Antibodies that bind to HLA-A2 / MAGE-A4

Abstract

The present invention generally relates to antibodies that bind to HLA-A2 / MAGE-A4, including, for example, multispecific antibodies for use in activating T cells. Furthermore, the present invention relates to polynucleotides encoding such antibodies, as well as vectors and host cells comprising such polynucleotides. The present invention further relates to methods for producing such antibodies, and to methods of using such antibodies in the treatment of diseases.

Description

Technical Field

[0001] The present invention generally relates to antibodies that bind to HLA-A2 / MAGE-A4, including, for example, multispecific antibodies for use in activating T cells. Furthermore, the present invention relates to polynucleotides encoding such antibodies, as well as vectors and host cells comprising such polynucleotides. The present invention further relates to methods for producing such antibodies, and to methods for using such antibodies in the treatment of diseases. Background Art

[0002] MAGE-A4 (melanoma-associated antigen 4) is a member of the cancer testis antigen (CTA) MAGE family. The MAGE-A protein family encompasses 12 highly homologous genes that are clustered at Xq26-28 and are characterized by the presence of a conserved domain (MAGE homology domain, MHD). Although MAGE proteins were discovered more than 20 years ago, their biological functions are still poorly understood. Based on their expression patterns, the MAGE family can be divided into two subfamilies, type I and type II, of which the MAGE-A group belongs to the type I subfamily, whose expression is limited to germ cell lines and cancer cells. Many reports have shown that overexpression of type I MAGE is associated with 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 as T cell epitopes via the major histocompatibility complex (MHC) I. Therefore, MAGE-A4-derived peptides such as MAGE-A4 p230-239 (GVYDGREHTV) is presented on the cell surface in the context of HLA-A2 and can trigger T cell recognition.

[0003] Given its expression pattern, MAGE-A4 may be a promising target for cancer therapy. To date, approaches to exploiting MAGE-A4 as a target for cancer (immuno)therapy have primarily focused on the development of T cell receptors (TCRs) targeting HLA-A2 / MAGE-A4 and their use in genetically engineered T cells (WO 2017 / 174824) or fusion molecules (WO 2017 / 175006). TCR-like antibodies against the HLA-A2 / MAGE-A4 complex have also been generated, including bispecific derivatives thereof that additionally target CD3 (WO 2016 / 199141).

[0004] Bispecific antibodies (also referred to herein as T cell bispecific antibodies or "TCBs") that bind to surface antigens on target cells and activating T cell antigens (such as CD3 on T cells) have great prospects for treating various cancers. The simultaneous binding of such an antibody to its two targets will force a temporary interaction between the target cell and the T cell, resulting in cross-linking of the T cell receptors and subsequent activation of any cytotoxic T cells, which then lyse the target cell. Given their effectiveness in killing target cells, the selection of targets and the specificity of the targeting antibodies are crucial for T cell bispecific antibodies to avoid on-target and off-target toxicity. Intracellular proteins such as MAGE-A4 are attractive targets, but are only accessible to T cell receptor (TCR)-like antibodies that bind to the major histocompatibility complex (MHC), which presents peptide antigens derived from intracellular proteins to the cell surface. The inherent problem with TCR-like antibodies is that they may cross-react with the MHC molecule itself or with MHC molecules that present peptides other than the desired peptide, which may impair organ or tissue selectivity. Summary of the Invention

[0005] The present invention provides novel antibodies, including multispecific (e.g., bispecific) antibodies, that bind to HLA-A2 / MAGE-A4 and possess particularly advantageous properties for therapeutic purposes. In particular, the (multispecific) antibodies bind to HLA-A2 / MAGE-A4 with good affinity and remarkable specificity, combined with good efficacy and manufacturability, low toxicity, and favorable pharmacokinetic properties.

[0006] In one aspect, the present invention provides an antibody that binds to HLA-A2 / MAGE-A4, wherein the antibody comprises a first antigen-binding domain comprising

[0007] (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0008] (ii) a VH comprising HCDR 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 3; and a VL comprising LCDR 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;

[0009] (iii) a VH comprising HCDR 1 of SEQ ID NO: 41, HCDR 2 of SEQ ID NO: 42, and HCDR 3 of SEQ ID NO: 43; and a VL comprising LCDR 1 of SEQ ID NO: 44, LCDR 2 of SEQ ID NO: 45, and LCDR 3 of SEQ ID NO: 46;

[0010] (iv) a VH comprising HCDR 1 of SEQ ID NO: 9, HCDR 2 of SEQ ID NO: 10, and HCDR 3 of SEQ ID NO: 11; and a VL comprising LCDR 1 of SEQ ID NO: 12, LCDR 2 of SEQ ID NO: 13, and LCDR 3 of SEQ ID NO: 14;

[0011] (v) a VH comprising HCDR 1 of SEQ ID NO: 17, HCDR 2 of SEQ ID NO: 18, and HCDR 3 of SEQ ID NO: 19; and a VL comprising LCDR 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21, and LCDR 3 of SEQ ID NO: 22; or

[0012] (vi) a VH comprising HCDR 1 of SEQ ID NO: 33, HCDR 2 of SEQ ID NO: 34, and HCDR 3 of SEQ ID NO: 35; and a VL comprising LCDR 1 of SEQ ID NO: 36, LCDR 2 of SEQ ID NO: 37, and LCDR 3 of SEQ ID NO: 38.

[0013] In one aspect, the first antigen binding domain comprises

[0014] (i) a VH comprising 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: 31; and / or a VL comprising 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;

[0015] (ii) a VH comprising 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: 7; and / or a VL comprising 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: 8;

[0016] (iii) a VH comprising 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 / or a VL comprising 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;

[0017] (iv) a VH comprising 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: 15; and / or a VL comprising 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: 16;

[0018] (v) a VH comprising 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: 23; and / or a VL comprising 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: 24; or

[0019] (vi) a VH comprising 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: 39; and / or a VL comprising 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: 40.

[0020] In another aspect, the present invention provides an antibody that binds to HLA-A2 / MAGE-A4, wherein the antibody comprises a first antigen-binding domain comprising

[0021] (i) a VH comprising the amino acid sequence of SEQ ID NO: 31; and / or a VL comprising the amino acid sequence of SEQ ID NO: 32;

[0022] (ii) VH comprising the amino acid sequence of SEQ ID NO: 7; and / or VL comprising the amino acid sequence of SEQ ID NO: 8;

[0023] (iii) VH comprising the amino acid sequence of SEQ ID NO: 47; and / or VL comprising the amino acid sequence of SEQ ID NO: 48;

[0024] (iv) a VH comprising the amino acid sequence of SEQ ID NO: 15; and / or a VL comprising the amino acid sequence of SEQ ID NO: 16;

[0025] (v) VH comprising the amino acid sequence of SEQ ID NO: 23; and / or VL comprising the amino acid sequence of SEQ ID NO: 24; or

[0026] (vi) VH comprising the amino acid sequence of SEQ ID NO: 39; and / or VL comprising the amino acid sequence of SEQ ID NO: 40.

[0027] In one aspect, the antibody is a multispecific antibody, in particular a bispecific antibody.

[0028] In one aspect, the antibody comprises a second antigen binding domain that binds to a second antigen.

[0029] In one aspect, the second antigen is an activating T cell antigen, particularly CD3, most particularly CD3ε.

[0030] In one aspect, the second antigen binding domain comprises

[0031] (i) a VH comprising HCDR 1 of SEQ ID NO: 59, HCDR 2 of SEQ ID NO: 60, and HCDR 3 of SEQ ID NO: 61; and a VL comprising LCDR 1 of SEQ ID NO: 62, LCDR 2 of SEQ ID NO: 63, and LCDR 3 of SEQ ID NO: 64; or

[0032] (ii) a VH comprising HCDR 1 of SEQ ID NO: 51, HCDR 2 of SEQ ID NO: 52, and HCDR 3 of SEQ ID NO: 53; and a VL comprising LCDR 1 of SEQ ID NO: 54, LCDR 2 of SEQ ID NO: 55, and LCDR 3 of SEQ ID NO: 56.

[0033] In one aspect, the second antigen binding domain comprises

[0034] (i) a VH comprising 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: 65; and / or a VL comprising 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: 66; or

[0035] (ii) a VH comprising 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: 57; and / or a VL comprising 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: 58.

[0036] In one aspect, the second antigen binding domain comprises

[0037] (i) a VH comprising the amino acid sequence of SEQ ID NO: 65; and / or a VL comprising the amino acid sequence of SEQ ID NO: 66;

[0038] (ii) VH comprising the amino acid sequence of SEQ ID NO: 57; and / or VL comprising the amino acid sequence of SEQ ID NO: 58.

[0039] In one aspect, the antibody comprises a third antigen binding domain. In one aspect, the third antigen binding domain binds to HLA-A2 / MAGE-A4. The third antigen binding domain can incorporate any of the features described herein, alone or in combination, in connection with the first antigen binding domain. In one aspect, the third antigen binding domain is identical to the first antigen binding domain.

[0040] In one aspect, the first antigen binding domain, the second antigen binding domain (if present), and / or the third antigen binding domain (if present) is a Fab molecule.

[0041] In one aspect, the second antigen binding domain is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other or the constant domains CL and CH1 are replaced with each other, in particular the variable domains VL and VH are replaced with each other.

[0042] In one aspect, the first antigen binding domain and, when present, the third antigen binding domain are conventional Fab molecules.

[0043] In one aspect, the first antigen binding domain and, if present, the third antigen binding domain are Fab molecules in which, in the constant domain CL, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering); and in the constant domain CH1, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index).

[0044] In one aspect, the first antigen binding domain and the second antigen binding domain are fused to each other, optionally via a peptide linker.

[0045] In one aspect, the first and second antigen binding domains are each a Fab molecule and (i) the first antigen binding domain 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 domain, or (ii) the second antigen binding domain 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 domain.

[0046] In one aspect, the antibody comprises an Fc domain composed of a first subunit and a second subunit.

[0047] In one aspect, the first antigen binding domain, the second antigen binding domain, and, if present, the third antigen binding domain are each a Fab molecule, and the antibody comprises an Fc domain composed of a first subunit and a second subunit; and (i) the first antigen binding domain 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 domain, and the second antigen binding domain 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 second antigen binding domain 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 domain, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; and the third antigen binding domain, when present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0048] In one aspect, the Fc domain is an IgG, particularly an IgG1 Fc domain. In one aspect, the Fc domain is a human Fc domain. In one aspect, the Fc domain comprises modifications that promote association of the first and second subunits of the Fc domain. In one aspect, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector function.

[0049] According to another aspect of the present invention, an isolated polynucleotide encoding the antibody of the present invention and a host cell comprising the isolated polynucleotide of the present invention are provided.

[0050] In another aspect, a method for producing an antibody that binds to HLA-A2 / MAGE-A4 is provided, comprising the steps of: (a) culturing a host cell of the invention under conditions suitable for expression of the antibody, and optionally (b) recovering the antibody. The invention also encompasses antibodies that bind to HLA-A2 / MAGE-A4 produced by the methods of the invention.

[0051] The present invention further provides a pharmaceutical composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier.

[0052] The present invention also encompasses methods of using the antibodies and pharmaceutical compositions of the present invention. In one aspect, the present invention provides an antibody or pharmaceutical composition according to the present invention for use as a medicament. In one aspect, an antibody or pharmaceutical composition according to the present invention for use in treating a disease is provided. In a specific aspect, the disease is cancer.

[0053] Also provided are uses of antibodies or pharmaceutical compositions according to the present invention in the manufacture of medicaments, and uses of antibodies or pharmaceutical compositions according to the present invention in the manufacture of medicaments for treating diseases, particularly cancer. The present invention also provides a method of treating a disease in an individual, comprising administering to the individual an effective amount of an antibody or pharmaceutical composition according to the present invention. In a specific aspect, the disease is cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1Exemplary configurations of bispecific antigen-binding molecules of the present invention. (A, D) Schematic diagrams of a "1+1 CrossMab" molecule. (B, E) Schematic diagrams of a "2+1 IgG Crossfab" molecule, in which the order of the Crossfab and Fab components alternates ("inverted"). (C, F) Schematic diagrams of a "2+1 IgG Crossfab" molecule. (G, K) Schematic diagrams of a "1+1 IgG Crossfab" molecule, in which the order of the Crossfab and Fab components alternates ("inverted"). (H, L) Schematic diagrams of a "1+1 IgG Crossfab" molecule. (I, M) Schematic diagrams of a "2+1 IgG Crossfab" molecule with two CrossFabs. (J, N) Schematic diagrams of a "2+1 IgG Crossfab" molecule with two CrossFabs, in which the order of the Crossfab and Fab components alternates ("inverted"). (O, S) Schematic diagrams of a "Fab-Crossfab" molecule. (P, T) Schematic diagrams of a "Crossfab-Fab" molecule. (Q, U) Schematic diagram of a "(Fab)2-Crossfab" molecule. (R, V) Schematic diagram of a "Crossfab-(Fab)2" molecule. (W, Y) Schematic diagram of a "Fab-(Crossfab)2" molecule. (X, Z) Schematic diagram of a "(Crossfab)2-Fab" molecule. Black dots: optional modifications in the Fc domain that promote heterodimerization. ++, --: amino acids with opposite charges optionally introduced into the CH1 and CL domains. The Crossfab molecule is described as comprising an exchange of the VH and VL regions, but may - in aspects in which no charge modifications are introduced into the CH1 and CL domains - alternatively comprise an exchange of the CH1 and CL domains.

[0055] Figure 2 Schematic representation of the T cell bispecific (TCB) antibody molecules prepared in the Examples. All TCB antibody molecules tested were generated as "2+1 IgG CrossFab, inverted" with charge modifications (VH / VL exchange in the CD3 binder, charge modification in the MAGE-A4 binder, EE=147E, 213E; RK=123R, 124K).

[0056] Figure 3 .T cell-mediated lysis of T2 cells, T2 cells with MAGE-A4 p230-239 peptide (GVYDGREHTV) (A) or an unrelated peptide (NY-ESO1 p157-165 :(SLLMWITQC)) (B) Pulsed and induced with different MAGE-A4 CD3 TCB molecules (E:T=10:1, human PBMC effector cells). Depicted are three replicates with SD.

[0057] Figure 4 Different MAGE-A4 CD3 TCB molecules were combined with Jurkat-NFAT reporter cells and T2 cells (using MAGE-A4 p230-239 peptide (GVYDGREHTV) (A) or an unrelated peptide (NY-ESO1 p157-165 :(SLLMWITQC)) (B) Jurkat activation (as measured by luminescence) upon simultaneous binding of human CD3 on (pulse) . Depicted are three replicates with SD.

[0058] Figure 5 T cell-mediated lysis of HLA-A2+ / MAGE-A4+ tumor cell lines from different indications and HLA-A2+ / MAGE-A4-MDA-MB-231 cells (E:T = 10:1, human PBMC effector cells) induced by different MAGE-A4 CD3 TCB molecules. (A) Molecule A, (B) Molecule D, (C) Molecule E, (D) Molecule B, (E) Molecule C, (F) Molecule F. Depicted are three replicates with SD.

[0059] Figure 6 T cell-mediated lysis of HLA-A2+ / MAGE-A4- tumor cell lines of different indications and HLA-A2+ / MAGE-A4+ NCI-H1755 cells (E:T = 10:1, human PBMC effector cells) induced by different MAGE-A4 CD3 TCB molecules. (A) Molecule A, (B) Molecule C, (C) Molecule E, (D) Molecule B, (E) Molecule D, (F) Molecule F. Depicted are three replicates with SD.

[0060] Figure 7 Identification of binding residues of the MAGE-A4 peptides of the MAGE-A4 CD3 TCB by alanine scanning. (A) EC50 values ​​determined for Jurkat activation of different MAGE-A4 CD3 TCB molecules for simultaneous binding to human CD3 on Jurkat-NFAT reporter cells and T2 cells pulsed with different ALA scanning peptides. Panel (B) depicts the x-fold change in EC50 for different mutant ALA scanning peptides. Key contact residues are marked in bold.

[0061] Figure 8 Different MAGE-A4 CD3 TCB molecules were combined with Jurkat-NFAT reporter cells and T2 cells (using MAGE-A4 p230-239Jurkat activation (as measured by luminescence) upon simultaneous binding of human CD3 to a peptide (GVYDGREHTV) or one of the 33 predicted off-target peptides shown. Plotted is the percentage of MAGE-A4-induced signal to POTP-induced signal ((CPS[POTP]–CPS[unpulsed cells]) / (CPS[MAGE-A4]–CPS[unpulsed cells])*100). Off-target peptides with ratios >2% are considered relevant and are highlighted in gray.

[0062] Figure 9 Summary of identified off-target peptides for Molecule D evaluated by the Jurkat activation assay against 509 predicted off-target peptides. Depicted is the percentage of MAGE-A4-induced signal to POTP-induced signal ((CPS[POTP]–CPS[unpulsed cells]) / (CPS[MAGE-A4]–CPS[unpulsed cells])*100). Depicted are all off-target peptides that showed a signal >2% in either of the two assays performed.

[0063] Figure 10 Validation of identified off-target peptides of molecule D in the Jurkat activation assay. Depicted is the luminescence measured upon simultaneous binding of molecule D to human CD3 on Jurkat-NFAT reporter cells and T2 cells pulsed with the off-target peptide of interest.

[0064] Figure 11 Summary of identified off-target peptides for Molecule G evaluated by the Jurkat activation assay against 509 predicted off-target peptides. Depicted is the percentage of MAGE-A4-induced signal to POTP-induced signal ((CPS[POTP]–CPS[unpulsed cells]) / (CPS[MAGE-A4]–CPS[unpulsed cells])*100). Depicted are all off-target peptides showing a signal >2%.

[0065] Figure 12 The PGLALA-modified Fc domains of different MAGE-A4 IgG molecules were co-cultured with Jurkat-NFAT reporter cells (genetically engineered to express a TCR directed against the PGLALA mutation in the Fc portion of the IgG molecule) and T2 cells (expressed with MAGE-A4 p230-239 PGLALA-CAR-J activation upon simultaneous binding of peptide (GVYDGREHTV) (A) or MAGE-A4- / HLA-A2+ MDA-MB231 cells (B). Depicted are three replicates with SD.

[0066] Figure 13T cell-mediated lysis of various HLA-A2+ / MAGE-A4+ tumor cell lines for different indications induced by different MAGE-A4CD3 TCB molecules containing different combinations of MAGE-A4 and CD3 binders (E:T = 10:1, human PBMC effector cells). Depicted are three replicates with SD. (A) UMUC-3 cells, (B) A375 cells, (C) NCI-H2023 cells.

[0067] Figure 14 T cell-mediated lysis of A375 cells, A375 cells engineered to overexpress HLA-A2, and A375 cells with MAGE-A4 knockout induced by the MAGE-A4 CD3 TCB molecule R (E:T=10:1, human PBMC effector cells).

[0068] Figure 15 Cytokine release upon T cell-mediated lysis of A375 cells, A375 cells engineered to overexpress HLA-A2, and A375 cells with MAGE-A4 knockout (ko) induced by the MAGE-A4 CD3 TCB molecule R (E:T=10:1, human PBMC effector cells).

[0069] Figure 16 Real-time evaluation of different HLA-A2 + / MAGE-A4 + Growth of tumor cell lines: (A) NCI-H1755; (B) UMUC-3; (C) A375; (D) ScaBer; (E) NCI-H1703; (F) NCI-H2023.

[0070] Figure 17 Study design and cohorts for a dose-finding efficacy study of the MAGE-A4CD3 TCB molecule R in IM-9 xenografts in humanized mice as described in Example 13.

[0071] Figure 18 Tumor growth kinetics for all groups in a dose-finding efficacy study of the MAGE-A4CD3 TCB molecule R in humanized IM-9 xenografts in mice as described in Example 13. (A) Mean + / - SEM; (B-E) Individual tumor growth per mouse.

[0072] Figure 19 T cell infiltration in tumors in a dose-finding efficacy study of the MAGE-A4CD3 TCB molecule R in IM-9 xenografts in humanized mice as described in Example 13 (B). DETAILED DESCRIPTION

[0073] I. Definition

[0074] Unless otherwise defined below, the terms used herein are generally as used in the art.

[0075] As used herein, the terms "first," "second," or "third" with respect to antigen-binding domains, etc., are used for convenience in distinguishing when there is more than one moiety of each type. Unless explicitly stated, the use of these terms is not intended to confer a particular order or orientation of the moieties. However, in general, the antigen-binding domain that binds to HLA-A2 / MAGE-A4 is referred to herein as the "first antigen-binding domain" (and, if there is an additional antigen-binding domain that binds to HLA-A2 / MAGE-A4, the "third antigen-binding domain"), and the antigen-binding domain that binds to the second antigen is referred to herein as the "second antigen-binding domain."

[0076] The terms "anti-HLA-A2 / MAGE-A4 antibody" and "antibody that binds to HLA-A2 / MAGE-A4" refer to an antibody that binds to HLA-A2 / MAGE-A4 with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent targeting HLA-A2 / MAGE-A4. In one aspect, the extent of binding of the anti-HLA-A2 / MAGE-A4 antibody to unrelated, non-HLA-A2 / MAGE-A4 proteins is less than about 10% of the binding of the antibody to HLA-A2 / MAGE-A4, as measured, for example, by surface plasmon resonance (SPR). In certain aspects, the antibody that binds to HLA-A2 / MAGE-A4 has a dissociation constant (K) of ≤1 μM, ≤500 nM, ≤200 nM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. D ).

[0077] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. Suitable assays for determining the specificity of the antibodies of the invention are described herein, for example, as described in Example 9 below. In one aspect, the extent of binding of the antibody to unrelated proteins is less than about 10% of the binding of the antibody to the antigen, as measured, for example, by SPR.

[0078] The term "antibody" herein is used in the broadest sense and encompasses various 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.

[0079] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies; and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0080] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure.

[0081] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for possible variant antibodies, each antibody included in the population is identical and / or binds to the same epitope, wherein the possible variant antibodies are, for example, those containing naturally occurring mutations or produced during the production process of monoclonal antibody preparations, and such variants are typically present in trace amounts. Contrary to the polyclonal antibody preparations typically comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is directed against a single determinant on the antigen. Therefore, the modifier "monoclonal" represents that the characteristic of an antibody is obtained from a substantially homogeneous antibody population, and should not be construed as requiring antibody production by any ad hoc method. For example, monoclonal antibodies can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0082] "Isolated" antibodies are antibodies that have been separated from the components of their natural environment. In some aspects, the antibody is purified to a purity greater than 95% or 99%, and purity is determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC, affinity chromatography, size exclusion chromatography) methods. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007). In some aspects, the antibodies provided by the present invention are isolated antibodies.

[0083] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0084] "Humanized" antibody refers to such chimeric antibodies, which comprise amino acid residues from non-human CDR and amino acid residues from people FR. In some aspects, humanized antibody will substantially comprise at least one, usually two variable domains, wherein all or substantially all CDRs correspond to the CDRs of non-human antibodies, and all or substantially all FRs correspond to the FRs of people antibodies. Such variable domains are referred to as "humanized variable regions" in this article. Humanized antibodies optionally can comprise at least a portion of the antibody constant region derived from people antibodies. In some aspects, some FR residues in the humanized antibody are replaced by corresponding residues from non-human antibodies (for example, the antibody from which CDR residues are derived), for example, to restore or improve antibody specificity or affinity. "Humanized form" of an antibody (for example, a non-human antibody) refers to an antibody that has undergone humanization.

[0085] "Human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from an amino acid sequence of an antibody derived from a non-human source utilizing a human antibody library or other human antibody encoding sequences. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. In some aspects, human antibodies are derived from non-human transgenic mammals, such as mice, rats, or rabbits. In some aspects, human antibodies are derived from hybridoma cell lines. Antibodies or antibody fragments isolated from human antibody libraries are also considered to be human antibodies or human antibody fragments herein.

[0086] The term "antigen binding domain" refers to a portion of an antibody that includes a region that binds to and is complementary to part or all of an antigen. The antigen binding domain can be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). In preferred aspects, the antigen binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0087] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in binding of the antibody to the antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and complementarity determining regions (CDRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman & Co, p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using the VH or VL domains from antibodies that bind to the antigen, respectively, to screen for libraries of complementary VL or VH domains. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991). As used herein, "Kabat numbering" with respect to variable region sequences refers to the numbering system set forth by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0088] As used herein, the amino acid positions of all constant regions and constant domains of heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "numbered according to Kabat" or "Kabat numbering." Specifically, the Kabat numbering system (see pages 647 to 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 the kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661 to 723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3), which is further clarified herein by being referred to in this context as "numbered according to the Kabat EU index" or "Kabat EU index numbering."

[0089] As used herein, the term "hypervariable region" or "HVR" refers to the individual regions of an antibody variable domain that are highly variable in sequence and determine antigen binding specificity, such as the "complementarity determining regions" ("CDRs"). Generally, an antibody comprises six CDRs; three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include:

[0090] (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));

[0091] (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

[0092] (c) Antigenic contacts 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)).

[0093] Unless otherwise indicated, CDRs are determined according to the method described in Kabat et al. (supra). One skilled in the art will appreciate that CDR names may also be determined according to the method described in Chothia (supra), McCallum (supra), or any other scientifically accepted nomenclature system.

[0094] "Framework" or "FR" refers to the variable domain residues excluding the complementarity determining regions (CDRs). The FR of a variable domain is typically composed of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following order in VH (or VL): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

[0095] Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0096] " acceptor people framework " for the purposes of this paper is such a framework, it comprises the amino acid sequence of light chain variable domain (VL) framework or heavy chain variable domain (VH) framework derived from human immunoglobulin framework or people's total framework as defined below.The acceptor people framework "derived from" human immunoglobulin framework or people's total framework can comprise the amino acid sequence identical with described human immunoglobulin framework or people's total framework, or it can comprise amino acid sequence change.In some aspects, the quantity of amino acid change is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less or 2 or less.In some aspects, VL acceptor people framework is identical with VL human immunoglobulin framework sequence or people's total framework sequence in sequence.

[0097] A "human consensus framework" is a framework that represents the most common amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Generally, the subset of sequences is a subset as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3.

[0098] The term "immunoglobulin molecule" herein refers to a protein with the structure of a naturally occurring antibody. For example, the IgG class immunoglobulin is a heterotetrameric glycoprotein of approximately 150,000 daltons, consisting of two light chains and two heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable domain (VH) (also referred to as a variable heavy chain domain or a heavy chain variable region), followed by three constant domains (CH1, CH2, and CH3) (also referred to as a heavy chain constant region). Similarly, from N-terminus to C-terminus, each light chain has a variable domain (VL) (also referred to as a variable light chain domain or a light chain variable region), followed by a constant light chain (CL) domain (also referred to as a light chain constant region). The heavy chains of immunoglobulins can be assigned to one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which are further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of two types: kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Immunoglobulins are essentially composed of two Fab molecules and an Fc domain connected by the immunoglobulin hinge region.

[0099] 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 them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0100] "Fab molecule" refers to a protein composed of the VH and CH1 domains of an immunoglobulin heavy chain ("Fab heavy chain") and the VL and CL domains of a light chain ("Fab light chain").

[0101] The so-called "crossover" Fab molecule (also referred to as "Crossfab") refers to the following Fab molecules: wherein the variable domains or constant domains of the Fab heavy chain and light chain are exchanged (i.e., replaced with each other), i.e., the crossover Fab molecule comprises a peptide chain (VL-CH1, in the N-terminal to C-terminal direction) consisting of a light chain variable domain VL and a heavy chain constant domain 1CH1, and a peptide chain (VH-CL, in the N-terminal to C-terminal direction) consisting of a heavy chain variable domain VH and a light chain constant domain CL. For clarity, in the crossover Fab molecule in which the variable domains of the Fab light chain and the variable domains of the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in the crossover Fab molecule in which the constant domains of the Fab light chain and the constant domains of the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0102] In contrast, by "conventional" Fab molecules are meant Fab molecules in their native form, i.e., comprising a heavy chain consisting of a heavy chain variable domain and a constant domain (VH-CH1, in N-terminal to C-terminal direction), and a light chain consisting of a light chain variable domain and a constant domain (VL-CL, in N-terminal to C-terminal direction).

[0103] The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the antibody produced by the host cell can undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, the antibody produced by the host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain can include a full-length heavy chain, or the antibody can include a cleavage variant of the full-length heavy chain. This may be a situation where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) in the Fc region may be present or absent. In one aspect, the heavy chain comprising the Fc region (subunit) as specified herein is included in an antibody according to the present invention, and the heavy chain comprises other C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one aspect, the heavy chain comprising the Fc region (subunit) as specified herein is included in an antibody according to the present invention, and the heavy chain comprises other C-terminal glycine residues (G446, numbered according to the Kabat EU index). Unless otherwise specified herein, the numbering of amino acid residues in Fc region or constant region is numbered according to the EU numbering system (also referred to as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (also referring to 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 comprising the C-terminal constant region of an immunoglobulin heavy chain, which polypeptide is capable of stably self-association. For example, a subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

[0104] By "fused", it is meant that the components (eg, Fab molecule and Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0105] The term "multispecific" means that the antibody is able to specifically bind to at least two different antigenic determinants. A multispecific antibody can be, for example, a bispecific antibody. Typically, a bispecific antibody comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In some aspects, the multispecific (e.g., bispecific) antibody is able to simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0106] As used herein, the term "valence" refers to the presence of a specified number of antigen binding sites in an antigen binding molecule. Thus, the term "monovalently binds to an antigen" refers to the presence of one (and no more than one) antigen binding site specific for an antigen in an antigen binding molecule.

[0107] "Antigen binding site" refers to the site, i.e., one or more amino acid residues, of an antigen-binding molecule that provides interaction with an antigen. For example, the antigen-binding site of an antibody comprises amino acid residues from the complementarity determining region (CDR). Natural immunoglobulin molecules typically have two antigen-binding sites, while Fab molecules typically have a single antigen-binding site.

[0108] As used herein, the term "antigenic determinant" or "antigen" refers to a site on a polypeptide macromolecule (e.g., a stretch of continuous amino acids or a conformational configuration consisting of different regions of non-continuous amino acids) to which an antigen binding domain binds, thereby forming an antigen binding domain-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, in free matter in serum, and / or in the extracellular matrix (ECM). In a preferred aspect, the antigen is a human protein.

[0109] As used herein, "activating T cell antigen" refers to an antigenic determinant expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, which can induce T cell activation when interacting with an antigen binding molecule. Specifically, the interaction of an antigen binding molecule with an activating T cell antigen can induce T cell activation by triggering the signaling cascade of the T cell receptor complex. In particular aspects, the activating T cell antigen is CD3, particularly the epsilon subunit of CD3.

[0110] Unless otherwise indicated, "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). The term encompasses "full-length" unprocessed CD3, as well as any form of CD3 produced by processing in cells. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one aspect, CD3 is human CD3, particularly the epsilon subunit (CD3 epsilon) of human CD3. The amino acid sequence of human CD3 epsilon is shown in SEQ ID NO: 76 (without signal peptide). See also UniProt (www.uniprot.org) accession number P07766 (version 189), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. On the other hand, CD3 is cynomolgus monkey (Macaca fascicularis) CD3, particularly cynomolgus monkey CD3 epsilon. The amino acid sequence of cynomolgus monkey CD3ε is shown in SEQ ID NO: 77 (without the signal peptide). See also NCBI GenBank no. BAB71849.1. In certain aspects, the antibodies of the present invention bind to an epitope of CD3 (particularly human and cynomolgus monkey CD3) that is conserved among CD3 antigens from different species. In preferred aspects, the antibodies bind to human CD3.

[0111] As used herein, "target cell antigen" refers to an antigenic determinant present on the surface of a target cell, for example a cell in a tumor (such as a cancer cell or a cell of the tumor stroma) (in this case, a "tumor cell antigen"). According to the present invention, the target cell antigen is HLA-A2 / MAGE-A4, in particular HLA-A2 / MAGE-A4. p230-239 .

[0112] "MAGE-A4" stands for "melanoma-associated antigen 4," a member of the cancer testis antigen (CTA) MAGE family. The MAGE-A protein family 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 (entry version 163), and the amino acid sequence of human MAGE-A4 is also shown herein in SEQ ID NO: 74. Unless otherwise indicated, "MAGE-A4," as used herein, 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). The term includes "full-length," unprocessed MAGE-A4, as well as any form of MAGE-A4 produced by processing in cells. The term also encompasses naturally occurring variants of MAGE-A4, such as splice variants or allelic variants. In one aspect, MAGE-A4 is human MAGE-A4, in particular the protein of SEQ ID NO:74.

[0113] "MAGE-A4 p230-239 " or "p230-239 peptide" means a MAGE-A4 derived peptide having the amino acid sequence GVYDGREHTV (SEQ ID NO: 73; positions 230-239 of the MAGE-A4 protein of SEQ ID NO: 74).

[0114] "HLA-A2," "HLA-A*02," "HLA-A02," or "HLA-A*2" (used interchangeably) refers to a human leukocyte antigen serotype in the HLA-A serotype group. The HLA-A2 protein (encoded by the respective HLA gene) comprises the α chain of the respective class I MHC (major histocompatibility complex) protein, which further comprises a β2 microglobulin subunit. A specific HLA-A2 protein is HLA-A201 (also referred to as HLA-A0201, HLA-A02.01, or HLA-A*02:01). In specific aspects, the HLA-A2 protein described herein is HLA-A201.

[0115] "HLA-A2 / MAGE-A4" refers to HLA-A2 molecules and MAGE-A4-derived peptides (also referred to herein as "MAGE-A4 peptides"), specifically the p230-239 peptide ("HLA-A2 / MAGE-A4 p230-239 ”) complex.

[0116] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ). Affinity can be measured by well-established methods known in the art, including those described herein. A preferred method for measuring affinity is surface plasmon resonance (SPR).

[0117] An "affinity matured" antibody is one with one or more alterations in one or more complementarity determining regions (CDRs) which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess such alteration(s).

[0118] "Decreased binding" (e.g., reduced binding to an Fc receptor) refers to a decrease in affinity for the corresponding interaction, as measured, for example, by SPR. For clarity, the term also includes a decrease in affinity to zero (or below the detection limit of the analytical method), i.e., complete elimination of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for the corresponding interaction.

[0119] As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and described herein.

[0120] A “modification that promotes the association of the first and second subunits of the Fc domain” is a manipulation of the peptide backbone or a post-translational modification of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with the same polypeptide to form a homodimer. As used herein, a “modification that promotes association” preferably includes a separate modification of each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, wherein the modifications are complementary to each other to promote the association of the two Fc domain subunits. For example, the modification that promotes association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (hetero) dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which may be different in terms of the additional components (e.g., antigen binding domains) fused to each subunit. In some aspects, the modification that promotes the association of the first and second subunits of the Fc domain comprises an amino acid mutation, particularly an amino acid substitution, in the Fc domain. In a preferred aspect, the modification promoting the association of the first and the second subunit of the Fc domain comprises a single amino acid mutation, in particular an amino acid substitution, in each of the two subunits of the Fc domain.

[0121] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with 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 B cell activation.

[0122] "Activating Fc receptors" are Fc receptors that, upon engagement by the Fc domain of an antibody, initiate signaling events that stimulate cells bearing the receptor to perform effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0123] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that causes immune effector cells to lyse antibody-coated target cells. Target cells are cells that specifically bind to antibodies or derivatives thereof comprising an Fc region, and this specific binding is typically through the protein portion of the N-terminus of the Fc region. As used herein, the term "reduced ADCC" is defined as a reduction in the number of target cells lysed by the ADCC mechanism defined above within a given time at a given antibody concentration in the culture medium surrounding the target cells, and / or an increase in the antibody concentration necessary to achieve lysis of a given number of target cells within a given time by the ADCC mechanism in the culture medium surrounding the target cells. ADCC reduction is relative to ADCC mediated by the same antibody produced by the same type of host cell but not yet engineered using the same standard production, purification, formulation and storage methods (such methods are known to those skilled in the art). For example, the reduction in ADCC mediated by an antibody comprising an amino acid substitution that reduces ADCC in the Fc domain is relative to ADCC mediated by the same antibody without the amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, eg, PCT Publication No. WO 2006 / 082515 or PCT Publication No. WO 2012 / 130831).

[0124] As used herein, the terms "engineering, engineered, engineered" are considered to include any manipulation of the peptide backbone, or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications to the amino acid sequence, glycosylation pattern, or side chain groups of individual amino acids, as well as combinations of these approaches.

[0125] As used herein, the term "amino acid mutation" means encompassing amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed to obtain the final construct, provided that the final construct has the desired characteristics, such as reduced binding to Fc receptors, or increased association with another peptide. Amino acid sequence deletions and insertions include amino acid amino-terminal and / or carboxyl-terminal deletions and insertions. Preferred amino acid mutations are amino acid substitutions. For the purpose of changing, for example, the binding characteristics of the Fc region, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include substitutions with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be produced using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is also useful to envision methods for changing amino acid side chain groups by methods other than genetic engineering (such as chemical modification). Various names can be used herein to indicate the same amino acid mutation. For example, substitution of proline at position 329 of the Fc domain with glycine can be represented as 329G, G329, G 329 , P329G or Pro329Gly.

[0126] " amino acid sequence identical percentage (%) " relative to a reference polypeptide sequence is defined as after comparing candidate sequence with the reference polypeptide sequence and introducing room (if necessary) to realize maximum sequence identical percentage, and without considering using any conservative substitution as a component of sequence identity, the percentage that the amino acid residue in the candidate sequence is identical with the amino acid residue in the reference polypeptide sequence. Comparison for determining amino acid sequence identical percentage can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA program package. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for realizing maximum alignment over the full length of the compared sequence. Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate identical percentage values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code has been filed with user documentation in the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087 and is described in WO 2001 / 007611.

[0127] Unless otherwise indicated, for purposes herein, the BLOSUM50 comparison matrix is ​​used and the ggsearch program of the FASTA package, version 36.3.8c or higher, is used to generate % amino acid sequence identity. The FASTA package was created by W. R. Pearson and D. J. Lipman, "Improved Tools for Biological Sequence Analysis," PNAS 85 (1988): 2444-2448; W. R. Pearson, "Effective protein sequence comparison," Meth. Enzymol. 266 (1996): 227-258; and Pearson et al., (Genomics 46 (1997) 24-36) and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global rather than a local alignment. The percentage of amino acid identity is given in the alignment header of the output.

[0128] The term "polynucleotide" or "nucleic acid molecule" includes any compound and / or substance comprising a nucleotide polymer. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a base sequence, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically expressed as from 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes sense and antisense strands, as well as single-stranded and double-stranded forms. In addition, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. The example of non-naturally occurring nucleotides includes modified nucleotide bases with derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for direct expression in vitro and / or in vivo (e.g., in a host or patient) of the antibodies of the present invention. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the coding molecule so that mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., (2017) Nature Medicine 23:815-817, or EP 2 101823B1).

[0129] An "isolated" nucleic acid molecule is one that has been separated from a component of its natural environment. An isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0130] An "isolated polynucleotide (or nucleic acid) encoding an antibody" refers to one or more polynucleotide molecules encoding an antibody heavy and light chain (or fragments thereof), including such polynucleotide molecules in a single vector or different vectors, and such polynucleotide molecules present in one or more locations in a host cell.

[0131] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0132] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived from the primary transformed cells, regardless of the number of passages. Progeny may not be completely identical to the nucleic acid content of the parent cell, but may contain mutations. This article includes mutant progeny with the same function or biological activity as screened or selected in the original transformed cells. Host cells are any type of cell system that can be used to produce the antibodies of the present invention. Host cells include cultured cells, for example, mammalian cultured cells, such as HEK cells, CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues. In one aspect, the host cells of the present invention are eukaryotic cells, particularly mammalian cells. In one aspect, the host cell is not a cell in the human body.

[0133] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered.

[0134] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0135] As used herein, "treatment" (and grammatical variants thereof, such as "treat" or "treating") refers to an attempt to alter the natural course of a disease in the individual being treated, and may be performed for prevention or as a clinical intervention performed during clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some aspects, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0136] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, an individual or subject is a human.

[0137] An "effective amount" of a pharmaceutical agent (eg, a pharmaceutical composition) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0138] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0139] II. Compositions and Methods

[0140] The present invention provides antibodies that bind to HLA-A2 / MAGE-A4, including multispecific antibodies that bind to HLA-A2 / MAGE-A4 and a second antigen. These (multispecific) antibodies exhibit good affinity and remarkable specificity, combined with other properties that are advantageous for therapeutic applications, such as efficacy and safety, pharmacokinetics, and manufacturability. The antibodies of the present invention can be used, for example, to treat diseases such as cancer.

[0141] A. Anti-HLA-A2 / MAGE-A4 Antibodies

[0142] In one aspect, the present invention provides antibodies that bind to HLA-A2 / MAGE-A4. In one aspect, the present invention provides isolated peptides that bind to HLA-A2 / MAGE-A4. In one aspect, the present invention provides antibodies that specifically bind to HLA-A2 / MAGE-A4.

[0143] In one aspect, the present invention provides an antibody that binds to HLA-A2 / MAGE-A4, wherein the antibody comprises a first antigen-binding domain comprising

[0144] (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0145] (ii) a VH comprising HCDR 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 3; and a VL comprising LCDR 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;

[0146] (iii) a VH comprising HCDR 1 of SEQ ID NO: 41, HCDR 2 of SEQ ID NO: 42, and HCDR 3 of SEQ ID NO: 43; and a VL comprising LCDR 1 of SEQ ID NO: 44, LCDR 2 of SEQ ID NO: 45, and LCDR 3 of SEQ ID NO: 46;

[0147] (iv) a VH comprising HCDR 1 of SEQ ID NO: 9, HCDR 2 of SEQ ID NO: 10, and HCDR 3 of SEQ ID NO: 11; and a VL comprising LCDR 1 of SEQ ID NO: 12, LCDR 2 of SEQ ID NO: 13, and LCDR 3 of SEQ ID NO: 14;

[0148] (v) a VH comprising HCDR 1 of SEQ ID NO: 17, HCDR 2 of SEQ ID NO: 18, and HCDR 3 of SEQ ID NO: 19; and a VL comprising LCDR 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21, and LCDR 3 of SEQ ID NO: 22; or

[0149] (vi) a VH comprising HCDR 1 of SEQ ID NO: 33, HCDR 2 of SEQ ID NO: 34, and HCDR 3 of SEQ ID NO: 35; and a VL comprising LCDR 1 of SEQ ID NO: 36, LCDR 2 of SEQ ID NO: 37, and LCDR 3 of SEQ ID NO: 38.

[0150] In one aspect, the present invention provides an antibody that binds to HLA-A2 / MAGE-A4, wherein the antibody comprises a first antigen-binding domain, the first antigen-binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27, and the light chain variable region comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 28, LCDR2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30.

[0151] In one aspect, the antibody is a humanized antibody. In one aspect, the antigen binding domain is a humanized antigen binding domain (i.e., the antigen binding domain of a humanized antibody). In one aspect, VH and / or VL are humanized variable regions.

[0152] In one aspect, the VH and / or VL comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0153] In one aspect, the antibody is a human antibody. In one aspect, the antigen binding domain is a human antigen binding domain (i.e., the antigen binding domain of a human antibody). In one aspect, the VH and / or VL are human variable regions.

[0154] In one aspect, the VH comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) selected from the group consisting of heavy chain variable region sequences of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39, and SEQ ID NO: 47, in particular the heavy chain variable region sequence of SEQ ID NO: 31. In one aspect, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39, and SEQ ID NO: 47, in particular the amino acid sequence of SEQ ID NO: 31. In one aspect, the VH comprises an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39, and SEQ ID NO: 47, particularly the amino acid sequence of SEQ ID NO: 31. In one aspect, the VH comprises an amino acid sequence that is at least about 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39, and SEQ ID NO: 47, particularly the amino acid sequence of SEQ ID NO: 31. In certain aspects, a VH sequence that is at least 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising the sequence retains the ability to bind to HLA-A2 / MAGE-A4. In some aspects, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39 or SEQ ID NO: 47. In some aspects, the substitutions, insertions or deletions occur in regions outside of the CDRs (i.e., in the FRs). In one aspect, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39 and SEQ ID NO: 47, particularly the amino acid sequence of SEQ ID NO: 31.Optionally, VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39 and SEQ ID NO: 47, in particular the amino acid sequence of SEQ ID NO: 31, including post-translational modifications of that sequence.

[0155] In one aspect, the VL comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) selected from the group consisting of light chain variable region sequences of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, and SEQ ID NO: 48, in particular the light chain variable region sequence of SEQ ID NO: 32. In one aspect, the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, and SEQ ID NO: 48, in particular the amino acid sequence of SEQ ID NO: 32. In one aspect, the VL comprises an amino acid sequence that is at least about 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, and SEQ ID NO: 48, particularly the amino acid sequence of SEQ ID NO: 32. In one aspect, the VL comprises an amino acid sequence that is at least about 98% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, and SEQ ID NO: 48, particularly the amino acid sequence of SEQ ID NO: 32. In certain aspects, a VL sequence that is at least 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising the sequence retains the ability to bind to HLA-A2 / MAGE-A4. In some aspects, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40 or SEQ ID NO: 48. In some aspects, the substitutions, insertions or deletions occur in regions outside the CDRs (i.e., in the FRs). In one aspect, the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40 and SEQ ID NO: 48, particularly the amino acid sequence of SEQ ID NO: 32.Optionally, VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40 and SEQ ID NO: 48, in particular the amino acid sequence of SEQ ID NO: 32, including post-translational modifications of that sequence.

[0156] In one aspect, the first antigen binding domain comprises

[0157] (i) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31; and / or a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 32;

[0158] (ii) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7; and / or a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8;

[0159] (iii) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 47; and / or a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 48;

[0160] (iv) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 15; and / or a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 16;

[0161] (v) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 23; and / or a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 24; or

[0162] (vi) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 39; and / or a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 40.

[0163] In one aspect, the first antigen binding domain comprises

[0164] (i) a VH comprising the amino acid sequence of SEQ ID NO: 31; and / or a VL comprising the amino acid sequence of SEQ ID NO: 32;

[0165] (ii) VH comprising the amino acid sequence of SEQ ID NO: 7; and / or VL comprising the amino acid sequence of SEQ ID NO: 8;

[0166] (iii) VH comprising the amino acid sequence of SEQ ID NO: 47; and / or VL comprising the amino acid sequence of SEQ ID NO: 48;

[0167] (iv) a VH comprising the amino acid sequence of SEQ ID NO: 15; and / or a VL comprising the amino acid sequence of SEQ ID NO: 16;

[0168] (v) VH comprising the amino acid sequence of SEQ ID NO: 23; and / or VL comprising the amino acid sequence of SEQ ID NO: 24; or

[0169] (vi) VH comprising the amino acid sequence of SEQ ID NO: 39; and / or VL comprising the amino acid sequence of SEQ ID NO: 40.

[0170] In one aspect, the first antigen-binding domain comprises a VH and a VL, wherein the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31; and the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 32. In one aspect, the first antigen-binding domain comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 31 and the VL comprises the amino acid sequence of SEQ ID NO: 32.

[0171] In another aspect, the present invention provides an antibody that binds to HLA-A2 / MAGE-A4, wherein the antibody comprises a first antigen-binding domain comprising

[0172] (i) a VH comprising the amino acid sequence of SEQ ID NO: 31; and / or a VL comprising the amino acid sequence of SEQ ID NO: 32;

[0173] (ii) VH comprising the amino acid sequence of SEQ ID NO: 7; and / or VL comprising the amino acid sequence of SEQ ID NO: 8;

[0174] (iii) VH comprising the amino acid sequence of SEQ ID NO: 47; and / or VL comprising the amino acid sequence of SEQ ID NO: 48;

[0175] (iv) a VH comprising the amino acid sequence of SEQ ID NO: 15; and / or a VL comprising the amino acid sequence of SEQ ID NO: 16;

[0176] (v) VH comprising the amino acid sequence of SEQ ID NO: 23; and / or VL comprising the amino acid sequence of SEQ ID NO: 24; or

[0177] (vi) VH comprising the amino acid sequence of SEQ ID NO: 39; and / or VL comprising the amino acid sequence of SEQ ID NO: 40.

[0178] In one aspect, the present invention provides an antibody that binds to HLA-A2 / MAGE-A4, wherein the antibody comprises a first antigen-binding domain, the first antigen-binding domain comprises VH and VL, VH comprises the amino acid sequence of SEQ ID NO: 31, and VL comprises the amino acid sequence of SEQ ID NO: 32.

[0179] In another aspect, the present invention provides an antibody that binds to HLA-A2 / MAGE-A4, wherein the antibody comprises a first antigen-binding domain comprising

[0180] (i) a VH comprising the heavy chain CDR sequence of VH of SEQ ID NO: 31 and a VL comprising the light chain CDR sequence of VL of SEQ ID NO: 32;

[0181] (ii) a VH comprising the heavy chain CDR sequence of VH of SEQ ID NO: 7 and a VL comprising the light chain CDR sequence of VL of SEQ ID NO: 8;

[0182] (iii) a VH comprising the heavy chain CDR sequence of VH of SEQ ID NO: 47 and a VL comprising the light chain CDR sequence of VL of SEQ ID NO: 48;

[0183] (iv) a VH comprising the heavy chain CDR sequence of VH of SEQ ID NO: 15 and a VL comprising the light chain CDR sequence of VL of SEQ ID NO: 16;

[0184] (v) a VH comprising the heavy chain CDR sequence of VH of SEQ ID NO: 23 and a VL comprising the light chain CDR sequence of VL of SEQ ID NO: 24; or

[0185] (vi) a VH comprising the heavy chain CDR sequence of VH of SEQ ID NO: 39 and a VL comprising the light chain CDR sequence of VL of SEQ ID NO: 40.

[0186] In one aspect, the present invention provides an antibody that binds to HLA-A2 / MAGE-A4, wherein the antibody comprises a first antigen-binding domain, the first antigen-binding domain comprising VH and VL, VH comprising the heavy chain CDR sequence of VH of SEQ ID NO: 31, and VL comprising the light chain CDR sequence of VL of SEQ ID NO: 32.

[0187] In another aspect, the first antigen binding domain comprises

[0188] (i) the HCDR1, HCDR2, and HCDR3 amino acid sequences of VH of SEQ ID NO: 31, and the LCDR1, LCDR2, and LCDR3 amino acid sequences of VL of SEQ ID NO: 32;

[0189] (ii) the HCDR1, HCDR2, and HCDR3 amino acid sequences of VH of SEQ ID NO: 7, and the LCDR1, LCDR2, and LCDR3 amino acid sequences of VL of SEQ ID NO: 8;

[0190] (iii) the HCDR1, HCDR2, and HCDR3 amino acid sequences of VH of SEQ ID NO: 47, and the LCDR1, LCDR2, and LCDR3 amino acid sequences of VL of SEQ ID NO: 48;

[0191] (iv) the HCDR1, HCDR2, and HCDR3 amino acid sequences of VH of SEQ ID NO: 15, and the LCDR1, LCDR2, and LCDR3 amino acid sequences of VL of SEQ ID NO: 16;

[0192] (v) the HCDR1, HCDR2, and HCDR3 amino acid sequences of VH of SEQ ID NO: 23, and the LCDR1, LCDR2, and LCDR3 amino acid sequences of VL of SEQ ID NO: 24; or

[0193] (vi) the HCDR1, HCDR2 and HCDR3 amino acid sequences of VH of SEQ ID NO: 39, and the LCDR1, LCDR2 and LCDR3 amino acid sequences of VL of SEQ ID NO: 40.

[0194] In one aspect, the first antigen binding domain comprises the HCDR1, HCDR2, and HCDR3 amino acid sequence of VH of SEQ ID NO: 31 and the LCDR1, LCDR2, and LCDR3 amino acid sequence of VL of SEQ ID NO: 32.

[0195] In one aspect, the first antigen binding domain comprises

[0196] (i) a VH and / or VL, wherein the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 31, and a framework sequence that is at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98% identical to the framework sequence of the VH of SEQ ID NO: 31; and the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 32, and a framework sequence that is at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98% identical to the framework sequence of the VL of SEQ ID NO: 32;

[0197] (i) a VH and / or VL, wherein the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 7, and a framework sequence that is at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98% identical to the framework sequence of the VH of SEQ ID NO: 7; and the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 8, and a framework sequence that is at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98% identical to the framework sequence of the VL of SEQ ID NO: 8;

[0198] (i) a VH and / or VL comprising the heavy chain CDR sequence of the VH of SEQ ID NO: 47, and a framework sequence that is at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98% identical to the framework sequence of the VH of SEQ ID NO: 47; and a VL comprising the light chain CDR sequence of the VL of SEQ ID NO: 48, and a framework sequence that is at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98% identical to the framework sequence of the VL of SEQ ID NO: 48;

[0199] (i) a VH and / or VL, wherein the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 15, and a framework sequence that is at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98% identical to the framework sequence of the VH of SEQ ID NO: 15; and the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 16, and a framework sequence that is at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98% identical to the framework sequence of the VL of SEQ ID NO: 16;

[0200] (i) a VH and / or VL comprising the heavy chain CDR sequence of the VH of SEQ ID NO: 23, and a framework sequence that is at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98% identical to the framework sequence of the VH of SEQ ID NO: 23; and a VL comprising the light chain CDR sequence of the VL of SEQ ID NO: 24, and a framework sequence that is at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98% identical to the framework sequence of the VL of SEQ ID NO: 24; or

[0201] (i) a VH and / or a VL, wherein the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 39, and a framework sequence that is at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98% identical to the framework sequence of the VH of SEQ ID NO: 39; and the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 40, and a framework sequence that is at least 95%, 96%, 97%, 98% or 99%, particularly at least 95% or at least 98% identical to the framework sequence of the VL of SEQ ID NO: 40.

[0202] In one aspect, the VH comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 31, and a framework that is at least 95%, 96%, 97%, 98% or 99% identical to the framework sequences of the VH of SEQ ID NO: 31. In one aspect, the VH comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 31, and a framework that is at least 95% identical to the framework sequences of the VH of SEQ ID NO: 31. In another aspect, the VH comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 31, and a framework that is at least 98% identical to the framework sequences of the VH of SEQ ID NO: 31.

[0203] In one aspect, the VL comprises the light chain CDRs of the VL of SEQ ID NO: 32 and a framework that is at least 95%, 96%, 97%, 98% or 99% identical to the framework sequence of the VL of SEQ ID NO: 32. In one aspect, the VL comprises the light chain CDR sequences of the VL of SEQ ID NO: 32 and a framework that is at least 95% identical to the framework sequence of the VL of SEQ ID NO: 32. In another aspect, the VL comprises the light chain CDR sequences of the VL of SEQ ID NO: 32 and a framework that is at least 98% identical to the framework sequence of the VL of SEQ ID NO: 32.

[0204] In one aspect, the present invention provides an antibody that binds to HLA-A2 / MAGE-A4, wherein the antibody comprises a first antigen-binding domain, the first antigen-binding domain comprising a VH sequence and a VL sequence, the VH sequence being as described in any of the aspects provided above, and the VL sequence being as described in any of the aspects provided above.

[0205] In one aspect, the antibody comprises a human constant region. In one aspect, the antibody is an immunoglobulin molecule comprising a human constant region, in particular an IgG class immunoglobulin molecule comprising a human CH1, CH2, CH3 and / or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NO: 79 and SEQ ID NO: 80 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 81 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In one aspect, the antibody comprises a light chain constant region comprising 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: 79 or SEQ ID NO: 80, in particular the amino acid sequence of SEQ ID NO: 79. In one aspect, the antibody comprises a heavy chain constant region comprising 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: 81. In particular, the heavy chain constant region may comprise amino acid mutations in the Fc domain as described herein.

[0206] In one aspect, the first antigen-binding domain comprises a human constant region. In one aspect, the first antigen-binding moiety is a Fab molecule comprising a human constant region, particularly a human CH1 and / or CL domain. In one aspect, the first antigen-binding domain comprises a light chain constant region, the amino acid sequence included in the light chain constant region, and SEQ ID NO: 79 or SEQ ID NO: 80 amino acid sequence, particularly SEQ ID NO: 79 amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical. Specifically, the light chain constant region can include amino acid mutations under "charge modification" as described herein and / or if in a cross Fab molecule, the deletion or substitution of one or more (particularly two) N-terminal amino acids can be included. In some aspects, the first antigen-binding domain comprises a heavy chain constant region, the heavy chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 81. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise amino acid mutations under "charge modification" as described herein.

[0207] In one aspect, the antibody is a monoclonal antibody.

[0208] In one aspect, the antibody is an IgG, particularly an IgG1 antibody. In one aspect, the antibody is a full-length antibody.

[0209] In another aspect, the antibody is an antibody fragment selected from the group consisting of an Fv molecule, a scFv molecule, a Fab molecule and a F(ab')2 molecule; in particular a Fab molecule. In another aspect, the antibody fragment is a diabody, a triabody or a tetrabody.

[0210] In one aspect, the first antigen-binding domain is a Fab molecule. In a preferred aspect, the first antigen-binding domain is a conventional Fab molecule. In alternative aspects, the first antigen-binding domain is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain replace each other or the constant domains CL and CH1 replace each other, particularly the variable domains VL and VH replace each other (i.e., the first antigen-binding domain is a cross Fab molecule).

[0211] In another aspect, the antibody according to any of the above aspects may incorporate the features described below in Section II A.1.-10, alone or in combination.

[0212] In preferred aspects, the antibody comprises an Fc domain, particularly an IgG Fc domain, more particularly an IgG1 Fc domain. In one aspect, the Fc domain is a human Fc domain. In one aspect, the Fc domain is an IgG1 Fc domain. The Fc domain is composed of a first subunit and a second subunit, and may incorporate any of the features described below for Fc domain variants (II. Section A.10.) either alone or in combination.

[0213] In another preferred aspect, the antibody comprises a second antigen-binding domain that binds to a second antigen (ie, the antibody is a multispecific antibody, as further described below (Section II.A.9.)).

[0214] 1. Antibody fragments

[0215] In certain aspects, the antibodies provided herein are antibody fragments.

[0216] In one aspect, the antibody fragment is a Fab', Fab'-SH or F(ab')2 molecule, in particular a Fab molecule as described herein. A "Fab' molecule" differs from a Fab molecule in that the Fab' fragment has residues added to the carboxyl terminus of the CH1 domain, which residues include one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' molecule in which the cysteine ​​residues of the constant domains have free sulfhydryl groups. Pepsin treatment produces a F(ab')2 molecule that has two antigen binding sites (two Fab molecules) and a portion of the Fc region.

[0217] In another aspect, the antibody fragment is a diabody, a triabody, or a tetrabody. A "diabody" is an antibody fragment 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). Hudson et al. also describe triabodies and tetrabodies in Nat. Med. 9:129-134 (2003).

[0218] In another aspect, the antibody fragment is a single-chain Fab molecule. A "single-chain Fab molecule" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In particular, the linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab molecule is stabilized via a natural disulfide bond between the CL domain and the CH1 domain. Furthermore, these single-chain Fab molecules can be further stabilized by generating interchain disulfide bonds via the insertion of cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0219] On the other hand, the antibody fragment is a single-chain variable fragment (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the heavy chain variable domain (VH) and the light chain variable domain (VL) of an antibody, connected by a linker. In particular, the linker is a short polypeptide of 10 to about 25 amino acids, and is generally rich in glycine to obtain flexibility, and rich in serine or threonine to obtain solubility, and the N-terminus of VH can be connected to the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of a linker, the protein still retains the specificity of the original antibody. For review of scFv fragments, see, e.g., Plückthun in The harmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patents 5,571,894 and 5,587,458.

[0220] On the other hand, the antibody fragment is a single domain antibody. A "single domain antibody" is an antibody fragment that contains all or part of the heavy chain variable domain of an antibody or all or part of the light chain variable domain of an antibody. In certain aspects, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516B1).

[0221] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and recombinant production by recombinant host cells (eg, E. coli), as described herein.

[0222] 2. Humanized Antibodies

[0223] In some aspects, the antibody provided herein is a humanized antibody. Generally, non-human antibodies are humanized to reduce immunogenicity to people, while retaining the specificity and avidity of the parent non-human antibody. Generally, humanized antibodies comprise one or more variable domains, wherein CDR (or its part) is derived from non-human antibodies, and FR (or its part) is derived from human antibody sequence. Humanized antibodies optionally also will comprise at least a portion of human constant region. In some aspects, some FR residues in the humanized antibody are replaced by corresponding residues from non-human antibodies (for example, the antibody from which CDR residues are derived), for example, to recover or improve antibody specificity or avidity.

[0224] Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" method for FR shuffling).

[0225] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (199 3)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0226] 3. Human Antibodies

[0227] In certain aspects, the antibodies provided herein are human antibodies. Various techniques known in the art can be used to produce human antibodies. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol. 5: 368-74 (2001) and Lonberg, Curr Opin Immunol. 20: 450-459 (2008).

[0228] Human antibodies can be prepared in the following manner: an immunogen is administered to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic stimulation. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or exists outside the chromosome of the animal or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, description of XENOMOUSE TM US Patent Nos. 6,075,181 and 6,150,584; Description US Patent No. 5,770,429; describes KM US Patent No. 7,041,870, and describes Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0229] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include, for example, those described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0230] Human antibodies can also be produced by isolating variable domain sequences selected from human phage display libraries. Such variable domain sequences can then be combined with expected human constant domains. The technology for selecting human antibodies from antibody libraries is described below.

[0231] 4. Antibodies from the library

[0232] In some aspects, the antibodies provided herein are derived from libraries. Antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies with one or more desired activities. Methods for screening combinatorial libraries are reviewed in, for example, Lerner et al., Nature Reviews 16: 498-508 (2016). For example, various methods are known in the art for generating phage display libraries and screening such libraries to obtain antibodies with desired binding characteristics. Such methods are reviewed, for example, in Frenzel et al., mAbs 8: 1177-1194 (2016); Bazan et al., Human Vaccines and Immunotherapeutics 8: 1817-1828 (2012), and Zhao et al., Critical Reviews in Biotechnology 36: 276-289 (2016), and in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ 2001), and Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0233] In certain phage display methods, the repertoire of VH and VL genes is individually cloned by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phage, as described in Winter et al., Annual Review of Immunology 12:433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, an initial library (e.g., from humans) can be cloned to provide a single source of antibodies for a variety of non-self and self antigens without any immunization, as described by Griffiths et al. in EMBO Journal 12:725-734 (1993). In addition, natural libraries are synthesized by cloning unrearranged V gene segments from stem cells; and using PCR primers containing random sequences to encode highly variable CDR3 regions and complete in vitro rearrangement, as described by Hoogenboom and Winter in Journal of Molecular Biology 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent Nos. 5,750,373; 7,985,840; 7,785,903 and 8,679,490 and U.S. Patent Publication Nos. 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936.

[0234] Other examples of methods for screening combinatorial libraries of antibodies with one or more desired activities known in the art include ribosome and mRNA display, and methods for displaying antibodies and selecting antibodies to bacteria, mammalian cells, insect cells or yeast cells. Methods for yeast surface display are summarized in, for example, Scholler et al., Methods in Molecular Biology 503:135-56 (2012) and Cherf et al., Methods in Molecular biology 1319:155-175 (2015) and Zhao et al., Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described in, for example, He et al., Nucleic Acids Research 25:5132-5134 (1997) and Hanes et al., PNAS 94:4937-4942 (1997).

[0235] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0236] 5. Glycosylation variants

[0237] In certain aspects, the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation. Addition or deletion of glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0238] In some aspects, the oligosaccharide in the antibody of the present invention can be modified to produce an antibody having a CH2 domain. In some aspects, the oligosaccharide in the antibody of the present invention can be modified to produce an antibody having a CH2 domain. In some aspects, the oligosaccharide in the antibody of the present invention can be modified to produce an antibody having a CH2 domain. In some aspects, the oligosaccharide in the antibody of the present invention can be modified to produce an antibody having a CH2 domain. In some aspects, the oligosaccharide in the antibody of the present invention can be modified to produce an antibody having a CH2 domain. In some aspects, the oligosaccharide in the antibody of the present invention can be modified to produce an antibody having a CH2 domain.

[0239] In one aspect, antibody variants are provided that have non-fucosylated oligosaccharides, i.e., oligosaccharide structures that lack (directly or indirectly) fucose attached to the Fc region. Such non-fucosylated oligosaccharides (also referred to as "defucosylated" oligosaccharides) are particularly N-linked oligosaccharides that lack the fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure. In one aspect, antibody variants are provided that have an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to a native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., the absence of fucosylated oligosaccharides). The percentage of non-fucosylated oligosaccharides, as described, for example, in WO 2006 / 082515, as measured by MALDI-TOF mass spectrometry, is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g., complex, hybrid, and high mannose structures) linked to Asn 297. Asn 297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations in antibodies, Asn 297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such antibodies having an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, US 2003 / 0157108; US 2004 / 0093621.

[0240] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108; and WO 2004 / 056312, particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2004 / 056312). 2003 / 085107), or cells with reduced or abolished GDP-fucose synthesis or transporter activity (see, e.g., US2004259150, US2005031613, US2004132140, US2004110282).

[0241] On the other hand, antibody variants provide bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. As described above, such antibody variants can have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342; WO 2004 / 065540, WO 2003 / 011878.

[0242] Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0243] 6. Cysteine-engineered antibody variants

[0244] In certain aspects, it may be desirable to generate antibodies with engineered cysteine ​​residues, such as THIOMAB. TM Antibodies, wherein one or more residues of the antibody are replaced by cysteine ​​residues. In preferred aspects, the substituted residues are present in accessible sites of the antibody. As further described herein, by replacing those residues with cysteine, reactive thiol groups are positioned in accessible sites of the antibody, and can be used to conjugate the antibody with other moieties (such as drug moieties or linker-drug moieties) to produce immunoconjugates. Cysteine ​​engineered antibodies can be produced as described in, for example, U.S. Patent Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130 or WO 2016040856.

[0245] 7. Antibody derivatives

[0246] In some aspects, the antibody provided herein can be further modified so that it contains other non-protein parts known in the art and easily obtainable. Parts suitable for antibody derivatization include but are not limited to water-soluble polymers. The non-limiting example of water-soluble polymers includes but is not limited to polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly-(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (such as glycerol), polyvinyl alcohol and their mixture. Due to its stability in water, polyethylene glycol propionaldehyde can have advantages in manufacturing. Polymer can have any molecular weight, and can have side chains or not. The number of the polymer attached to the antibody is variable, and if more than one polymer is attached, they can be identical or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0247] 8. Immunoconjugates

[0248] The invention also provides immunoconjugates comprising an anti-HLA-A2 / MAGE-A4 antibody herein conjugated (chemically bonded) to one or more therapeutic agents, such as a cytotoxic agent, a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope.

[0249] On the one hand, immunoconjugates are antibody-drug conjugates (ADCs), in which an antibody is conjugated to one or more therapeutic agents. A linker is typically used to connect the antibody to one or more therapeutic agents. An overview of ADC technology is provided in Pharmacol Review 68:3-19 (2016), which includes examples of therapeutic agents, drugs, and linkers.

[0250] In another aspect, the immunoconjugate comprises an antibody of the invention conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelatin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[0251] In another aspect, immunoconjugates include antibodies of the invention conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes can be used to prepare radioconjugates. Examples include At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 , Pb 212 When the radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, e.g., Tc 99m or I 123 , or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as I 123 , I 131 、In 111 、F 19 、C 13 、N 15 , O 17 , gadolinium, manganese, or iron.

[0252] Conjugates of the antibody and cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that promotes the release of the cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0253] The immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates prepared with cross-linking agents including, but not limited to, commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate).

[0254] 9. Multispecific Antibodies

[0255] In some aspects, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity for at least two different antigenic determinants (e.g., two different proteins, or two different epitopes on the same protein). In some aspects, multispecific antibodies have three or more binding specificities. In some aspects, one of the binding specificities is for HLA-A2 / MAGE-A4, and the other specificity is for any other antigen. In some aspects, multispecific antibodies can bind to two (or more) different epitopes of HLA-A2 / MAGE-A4. Multispecific (e.g., bispecific) antibodies can also be used to localize cytotoxic agents or cells to cells expressing HLA-A2 / MAGE-A4. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0256] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, 305:537 (1983)) and “knob-in-hole” engineering (see, e.g., U.S. Patent 5,731,168, and Atwell et al., J. Mol. Biol., 270:26 (1997)). Multispecific antibodies can also be prepared by engineering electrostatic manipulation effects for preparing antibody Fc-heterodimer molecules (see, e.g., WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to create bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); using common light chain technology to avoid light chain mispairing problems (see, e.g., WO 98 / 50431); using "diabody" technology for preparing bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described in Tutt et al., J. Immunol. 147:60 (1991).

[0257] Also included herein are engineered antibodies with three or more antigen binding sites, including, for example, "octopus antibodies" or DVD-Igs (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. Multispecific antibodies or antigen-binding fragments thereof also include "dual-action FAbs" or "DAFs," which comprise an antigen binding site that binds to CD3 and another different antigen or to two different epitopes of CD3 (see, for example, US 2008 / 0069820 and WO 2015 / 095539).

[0258] Multispecific antibodies can also be provided in an asymmetric format, where domains are crossed in one or more binding arms with the same antigen specificity (so-called "CrossMab" technology), i.e., by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253), or complete Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299, see also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interface to direct correct Fab pairing. See, e.g., WO 2016 / 172485.

[0259] Various other molecular formats of multispecific antibodies are known in the art and are encompassed herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

[0260] A specific type of multispecific antibody also included herein is a bispecific antibody that is designed to simultaneously bind to a surface antigen on a target cell (e.g., a tumor cell) and an activation-invariant component of the T cell receptor (TCR) complex (such as CD3) for retargeting T cells to kill the target cell. Thus, in preferred aspects, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies, wherein one of the binding specificities is for HLA-A2 / MAGE-A4 and the other is for an activating T cell antigen, particularly CD3.

[0261] Examples of bispecific antibody formats that can be used for this purpose include, but are not limited to, so-called "BiTE" (bispecific T-cell engager) molecules in which two scFv molecules are fused via a flexible linker (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261, and WO 2008 / 119567; Nagorsen and Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies ("TandAb"; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); "DART" (dual affinity retargeting) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which are fully hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Specific T cell bispecific antibody formats encompassed herein are described in WO 2013 / 026833; WO 2013 / 026839; WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0262] Preferred aspects of the multispecific antibodies of the present invention are described below.

[0263] In one aspect, the invention provides an antibody that binds to HLA-A2 / MAGE-A4, comprising a first antigen-binding domain that binds to HLA-A2 / MAGE-A4 as described herein, and comprising a second antigen-binding domain that binds to a second antigen (and optionally a third antigen-binding domain that binds to HLA-A2 / MAGE-A4).

[0264] According to a preferred aspect of the present invention, the antigen binding domain contained in the antibody is a Fab molecule (i.e., an antigen binding domain consisting of a heavy chain and a light chain, each antigen binding domain comprising a variable domain and a constant domain). In one aspect, the first antigen binding domain, the second antigen binding domain, and / or the third antigen binding domain when present are Fab molecules. In one aspect, the Fab molecules are human. On the other hand, the Fab molecules are humanized. On the other hand, the Fab molecules comprise human heavy and light chain constant domains.

[0265] Preferably, at least one antigen-binding domain in the antigen-binding domains is a cross Fab molecule. This modification reduces the mispairing of the heavy chain and light chain from different Fab molecules, thereby improving the yield and purity of the (multi-specific) antibody of the present invention in recombinant production. In the preferred cross Fab molecules that can be used for being included in the (multi-specific) antibody of the present invention, the variable domains (VL and VH, respectively) of the Fab light chain and the Fab heavy chain are exchanged. However, even if this domain exchange is carried out, due to the so-called Bence Jones type interaction between the heavy chain and the light chain of the mispairing, the preparation of (multi-specific) antibodies may include some by-products (see Schaefer et al., PNAS, 108 (2011) 11187-11191). In order to further reduce the mispairing of heavy and light chains from different Fab molecules and thereby improve the purity and yield of the desired (multispecific) antibodies, oppositely charged amino acids can be introduced at specific amino acid positions of the CH1 and CL domains of either the Fab molecule binding to the first antigen (HLA-A2 / MAGE-A4) or the Fab molecule binding to the second antigen (e.g. CD3), as further described herein. In conventional Fab molecules comprised in a (multispecific) antibody (such as, for example, Figure 1 shown in AC, GJ) or in a VH / VL crossover Fab molecule comprised in a (multispecific) antibody (such as e.g. Figure 1 In a preferred aspect, the charge modification is performed in a conventional Fab molecule comprised in a (multispecific) antibody (which in a preferred aspect binds to the first antigen, i.e. HLA-A2 / MAGE-A4).

[0266] In a preferred aspect according to the present invention, the (multispecific) antibody is capable of simultaneously binding to a first antigen (i.e., HLA-A2 / MAGE-A4) and a second antigen (e.g., an activating T cell antigen, such as CD3). In one aspect, the (multispecific) antibody is capable of crosslinking T cells and target cells by simultaneously binding to the second antigen (e.g., an activating T cell antigen, such as CD3) and HLA-A2 / MAGE-A4. In an even more preferred aspect, such simultaneous binding results in lysis of target cells, in particular tumor cells expressing the target cell antigen (i.e., HLA-A2 / MAGE-A4). In one aspect, such simultaneous binding results in activation of T cells. In other aspects, such simultaneous binding results in a cellular response of T lymphocytes, in particular cytotoxic T lymphocytes, selected from the group consisting of: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In one aspect, binding of the (multispecific) antibody to the second antigen (e.g., an activating T cell antigen, such as CD3) without simultaneous binding to the target cell antigen (i.e., HLA-A2 / MAGE-A4) does not result in T cell activation.

[0267] In one aspect, the (multispecific) antibody is capable of redirecting the cytotoxic activity of T cells to target cells. In a preferred aspect, the redirection is independent of MHC-mediated peptide antigen presentation by the target cells and / or the specificity of the T cells.

[0268] Preferably, the T cells according to any aspect of the invention are cytotoxic T cells. In some aspects, the T cells are CD4 + or CD8 + T cells, especially CD8 + T cells.

[0269] a) First (and third) antigen binding domain

[0270] The (multispecific) antibodies of the present invention comprise at least one antigen-binding domain (first antigen-binding domain) that binds to HLA-A2 / MAGE-A4. In a preferred aspect, HLA-A2 / MAGE-A4 is human HLA-A2 / MAGE-A4. In a specific aspect, HLA-A2 / MAGE-A4 is HLA-A2 / MAGE-A4. p230-239 .

[0271] The first antigen binding domain is capable of directing the (multispecific) antibody to the target site, for example to a specific type of tumor cell expressing HLA-A2 / MAGE-A4.

[0272] In a preferred aspect, the (multispecific) antibody comprises two antigen binding domains that bind to HLA-A2 / MAGE-A4. In one aspect, the (multispecific) antibody provides bivalent binding to HLA-A2 / MAGE-A4.

[0273] In one aspect, the antigen-binding domain that binds to HLA-A2 / MAGE-A4 is an antibody fragment selected from the group consisting of an Fv molecule, a scFv molecule, a Fab molecule, and a F(ab')2 molecule. In a preferred aspect, the antigen-binding domain that binds to HLA-A2 / MAGE-A4 is a Fab molecule.

[0274] In certain aspects, the (multispecific) antibody comprises two antigen-binding domains, particularly Fab molecules, that bind to HLA-A2 / MAGE-A4. In a specific aspect, preferably all of these antigen-binding domains are identical, i.e., they have the same molecular format (e.g., conventional or cross-linked Fab molecules) and comprise the same amino acid sequence (including the same amino acid substitutions in the CH1 and CL domains, as described herein (if any)). In one aspect, the (multispecific) antibody comprises no more than two antigen-binding domains, particularly Fab molecules, that bind to HLA-A2 / MAGE-A4.

[0275] In a preferred aspect, the antigen binding domain that binds to HLA-A2 / MAGE-A4 is a conventional Fab molecule. In such aspects, the antigen binding domain that binds to the second antigen is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy and light chains are exchanged / replaced with each other or the constant domains CH1 and CL are exchanged / replaced with each other.

[0276] In alternative aspects, the antigen binding domain that binds to HLA-A2 / MAGE-A4 is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy and light chains are exchanged / replaced with each other or the constant domains CH1 and CL are exchanged / replaced with each other. In such aspects, the antigen binding domain that binds to the second antigen is a conventional Fab molecule.

[0277] In one aspect, the first antigen-binding domains (and the third antigen-binding domains when present) include human constant regions. In one aspect, the first antigen-binding domains (and the third antigen-binding domains when present) are Fab molecules comprising human constant regions, particularly human CH1 and / or CL domains. Exemplary sequences of human constant domains are given in SEQ ID NO: 79 and SEQ ID NO: 80 (people κ and λ CL domains, respectively) and SEQ ID NO: 81 (human IgG1 heavy chain constant domains CH1-CH2-CH3). In one aspect, the first antigen-binding domains (and the third antigen-binding domains when present) include a light chain constant region comprising an amino acid sequence of at least about 95%, 96%, 97%, 98%, 99% or 100% the same as SEQ ID NO: 79 or SEQ ID NO: 80, particularly SEQ ID NO: 79. In some aspects, the first antigen-binding domain (and the third antigen-binding domain when present) comprises a heavy chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the CH1 domain sequence in the amino acid sequence of SEQ ID NO: 81. In some aspects, the light chain constant region (particularly the CH1 domain) may comprise an amino acid mutation under "charge modification" as described herein and / or may comprise a deletion or substitution of one or more (particularly two) N-terminal amino acids if in a cross-Fab molecule. In some aspects, the first antigen-binding domain (and the third antigen-binding domain when present) comprises a heavy chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the CH1 domain sequence in the amino acid sequence of SEQ ID NO: 81. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise an amino acid mutation under "charge modification" as described herein.

[0278] b) Second antigen binding domain

[0279] In certain aspects, the (multispecific) antibodies of the present invention comprise at least one antigen binding domain, particularly a Fab molecule, that binds to a second antigen. The second antigen is preferably not HLA-A2 / MAGE-A4, i.e., different from HLA-A2 / MAGE-A4. In one aspect, the second antigen is an antigen expressed on cells different from HLA-A2 / MAGE-A4 (e.g., expressed on cells other than target cells such as tumor cells). In one aspect, the second antigen is a T cell antigen, particularly an activating T cell antigen. In specific aspects, the second antigen is CD3. In preferred aspects, CD3 is human CD3 (SEQ ID NO: 76) or cynomolgus monkey CD3 (SEQ ID NO: 77), most particularly human CD3. In one aspect, the second antigen binding domain cross-reacts (i.e., specifically binds) with human and cynomolgus monkey CD3. In some aspects, CD3 is the epsilon subunit of CD3 (CD3 epsilon).

[0280] In a preferred aspect, the (multispecific) antibody comprises no more than one antigen binding domain that binds to a second antigen (e.g., an activating T cell antigen, such as CD3). In one aspect, the (multispecific) antibody provides monovalent binding to a second antigen (e.g., an activating T cell antigen, such as CD3).

[0281] In one aspect, the antigen binding domain that binds to the second antigen is an antibody fragment selected from the group consisting of an Fv molecule, a scFv molecule, a Fab molecule, and a F(ab')2 molecule. In a preferred aspect, the antigen binding domain that binds to the second antigen is a Fab molecule.

[0282] In preferred aspects, the antigen-binding domain that binds to a second antigen (e.g., an activating T cell antigen such as CD3) is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy and light chains are exchanged / replaced with each other or the constant domains CH1 and CL are exchanged / replaced with each other. In such aspects, the antigen-binding domain that binds to HLA-A2 / MAGE-A4 is preferably a conventional Fab molecule. In aspects where there is more than one antigen-binding domain that binds to HLA-A2 / MAGE-A4 in a (multispecific) antibody, particularly a Fab molecule, the antigen-binding domain that binds to the second antigen is preferably a crossover Fab molecule, and the antigen-binding domain that binds to HLA-A2 / MAGE-A4 is a conventional Fab molecule.

[0283] In alternative aspects, the antigen-binding domain that binds to a second antigen (e.g., an activating T cell antigen such as CD3) is a conventional Fab molecule. In such aspects, the antigen-binding domain that binds to HLA-A2 / MAGE-A4 is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy and light chains are exchanged / replaced with each other or the constant domains CH1 and CL are exchanged / replaced with each other. In aspects where there is more than one antigen-binding domain, particularly a Fab molecule, that binds to a second antigen in a (multispecific) antibody, the antigen-binding domain that binds to HLA-A2 / MAGE-A4 is preferably a crossover Fab molecule, and the antigen-binding domain that binds to the second antigen is a conventional Fab molecule.

[0284] In a preferred aspect, the second antigen binding domain is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other or the constant domains CL and CH1 are replaced with each other, in particular the variable domains VL and VH are replaced with each other (i.e., according to such aspects, the second antigen binding domain is a crossover Fab molecule, wherein the variable or constant domains of the Fab light chain and the Fab heavy chain are exchanged). In one such aspect, the first (and third, if any) antigen binding domain is a conventional Fab molecule.

[0285] In a preferred aspect, the second antigen is CD3, as described above.

[0286] In one aspect, the second antigen binding domain comprises

[0287] (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 59, HCDR 2 of SEQ ID NO: 60, and HCDR 3 of SEQ ID NO: 61, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 62, LCDR 2 of SEQ ID NO: 63, and LCDR 3 of SEQ ID NO: 64; or

[0288] (ii) VH and a light chain variable region (VL), wherein the VH comprises HCDR 1 of SEQ ID NO: 51, HCDR 2 of SEQ ID NO: 52, and HCDR 3 of SEQ ID NO: 53, and the light chain variable region (VL) comprises a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 54, LCDR 2 of SEQ ID NO: 55, and LCDR 3 of SEQ ID NO: 56.

[0289] In one aspect, the second antigen-binding domain is (or is derived from) a humanized antibody. In one aspect, the second antigen-binding domain is a humanized antigen-binding domain (i.e., the antigen-binding domain of a humanized antibody). In one aspect, the VH and / or VL of the second antigen-binding domain are humanized variable regions.

[0290] In one aspect, the VH and / or VL of the second antigen binding domain comprises an acceptor human framework, eg, a human immunoglobulin framework or a human consensus framework.

[0291] In some aspects, the second antigen binding domain comprises

[0292] (i) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 59, HCDR 2 of SEQ ID NO: 60, and HCDR 3 of SEQ ID NO: 61, and the light chain variable region comprises a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 62, LCDR 2 of SEQ ID NO: 63, and LCDR 3 of SEQ ID NO: 64.

[0293] In one aspect, the VH of the second antigen-binding domain comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of SEQ ID NO: 65. In one aspect, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 65. In one aspect, the VH comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 65. In one aspect, the VH comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 65. In certain aspects, the VH sequence that is at least 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the antibody comprising the sequence retains the ability to bind to the second antigen (e.g., an activated T cell antigen, such as CD3). In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 65. In some aspects, the substitution, insertion or deletion occurs in a region outside of the CDRs (i.e., in the FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 65. Optionally, the VH comprises the amino acid sequence of SEQ ID NO: 65, including post-translational modifications of the sequence.

[0294] In one aspect, the VL of the second antigen-binding domain comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of SEQ ID NO: 66. In one aspect, the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 66. In one aspect, the VL comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 66. In one aspect, the VL comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 66. In certain aspects, the VL sequence that is at least 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the antibody comprising the sequence retains the ability to bind to the second antigen (e.g., an activated T cell antigen, such as CD3). In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 66. In some aspects, the substitution, insertion or deletion occurs in a region outside of the CDRs (i.e., in the FRs). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 66. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 66, including post-translational modifications of that sequence.

[0295] In one aspect, the VH of the second antigen binding domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 65, and the VL of the second antigen binding domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 66. In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 65, and the VL comprises the amino acid sequence of SEQ ID NO: 66.

[0296] In another aspect, the second antigen binding domain comprises a VH comprising the sequence of SEQ ID NO: 65 and a VL comprising the sequence of SEQ ID NO: 66.

[0297] In another aspect, the second antigen binding domain comprises the VH sequence of SEQ ID NO:65 and the VL sequence of SEQ ID NO:66.

[0298] In another aspect, the second antigen binding domain comprises a VH comprising the heavy chain CDR sequence of the VH of SEQ ID NO: 65 and a VL comprising the light chain CDR sequence of the VL of SEQ ID NO: 66.

[0299] In another aspect, the second antigen binding domain comprises the HCDR1, HCDR2, and HCDR3 amino acid sequence of VH of SEQ ID NO: 65 and the LCDR1, LCDR2, and LCDR3 amino acid sequence of VL of SEQ ID NO: 66.

[0300] In one aspect, the VH of the second antigen binding domain comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 65, and a framework that is at least 95%, 96%, 97%, 98% or 99% identical to the framework sequences of the VH of SEQ ID NO: 65. In one aspect, the VH comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 65, and a framework that is at least 95% identical to the framework sequences of the VH of SEQ ID NO: 65. In another aspect, the VH comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 65, and a framework that is at least 98% identical to the framework sequences of the VH of SEQ ID NO: 65.

[0301] In one aspect, the VL of the second antigen binding domain comprises the light chain CDR sequences of the VL of SEQ ID NO: 66, and a framework that is at least 95%, 96%, 97%, 98%, or 99% identical to the framework sequences of the VL of SEQ ID NO: 66. In one aspect, the VL comprises the light chain CDR sequences of the VL of SEQ ID NO: 66, and a framework that is at least 95% identical to the framework sequences of the VL of SEQ ID NO: 66. In another aspect, the VL comprises the light chain CDR sequences of the VL of SEQ ID NO: 66, and a framework that is at least 98% identical to the framework sequences of the VL of SEQ ID NO: 66.

[0302] In some aspects, the second antigen binding domain comprises

[0303] (i) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 51, HCDR 2 of SEQ ID NO: 52, and HCDR 3 of SEQ ID NO: 53, and the light chain variable region comprises a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 54, LCDR 2 of SEQ ID NO: 55, and LCDR 3 of SEQ ID NO: 56.

[0304] In one aspect, the VH of the second antigen-binding domain comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of SEQ ID NO: 57. In one aspect, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57. In one aspect, the VH comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 57. In one aspect, the VH comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 57. In certain aspects, the VH sequence that is at least 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the antibody comprising the sequence retains the ability to bind to the second antigen (e.g., an activated T cell antigen, such as CD3). In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 57. In some aspects, the substitution, insertion or deletion occurs in a region outside of the CDRs (i.e., in the FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 57. Optionally, the VH comprises the amino acid sequence of SEQ ID NO: 57, including post-translational modifications of the sequence.

[0305] In one aspect, the VL of the second antigen-binding domain comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of SEQ ID NO: 58. In one aspect, the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In one aspect, the VL comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 58. In one aspect, the VL comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 58. In certain aspects, the VL sequence that is at least 95%, 96%, 97%, 98%, or 99% identical contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the antibody comprising the sequence retains the ability to bind to the second antigen (e.g., an activated T cell antigen, such as CD3). In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 58. In some aspects, the substitution, insertion or deletion occurs in a region outside of the CDRs (i.e., in the FRs). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 58. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 58, including post-translational modifications of that sequence.

[0306] In one aspect, the VH of the second antigen binding domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57, and the VL of the second antigen binding domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 58. In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 57, and the VL comprises the amino acid sequence of SEQ ID NO: 58.

[0307] In another aspect, the second antigen binding domain comprises a VH comprising the sequence of SEQ ID NO:57 and a VL comprising the sequence of SEQ ID NO:58.

[0308] In another aspect, the second antigen binding domain comprises the VH sequence of SEQ ID NO:57 and the VL sequence of SEQ ID NO:58.

[0309] In another aspect, the second antigen binding domain comprises a VH comprising the heavy chain CDR sequence of the VH of SEQ ID NO: 57 and a VL comprising the light chain CDR sequence of the VL of SEQ ID NO: 58.

[0310] In another aspect, the second antigen binding domain comprises the HCDR1, HCDR2, and HCDR3 amino acid sequence of VH of SEQ ID NO: 57 and the LCDR1, LCDR2, and LCDR3 amino acid sequence of VL of SEQ ID NO: 58.

[0311] In one aspect, the VH of the second antigen binding domain comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 57, and a framework that is at least 95%, 96%, 97%, 98% or 99% identical to the framework sequences of the VH of SEQ ID NO: 57. In one aspect, the VH comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 57, and a framework that is at least 95% identical to the framework sequences of the VH of SEQ ID NO: 57. In another aspect, the VH comprises the heavy chain CDR sequences of the VH of SEQ ID NO: 57, and a framework that is at least 98% identical to the framework sequences of the VH of SEQ ID NO: 57.

[0312] In one aspect, the VL of the second antigen binding domain comprises the light chain CDR sequences of the VL of SEQ ID NO: 58, and a framework that is at least 95%, 96%, 97%, 98%, or 99% identical to the framework sequences of the VL of SEQ ID NO: 58. In one aspect, the VL comprises the light chain CDR sequences of the VL of SEQ ID NO: 58, and a framework that is at least 95% identical to the framework sequences of the VL of SEQ ID NO: 58. In another aspect, the VL comprises the light chain CDR sequences of the VL of SEQ ID NO: 58, and a framework that is at least 98% identical to the framework sequences of the VL of SEQ ID NO: 58.

[0313] c) Charge modification

[0314] The (multispecific) antibodies of the present invention may comprise amino acid substitutions in the Fab molecules contained therein that are particularly effective in reducing mispairing of the light chain with an unmatched heavy chain (Bence-Jones type byproducts), which mispairing can occur in the production of Fab-based multispecific antibodies, wherein the bi / multispecific antigen-binding molecule has a VH / VL exchange in one of its binding arms (or multiple binding arms in the case where the molecule comprises more than two antigen-binding Fab molecules) (see also PCT Publication No. WO 2015 / 150447, in particular for examples therein, the entire contents of which are incorporated herein by reference). The ratio of desired (multispecific) antibodies to undesirable byproducts, in particular Bence Jones type byproducts that occur in multispecific antibodies with a VH / VL domain exchange in one of the binding arms of the (multispecific) antibody, can be improved by introducing oppositely charged amino acids at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modification").

[0315] Thus, in some aspects, in which the first and second antigen-binding domains (and the third antigen-binding domain when present) of the (multispecific) antibody are both Fab molecules, and in one of the antigen-binding domains (particularly the second antigen-binding domain), the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other,

[0316] i) in the constant domain CL of the first antigen-binding domain (and the third antigen-binding domain, when present), the amino acid at position 124 is substituted by a positively charged amino acid (according to Kabat numbering), and wherein in the constant domain CH1 of the first antigen-binding domain (and the third antigen-binding domain, when present), the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbering according to the Kabat EU index); or

[0317] ii) in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is substituted by a positively charged amino acid (according to Kabat numbering), and wherein in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (according to the Kabat EU index).

[0318] The (multispecific) antibody does not comprise the two modifications mentioned in i) and ii). The constant domains CL and CH1 of the antigen binding domain having the VH / VL exchange are not replaced with each other (ie remain unexchanged).

[0319] In a more specific aspect,

[0320] i) in the constant domain CL of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index); or

[0321] ii) in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0322] In one such aspect, in the constant domain CL of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 147 or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

[0323] In another aspect, in the constant domain CL of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0324] In a preferred aspect, in the constant domain CL of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index) and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

[0325] In a more preferred aspect, in the constant domain CL of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering) and the amino acid at position 123 is substituted by lysine (K) (according to Kabat numbering), and in the constant domain CH1 of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the Kabat EU index).

[0326] In an even more preferred aspect, in the constant domain CL of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the Kabat EU index).

[0327] In a preferred aspect, if the amino acid substitutions according to the above aspects are made in the constant domain CL and constant domain CH1 of the first antigen binding domain (and the third antigen binding domain when present), the constant domain CL of the first antigen binding domain (and the third antigen binding domain when present) is of the κ isotype.

[0328] Alternatively, the amino acid substitutions according to the above aspects may be made in the constant domain CL and constant domain CH1 of the second antigen-binding domain, rather than in the constant domain CL and constant domain CH1 of the first antigen-binding domain (and, if present, the third antigen-binding domain). In preferred aspects of this type, the constant domain CL of the second antigen-binding domain is of the kappa isotype.

[0329] Thus, in one aspect, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding domain, the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0330] In another aspect, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding domain, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index numbering).

[0331] In a further aspect, in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index) and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index).

[0332] In one aspect, in the constant domain CL of the second antigen binding domain, the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and 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 domain, the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the Kabat EU index).

[0333] On the other hand, in the constant domain CL of the second antigen-binding domain, the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering) and the amino acid at position 123 is substituted by arginine (R) (according to Kabat numbering), and in the constant domain CH1 of the second antigen-binding domain, the amino acid at position 147 is substituted by glutamic acid (E) (according to the Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (according to the Kabat EU index).

[0334] In a preferred aspect, the (multispecific) antibody of the invention comprises

[0335] (A) a first and, optionally, a third antigen-binding domain that binds to HLA-A2 / MAGE-A4;

[0336] wherein the first antigen binding domain (and the third antigen binding domain when present) is a conventional Fab molecule, and the conventional Fab molecule comprises

[0337] (i) a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0338] (ii) a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0339] (iii) a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0340] (iv) a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0341] (v) a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0342] (vi) a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30; and

[0343] wherein in the constant domain CL of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) (in a preferred aspect, is independently substituted by lysine (K) or arginine (R)) and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) (in a preferred aspect, is independently substituted by lysine (K) or arginine (R)); and in the constant domain CH1 of the second antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index) and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index);

[0344] as well as

[0345] (B) a second antigen-binding domain that binds to a second antigen (preferably CD3), wherein the second antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other.

[0346] d) Multispecific antibody formats

[0347] The (multispecific) antibodies according to the invention may have various configurations. Figure 1 described in .

[0348] In preferred aspects, the antigen binding domains comprised in the (multispecific) antibody are Fab molecules. In such aspects, the first, second, third antigen binding domains, etc., may be referred to herein as the first, second, third Fab molecules, etc., respectively.

[0349] In one aspect, the first antigen-binding domains and the second antigen-binding domains of (multi-specific) antibody are fused to each other, optionally fused via a peptide linker. In a preferred aspect, the first antigen-binding domains and the second antigen-binding domains are each a Fab molecule. In one such aspect, the first antigen-binding domains are fused to the N-terminal of the Fab heavy chain of the second antigen-binding domains at the C-terminal of the Fab heavy chain. In another such aspect, the second antigen-binding domains are fused to the N-terminal of the Fab heavy chain of the first antigen-binding domains at the C-terminal of the Fab heavy chain. In the aspect that (i) the first antigen-binding domains are fused to the N-terminal of the Fab heavy chain of the second antigen-binding domains at the C-terminal of the Fab heavy chain or (ii) the second antigen-binding domains are fused to the N-terminal of the Fab heavy chain of the first antigen-binding domains at the C-terminal of the Fab heavy chain, additionally the Fab light chain of the first antigen-binding domains and the Fab light chain of the second antigen-binding domains can optionally be fused to each other via a peptide linker.

[0350] have the ability to specifically bind to HLA-A2 / MAGE-A4 (e.g. Figure 1 A, D, G, H, K, L) are useful, particularly when antigen internalization is expected after binding of a high-affinity antigen-binding domain. In this case, the presence of more than one antigen-binding domain specific for HLA-A2 / MAGE-A4 may enhance internalization of the antigen, thereby reducing its availability.

[0351] However, in other cases it would be advantageous to have a (multispecific) antibody (such as a Fab molecule) comprising two or more antigen binding domains for HLA-A2 / MAGE-A4 (see Figure 1 B, 1C, 1E, 1F, 1I, 1J, 1M or 1N), for example, to optimize targeting to a target site or to allow cross-linking of target cell antigens.

[0352] Thus, in a preferred aspect, the (multispecific) antibody according to the invention comprises a third antigen binding domain.

[0353] In one aspect, the third antigen binding domain binds to HLA-A2 / MAGE-A4. In one aspect, the third antigen binding domain is a Fab molecule.

[0354] In one aspect, the third antigen binding domain is identical to the first antigen binding domain.

[0355] In some aspects, the third and first antigen-binding domains are each a Fab molecule and the third antigen-binding domains are identical to the first antigen-binding domains. Therefore, in these aspects, the first antigen-binding domains and the third antigen-binding domains comprise identical heavy chain and light chain amino acid sequences and have identical domain arrangement (i.e., conventional or crossover). In addition, in these aspects, the third antigen-binding domains comprise amino acid replacements identical to the first antigen-binding domains (if any). For example, amino acid replacements described herein as "charge modified" will be carried out in the constant domains CL and constant domain CH1 of the first and third antigen-binding domains. Alternatively, the amino acid replacements can be carried out in the constant domains CL and constant domain CH1 of the second antigen-binding domains (which are also Fab molecules in preferred aspects), but not in the constant domains CL and constant domain CH1 of the first and third antigen-binding domains.

[0356] The same as the first antigen-binding domains, the third antigen-binding domains are preferably conventional Fab molecules. However, the aspect that the first antigen-binding domains and the third antigen-binding domains are cross Fab molecules (and the second antigen-binding domains are conventional Fab molecules) is also considered. Therefore, in preferred aspects, the first antigen-binding domains and the third antigen-binding domains are each conventional Fab molecules, and the second antigen-binding domains are cross Fab molecules as described herein, i.e., such Fab molecules, wherein in the Fab molecule, the variable domains VH and VL or constant domains CL and CH1 of Fab heavy chain and light chain exchange / replace each other. In other aspects, the first antigen-binding domains and the third antigen-binding domains are each cross Fab molecules and the second antigen-binding domains are conventional Fab molecules.

[0357] If a third antigen binding domain is present, in preferred aspects, the first and third antigen binding domains bind to HLA-A2 / MAGE-A4, and the second antigen domain binds to a second antigen (e.g., an activating T cell antigen such as CD3).

[0358] In a preferred aspect, the (multispecific) antibody of the present invention comprises an Fc domain composed of a first subunit and a second subunit. The first subunit and the second subunit of the Fc domain are capable of stably associating.

[0359] The (multispecific) antibodies according to the present invention can have different configurations, i.e. the first antigen binding domain, the second antigen binding domain (and optionally the third antigen binding domain) can be fused to each other and to the Fc domain in different ways. The components can be fused directly to each other, or preferably fused via one or more suitable peptide linkers. When a Fab molecule is fused to the N-terminus of a subunit of the Fc domain, the fusion is typically via an immunoglobulin hinge region.

[0360] In some aspects, the first antigen-binding domain and the second antigen-binding domain are each a Fab molecule, and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In such aspects, the first antigen-binding domain can be fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain at the C-terminus of the Fab heavy chain or fused to the N-terminus of another subunit of the Fc domain. In preferred such aspects, the first antigen-binding domain is a conventional Fab molecule, and the second antigen-binding domain is a cross Fab molecule as described herein, i.e., such a Fab molecule, wherein in the Fab molecule, the variable domains VH and VL or constant domains CL and CH1 of the Fab heavy chain and light chain exchange / replace each other. In other such aspects, the first antigen-binding domain is a cross Fab molecule, and the second antigen-binding domain is a conventional Fab molecule.

[0361] In one aspect, the first antigen binding domain and the second antigen binding domain are each a Fab molecule, the second antigen binding domain is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain, and the first antigen binding domain is fused to the N-terminus of the Fab heavy chain of the second antigen binding domain at the C-terminus of the Fab heavy chain. In a specific aspect, the (multispecific) antibody is essentially composed of the first and second Fab molecules, the Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, and the second Fab molecule is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. This configuration is Figure 1 G and 1K (the second antigen binding domain in these examples is a VH / VL crossover Fab molecule). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.

[0362] In another aspect, the first antigen binding domain and the second antigen binding domain are each a Fab molecule, and the first antigen binding domain and the second antigen binding domain are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. In a specific aspect, the (multispecific) antibody essentially consists of a first Fab molecule and a second Fab molecule, an Fc domain consisting of a first subunit and a second subunit, and optionally one or more peptide linkers, wherein the first Fab molecule and the second Fab molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. This configuration is Figure 1 A and 1D are schematically depicted (in these examples, the second antigen binding domain is a VH / VL cross Fab molecule and the first antigen binding domain is a conventional Fab molecule). The first Fab molecule and the second Fab molecule can be fused to the Fc domain directly or through a peptide linker. In preferred aspects, the first Fab molecule and the second Fab molecule are each fused to the Fc domain via an immunoglobulin hinge region. In specific aspects, the immunoglobulin hinge region is a human IgG1 hinge region, particularly when the Fc domain is an IgG1 Fc domain.

[0363] In some aspects, the first antigen-binding domains and the second antigen-binding domains are each a Fab molecule, and the first antigen-binding domains are fused at the C-terminus of the Fab heavy chain and the N-terminus of the first or second subunit of the Fc domains. In such aspects, the second antigen-binding domains can be fused to the N-terminus of the Fab heavy chain of the second antigen-binding domains at the C-terminus of the Fab heavy chain or (as described above) are fused to the N-terminus of another subunit of the Fc domains. In such aspects preferably, the first antigen-binding domains are conventional Fab molecules, and the second antigen-binding domains are cross Fab molecules as described herein, i.e., such Fab molecules, wherein in the Fab molecule, the variable domains VH and VL or constant domains CL and CH1 of Fab heavy chain and light chain exchange / replace each other. In other such aspects, the first antigen-binding domains are cross Fab molecules, and the second antigen-binding domains are conventional Fab molecules.

[0364] In one aspect, the first antigen binding domain and the second antigen binding domain are each a Fab molecule, the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the second antigen binding domain 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 domain. In a specific aspect, the (multispecific) antibody essentially consists of the first and second Fab molecules, the Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. This configuration is Figure 1 H and 1 L (in these examples, the second antigen binding domain is a VH / VL crossover Fab molecule and the first antigen binding domain is a conventional Fab molecule). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can additionally be fused to each other.

[0365] In some aspects, the third antigen-binding domain, in particular the third Fab molecule, is fused to the N-terminus of the first or second subunit of the Fc domain at the C-terminus of the Fab heavy chain. In preferred aspects of this type, the first and third antigen-binding domains are each a conventional Fab molecule, and the second antigen-binding domain is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged / replaced with each other in the Fab molecule. In other aspects of this type, the first and third antigen-binding domains are each a crossover Fab molecule and the second antigen-binding domain is a conventional Fab molecule.

[0366] In preferred such aspects, the second antigen binding domain and the third antigen binding domain are each fused to the N-terminus of one of the subunits of the Fc domain at the C-terminus of the Fab heavy chain, and the first antigen binding domain is fused to the N-terminus of the Fab heavy chain of the second antigen binding domain at the C-terminus of the Fab heavy chain. In specific aspects, (multi-specific) antibodies are essentially composed of the first, second and third Fab molecules, the Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused to the N-terminus of the Fab heavy chain of the second Fab molecule at the C-terminus of the Fab heavy chain, and the second Fab molecule is fused to the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and wherein the third Fab molecule is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain. This configuration is schematically described in Figure 1B and IE (in these examples, the second antigen binding domain is a VH / VL crossover Fab molecule and the first and third antigen binding domains are conventional Fab molecules), and Figure 1 J and 1N (in these examples, the second antigen binding domain is a conventional Fab molecule, and the first and third antigen binding domains are VH / VL cross Fab molecules). The second and third Fab molecules can be fused to the Fc domain directly or through a peptide linker. In a preferred aspect, the second Fab molecule and the third Fab molecule are each fused to the Fc domain via an immunoglobulin hinge region. In a specific aspect, the immunoglobulin hinge region is a human IgG1 hinge region, particularly when the Fc domain is an IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can additionally be fused to each other.

[0367] In another such aspect, the first antigen binding domain and the third antigen binding domain are each fused to the N-terminus of one of the subunits of the Fc domain at the C-terminus of the Fab heavy chain, and the second antigen binding domain is fused to the N-terminus of the Fab heavy chain of the first antigen binding domain at the C-terminus of the Fab heavy chain. In specific aspects, the (multi-specific) antibody is essentially composed of the first, second and third Fab molecules, the Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the first Fab molecule is fused to the first subunit of the Fc domain at the C-terminus of the Fab heavy chain, and wherein the third Fab molecule is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain. This configuration is schematically described in Figure 1 C and 1F (in these examples, the second antigen binding domain is a VH / VL crossover Fab molecule and the first and third antigen binding domains are conventional Fab molecules), and Figure 1 1 and 1M (in these examples, the second antigen binding domain is a conventional Fab molecule, the first antigen binding domain and the third antigen binding domain are VH / VL cross Fab molecules). The first and third Fab molecules can be fused to the Fc domain directly or through a peptide linker. In a preferred aspect, the first Fab molecule and the third Fab molecule are each fused to the Fc domain via an immunoglobulin hinge region. In a specific aspect, the immunoglobulin hinge region is a human IgG1 hinge region, particularly when the Fc domain is an IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can additionally be fused to each other.

[0368] In the configuration of a (multispecific) antibody, wherein the Fab molecule is fused to the N-terminus of each of the subunits of the Fc domain at the C-terminus of the Fab heavy chain via an immunoglobulin hinge region, the two Fab molecules, the hinge region, and the Fc domain essentially form an immunoglobulin molecule. In a preferred aspect, the immunoglobulin molecule is an IgG class immunoglobulin. In an even more preferred aspect, the immunoglobulin is an IgG1 subclass immunoglobulin. On the other hand, the immunoglobulin is an IgG4 subclass immunoglobulin. In another preferred aspect, the immunoglobulin is a human immunoglobulin. In other aspects, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one aspect, the immunoglobulin comprises a human constant region, particularly a human Fc region.

[0369] In some (multi-specific) antibodies of the present invention, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are fused to each other, optionally through a peptide linker. Depending on the configuration of the first and second Fab molecules, the Fab light chain of the first Fab molecule can be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule can be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. The fusion of the Fab light chains of the first and second Fab molecules further reduces the mispairing of unmatched Fab heavy and light chains and also reduces the number of plasmids required to express some (multi-specific) antibodies of the present invention.

[0370] The antigen binding domain can be fused to the Fc domain directly or via a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n 、(SG4) n 、(G4S) n or G4 (SG4) n Peptide linker. "n" is typically an integer from 1 to 10, typically from 2 to 4. In one aspect, the peptide linker is at least 5 amino acids in length, in one aspect, from 5 to 100 amino acids in length, in another aspect, from 10 to 50 amino acids in length. In one aspect, the peptide linker is (GxS) n or (GxS) n G m, wherein G = glycine, S = serine, and (x = 3, n = 3, 4, 5 or 6, and m = 0, 1, 2 or 3) or (x = 4, n = 2, 3, 4 or 5 and m = 0, 1, 2 or 3), in one aspect, x = 4 and n = 2 or 3, in another aspect, x = 4 and n = 2. In one aspect, the peptide linker is (G4S)2. A particularly suitable peptide linker for fusing the Fab light chains of the first and second Fab molecules to each other is (G4S)2. An exemplary peptide linker suitable for linking the Fab heavy chains of the first and second Fab fragments comprises the sequence (D)-(G4S)2 (SEQ ID NOs 82 and 83). Another suitable such linker comprises the sequence (G4S)4. Additionally, the linker may comprise (a portion of) an immunoglobulin hinge region. In particular, in case of fusion of a Fab molecule to the N-terminus of an Fc domain subunit, the fusion may be via the immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

[0371] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide in which the Fab light chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL (2) -CH1 (2) -CH2-CH3(-CH4)), and polypeptides in which the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond (VH (1) -CH1 (1) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH (2) -CL (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) ). In certain aspects, the polypeptides are covalently linked, for example, via disulfide bonds.

[0372] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide in which the Fab heavy chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CL (2) -CH2-CH3(-CH4)), and polypeptides in which the Fab heavy chain of the first Fab molecule shares a carboxyl-terminal peptide bond (VH (1) -CH1 (1) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL (2) -CH1 (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) ). In certain aspects, the polypeptides are covalently linked, for example, via disulfide bonds.

[0373] In some aspects, the (multispecific) antibody comprises a polypeptide in which the Fab light chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL (2) -CH1 (2) -VH (1) -CH1 (1) -CH2-CH3(-CH4)). In other aspects, the (multispecific) antibody comprises a polypeptide in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (1) -CH1 (1) -VL (2) -CH1 (2)In some of these aspects, the (multispecific) antibody further comprises a crossover Fab light chain polypeptide of a second Fab molecule, wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH) with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) In other such aspects, the (multispecific) antibody further comprises a polypeptide wherein the Fab heavy chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the first Fab molecule (VH (2) -CL (2) -VL (1) -CL (1) ), or wherein the Fab light chain polypeptide of a first Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxyl-terminal peptide bond (VL (1) -CL (1) -VH (2) -CL (2) ), as appropriate. The (multispecific) antibody according to these aspects may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxyl-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)), and the Fab light chain polypeptide (VL (3) -CL (3) ). In certain aspects, the polypeptides are covalently linked, for example, via disulfide bonds.

[0374] In some aspects, the (multispecific) antibody comprises a polypeptide in which the Fab heavy chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CL (2) -VH (1) -CH1(1) -CH2-CH3(-CH4)). In other aspects, the (multispecific) antibody comprises a polypeptide in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (1) -CH1 (1) -VH (2) -CL (2) In some of these aspects, the (multispecific) antibody further comprises a crossover Fab light chain polypeptide of a second Fab molecule, wherein the Fab light chain variable region of the second Fab molecule shares a carboxyl-terminal peptide bond (VL (2) -CH1 (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) In other such aspects, the (multispecific) antibody further comprises a polypeptide wherein the Fab light chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond (VL (2) -CH1 (2) -VL (1) -CL (1) ), or wherein the Fab light chain polypeptide of a first Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxyl-terminal peptide bond (VL (1) -CL (1) -VH (2) -CL (2) ), as appropriate. The (multispecific) antibody according to these aspects may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxyl-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)), and the Fab light chain polypeptide (VL (3) -CL (3)). In certain aspects, the polypeptides are covalently linked, for example, via disulfide bonds.

[0375] In some aspects, the (multispecific) antibody does not comprise an Fc domain. In preferred aspects of this type, the first antigen-binding domain and (if present) the third antigen-binding domain are each conventional Fab molecules, and the second antigen-binding domain is a cross Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are exchanged / replaced with each other in the Fab molecule. In other aspects of this type, the first antigen-binding domain and (if present) the third antigen-binding domain are each cross Fab molecules and the second antigen-binding domain is a conventional Fab molecule.

[0376] In one such aspect, the (multispecific) antibody essentially consists of a first antigen binding domain and a second antigen binding domain and optionally one or more peptide linkers, wherein both the first antigen binding domain and the second antigen binding domain are Fab molecules and the first antigen binding domain 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 domain. Figure 1 O and 1S (in these examples, the second antigen binding domain is a VH / VL crossover Fab molecule and the first antigen binding domain is a conventional Fab molecule).

[0377] In another such aspect, the (multispecific) antibody consists essentially of a first antigen binding domain and a second antigen binding domain and optionally one or more peptide linkers, wherein both the first antigen binding domain and the second antigen binding domain are Fab molecules and the second antigen binding domain 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 domain. Figure 1 P and 1T (in these examples, the second antigen binding domain is a VH / VL crossover Fab molecule and the first antigen binding domain is a conventional Fab molecule).

[0378] In some aspects, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the (multispecific) antibody further comprises a third antigen binding domain, in particular a third Fab molecule, wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In some such aspects, the (multispecific) antibody essentially consists of the first, second and third Fab molecules, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. This configuration is schematically depicted in Figure 1 Q and 1U (in these examples, the second antigen binding domain is a VH / VL crossover Fab molecule and the first and third antigen binding domains are each conventional Fab molecules), or Figure 1 X and 1Z (in these examples, the second antigen binding domain is a conventional Fab molecule and the first and third antigen binding domains are each VH / VL crossover Fab molecules).

[0379] In some aspects, the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the (multispecific) antibody further comprises a third antigen binding domain, in particular a third Fab molecule, wherein the third Fab molecule is fused to the C-terminus of the Fab heavy chain of the first Fab molecule at the N-terminus of the Fab heavy chain. In some such aspects, the (multispecific) antibody essentially consists of the first, second and third Fab molecules and optionally one or more peptide linkers, wherein the second Fab molecule is fused to the N-terminus of the Fab heavy chain of the first Fab molecule at the C-terminus of the Fab heavy chain, and the third Fab molecule is fused to the C-terminus of the Fab heavy chain of the first Fab molecule at the N-terminus of the Fab heavy chain. This configuration is schematically described in Figure 1 R and IV (in these examples, the second antigen binding domain is a VH / VL crossover Fab molecule and the first and third antigen binding domains are each conventional Fab molecules), or Figure 1 I and 1Y (in these examples, the second antigen binding domain is a conventional Fab molecule and the first and third antigen binding domains are each VH / VL crossover Fab molecules).

[0380] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain wherein the heavy chain variable region is replaced by a light chain variable region) (VH (1) -CH1 (1) -VL (2) -CH1 (2) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH (2) -CL (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) ).

[0381] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond (VL (2) -CH1 (2) -VH (1) -CH1 (1) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH (2) -CL (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) ).

[0382] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of a second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain wherein the heavy chain constant region is replaced by a light chain constant region) (VH (1) -CH1(1) -VH (2) -CL (2) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL (2) -CH1 (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) ).

[0383] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond (VH) with the Fab heavy chain of the first Fab molecule. (2) -CL (2) -VH (1) -CH1 (1) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL (2) -CH1 (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) ).

[0384] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises crossover Fab heavy chains, wherein the heavy chain variable region is replaced by a light chain variable region) (VH (3) -CH1 (3) -VH (1) -CH1 (1) -VL (2) -CH1 (2)In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH (2) -CL (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) In some aspects, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL (3) -CL (3) ).

[0385] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region) (VH (3) -CH1 (3) -VH (1) -CH1 (1) -VH (2) -CL (2) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL (2) -CH1 (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) In some aspects, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL (3) -CL (3) ).

[0386] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide in which the Fab light chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule (VL (2) -CH1 (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH (2) -CL (2) ) and shares a carboxyl-terminal peptide bond (VL (1) -CL (1) In some aspects, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL (3) -CL (3) ).

[0387] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide in which the Fab heavy chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule (VH (2) -CL (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL (2) -CH1 (2) ) and shares a carboxyl-terminal peptide bond (VL (1)-CL (1) In some aspects, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL (3) -CL (3) ).

[0388] In certain aspects, a multispecific antibody according to the invention comprises a polypeptide in which the Fab heavy chain of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of a third Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region) (VH (2) -CH1 (2) -VL (1) -CH1 (1) -VL (3) -CH1 (3) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond (VH (1) -CL (1) ) and shares a carboxyl-terminal peptide bond (VL) with the Fab light chain polypeptide of the second Fab molecule (2) -CL (2) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the third Fab molecule shares a carboxyl-terminal peptide bond (VH (3) -CL (3) ).

[0389] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide in which the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e. the first Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the third Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (i.e. the third Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by the light chain constant region) (VH (2) -CH1 (2) -VH (1) -CL (1) -VH (3) -CL (3) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond (VL (1) -CH1 (1) ) and shares a carboxyl-terminal peptide bond (VL) with the Fab light chain polypeptide of the second Fab molecule (2) -CL (2) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the third Fab molecule shares a carboxyl-terminal peptide bond (VL (3) -CH1 (3) ).

[0390] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide in which the Fab light chain variable region of the third Fab molecule shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e. the third Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxyl-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e. the first Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxyl-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (3) -CH1 (3) -VL(1) -CH1 (1) -VH (2) -CH1 (2) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond (VH (1) -CL (1) ) and shares a carboxyl-terminal peptide bond (VL) with the Fab light chain polypeptide of the second Fab molecule (2) -CL (2) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the third Fab molecule shares a carboxyl-terminal peptide bond (VH (3) -CL (3) ).

[0391] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (i.e. the third Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e. the third Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule (VH (3) -CL (3) -VH (1) -CL (1) -VH (2) -CH1 (2) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond (VL (1) -CH1 (1) ) and shares a carboxyl-terminal peptide bond (VL) with the Fab light chain polypeptide of the second Fab molecule (2) -CL (2) In some aspects, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the third Fab molecule shares a carboxyl-terminal peptide bond (VL (3)-CH1 (3) ).

[0392] In one aspect, the present invention provides a (multispecific) antibody comprising

[0393] (A) a first antigen-binding domain that binds to HLA-A2 / MAGE-A4, wherein the first antigen-binding domain is a (conventional) Fab molecule, and the (conventional) Fab molecule comprises

[0394] (i) a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0395] (ii) a VH comprising HCDR 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 3; and a VL comprising LCDR 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;

[0396] (iii) a VH comprising HCDR 1 of SEQ ID NO: 41, HCDR 2 of SEQ ID NO: 42, and HCDR 3 of SEQ ID NO: 43; and a VL comprising LCDR 1 of SEQ ID NO: 44, LCDR 2 of SEQ ID NO: 45, and LCDR 3 of SEQ ID NO: 46;

[0397] (iv) a VH comprising HCDR 1 of SEQ ID NO: 9, HCDR 2 of SEQ ID NO: 10, and HCDR 3 of SEQ ID NO: 11; and a VL comprising LCDR 1 of SEQ ID NO: 12, LCDR 2 of SEQ ID NO: 13, and LCDR 3 of SEQ ID NO: 14;

[0398] (v) a VH comprising HCDR 1 of SEQ ID NO: 17, HCDR 2 of SEQ ID NO: 18, and HCDR 3 of SEQ ID NO: 19; and a VL comprising LCDR 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21, and LCDR 3 of SEQ ID NO: 22; or

[0399] (vi) a VH comprising HCDR 1 of SEQ ID NO: 33, HCDR 2 of SEQ ID NO: 34, and HCDR 3 of SEQ ID NO: 35; and a VL comprising LCDR 1 of SEQ ID NO: 36, LCDR 2 of SEQ ID NO: 37, and LCDR 3 of SEQ ID NO: 38;

[0400] (particularly a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30);

[0401] (B) a second antigen-binding domain that binds to a second antigen, particularly an activating T cell antigen, more particularly CD3, wherein the second antigen-binding domain is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain are replaced with each other;

[0402] (C) an Fc domain consisting of a first subunit and a second subunit;

[0403] in

[0404] (i) the first antigen binding domain according to (A) 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 domain according to (B), and the second antigen binding domain according to (B) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to (C), or

[0405] (ii) the second antigen binding domain according to (B) 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 domain according to (A), and the first antigen binding domain according to (A) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to (C).

[0406] In a preferred aspect, the present invention provides a (multispecific) antibody comprising

[0407] (A) a first antigen-binding domain and a third antigen-binding domain that bind to HLA-A2 / MAGE-A4, wherein each of the first antigen-binding domain and the third antigen-binding domain is a (conventional) Fab molecule and comprises

[0408] (i) a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0409] (ii) a VH comprising HCDR 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 3; and a VL comprising LCDR 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;

[0410] (iii) a VH comprising HCDR 1 of SEQ ID NO: 41, HCDR 2 of SEQ ID NO: 42, and HCDR 3 of SEQ ID NO: 43; and a VL comprising LCDR 1 of SEQ ID NO: 44, LCDR 2 of SEQ ID NO: 45, and LCDR 3 of SEQ ID NO: 46;

[0411] (iv) a VH comprising HCDR 1 of SEQ ID NO: 9, HCDR 2 of SEQ ID NO: 10, and HCDR 3 of SEQ ID NO: 11; and a VL comprising LCDR 1 of SEQ ID NO: 12, LCDR 2 of SEQ ID NO: 13, and LCDR 3 of SEQ ID NO: 14;

[0412] (v) a VH comprising HCDR 1 of SEQ ID NO: 17, HCDR 2 of SEQ ID NO: 18, and HCDR 3 of SEQ ID NO: 19; and a VL comprising LCDR 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21, and LCDR 3 of SEQ ID NO: 22; or

[0413] (vi) a VH comprising HCDR 1 of SEQ ID NO: 33, HCDR 2 of SEQ ID NO: 34, and HCDR 3 of SEQ ID NO: 35; and a VL comprising LCDR 1 of SEQ ID NO: 36, LCDR 2 of SEQ ID NO: 37, and LCDR 3 of SEQ ID NO: 38;

[0414] (particularly a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30);

[0415] (B) a second antigen-binding domain that binds to a second antigen, particularly an activating T cell antigen, more particularly CD3, wherein the second antigen-binding domain is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain are replaced with each other; and

[0416] (C) an Fc domain consisting of a first subunit and a second subunit;

[0417] in

[0418] (i) the first antigen-binding domain according to (A) 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 domain according to (B), and the second antigen-binding domain according to (B) and the third antigen-binding domain according to (A) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to (C), or

[0419] (ii) the second antigen binding domain according to (B) 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 domain according to (A), and the first antigen binding domain according to (A) and the third antigen binding domain according to (A) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to (C).

[0420] In another aspect, the present invention provides a (multispecific) antibody comprising

[0421] (A) a first antigen-binding domain that binds to HLA-A2 / MAGE-A4, wherein the first antigen-binding domain is a (conventional) Fab molecule, and the (conventional) Fab molecule comprises

[0422] (i) a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0423] (ii) a VH comprising HCDR 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 3; and a VL comprising LCDR 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;

[0424] (iii) a VH comprising HCDR 1 of SEQ ID NO: 41, HCDR 2 of SEQ ID NO: 42, and HCDR 3 of SEQ ID NO: 43; and a VL comprising LCDR 1 of SEQ ID NO: 44, LCDR 2 of SEQ ID NO: 45, and LCDR 3 of SEQ ID NO: 46;

[0425] (iv) a VH comprising HCDR 1 of SEQ ID NO: 9, HCDR 2 of SEQ ID NO: 10, and HCDR 3 of SEQ ID NO: 11; and a VL comprising LCDR 1 of SEQ ID NO: 12, LCDR 2 of SEQ ID NO: 13, and LCDR 3 of SEQ ID NO: 14;

[0426] (v) a VH comprising HCDR 1 of SEQ ID NO: 17, HCDR 2 of SEQ ID NO: 18, and HCDR 3 of SEQ ID NO: 19; and a VL comprising LCDR 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21, and LCDR 3 of SEQ ID NO: 22; or

[0427] (vi) a VH comprising HCDR 1 of SEQ ID NO: 33, HCDR 2 of SEQ ID NO: 34, and HCDR 3 of SEQ ID NO: 35; and a VL comprising LCDR 1 of SEQ ID NO: 36, LCDR 2 of SEQ ID NO: 37, and LCDR 3 of SEQ ID NO: 38;

[0428] (particularly a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30);

[0429] (B) a second antigen-binding domain that binds to a second antigen, particularly an activating T cell antigen, more particularly CD3, wherein the second antigen-binding domain is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain are replaced with each other; and

[0430] (C) an Fc domain consisting of a first subunit and a second subunit;

[0431] in

[0432] (i) The first antigen binding domain according to (A) and the second antigen binding domain according to (B) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to (C).

[0433] In all different configurations of the (multispecific) antibodies according to the present invention, the amino acid substitutions ("charge modifications") described herein (if any) may be in the CH1 and CL domains of the first and (if any) third antigen-binding domains / Fab molecules, or in the CH1 and CL domains of the second antigen-binding domains / Fab molecules. Preferably, they are in the CH1 and CL domains of the first and (if any) third antigen-binding domains / Fab molecules. According to the concept of the present invention, if an amino acid substitution as described herein is performed in the first antigen-binding domain (and the third antigen-binding domain if present) / Fab molecule, such an amino acid substitution is not performed in the second antigen-binding domain / Fab molecule. Conversely, if an amino acid substitution as described herein is performed in the second antigen-binding domain / Fab molecule, such an amino acid substitution is not performed in the first antigen-binding domain (and the third antigen-binding domain if present) / Fab molecule. Amino acid substitutions are preferably performed in (multispecific) antibodies comprising Fab molecules in which the variable domains VL and VH1 of the Fab light chain and the Fab heavy chain replace each other.

[0434] In preferred aspects of the (multispecific) antibodies according to the invention, in particular wherein the amino acid substitutions as described herein are made in the first (and if present, the third) antigen binding domain / Fab molecule, and the constant domain CL of the first (and if present, the third) Fab molecule is of the kappa isotype. In other aspects of the (multispecific) antibodies according to the invention, in particular wherein the amino acid substitutions as described herein are made in the second antigen binding domain / Fab molecule, and the constant domain CL of the second antigen binding domain / Fab molecule is of the kappa isotype. In some aspects, the constant domain CL of the first (and if present, the third) antigen binding domain / Fab molecule and the constant domain CL of the second antigen binding domain / Fab molecule are of the kappa isotype.

[0435] In one aspect, the present invention provides a (multispecific) antibody comprising

[0436] (A) a first antigen-binding domain that binds to HLA-A2 / MAGE-A4, wherein the first antigen-binding domain is a (conventional) Fab molecule, and the (conventional) Fab molecule comprises

[0437] (i) a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0438] (ii) a VH comprising HCDR 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 3; and a VL comprising LCDR 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;

[0439] (iii) a VH comprising HCDR 1 of SEQ ID NO: 41, HCDR 2 of SEQ ID NO: 42, and HCDR 3 of SEQ ID NO: 43; and a VL comprising LCDR 1 of SEQ ID NO: 44, LCDR 2 of SEQ ID NO: 45, and LCDR 3 of SEQ ID NO: 46;

[0440] (iv) a VH comprising HCDR 1 of SEQ ID NO: 9, HCDR 2 of SEQ ID NO: 10, and HCDR 3 of SEQ ID NO: 11; and a VL comprising LCDR 1 of SEQ ID NO: 12, LCDR 2 of SEQ ID NO: 13, and LCDR 3 of SEQ ID NO: 14;

[0441] (v) a VH comprising HCDR 1 of SEQ ID NO: 17, HCDR 2 of SEQ ID NO: 18, and HCDR 3 of SEQ ID NO: 19; and a VL comprising LCDR 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21, and LCDR 3 of SEQ ID NO: 22; or

[0442] (vi) a VH comprising HCDR 1 of SEQ ID NO: 33, HCDR 2 of SEQ ID NO: 34, and HCDR 3 of SEQ ID NO: 35; and a VL comprising LCDR 1 of SEQ ID NO: 36, LCDR 2 of SEQ ID NO: 37, and LCDR 3 of SEQ ID NO: 38;

[0443] (particularly a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30);

[0444] (B) a second antigen-binding domain that binds to a second antigen (particularly an activating T cell antigen, more particularly CD3), wherein the second antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other; and

[0445] (C) an Fc domain consisting of a first subunit and a second subunit;

[0446] wherein in the constant domain CL of the first antigen-binding domain according to (A), the amino acid at position 124 is substituted by lysine (K) (according to Kabat numbering) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (according to Kabat numbering) (most preferably by arginine (R)); and wherein in the constant domain CH1 of the second antigen-binding domain according to (A), the amino acid at position 147 is substituted by glutamic acid (E) (according to Kabat EU index numbering) and the amino acid at position 213 is substituted by glutamic acid (E) (according to Kabat EU index numbering); and

[0447] in

[0448] (i) the first antigen binding domain according to (A) 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 domain according to (B), and the second antigen binding domain according to (B) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to (C), or

[0449] (ii) the second antigen binding domain according to (B) 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 domain according to (A), and the first antigen binding domain according to (A) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to (C).

[0450] In a preferred aspect, the present invention provides a (multispecific) antibody comprising

[0451] (A) a first antigen-binding domain and a third antigen-binding domain that bind to HLA-A2 / MAGE-A4, wherein each of the first antigen-binding domain and the third antigen-binding domain is a (conventional) Fab molecule and comprises

[0452] (i) a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0453] (ii) a VH comprising HCDR 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 3; and a VL comprising LCDR 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;

[0454] (iii) a VH comprising HCDR 1 of SEQ ID NO: 41, HCDR 2 of SEQ ID NO: 42, and HCDR 3 of SEQ ID NO: 43; and a VL comprising LCDR 1 of SEQ ID NO: 44, LCDR 2 of SEQ ID NO: 45, and LCDR 3 of SEQ ID NO: 46;

[0455] (iv) a VH comprising HCDR 1 of SEQ ID NO: 9, HCDR 2 of SEQ ID NO: 10, and HCDR 3 of SEQ ID NO: 11; and a VL comprising LCDR 1 of SEQ ID NO: 12, LCDR 2 of SEQ ID NO: 13, and LCDR 3 of SEQ ID NO: 14;

[0456] (v) a VH comprising HCDR 1 of SEQ ID NO: 17, HCDR 2 of SEQ ID NO: 18, and HCDR 3 of SEQ ID NO: 19; and a VL comprising LCDR 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21, and LCDR 3 of SEQ ID NO: 22; or

[0457] (vi) a VH comprising HCDR 1 of SEQ ID NO: 33, HCDR 2 of SEQ ID NO: 34, and HCDR 3 of SEQ ID NO: 35; and a VL comprising LCDR 1 of SEQ ID NO: 36, LCDR 2 of SEQ ID NO: 37, and LCDR 3 of SEQ ID NO: 38;

[0458] (particularly a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30);

[0459] (B) a second antigen-binding domain that binds to a second antigen (particularly an activating T cell antigen, more particularly CD3), wherein the second antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other; and

[0460] (C) an Fc domain consisting of a first subunit and a second subunit;

[0461] wherein in the constant domain CL of the first antigen-binding domain according to (A) and the third antigen-binding domain according to (A), the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (numbering according to Kabat) (most preferably by arginine (R)), and wherein in the constant domain CH1 of the first antigen-binding domain according to (A) and the third antigen-binding domain according to (A), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the EU index as in Kabat) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the EU index as in Kabat); and

[0462] in

[0463] (i) the first antigen-binding domain according to (A) 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 domain according to (B), and the second antigen-binding domain according to (B) and the third antigen-binding domain according to (A) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to (C), or

[0464] (ii) the second antigen binding domain according to (B) 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 domain according to (A), and the first antigen binding domain according to (A) and the third antigen binding domain according to (A) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to (C).

[0465] In another aspect, the present invention provides a (multispecific) antibody comprising

[0466] (A) a first antigen-binding domain that binds to HLA-A2 / MAGE-A4, wherein the first antigen-binding domain is a (conventional) Fab molecule, and the (conventional) Fab molecule comprises

[0467] (i) a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0468] (ii) a VH comprising HCDR 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 3; and a VL comprising LCDR 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;

[0469] (iii) a VH comprising HCDR 1 of SEQ ID NO: 41, HCDR 2 of SEQ ID NO: 42, and HCDR 3 of SEQ ID NO: 43; and a VL comprising LCDR 1 of SEQ ID NO: 44, LCDR 2 of SEQ ID NO: 45, and LCDR 3 of SEQ ID NO: 46;

[0470] (iv) a VH comprising HCDR 1 of SEQ ID NO: 9, HCDR 2 of SEQ ID NO: 10, and HCDR 3 of SEQ ID NO: 11; and a VL comprising LCDR 1 of SEQ ID NO: 12, LCDR 2 of SEQ ID NO: 13, and LCDR 3 of SEQ ID NO: 14;

[0471] (v) a VH comprising HCDR 1 of SEQ ID NO: 17, HCDR 2 of SEQ ID NO: 18, and HCDR 3 of SEQ ID NO: 19; and a VL comprising LCDR 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21, and LCDR 3 of SEQ ID NO: 22; or

[0472] (vi) a VH comprising HCDR 1 of SEQ ID NO: 33, HCDR 2 of SEQ ID NO: 34, and HCDR 3 of SEQ ID NO: 35; and a VL comprising LCDR 1 of SEQ ID NO: 36, LCDR 2 of SEQ ID NO: 37, and LCDR 3 of SEQ ID NO: 38;

[0473] (particularly a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30);

[0474] (B) a second antigen-binding domain that binds to a second antigen (particularly an activating T cell antigen, more particularly CD3), wherein the second antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other; and

[0475] (C) an Fc domain consisting of a first subunit and a second subunit;

[0476] wherein in the constant domain CL of the first antigen-binding domain according to (A), the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (numbering according to Kabat) (most preferably by arginine (R)), and wherein in the constant domain CH1 of the first antigen-binding domain according to (A), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat); and

[0477] wherein the first antigen binding domain according to (A) and the second antigen binding domain according to (B) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to (C).

[0478] According to any of the above aspects, the components of the (multispecific) antibodies (e.g., Fab molecules, Fc domains) can be fused directly or via various linkers, in particular peptide linkers comprising one or more amino acids, typically about 2-20 amino acids, which are described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S) n 、(SG4) n 、(G4S) n or G4 (SG4) n A peptide linker wherein "n" is typically an integer from 1 to 10, usually from 2 to 4.

[0479] Furthermore, according to any of the above aspects, the second antigen binding domain may comprise (i) a VH comprising HCDR 1 of SEQ ID NO: 59, HCDR 2 of SEQ ID NO: 60, and HCDR 3 of SEQ ID NO: 61; and a VL comprising LCDR 1 of SEQ ID NO: 62, LCDR 2 of SEQ ID NO: 63, and LCDR 3 of SEQ ID NO: 64, or (ii) a VH comprising HCDR 1 of SEQ ID NO: 51, HCDR 2 of SEQ ID NO: 52, and HCDR 3 of SEQ ID NO: 53; and a VL comprising LCDR 1 of SEQ ID NO: 54, LCDR 2 of SEQ ID NO: 55, and LCDR 3 of SEQ ID NO: 56 (particularly a VH comprising HCDR 1 of SEQ ID NO: 59, HCDR 2 of SEQ ID NO: 60, and HCDR 3 of SEQ ID NO: 61; and a VL comprising LCDR 1 of SEQ ID NO: 62). 1, LCDR 2 of SEQ ID NO: 63 and LCDR 3 of SEQ ID NO: 64); and / or (i) a VH comprising the amino acid sequence of SEQ ID NO: 65; and a VL comprising the amino acid sequence of SEQ ID NO: 66, or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 57; and a VL comprising the amino acid sequence of SEQ ID NO: 58 (particularly a VH comprising the amino acid sequence of SEQ ID NO: 65; and a VL comprising the amino acid sequence of SEQ ID NO: 66).

[0480] In a preferred aspect, the present invention provides a (multispecific) antibody comprising

[0481] (A) a first antigen-binding domain and a third antigen-binding domain that bind to HLA-A2 / MAGE-A4, wherein each of the first antigen-binding domain and the third antigen-binding domain is a (conventional) Fab molecule and comprises

[0482] (i) a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30;

[0483] (ii) a VH comprising HCDR 1 of SEQ ID NO: 1, HCDR 2 of SEQ ID NO: 2, and HCDR 3 of SEQ ID NO: 3; and a VL comprising LCDR 1 of SEQ ID NO: 4, LCDR 2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6;

[0484] (iii) a VH comprising HCDR 1 of SEQ ID NO: 41, HCDR 2 of SEQ ID NO: 42, and HCDR 3 of SEQ ID NO: 43; and a VL comprising LCDR 1 of SEQ ID NO: 44, LCDR 2 of SEQ ID NO: 45, and LCDR 3 of SEQ ID NO: 46;

[0485] (iv) a VH comprising HCDR 1 of SEQ ID NO: 9, HCDR 2 of SEQ ID NO: 10, and HCDR 3 of SEQ ID NO: 11; and a VL comprising LCDR 1 of SEQ ID NO: 12, LCDR 2 of SEQ ID NO: 13, and LCDR 3 of SEQ ID NO: 14;

[0486] (v) a VH comprising HCDR 1 of SEQ ID NO: 17, HCDR 2 of SEQ ID NO: 18, and HCDR 3 of SEQ ID NO: 19; and a VL comprising LCDR 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21, and LCDR 3 of SEQ ID NO: 22; or

[0487] (vi) a VH comprising HCDR 1 of SEQ ID NO: 33, HCDR 2 of SEQ ID NO: 34, and HCDR 3 of SEQ ID NO: 35; and a VL comprising LCDR 1 of SEQ ID NO: 36, LCDR 2 of SEQ ID NO: 37, and LCDR 3 of SEQ ID NO: 38;

[0488] (particularly a VH comprising HCDR 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and a VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30);

[0489] (B) a second antigen-binding domain that binds to a second antigen (particularly an activating T cell antigen, more particularly CD3), wherein the second antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other and comprise

[0490] (i) a VH comprising HCDR 1 of SEQ ID NO: 59, HCDR 2 of SEQ ID NO: 60, and HCDR 3 of SEQ ID NO: 61; and a VL comprising LCDR 1 of SEQ ID NO: 62, LCDR 2 of SEQ ID NO: 63, and LCDR 3 of SEQ ID NO: 64; or

[0491] (ii) a VH comprising HCDR 1 of SEQ ID NO: 51, HCDR 2 of SEQ ID NO: 52, and HCDR 3 of SEQ ID NO: 53; and a VL comprising LCDR 1 of SEQ ID NO: 54, LCDR 2 of SEQ ID NO: 55, and LCDR 3 of SEQ ID NO: 56;

[0492] (particularly a VH comprising HCDR 1 of SEQ ID NO: 59, HCDR 2 of SEQ ID NO: 60, and HCDR 3 of SEQ ID NO: 61; and a VL comprising LCDR 1 of SEQ ID NO: 62, LCDR 2 of SEQ ID NO: 63, and LCDR 3 of SEQ ID NO: 64); and

[0493] (C) an Fc domain consisting of a first subunit and a second subunit;

[0494] in

[0495] in the constant domain CL of the first and third antigen-binding domains according to (A), the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (numbering according to Kabat) (most preferably by arginine (R)), and wherein in the constant domain CH1 of the first and third antigen-binding domains according to (A), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat);

[0496] And further

[0497] The first antigen binding domain according to (A) 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 domain according to (B), and the second antigen binding domain according to (B) and the third antigen binding domain according to (A) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to (C).

[0498] In another preferred aspect, the present invention provides a (multispecific) antibody comprising

[0499] (A) a first antigen-binding domain and a third antigen-binding domain that bind to HLA-A2 / MAGE-A4, wherein each of the first antigen-binding domain and the third antigen-binding domain is a (conventional) Fab molecule and comprises

[0500] (i) a VH comprising the amino acid sequence of SEQ ID NO: 31; and a VL comprising the amino acid sequence of SEQ ID NO: 32;

[0501] (i) a VH comprising the amino acid sequence of SEQ ID NO: 7; and a VL comprising the amino acid sequence of SEQ ID NO: 8;

[0502] (i) a VH comprising the amino acid sequence of SEQ ID NO: 47; and a VL comprising the amino acid sequence of SEQ ID NO: 48;

[0503] (i) a VH comprising the amino acid sequence of SEQ ID NO: 15; and a VL comprising the amino acid sequence of SEQ ID NO: 16;

[0504] (i) a VH comprising the amino acid sequence of SEQ ID NO: 23; and a VL comprising the amino acid sequence of SEQ ID NO: 24;

[0505] (i) a VH comprising the amino acid sequence of SEQ ID NO: 39; and a VL comprising the amino acid sequence of SEQ ID NO: 40;

[0506] (particularly VH comprising the amino acid sequence of SEQ ID NO: 31; and VL comprising the amino acid sequence of SEQ ID NO: 32);

[0507] (B) a second antigen-binding domain that binds to a second antigen (particularly an activating T cell antigen, more particularly CD3), wherein the second antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other and comprise

[0508] (i) a VH comprising the amino acid sequence of SEQ ID NO: 65; and a VL comprising the amino acid sequence of SEQ ID NO: 66; or

[0509] (ii) VH comprising the amino acid sequence of SEQ ID NO: 57; and VL comprising the amino acid sequence of SEQ ID NO: 58;

[0510] (particularly VH comprising the amino acid sequence of SEQ ID NO: 65; and VL comprising the amino acid sequence of SEQ ID NO: 66); and

[0511] (C) an Fc domain consisting of a first subunit and a second subunit;

[0512] in

[0513] in the constant domain CL of the first and third antigen-binding domains according to (A), the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (numbering according to Kabat) (most preferably by arginine (R)), and wherein in the constant domain CH1 of the first and third antigen-binding domains according to (A), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the EU index of Kabat);

[0514] And further

[0515] The first antigen binding domain according to (A) 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 domain according to (B), and the second antigen binding domain according to (B) and the third antigen binding domain according to (A) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to (C).

[0516] In one aspect according to these aspects of the invention, in said first subunit of said Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W); and in said second subunit of said Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), and 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).

[0517] In another aspect according to these aspects of the invention, 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).

[0518] In yet another aspect according to these aspects of the invention, in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (numbering according to the KabatEU index).

[0519] In yet another aspect according to these aspects of the invention, the Fc domain is a human IgG1 Fc domain.

[0520] In a specific aspect, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 68, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 69, a polypeptide (particularly two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 70, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 72. In another specific aspect, the (multispecific) antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 68, a polypeptide comprising the amino acid sequence of SEQ ID NO: 69, a polypeptide (particularly two polypeptides) comprising the amino acid sequence of SEQ ID NO: 70, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 72.

[0521] In one aspect, the invention provides (multispecific) antibodies that bind to HLA-A2 / MAGE-A4 and CD3, the antibodies comprising a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:68, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:69, a polypeptide (particularly two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:70, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:72. In one aspect, the present invention provides a (multispecific) antibody that binds to HLA-A2 / MAGE-A4 and CD3, the antibody comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 68, a polypeptide comprising the amino acid sequence of SEQ ID NO: 69, a polypeptide comprising the amino acid sequence of SEQ ID NO: 70 (in particular, two polypeptides), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 72.

[0522] In another specific aspect, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 67, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 69, a polypeptide (particularly two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 70, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 71. In another specific aspect, the (multispecific) antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 67, a polypeptide comprising the amino acid sequence of SEQ ID NO: 69, a polypeptide (particularly two polypeptides) comprising the amino acid sequence of SEQ ID NO: 70, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 71.

[0523] In one aspect, the invention provides (multispecific) antibodies that bind to HLA-A2 / MAGE-A4 and CD3, the antibodies comprising a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:67, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:69, a polypeptide (particularly two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:70, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:71. In one aspect, the present invention provides a (multispecific) antibody that binds to HLA-A2 / MAGE-A4 and CD3, the antibody comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 67, a polypeptide comprising the amino acid sequence of SEQ ID NO: 69, a polypeptide comprising the amino acid sequence of SEQ ID NO: 70 (in particular, two polypeptides), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 71.

[0524] 10. Fc domain variants

[0525] In a preferred aspect, the (multispecific) antibody of the present invention comprises an Fc domain composed of a first subunit and a second subunit.

[0526] The Fc domain of a (multispecific) antibody is composed of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises a CH2 and a CH3 IgG heavy chain constant domain. The two subunits of the Fc domain are capable of stably associating with each other. In one aspect, the (multispecific) antibody of the present invention comprises no more than one Fc domain.

[0527] In one aspect, the Fc domain of (multispecific) antibody is IgG Fc domain. In a preferred aspect, the Fc domain is IgG1 Fc domain. In another aspect, the Fc domain is IgG4 Fc domain. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution (particularly amino acid substitution S228P) at position S228 (Kabat EU index numbering). This amino acid substitution reduces the in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In another preferred aspect, the Fc domain is a human Fc domain. In an even more preferred aspect, the Fc domain is a human IgG1 Fc domain. The exemplary sequence of the human IgG1 Fc region is provided with SEQ ID NO:78.

[0528] a) Fc domain modifications that promote heterodimerization

[0529] The (multispecific) antibodies according to the present invention comprise different antigen-binding domains that can be fused to one or the other of the two subunits of the Fc domain, whereby the two subunits of the Fc domain are typically contained in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. In order to increase the yield and purity of (multispecific) antibodies in recombinant production, it would therefore be advantageous to introduce modifications in the Fc domain of the (multispecific) antibodies that promote the association of the desired polypeptides.

[0530] Thus, in a preferred aspect, the Fc domain of a (multispecific) antibody according to the invention comprises a modification that promotes the association of the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one aspect, the modification is in the CH3 domain of the Fc domain.

[0531] There are several methods for modifying the CH3 domain of the Fc domain to achieve heterodimerization, which are described in detail in, for example, WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, and WO 2013096291. Generally, in all such approaches, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are engineered in a complementary manner so that each CH3 domain (or heavy chain comprising it) can no longer homodimerize with itself, but is forced to heterodimerize with the other complementary engineered CH3 domain (such that the first and second CH3 domains heterodimerize and no homodimers are formed between the two first or two second CH3 domains). These different approaches to achieving improved heavy chain heterodimerization are considered to be different replacement schemes combined with heavy-light chain modifications in (multispecific) antibodies (e.g., VH and VL exchange / replacement in one binding arm and introduction of oppositely charged amino acid substitutions at the CH1 / CL interface) that reduce heavy chain / light chain mispairing and Bence Jones-type side products.

[0532] In one specific aspect, the modification that promotes the association of the first and second subunits of the Fc domain is a so-called "knob-to-hole" modification, which comprises a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain.

[0533] The knob-in-hole technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide so that the protrusion can be positioned in the cavity to promote the formation of heterodimers and hinder the formation of homodimers. The protrusion is constructed by replacing the small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A compensatory cavity having the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0534] Therefore, in a preferred aspect, in the CH3 domain of the first subunit of the Fc domain of the (multispecific) antibody, amino acid residues are substituted by amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit, and the protrusion can be positioned in a cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, amino acid residues are substituted by amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, and the protrusion in the CH3 domain of the first subunit can be positioned in the cavity.

[0535] Preferably, the amino acid residue with a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

[0536] Preferably, the amino acid residue with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V).

[0537] Protrusions and cavities can be produced by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or by peptide synthesis.

[0538] In a specific aspect, in the first subunit (the "knob" subunit) of the Fc domain (the CH3 domain), the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit (the "hole" subunit) of the Fc domain (the CH3 domain), the tyrosine residue at position 407 is replaced by a valine residue (Y407V). In one aspect, in the second subunit of the Fc domain, 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).

[0539] In another aspect, 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) (particularly 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) (numbered according to the Kabat EU index). Introducing these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0540] In a preferred aspect, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0541] In a preferred aspect, a second antigen-binding domain that preferably binds to an activating T cell antigen (e.g., CD3) is fused to the first subunit of the Fc domain (comprising a "knob" modification) (optionally via a first antigen-binding domain that binds to HLA-A2 / MAGE-A4 and / or a peptide linker). Without wishing to be bound by theory, fusion of an antigen-binding domain that binds to a second antigen (e.g., CD3) to a knob-containing subunit of the Fc domain will (further) minimize the production of antibodies comprising two antigen-binding domains that bind to a second antigen (steric hindrance of the two knob-containing polypeptides).

[0542] Other CH3 modification techniques for implementing heterodimerization are envisioned as alternatives according to the present invention and are described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291.

[0543] In one aspect, the heterodimerization method described in EP 1870459 can be used instead. This method is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. A specific aspect of the (multispecific) antibodies of the invention is the amino acid mutation R409D; K370E in one of the two CH3 domains (of the Fc domain), and the amino acid mutation D399K; E357K in the other CH3 domain of the Fc domain (numbering according to the Kabat EU index).

[0544] In another aspect, the (multispecific) antibody of the invention comprises the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations T366S, L368A, Y407V, and additionally the amino acid mutation R409D in the CH3 domain of the second subunit of the Fc domain; K370E in the CH3 domain of the first subunit of the Fc domain, and the amino acid mutation D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numbering according to the Kabat EU index).

[0545] In another aspect, the (multispecific) antibody of the invention comprises the amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the (multispecific) antibody comprises the amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain and additionally the amino acid mutation R409D; K370E in the CH3 domain of the first subunit of the Fc domain, and the amino acid mutation D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numbering according to the Kabat EU index).

[0546] In one aspect, the heterodimerization method described in WO 2013 / 157953 can be used alternatively. In one aspect, the first CH3 domain comprises the amino acid mutation T366K, and the second CH3 domain comprises the amino acid mutation L351D (numbering according to the Kabat EU index). In another aspect, the first CH3 domain comprises the additional amino acid mutation L351K. In another aspect, the second CH3 domain further comprises an amino acid mutation selected from the group consisting of Y349E, Y349D, and L368E (particularly L368E) (numbering according to the Kabat EU index).

[0547] In one aspect, the heterodimerization method described in WO 2012 / 058768 can be used alternatively. In one aspect, the first CH3 domain comprises amino acid mutations L351Y, Y407A, and the second CH3 domain comprises amino acid mutations T366A, K409F. In another aspect, the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390 or K392, for example selected from the group consisting of: a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y or D 399K, c) S400E, S400D, S400R or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numbering according to the Kabat EU index). In another aspect, the first CH3 domain comprises amino acid mutations L351Y, Y407A, and the second CH3 domain comprises amino acid mutations T366V, K409F. In another aspect, the first CH3 domain comprises amino acid mutations Y407A, and the second CH3 domain comprises amino acid mutations T366A, K409F. In another aspect, the second CH3 domain further comprises amino acid mutations K392E, T411E, D399R and S400R (numbered according to the Kabat EU index).

[0548] In one aspect, the heterodimerization method described in WO 2011 / 143545 may be used instead, e.g., with amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to the Kabat EU index).

[0549] In one aspect, the heterodimerization method described in WO 2011 / 090762 can be used alternatively, which also uses the above-mentioned knob-in-hole structure technology. In one aspect, the first CH3 domain comprises the amino acid mutation T366W, and the second CH3 domain comprises the amino acid mutation Y407A. In one aspect, the first CH3 domain comprises the amino acid mutation T366Y, and the second CH3 domain comprises the amino acid mutation Y407T (numbering according to the Kabat EU index).

[0550] In one aspect the (multispecific) antibody or its Fc domain is of IgG2 subclass and the heterodimerization approach described in WO2010 / 129304 may alternatively be used.

[0551] In one alternative aspect, modifications that promote association of the first and second subunits of the Fc domain include modifications that mediate an electrostatic steering effect, such as described in PCT Publication WO 2009 / 089004. Typically, this approach involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues such that homodimer formation becomes electrostatically unfavorable, but heterodimerization is electrostatically favorable. In one such aspect, the first CH3 domain comprises an amino acid substitution with a negatively charged amino acid pair K392 or N392 (e.g., glutamic acid (E) or aspartic acid (D), particularly K392D or N392D), and the second CH3 domain comprises an amino acid substitution with a positively charged amino acid pair D399, E356, D356 or E357 (e.g., lysine (K) or arginine (R), particularly D399K, E356K, D356K or E357K, more particularly D399K and E356K). In another aspect, the first CH3 domain further comprises an amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), in particular K409D or R409D). In another aspect, the first CH3 domain further or alternatively comprises an amino acid substitution of K439 and / or K370 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)) (all numbering according to the Kabat EU index).

[0552] In yet another aspect, the heterodimerization method described in WO 2007 / 147901 may be used instead.In one aspect, the first CH3 domain comprises the amino acid mutations K253E, D282K and K322D, and the second CH3 domain comprises the amino acid mutations D239K, E240K and K292D (numbered according to the Kabat EU index).

[0553] In yet another aspect, the heterodimerization method described in WO 2007 / 110205 may be used instead.

[0554] In one aspect, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbering according to the Kabat EU index).

[0555] b) Fc domain modifications that reduce Fc receptor binding and / or effector function

[0556] The Fc domain confers favorable pharmacokinetic properties to (multispecific) antibodies, including a long serum half-life and a favorable tissue-blood distribution ratio that contribute to good accumulation in target tissues. However, at the same time, it may cause the (multispecific) antibodies to be undesirably targeted to cells expressing Fc receptors, rather than preferred antigen-bearing cells. In addition, co-activation of Fc receptor signaling pathways can lead to cytokine release, which, combined with the T cell activation properties and the long half-life of the (multispecific) antibodies, leads to excessive activation of cytokine receptors and severe side effects after systemic administration. Due to the potential destruction of T cells (e.g., by NK cells), the activation of immune cells other than T cells (with Fc receptors) may even reduce the efficacy of the (multispecific) antibodies.

[0557] Thus, in a preferred aspect, the Fc domain of the (multispecific) antibody according to the present invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain. In one such aspect, the Fc domain (or a (multispecific) antibody comprising said Fc domain) exhibits less than 50%, particularly less than 20%, more particularly less than 10% and most particularly less than 5% of the binding affinity compared to a native IgG1 Fc domain (or a (multispecific) antibody comprising a native IgG1 Fc domain), and / or less than 50%, particularly less than 20%, more particularly less than 10% and most particularly less than 5% of the effector function compared to a native IgG1 Fc domain (or a (multispecific) antibody comprising a native IgG1 Fc domain). In one aspect, the Fc domain domain (or a (multispecific) antibody comprising said Fc domain) does not substantially bind to an Fc receptor and / or induce effector function. In a preferred aspect, the Fc receptor is an Fcγ receptor. In one aspect, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one aspect, the effector function is one or more effector functions selected from the group consisting of CDC, ADCC, ADCP and cytokine secretion. In a preferred aspect, the effector function is ADCC. In one aspect, the Fc domain domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the native IgG1 Fc domain domain. When the Fc domain (or (multispecific) antibody comprising the Fc domain) exhibits greater than about 70% of the binding affinity of the native IgG1 Fc domain (or (multispecific) antibody comprising the native IgG1 Fc domain) to FcRn, particularly greater than about 80%, more particularly greater than about 90%, substantially similar binding to FcRn is achieved.

[0558] In some aspects, the Fc domain is engineered to have a reduced binding affinity to Fc receptors and / or reduced effector functions compared to a non-engineered Fc domain. In preferred aspects, the Fc domain of a (multi-specific) antibody comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to the Fc receptors and / or the effector functions. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one aspect, the amino acid mutations reduce the binding affinity of the Fc domain to the Fc receptors. In one aspect, the amino acid mutations reduce the binding affinity of the Fc domain to the Fc receptors by at least 2 times, at least 5 times, or at least 10 times. In the presence of more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptors, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptors by at least 10 times, at least 20 times, or even at least 50 times. In one aspect, compared to a (multi-specific) antibody comprising a non-engineered Fc domain, a (multi-specific) antibody comprising an engineered Fc domain exhibits less than 20% of the binding affinity to an Fc receptor, particularly less than 10%, more particularly less than 5%. In a preferred aspect, the Fc receptor is an Fcγ receptor. In some aspects, the Fc receptor is a human Fc receptor. In some aspects, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, the binding to each of these receptors is reduced. In some aspects, the binding affinity to the complementary component and the specific binding affinity to C1q are also reduced. In one aspect, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e., the binding affinity of the Fc domain for the receptor is maintained, is achieved when the Fc domain (or (multispecific) antibody comprising the Fc domain) exhibits greater than about 70% of the binding affinity of the non-engineered form of the Fc domain (or (multispecific) antibody comprising the non-engineered form of the Fc domain) for FcRn. The Fc domain or the (multispecific) antibody of the invention comprising the Fc domain may exhibit greater than about 80%, and even greater than about 90%, of this affinity. In certain aspects, the Fc domain of the (multispecific) antibody is engineered to have reduced effector function compared to the non-engineered Fc domain.The effector function of reduction can include but is not limited to one or more of the following items: the complement dependent cytotoxicity (CDC) of reduction, the antibody dependent cell-mediated cytotoxicity (ADCC) of reduction, the antibody dependent cellular phagocytosis (ADCP) of reduction, the cytokine secretion of reduction, the antigen presenting cells of the immune complex mediation of reduction to the uptake of antigen, the combination of reduction with NK cells, the combination of reduction with macrophages, the combination of reduction with monocytes, the combination of reduction with polymorphonuclear cells, the apoptosis induced by direct signaling of reduction, the cross-linking of the target binding antibodies of reduction, the dendritic cell maturation of reduction, or the T cell sensitization of reduction. In one aspect, the effector function of reduction is the effector function of one or more reductions selected from the group of the CDC of reduction, the ADCC of reduction, the ADCP of reduction and the cytokine secretion of reduction. In a preferred aspect, the effector function of reduction is the ADCC of reduction. In one aspect, the ADCC of reduction is less than 20% of the ADCC induced by non-engineered Fc domains (or (multi-specific) antibodies comprising the non-engineered Fc domains).

[0559] In one aspect, the amino acid mutation that reduces the binding affinity of Fc domain to Fc acceptor and / or effector function is amino acid replacement.In one aspect, described Fc domain comprises amino acid replacement at the position selected from the group comprising E233, L234, L235, N297, P331 and P329 (according to Kabat EU index numbering).In a more specific aspect, described Fc domain comprises amino acid replacement at the position selected from the group comprising L234, L235 and P329 (according to Kabat EU index numbering).In some aspects, described Fc domain comprises amino acid replacement L234A and L235A (according to Kabat EU index numbering).In such aspect, Fc domain is IgG1 Fc domain, particularly human IgG1 Fc domain.In one aspect, described Fc domain comprises amino acid replacement at position P329.In a more specific aspect, amino acid replacement is P329A or P329G, particularly P329G (according to Kabat EU index numbering). In one aspect, the Fc domains comprise amino acid replacement at position P329, and comprise other amino acid replacement at the position selected from E233, L234, L235, N297 and P331 (according to Kabat EU index numbering).In a more specific aspect, described other amino acid replacement is E233P, L234A, L235A, L235E, N297A, N297D or P331S.In preferred aspects, the Fc domains comprise amino acid replacement at position P329, L234 and L235 (according to Kabat EU index numbering).In more preferred aspects, the Fc domains comprise amino acid mutation L234A, L235A and P329G (" P329G LALA ", " PGLALA " or " LALAPG "). In particular, in a preferred aspect, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e., in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to the Kabat EU index).

[0560] In one such aspect, the Fc domain is an IgG1 Fc domain, in particular a human IgG1 Fc domain. The amino acid substitution "P329G LALA" combination almost completely abolishes Fcγ receptor (and complement) binding of the human IgG1 Fc domain, as described in PCT Publication No. WO 2012 / 130831, the entire contents of which are incorporated herein by reference. WO 2012 / 130831 also describes methods for preparing such mutant Fc domains and methods for determining their properties (such as Fc receptor binding or effector function).

[0561] Compared with IgG1 antibody, IgG4 antibody shows reduced binding affinity to Fc acceptors and reduced effector function.Therefore, in some aspects, the Fc domain of (multi-specific) antibody of the present invention is IgG4 Fc domain, particularly human IgG4 Fc domain.In one aspect, IgG4 Fc domain is included in the amino acid replacement at S228 position, particularly amino acid replacement S228P (according to Kabat EU index numbering).In order to further reduce its binding affinity to Fc acceptors and / or its effector function, in one aspect, IgG4 Fc domain comprises the amino acid replacement at L235 position, particularly amino acid replacement L235E (according to Kabat EU index numbering).On the other hand, IgG4 Fc domain comprises the amino acid replacement at P329 position, particularly amino acid replacement P329G (according to Kabat EU index numbering). In a preferred aspect, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235, and P329, particularly amino acid substitutions S228P, L235E, and P329G (numbered according to the Kabat EU index). Such IgG4 Fc domain mutants and their Fcγ receptor binding properties are described in PCT Publication No. WO 2012 / 130831, the entire contents of which are incorporated herein by reference.

[0562] In a preferred aspect, the Fc domain that exhibits reduced binding affinity to an Fc receptor and / or reduced effector function compared to a native IgG1 Fc domain is a human IgG1 Fc domain comprising amino acid substitutions L234A, L235A and optionally P329G, or is a human IgG4 Fc domain comprising amino acid substitutions S228P, L235E and optionally P329G (numbering according to the Kabat EU index).

[0563] In certain aspects, N-glycosylation of the Fc domain has been eliminated. In one such aspect, the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine with alanine (N297A) or aspartic acid (N297D) (numbering according to the Kabat EU index).

[0564] In addition to the Fc domains described above and in PCT Publication No. WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or decreased effector function also include those with substitutions of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (numbered according to the EU index of Kabat). Such Fc mutants include Fc mutants with substitutions at two or more of amino acids 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0565] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods can include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The correct nucleotide changes can be verified, for example, by sequencing.

[0566] Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instruments (such as BIAcore instruments (GE Healthcare)), and Fc receptors can be obtained, for example, by recombinant expression. Alternatively, cell lines known to express specific Fc receptors (e.g., human NK cells expressing FcγIIIa receptors) can be used to assess the binding affinity of Fc domains or (multispecific) antibodies comprising Fc domains to Fc receptors.

[0567] The effector functions of an Fc domain or a (multispecific) antibody comprising an Fc domain can be measured by methods known in the art. Examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al., Proc Natl Acad Sci USA 83,7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82,1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166,1351-1361 (1987). Alternatively, a non-radioactive assay can be used (see, for example, ACTI for flow cytometry). TM Non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0568] In some aspects, the binding of the Fc domain to complement components, particularly C1q, is reduced. Thus, in some aspects, wherein the Fc domain is engineered to have reduced effector function, the reduced effector function includes reduced CDC. C1q binding assays can be performed to determine whether the Fc domain or a (multispecific) antibody comprising an Fc domain is able to bind to C1q and therefore has CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0569] FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, eg, Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006); WO 2013 / 120929).

[0570] B. Polynucleotides

[0571] The present invention further provides an isolated polynucleotide encoding an antibody of the present invention. The isolated polynucleotide may be a single polynucleotide or a plurality of polynucleotides.

[0572] The polynucleotide encoding the (multi-specific) antibody of the present invention can be expressed as a single polynucleotide encoding a complete antibody, or as a plurality of (e.g., two or more) polynucleotides of co-expression. The polypeptide encoded by the co-expressed polynucleotide can be associated to form a functional antibody via, for example, a disulfide bond or other means. For example, the light chain portion of an antibody can be encoded by a polynucleotide separate from the portion of the antibody comprising the heavy chain of the antibody. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an antibody. In another example, an antibody comprising one of the subunits in two Fc domain subunits and optionally a part for one or more Fab molecules can be encoded by a polynucleotide separate from the antibody comprising another subunit in the two Fc domain subunits and optionally a part for a Fab molecule. When co-expressed, the Fc domain subunits will associate to form an Fc domain.

[0573] In some aspects, the isolated polynucleotide encodes a complete antibody molecule according to the invention as described herein. In other aspects, the isolated polynucleotide encodes a polypeptide contained in an antibody according to the invention as described herein.

[0574] In some aspects, the polynucleotide or nucleic acid is DNA. In other aspects, the polynucleotide of the present invention is RNA, for example in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

[0575] C. Recombination Methods

[0576] The antibodies of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding antibodies, such as those described above, are separated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such polynucleotides can be easily separated and sequenced using conventional methods. In one aspect, a vector comprising a polynucleotide of the present invention (i.e., a single polynucleotide or multiple polynucleotides) is provided, particularly an expression vector. Methods well known to those skilled in the art can be used to construct an expression vector containing the coding sequence of the antibody and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo recombination / genetic recombination. See, for example, the technology described in the following documents: Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY (1989). The expression vector can be a part of a plasmid, a virus, or can be a nucleic acid fragment. The expression vector includes an expression cassette, and the polynucleotide encoding the antibody (i.e., coding region) is cloned into the expression cassette in an operably associated manner with a promoter and / or other transcription or translation control elements. As used herein, a "coding region" is a portion of a nucleic acid that is composed of codons that are translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into amino acids, it (if present) can be considered to be a portion of the coding region, and any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc., are not a portion of the coding region. Two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector), or in a separate polynucleotide construct (e.g., on a separate (different) vector). In addition, any vector may contain a single coding region, or may comprise two or more coding regions, such as a vector of the present invention may encode one or more polypeptides that are separated into final proteins by proteolytic cleavage after translation or during translation. In addition, the vectors, polynucleotides or nucleic acids of the present invention may encode heterologous coding regions, which may be fused or unfused to a polynucleotide encoding an antibody of the present invention or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains.Operable association is when the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences in a manner such that the expression of the gene product is under the influence or control of the regulatory sequences. If the induction of promoter function results in the transcription of mRNA encoding the desired gene product, and if the nature of the bond between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or interfere with the ability of the gene template to be transcribed, then the two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated". Thus, if the promoter is able to affect the transcription of the nucleic acid, the promoter region will be operably associated with the nucleic acid encoding the polypeptide. The promoter can be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. In addition to the promoter, other transcription control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These transcription control regions include, but are not limited to, those that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., immediate early promoter in conjunction with intron-A), simian virus 40 (e.g., early promoter), and retroviruses (such as, for example, Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes (such as actin, heat shock protein, bovine growth hormone, and rabbit beta-globin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcription control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translation control elements are known to those of ordinary skill in the art. These translation control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites, or IRES, also known as CITE sequences). The expression cassette may also include other features, such as an origin of replication, and / or chromosomal integration elements, such as retroviral long terminal repeats (LTRs), or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0577] The polynucleotides and nucleic acid coding regions of the present invention can be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of the polypeptides encoded by the polynucleotides of the present invention. For example, if secretory antibodies are desired, DNA encoding a signal sequence can be placed upstream of the nucleic acid of the antibody of the present invention or its fragment. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once the growing protein chain has been exported across the rough endoplasmic reticulum. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the translated polypeptide to produce a secreted or "mature" form of the polypeptide. In certain aspects, a native signal peptide (e.g., an immunoglobulin heavy or light chain signal peptide) is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide to which it is operably associated. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse beta-glucuronidase.

[0578] DNA encoding a short protein sequence that can be used to facilitate subsequent purification (eg, a histidine tag) or to aid in labeling the antibody can be included internally or at the end of the antibody (fragment) encoding polynucleotide.

[0579] In another aspect, a host cell comprising a polynucleotide of the present invention (i.e., a single polynucleotide or multiple polynucleotides) is provided. In certain aspects, a host cell comprising a vector of the present invention is provided. The polynucleotides and vectors may be incorporated herein, alone or in combination, into any of the features described herein for the polynucleotides and vectors, respectively. In one such aspect, the host cell comprises one or more vectors (e.g., transformed or transfected with one or more vectors), the one or more vectors comprising one or more polynucleotides encoding (a portion of) an antibody of the present invention. As used herein, the term "host cell" refers to any type of cell system that can be engineered to produce an antibody of the present invention or a fragment thereof. Host cells suitable for replication and support antibody expression are well known in the art. Such cells can be appropriately transfected or transduced with specific expression vectors, and large quantities of cells containing the vector can be grown to inoculate large-scale fermenters to obtain sufficient amounts of antibodies for clinical applications. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, polypeptides can be produced in bacteria, particularly when glycosylation is not required. After expression, the polypeptide can be separated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptides, including fungi and yeast strains whose glycosylation pathways have been "humanized", thereby resulting in the production of polypeptides with partial or complete human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for expressing (glycosylated) polypeptides also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains that can be used with insect cells have been identified, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TMVertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells, as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT060562), TRI cells (as described, e.g., in Mather et al., Annals NY Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr - CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines, such as YO, NS0, P3X63 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as, for example, mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, and also include cells contained in transgenic animals, transgenic plants or cultured plants or animal tissues. In one aspect, host cells are eukaryotic cells, particularly mammalian cells, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells or lymphocytes (e.g., Y0, NS0, Sp20 cells). In one aspect, host cells are not cells in the human body.

[0580] Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing polypeptides comprising antigen binding domains, such as heavy or light chains of antibodies, can be engineered to also express the other antibody chain so that the expressed product is an antibody having both heavy and light chains.

[0581] In one aspect, a method of producing an antibody according to the invention is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding an antibody as provided herein under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0582] The components of the (multispecific) antibodies of the present invention can be genetically fused to each other. The (multispecific) antibodies can be designed so that their components are fused to each other directly or indirectly through a linker sequence. The composition and length of the linker can be determined according to methods well known in the art, and the efficacy of the linker can be tested. Examples of linker sequences between the different components of the (multispecific) antibodies are provided herein. If necessary, additional sequences (e.g., endopeptidase recognition sequences) can also be included to incorporate cleavage sites to separate the fused components.

[0583] Antibodies prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen that binds to the antibody can be used. For example, for affinity chromatography purification of the antibodies of the present invention, a matrix with protein A or protein G can be used. Substantially as described in the examples, sequential protein A or G affinity chromatography and size exclusion chromatography can be used to separate the antibodies. The purity of the antibody can be determined by any of the various analytical methods known to all, including gel electrophoresis, high pressure liquid chromatography, and the like.

[0584] D. Determination

[0585] The physical / chemical properties and / or biological activities of the antibodies provided herein can be identified, screened, or characterized by various assays known in the art.

[0586] 1. Binding Assay

[0587] The binding (affinity) of an antibody to a target antigen or Fc receptor can be determined, for example, by surface plasmon resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare) and receptors or target antigens such as those that can be obtained by recombinant expression. Alternatively, binding of an antibody to a different receptor or target antigen can be assessed, for example, by flow cytometry (FACS) using cell lines expressing a specific receptor or target antigen. Specific illustrative and exemplary aspects for measuring binding activity to HLA-A2 / MAGE-A4 are described below.

[0588] In one aspect, the binding affinity of the (multispecific) antibodies of the invention to HLA-A2 / MAGE-A4 is determined by SPR as follows:

[0589] SPR was performed on a Biacore T200 instrument (GE Healthcare) at 25°C using HBS-EP+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.005% surfactant P20 (GE Healthcare, #BR-1006-69)) as a running buffer. Anti-human Fc-specific antibodies (GE Healthcare, #BR-1008-39) were directly immobilized on a CM5 chip (GE Healthcare) via amine coupling. HLA-A2 / MAGE-A4 antibodies were captured at 2.5 nM for 60 seconds. HLA-A2 / MAGE-A4 p230-239 A three-fold dilution series of the complex in HBS-EP (6.17 to 1500 nM) was passed over the ligand at a rate of 30 μl / min for 240 seconds to record the association phase. The dissociation phase was monitored for 240 seconds and triggered by switching from the sample solution to HBS-EP+. After each cycle, the chip surface was regenerated using a 30-second injection of 3M MgCl2 at a rate of 10 μl / min. Bulk refractive index differences were corrected by subtracting the response obtained on a reference flow cell containing anti-human Fc antibodies but without HLA-A2 / MAGE-A4 antibodies captured thereon. Affinity constants were derived from kinetic rate constants by fitting to 1:1 Langmuir binding using BIAeval software (GE Healthcare).

[0590] 2. Activity Assay

[0591] The biological activity of the (multispecific) antibodies of the invention can be measured by various assays as described in the Examples. Biological activities can, for example, include induction of T cell proliferation, induction of signaling in T cells, induction of expression of activation markers in T cells, induction of T cell cytokine secretion, induction of target cell (e.g., tumor cell) lysis, and induction of tumor regression and / or improved survival.

[0592] E. Compositions, Formulations, and Routes of Administration

[0593] In another aspect, the present invention provides a pharmaceutical composition comprising any of the antibodies provided herein, for example, for use in any of the following methods of treatment. In one aspect, the pharmaceutical composition comprises an antibody according to the present invention and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises an antibody according to the present invention and at least one additional therapeutic agent as described below.

[0594] Also provided is a method of producing an antibody of the invention in a form suitable for in vivo administration, the method comprising (a) obtaining an antibody according to the invention, and (b) formulating the antibody with at least one pharmaceutically acceptable carrier, thereby formulating the antibody preparation for in vivo administration.

[0595] The pharmaceutical composition of the present invention comprises an effective amount of antibody dissolved or dispersed in a pharmaceutical carrier. The term "pharmaceutically acceptable" refers to that molecular entities and compositions are generally nontoxic to the recipient at the dosage and concentration employed, i.e., do not produce adverse, allergic or other adverse reactions when applied to animals (e.g., humans) as appropriate. According to the present disclosure, the preparation of pharmaceutical compositions containing antibodies and optionally other active ingredients will be known to those skilled in the art, as illustrated by Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, which is incorporated herein by reference. In addition, for animal (e.g., humans) administration, it should be understood that the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards or other national / regional authorities. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, similar substances and combinations thereof, as would be known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). Except in the case where any conventional carrier is incompatible with the active ingredient, the use of such carriers in pharmaceutical compositions is contemplated.

[0596] The antibodies of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if desired for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Dosing can be performed by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or chronic.

[0597] Parenteral compositions include those designed for injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection). For injection, the antibodies of the present invention can be formulated in an aqueous solution, particularly in a physiologically compatible buffer (e.g., Hanks solution, Ringer solution or physiological saline buffer). The solution may contain a formulation agent (formulatory agent), such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, the antibody can be in powder form for construction with a suitable vehicle (e.g., sterile pyrogen-free water) before use. Sterile injectable solutions are prepared by incorporating the antibodies of the present invention into a suitable solvent in the desired amount together with the various other ingredients listed below as needed. For example, sterility can be easily achieved by filtration through a sterile filtration membrane. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and / or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred preparation method is vacuum drying or lyophilization techniques, which produce a powder of the active ingredient plus any additional required ingredients from a previously sterile-filtered liquid medium. If necessary, the liquid medium should be suitably buffered, and sufficient saline or glucose should first be used to make the liquid diluent isotonic before injection. The composition must be stable under the conditions of manufacture and storage and be preserved to resist the contaminating effects of microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to a minimum, for example, at a safety level below 0.5 ng / mg protein. Suitable pharmaceutical carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as Serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, etc. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow the preparation of high concentration solutions.Alternatively, suspensions of the active compound may be prepared ...

Claims

1. An antibody that binds to HLA-A2 / MAGE-A4, wherein the antibody comprises a first antigen-binding domain comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 25, HCDR 2 of SEQ ID NO: 26, and HCDR 3 of SEQ ID NO: 27; and A light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO:

30.

2. The antibody of claim 1, wherein the first antigen binding domain comprises a VH comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 31; and / or a VL comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

32.

3. An antibody that binds to HLA-A2 / MAGE-A4, wherein the antibody comprises a first antigen-binding domain comprising VH, which is shown in SEQ ID NO:31; and VL, which is shown in SEQ ID NO:

32. 4 . The antibody according to claim 1 , wherein the antibody is a bispecific antibody that binds to HLA-A2 / MAGE-A4 and CD3.

5. The antibody of any one of claims 1-3, wherein the antibody comprises a second antigen binding domain that binds to CD3.

6. The antibody of claim 5, wherein the second antigen binding domain comprises (i) a VH comprising HCDR1 of SEQ ID NO: 59, HCDR 2 of SEQ ID NO: 60, and HCDR3 of SEQ ID NO: 61; and a VL comprising LCDR 1 of SEQ ID NO: 62, LCDR 2 of SEQ ID NO: 63, and LCDR 3 of SEQ ID NO: 64; or (ii) a VH comprising HCDR 1 of SEQ ID NO: 51, HCDR 2 of SEQ ID NO: 52, and HCDR 3 of SEQ ID NO: 53; and a VL comprising LCDR 1 of SEQ ID NO: 54, LCDR 2 of SEQ ID NO: 55, and LCDR 3 of SEQ ID NO:

56.

7. The antibody of claim 6, wherein the second antigen binding domain comprises (i) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 65; and / or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 66; or (ii) a VH comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 57; and / or a VL comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

58.

8. The antibody of claim 5, wherein the second antigen binding domain comprises (i) VH, which is set forth in SEQ ID NO: 65; and VL, which is set forth in SEQ ID NO: 66; (ii) VH, which is shown in SEQ ID NO: 57; and VL, which is shown in SEQ ID NO:

58.

9. The antibody of claim 5, wherein the antibody comprises a third antigen binding domain that binds to HLA-A2 / MAGE-A4.

10. The antibody of claim 9, wherein the third antigenic domain comprises VH, which comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:25, HCDR2 of SEQ ID NO:26, and HCDR 3 of SEQ ID NO:27; and a light chain variable region (VL), which comprises a light chain complementarity determining region (LCDR) 1 of SEQ ID NO:28, LCDR 2 of SEQ ID NO:29, and LCDR 3 of SEQ ID NO:

30.

11. The antibody of claim 10, wherein the third antigen binding domain comprises a VH comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 31; and / or a VL comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

32.

12. The antibody of claim 9, wherein the third antigen binding domain comprises VH, which is shown in SEQ ID NO:31; and VL, which is shown in SEQ ID NO:

32.

13. The antibody of claim 9, wherein the third antigen binding domain is identical to the first antigen binding domain.

14. The antibody of claim 1, wherein the first antigen binding domain is a Fab molecule.

15. The antibody of claim 5, wherein the second antigen binding domain is a Fab molecule.

16. The antibody of claim 9, wherein the third antigen binding domain is a Fab molecule. 17 . The antibody according to claim 5 , wherein the second antigen-binding domain is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other or the constant domains CL and CH1 are replaced with each other.

18. The antibody of claim 17, wherein the first antigen binding domain is a conventional Fab molecule.

19. The antibody of claim 18, wherein in the first antigen-binding domain, in the constant domain CL, the amino acid at position 124 according to the Kabat numbering is independently substituted by lysine (K), arginine (R) or histidine (H), and the amino acid at position 123 according to the Kabat numbering is independently substituted by lysine (K), arginine (R) or histidine (H); and in the constant domain CH1, the amino acid at position 147 according to the Kabat EU index is independently substituted by glutamic acid (E) or aspartic acid (D), and the amino acid at position 213 according to the Kabat EU index is independently substituted by glutamic acid (E) or aspartic acid (D).

20. The antibody of claim 9, wherein the second antigen-binding domain is a Fab molecule, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other or the constant domains CL and CH1 are replaced with each other.

21. The antibody of claim 20, wherein the first antigen binding domain and the third antigen binding domain are each conventional Fab molecules.

22. The antibody of claim 21 , wherein in the first antigen-binding domain and in the third antigen-binding domain, in the constant domain CL, the amino acid at position 124 according to the Kabat numbering is independently substituted by lysine (K), arginine (R) or histidine (H), and the amino acid at position 123 according to the Kabat numbering is independently substituted by lysine (K), arginine (R) or histidine (H); and in the constant domain CH1, the amino acid at position 147 according to the Kabat EU index is independently substituted by glutamic acid (E) or aspartic acid (D), and the amino acid at position 213 according to the Kabat EU index is independently substituted by glutamic acid (E) or aspartic acid (D).

23. The antibody of claim 5, wherein the first antigen-binding domain and the second antigen-binding domain are fused to each other.

24. The antibody of claim 23, wherein the first antigen-binding domain and the second antigen-binding domain are fused to each other via a peptide linker.

25. The antibody of claim 23 or 24, wherein the first antigen binding domain and the second antigen binding domain are each a Fab molecule, and (i) the first antigen binding domain 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 domain, or (ii) the second antigen binding domain 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 domain.

26. The antibody of claim 1, wherein the antibody comprises an Fc domain composed of a first subunit and a second subunit.

27. The antibody of claim 5, wherein the first antigen-binding domain and the second antigen-binding domain are each a Fab molecule, and the antibody comprises an Fc domain composed of a first subunit and a second subunit; and (i) the first antigen-binding domain 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 domain, and the second antigen-binding domain 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 second antigen-binding domain 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 domain, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain.

28. The antibody of claim 9, wherein the first, second, and third antigen-binding domains are each a Fab molecule, and the antibody comprises an Fc domain composed of a first and a second subunit; and (i) the first antigen-binding domain 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 domain, and the second antigen-binding domain 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 second antigen-binding domain 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 domain, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; and the third antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

29. The antibody of any one of claims 26 to 28, wherein the Fc domain is an IgG Fc domain.

30. The antibody of claim 29, wherein the Fc domain is an IgG1 Fc domain.

31. The antibody of any one of claims 26 to 28, wherein the Fc domain is a human Fc domain.

32. The antibody of any one of claims 26 to 28, wherein the Fc domain is a human IgG1 Fc domain.

33. The antibody of any one of claims 26 to 28, wherein the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain.

34. The antibody of any one of claims 26 to 28, wherein amino acid residues in the CH3 domain of the first subunit of the Fc domain are substituted with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit, and the protrusion is positionable in a cavity in the CH3 domain of the second subunit, and amino acid residues in the CH3 domain of the second subunit of the Fc domain are substituted with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, and the protrusion in the CH3 domain of the first subunit is positionable in the cavity.

35. The antibody according to any one of claims 26 to 28, wherein in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain, The tyrosine residue at position 407 was replaced by a valine residue (Y407V), numbered according to the Kabat EU index.

36. The antibody according to claim 35, wherein in the second subunit of the Fc domain, additionally, 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), numbered according to the Kabat EU index.

37. The antibody of claim 35 or 36, wherein 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), and in the second subunit of the Fc domain, additionally, the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C), numbered according to the Kabat EU index.

38. The antibody of any one of claims 26 to 28, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or decrease effector function.

39. The antibody of claim 38, wherein the one or more amino acid substitutions are at a position selected from the group consisting of E233, L234, L235, N297, P331 and P329, numbered according to the Kabat EU index.

40. The antibody of any one of claims 26 to 28, wherein each subunit of the Fc domain comprises amino acid substitutions L234A, L235A, and P329G, numbered according to the Kabat EU index.

41. The antibody of claim 3, comprising: a) a first antigen-binding domain and a third antigen-binding domain that bind to HLA-A2 / MAGE-A4, wherein each of the first antigen-binding domain and the third antigen-binding domain is a conventional Fab molecule comprising a heavy chain variable region as shown in SEQ ID NO: 31 and a light chain variable region as shown in SEQ ID NO: 32; b) a second antigen-binding domain that binds to CD3, wherein the second antigen-binding domain is a Fab molecule in which the variable regions VL and VH of the Fab light chain and Fab heavy chain are replaced with each other, comprising the heavy chain variable region set forth in SEQ ID NO: 65 and the light chain variable region set forth in SEQ ID NO: 66; c) a human IgG1 Fc domain, which consists of a first subunit and a second subunit; in (i) in the constant domain CL of the first antigen-binding domain and the third antigen-binding domain, the amino acid at position 124 according to Kabat numbering is substituted by lysine (K), and the amino acid at position 123 according to Kabat numbering is substituted by arginine (R); and in the constant domain CH1 of the first antigen-binding domain and the third antigen-binding domain, the amino acid at position 147 according to the Kabat EU index is substituted by glutamic acid (E), and the amino acid at position 213 according to the Kabat EU index is substituted by glutamic acid (E); (ii) the first antigen-binding domain 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 domain, and the second and third antigen-binding domains are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain; and (iii) the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V, and each subunit of the Fc domain comprises amino acid substitutions L234A, L235A, and P329G, as numbered according to the Kabat EU index.

42. The antibody of claim 41 , comprising: a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 68, a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 69, two polypeptides comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 70, and a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

72.

43. The antibody of claim 4, comprising: the polypeptide shown in SEQ ID NO: 68, the polypeptide shown in SEQ ID NO: 69, two polypeptides shown in SEQ ID NO: 70, and the polypeptide shown in SEQ ID NO:

72.

44. An isolated polynucleotide encoding the antibody of any one of claims 1 to 43.

45. A host cell comprising the isolated polynucleotide of claim 44.

46. ​​A method for producing an antibody that binds to HLA-A2 / MAGE-A4, the method comprising the steps of: (a) culturing the host cell according to claim 45 under conditions suitable for expressing the antibody.

47. The method according to claim 46, further comprising the steps of: (b) recovering the antibody.

48. An antibody that binds to HLA-A2 / MAGE-A4, produced by the method according to claim 46 or 47.

49. A pharmaceutical composition comprising the antibody of any one of claims 1 to 43 or claim 48 and a pharmaceutically acceptable carrier.

50. The antibody according to any one of claims 1 to 43 or claim 48 or the pharmaceutical composition according to claim 49, characterized in that The antibody or the pharmaceutical composition is used as a medicament.

51. The antibody according to any one of claims 1 to 43 or claim 48 or the pharmaceutical composition according to claim 49, characterized in that The antibody or the pharmaceutical composition is used in the treatment of cancer.

52. The antibody according to any one of claims 1 to 43 or claim 48 or the pharmaceutical composition according to claim 49, characterized in that The antibody or the pharmaceutical composition is used in the treatment of cancer expressing HLA-A2 / MAGE-A4.

53. The antibody according to any one of claims 1 to 43 or claim 48 or the pharmaceutical composition according to claim 49, characterized in that The antibody or the pharmaceutical composition is used in the treatment of a cancer selected from the group consisting of lung cancer, head and neck cancer, bladder cancer, esophageal cancer, skin cancer, gastric cancer, and ovarian cancer.

54. Use of an antibody according to any one of claims 1 to 43 or claim 48 or a pharmaceutical composition according to claim 49 in the manufacture of a medicament for treating lung cancer, bladder cancer, or melanoma.

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