Treatment of cancer using HLA-A2 / MAGE-A4×CD3 bispecific antibodies and 4-1BB (CD137) agonists

Abstract

The present invention relates to the treatment of cancer, in particular using HLA-A2 / MAGE-A4xCD3 bispecific antibodies and 4-1BB (CD137) agonists.

Description

[Technical field]

[0001] The present invention relates to the treatment of cancer, in particular using HLA-A2 / MAGE-A4xCD3 bispecific antibodies and 4-1BB (CD137) agonists. [Background technology]

[0002] T cell-activating bispecific antibodies are promising cancer therapies designed to target cytotoxic T cells against tumor cells. The simultaneous binding of such antibodies to CD3 on T cells and to antigens expressed on tumor cells forces a transient interaction between the tumor cell and the T cell, leading to T cell activation and subsequent tumor cell lysis.

[0003] MAGE-A4 (melanoma-associated antigen 4) is a member of the MAGE family of cancer-testis antigens (CTAs). The MAGE-A family of proteins encompasses 12 highly homologous genes, clustered at Xq26-28, characterized by the presence of a conserved domain (MAGE homology domain, MHD). Despite being discovered more than 20 years ago, the biological functions of MAGE proteins remain poorly understood. Based on their expression patterns, the MAGE family can be divided into two subfamilies, namely type-I and type-II, and the MAGE-A group belongs to the type-I subfamily, whose expression is restricted to the germline and cancer cells. Many reports have suggested a correlation between overexpression of type-I MAGEs and cancer malignancy, tumor growth, and poor patient prognosis. Intracellular proteins such as MAGE-A4 can be degraded in the proteasome, processed, and presented on the cell surface by major histocompatibility complex (MHC) I as a T cell epitope. Thus, MAGE-A4 p230-239 MAGE-A4-derived peptides, such as (GVYDGREHTV), are presented on the cell surface in the context of HLA-A2 and can trigger T cell recognition. Considering its expression pattern, MAGE-A4 may be a promising target for cancer therapy.

[0004] T cell activating bispecific antibodies targeting HLA-A2 / MAGE-A4 are described in WO 2021 / 122875. Such T cell activating bispecific antibodies may be useful, for example, in the treatment of MAGE-A4-expressing cancers. HLA-A2 / MAGE-A4×CD3 bispecific antibody ("MAGE-A4-TCB") is currently being studied in a Phase I clinical trial in patients with MAGE-A4-expressing solid tumors carrying the HLA-A*02:01 allele (NCT05129280).

[0005] Unfortunately, the treatment of solid tumors with T cell-based immunotherapy still faces many challenges, the most prominent of which is overcoming the unfavorable tumor microenvironment (TME). Fibroblast activation protein (FAP) is highly expressed by cancer-associated fibroblasts (CAFs) and has the ability to regulate the tumor microenvironment (TME) by remodeling the extracellular matrix (ECM). FAP overexpression on CAFs is associated with poor prognosis in various cancers.

[0006] Therefore, to maximize the therapeutic effect of HLA-A2 / MAGE-A4-targeted T cell activating antibodies, particularly in solid tumor cancers, it would be desirable to enhance the effect of those antibodies in the TME despite its presence. Summary of the Invention

[0007] The present invention enhances T cell responses in the TME, despite the presence of TME (e.g. solid tumor cancer), by combination treatment of HLA-A2 / MAGE-A4-targeting T cell activating bispecific antibodies with 4-1BB (CD137) agonists (4-1BBL) that provide positive costimulatory signals to T cells, particularly 4-1BB (CD137) agonists that target stromal antigens such as fibroblast activation proteins (FAPs). As mentioned above, FAPs are upregulated on cancer-associated fibroblasts in tumors, and thus the FAP specificity of 4-1BB (CD137) agonists provides T cell costimulation strictly restricted to tumors.

[0008] The inventors have discovered that the combination of an HLA-A2 / MAGE-A4 targeted T cell activating bispecific antibody with a 4-1BB (CD137) agonist leads to enhanced activity in MAGE-A4 expressing cancers compared to the bispecific antibody alone activated by HLA-A2 / MAGE-A4 targeted T cells.

[0009] Using human cancer cell lines, the inventors surprisingly found that tumor cell lysis induced by HLA-A2 / MAGE-A4xCD3 bispecific antibody in the presence of FAP-expressing fibroblasts was enhanced by the addition of the 4-1BB agonist FAP-4-1BBL, even in cell lines and / or at doses where the HLA-A2 / MAGE-A4xCD3 bispecific antibody alone showed no activity.

[0010] Thus, in a first aspect, the present invention provides an HLA-A2 / MAGE-A4×CD3 bispecific antibody for use in the treatment of cancer in an individual, the treatment comprising administration of the HLA-A2 / MAGE-A4×CD3 bispecific antibody in combination with a 4-1BB (CD137) agonist.

[0011] In a further aspect, the present invention provides a 4-1BB (CD137) agonist for use in treating cancer in an individual, the treatment comprising administration of a 4-1BB (CD137) agonist in combination with an HLA-A2 / MAGE-A4×CD3 bispecific antibody.

[0012] In one aspect, the invention provides the use of an HLA-A2 / MAGE-A4×CD3 bispecific antibody in the manufacture of a medicament for the treatment of cancer in an individual, the treatment comprising administration of an HLA-A2 / MAGE-A4×CD3 bispecific antibody in combination with a 4-1BB (CD137) agonist.

[0013] In a further aspect, the present invention provides the use of a 4-1BB (CD137) agonist in the manufacture of a medicament for the treatment of cancer in an individual, the treatment comprising administration of a 4-1BB (CD137) agonist in combination with an HLA-A2 / MAGE-A4×CD3 bispecific antibody.

[0014] In a still further aspect, the present invention provides a method for treating cancer in an individual comprising administering to the individual an HLA-A2 / MAGE-A4xCD3 bispecific antibody and a 4-1BB (CD137) agonist.

[0015] In one aspect, the invention also provides a kit comprising a first medicament comprising an HLA-A2 / MAGE-A4xCD3 bispecific antibody and a second medicament comprising a 4-1BB (CD137) agonist, optionally further comprising a package insert comprising instructions for administration of the first medicament in combination with the second medicament to treat cancer in an individual.

[0016] The HLA-A2 / MAGE-A4×CD3 bispecific antibodies, 4-1BB (CD137) agonists, methods, uses or kits described above and herein may incorporate any of the features described below, either alone or in combination (unless the context dictates otherwise).

[0017] The HLA-A2 / MAGE-A4×CD3 bispecific antibody herein is a combination of CD3 and HLA-A2 / MAGE-A4, particularly HLA-A2 / MAGE-A4 p230-239 A bispecific antibody that specifically binds to HLA-A2 / MAGE-A4×CD3. Particularly useful HLA-A2 / MAGE-A4×CD3 bispecific antibodies for use in the present invention are described, for example, in WO 2021 / 122875 (incorporated herein by reference in its entirety). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The term "bispecific" means that an antibody is capable of specifically binding to at least two distinct antigenic determinants. Typically, a bispecific antibody contains two antigen binding sites, each of which is specific for a different antigenic determinant. In some embodiments, a bispecific antibody is capable of specifically binding to two antigenic determinants,

[0019] In particular, it is capable of binding together two antigenic determinants expressed on two separate cells.

[0020] The term "antigenic determinant" as used herein is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a contiguous stretch of amino acids or a conformation composed of distinct regions of non-contiguous amino acids) to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM).

[0021] The term "antigen-binding moiety" as used herein refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding moiety can target the moiety to which it binds (e.g., a second antigen-binding moiety) to a target site, e.g., a particular type of tumor cell that bears the antigenic determinant. In another embodiment, an antigen-binding moiety can activate signaling through its target antigen, e.g., a T-cell receptor complex antigen. Antigen-binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen-binding moieties include the antigen-binding domain of an antibody, including an antibody heavy chain variable region and an antibody light chain variable region. In some embodiments, an antigen-binding moiety may include an antibody constant region known in the art, as further defined below. Useful heavy chain constant regions include any of the five isotypes: alpha, delta, epsilon, gamma, or mu. Useful light chain constant regions include any of the two isotypes: kappa and lambda.

[0022] "Specifically binds" means that the binding is antigen-selective and can be distinguished from undesired or non-specific interactions. The binding ability of an antigen-binding moiety to a specific antigenic 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) techniques (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of the antigen-binding moiety to an unrelated protein is less than about 10% of the binding of the antigen-binding moiety to the antigen, e.g., as measured by SPR. In some aspects, an antigen-binding portion that binds to an antigen, or an antibody comprising an antigen-binding portion, has a specific binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 Dissociation constant (K D ).

[0023] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding moiety and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ), which can be expressed as the dissociation rate constant and the association rate constant (k off and k on) is the ratio of the rate constants. Thus, equivalent affinities may include different rate constants, as long as the ratio of the rate constants is the same. Affinity can be measured by well-established methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0024] "CD3" refers to any native CD3 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 indicated. The term encompasses "full-length", unprocessed CD3, as well as any form of CD3 resulting from processing within a cell. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, the CD3 is human CD3, in particular the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in UniProt (www.uniprot.org) under accession number P07766 (accession version 217), or in NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. See also SEQ ID NO: 24. The amino acid sequence of Macaca fascicularis CD3ε is shown in NCBI GenBank No. BAB71849.1. See also SEQ ID NO:25.

[0025] "MAGE-A4" stands for "melanoma associated antigen 4", a member of the MAGE family of cancer testis antigens (CTAs). The MAGE-A family of proteins encompasses 12 highly homologous genes clustered at Xq26-28 and characterized by the presence of a conserved domain (MAGE homology domain, MHD). Human MAGE-A4 is described in UniProt (www.uniprot.org) accession number P43358 (accession version 174), and the amino acid sequence of human MAGE-A4 is also set forth in SEQ ID NO: 21 herein. As used herein, "MAGE-A4" refers to any native MAGE-A4 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 indicated. The term encompasses "full-length," unprocessed MAGE-A4 as well as any form of MAGE-A4 that results from processing within the cell. The term also encompasses naturally occurring variants of MAGE-A4, such as splice variants or allelic variants. In one embodiment, the MAGE-A4 is human MAGE-A4, in particular the protein of SEQ ID NO:21.

[0026] "MAGE-A4 p230-239 or "p230-239 peptide" means a MAGE-A4 derived peptide having the amino acid sequence GVYDGREHTV (SEQ ID NO:22; positions 230-239 of the MAGE-A4 protein of SEQ ID NO:21).

[0027] "HLA-A2", "HLA-A * 02," "HLA-A02," or "HLA-A *"HLA-A2" (used interchangeably) refers to a human leukocyte antigen serotype in the HLA-A serogroup. HLA-A2 proteins (encoded by the respective HLA genes) constitute the α chain of the respective class I MHC (major histocompatibility complex) protein, which further comprises the β2 microglobulin subunit. A particular HLA-A2 protein is also called HLA-A201 (HLA-A0201, HLA-A02.01, or HLA-A * 02:01). In a particular embodiment, the HLA-A2 protein described herein is HLA-A201. An exemplary sequence of human HLA-A2 is given in SEQ ID NO:23.

[0028] "HLA-A2 / MAGE-A4" refers to a peptide derived from HLA-A2 molecule and MAGE-A4 (also referred to herein as "MAGE-A4 peptide"), specifically the p230-239 peptide ("HLA-A2 / MAGE-A4 p230-239 ")

[0029] As used herein, the terms "first," "second," or "third," etc., in reference to Fab molecules, are used for ease of distinction when more than one of each type of moiety is present. The use of these terms is not intended to confer a particular order or orientation of the bispecific antibody, unless specifically stated otherwise.

[0030] The term "valency" as used herein refers to the presence of a specific number of antigen-binding sites in an antibody. Thus, the term "monovalent binding to an antigen" refers to the presence of one (and not more than one) antigen-binding site in an antibody that is specific for the antigen.

[0031] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0032] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure.

[0033] "Antibody fragment" refers to a molecule other than an intact antibody that binds to an antigen bound by the intact antibody and contains a portion of the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 Antibody fragments include Fab and F(ab') fragments, which contain salvage receptor binding epitope residues and have extended in vivo half-lives. 2For a description of fragments, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097, WO 1993 / 01161, Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In some aspects, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 B1). Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0034] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have a similar structure, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6 thed., W. H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. "Kabat numbering," as used herein with respect to variable region sequences, refers to the numbering system established by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0035] As used herein, amino acid positions of all heavy and light chain constant regions and domains 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), referred to herein as "Kabat numbering" or "Kabat numbering". Specifically, the Kabat numbering system (see pages 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 domains CL of kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3), which is further specified by being referred to as "Kabat EU index numbering".

[0036] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence and determine antigen binding specificity, e.g., "complementarity determining regions" (CDRs). Typically, antibodies contain six CDRs; three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include the following: (a) hypervariable loops occurring 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 contacts 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)).

[0037] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. One of skill in the art will appreciate that the designations of CDRs can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0038] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0039] The "class" of an antibody or immunoglobulin refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these have subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0040] A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain (a "Fab heavy chain") and the VL and CL domains of an immunoglobulin light chain (a "Fab light chain").

[0041] A "crossover" Fab molecule (also called "Crossfab") refers to a Fab molecule in which the variable or constant domains of the Fab heavy and light chains have been exchanged (i.e., replaced by each other), i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CH1 (VL-CH1, in the N-terminal to C-terminal direction) and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in the N-terminal to C-terminal direction). For the sake of clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising 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 the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0042] In contrast, a "conventional" Fab molecule is a Fab molecule that contains a heavy chain in its native form, i.e. composed of a heavy chain variable domain and a constant domain (VH-CH1, in the N-terminal to C-terminal direction), and a light chain that contains a light chain variable domain and a constant domain (VL-CL, in the N-terminal to C-terminal direction).

[0043] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons composed of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called the variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called the variable light domain or light chain variable region, followed by a constant light (CL) domain, also called the light chain constant region. The heavy chains of immunoglobulins are assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which are classified as γ, ... 1 (IgG 1 ), γ 2 (IgG 2 ), γ 3 (IgG 3 ), γ 4 (IgG 4 ), α 1 (IgA 1 ) and α 2 (IgA 2 ). The light chains of immunoglobulins can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain. Immunoglobulins essentially consist of two Fab molecules and an Fc domain linked via an immunoglobulin hinge region.

[0044] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined as extending from Cys226, or from Pro230, to the carboxy terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational truncation of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, antibodies produced by host cells by expression of a particular nucleic acid molecule encoding a full-length heavy chain may contain a full-length heavy chain or a truncated variant of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called 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 dimeric Fc domain, i.e., a polypeptide that comprises the C-terminal constant region of an immunoglobulin heavy chain and has the ability to stably self-associate. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

[0045] A "modification that promotes the association of a first subunit of an Fc domain with a second subunit" is a peptide backbone manipulation or post-translational modification of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. As used herein, a modification that promotes association particularly includes separate modifications made to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to be associated, which are complementary to each other to promote the association of the two Fc domain subunits. For example, a modification that promotes association can change the structure or charge of one or both of the Fc domain subunits to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may not be identical in the sense that the additional components (e.g., antigen-binding moieties) fused to each of the subunits are not the same. In some embodiments, a modification that promotes association includes amino acid mutations, particularly amino acid substitutions, within the Fc domain. In certain embodiments, the association-promoting modifications comprise separate amino acid mutations, specifically amino acid substitutions, in each of the two subunits of the Fc domain.

[0046] The term "effector function" refers to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); cytokine secretion; immune complex-mediated antigen uptake by antigen-presenting cells; downregulation of cell surface receptors (e.g., B cell receptor); and activation of B cells.

[0047] "Percent (%) amino acid sequence identity" with respect 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 obtain the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished using a variety of methods within the skill of the art, such as publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, or followed by the BLOSUM50 comparison matrix. The FASTA program package was developed by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:24442448; WR Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et.al. (1997) Genomics 46:2436, and is publicly available at http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml.Alternatively, the sequences can be compared using the public server accessible at http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch(globalprotein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2) to perform a global, rather than local, alignment. The percent amino acid identity is given in the output alignment header.

[0048] An "activating Fc receptor" is an Fc receptor that, following engagement by the Fc domain of an antibody, triggers signaling events that stimulate the receptor-bearing cell to carry out an effector function. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32) and FcαRI (CD89).

[0049] "Reduced binding", e.g., reduced binding to Fc receptors, refers to a reduced affinity for the respective interaction, e.g., measured by SPR. For clarity, the term also includes a reduction in affinity to zero (or below the detection limit of the analytical method), i.e., a complete loss of interaction. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0050] By "fused" it is meant that the components (eg, a Fab molecule and an Fc domain subunit) are linked by a peptide bond, either directly or via one or more peptide linkers.

[0051] The HLA-A2 / MAGE-A4×CD3 bispecific antibody comprises a first antigen-binding portion that specifically binds to CD3 and a second antigen-binding portion that specifically binds to HLA-A2 / MAGE-A4, particularly HLA-A2 / MAGE-A4. p230-239 and a second antigen-binding moiety that specifically binds to

[0052] In one embodiment, the first antigen binding portion comprises a heavy chain variable region comprising a heavy chain CDR (HCDR)1 of SEQ ID NO: 1, an HCDR2 of SEQ ID NO: 2, and an HCDR3 of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR (LCDR)1 of SEQ ID NO: 4, an LCDR2 of SEQ ID NO: 5, and an LCDR3 of SEQ ID NO: 6.

[0053] In one embodiment, the second antigen binding portion comprises a heavy chain variable region comprising a heavy chain CDR (HCDR)1 of SEQ ID NO: 9, an HCDR2 of SEQ ID NO: 10, and an HCDR3 of SEQ ID NO: 11, and a light chain variable region comprising a light chain CDR (LCDR)1 of SEQ ID NO: 12, an LCDR2 of SEQ ID NO: 13, and an LCDR3 of SEQ ID NO: 14.

[0054] In a particular embodiment, the HLA-A2 / MAGE-A4×CD3 bispecific antibody comprises (i) a first antigen-binding moiety that specifically binds to CD3, the first antigen-binding moiety comprising a heavy chain variable region comprising a heavy chain CDR (HCDR)1 of SEQ ID NO: 1, an HCDR2 of SEQ ID NO: 2, and an HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR (LCDR)1 of SEQ ID NO: 4, an LCDR2 of SEQ ID NO: 5, and an LCDR3 of SEQ ID NO: 6; (ii) a second antigen-binding portion that specifically binds to HLA-A2 / MAGE-A4, the second antigen-binding portion comprising a heavy chain variable region comprising a heavy chain CDR (HCDR)1 of SEQ ID NO: 9, an HCDR2 of SEQ ID NO: 10, and an HCDR3 of SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR (LCDR)1 of SEQ ID NO: 12, an LCDR2 of SEQ ID NO: 13, and an LCDR3 of SEQ ID NO: 14. Includes.

[0055] In one embodiment, the first antigen-binding portion comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8.

[0056] In one embodiment, the first antigen binding portion comprises a heavy chain variable region sequence of SEQ ID NO:7 and a light chain variable region sequence of SEQ ID NO:8.

[0057] In one embodiment, the second antigen-binding portion comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:15, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:16.

[0058] In one embodiment, the second antigen binding portion comprises the heavy chain variable region sequence of SEQ ID NO:15 and the light chain variable region sequence of SEQ ID NO:16.

[0059] In some embodiments, the first and / or second antigen-binding moiety is a Fab molecule. In some embodiments, the first antigen-binding moiety is a crossover Fab molecule in which the variable or constant regions of the Fab light chain and the Fab heavy chain are exchanged. In such embodiments, the second antigen-binding moiety is preferably a conventional Fab molecule.

[0060] In some embodiments, where the first and second antigen-binding moieties of the bispecific antibody are both Fab molecules and in one of the antigen-binding moieties (particularly the first antigen-binding moiety), the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other, i) in the constant domain CL of the first antigen-binding moiety the amino acid at position 124 is substituted by a positively charged amino acid (Kabat numbering) and in the constant domain CH1 of the first antigen-binding moiety the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (Kabat EU index numbering); or ii) in the constant domain CL of the second antigen-binding moiety the amino acid at position 124 is substituted by a positively charged amino acid (Kabat numbering) and in the constant domain CH1 of the second antigen-binding moiety the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (Kabat EU index numbering).

[0061] A bispecific antibody does not comprise both of the modifications described under i) and ii): the constant domains CL and CH1 of the antigen-binding portion having a VH / VL exchange are not replaced by each other (i.e. remain unexchanged).

[0062] In a more specific embodiment, i) in the constant domain CL of the first antigen-binding moiety the amino acid at position 124 is substituted independently by a lysine (K), an arginine (R) or a histidine (H) (Kabat numbering) and in the constant domain CH1 of the first antigen-binding moiety the amino acid at position 147 or the amino acid at position 213 is substituted independently by a glutamic acid (E) or an aspartic acid (D) (Kabat EU index numbering); or ii) in the constant domain CL of the second antigen-binding moiety the amino acid at position 124 is substituted independently by a lysine (K), an arginine (R) or a histidine (H) (Kabat numbering) and in the constant domain CH1 of the second antigen-binding moiety the amino acid at position 147 or the amino acid at position 213 is substituted independently by a glutamic acid (E) or an aspartic acid (D) (Kabat EU index numbering).

[0063] In one such embodiment, in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is independently substituted by a lysine (K), an arginine (R) or a histidine (H) (Kabat numbering) and in the constant domain CH1 of the second antigen binding moiety the amino acid at position 147 or the amino acid at position 213 is independently substituted by a glutamic acid (E) or an aspartic acid (D) (Kabat EU index numbering).

[0064] In a further embodiment, in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is independently substituted by a lysine (K), an arginine (R) or a histidine (H) (Kabat numbering) and in the constant domain CH1 of the second antigen binding moiety the amino acid at position 147 is independently substituted by a glutamic acid (E) or an aspartic acid (D) (Kabat EU index numbering).

[0065] In a preferred embodiment, in the constant domain CL of the second antigen-binding moiety the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), in the constant domain CH1 of the second antigen-binding moiety the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering) and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0066] In one embodiment, in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat), and in the constant domain CH1 of the second antigen binding moiety the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0067] In one embodiment, in the constant domain CL of the second antigen binding moiety the amino acid at position 124 is substituted by a lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by an arginine (R) (numbering according to Kabat), and in the constant domain CH1 of the second antigen binding moiety the amino acid at position 147 is substituted by a glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by a glutamic acid (E) (numbering according to Kabat EU index).

[0068] In a particular embodiment, when the amino acid substitutions according to the above embodiments are made in the constant domain CL and in the constant domain CH1 of the second antigen binding moiety, the constant domain CL of the second antigen binding moiety is of the kappa isotype.

[0069] In some embodiments, the first antigen-binding moiety and the second antigen-binding moiety are fused to one another, optionally via a peptide linker.

[0070] In some embodiments, each of the first and second antigen-binding moieties is a Fab molecule, and (i) the second 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 first antigen-binding moiety, or (ii) 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.

[0071] In some embodiments, the HLA-A2 / MAGE-A4xCD3 bispecific antibody provides monovalent binding to CD3.

[0072] In certain embodiments, the HLA-A2 / MAGE-A4xCD3 bispecific antibody comprises a single antigen-binding moiety that specifically binds to CD3 and two antigen-binding moieties that specifically bind to HLA-A2 / MAGE-A4. Thus, in some embodiments, the HLA-A2 / MAGE-A4xCD3 bispecific antibody comprises a third antigen-binding moiety, in particular a Fab molecule, in particular a conventional Fab molecule, that specifically binds to HLA-A2 / MAGE-A4. The third antigen-binding moiety can incorporate all of the features described above for the second antigen-binding moiety (e.g., CDR sequences, variable region sequences, and / or amino acid substitutions within the constant region), either alone or in combination. In some embodiments, the third antigen-binding moiety is identical to the first antigen-binding moiety (e.g., it is also a conventional Fab molecule and comprises the same amino acid sequence).

[0073] In a particular embodiment, the HLA-A2 / MAGE-A4xCD3 bispecific antibody further comprises an Fc domain composed of a first subunit and a second subunit. In one embodiment, the Fc domain is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG 1 In another embodiment, the Fc domain is an Fc domain of an IgG 4 In a more specific embodiment, the Fc domain is an IgG Fc domain comprising an amino acid substitution at position S228, in particular the amino acid substitution S228P. 4 Fc domain (Kabat EU index numbering). This amino acid substitution is 4 Reduces Fab arm exchange of antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In more detailed embodiments, the Fc domain is a human Fc domain. In certain preferred embodiments, the Fc domain is a human IgG 1 Fc domain. Human IgG 1 An exemplary sequence of an Fc region is provided in SEQ ID NO:26.

[0074] In some embodiments in which each of the first, second, and, if present, third antigen binding moieties is a Fab molecule, (a) (i) the second 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 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 Fab heavy chain 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 (b) 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.

[0075] In a particular embodiment, the Fc domain comprises a modification that promotes the association of the first subunit with the second subunit of the Fc domain. The most extensive site of protein-protein interaction between the two subunits of the human IgG Fc domain is in the CH3 domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0076] In a specific embodiment, the modification that promotes the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which comprises 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. The knob-into-hole technique is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). In general, the method involves introducing a protuberance ("knob") at the contact surface of a first polypeptide and a corresponding cavity ("hole") at the contact surface of a second polypeptide, such that the protuberance is located within the cavity, thereby promoting heterodimerization and preventing homodimerization. A protuberance is constructed by replacing a small amino acid side chain from the interface of a first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of identical or similar size to the protuberance is created in the interface of a second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0077] Thus, in some embodiments, an amino acid residue in the CH3 domain of a first subunit of an Fc domain is replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance within the CH3 domain of the first subunit that can be placed into a cavity within the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of a second subunit of an Fc domain is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit that can be placed into a cavity within the CH3 domain of the first subunit. 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). 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). The protuberances and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0078] In certain such embodiments, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), optionally the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbering according to the Kabat EU index). In a further embodiment, in the first subunit of the Fc domain, additionally the serine residue at position 354 is replaced by a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 is replaced by a cysteine ​​residue), and in the second subunit of the Fc domain, additionally the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). In a preferred embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the Kabat EU index).

[0079] In some aspects, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or decrease effector function.

[0080] In a particular embodiment, 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 activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In a particular embodiment, the effector function is ADCC.

[0081] Typically, the same amino acid substitution or substitutions are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid substitution or substitutions reduce the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the amino acid substitution or substitutions reduce the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold.

[0082] In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numbering according to Kabat EU index). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numbering according to Kabat EU index). In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A (numbering according to Kabat EU index). In one such embodiment, the Fc domain comprises an IgG 1 Fc domain, especially human IgG 1It is an Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, in particular P329G (numbering according to Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297 and P331 (numbering according to 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 comprises amino acid substitutions at positions P329, L234 and L235 (numbering according to Kabat EU index). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG"). Specifically, in a preferred embodiment, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e., 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) in each of the first and second subunits of the Fc domain (Kabat EU index numbering). In one such embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G (Kabat EU index numbering). 1 Fc domain, especially human IgG 1 The Fc domain.

[0083] In a preferred embodiment, the HLA-A2 / MAGE-A4×CD3 bispecific antibody comprises (i) a first antigen-binding moiety that specifically binds to CD3, the first antigen-binding moiety comprising a heavy chain variable region comprising a heavy chain CDR (HCDR)1 of SEQ ID NO: 1, an HCDR2 of SEQ ID NO: 2, and an HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR (LCDR)1 of SEQ ID NO: 4, an LCDR2 of SEQ ID NO: 5, and an LCDR3 of SEQ ID NO: 6, wherein the variable or constant regions, particularly the variable regions, of the Fab light chain and the Fab heavy chain are exchanged; (ii) second and third antigen-binding moieties that specifically bind to HLA-A2 / MAGE-A4, the second and third antigen-binding moieties comprising a heavy chain variable region comprising a heavy chain CDR (HCDR)1 of SEQ ID NO: 9, an HCDR2 of SEQ ID NO: 10, and an HCDR3 of SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR (LCDR)1 of SEQ ID NO: 12, an LCDR2 of SEQ ID NO: 13, and an LCDR3 of SEQ ID NO: 14, wherein each of the second and third antigen-binding moieties is a Fab molecule, in particular a conventional Fab molecule; (iii) an Fc domain composed of a first subunit and a second subunit, an Fc domain, wherein a second 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 first antigen-binding portion, wherein the first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of a first subunit of the Fc domain, and wherein a third antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of a second subunit of the Fc domain; Includes.

[0084] In one embodiment, the first antigen-binding portion comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8.

[0085] In one embodiment, the first antigen binding portion comprises a heavy chain variable region sequence of SEQ ID NO:7 and a light chain variable region sequence of SEQ ID NO:8.

[0086] In one embodiment, the second and third antigen binding portions comprise a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:15 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:16.

[0087] In one embodiment, the second and third antigen binding portions comprise a heavy chain variable region of SEQ ID NO:15 and a light chain variable region of SEQ ID NO:16.

[0088] The Fc domain according to the above embodiment may comprise all of the features described above in relation to the Fc domain, either alone or in combination.

[0089] In one embodiment the antigen binding portion and the Fc region are fused to each other by a peptide linker, in particular the peptide linkers in SEQ ID NO:18 and SEQ ID NO:20.

[0090] In one embodiment, in the constant domain CL of the second and third Fab molecule of (ii) the amino acid at position 124 is substituted by a lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by a lysine (K) or an arginine (R), in particular by an arginine (R) (numbering according to Kabat); and in the constant domain CHI of the second and third Fab molecule of (ii) the amino acid at position 147 is substituted by a glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by a glutamic acid (E) (numbering according to Kabat EU index).

[0091] In one embodiment the HLA-A2 / MAGE-A4xCD3 bispecific antibody comprises a polypeptide (in particular two polypeptides) comprising a sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 17, a polypeptide comprising a sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 18, a polypeptide comprising a sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 19, and a polypeptide comprising a sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 20.

[0092] In one embodiment the HLA-A2 / MAGE-A4xCD3 bispecific antibody comprises a polypeptide (in particular two polypeptides) comprising the sequence of SEQ ID NO: 17, a polypeptide comprising the sequence of SEQ ID NO: 18, a polypeptide comprising the sequence of SEQ ID NO: 19 and a polypeptide comprising the sequence of SEQ ID NO: 20.

[0093] The HLA-A2 / MAGE-A4xCD3 bispecific antibody herein is used in combination with a 4-1BB (CD137) agonist.

[0094] Unless otherwise indicated, the term "4-1BB" or "CD137" as used herein refers to any native form of 4-1BB from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice, rats). The term encompasses "full-length", unprocessed 4-1BB as well as any form of 4-1BB resulting from processing within a cell. The term also encompasses naturally occurring variants of 4-1BB, such as splice variants or allelic variants. The amino acid sequence of human 4-1BB is shown in UniProt accession number Q07011 (accession version 185).

[0095] "4-1BBL" or "4-1BB ligand" or "CD137L" is a family member of costimulatory TNF ligands that can costimulate T cell proliferation and cytokine production. Costimulatory TNF family ligands can costimulate TCR signals when interacting with the corresponding TNF receptor, which interaction with the receptor leads to recruitment of TNFR-associated factors (TRAFs), which initiate a signaling cascade leading to T cell activation. 4-1BBL is a type II transmembrane protein. It has been described that complete or full-length 4-1BBL, having the amino acid sequence shown in UniProt accession number P41273 (accession version 153), forms trimers on the surface of cells. Trimer formation is made possible by a specific motif in the ectodomain of 4-1BBL. Said motif is herein named "trimerization region". Amino acids 50 to 254 of the human 4-1BBL sequence (SEQ ID NO: 27) form the ectodomain of 4-1BBL, but even a fragment thereof is capable of forming a trimer.

[0096] An "ectodomain" is a domain of a membrane protein that extends into the extracellular space (i.e. the space outside the cell), also called the "ectodomain". The ectodomain of 4-1BBL as defined herein refers not only to the part of the 4-1BBL protein, in particular the human 4-1BBL protein (UniProt accession number P41273 (accession version 153)) that extends into the extracellular space (ectodomain), but also includes shorter parts or fragments thereof that are responsible for trimerization and binding to the corresponding receptor 4-1BB.

[0097] Thus, the term "ectodomain of 4-1BBL or a fragment thereof" refers to the extracellular domain of 4-1BBL or a portion thereof capable of binding to 4-1BB and having trimerization ability. In a particular embodiment of the present invention, the term "ectodomain of 4-1BBL or a fragment thereof" refers to a polypeptide having amino acids selected from SEQ ID NO: 31 (amino acids 52-254 of human 4-1BBL), SEQ ID NO: 28 (amino acids 71-254 of human 4-1BBL), SEQ ID NO: 30 (amino acids 80-254 of human 4-1BBL), SEQ ID NO: 29 (amino acids 85-254 of human 4-1BBL), SEQ ID NO: 32 (amino acids 71-248 of human 4-1BBL), SEQ ID NO: 33 (amino acids 85-248 of human 4-1BBL), SEQ ID NO: 34 (amino acids 80-248 of human 4-1BBL) and SEQ ID NO: 35 (amino acids 52-248 of human 4-1BBL).

[0098] The term "antigen-binding molecule" as used herein in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are antibodies, antibody fragments and scaffold antigen-binding proteins.

[0099] "Fibroblast activation protein (FAP)", also known as prolyl endopeptidase FAP or seprase (EC 3.4.21), refers to any native FAP 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 indicated. The term encompasses "full-length", unprocessed FAPs, as well as any form of FAP resulting from processing within a cell. The term also encompasses naturally occurring variants of FAPs, such as splice variants or allelic variants. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884 (accession version 197). The extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760. An antigen-binding moiety that binds to a FAP as used herein preferably binds to the extracellular domain of a FAP. Exemplary anti-FAP binding molecules are described, for example, in WO 2012 / 020006.

[0100] Particularly useful 4-1BB (CD137) agonists for use in the present invention are described, for example, in WO 2016 / 075278 or WO 2016 / 156291, which are hereby incorporated by reference in their entireties.

[0101] In one embodiment, the 4-1BB (CD137) agonist comprises 4-1BBL (particularly human 4-1BBL) or a fragment thereof, particularly the ectodomain of 4-1BBL or a fragment thereof. In one embodiment, the 4-1BB (CD137) agonist comprises the three ectodomains of 4-1BBL or a fragment thereof (i.e. the first, second and third ectodomains of 4-1BBL or a fragment thereof). In one embodiment, the ectodomain of 4-1BBL or a fragment thereof 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 NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35. In one embodiment, the ectodomain of 4-1BBL or a fragment thereof comprises 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:32. In one embodiment, the ectodomain of 4-1BBL or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 32. In one embodiment, the ectodomain of 4-1BBL or a fragment thereof consists of the amino acid sequence of SEQ ID NO:32.

[0102] In a particular aspect, the 4-1BB (CD137) agonist is a molecule comprising the three ectodomains of 4-1BBL or fragments thereof, wherein the ectodomains of 4-1BBL or fragments thereof comprise (or consist of) the amino acid sequence of SEQ ID NO:32.

[0103] In one embodiment, the 4-1BB (CD137) agonist comprises three ectodomains of 4-1BBL or fragments thereof (i.e., the first, second and third ectodomains or fragments thereof of 4-1BBL), where the first ectodomain of 4-1BBL or fragment thereof and the second ectodomain of 4-1BBL or fragment thereof are fused to each other, optionally via a peptide linker (i.e., the first and second ectodomains of 4-1BBL or fragments thereof are on the same polypeptide), and the third ectodomain of 4-1BBL or fragment thereof is not fused to the first or second ectodomain of 4-1BBL or fragment thereof (i.e., the third ectodomain of 4-1BBL or fragment thereof is on a separate polypeptide from the first and second ectodomains of 4-1BBL or fragments thereof).

[0104] In one embodiment, the 4-1BB (CD137) agonist is a molecule comprising a first polypeptide comprising the first and second ectodomains of 4-1BBL or fragments thereof, and a second polypeptide comprising the third ectodomain of 4-1BBL or fragments thereof. In one embodiment, the first and second ectodomains of 4-1BBL or fragments thereof are linked by a peptide linker, in particular (G 4 S) 2 The first polypeptide is fused via a peptide linker. In one embodiment, the first and second polypeptides are linked by a disulfide bond. In one embodiment, the first polypeptide comprises 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: 44. In one embodiment, the first polypeptide comprises an amino acid sequence of SEQ ID NO: 44. In one embodiment, the second polypeptide comprises 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: 32. In one embodiment, the second polypeptide comprises an amino acid sequence of SEQ ID NO: 32.

[0105] In a further aspect, the 4-1BB (CD137) agonist comprises an antigen-binding portion that specifically binds to a tumor-associated antigen, in particular a tumor stromal antigen (i.e., a tumor stromal-associated antigen), and more particularly a tumor fibroblast antigen (i.e., an antigen expressed on cancer-associated fibroblasts).

[0106] In a preferred embodiment, the antigen-binding portion specifically binds to a fibroblast activation protein (FAP), particularly a human FAP.

[0107] In one embodiment the antigen binding portion is a Fab molecule, in particular a conventional Fab molecule.

[0108] Thus, in a particular embodiment, a 4-1BB (CD137) agonist is an antigen-binding molecule comprising the three ectodomains of 4-1BBL or a fragment thereof and at least one antigen-binding portion that specifically binds to a tumor-associated antigen, in particular an antigen-binding portion that specifically binds to a FAP.

[0109] In one embodiment, an antigen binding portion that specifically binds to a FAP comprises a heavy chain variable region (VH) comprising a heavy chain CDR (HCDR)1 of SEQ ID NO: 36, an HCDR2 of SEQ ID NO: 37, and an HCDR3 of SEQ ID NO: 38, and a light chain variable region comprising a light chain CDR (LCDR)1 of SEQ ID NO: 39, an LCDR2 of SEQ ID NO: 40, and an LCDR3 of SEQ ID NO: 41.

[0110] In one embodiment, an antigen binding portion that specifically binds to a FAP comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:42 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:43.

[0111] In one embodiment, an antigen binding portion that specifically binds to a FAP comprises a heavy chain variable region sequence of SEQ ID NO:42 and a light chain variable region sequence of SEQ ID NO:43.

[0112] In a further aspect, the 4-1BB (CD137) agonist comprises an Fc domain composed of a first and a second subunit.

[0113] The Fc domain contained in the 4-1BB (CD137) agonist may incorporate all of the features described herein above in relation to the Fc domain contained in the HLA-A2 / MAGE-A4×CD3 bispecific antibody, either alone or in combination.

[0114] In particular, in one embodiment, the Fc domain contained in the 4-1BB (CD137) agonist is an IgG Fc domain. 1 In further preferred embodiments, the Fc domain is a human Fc domain. In certain preferred embodiments, the Fc domain is a human IgG 1 The Fc domain.

[0115] In certain embodiments, the Fc domain comprises a modification that promotes association of a first subunit with a second subunit of the Fc domain (e.g., a "knob-into-hole" modification), as described herein above in connection with the HLA-A2 / MAGE-A4 x CD3 bispecific antibody.

[0116] In further particular aspects, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or decrease effector function as described herein above in relation to the HLA-A2 / MAGE-A4xCD3 bispecific antibody (e.g., the "P329G LALA", "PGLALA" or "LALAPG" amino acid substitutions).

[0117] In a particularly preferred embodiment, the Fc domain contained in the 4-1BB (CD137) agonist is human IgG 1 Fc domain, each subunit of the Fc domain contains the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).

[0118] In one embodiment, the 4-1BBL (CD137) agonist is (i) the three ectodomains of 4-1BBL or fragments thereof; (ii) an antigen-binding portion that specifically binds to a FAP, in particular an antigen-binding portion that is a Fab molecule; and (iii) an Fc domain composed of a first and a second subunit, the Fc domain comprising a modification that in particular facilitates association of the first and second subunits of the Fc domain and / or one or more amino acid substitutions that reduce binding to an Fc receptor and / or reduce effector function; It is an antigen-binding molecule comprising:

[0119] In certain embodiments, the 4-1BBL (CD137) agonist is (i) the first, second and third ectodomains of 4-1BBL or fragments thereof; (ii) an antigen-binding portion that specifically binds to a FAP, the antigen-binding portion being a Fab molecule; (iii) an Fc domain composed of a first and a second subunit, the Fc domain comprising a modification that in particular facilitates association of the first and second subunits of the Fc domain and / or one or more amino acid substitutions that reduce binding to an Fc receptor and / or decrease effector function; (iv) CL domain and CH1 domain An antigen-binding molecule comprising: This antigen-binding molecule is (a) a first polypeptide comprising: (a1) a first ectodomain of 4-1BBL or a fragment thereof fused at its C-terminus to the N-terminus of a first ectodomain of 4-1BBL or a fragment thereof; (a2) a second ectodomain of 4-1BBL or a fragment thereof fused at its C-terminus to the N-terminus of a CL domain; (a3) ​​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 (a4) one of the subunits of the Fc domain (e.g., the first subunit); (b) (b1) a third ectodomain of 4-1BBL or a fragment thereof fused at its C-terminus to the N-terminus of the CH1 domain, and (b2) a second polypeptide comprising the CH1 domain; (c) (c1) a heavy chain of a Fab molecule 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 (c2) a third polypeptide comprising the other of the subunits of the Fc domain (e.g., the second subunit); (d) a fourth polypeptide comprising a light chain of a Fab molecule; It consists of:

[0120] The fusion between the various domains of the antigen-binding molecule is preferably via a peptide linker, which may also comprise or consist of (part of) the immunoglobulin hinge region. In particular, the fusion of the first ectodomain of 4-1BBL or a fragment thereof with the second ectodomain of 4-1BBL or a fragment thereof may be mediated by a peptide linker, in particular a(G4S) 2 Through the linker.

[0121] In one embodiment, the first and second polypeptides are linked to each other by a disulfide bond, in particular a disulfide bond between the CL domain and the CH1 domain.

[0122] In one aspect, in the CL domain of the first polypeptide, 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), and in the CH1 domain of the second polypeptide, 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).

[0123] In one embodiment, in the CL domain of the first polypeptide, the amino acid at position 124 is substituted by lysine (K) (Kabat numbering), the amino acid at position 123 is substituted by lysine (K) (Kabat numbering), and in the CH1 domain of the second polypeptide, the amino acid at position 147 is substituted by glutamic acid (E) (Kabat EU index numbering), and the amino acid at position 213 is substituted by glutamic acid (E) (Kabat EU index numbering).

[0124] In a particular embodiment, in the CL domain of the first polypeptide, the amino acid at position 124 is substituted with a lysine (K) (Kabat numbering) and the amino acid at position 123 is substituted with an arginine (R) (Kabat numbering), and in the CH1 domain of the second polypeptide, the amino acid at position 147 is substituted with a glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is substituted with a glutamic acid (E) (Kabat EU index numbering).

[0125] In a particular embodiment, when the amino acid substitutions according to the above embodiments are made in the CL and CH1 domains of the first and second polypeptides, the CL domain of the first polypeptide is of the kappa isotype.

[0126] In one embodiment, the CL domain is a human CL domain, particularly a human CL domain of the kappa isotype. In a further embodiment, the CH1 domain is a human CH1 domain, particularly a human CH1 domain of the gamma isotype, most particularly a human CH1 domain of the gamma 1 isotype.

[0127] In one embodiment, the first polypeptide comprises 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.

[0128] In one embodiment, the second polypeptide comprises 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.

[0129] In one embodiment, the third polypeptide comprises 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.

[0130] In one embodiment, the fourth polypeptide comprises 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.

[0131] In one embodiment, the 4-1BBL (CD137) agonist is an antigen-binding molecule comprising a first polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 45, a second polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 46, a third polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47, and a fourth polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 48.

[0132] In an alternative embodiment, the 4-1BB agonist may be an anti-4-1BB antibody, in particular an anti-FAP / anti-4-1BB bispecific antibody.

[0133] The term "cancer" refers to a physiological condition in mammals that is typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Non-limiting examples of cancer include blood cancers such as leukemia, bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, bile duct cancer, thyroid 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, skin cancer, squamous cell carcinoma, sarcoma, bone cancer, and kidney cancer. Other cell proliferative disorders include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, breast, and genitourinary system. Precancerous conditions or lesions and cancer metastases are also included.

[0134] In some embodiments of the HLA-A2 / MAGE-A4×CD3 bispecific antibodies, 4-1BB (CD137) agonists, methods, uses and kits of the invention, the cancer is a solid tumor cancer. By "solid tumor cancer" is meant a malignant tumor, such as a sarcoma or carcinoma, that forms a discrete tumor mass (including tumor metastases) in a specific location in the patient's body (as opposed to blood cancers, such as leukemia, which do not usually form solid tumors). Non-limiting examples of solid tumor cancers 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, skin cancer, squamous cell carcinoma, bone cancer, liver cancer and kidney cancer. Other solid tumor cancers contemplated in the context of the present invention include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, muscle, spleen, thoracic region, and genitourinary system. Precancerous conditions or lesions and cancer metastases are also included.

[0135] In one aspect, the cancer is selected from the group consisting of lung cancer, head and neck cancer, bladder cancer, esophageal cancer, skin cancer, soft tissue cancer, gastric cancer, cervical cancer and ovarian cancer. In one aspect, the cancer is selected from the group consisting of lung cancer, head and neck cancer, bladder cancer, esophageal cancer, soft tissue cancer and ovarian cancer. In one aspect, the cancer is bladder cancer. In another aspect, the cancer is cervical cancer.

[0136] In some embodiments, the cancer is a MAGE-A4 positive cancer. "MAGE-A4 positive cancer" or "MAGE-A4 expressing cancer" refers to a cancer characterized by expression or overexpression of MAGE-A4 in cancer cells. Expression of MAGE-A4 can be determined, for example, by quantitative real-time PCR (measuring MAGE-A4 mRNA levels), immunohistochemistry (IHC), or Western blot assay. In one embodiment, the cancer expresses MAGE-A4. In one embodiment, the cancer expresses MAGE-A4 in at least 20%, preferably at least 50%, or at least 80% of tumor cells as determined by immunohistochemistry (IHC) using an antibody specific for MAGE-A4.

[0137] In some embodiments, the cancer comprises cells (e.g., fibroblasts) that express a FAP. In some embodiments, the cancer expresses a FAP, particularly in the tumor stroma.

[0138] A "patient", "subject" or "individual" herein is any single human subject who is experiencing or has experienced one or more signs, symptoms, or other indicators of cancer and is eligible for treatment. In some embodiments, the patient has cancer or has been diagnosed with cancer. The patient may or may not have been previously treated with HLA-A2 / MAGE-A4xCD3 bispecific antibody or another drug. In certain embodiments, the patient has not been previously treated with HLA-A2 / MAGE-A4xCD3 bispecific antibody. The patient may have been treated with a therapy including one or more drugs other than HLA-A2 / MAGE-A4xCD3 bispecific antibody before HLA-A2 / MAGE-A4xCD3 bispecific antibody therapy is initiated. In certain embodiments, the patient carries an HLA-A2 allele, in particular the HLA-A*02:01 allele.

[0139] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing the direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of disease progression, ameliorating or remitigating the condition, and remission or improving prognosis.

[0140] The HLA-A2 / MAGE-A4xCD3 bispecific antibody and the 4-1BB (CD137) agonist are administered in effective amounts.

[0141] An "effective amount" of an agent, eg, a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0142] In one embodiment, administration of the HLA-A2 / MAGE-A4xCD3 bispecific antibody results in activation of T cells, particularly cytotoxic T cells, particularly at the site of the cancer. Said activation may include T cell proliferation, T cell differentiation, cytokine secretion by the T cells, release of cytotoxic effector molecules from the T cells, cytotoxic activity of the T cells, and expression of activation markers by the T cells. In one embodiment, administration of the HLA-A2 / MAGE-A4xCD3 bispecific antibody results in an increase in the number of T cells, particularly cytotoxic T cells, at the site of the cancer.

[0143] In some embodiments of the HLA-A2 / MAGE-A4xCD3 bispecific antibodies, 4-1BB (CD137) agonists, methods, uses or kits described above and herein, treatment with or administration of the HLA-A2 / MAGE-A4xCD3 bispecific antibodies and 4-1BB (CD137) agonists results in increased activation of T cells, particularly cytotoxic T cells, particularly at the site of the cancer, compared to treatment with or administration of the HLA-A2 / MAGE-A4xCD3 bispecific antibody alone. In certain embodiments, activation comprises the cytotoxic activity of the T cells (particularly lysis of cancer cells) and / or secretion of cytokines (particularly IL-2, TNF-α, and / or interferon-γ) by the T cells.

[0144] In some embodiments of the HLA-A2 / MAGE-A4xCD3 bispecific antibody, 4-1BB (CD137) agonist, methods, uses or kits described above and herein, treatment with or administration of the HLA-A2 / MAGE-A4xCD3 bispecific antibody and 4-1BB (CD137) agonist results in increased differentiation of naive T cells into memory T cells, particularly at the site of the cancer, compared to treatment with or administration of the HLA-A2 / MAGE-A4xCD3 bispecific antibody alone. In one embodiment, differentiation is detected by measurement of CD45RA expression, for example using flow cytometry.

[0145] In some aspects of the HLA-A2 / MAGE-A4xCD3 bispecific antibodies, 4-1BB (CD137) agonists, methods, uses or kits described above and herein, treatment with or administration of the HLA-A2 / MAGE-A4xCD3 bispecific antibodies and 4-1BB (CD137) agonists may result in a response in an individual. In some aspects, the response may be a complete remission. In some aspects, the response may be a sustained response after cessation of treatment. In some aspects, the response may be a complete remission that is sustained after cessation of treatment. In other aspects, the response may be a partial remission. In some aspects, the response may be a partial remission that is sustained after cessation of treatment. In some aspects, treatment with or administration of an HLA-A2 / MAGE-A4xCD3 bispecific antibody and a 4-1BB (CD137) agonist may improve the response compared to treatment with or administration of an HLA-A2 / MAGE-A4xCD3 bispecific antibody alone (i.e. without a 4-1BB (CD137) agonist).

[0146] In some aspects, treatment with or administration of an HLA-A2 / MAGE-A4×CD3 bispecific antibody and a 4-1BB (CD137) agonist may increase the response rate of a patient population compared to a corresponding patient population treated with only an HLA-A2 / MAGE-A4×CD3 bispecific antibody (i.e., without a 4-1BB (CD137) agonist).

[0147] The combination therapy of the present invention comprises the administration of an HLA-A2 / MAGE-A4xCD3 bispecific antibody and a 4-1BB (CD137) agonist.

[0148] As used herein, "combination" (and grammatical variations thereof such as "combine" or "combining") encompasses the combination of an HLA-A2 / MAGE-A4xCD3 bispecific antibody according to the invention with a 4-1BB (CD137) agonist, wherein the HLA-A2 / MAGE-A4xCD3 bispecific antibody and the 4-1BB (CD137) agonist are in the same or different containers, in the same or different pharmaceutical formulations, administered together or separately, administered together or sequentially (in any order), administered by the same or different routes, provided that the HLA-A2 / MAGE-A4xCD3 bispecific antibody and the 4-1BB (CD137) agonist are capable of exerting their biological effect together in the body. For example, "combining" an HLA-A2 / MAGE-A4×CD3 bispecific antibody according to the invention with a 4-1BB (CD137) agonist means administering first the HLA-A2 / MAGE-A4×CD3 bispecific antibody in a particular pharmaceutical formulation, followed by the 4-1BB (CD137) agonist in another pharmaceutical formulation, or vice versa.

[0149] The HLA-A2 / MAGE-A4×CD3 bispecific antibody and the 4-1BB (CD137) agonist may be administered in any suitable manner known in the art. In one embodiment, the HLA-A2 / MAGE-A4×CD3 bispecific antibody and the 4-1BB (CD137) agonist are administered sequentially (at different times). In another embodiment, the HLA-A2 / MAGE-A4×CD3 bispecific antibody and the 4-1BB (CD137) agonist are administered simultaneously (at the same time). Without wishing to be bound by theory, it may be advantageous to administer the 4-1BB (CD137) agonist prior to and / or simultaneously with the HLA-A2 / MAGE-A4×CD3 bispecific antibody. In some embodiments, the HLA-A2 / MAGE-A4×CD3 bispecific antibody is in a separate composition from the 4-1BB (CD137) agonist. In some embodiments, the HLA-A2 / MAGE-A4xCD3 bispecific antibody is in the same composition as the 4-1BB (CD137) agonist.

[0150] The HLA-A2 / MAGE-A4xCD3 bispecific antibody and the 4-1BB (CD137) agonist can be administered by any suitable route, and may be administered by the same or different routes of administration. In some embodiments, the HLA-A2 / MAGE-A4xCD3 bispecific antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In a particular embodiment, the HLA-A2 / MAGE-A4xCD3 bispecific antibody is administered intravenously. In some embodiments, the 4-1BB (CD137) agonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In a particular embodiment, the 4-1BB (CD137) agonist is administered intravenously. Effective amounts of HLA-A2 / MAGE-A4xCD3 bispecific antibodies and 4-1BB (CD137) agonists may be administered for the prevention or treatment of disease. The appropriate route of administration and dosage of HLA-A2 / MAGE-A4xCD3 bispecific antibodies and / or 4-1BB (CD137) agonists may be determined based on the type of disease being treated, the type of HLA-A2 / MAGE-A4xCD3 bispecific antibodies, the type of 4-1BB (CD137) agonist, the severity and course of the disease, the clinical condition of the individual, the individual's medical history and response to treatment, and the discretion of the attending physician. Dosing may be by any suitable route, for example, injections such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion. The HLA-A2 / MAGE-A4xCD3 bispecific antibody and the 4-1BB (CD137) agonist are suitably administered to the patient at one time or over a series of treatments.

[0151] The combination of the present invention can be used in therapy alone or in combination with other drugs.For example, the combination of the present invention can be co-administered with at least one additional therapeutic agent.In some embodiments, the additional therapeutic agent is an anti-cancer agent, such as a chemotherapeutic agent, an inhibitor of tumor cell proliferation, or an activator of tumor cell apoptosis.The combination of the present invention can also be combined with radiation therapy.

[0152] The kits provided herein typically include one or more containers and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV infusion bags, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The containers hold a composition, by itself or in combination with another composition, that is effective in treating, preventing, and / or diagnosing a disease, and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is the HLA-A2 / MAGE-A4×CD3 bispecific antibody used in the combination of the invention. Another active agent is a 4-1BB (CD137) agonist used in the combination of the invention, which can be in the same composition and container as the bispecific antibody, or can be provided in a different composition and container. The label or package insert indicates that the one or more compositions are used to treat a selected condition, such as cancer.

[0153] In one aspect, the invention provides a kit intended for the treatment of cancer, the kit comprising, in the same or separate containers, (a) an HLA-A2 / MAGE-A4xCD3 bispecific antibody, and (b) a 4-1BB (CD137) agonist, and optionally (c) a package insert comprising printed instructions directing the use of the combination treatment as a method for treating cancer. The kit further comprises a first container comprising a composition in (a), the composition comprising an HLA-A2 / MAGE-A4xCD3 bispecific antibody; a second container comprising a composition in (b), the composition comprising a 4-1BB (CD137) agonist; and optionally a third container comprising a composition in (c), the composition comprising an additional cytotoxic or otherwise therapeutic agent. The kits of these aspects of the invention may further comprise a package insert indicating that the composition can be used to treat cancer. Alternatively, or additionally, the kit may further comprise a third (or fourth) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. Amino acid sequence TIFF2024546589000001.tif255170TIFF2024546589000002.tif255170TIFF20245465890 00003.tif255169TIFF2024546589000004.tif255169TIFF2024546589000005.tif244170 [Brief description of the drawings]

[0154] [Figure 1](A) Schematic of the HLA-A2 / MAGE-A4 targeting T cell bispecific (TCB) antibody molecule ("MAGE-A4 TCB") used in the examples. The molecule contains a single antigen binding moiety for CD3, two antigen binding moieties for HLA-A2 / MAGE-A4, and an Fc domain. (B) Schematic of the FAP targeting 4-1BB (CD137) agonist ("FAP-4-1-BBL") used in the examples. The molecule contains a 4-1BBL ectodomain trimer, an antigen binding moiety for FAP, and an Fc domain. Black dots: modifications in the Fc domain that promote heterodimerization. *: oppositely charged amino acids introduced into the CH and CL domains. [Diagram 2] Continuous live cell imaging of HLA-A*02+ / MAGE-A4+ tumor cell line SCaBER treated with the combination of MAGE-A4 TCB+FAP-4-1-BBL. SCaBER cells were incubated with human PBMCs at a 5:1 E:T ratio in the presence of irradiated NIH / 3T3-huFAP cells and treated with 2 nM FAP-4-1-BBL in combination with various concentrations of MAGE-A4 TCB ((A) 50 nM, (B) 8.33 nM, (C) 1.39 nM, (D) 0.23 nM, (E) 0.039 nM). Tumor cell proliferation curves (measured as number of red cells per image) were monitored by continuous live cell imaging over a period of 120 h. As controls, SCaBER cells were incubated with PBMCs (in the presence of irradiated NIH / 3T3-huFAP cells) in the absence of treatment (No Treatment), with 2 nM FAP-4-1-BBL alone (FAP-4-1-BBL) or with various concentrations of MAGE-A4 TCB without FAP-4-1-BBL (MAGE-A4 TCB). [Diagram 3]Continuous live-cell imaging of HLA-A*02+ / MAGE-A4+ tumor cell line C-33a treated with the combination of MAGE-A4 TCB+FAP-4-1-BBL. C-33a cells were incubated with human PBMCs at a 5:1 E:T ratio in the presence of irradiated NIH / 3T3-huFAP cells and treated with 2 nM FAP-4-1-BBL in combination with various concentrations of MAGE-A4 TCB ((A) 50 nM, (B) 8.33 nM, (C) 1.39 nM, (D) 0.23 nM, (E) 0.039 nM). Tumor cell proliferation curves (measured as number of red cells per image) were monitored by continuous live-cell imaging over a period of 120 hours. As controls, C-33a cells were incubated with PBMCs (in the presence of irradiated NIH / 3T3-huFAP cells) in the absence of treatment (No Treatment), with 2 nM FAP-4-1-BBL alone (FAP-4-1-BBL) or with various concentrations of MAGE-A4 TCB without FAP-4-1-BBL (MAGE-A4 TCB). [Figure 4A-F] Quantification of cytokines and granzyme B released by MAGE-A4 TCB-activated T cells. SCaBER cells were incubated with human PBMCs at a 5:1 E:T ratio in the presence of irradiated NIH / 3T3-huFAP cells and treated with a combination of 2 nM FAP-4-1-BBL and various concentrations (50 nM to 1 pM) of MAGE-A4 TCB. As controls, SCaBER cells were incubated with PBMCs (in the presence of irradiated NIH / 3T3-huFAP cells) in the absence of treatment (No Treatment), with 2 nM FAP-4-1-BBL alone (FAP-4-1-BBL) or with various concentrations of MAGE-A4 TCB without FAP-4-1-BBL (MAGE-A4 TCB). Shown are dose-response curves of cytokine release (pg / mL) into the supernatant after 48 h of incubation as determined by Luminex immunoassay. (A) Interferon (IFN)-γ, (B) interleukin (IL)-2, (C) tumor necrosis factor (TNF)-α, (D) IL-6, (E) IL-8, (F) IL-10. [Figure 4G] Quantification of cytokines and granzyme B released by MAGE-A4 TCB-activated T cells. SCaBER cells were incubated with human PBMCs at a 5:1 E:T ratio in the presence of irradiated NIH / 3T3-huFAP cells and treated with a combination of 2 nM FAP-4-1-BBL and various concentrations (50 nM to 1 pM) of MAGE-A4 TCB. As controls, SCaBER cells were incubated with PBMCs (in the presence of irradiated NIH / 3T3-huFAP cells) in the absence of treatment (No Treatment), with 2 nM FAP-4-1-BBL alone (FAP-4-1-BBL) or with various concentrations of MAGE-A4 TCB without FAP-4-1-BBL (MAGE-A4 TCB). Shown are dose-response curves of cytokine release (pg / mL) into the supernatant after 48 h of incubation as determined by Luminex immunoassay. (G) Granzyme B. [Figure 5A-F] Quantification of cytokines and granzyme B released by MAGE-A4 TCB-activated T cells. C33-a cells were incubated with human PBMCs at an E:T ratio of 5:1 in the presence of irradiated NIH / 3T3-huFAP cells and treated with a combination of 2 nM FAP-4-1-BBL and various concentrations (50 nM to 1 pM) of MAGE-A4 TCB. As controls, C-33a cells were incubated with PBMCs (in the presence of irradiated NIH / 3T3-huFAP cells) in the absence of treatment (No Treatment), with 2 nM FAP-4-1-BBL alone (FAP-4-1-BBL) or with various concentrations of MAGE-A4 TCB without FAP-4-1-BBL (MAGE-A4 TCB). Shown are dose-response curves (pg / mL) of cytokine release into the supernatant after 48 h of incubation as determined by Luminex immunoassay: (A) interferon (IFN)-γ, (B) interleukin (IL)-2, (C) tumor necrosis factor (TNF)-α, (D) IL-6, (E) IL-8, (F) IL-10, (G) granzyme B. [Figure 5G]Quantification of cytokines and granzyme B released by MAGE-A4 TCB-activated T cells. C33-a cells were incubated with human PBMCs at an E:T ratio of 5:1 in the presence of irradiated NIH / 3T3-huFAP cells and treated with a combination of 2 nM FAP-4-1-BBL and various concentrations (50 nM to 1 pM) of MAGE-A4 TCB. As controls, C-33a cells were incubated with PBMCs (in the presence of irradiated NIH / 3T3-huFAP cells) in the absence of treatment (No Treatment), with 2 nM FAP-4-1-BBL alone (FAP-4-1-BBL) or with various concentrations of MAGE-A4 TCB without FAP-4-1-BBL (MAGE-A4 TCB). Shown are dose-response curves of cytokine release (pg / mL) into the supernatant after 48 h of incubation as determined by Luminex immunoassay. (G) Granzyme B. EXAMPLES

[0155] The following are examples of methods and compositions of the present invention. Given the general description above, it will be understood that various other embodiments may be practiced.

[0156] Example 1 - Continuous live cell imaging to monitor MAGE-A4 TCB+FAP-4-1-BBL mediated tumor cell lysis To monitor the dynamic tumor cell lysis induced by the MAGE-A4 TCB+FAP-4-1-BBL combination over several days, continuous live-cell imaging was performed using two different MAGE-A4 + / HLA-A*02:01 +The study was performed with cell lines (SCaBER, a cell line derived from a human squamous cell carcinoma of the bladder (O'Toole et al., Int. J. Cancer 17:707-714 (1976)) and C-33a, a cell line derived from a human cervical carcinoma (Auersperg, J. Natl. Cancer Inst. 32, 135-163 (1964)). Cells were incubated with human PBMCs in the presence of irradiated NIH / 3T3-huFAP fibroblast cells (NIH / 3T3 cells stably expressing human FAP, irradiated to prevent proliferation) and treated with a combination of 2 nM FAP-4-1-BBL and various concentrations (50 nM to 1 pM) of MAGE-A4 TCB. As controls, tumor cells were treated in the absence of treatment (no treatment), with 2 nM FAP-4-1-BBL alone, or with various concentrations of MAGE-A4 TCB. TCB alone was used for incubation with PBMCs.

[0157] One day prior to assay setup, healthy donor PBMCs (Lonza) were thawed and resuspended in RPMI 1640 medium containing 10% FCS and 1% GlutaMAX. Cells were incubated at 37°C and 5% CO 2 The plates were then kept in an incubator overnight.

[0158] In parallel, target tumor cell lines were collected and their viability and cell number were assessed using a ViCell XR cell counter (Beckman Coulter). × After 5 min at 4 °C for 1 h, the cells were counted in RPMI 1640 containing 10% FCS and 1% GlutaMAX. × 10 4 cells / mL (transduced with lentivirus stably expressing SCaBER H2BRFP, i.e., nuclear red fluorescent protein (Essen Bioscience, #4476)), 24 × 10 4 cells / mL (C-33a H2BRFP), or 16 × 10 4The cells were adjusted to 1000 cells / mL (NIH / 3T3-huFAP clone 19 irradiated). 62.5 μL of the cell suspension was plated to obtain a final cell density of 5000, 15,000, or 10,000 cells / well. All edge wells were filled with assay medium to avoid evaporation of liquid in the assay wells. The assay plates containing the target cells were incubated at 37° C. and 5% CO. 2 The mixture was incubated overnight at RT.

[0159] On the day of assay, 62.5 μL of MAGE-A4 TCB was added to target cells to give final concentrations of 50 nM to 1 pM per well. Treatments with 2 nM FAP-4-1BBL or assay medium alone for MAGE-A4 TCB were added. "No antibody" controls received assay medium.

[0160] Previously thawed PBMCs (see above) were harvested from the incubator, counted using a ViCell XR cell counter (Beckman Coulter), and centrifuged (350 × g, 5 min). PBMCs were cultured in assay medium for 4 h. × 10 5 cells / mL (for SCaBER H2BRFP) or 1.2 × 10 6 The cells were resuspended at 1000 cells / mL (for C33a H2BRFP). Each PBMC solution was plated per well to give an effector to target (E:T) ratio of 5:1 in a final volume of 250 μL.

[0161] 200 × After centrifugation at 4× g for 1 min, the plates were incubated at 37°C and 5% CO 2The plates were placed in the incubator of an Incucyte S3 Live-Cell Analysis System (Essen Bioscience, Ltd.) at 100° C. and scanning was started 1 hour later (time 0 h). Plates were scanned every 3 h for a total of 120 h by taking 4 pictures per well (phase contrast, red channel [i.e., target cells]). The average signal at time 0 h was calculated from the three replicates of each condition. This value was subtracted from each value of the same condition at the subsequent time points to obtain the averaged value.

[0162] After 48 hours, 25 μL of supernatant from each well was transferred to a fresh plate and frozen at −80° C. for Luminex cytokine analysis (see Example 0).

[0163] The results are shown in Figures 2 and 3. The normalized values ​​of the number of tumor cells per image (i.e., red signals per image) were graphed.

[0164] As shown in Figures 2 and 3, the combination of MAGE-A4 TCB with the targeted costimulatory molecule FAP-4-1-BBL leads to a strong inhibition of tumor cell proliferation as monitored by continuous live cell imaging. In contrast, MAGE-A4 TCB, when used as a monotherapy, induces no or only very weak inhibition of tumor growth (at high concentrations). This indicates that the non-responsiveness of SCaBER and C-33a cells to MAGE-A4 TCB monotherapy can be overcome by combination therapy with FAP-4-1-BBL.

[0165] Example 2 - Luminex immunoassay to quantify cytokine release induced by the combination of MAGE-A4 TCB + FAP-4-1-BBL In addition to continuous live cell imaging, the pharmacological activity of the MAGE-A4 TCB+FAP-4-1-BBL combination was further evaluated by quantifying the levels of cytokines and cytotoxic granules (granzyme B) released into the cell supernatant by activated T cells. Cytokines released into the cell culture supernatant were quantified via Luminex immunoassay by using the Human Custom ProcartaPlex 7-plex kit (Thermo Fisher Scientific # PPX-07-MXFVK4Y).

[0166] On the day of the assay, frozen cell supernatants from tumor cell lysis experiments were thawed on ice. Three replicates were mixed well by vortexing and pooled into one well. Samples were used diluted 1:3 in assay medium (RPMI1640, 10% FBS, 1% GlutaMAX).

[0167] The standard vial (provided in the kit) was × The plates were centrifuged at g for 10 seconds and reconstituted by adding assay medium according to the manufacturer's instructions (ThermoFisher Scientific). A 3-fold standard dilution series in assay medium and blank (medium only) samples were added into the suspension plate according to the plate layout.

[0168] The magnetic beads were vortexed for 30 seconds, diluted 1:2 in assay medium and protected from light (by aluminum foil).

[0169] The filter plates were pre-wetted by adding 100 μL / well of PBS for 5 minutes and dried on a vacuum manifold.

[0170] The bead solution was vortexed for 30 seconds and 50 μL / well was added to the filter assay plate. After washing twice with 100 μL / well wash buffer, 50 μL / well of sample, standard, or blank was added and the plate was incubated for >60 minutes on a plate shaker (500 rpm) at room temperature in the dark.

[0171] The filter plate was washed twice with 100 μL / well of wash buffer. × The detection antibody mixture was diluted 1:2 in PBS or Universal Assay Buffer and 25 μL was added to each well and incubated for 30 minutes on a plate shaker (500 rpm) at room temperature in the dark.

[0172] After washing twice with 100 μL / well of wash buffer, 50 μL / well of SA-RPE solution (diluted 1:2 in PBS or Universal Assay Buffer) was added to each well. The plate was incubated for 30 minutes on a plate shaker (500 rpm) at room temperature in the dark. After washing twice with 100 μL / well of wash buffer, 100 μL / well of read buffer was added to each well and the plate was incubated for another 5 minutes under the same conditions.

[0173] Plates were read on a BioPlex® (BioRad) plate reader and the resulting signal was automatically quantified by Bio-Plex Manager 6.1.1 software.

[0174] The results are shown in Figures 4 and 5. The cytokine concentration values ​​were transferred to GraphPad Prism and graphed. Each cytokine concentration value was corrected by baseline subtraction. The baseline refers to the cytokine concentration accumulated in the supernatant of wells co-cultured with target cells and PBMCs without any therapeutic molecule. Sigmoidal dose-response, four-parameter logistic equation, and least squares (conventional) fitting analysis were applied to the baseline-subtracted values ​​to perform dose-response curve fitting.

[0175] Consistent with the results obtained with continuous live cell imaging, dose-dependent cytokine secretion could be observed for MAGE-A4 TCB+FAP-4-1BBL-activated T cells (Figures 4 and 5), whereas for MAGE-A4 TCB monotherapy, no cytokine release could be observed or the observed cytokine release was very small.

[0176] The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, but such illustration and description are not intended to limit the scope of the invention. The disclosures of all patent and scientific literature cited herein are incorporated by reference in their entireties.

Claims

1. 1. An HLA-A2 / MAGE-A4xCD3 bispecific antibody for use in the treatment of cancer in an individual, the treatment comprising administration of the HLA-A2 / MAGE-A4xCD3 bispecific antibody in combination with a 4-1BB (CD137) agonist.

2. 1. A 4-1BB (CD137) agonist for use in treating cancer in an individual, the treatment comprising administration of the 4-1BB (CD137) agonist in combination with an HLA-A2 / MAGE-A4xCD3 bispecific antibody.

3. 1. Use of an HLA-A2 / MAGE-A4xCD3 bispecific antibody in the manufacture of a medicament for the treatment of cancer in an individual, wherein the treatment comprises administration of an HLA-A2 / MAGE-A4xCD3 bispecific antibody in combination with a 4-1BB (CD137) agonist.

4. 20. The use of a 4-1BB (CD137) agonist in the manufacture of a medicament for the treatment of cancer in an individual, wherein the treatment comprises administration of a 4-1BB (CD137) agonist in combination with an HLA-A2 / MAGE-A4xCD3 bispecific antibody.

5. A method for treating cancer in an individual comprising administering to the individual an HLA-A2 / MAGE-A4xCD3 bispecific antibody and a 4-1BB (CD137) agonist.

6. 1. A kit comprising a first medicament comprising an HLA-A2 / MAGE-A4 x CD3 bispecific antibody and a second medicament comprising a 4-1BB (CD137) agonist, optionally further comprising a package insert comprising instructions for administration of the first medicament in combination with the second medicament to treat cancer in an individual.

7. HLA-A2 / MAGE-A4×CD3 bispecific antibody (i) a first antigen-binding moiety that specifically binds to CD3, the first antigen-binding moiety comprising a heavy chain variable region comprising a heavy chain CDR (HCDR)1 of SEQ ID NO:1, a HCDR2 of SEQ ID NO:2, and a HCDR3 of SEQ ID NO:3; and a light chain variable region comprising a light chain CDR (LCDR)1 of SEQ ID NO:4, a LCDR2 of SEQ ID NO:5, and a LCDR3 of SEQ ID NO:6; (ii) a second antigen-binding portion that specifically binds to HLA-A2 / MAGE-A4, the second antigen-binding portion comprising a heavy chain variable region comprising a heavy chain CDR (HCDR)1 of SEQ ID NO:9, a HCDR2 of SEQ ID NO:10, and a HCDR3 of SEQ ID NO:11; and a light chain variable region comprising a light chain CDR (LCDR)1 of SEQ ID NO:12, a LCDR2 of SEQ ID NO:13, and a LCDR3 of SEQ ID NO:

14.

7. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, the 4-1BBL (CD137) agonist for use, the use, the method or the kit according to any one of claims 1 to 6, comprising:

8. The HLA-A2 / MAGE-A4×CD3 bispecific antibody for use, the use, the 4-1BBL (CD137) agonist for use, the use, the method or the kit according to any one of claims 1 to 7, wherein the HLA-A2 / MAGE-A4×CD3 bispecific antibody comprises a third antigen-binding portion that specifically binds to HLA-A2 / MAGE-A4 and / or an Fc domain composed of the first and second subunits.

9. HLA-A2 / MAGE-A4×CD3 bispecific antibody (i) a first antigen-binding moiety that specifically binds to CD3, the first antigen-binding moiety comprising a heavy chain variable region comprising a heavy chain CDR (HCDR)1 of SEQ ID NO: 1, a HCDR2 of SEQ ID NO: 2, and a HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR (LCDR)1 of SEQ ID NO: 4, a LCDR2 of SEQ ID NO: 5, and a LCDR3 of SEQ ID NO: 6, the first antigen-binding moiety being a crossover Fab molecule in which the variable or constant regions of the Fab light chain and the Fab heavy chain have been swapped; (ii) second and third antigen-binding moieties that specifically bind to HLA-A2 / MAGE-A4, the second and third antigen-binding moieties comprising a heavy chain variable region comprising a heavy chain CDR (HCDR)1 of SEQ ID NO:9, a HCDR2 of SEQ ID NO:10, and a HCDR3 of SEQ ID NO:11; and a light chain variable region comprising a light chain CDR (LCDR)1 of SEQ ID NO:12, a LCDR2 of SEQ ID NO:13, and a LCDR3 of SEQ ID NO:14, wherein each of the second and third antigen-binding moieties is a Fab molecule, in particular a conventional Fab molecule; (iii) an Fc domain composed of a first subunit and a second subunit, an Fc domain, wherein a second 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 first antigen-binding portion, a first antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of a first subunit of the Fc domain, and a third antigen-binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of a second subunit of the Fc domain; 9. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, the 4-1BBL (CD137) agonist for use, the use, the method or the kit according to any one of claims 1 to 8.

10. a first antigen-binding portion of the HLA-A2 / MAGE-A4xCD3 bispecific antibody comprising a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8; and / or a second antigen-binding portion and (if present) a third antigen-binding portion of the HLA-A2 / MAGE-A4xCD3 bispecific antibody comprising a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

8.

10. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, use, 4-1BBL (CD137) agonist for use, use, method or kit according to any one of claims 1 to 9, wherein the portion comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

16.

11. The first antigen-binding portion of the HLA-A2 / MAGE-A4×CD3 bispecific antibody is a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, and the second antigen-binding portion and, if present, the third antigen-binding portion of the HLA-A2 / MAGE-A4×CD3 bispecific antibody are each independently substituted with lysine (K), arginine (R) or histidine (H) in the constant domain CL (numbering according to Kabat) at amino acid position 124 (K), arginine (R) or histidine (H), and independently substituted with lysine (K), arginine (R) or histidine (H) in the constant domain CL (numbering according to Kabat).

11. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, 4-1BBL (CD137) agonist for use, use, method or kit according to any one of claims 1 to 10, which is a conventional Fab molecule in which, in the constant domain CH1, the amino acid at position 147 is independently substituted by a glutamic acid (E) or an aspartic acid (D) (Kabat EU index numbering) and the amino acid at position 213 is independently substituted by a glutamic acid (E) or an aspartic acid (D) (Kabat EU index numbering).

12. 12. The HLA-A2 / MAGE-A4xCD3 bispecific antibody for use, the 4-1BBL (CD137) agonist for use, the use, the method or the kit according to any one of claims 1 to 11, wherein the Fc domain of the HLA-A2 / MAGE-A4xCD3 bispecific antibody comprises a modification that facilitates 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 an Fc receptor and / or reduce effector function.

13. 13. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, the 4-1BBL (CD137) agonist for use, the use, the method or the kit according to any one of claims 1 to 12, wherein the 4-1BB (CD137) agonist comprises the ectodomain of 4-1BBL or a fragment thereof, in particular the three ectodomains of 4-1BBL or a fragment thereof.

14. 14. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, 4-1BBL (CD137) agonist for use, use, method or kit according to any one of claims 1 to 13, wherein the ectodomain of 4-1BBL or a fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, in particular an amino acid sequence which is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

32.

15. 15. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, the 4-1BBL (CD137) agonist for use, the use, the method or the kit according to any one of claims 1 to 14, wherein the 4-1BB (CD137) agonist comprises an antigen-binding portion which specifically binds to a tumor-associated antigen, in particular fibroblast activation protein (FAP).

16. 16. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, 4-1BBL (CD137) agonist for use, use, method or kit according to claim 15, wherein the antigen binding portion that specifically binds to FAP comprises a heavy chain variable region (VH) comprising a heavy chain CDR (HCDR) 1 of SEQ ID NO: 36, a HCDR2 of SEQ ID NO: 37, and a HCDR3 of SEQ ID NO: 38; and a light chain variable region comprising a light chain CDR (LCDR) 1 of SEQ ID NO: 39, a LCDR2 of SEQ ID NO: 40, and a LCDR3 of SEQ ID NO:

41.

17. 17. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, 4-1BBL (CD137) agonist for use, use, method or kit of claim 15 or 16, wherein the antigen binding portion that specifically binds to FAP comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 42 and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

43.

18. 18. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, the 4-1BBL (CD137) agonist for use, the use, the method or the kit according to any one of claims 1 to 17, wherein the 4-1BB (CD137) agonist comprises an Fc domain composed of a first and a second subunit.

19. 19. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, the 4-1BBL (CD137) agonist for use, the use, the method or the kit according to claim 18, wherein the Fc domain of the 4-1BB (CD137) agonist comprises a modification that facilitates 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 an Fc receptor and / or reduce effector function.

20. 4-1BBL (CD137) agonist, (i) the first, second and third ectodomains of 4-1BBL or fragments thereof; (ii) an antigen-binding portion that specifically binds to FAP, the antigen-binding portion being a Fab molecule; (iii) an Fc domain composed of a first subunit and a second subunit; (iv) CL domain and CH1 domain An antigen-binding molecule comprising: The antigen-binding molecule, (a) a first polypeptide comprising: (a1) a first ectodomain of 4-1BBL or a fragment thereof fused at its C-terminus to the N-terminus of a second ectodomain of 4-1BBL or a fragment thereof; (a2) a second ectodomain of 4-1BBL or a fragment thereof fused at its C-terminus to the N-terminus of a CL domain; (a3) ​​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 (a4) one of the subunits of the Fc domain (e.g., the first subunit); (b) (b1) a third ectodomain of 4-1BBL or a fragment thereof fused at its C-terminus to the N-terminus of the CH1 domain, and (b2) a second polypeptide comprising the CH1 domain; (c) a third polypeptide comprising (c1) a heavy chain of a Fab molecule 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 (c2) the other of the subunits of the Fc domain (e.g., the second subunit); (d) a fourth polypeptide comprising a light chain of a Fab molecule; and 20. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, the 4-1BBL (CD137) agonist for use, the use, the method or the kit according to any one of claims 1 to 19, comprising:

21. 21. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, 4-1BBL (CD137) agonist for use, use, method or kit according to claim 20, wherein in the CL domain of the first polypeptide, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and in the CH1 domain of the second polypeptide, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

22. 22. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, 4-1BBL (CD137) agonist for use, use, method or kit according to claim 20 or 21, wherein the first polypeptide comprises 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, the second polypeptide comprises 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, the third polypeptide comprises 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 the fourth polypeptide comprises 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.

23. 13. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, the 4-1BBL (CD137) agonist for use, the use, the method or the kit according to any one of claims 1 to 12, wherein the 4-1BB (CD137) agonist is an anti-4-1BB antibody, in particular an anti-FAP / anti-4-1BB bispecific antibody.

24. 24. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, the 4-1BBL (CD137) agonist for use, the use, the method or the kit according to any one of claims 1 to 23, wherein the cancer is a MAGE-A4 positive cancer.

25. 25. The HLA-A2 / MAGE-A4 x CD3 bispecific antibody for use, the 4-1BBL (CD137) agonist for use, the use, the method or the kit according to any one of claims 1 to 24, wherein the cancer is selected from the group consisting of lung cancer, head and neck cancer, bladder cancer, esophageal cancer, skin cancer, soft tissue cancer, gastric cancer, cervical cancer and ovarian cancer.

26. 13. The invention as hereinbefore described.

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