sterile PBS (centrifugation stage 350 × g, 10 min). The resulting PBMC population was automatically counted (ViCell) and further stored in a cell incubator at 37°C, 5% CO2 in RPMI1640 medium containing 2% FBS and 1% GlutaMAX.

[0299] T cell-mediated tumor cell killing In short, U87MG glioblastoma cells (target cells) stably transfected with human EGFRvIII were harvested with trypsin / EDTA, washed, and resuspended in RPMI1640 medium containing 2% FBS and 1% GlutaMAX. Cells were plated at a density of 50,000 cells / well using flat-bottom 96-well plates. For the killing assay, EGFRvIII-TCB (SEQ ID NOs. 17, 18, 19, 20) was added in triplicate at the indicated concentrations, either alone or in combination with 1 nM FAP-4-1BBL (SEQ ID NOs. 45, 46, 47, 48) or 1 nM untargeted DP47-4-1BBL (SEQ ID NOs. 54, 55, 56, 57). PBMCs (effector cells) were added to the target cells in a final effector-to-target (E:T) ratio of 2:1. Target cell death was assessed by quantifying LDH released into the cell supernatant by apoptotic / necrotic cells after incubation at 37°C and 5% CO2 for 24, 48, and 72 hours (LDH detection kit, Roche Applied Science, #11644793001). Maximum target cell lysis (=100%) was achieved by incubation of target cells with 1% Triton X-100. Minimum lysis (=0%) refers to target cells co-incubated with effector cells without the bispecific construct.

[0300] Cytokine secretion in the supernatant was measured by flow cytometry after 72 hours using a cytometry bead array (CBA) according to the manufacturer's instructions, but 25 μl of supernatant and beads were used instead of 50 μl of beads and sample. The following CBA kits (BD Biosciences) were used: CBA Human Interferon Gamma (IFNγ) Flex Set, CBA Human IL-2 Flex Set, CBA Human IL-10 Flex Set, CBA Human Granzyme B Flex Set, and CBA Human TNF Flex Set. Samples were measured using BD FACS Canto II or BD FACS Fortessa, and analysis was performed using Diva Software (BD Biosciences). Characterization of EGFRvIII-TCB in combination with FAP-4-1BBL

[0301] Using FAP-expressing U87MG-huEGFRvIII glioblastoma cells as targets and PBMCs as effector cells, the functional activity of EGFRvIII-TCB was compared to EGFRvIII-TCB combined with FAP-4-1BBL or untargeted control DP47-4-1BBL in T cell death assays at several time points. All three conditions (EGFRvIII-TCB alone, EGFRvIII-TCB + FAP-4-1BBL, and EGFRvIII-TCB + DP47-4-1BBL) induced strong tumor cell death, and no differences in tumor cell death were observed between the conditions (Figure 1). In parallel, cytokine release into the supernatant after 72 hours was measured from the same experiment. Compared to EGFRvIII-TCB alone or EGFRvIII-TCB in combination with DP47-4-1BBL, treatment with EGFRvIII-TCB and FAP-4-1BBL resulted in increased release of IL2, TNFα, IFNγ, and IL10, indicating increased T cell activation due to the addition of FAP-4-1BBL (Figure 2). Addition of untargeted control DP47-4-1BBL to EGFRvIII-TCB did not increase cytokine release, suggesting that crosslinking via bound FAP is necessary for increased activation. No difference in granzyme B release was detected between EGFRvIII-TCB alone and the combination of EGFRvIII-TCB and FAP-4-1BBL, which is consistent with the cell death data shown in Figure 1.

[0302] Example 2: In vivo efficacy of EGFRvIII-TCB as a monotherapy and in combination with FAP-41BBL in a xenograft model of human tumor cell lines. The antitumor efficacy of a human EGFRvIII-TCB bispecific antibody was tested both as a monotherapy and in combination with FAP-41BBL in a xenograft model in which the human glioblastoma U87-EGFRvIII cell line was subcutaneously injected into fully humanized NSG mice.

[0303] U87 cells (human glioblastoma) were initially obtained from ATCC (Manassas, USA) and stably transfected to express the huEGFRvIII protein (Roche-Glycart, Schlieren, Switzerland). After proliferation, the cells were stored in the Roche-Glycart internal cell bank. The U87-EGFRvIII cell line was cultured in DMEM medium containing 10% FCS (Sigma) and 0.5 μg / ml puromycin (Invitrogen, Germany). Cells were cultured at 37°C in a saturated steam atmosphere with 5% CO2. Passage 9 was used for transplantation. Cell viability was 96.3%. 5 x 10⁶ cells per animal. 5 The cells were subcutaneously injected into the flank of mice in 100 μl of RPMI cell culture medium (Gibco) using a 1 ml tuberculin syringe (BD Biosciences, Germany).

[0304] Fully humanized NSG female mice (Roche-Glycart, Schlieren, Switzerland) were maintained under specific pathogen-free conditions in a daily 12-hour light / 12-hour dark cycle according to established guidelines (GV-Solas; Ferasa; Tiersch). The experimental research protocol was reviewed and approved by the local authority. Continuous health monitoring was performed regularly.

[0305] On day 0 of the study, mice were given 5 x 10 5 Individual U87-EGFRvIII cells were subcutaneously injected and randomized for weighing. Two weeks after tumor cell injection (tumor volume > 150 mm²) 3Mice were intravenously (iv) injected once a week for 4 weeks with EGFRvIII-TCB 0.03 mg / kg (SEQ ID NO: 17, 18, 19, 20), FAP-41BBL 1 mg / kg (SEQ ID NO: 45, 46, 47, 48), or a combination thereof. All mice received 200 μl of the appropriate solution via iv injection. The vehicle group mice were injected with histidine buffer, and the treatment group mice were injected with EGFRvIII-TCB, FAP-41BBL, or a combination thereof. Stock solutions were diluted with histidine buffer as needed to obtain the appropriate amount of compound per 200 μl. Tumor growth measurements were assessed three times a week using calipers and measured by volume (mm³) using GrahPad Prism software. 3 The plot was generated using + / -SEM.

[0306] Figure 3 shows that the EGFRvIII-TCB + FAP-41BBL combination mediated superior efficacy in inhibiting tumor growth compared to EGFRvIII-TCB and FAP-41BBL alone. [Table 1] TIFF2026510318000002.tif252170TIFF2026510318000003.tif253170TIFF202 6510318000004.tif255170TIFF2026510318000005.tif255170TIFF20265103180 00006.tif253170TIFF2026510318000007.tif255170TIFF2026510318000008.t if253170TIFF2026510318000009.tif253170TIFF2026510318000010.tif124170

Claims

1. An anti-EGFRvIII / anti-CD3 bispecific antibody combined with a tumor-targeted 4-1BB (CD137) agonist for use as an adjunct therapy in cancer treatment.

2. Use of anti-EGFRvIII / anti-CD3 bispecific antibodies combined with tumor-targeted 4-1BB agonists in the manufacture of pharmaceuticals for the treatment of cancer.

3. A method for treating cancer in an individual, comprising administering to the individual an anti-EGFRvIII / anti-CD3 bispecific antibody in combination with a tumor-targeting 4-1BB agonist.

4. A kit comprising a first pharmaceutical product containing an anti-EGFRvIII / anti-CD3 bispecific antibody and a second pharmaceutical product containing a tumor-targeting 4-1BB agonist, further comprising, optionally, a package insert containing instructions for administering the first pharmaceutical product in combination with the second pharmaceutical product to treat cancer in an individual.

5. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, wherein the anti-EGFRvIII / anti-CD3 bispecific antibody is: (i) A first antigen-binding moiety that specifically binds to EGFRvIII and includes a heavy chain variable region (VH) containing heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 3, and a light chain variable region (VL) containing light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and LCDR 3 of SEQ ID NO: 6, and (ii) A second antigen-binding region that specifically binds to CD3 and includes a heavy chain variable region (VH) containing the heavy chain complementarity-determining region (HCDR) 1 of SEQ ID NO: 9, HCDR 2 of SEQ ID NO: 10, and HCDR 3 of SEQ ID NO: 11, and a light chain variable region (VL) containing the light chain complementarity-determining region (LCDR) 1 of SEQ ID NO: 12, LCDR 2 of SEQ ID NO: 13, and LCDR 3 of SEQ ID NO:

14. Anti-EGFRvIII / anti-CD3 bispecific antibody combined with a tumor-targeting 4-1BB agonist for use, including, methods, or kits.

6. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, wherein the anti-EGFRvIII / anti-CD3 bispecific antibody is: (i) A first antigen-binding moiety that specifically binds to EGFRvIII, comprising VH which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7, and VL which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8, and (ii) A second antigen-binding moiety that specifically binds to CD3, comprising VH which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15, and VL which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

16. Anti-EGFRvIII / anti-CD3 bispecific antibody combined with a tumor-targeting 4-1BB agonist for use, including, methods, or kits.

7. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist according to any one of claims 1 to 6, wherein the first antigen-binding portion and / or the second antigen-binding portion of the anti-EGFRvIII / anti-CD3 bispecific antibody is a Fab molecule.

8. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist according to any one of claims 1 to 7, wherein the second antigen-binding portion is a Fab molecule, and the variable domains VL and VH or constant domains CL and CH1 of the Fab light chain and Fab heavy chain, particularly the variable domains VL and VH, are substituted for each other.

9. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist according to any one of claims 5 to 8, wherein the first antigen-binding portion is a Fab molecule, and in the constant domain, the amino acid at position 124 (numbered according to Kabat) is independently substituted with lysine (K), arginine (R), or histidine (H), and in the constant domain CH1, the amino acid at position 147 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D), and the amino acid at position 213 (numbered according to the Kabat EU index) is independently substituted with glutamic acid (E) or aspartic acid (D).

10. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist according to any one of claims 5 to 9, wherein the first antigen-binding portion and the second antigen-binding portion are optionally fused to each other via a peptide linker.

11. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use in combination with a tumor-targeted 4-1BB agonist according to any one of claims 5 to 10, wherein the first antigen-binding portion and the second antigen-binding portion are each a Fab molecule, and either (i) the second antigen-binding portion is fused at the C-terminus of the Fab heavy chain of the first antigen-binding portion to the N-terminus of the Fab heavy chain of the first antigen-binding portion, or (ii) the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding portion.

12. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist for use, according to any one of claims 5 to 11, wherein the anti-EGFRvIII / anti-CD3 bispecific antibody comprises a third antigen-binding moiety.

13. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist according to claim 12, wherein the third antigen portion is identical to the first antigen-binding portion.

14. The anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit, wherein the anti-EGFRvIII / anti-CD3 bispecific antibody comprises an Fc domain composed of first and second subunits, combined with a tumor-targeted 4-1BB agonist for use according to any one of claims 1 to 13.

15. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist, according to any one of claims 1 to 14, wherein the first, second, and, if present, third antigen-binding moieties are each Fab molecules, and (i) the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first antigen-binding moiety, and the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second antigen-binding moiety, and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and, if present, the third antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

16. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist according to claim 14 or 15, wherein the Fc domain is an IgG Fc domain, particularly an IgG1 Fc domain.

17. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist for use, according to any one of claims 14 to 16, wherein the Fc domain is a human Fc domain.

18. Anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use in combination with a tumor-targeted 4-1BB agonist according to any one of claims 14 to 17, wherein the Fc domain comprises a modification that promotes the association of the first and second subunits of the Fc domain, and / or the Fc domain comprises one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

19. The anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit, in combination with a tumor-targeted 4-1BB agonist for use according to any one of claims 1 to 18, comprising the polypeptide sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:

20.

20. Anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist according to any one of claims 1 to 19, wherein the tumor-targeted 4-1BB agonist comprises an antigen-binding moiety that specifically binds to fibroblast-activating protein (FAP) or carcinoembryonic antigen (CEA).

21. An anti-EGFRvIII / anti-CD3 bispecific antibody in combination with a tumor-targeted 4-1BB agonist for use, use, method, or kit, wherein the antigen-binding moiety that specifically binds to FAP comprises a heavy chain variable region (VH) including heavy chain CDR (HCDR) 1 of SEQ ID NO: 21, HCDR 2 of SEQ ID NO: 22, and HCDR 3 of SEQ ID NO: 23, and a light chain variable region including light chain CDR (LCDR) 1 of SEQ ID NO: 24, LCDR 2 of SEQ ID NO: 25, and LCDR 3 of SEQ ID NO: 26, according to claim 20.

22. Anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist according to claim 20 or 21, comprising VH, whose antigen-binding moiety specifically binding to FAP is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 27, and VL, which is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

28.

23. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist for use, according to any one of claims 20 to 22, wherein the antigen-binding portion that specifically binds to FAP is a Fab molecule.

24. Anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist according to any one of claims 1 to 23, wherein the tumor-targeted 4-1BB agonist comprises three ectodomains or fragments of 4-1BBL.

25. Anti-EGFRvIII / anti-CD3 bispecific antibody, use, method or kit, in combination with a tumor-targeted 4-1BB agonist for use according to claim 24, wherein the three ectodomains of the 4-1BBL include an amino acid sequence selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, in particular an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO:

33.

26. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist for use according to any one of claims 1 to 25, wherein the tumor-targeted 4-1BB agonist comprises first and second polypeptides linked to each other by disulfide bonds, An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit, in combination with a tumor-targeted 4-1BB agonist for use, wherein the first polypeptide comprises two ectodomains or fragments of 4-1BBL linked together by a peptide linker, and the second polypeptide comprises one ectodomain or fragment of 4-1BBL.

27. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist according to claim 26, wherein the first polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 37, SEQ ID NOs. 38, SEQ ID NOs. 39, SEQ ID NOs. 40, SEQ ID NOs. 41, SEQ ID NOs. 42, SEQ ID NOs. 43, and SEQ ID NOs. 44, and the second polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 29, SEQ ID NOs. 30, SEQ ID NOs. 31, SEQ ID NOs. 32, SEQ ID NOs. 33, SEQ ID NOs. 34, SEQ ID NOs. 35, and SEQ ID NOs.

28. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist according to any one of claims 1 to 27, wherein the tumor-targeted 4-1BB agonist comprises an Fc domain composed of first and second subunits.

29. An anti-EGFRvIII / anti-CD3 bispecific antibody in combination with a tumor-targeted 4-1BB agonist for use, use, method, or kit, wherein the Fc domain of the tumor-targeted 4-1BB agonist comprises a modification that promotes the association of the first and second subunits of the Fc domain, and / or the Fc domain comprises one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor, according to claim 28.

30. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, wherein the tumor-targeted 4-1BB agonist is, (i) First and second polypeptides, wherein the first polypeptide comprises two ectodomains or fragments of 4-1BBL linked to each other by a peptide linker, and the second polypeptide comprises one ectodomain or fragment of 4-1BBL, (ii) An antigen-binding portion that specifically binds to FAP, and is a Fab molecule, (iii) Fc domain consisting of the first and second subunits, (iv) CL domain and CH1 domain An antigen-binding molecule containing, (a) A first heavy chain comprising the first polypeptide fused at its C-terminus to the N-terminus of the CL domain, the CL domain fused at its C-terminus to the N-terminus of one of the subunits of the Fc domain (e.g., the first subunit), (b) A first light chain comprising the second polypeptide fused to the N-terminus of the CH1 domain at its C-terminus, and the CH1 domain, (c) A second heavy chain comprising the heavy chain of the Fab molecule, the heavy chain of the Fab molecule, the heavy chain of the Fab molecule, the heavy chain of the Fab molecule, the heavy chain of the Fab molecule, the heavy chain of the Fab domain, the heavy chain, and the heavy chain, the heavy chain, and (d) A second light chain including the light chain of the Fab molecule, An anti-EGFRvIII / anti-CD3 bispecific antibody, consisting of a tumor-targeting 4-1BB agonist for use, in combination with a kit, for use, method, or kit.

31. The tumor-targeting 4-1BB agonist comprises a first heavy chain containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 45; a first light chain containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 46; and at least about 95%, 96%, 97%, 98%, 99%, or 9% identical to the amino acid sequence of SEQ ID NO:

47. An anti-EGFRvIII / anti-CD3 bispecific antibody, use, method, or kit for use, in combination with a tumor-targeted 4-1BB agonist according to any one of claims 1 to 30, comprising an antigen-binding molecule comprising a second heavy chain containing an amino acid sequence that is 9% or 100% identical, and a second light chain containing an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 48.

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