Antibodies binding to cd3, production methods, pharmaceutical compositions and uses thereof

Abstract

This invention generally relates to antibodies that bind to CD3, including, for example, multispecific antibodies for activating T cells. Additionally, this invention relates to polynucleotides encoding such antibodies, as well as vectors and host cells containing such polynucleotides. The invention further relates to methods for generating such antibodies, and methods for treating diseases using such antibodies.

Description

Technical field

[0001] The present invention generally relates to antibodies that bind to CD3, including, for example, multispecific antibodies for the activation of T cells. In addition, the present invention relates to polynucleotides encoding the antibodies, as well as vectors and host cells comprising the polynucleotides. The invention further relates to methods of producing such antibodies, and methods of using such antibodies to treat diseases.

Prior Technology

[0002] CD3 (cluster of differentiation 3) is a protein complex composed of four subunits, CD3γ chain, CD3δ chain and two CD3ɛ chains. CD3 associates with the T cell receptor and ζ chain, generating an activation signal in T lymphocytes.

[0003] CD3 has been explored as a drug target. Monoclonal antibodies targeting CD3 have been used as immunosuppressant therapy in autoimmune diseases such as type 1 diabetes or in the treatment of transplant rejection. In 1985, the CD3 antibody murozumab-CD3 (OKT3) became the first monoclonal antibody approved for clinical use in humans.

[0004] A more recent application of CD3 antibodies is in the form of bispecific antibodies, which bind CD3 on the one hand and tumor cell antigens on the other. Simultaneous binding of such an antibody to its two targets will force a transient interaction between the target cell and the T cell, resulting in the activation of any cytotoxic T cells and subsequent lysis of the target cell.

[0005] An important requirement that antibodies must fulfill for therapeutic purposes is to have sufficiently high stability both in vitro (for drug storage) and in vivo (after administration to a patient).

[0006] Modification (such as asparagine deamidation) is a typical degradation pathway of recombinant antibodies, which may affect the stability in vitro and biological function in vivo.

[0007] Given the enormous therapeutic potential of antibodies, in particular bispecific antibodies that activate T cells, there is a need for CD3 antibodies with optimized properties.

Content of invention

[0008] The present invention provides antibodies, including multispecific (eg, bispecific) antibodies, that bind CD3 with good affinity and are resistant to degradation such as asparagine deamidation, thus Especially stable when required for therapeutic purposes. The provided (multispecific) antibodies combine good efficacy (e.g. target cell cytotoxicity) with manufacturability and low toxicity (e.g. no activation of T cells in the absence of target cells) with favorable drug Metabolokinetic properties.

[0009] As shown herein, the CD3-binding antibodies (including multispecific antibodies) provided by the present invention have higher binding activity to CD3 after being placed at pH 7.4 and 37°C for 2 weeks than at pH 6 and -80°C About 95% or more of the binding activity was retained after 2 weeks of storage, as measured by surface plasmon resonance (SPR).

[0010] In one aspect, the present invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen binding domain comprising (i) a heavy chain variable region (VH) selected from the group consisting of Groups: (a) VHs comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10, (b) VHs of HCDR 1 comprising SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4, and HCDR 3 of SEQ ID NO: 12, (c) a VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 1 of SEQ ID NO: 5 HCDR 2 and HCDR 3 of SEQ ID NO: 9, (d) a VH comprising HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 6 and HCDR 3 of SEQ ID NO: 11, or (e) VH comprising HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 7 and HCDR 3 of SEQ ID NO: 13, and (ii) a light chain variable region (VL) comprising SEQ ID NO: Light chain complementarity determining region (LCDR) 1 of 20, LCDR 2 of SEQ ID NO: 21 and LCDR 3 of SEQ ID NO: 22.

[0011] In one aspect, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19, and / or VL comprises at least about 95% of the amino acid sequence of SEQ ID NO: 23 , 96%, 97%, 98%, 99% or 100% identical amino acid sequences.

[0012] In yet another aspect, the present invention provides an antibody binding to CD3, wherein the antibody comprises a first antigen binding domain comprising a VH sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19; and the VL sequence of SEQ ID NO: 23.

[0013] In one aspect, the first antigen binding domain is a Fab molecule.

[0014] In one aspect, the antibody comprises an Fc domain consisting of a first unit and a second unit.

[0015] In one aspect, an antibody comprises a second antigen binding domain that binds a second antigen and optionally a third antigen binding domain.

[0016] In one aspect, the second antigen binding domain and / or the third antigen binding domain, if present, is a Fab molecule.

[0017] In one aspect, 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 or the constant domains CL and CH1, in particular the variable domains VL and VH are mutually replace.

[0018] In one aspect, the second antigen binding domain and, if present, the third antigen binding domain are conventional Fab molecules.

[0019] In one aspect, the second antigen binding domain and, when present, the third antigen binding domain are Fab molecules wherein, in the constant domain CL, the amino acid at position 124 is independently replaced by lysine (K ), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index), And the amino acid at position 213 was independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

[0020] In one aspect, the first antigen binding domain and the second antigen binding domain are fused to each other, optionally via a peptide linker.

[0021] In one aspect, the first antigen-binding domain and the second antigen-binding domain are each a Fab molecule, and (i) the second antigen-binding domain is at the C-terminus of the Fab heavy chain and the Fab heavy chain of the first antigen-binding domain or (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.

[0022] In one aspect, each of the first antigen binding domain, the second antigen binding domain and, when present, the third antigen binding domain is a Fab molecule, and the antibody comprises an Fc composed of the first unit and the second unit and wherein, (i) 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 at the C-terminus of the Fab heavy chain fused to the N-terminus of the first unit of the Fc domain, or (ii) 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, and the second antigen-binding domain The domain is fused to the N-terminus of the first unit of the Fc domain at the C-terminus of the Fab heavy chain; and the third antigen-binding domain, when present, is N-terminal to the second unit of the Fc domain at the C-terminus of the Fab heavy chain fusion.

[0023] In one aspect, the Fc domain is an IgG Fc domain, in particular an IgG1 Fc domain. In one aspect, the Fc domain is a human Fc domain. In one aspect, the Fc comprises modifications that facilitate the association of the first and second units of the Fc domain. In one aspect, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

[0024] In one aspect, the second antigen is a target cell antigen, in particular a tumor cell antigen.

[0025] In one aspect, the second antigen is TYRP-1. In one aspect, the second antigen binding domain and, when present, the third antigen binding domain comprise: VH comprising HCDR 1 of SEQ ID NO: 24, HCDR 2 of SEQ ID NO: 25 and HCDR 2 of SEQ ID NO: 26 HCDR 3; and VL comprising LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29, and LCDR 3 of SEQ ID NO: 30. In one aspect, the second antigen binding domain and, when present, the third antigen binding domain comprise: VH comprising at least about 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO: 27 , 99% or 100% identical amino acid sequence; and / or VL comprising at least about 95%, 96%, 97%, 98%, 99% or 100% of the amino acid sequence of SEQ ID NO: 31 the same amino acid sequence.

[0026] In one aspect, the second antigen is CEA. In one aspect, the second antigen binding domain and, when present, the third antigen binding domain comprise: (i) a VH comprising HCDR 1 of SEQ ID NO: 53, HCDR 2 of SEQ ID NO: 54 and SEQ ID NO HCDR 3 of : 55, and VL, which comprises LCDR 1 of SEQ ID NO: 57, LCDR 2 of SEQ ID NO: 58, and LCDR 3 of SEQ ID NO: 59; (ii) VH, which comprises SEQ ID NO: 105 HCDR 1 of SEQ ID NO: 106 and HCDR 3 of SEQ ID NO: 107, and VL comprising LCDR 1 of SEQ ID NO: 109, LCDR 2 of SEQ ID NO: 110 and SEQ ID NO: 111 or (iii) VH comprising HCDR 1 of SEQ ID NO: 113, HCDR 2 of SEQ ID NO: 114 and HCDR 3 of SEQ ID NO: 115, and VL comprising of SEQ ID NO: 117 LCDR 1, LCDR 2 of SEQ ID NO: 118 and LCDR 3 of SEQ ID NO: 119. In one aspect, the second antigen binding domain and, when present, the third antigen binding domain comprise: (i) VH comprising at least about 95%, 96%, 97% of the amino acid sequence of SEQ ID NO: 56 , 98%, 99% or 100% identical amino acid sequence, and / or VL comprising at least about 95%, 96%, 97%, 98%, 99% of the amino acid sequence of SEQ ID NO: 60 or a 100% identical amino acid sequence; (ii) a VH comprising an amine that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 108 An amino acid sequence, and / or a VL comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 112; or ( iii) VH comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 116, and / or 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: 120.

[0027] In one aspect, the second antigen is GPRC5D. In one aspect, the second antigen binding domain and, when present, the third antigen binding domain comprise: VH comprising HCDR 1 of SEQ ID NO: 61, HCDR 2 of SEQ ID NO: 62 and HCDR 2 of SEQ ID NO: 63 HCDR 3; and VL comprising LCDR 1 of SEQ ID NO: 65, LCDR 2 of SEQ ID NO: 66, and LCDR 3 of SEQ ID NO: 67. In one aspect, the second antigen binding domain and, when present, the third antigen binding domain comprise: VH comprising at least about 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO: 64 , 99% or 100% identical amino acid sequence; and / or VL comprising at least about 95%, 96%, 97%, 98%, 99% or 100% of the amino acid sequence of SEQ ID NO: 68 the same amino acid sequence.

[0028] In one aspect, the second antigen is CD19. In one aspect, the second antigen binding domain and, when present, the third antigen binding domain comprise: (i) a VH comprising HCDR 1 of SEQ ID NO: 75, HCDR 2 of SEQ ID NO: 76 and SEQ ID NO HCDR 3 of : 77, and VL comprising LCDR 1 of SEQ ID NO: 79, LCDR 2 of SEQ ID NO: 80, and LCDR 3 of SEQ ID NO: 81; or (ii) VH comprising SEQ ID NO: HCDR 1 of 83, HCDR 2 of SEQ ID NO: 84 and HCDR 3 of SEQ ID NO: 85, and VL comprising LCDR 1 of SEQ ID NO: 87, LCDR 2 of SEQ ID NO: 88 and SEQ ID NO: LCDR 3 of 89. In one aspect, the second antigen binding domain and, when present, the third antigen binding domain comprise: (i) VH comprising at least about 95%, 96%, 97% of the amino acid sequence of SEQ ID NO: 78 , 98%, 99% or 100% identical amino acid sequence, and / or VL comprising at least about 95%, 96%, 97%, 98%, 99% of the amino acid sequence of SEQ ID NO: 82 or a 100% identical amino acid sequence; or (ii) a VH comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 86 An amino acid sequence, and / or VL, comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 90.

[0029] According to another aspect of the present invention, there is provided an isolated polynucleotide encoding the antibody of the present invention, and a host cell comprising the isolated polynucleotide of the present invention.

[0030] In another aspect, there is provided a method of producing an antibody that binds to CD3, comprising the steps of: (a) cultivating the host cell of the invention under conditions suitable for expressing the antibody, and optionally (b ) to recover the antibody. The invention also encompasses an antibody that binds to CD3 produced by the method of the invention.

[0031] The present invention also provides a pharmaceutical composition comprising the antibody of the present invention and a pharmaceutically acceptable carrier.

[0032] The invention also encompasses methods of using the antibodies and pharmaceutical compositions of the invention. In one aspect, the invention provides an antibody or pharmaceutical composition for use as a medicament according to the invention. In one aspect, there is provided an antibody or pharmaceutical composition for use in treating a disease according to the present invention. In a specific aspect, the disease is cancer.

[0033] Also provided is the use of the antibody or pharmaceutical composition according to the present invention for the manufacture of a medicament, and the use of the antibody or pharmaceutical composition according to the present invention for the manufacture of a medicament for treating diseases, particularly cancer. The present invention also provides a method of treating a disease in a subject, which comprises administering to the subject an effective amount of the antibody or pharmaceutical composition according to the present invention.

Implementation

[0035] I. Define

[0036] Definitions Unless otherwise defined below, terms used herein are those of ordinary usage in the art.

[0037] The terms "first", "second" or "third" as used herein with respect to antigen binding domains etc. are used for convenience to distinguish when more than one of each type of moiety is present. Use of these terms is not intended to impart a particular order or direction to the parts unless explicitly stated.

[0038] The terms "anti-CD3 antibody" and "antibody that binds to CD3" refer to an antibody that is capable of binding CD3 with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting CD3. In one aspect, the anti-CD3 antibody binds to an irrelevant, non-CD3 protein to a degree that is about 10% less than the antibody binds to CD3, as measured by, for example, surface plasmon resonance (SPR). In certain aspects, antibodies that bind to CD3 have a dissociation constant (KD) of ≤ 1 μM, ≤ 500 nM, ≤ 200 nM, or ≤ 100 nM. An antibody is said to "specifically bind" CD3 when the antibody has a KD of 1 μM or less (e.g., as measured by SPR). In certain aspects, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3s of different species.

[0039] 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 (such as bispecific antibodies), and antibody fragments, so long as they are It is sufficient if the expected antigen-binding activity is exhibited.

[0040] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds 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 (such as scFv and scFab), single domain antibodies, and antibody-derived antibodies. Multispecific antibodies formed from fragments. For a review of certain antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0041] The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody.

[0042] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies contained in the population are identical and / or bind to the same antigenic determination Genes, but do not include, for example, antibodies containing naturally occurring mutations or possible variants arising during the production of monoclonal antibody preparations, which variants usually exist in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinant sites (epitopes), monoclonal antibody preparations have each monoclonal antibody directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques including, but not limited to, fusionoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin loci, These methods and other exemplary methods for making monoclonal antibodies are described herein.

[0043] An "isolated" antibody is one that has been separated from a component of its natural environment. In some aspects, antibodies are purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC, Affinity chromatography, particle size screening chromatography) method was determined. For a review of methods for assessing antibody purity, see, eg, Flatman et al., J. Chromatogr. B848:79-87 (2007). In certain aspects, the antibodies provided herein are isolated antibodies.

[0044] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0045] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one (and usually two) variable domains, wherein all or substantially all of the CDRs correspond to those of the non-human antibody, and all or substantially all of the FRs correspond to And so on for human antibodies. Such variable domains are referred to herein as "humanized variable regions". A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (eg, an antibody from which CDR residues are derived), eg, to restore or improve antibody specificity or affinity. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.

[0046] "Human antibody (human antibody)" is an antibody having an amino acid sequence corresponding to a human antibody coding sequence produced by a human or human cell or obtained from an antibody repertoire or other human antibody The amino acid sequence of an antibody derived from a non-human source. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. In certain aspects, human antibodies are derived from non-human transgenic mammals, such as mice, rats or rabbits. In certain aspects, human antibodies are derived from fusionoma cell lines. Antibodies or antibody fragments isolated from human antibody repertoires are also considered human antibodies or human antibody fragments herein.

[0047] The term "antigen-binding domain" refers to a portion of an antibody comprising a region that binds to and is complementary to part or all of an antigen. An antigen binding domain may be provided, for example, by one or more antibody variable domains (also known 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).

[0048] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of native antibodies generally have a similar structure, with each domain comprising four conserved framework regions (FR) and complementarity determining regions (CDR). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman & Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, VH or VL domains can be used to isolate antigen-binding antibodies from antigen-binding antibodies to screen libraries of complementary VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). "Kabat numbering" as used herein in connection with variable region sequences refers to that described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD (1991) numbering system.

[0049] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are according to those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) Kabat numbering system (referred to herein as "according to Kabat numbering" or "Kabat numbering") numbering. Specifically, the Kabat numbering system (see pages 647-660 of Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for kappa and The light chain constant domains of the lambda isotype CL and the Kabat and EU index numbering system (see pages 661-723) are used for the heavy chain constant domains (CH1, hinge, CH2 and CH3), which in this case are referred to herein by Refer to "According to Kabat EU Index Number" or "Kabat EU Index Number" for further clarification.

[0050] As used herein, the term "hypervariable region" or "HVR" refers to each region in the variable domain of an antibody that is highly variable in sequence and determines antigen-binding specificity, such as "complementarity determining regions" ("CDR") . Typically, antibodies include six CDRs: three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3). Herein, exemplary CDRs include: (a) A hypervariable loop exists at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1) , 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) the CDR is present at amino acid residue 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 Edition Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) antigen contacts are present 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)).

[0051] Unless otherwise stated, CDRs were determined according to the method described by Kabat et al., supra. Those skilled in the art will appreciate that CDR names may also be determined according to the methods described by Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0052] "Framework" or "FR" refers to the variable domain residues outside the complementarity determining regions (CDRs). The FR of a variable domain usually consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0053] Unless otherwise stated, CDR residues and other residues (eg, FR residues) in variable domains are numbered herein according to Kabat et al. (supra).

[0054] For the purposes herein, an "acceptor human framework" is a light chain variable domain (VL) framework or a heavy chain variable domain ( The backbone of the amino acid sequence of the VH) backbone is defined below. A recipient human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some aspects, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or more Less or 2 or less. In some aspects, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0055] A "human consensus framework" is a framework representing the most common amino acid residues in a series of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is that described by Kabat et al. in Sequences of Proteins of Immunological Interest (5th edition, NIH Publication 91-3242, Bethesda MD (1991), vol. 1-3).

[0056] As used herein the term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 daltons consisting of two light chains and two heavy chains that are disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable domain (VH), also known as heavy chain variable domain or heavy chain variable region, followed by three constant domains (CH1, CH2 and CH3), also known as for the heavy chain constant region. Similarly, from N-terminus to C-terminus, each light chain has a variable domain (VL), also known as light chain variable domain or light chain variable region, followed by a light chain constant (CL) domain, also known as Light chain constant region. The heavy chains of immunoglobulins can be classified as one of five classes, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), some of which can be further classified into Divided into subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). Based on the amino acid sequence of their constant domains, the light chains of immunoglobulins can be classified into one of two types, called kappa (κ) and lambda (λ). Immunoglobulins essentially consist of two Fab molecules and an Fc domain linked by the immunoglobulin hinge region.

[0057] 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 several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0058] "Fab molecule" means a protein consisting 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").

[0059] "Crossover" Fab molecule (also known as "Crossfab") means a Fab molecule in which the variable or constant domains of the Fab heavy chain and the Fab light chain are exchanged (i.e. replaced with each other), i.e., The exchanged Fab molecule consists of a peptide chain consisting of the light chain variable domain VL and the heavy chain constant domain 1 CH1 (VL-CH1, in the N-terminal to C-terminal direction), and the heavy chain variable domain VH and the light chain constant domain Peptide chain composed of domain CL (VH-CL, in N-terminal to C-terminal direction). For clarity, in a swapped Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain are swapped, the peptide chain comprising the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (swapped) Fab molecule. ". Conversely, in a swapped Fab molecule in which the constant domains of the Fab light chain and the Fab heavy chain are swapped, the peptide chain comprising the variable domain VH of the heavy chain is referred to herein as the "heavy chain" of the (swapped) Fab molecule.

[0060] In contrast, a "conventional" Fab molecule is meant in its natural form (i.e. comprising a heavy chain consisting of a heavy chain variable domain and a constant domain (VH-CH1, in the N-terminal to C-terminal direction) Fab molecule of a light chain (VL-CL, in N-terminal to C-terminal direction) consisting of chain variable and constant domains.

[0061] The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region. The term includes native sequence Fc regions as well as variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxy-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage at one or more, particularly one or two, amino acids at the C-terminus of the heavy chain. Thus, an antibody produced by a host cell expressing a particular nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or may include cleavage variants of the full-length heavy chain. This may be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the amino acid sequence of the heavy chain comprising the Fc region (or a subunit of an Fc domain as defined herein) is expressed herein without the C-terminal glycine-lysine dipeptide. In one aspect, the heavy chain comprising an Fc region (subunit) as described herein comprising an additional C-terminal glycine-lysine dipeptide (G446 and K447 according to Kabat EU index number). In one aspect, the heavy chain comprising the Fc region (subunit) described herein comprised in an antibody according to the invention comprises an additional C-terminal glycine residue (G446, numbered according to the Kabat EU index). Unless otherwise indicated herein, the numbering of amino acid residues in the Fc or constant regions is according to the EU numbering system (also known 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 (see also above). A "subunit" of an Fc domain, as used herein, refers to one of the two polypeptides forming a dimeric Fc domain, ie, a polypeptide comprising a C-terminal constant region capable of stabilizing self-associated heavy chain immunoglobulins. For example, the subunit of the IgG Fc domain contains IgG CH2 and IgG CH3 constant domains.

[0062] "Fusion" means that components (such as a Fab molecule and an Fc domain subunit) are linked via peptide bonds, either directly or via one or more peptide linkers.

[0063] The term "multispecific" means that an antibody is capable of specifically binding to at least two different epitopes. A multispecific antibody can be, for example, a bispecific antibody. Typically, bispecific antibodies comprise two antigen-binding sites, where each antigen-binding site is specific for a different epitope. In certain aspects, a multispecific (eg, bispecific) antibody is capable of simultaneously binding two epitopes, specifically two epitopes expressed on two different cells.

[0064] The term "valent" as used herein means the presence of a specified number of antigen binding sites in an antigen binding molecule. Thus, the term "monovalent binding to an antigen" indicates the presence of one (and not more than one) antigen-binding site specific for the antigen in the antigen-binding molecule.

[0065] "Antigen binding site" refers to a site, ie, 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 regions (CDRs). Native immunoglobulin molecules usually have two antigen-binding sites and Fab molecules usually have a single antigen-binding site.

[0066] As used herein, the term "antigenic determinant" or "epitope" refers to a portion of a polypeptide macromolecule that binds to an antigen-binding domain to form an antigen-binding domain-antigen complex. A site (eg, a continuous stretch of amino acids or a conformational configuration composed of distinct regions of non-contiguous amino acids). For example, available epitopes may be present 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, absent in serum, and / or present in in the extracellular matrix (ECM). In a preferred aspect, the antigen is a human protein.

[0067] Unless otherwise stated, "CD3" refers to any native CD3 derived from any vertebrate, including mammals, such as primates (e.g., humans), non-human primates (e.g., carnivorous cynomolgus monkeys) and rodents (such as mice and rats). The term encompasses "full-length", unprocessed CD3 as well as any form of CD3 that has been obtained by processing the cells. The term also covers naturally occurring CD3 variants, eg, splice variants or allele variants. In one aspect, the CD3 is human CD3, specifically the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in SEQ ID NO: 45 (no signal peptide). See also UniProt (www.uniprot.org) accession number P07766 (version 189), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. In another aspect, the CD3 is cynomolgus / Macaca fascicularis CD3, specifically cynomolgus CD3ɛ. The amino acid sequence of cynomolgus CD3ε is shown in SEQ ID NO: 46 (no signal peptide). See also NCBI GenBank No. BAB71849.1. In certain aspects, the antibodies of the invention bind to an epitope of CD3 that is conserved among CD3 antigens from different species, particularly human and cynomolgus CD3. In preferred aspects, the antibody binds to human CD3.

[0068] "Target cell antigen" as used herein refers to an epitope presented on the surface of a target cell, such as a cell in a tumor, such as a cancer cell or a cell of the tumor stroma (in In this case "tumor cell antigen"). Preferably, the target cell antigen is not CD3 and / or is expressed on a different cell than CD3. In one aspect, the target cell antigen is TYRP-1, specifically TYRP-1. In another aspect, the target cell antigen is CEA, specifically CEA. In yet another aspect, the target cell antigen is GPRC5D, specifically GPRC5D. In yet another aspect, the target cell antigen is CD19, specifically CD19.

[0069] Unless otherwise stated, the term "TYRP1" or "TYRP-1" stands for Tyrosine-related protein 1 (also known as 5,6-dihydroxyindole-2-carboxylate oxidase), and refers to Any native TYRP-1 derived from any vertebrate including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats) mouse). The term encompasses "full-length", unprocessed TYRP1 as well as any form of TYRP-1 that has been obtained by processing the cells. The term also encompasses naturally occurring variants of TYRP-1, for example, splice variants or allele variants. In one aspect, TYRP-1 is human TYRP-1. See also Human Protein UniProt (www.uniprot.org) accession number P17643 (version 195), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000541.1. In certain aspects, the antibodies of the invention bind to an epitope of TYRP-1 that is found in TYRP-1 antigens from different species, specifically human and cynomolgus TYRP-1 is conservative. In preferred aspects, the antibody binds to human TYRP-1.

[0070] Unless otherwise stated, the term "CEA" stands for Carcinoembryonic Antigen (also known as Carcinoembryonic Antigen-Associated Cell Adhesion Molecule 5 (CEACAM5)), and refers to any native CEA derived from any vertebrate that Included are mammals such as primates (eg, humans), non-human primates (eg, cynomolgus monkeys), and rodents (eg, mice and rats). The term encompasses "full-length", unprocessed CEA as well as any form of CEA obtained by processing cells. The term also covers naturally occurring variants of CEA, for example, splice variants or allele variants. In one aspect, the CEA is a human CEA. See also Human Protein UniProt (www.uniprot.org) accession number P06731 (version 195), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004354.2. In certain aspects, an antibody of the invention binds to an epitope of CEA that is conserved among CEA antigens from different species, particularly human and cynomolgus CEA. In preferred aspects, the antibody binds to human CEA.

[0071] Unless otherwise stated, the term "GPRC5D" stands for G protein-coupled receptor family C group 5 member D and refers to any native GPRC5D derived from any vertebrate including mammals such as primates Animals (such as humans), non-human primates (such as cynomolgus monkeys) and rodents (such as mice and rats). The term encompasses "full-length", unprocessed GPRC5D as well as any form of GPRC5D that has been obtained from the treatment of cells. The term also encompasses naturally occurring GPRC5D variants, eg, splice variants or allele variants. In one aspect, the GPRC5D is human GPRC5D. See also Human Protein UniProt (www.uniprot.org) accession number Q9NZD1 (version 131), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_061124.1. In certain aspects, an antibody of the invention binds to an epitope of GPRC5D that is conserved among GPRC5D antigens from different species, particularly human and cynomolgus GPRC5D. In preferred aspects, the antibody binds to human GPRC5D.

[0072] Unless otherwise stated, the term "CD19" stands for Cluster of Differentiation 19 (also known as B-lymphocyte antigen CD19 or B-lymphocyte surface antigen B4) and refers to any native CD19 derived from any vertebrate that Animals include mammals, such as primates (eg, humans), non-human primates (eg, cynomolgus monkeys), and rodents (eg, mice and rats). The term encompasses "full-length", unprocessed CD19 as well as any form of CD19 that results from the treatment of cells. The term also encompasses naturally occurring variants of CD19, for example, splice variants or allele variants. In one aspect, CD19 is human CD19. See also Human Protein UniProt (www.uniprot.org) accession number P15391 (version 211), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_001761.3. In certain aspects, the antibodies of the invention bind to an epitope of CD19 that is conserved among CD19 antigens from different species, particularly human and cynomolgus CD19. In preferred aspects, the antibody binds to human CD19.

[0073] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise stated, "binding affinity" as used herein refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can usually be expressed by the dissociation constant (KD). Affinity can be measured by established methods known in the art, including those described herein. A preferred method for determining affinity is Surface Plasmon Resonance (SPR).

[0074] The term "affinity matured" antibody refers to an antibody that has one or more changes in one or more complementarity determining regions (CDRs) that cause such changes compared to a parent antibody that does not have such changes. Improvement of the affinity of an antibody for an antigen.

[0075] "Reduced binding", eg reduced binding to an Fc receptor, means a decrease in the affinity of the respective interaction as measured, for example, by SPR. For clarity, the term also includes the reduction of the affinity to zero (or below the detection limit of the analytical method), ie the complete abrogation of the interaction. In contrast, "increased binding" refers to an increase in the binding affinity of the respective interaction.

[0076] As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes (specifically cytotoxic T lymphocytes), selected from: 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.

[0077] "Modification that promotes the association of the first unit and the second unit of the Fc domain" refers to manipulation of the peptide backbone or post-translational modification of the Fc domain subunit, which reduces or prevents the inclusion of the Fc domain subunit. The association of polypeptides of a unit with the same polypeptide forms a homodimer. As used herein, the association-promoting modification preferably includes a separate modification to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) for which association is desired, wherein, The modifications are complementary to each other in order to facilitate the association of the two Fc domain subunits. For example, association-promoting modifications can alter the structure or charge of one or both Fc domain subunits to make them sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which in turn is fused to the other set of each subunit (e.g., an antigen-binding domain). may vary. In some aspects, the modification that facilitates the association of the first and second units of the Fc domain comprises amino acid mutations, specifically amino acid substitutions, in the Fc domain. In a preferred aspect, the modification that facilitates the association of the first and second subunits of the Fc domain comprises individual amino acid mutations in each of the two subunits of the Fc domain, specifically the amine group Acid substitution.

[0078] The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody, which vary with 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 uptake by antigen-presenting cells mediates antigen, downregulation of cell surface receptors (eg, B-cell receptor), and B-cell activation.

[0079] "Activating Fc receptors" are Fc receptors that, after the participation of the Fc domain of an antibody, cause signal transduction events that stimulate receptor-carrying cells to perform effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0080] 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 in which antibodies or derivatives thereof contain an Fc region, which typically binds specifically to the Fc region through a protein moiety as the N-terminus. The term "reducing ADCC" as used herein refers to the cleavage of a target within a given time with a given concentration of antibody in the medium surrounding the target cell by the ADCC mechanism defined above A decrease in the number of cells, and / or an increase in the antibody concentration in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time period by the ADCC mechanism. Reduction of ADCC relative to ADCC mediated by the same antibody (but not yet engineered) produced by the same type of host cell using the same standard production, purification, formulation and storage methods (methods known to those of ordinary skill in the art) . For example, the reduction in ADCC mediated by an antibody comprising an ADCC-reducing amino acid substitution in the Fc domain is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, e.g., PCT Publication No. WO 2006 / 082515 or PCT Publication No. WO 2012 / 130831).

[0081] The terms "engineer, engineered, engineering" as used herein 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 modifying the amino acid sequence, glycosylation pattern, or side chain groups of individual amino acids, as well as combinations of these methods.

[0082] The term "amino acid mutation" as used herein is meant to encompass amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to arrive at the final construct provided that the final construct possesses the desired characteristics, for example, reduced binding to an Fc receptor or increased association with another peptide. Amino acid sequence deletions and insertions include deletions and insertions at the amino and / or carboxy termini of amino acids. Preferred amino acid mutations are amino acid substitutions. In order to change, for example, the binding characteristics of the Fc region, non-conservative amino acid substitutions, that is, substitution of one amino acid with another amino acid with different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include non-naturally occurring amino acids or naturally occurring amino acid derivatives with twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine acid, homoserine, 5-hydroxylysine) replacement. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that methods other than genetic engineering, such as chemical modification, to alter the side chain groups of amino acids may also be useful. Various names may be used herein to refer to mutations of the same amino acid. For example, a substitution of proline to glycine at position 329 of the Fc domain could be represented as 329G, G329, G329, P329G, or Pro329Gly.

[0083] The "percentage (%) amino acid sequence identity" described with respect to the reference polypeptide sequence refers to the percentage of the amino acid residues in the candidate sequence identical to the amino acid residues in the reference polypeptide sequence, in The maximum percent sequence identity is achieved after aligning the sequences and introducing differences, if necessary, and does not consider any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by various means that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA program package implementation. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, percent identity values ​​can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and its source code is filed with user documentation in the United States Copyright Office, Washington, DC 20559, USA, and it is registered (US Copyright Registration No. TXU510087) and described in WO 2001 / 007611.

[0084] Unless otherwise stated, for the purposes of this article, the ggsearch program with FASTA package version 36.3.8c or later was used with the BLOSUM50 comparison matrix to generate amino acid sequence identity % values. The FASTA package was developed 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 available from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta is publicly available. Alternatively, the public server accessed at fasta.bioch.virginia.edu / fasta_www2 / index.cgi can be used using the ggsearch (global protein:protein) program with default options (BLOSUM50; open: -10; ext : -2; Ktup = 2) Compares sequences to ensure that global rather than local alignments are performed. The percent amino acid identity is provided in the output alignment header.

[0085] The term "polynucleotide" or "nucleic acid molecule" includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), Sugar (ie, deoxyribose or ribose) and phosphate groups. Generally, nucleic acid molecules are described by base sequences, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually represented by 5' to 3'. As used herein, the term nucleic acid molecule includes: deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), specifically messenger RNA (mRNA); synthetic forms of DNA or RNA and hybrid polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. Additionally, the term nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein may comprise naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars, phosphate linkages, or chemically modified residues. Nucleic acid molecules also include DNA and RNA molecules suitable as vectors for direct expression of antibodies of the invention in vitro and / or in vivo, such as in a host or patient. Such DNA (eg, cDNA) or RNA (eg, mRNA) vectors may 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 encoded molecule, so that the mRNA can be injected into a living individual to produce antibodies (see for example Stadler et al. (2017) Nature Medicine 23:815-817 or EP 2 101 823 B1).

[0086] An "isolated" nucleic acid molecule refers to a nucleic acid molecule that has been separated from components 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 different from the natural chromosomal location.

[0087] "Isolated polynucleotide (or nucleic acid) encoding an antibody" refers to one or more polynucleotide molecules encoding antibody heavy and light chains (or fragments thereof), included in a single vector or a separate vector Such polynucleotide molecules in, and such polynucleotide molecules present at one or more locations in the host cell.

[0088] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of the host cell that has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. These vehicles are referred to herein as "expression vehicles."

[0089] The terms "host cell", "host cell strain" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny cells derived therefrom, regardless of the number of passages. The nucleic acid content of the progeny cells may not be identical to that of the parent cells, but may contain mutations. Included herein is the screening or selection of mutant progeny cells having the same function or biological activity from the original transformed cells. A host cell is any type of cellular system that can be used to produce the antibodies of the invention. Host cells include cultured cells, such as cultured mammalian cells, such as HEK cells, CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 Cells or fusion tumor cells, yeast cells, insect cells and plant cells, etc., also include cells in transgenic animals, transgenic plants or cultured plant or animal tissues. In one aspect, host cells of the invention are eukaryotic cells, in particular mammalian cells. In one aspect, the host cell is not a cell in the human body.

[0090] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation in a form that permits the biological activity of the active ingredients contained therein to be effective and which does not contain substances that would be unacceptable to the subject to whom the composition is administered. other toxic components.

[0091] "Pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical composition or formulation other than active ingredients that are non-toxic to individuals. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0092] As used herein, "treatment" (and its grammatical variants, such as "course of treatment" or "in treatment"), refers to a clinical intervention that attempts to alter the natural course of a disease in a subject being treated, and may prevent Or performed during clinical pathology. Desired therapeutic effects include, but are not limited to, prevention of occurrence or recurrence of disease, alleviation of symptoms, alleviation of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of rate of disease progression, amelioration or alleviation of disease state, and remission or improved 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.

[0093] 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 some aspects, the subject or individual is human.

[0094] A "therapeutically effective amount" of a medicament, such as a pharmaceutical composition, refers to an amount effective to achieve a desired therapeutic or prophylactic effect at a desired dosage and time period.

[0095] The term "package insert" is used to refer to instructions commonly included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, route of administration, combination therapy, Information such as contraindications and / or warnings. II. Composition and method

[0096] The invention provides antibodies that bind CD3, including multispecific antibodies that bind CD3 and a second antigen. The antibody exhibits excellent binding and stability, combined with other favorable properties for therapeutic applications (eg, in terms of efficacy and safety, pharmacokinetics, and manufacturability). Antibodies of the invention are useful, for example, in the treatment of diseases such as cancer. A. Anti-CD3 antibody

[0097] In one aspect, the invention provides antibodies that bind to CD3. In one aspect, isolated antibodies that bind CD3 are provided. In one aspect, the invention provides antibodies that specifically bind CD3. In certain aspects, the anti-CD3 antibody retains more than about 90% of its binding activity to CD3 after 2 weeks at pH 7.4 at 37°C relative to the binding activity at pH 6 at -80°C for 2 weeks (specifically 95%), as measured by surface plasmon resonance (SPR).

[0098] In one aspect, the present invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen binding domain comprising (i) a heavy chain variable region (VH) selected from the group consisting of Groups: (a) VHs comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10, (b) VHs of HCDR 1 comprising SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4, and HCDR 3 of SEQ ID NO: 12, (c) a VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 1 of SEQ ID NO: 5 HCDR 2 and HCDR 3 of SEQ ID NO: 9, (d) a VH comprising HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 6 and HCDR 3 of SEQ ID NO: 11, or (e) VH comprising HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 7 and HCDR 3 of SEQ ID NO: 13, and (ii) a light chain variable region (VL) comprising SEQ ID NO: Light chain complementarity determining region (LCDR) 1 of 20, LCDR 2 of SEQ ID NO: 21 and LCDR 3 of SEQ ID NO: 22.

[0099] In a preferred aspect, the present invention provides an antibody binding to CD3, wherein the antibody comprises a first antigen-binding domain comprising: a heavy chain variable region (VH) comprising SEQ ID NO: Heavy chain complementarity determining region (HCDR) 1 of 2, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10; and a light chain variable region (VL) comprising the light chain of SEQ ID NO: 20 Complementarity Determining Region (LCDR) 1, LCDR 2 of SEQ ID NO: 21 and LCDR 3 of SEQ ID NO: 22.

[0100] In one aspect, the invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen binding domain comprising: a heavy chain variable region (VH) comprising the heavy chain of SEQ ID NO: 2 Chain complementarity determining region (HCDR) 1, HCDR 2 of SEQ ID NO: 5 and HCDR 3 of SEQ ID NO: 9; and a light chain variable region (VL) comprising the light chain complementarity determining region of SEQ ID NO: 20 (LCDR) 1, LCDR 2 of SEQ ID NO: 21 and LCDR 3 of SEQ ID NO: 22.

[0101] In one aspect, the invention provides an antibody binding to CD3, wherein the antibody comprises a first antigen binding domain comprising: a heavy chain variable region (VH) comprising the heavy chain of SEQ ID NO: 3 Chain complementarity determining region (HCDR) 1, HCDR 2 of SEQ ID NO: 7 and HCDR 3 of SEQ ID NO: 13; and a light chain variable region (VL) comprising the light chain complementarity determining region of SEQ ID NO: 20 (LCDR) 1, LCDR 2 of SEQ ID NO: 21 and LCDR 3 of SEQ ID NO: 22.

[0102] In one aspect, the antibody is a humanized antibody. In one aspect, the antigen binding domain is a humanized antigen binding domain (ie, the antigen binding domain of a humanized antibody). In one aspect, VH and / or VL are humanized variable regions.

[0103] In one aspect, the VH and / or VL comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0104] 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 heavy chain variable region sequences of the group consisting of: SEQ ID NO NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19. 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: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19. 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: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19. 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: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19. In certain aspects, a VH sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise Antibodies retain the ability to bind CD3. In certain aspects, in the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19, there are a total of 1 to 10 amines amino acid substitutions, insertions and / or deletions. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19 . Optionally, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19, including Post-translational modification of the sequence.

[0105] In one aspect, the VL comprises one or more light chain framework sequences (i.e. FR1, FR2, FR3 and / or FR4 sequences) of the light chain variable region sequence of SEQ ID NO: 23. 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: 23. 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: 23. 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: 23. In certain aspects, a VL sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise Antibodies retain the ability to bind CD3. In certain aspects, in the amino acid sequence of SEQ ID NO: 23, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 23. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 23, including post-translational modifications of that sequence.

[0106] 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 : 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19, and VL comprises at least about 95%, 96%, 97% of the amino acid sequence of SEQ ID NO: 23 %, 98% or 99% identical amino acid sequences. In one aspect, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19 , and VL comprises the amino acid sequence of SEQ ID NO: 23.

[0107] In a preferred aspect, VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 16, and 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: 23. In one aspect, VH comprises the amino acid sequence of SEQ ID NO: 16 and VL comprises the amino acid sequence of SEQ ID NO: 23.

[0108] In one aspect, VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 15, and VL comprises an amino acid sequence identical to that of SEQ ID NO: 15. The amino acid sequence of NO: 23 is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence. In one aspect, VH comprises the amino acid sequence of SEQ ID NO: 15 and VL comprises the amino acid sequence of SEQ ID NO: 23.

[0109] In one aspect, VH comprises an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19, and VL comprises an amino acid sequence identical to that of SEQ ID NO: 19. The amino acid sequence of NO: 23 is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence. In one aspect, VH comprises the amino acid sequence of SEQ ID NO: 19 and VL comprises the amino acid sequence of SEQ ID NO: 23.

[0110] In yet another aspect, the present invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen binding domain comprising: VH comprising a group selected from the group consisting of Amino acid sequences of groups: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19; and VL, which comprises the amino group of SEQ ID NO: 23 acid sequence.

[0111] In a preferred aspect, the present invention provides an antibody binding to CD3, wherein the antibody comprises a first antigen binding domain comprising: VH comprising the amine of SEQ ID NO: 16 amino acid sequence; and VL, which comprises the amino acid sequence of SEQ ID NO: 23.

[0112] In one aspect, the present invention provides an antibody binding to CD3, wherein the antibody comprises a first antigen binding domain, the first antigen binding domain comprising: VH comprising the amino acid sequence of SEQ ID NO: 15 and VL, which comprises the amino acid sequence of SEQ ID NO: 23.

[0113] In one aspect, the present invention provides an antibody binding to CD3, wherein the antibody comprises a first antigen binding domain, the first antigen binding domain comprising: VH comprising the amino acid sequence of SEQ ID NO: 19 and VL, which comprises the amino acid sequence of SEQ ID NO: 23.

[0114] In yet another aspect, the invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen binding domain comprising a VH sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19; and the VL sequence of SEQ ID NO: 23.

[0115] In a preferred aspect, the present invention provides an antibody binding to CD3, wherein the antibody comprises a first antigen-binding domain, the first antigen-binding domain comprising the VH sequence of SEQ ID NO: 16, and SEQ ID NO: 23 VL sequences.

[0116] In one aspect, the present invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen binding domain comprising the VH sequence of SEQ ID NO: 15, and SEQ ID NO: 23 The VL sequence.

[0117] In one aspect, the present invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen binding domain comprising the VH sequence of SEQ ID NO: 19 and the VH sequence of SEQ ID NO: 23 VL sequence.

[0118] In another aspect, the invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen binding domain comprising: a VH comprising a heavy chain CDR sequence of the VH, the The heavy chain CDR sequence is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19; and VL, which comprises VL Light chain CDR sequence, the light chain CDR sequence is SEQ ID NO: 23.

[0119] In a preferred aspect, the present invention provides an antibody binding to CD3, wherein the antibody comprises a first antigen binding domain comprising: VH comprising a heavy chain CDR sequence of VH , the heavy chain CDR sequence is SEQ ID NO: 16; and VL, which comprises the light chain CDR sequence of VL, and the light chain CDR sequence is SEQ ID NO: 23.

[0120] In one aspect, the invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen binding domain comprising: a VH comprising a heavy chain CDR sequence of the VH, the The heavy chain CDR sequence is SEQ ID NO: 15; and the VL, which comprises the light chain CDR sequence of the VL, the light chain CDR sequences are SEQ ID NO: 23.

[0121] In one aspect, the invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen binding domain comprising: a VH comprising a heavy chain CDR sequence of the VH, the The heavy chain CDR sequence is SEQ ID NO: 19; and the VL, which comprises the light chain CDR sequence of the VL, the light chain CDR sequences are SEQ ID NO: 23.

[0122] In yet another aspect, the first antigen binding domain comprises: HCDR1, HCDR2 and HCDR3 amino acid sequences of VH selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of VL, the amino acid sequences of which are SEQ ID NO: 23.

[0123] In a preferred aspect, the first antigen-binding domain comprises: the amino acid sequences of HCDR1, HCDR2 and HCDR3 of VH, the amino acid sequences of which are SEQ ID NO: 16; and LCDR1, LCDR2 and The amino acid sequence of LCDR3, the amino acid sequence is SEQ ID NO: 23.

[0124] In one aspect, the first antigen binding domain comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the VH of SEQ ID NO: 15 and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the VL of SEQ ID NO: 23.

[0125] In one aspect, the first antigen binding domain comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the VH of SEQ ID NO: 19 and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the VL of SEQ ID NO: 23.

[0126] In one aspect, the VH comprises: a heavy chain CDR sequence of a VH selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15 , SEQ ID NO: 17 and SEQ ID NO: 19; and a backbone having at least 95%, 96%, 97%, 98% or 99% sequence to a backbone sequence of VH selected from the group consisting of Identity: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19. In one aspect, the VH comprises: a heavy chain CDR sequence of a VH selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19; and a backbone, which has at least 95% sequence identity with the backbone sequence of VH selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19. In another aspect, the VH comprises: heavy chain CDR sequences of VH selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 15, SEQ ID NO: ID NO: 17 and SEQ ID NO: 19; and a backbone having at least 98% sequence identity to a backbone sequence of a VH selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18 , SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19.

[0127] In a preferred aspect, VH comprises: the heavy chain CDR sequence of VH, the heavy chain CDR sequence is SEQ ID NO: 16; and the backbone, the backbone and the VH backbone sequence of SEQ ID NO: 16 have at least 95%, 96%, 97%, 98% or 99% sequence identity. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 16 and a backbone having at least 95% sequence identity to the backbone sequence of the VH of SEQ ID NO: 16. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 16 and a backbone having at least 98% sequence identity to the backbone sequence of the VH of SEQ ID NO: 16.

[0128] In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 15 and at least 95%, 96%, 97%, 98% or 99% identical to the framework sequence of the VH of SEQ ID NO: 15 Sequence identity backbone. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 15 and a backbone having at least 95% sequence identity to the backbone sequence of the VH of SEQ ID NO: 15. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 15 and a backbone having at least 98% sequence identity to the backbone sequence of the VH of SEQ ID NO: 15.

[0129] In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 19 and at least 95%, 96%, 97%, 98% or 99% identical to the framework sequence of the VH of SEQ ID NO: 19 Sequence identity backbone. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 19 and a backbone having at least 95% sequence identity to the backbone sequence of the VH of SEQ ID NO: 19. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 19 and a backbone having at least 98% sequence identity to the backbone sequence of the VH of SEQ ID NO: 19.

[0130] In one aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 23 and at least 95%, 96%, 97%, 98% or 99% identical to the backbone sequence of the VL of SEQ ID NO: 23 Sequence identity backbone. In one aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 23 and a backbone having at least 95% sequence identity to the backbone sequence of the VL of SEQ ID NO: 23. In another aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 23 and a backbone with at least 98% sequence identity to the backbone sequence of the VL of SEQ ID NO: 23.

[0131] In one aspect, the invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen binding domain comprising a VH sequence as described in any aspect provided above and any aspect as provided above The VL sequence described.

[0132] 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 human CH1, CH2, CH3 and / or CL domains. Exemplary sequences of human constant domains are given in SEQ ID NOs 50 and 51 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 52 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In one aspect, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 51, specifically the amino acid sequence of SEQ ID NO: 50 is at least About 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences. In one aspect, the antibody comprises a heavy chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 52. amino acid sequence. In particular, the heavy chain constant region may comprise amino acid mutations in the Fc domain, as described herein.

[0133] 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, in particular a human CH1 and / or CL domain. In one aspect, the first antigen binding domain comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 51, in particular the amine of SEQ ID NO: 50 Amino acid sequences that are at least about 95%, 96%, 97%, 98%, 99%, or 100% identical in amino acid sequence. In particular, the light chain constant region may comprise amino acid mutations as described herein under "Charge Modification", and / or may comprise one or more (especially two) N-terminal amines in the exchanged Fab molecule amino acid deletions or substitutions. In some aspects, the first antigen binding domain comprises a heavy chain constant region comprising at least about 95%, 96%, 97% of the CH1 domain sequence comprised in the amino acid sequence of SEQ ID NO: 52 , 98%, 99% or 100% identical amino acid sequences. In particular, the heavy chain constant region (specifically the CH1 domain) may comprise amino acid mutations as described herein under "Charge Modifications".

[0134] In one aspect, the antibody is a monoclonal antibody.

[0135] In one aspect, the antibody is an IgG antibody, in particular an IgG1 antibody. In one aspect, the antibody is a full length antibody.

[0136] In another aspect, the antibody is an antibody fragment selected from the group of Fv molecules, scFv molecules, Fab molecules, and F(ab')2 molecules; in particular antibody fragments of Fab molecules. In another embodiment, the antibody fragment is a diabody, triabody or tetrabody.

[0137] In one aspect, the first antigen binding domain is a Fab molecule. In a preferred aspect, 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 or the constant domains CL and CH1, in particular the variable domains VL and VH are mutually Substitution (ie, the first antigen binding domain is an exchanged Fab molecule).

[0138] In a further aspect, the antibody according to any of the above aspects may bind any of the features, either alone or in combination, as described in Section II.A.1.-8. below.

[0139] In a preferred aspect, 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 a human IgG1 Fc domain. The Fc domain is composed of first and second subunits and may incorporate, alone or in combination, any of the features described below for Fc domain variants (Section II.A.8.).

[0140] In another preferred aspect, the antibody comprises a second antigen-binding domain that binds a second antigen and optionally a third antigen-binding domain (i.e., the antibody is a multispecific antibody, as further described below. ) (Section II.A. 7.). 1. Antibody fragments

[0141] In certain aspects, the antibodies provided herein are antibody fragments.

[0142] In one aspect, the antibody fragment is a Fab, Fab', Fab'-SH or F(ab')2 molecule, in particular a Fab molecule as described herein. A "Fab' molecule" differs from a Fab molecule by the addition of residues at the carboxy-terminus of the CH1 domain that include one or more cysteines from the antibody hinge region. Fab’-SH is a Fab’ molecule in which the cysteine ​​residue of the constant domain bears a free thiol group. Pepsin treatment yields an F(ab')2 molecule with two antigen-binding sites (two Fab molecules) and a portion of the Fc region.

[0143] In another embodiment, the antibody fragment is a diabody, triabody or tetrabody. Diabodies are antibody fragments that have two antigen-binding sites (which may be bivalent or bispecific). See, eg, 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). Trifunctional and tetrafunctional antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0144] In yet another aspect, the antibody fragment is a single chain Fab molecule. A "single-chain Fab molecule" or "scFab" is composed 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 domains 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 consisting of at least 30 amino acids and preferably 32 to 50 amino acids. These single-chain Fab molecules are stabilized by a natural disulfide bond between the CL and CH1 domains. In addition, these single-chain Fab molecules can be further stabilized by insertion of cysteine ​​residues to create interchain disulfide bonds (e.g. insertion at position 44 of the variant heavy chain and position 100 of the variant light chain according to Kabat numbering). ).

[0145] In another aspect, the antibody fragment is a single chain variable fragment (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy (VH) and light (VL) chains of an antibody, linked by a linker. In particular, linkers are short polypeptides of 10 to 25 amino acids, and are usually rich in glycine for flexibility and serine or threonine for solubility, and can link the VH The N-terminus is linked to the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of a linker, the protein retains the specificity of the original antibody. For a review of scFv fragments see e.g. Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185 and US Patent Nos. 5,571,894 and 5,587,458.

[0146] In another aspect, the antibody fragment is a single domain antibody. A single domain antibody is an antibody fragment comprising 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, eg, US Patent No. 6,248,516 B1).

[0147] Antibody fragments can be produced by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies as described herein and recombinant production by recombinant host cells such as E. coli. 2. Humanized Antibody

[0148] In certain aspects, the antibodies provided herein are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibody. Typically, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (eg, an antibody from which CDR residues are derived), eg, to restore or improve antibody specificity or affinity.

[0149] Humanized antibodies and methods for their preparation are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, e.g., in Riechmann et al., Nature 332:323-329 (1988) ; Queen et al., Proc. Nat'l Acad. Sci. USA86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Determining region (SDR) grafting); Padlan, Mol. Immunol.28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods36:43-60 (2005) ( describes "FR reorganization"); Osbourn et al., Methods36:61-68 (2005); and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing "directed selection" for FR reorganization Law).

[0150] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" approach (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); The framework region of the consensus sequence of human antibodies of a particular subgroup of chain or heavy chain variable regions (see for example: Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); Human mature (somatically mutated) framework or human germline framework (see e.g. Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and sources Framework regions in 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)) . 3. Glycosylation variants

[0151] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites in an antibody is conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0152] When the antibody comprises an Fc region, the oligosaccharide attached to it can be altered. Native antibodies produced by mammalian cells usually contain branched biantennary oligosaccharides, usually N-bonded to Asn297 of the CH2 domain of the Fc region. See, eg, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure . In some embodiments, the oligosaccharides in the antibodies of the invention can be modified to produce antibody variants with certain improved properties.

[0153] In one aspect, antibody variants are provided that have afucosylated oligosaccharides, ie, oligosaccharide structures that lack fucose attached (directly or indirectly) to the Fc region. These non-fucosylated oligosaccharides (also called "defucosylated" oligosaccharides) specifically lack a fucose residue in the stem of the biantennary oligosaccharide structure to which the first GlcNAc is attached N-linked oligosaccharides. In one aspect, antibody variants are provided that have an increased proportion of afucosylated oligosaccharides in the Fc region compared to a native or parental 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. no fucosylated oligosaccharides are present ). The percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking fucose residues relative to the sum of all oligosaccharides linked to Asn 297 (e.g. complex, hybrid and high mannose structures) , the percentage is determined by MALDI-TOF mass spectrometry, eg as described in WO 2006 / 082515. Asn297 refers to the asparagine residue located near position 297 of the Fc region (EU numbering for Fc region residues); however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e. due to A slight sequence change between positions 294 and 300. Such antibodies having an increased proportion of afucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector functions, in particular improved ADCC function. See eg US 2003 / 0157108; US 2004 / 0093621.

[0154] 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, especially in Example 11); and knockout cell lines, such as CHO cells knocked out of the α-1,6-fucosyltransferase gene FUT8 (see, for example, Yamane - Ohnuki et al. Biotech. Bioeng.87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107); or GDP - cells with reduced or abolished activity of fucose synthesis or transporters (see eg US2004259150, US2005031613, US2004132140, US2004110282).

[0155] In yet another aspect, the antibody variant is provided with bisected oligosaccharides, e.g., wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by the GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function as described above. Examples of such antibody variants are described e.g. in: 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.

[0156] Also provided are antibody variants having at least one galactose residue on the oligosaccharide linked 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. 4. Cysteine ​​engineered antibody variants

[0157] In certain aspects, it may be desirable to create cysteine ​​engineered antibodies, such as THIOMAB™ antibodies, in which one or more residues of the antibody are substituted with cysteine ​​residues. In preferred aspects, the substituted residue occurs at an accessible site of the antibody. By substituting those residues with cysteine, reactive thiol groups are thus positioned at accessible sites on the antibody and can be used to bind the antibody to other moieties (e.g., drug moieties or linker-drug moieties) combined to form an immunoconjugate, as further described herein. Cysteine-engineered antibodies can be produced, for example, according to methods described in US Patent Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130 or WO 2016040856. 5. Antibody Derivatives

[0158] In certain aspects, the antibodies provided herein can be further modified to include additional non-proteinaceous moieties known and readily available in the art. Moieties suitable for derivatization of antibodies include, but are not limited to, water soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1 ,3-dioxolane, poly-1,3,6-tris㗁𠮿, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly( N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the amount and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used under specified conditions The treatment is moderate. 6. Immunoconjugates

[0159] The present invention also provides immunoconjugates comprising an anti-CD3 antibody as described herein bound (chemically bonded) to one or more therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, drugs, growth inhibitory Agents, toxins (such as protein toxins, enzymatically active toxins or fragments thereof) or radioactive isotopes derived from bacteria, fungi, plants or animals.

[0160] In one aspect, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more of the therapeutic agents described above. Linkers are typically used to link the antibody to one or more therapeutic agents. An overview of ADC technology, including examples of therapeutics, drugs, and linkers, is presented in Pharmacol Review68:3-19 (2016).

[0161] 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, non-binding active fragments of diphtheria toxin, Exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modinus A chain, α-sarcinina, tung protein, carnation toxin, american Lupin (PAPI, PAPII and PAP-S), bitter melon inhibitor, curcumin, crotonin, saponaria inhibitor, gelonin, mitocetins, limitoxin, phenomycin, ionomycin and Trichothecenes.

[0162] In another aspect, an immunoconjugate comprises an antibody of the invention bound to a radioactive atom to form a radioconjugate. In another example, multiple radioactive isotopes can be used to generate radioconjugates. Examples include radioisotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu. When a radioconjugate is used for detection, it may contain a radioactive atom such as Tc99m or I123 for scintigraphy studies, or a spin label for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI) substances, such as I123, I131, In111, F19, C13, N15, O17, gadolinium, manganese or iron.

[0163] Conjugates of antibodies and cytotoxic agents 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), imidosulfane (IT), Bifunctional derivatives of amino acid esters (e.g., dimethyl adipate hydrochloride, HCl), active esters (e.g., disuccinimidyl suberic acid), aldehydes (e.g., glutaraldehyde), bis Azides (e.g., bis(p-azidobenzoyl)hexamethylenediamine), dinitrogen derivatives (e.g., bis-(p-azidobenzoyl)-ethylenediamine), diisocyanates (e.g. , toluene 2,6-diisocyanate) and bis-reactive fluorine compounds (eg, 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described by Vitetta et al. (Science 238:1098 (1987)). An exemplary chelator for conjugating radionucleotides to antibodies is carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) . See WO 94 / 11026. The linker may be a "cleavable linker" that facilitates the release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Res. 52:127 -131 (1992); US Patent No. 5,208,020).

[0164] The immunoconjugates or ADCs herein expressly 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., U.S.A) 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 and sulfo-SMPB and SVSB (succinimidyl-(4-vinylsulfone)benzoate). 7. Multispecific Antibodies

[0165] In certain aspects, the antibodies provided herein are multispecific antibodies, in particular bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different epitopes (eg, two different proteins, or two different epitopes on the same protein). In certain embodiments, multispecific antibodies have three or more binding specificities. In certain aspects, one of the binding specificities is for CD3, while the other specificity is for any other antigen. In certain aspects, a multispecific antibody can bind to two (or more) different epitopes of CD3. Multispecific (eg, bispecific) antibodies can also be used to localize cytotoxic agents or cells to CD3-expressing cells. Multispecific antibodies can be produced as full-length antibodies or antibody fragments.

[0166] 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 eg US Pat. No. 5,731,168, and Atwell et al. J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be prepared by engineering electrostatic steering effects for the preparation of antibody Fc-heterodimer molecules (see e.g. WO 2009 / 089004); cross-linking two or more antibodies or fragments ( See, e.g., U.S. Patent No. 4,676,980; and Brennan et al., Science, 229: 81 (1985)); generation of bispecific antibodies using a leucine zipper (see e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992); and WO 2011 / 034605); using common light chain technology to circumvent the light chain mismatch problem (see e.g. WO 98 / 50431); using "diabody" technology to prepare bispecific antibody fragments (see e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single-chain Fv (sFv) dimers (see e.g. Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0167] Also included herein are engineered antibodies having three or more antigen binding sites, including, for example, "Octopus antibodies" or DVD-Igs (see, eg, WO 2001 / 77342 and WO 2008 / 024715). Additional examples of multispecific antibodies having 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-acting FAbs" or "DAFs" that comprise an antigen-binding site that binds to CD3 and to another, different antigen or to two different epitopes of CD3 (see e.g. US 2008 / 0069820 and WO 2015 / 095539).

[0168] Multispecific antibodies can also be provided in an asymmetric format in which 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 eg WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see eg WO 2009 / 080253) or complete Fab arms (see eg 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 designed by introducing charged or uncharged amino acid mutations into domain interfaces to guide correct Fab pairing. See eg WO 2016 / 172485.

[0169] Various other molecular formats for multispecific antibodies are known in the art and are included herein (see e.g. Spiess et al., Mol Immunol 67 (2015) 95-106).

[0170] Also included herein are certain types of multispecific antibodies that are bispecific antibodies that are designed to bind to both surface antigens and T cell receptors on target cells (e.g., tumor cells) Activation-invariant components of (TCR) complexes such as CD3 are used to redirect T cells to kill target cells. Therefore, in a preferred aspect, the antibodies provided herein are multispecific antibodies, in particular bispecific antibodies, wherein one of the binding specificities is for CD3 and the other binding specificity is for a target cell antigen .

[0171] 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 Bäuerle, 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 with a C-terminal disulfide bond for further stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which are complete mouse / rat IgG hybrid molecules (see Seimetz et al. Review: 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; and Bacac et al. Oncoimmunology 5(8) (2016) e1203498.

[0172] Preferred aspects of the multispecific antibodies of the invention are described below.

[0173] In one aspect, the invention provides a CD3-binding antibody comprising a first antigen-binding domain that binds CD3, as described herein, and a second antigen-binding domain that binds a second antigen and Optionally a third antigen binding domain.

[0174] According to a preferred aspect of the invention, the antigen binding domain comprised in the antibody is a Fab molecule (i.e., an antigen binding domain consisting of a heavy chain and a light chain, each of which comprises 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 is a Fab molecule. In one aspect, the Fab molecule is a human Fab molecule. In a preferred aspect, the Fab molecule is a humanized Fab molecule. In yet another aspect, the Fab molecule comprises a human heavy chain constant domain and a human light chain constant domain.

[0175] Preferably, at least one of the antigen binding domains is an exchanged Fab molecule. These modifications reduce the mismatch of heavy and light chains from different Fab molecules, thereby increasing the yield and purity of recombinantly produced (multispecific) antibodies of the invention. In preferred exchanged Fab molecules for use in the (multispecific) antibodies of the invention, the variable domains of the Fab light chain and the Fab heavy chain (VL and VH, respectively) are exchanged. However, even with this domain exchange, the production of (multispecific) antibodies may contain certain by-products due to so-called Bence Jones-type interactions between mismatched heavy and light chains (see Schaefer et al., PNAS, 108 (2011) 11187-11191). To further reduce the mismatch of heavy and light chains from different Fab molecules, thereby increasing the purity and yield of desired (multispecific) antibodies, Fab molecules that bind to the first antigen (CD3) or bind to the second antigen Specific amino acid positions in the CH1 and CL domains of the Fab molecule bound by a secondary antigen (e.g., a target cell antigen such as TYRP-1, CEA, GPRC5D, or CD19) introduce oppositely charged amino acids, as further described herein. stated. In a conventional Fab molecule contained in a (multispecific) antibody (such as shown for example in A-C, G-J of Fig. 1 ) or in a VH / VL exchanged Fab molecule contained in a (multispecific) antibody (such as Charge modification is performed in (but not both) such as those shown in D-F, K-N of Figure 1. In a preferred aspect, charge modification is performed in a conventional Fab molecule comprised in a (multispecific) antibody (in a preferred aspect, the antibody binds to a second antigen, such as a target cell antigen, such as TYRP- 1. CEA, GPRC5D or CD19).

[0176] In a preferred aspect according to the present invention, the (multispecific) antibody is capable of simultaneously binding to the first antigen (ie CD3) and the second antigen (eg target cell antigen, such as TYRP-1, CEA, GPRC5D or CD19). In one aspect, the (multispecific) antibody is capable of crosslinking T cells and target cells by binding to CD3 and target cell antigens. In an even more preferred aspect, such simultaneous binding results in lysis of target cells, in particular target cell antigens expressing tumor cells (i.e. TYRP-1, CEA, GPRC5D or CD19). In one aspect, such simultaneous binding results in T cell activation. 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, secretion of cytokines, release of cytotoxic effector molecules, cytotoxic activity and expression of activation markers. In one aspect, the (multispecific) antibody binds to CD3 without simultaneously binding to the target cell antigen and does not result in T cell activation.

[0177] 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 presentation of the peptide antigen by the target cell and / or T cell specificity.

[0178] 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, specifically CD8+ T cells. a) The first antigen-binding domain

[0179] The (multispecific) antibody of the invention comprises at least one antigen-binding domain (first antigen-binding domain) that binds to CD3. In preferred aspects, the CD3 is human CD3 (SEQ ID NO: 45) or cynomolgus monkey CD3 (SEQ ID NO: 46), most specifically human CD3. In one aspect, the first antigen binding domain is cross-reactive with (i.e. specifically binds to) human and cynomolgus CD3. In some aspects, CD3 is the epsilon subunit of CD3 (CD3 epsilon).

[0180] In a preferred aspect, the (multispecific) antibody comprises no more than one antigen binding domain that binds to CD3. In one aspect, the (multispecific) antibody provides monovalent binding to CD3.

[0181] In one aspect, the antigen binding domain that binds CD3 is an antibody fragment selected from the group of Fv molecules, scFv molecules, Fab molecules and F(ab')2 molecules. In a preferred aspect, the antigen-binding domain that binds to CD3 is a Fab molecule.

[0182] In a preferred aspect, the antigen binding domain that binds to CD3 is an exchanged Fab molecule as described herein, i.e. wherein the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged for each other / replaced Fab molecules. In these aspects, the antigen binding domain that binds to the second antigen (e.g. a target cell antigen such as TYRP-1, CEA, GPRC5D or CD19) is preferably a conventional Fab molecule. In aspects wherein there is more than one antigen-binding domain, in particular a Fab molecule, which binds to a second antigen comprised in the (multispecific) antibody, the antigen-binding domain which binds to CD3 is preferably an exchanged Fab molecule, And the antigen-binding domain that binds to the second antigen is a conventional Fab molecule.

[0183] In an alternative aspect, the antigen binding domain that binds CD3 is a conventional Fab molecule. In these aspects, the antigen binding domain that binds to the second antigen (e.g., a target cell antigen, such as TYRP-1, CEA, GPRC5D or CD19) is an exchanged Fab molecule as described herein, i.e., wherein the Fab heavy chain and light Fab molecules in which the variable domains VH and VL or the constant domains CH1 and CL of the chains are exchanged / replaced with each other. In the aspect wherein there is more than one antigen-binding domain, in particular a Fab molecule, which binds to CD3 comprised in the (multispecific) antibody, the antigen-binding domain which binds to the second antigen is preferably an exchanged Fab molecule, And the antigen-binding domain that binds to CD3 is a conventional Fab molecule.

[0184] In a preferred aspect, 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 or the constant domains CL and CH1, in particular the variable domains VL and The VHs are exchanged for each other (ie, according to such aspects, the first antigen binding domain is a swapped Fab molecule in which the variable or constant domains of the Fab light chain and the Fab heavy chain are swapped). In one such aspect, the second antigen binding domain (and third antigen binding domain, if present) is a conventional Fab molecule.

[0185] In one aspect, no more than one antigen binding domain that binds CD3 is present in the (multispecific) antibody (i.e. the antibody provides monovalent binding to CD3). b) Second antigen binding domain (and third antigen binding domain)

[0186] In certain aspects, the (multispecific) antibody of the invention comprises at least one antigen binding domain, in particular a Fab molecule, which binds to a second antigen. The second antigen is preferably not CD3, i.e. different from CD3. In one aspect, the second antigen is an antigen expressed on cells other than CD3 (eg, expressed on cells other than T cells). In one aspect, the second antigen is a target cell antigen, in particular a tumor cell antigen. In a specific aspect, the second antigen is TYRP-1. In another specific aspect, the second antigen is CEA. In yet another specific aspect, the second antigen is GPRC5D. In another specific aspect, the second antigen is CD19. The second antigen binding domain is capable of directing the (multispecific) antibody to a target site, eg to a particular type of tumor cell expressing the second antigen.

[0187] In one aspect, the antigen binding domain that binds the second antigen is an antibody fragment selected from the group of Fv molecules, scFv molecules, Fab molecules and F(ab')2 molecules. In a preferred aspect, the antigen-binding domain that binds to the second antigen is a Fab molecule.

[0188] In certain aspects, a (multispecific) antibody comprises two antigen binding domains, in particular Fab molecules, which bind a second antigen. In a preferred such aspect, each of the antigen binding domains binds to the same epitope. In an even more preferred aspect, all of these antigen binding domains are identical, i.e. they are of the same molecular format (e.g. conventional or exchanged Fab molecules) and comprise the same amino acid sequence, including the same as described herein. The same amino acid substitutions (if any) in the CH1 and CL domains. In one aspect, a (multispecific) antibody comprises no more than two antigen binding domains, in particular a Fab molecule that binds a second antigen.

[0189] In a preferred aspect, the antigen binding domain that binds the second antigen is a conventional Fab molecule. In these aspects, the antigen binding domain that binds to CD3 is a swapped Fab molecule as described herein, i.e. a Fab in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged / replaced with each other molecular.

[0190] In an alternative aspect, the antigen binding domain that binds to the second antigen is an exchanged Fab molecule as described herein, i.e. wherein the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains are exchanged for each other / replaced Fab molecules. In these aspects, the antigen binding domain that binds CD3 is a conventional Fab molecule.

[0191] In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises a human constant region. In one aspect, the second antigen binding domain (and the third antigen binding domain when present) is a Fab molecule comprising a human constant region, in particular a human CH1 and / or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs 50 and 51 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 52 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In one aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises a light chain constant region comprising the amino acids of SEQ ID NO: 50 or SEQ ID NO: 51 Sequences, in particular, amino acid sequences that are at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 50. In particular, the light chain constant region may comprise amino acid mutations as described herein under "Charge Modification", and / or may comprise one or more (especially two) N-terminal amines in the exchanged Fab molecule amino acid deletions or substitutions. In some aspects, the second antigen binding domain (and, when present, the third antigen binding domain) comprises a heavy chain constant region comprising and comprising CH1 contained in the amino acid sequence of SEQ ID NO: 52 Domain sequences are amino acid sequences that are at least about 95%, 96%, 97%, 98%, 99%, or 100% identical. In particular, the heavy chain constant region (specifically the CH1 domain) may comprise amino acid mutations as described herein under "Charge Modifications".

[0192] In some aspects, the second antigen is TYRP-1, specifically human TYRP-1.

[0193] In one aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region of SEQ ID NO: 24 ( HCDR) 1, HCDR 2 of SEQ ID NO: 25 and HCDR 3 of SEQ ID NO: 26; and a light chain variable region (VL) comprising the 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.

[0194] In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is (derived from) a humanized antibody. In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is a humanized antigen binding domain (ie, the antigen binding domain of a humanized antibody). In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) are humanized variable regions.

[0195] In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0196] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more heavy chain framework sequences of the heavy chain variable region sequence of SEQ ID NO: 27 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 27. 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: 27. 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: 27. In certain aspects, a VH sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise Antibodies retain the ability to bind to TYRP-1. In certain aspects, in the amino acid sequence of SEQ ID NO: 27, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 27. The VH optionally comprises the amino acid sequence of SEQ ID NO: 27, including post-translational modifications of that sequence. In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more light chain framework sequences of the light chain variable region sequence of SEQ ID NO: 31 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 31. 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: 31. 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: 31. In certain aspects, a VL sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise Antibodies retain the ability to bind to TYRP-1. In certain aspects, in the amino acid sequence of SEQ ID NO: 31, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 31. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 31, including post-translational modifications of that sequence.

[0198] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequence, and the VL of the second antigen binding domain (and the third antigen binding domain when present) comprises at least about 95%, 96% of the amino acid sequence of SEQ ID NO: 31 %, 97%, 98% or 99% identical amino acid sequences. In one aspect, VH comprises the amino acid sequence of SEQ ID NO:27 and VL comprises the amino acid sequence of SEQ ID NO:31.

[0199] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: VH comprising the amino acid sequence of SEQ ID NO: 27; and VL comprising SEQ ID NO: Amino acid sequence of 31.

[0200] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises the VH sequence of SEQ ID NO: 27 and the VL sequence of SEQ ID NO: 31.

[0201] In another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises a VH comprising the heavy chain CDR sequence of the VH of SEQ ID NO: 27, and a VL comprising SEQ ID NO: 27 ID NO: 31 VL light chain CDR sequence.

[0202] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: the amino acid sequences of HCDR1, HCDR2 and HCDR3 of VH, the amino acid sequences of which are SEQ ID NO : 27; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of VL, these amino acid sequences are SEQ ID NO: 31.

[0203] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises: the heavy chain CDR sequences of the VH, the heavy chain CDR sequences being SEQ ID NO: 27; and A backbone having at least 95%, 96%, 97%, 98% or 99% sequence identity to the backbone sequence of the VH of SEQ ID NO: 27. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 27 and a backbone having at least 95% sequence identity to the backbone sequence of the VH of SEQ ID NO: 27. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 27 and a backbone having at least 98% sequence identity to the backbone sequence of the VH of SEQ ID NO: 27.

[0204] In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises: the light chain CDR sequence of the VL, the light chain CDR sequences being SEQ ID NO: 31; and A backbone having at least 95%, 96%, 97%, 98% or 99% sequence identity to the backbone sequence of the VL of SEQ ID NO: 31. In one aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 31 and a backbone having at least 95% sequence identity to the backbone sequence of the VL of SEQ ID NO: 31. In another aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 31 and a backbone having at least 98% sequence identity to the backbone sequence of the VL of SEQ ID NO: 31.

[0205] In some aspects, the second antigen is CEA, specifically human CEA.

[0206] In one aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region of SEQ ID NO: 53 ( HCDR) 1, HCDR 2 of SEQ ID NO: 54 and HCDR 3 of SEQ ID NO: 55; and a light chain variable region (VL) comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 57, LCDR 2 of SEQ ID NO: 58 and LCDR 3 of SEQ ID NO: 59.

[0207] In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is (derived from) a humanized antibody. In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is a humanized antigen binding domain (ie, the antigen binding domain of a humanized antibody). In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) are humanized variable regions.

[0208] In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more heavy chain framework sequences of the heavy chain variable region sequence of SEQ ID NO: 56 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 56. 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: 56. 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: 56. In certain aspects, a VH sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise Antibodies retain the ability to bind to CEA. In certain aspects, in the amino acid sequence of SEQ ID NO: 56, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 56. The VH optionally comprises the amino acid sequence of SEQ ID NO: 56, including post-translational modifications of that sequence. In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more light chain framework sequences of the light chain variable region sequence of SEQ ID NO: 60 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 60. 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: 60. 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: 60. In certain aspects, a VL sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise Antibodies retain the ability to bind to CEA. In certain aspects, in the amino acid sequence of SEQ ID NO: 60, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 60. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 60, including post-translational modifications of that sequence.

[0211] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequence, and the VL of the second antigen binding domain (and the third antigen binding domain when present) comprises at least about 95%, 96% of the amino acid sequence of SEQ ID NO: 60 %, 97%, 98% or 99% identical amino acid sequences. In one aspect, VH comprises the amino acid sequence of SEQ ID NO:56 and VL comprises the amino acid sequence of SEQ ID NO:60.

[0212] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: VH comprising the amino acid sequence of SEQ ID NO: 56; and VL comprising SEQ ID NO: Amino acid sequence of 60.

[0213] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises the VH sequence of SEQ ID NO: 56 and the VL sequence of SEQ ID NO: 60.

[0214] In another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises a VH comprising the heavy chain CDR sequence of the VH of SEQ ID NO: 56, and a VL comprising SEQ ID NO: 56 ID NO: 60 VL light chain CDR sequence.

[0215] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: the amino acid sequences of HCDR1, HCDR2 and HCDR3 of VH, the amino acid sequences of which are SEQ ID NO : 56; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of VL, these amino acid sequences are SEQ ID NO: 60.

[0216] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises: the heavy chain CDR sequences of the VH, the heavy chain CDR sequences being SEQ ID NO: 56; and A backbone having at least 95%, 96%, 97%, 98% or 99% sequence identity to the backbone sequence of the VH of SEQ ID NO: 56. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 56 and a backbone having at least 95% sequence identity to the backbone sequence of the VH of SEQ ID NO: 56. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 56 and a backbone having at least 98% sequence identity to the backbone sequence of the VH of SEQ ID NO: 56.

[0217] In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises: the light chain CDR sequence of the VL, the light chain CDR sequences being SEQ ID NO: 60; and A backbone having at least 95%, 96%, 97%, 98% or 99% sequence identity to the backbone sequence of the VL of SEQ ID NO: 60. In one aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 60 and a backbone having at least 95% sequence identity to the backbone sequence of the VL of SEQ ID NO: 60. In another aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 60 and a backbone with at least 98% sequence identity to the backbone sequence of the VL of SEQ ID NO: 60.

[0218] In another aspect, wherein the second antigen is CEA, specifically human CEA, the second antigen binding domain (and the third antigen binding domain when present) comprises: a heavy chain variable region (VH), It comprises the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 105, the HCDR 2 of SEQ ID NO: 106 and the HCDR 3 of SEQ ID NO: 107; and the light chain variable region (VL), which comprises SEQ ID Light chain complementarity determining region (LCDR) 1 of NO: 109, LCDR 2 of SEQ ID NO: 110 and LCDR 3 of SEQ ID NO: 111.

[0219] In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is (derived from) a humanized antibody. In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is a humanized antigen binding domain (ie, the antigen binding domain of a humanized antibody). In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) are humanized variable regions.

[0220] In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more heavy chain framework sequences of the heavy chain variable region sequence of SEQ ID NO: 108 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 108. 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: 108. 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: 108. In certain aspects, a VH sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise Antibodies retain the ability to bind to CEA. In certain aspects, in the amino acid sequence of SEQ ID NO: 108, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 108. The VH optionally comprises the amino acid sequence of SEQ ID NO: 108, including post-translational modifications of that sequence. In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more light chain framework sequences of the light chain variable region sequence of SEQ ID NO: 112 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 112. 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: 112. 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: 112. In certain aspects, a VL sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise Antibodies retain the ability to bind to CEA. In certain aspects, in the amino acid sequence of SEQ ID NO: 112, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 112. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 112, including post-translational modifications of that sequence.

[0223] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequence, and the VL of the second antigen binding domain (and the third antigen binding domain when present) comprises at least about 95%, 96% of the amino acid sequence of SEQ ID NO: 112 %, 97%, 98% or 99% identical amino acid sequences. In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 108 and the VL comprises the amino acid sequence of SEQ ID NO: 112.

[0224] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: VH comprising the amino acid sequence of SEQ ID NO: 108; and VL comprising SEQ ID NO: The amino acid sequence of 112.

[0225] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises the VH sequence of SEQ ID NO: 108 and the VL sequence of SEQ ID NO: 112.

[0226] In another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises a VH comprising the heavy chain CDR sequence of the VH of SEQ ID NO: 108, and a VL comprising SEQ ID NO: 108 VL light chain CDR sequence of ID NO: 112.

[0227] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: the amino acid sequences of HCDR1, HCDR2 and HCDR3 of VH, the amino acid sequences of which are SEQ ID NO : 108; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of VL, these amino acid sequences are SEQ ID NO: 112.

[0228] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises: the heavy chain CDR sequences of the VH, the heavy chain CDR sequences being SEQ ID NO: 108; and A backbone having at least 95%, 96%, 97%, 98% or 99% sequence identity to the backbone sequence of the VH of SEQ ID NO: 108. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 108 and a backbone having at least 95% sequence identity to the backbone sequence of the VH of SEQ ID NO: 108. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 108 and a backbone having at least 98% sequence identity to the backbone sequence of the VH of SEQ ID NO: 108.

[0229] In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises: the light chain CDR sequence of the VL, the light chain CDR sequences being SEQ ID NO: 112; and A backbone having at least 95%, 96%, 97%, 98% or 99% sequence identity to the backbone sequence of the VL of SEQ ID NO: 112. In one aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 112 and a backbone with at least 95% sequence identity to the backbone sequence of the VL of SEQ ID NO: 112. In another aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 112 and a backbone having at least 98% sequence identity to the backbone sequence of the VL of SEQ ID NO: 112.

[0230] In another aspect, wherein the second antigen is CEA, specifically human CEA, the second antigen binding domain (and the third antigen binding domain when present) comprises: a heavy chain variable region (VH), It comprises the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 113, the HCDR 2 of SEQ ID NO: 114 and the HCDR 3 of SEQ ID NO: 115; and the light chain variable region (VL), which comprises SEQ ID Light chain complementarity determining region (LCDR) 1 of NO: 117, LCDR 2 of SEQ ID NO: 118 and LCDR 3 of SEQ ID NO: 119.

[0231] In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is (derived from) a humanized antibody. In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is a humanized antigen binding domain (ie, the antigen binding domain of a humanized antibody). In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) are humanized variable regions.

[0232] In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more heavy chain framework sequences of the heavy chain variable region sequence of SEQ ID NO: 116 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 116. 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: 116. 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: 116. In certain aspects, a VH sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise Antibodies retain the ability to bind to CEA. In certain aspects, in the amino acid sequence of SEQ ID NO: 116, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 116. The VH optionally comprises the amino acid sequence of SEQ ID NO: 116, including post-translational modifications of that sequence. In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more light chain framework sequences of the light chain variable region sequence of SEQ ID NO: 120 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 120. 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: 120. 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: 120. In certain aspects, a VL sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise Antibodies retain the ability to bind to CEA. In certain aspects, in the amino acid sequence of SEQ ID NO: 120, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 120. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 120, including post-translational modifications of that sequence.

[0235] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequence, and the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises at least about 95%, 96% of the amino acid sequence of SEQ ID NO: 120 %, 97%, 98% or 99% identical amino acid sequences. In one aspect, the VH comprises the amino acid sequence of SEQ ID NO:116 and the VL comprises the amino acid sequence of SEQ ID NO:120.

[0236] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: VH comprising the amino acid sequence of SEQ ID NO: 116; and VL comprising SEQ ID NO: The amino acid sequence of 120.

[0237] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises the VH sequence of SEQ ID NO: 116 and the VL sequence of SEQ ID NO: 120.

[0238] In another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises a VH comprising the heavy chain CDR sequence of the VH of SEQ ID NO: 116, and a VL comprising SEQ ID NO: 116 ID NO: 120 VL light chain CDR sequence.

[0239] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: the amino acid sequences of HCDR1, HCDR2 and HCDR3 of VH, the amino acid sequences of which are SEQ ID NO : 116; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of VL, these amino acid sequences are SEQ ID NO: 120.

[0240] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises: the heavy chain CDR sequences of the VH, the heavy chain CDR sequences being SEQ ID NO: 116; and A backbone having at least 95%, 96%, 97%, 98% or 99% sequence identity to the backbone sequence of the VH of SEQ ID NO: 116. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 116 and a backbone having at least 95% sequence identity to the backbone sequence of the VH of SEQ ID NO: 116. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 116 and a backbone having at least 98% sequence identity to the backbone sequence of the VH of SEQ ID NO: 116.

[0241] In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises: the light chain CDR sequence of the VL, the light chain CDR sequences being SEQ ID NO: 120; and A backbone having at least 95%, 96%, 97%, 98% or 99% sequence identity to the backbone sequence of the VL of SEQ ID NO: 120. In one aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 120 and a backbone with at least 95% sequence identity to the backbone sequence of the VL of SEQ ID NO: 120. In another aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 120 and a backbone having at least 98% sequence identity to the backbone sequence of the VL of SEQ ID NO: 120.

[0242] In some aspects, the second antigen is GPRC5D, in particular human GPRC5D.

[0243] In one aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region of SEQ ID NO: 61 ( HCDR) 1, HCDR 2 of SEQ ID NO: 62 and HCDR 3 of SEQ ID NO: 63; and a light chain variable region (VL) comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 65, LCDR 2 of SEQ ID NO: 66 and LCDR 3 of SEQ ID NO: 67.

[0244] In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is (derived from) a humanized antibody. In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is a humanized antigen binding domain (ie, the antigen binding domain of a humanized antibody). In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) are humanized variable regions.

[0245] In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more heavy chain framework sequences of the heavy chain variable region sequence of SEQ ID NO: 64 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 64. 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: 64. 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: 64. In certain aspects, a VH sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise Antibodies retain the ability to bind GPRC5D. In certain aspects, in the amino acid sequence of SEQ ID NO: 64, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 64. VH optionally comprises the amino acid sequence of SEQ ID NO: 64, including post-translational modifications of that sequence. In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more light chain framework sequences of the light chain variable region sequence of SEQ ID NO: 68 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 68. 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: 68. 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: 68. In certain aspects, a VL sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise Antibodies retain the ability to bind GPRC5D. In certain aspects, in the amino acid sequence of SEQ ID NO: 68, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 68. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 68, including post-translational modifications of that sequence.

[0248] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequence, and the VL of the second antigen binding domain (and the third antigen binding domain when present) comprises at least about 95%, 96% of the amino acid sequence of SEQ ID NO: 68 %, 97%, 98% or 99% identical amino acid sequences. In one aspect, VH comprises the amino acid sequence of SEQ ID NO:64 and VL comprises the amino acid sequence of SEQ ID NO:68.

[0249] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: VH comprising the amino acid sequence of SEQ ID NO: 64; and VL comprising SEQ ID NO: The amino acid sequence of 68.

[0250] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises the VH sequence of SEQ ID NO: 64 and the VL sequence of SEQ ID NO: 68.

[0251] In another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises a VH comprising the heavy chain CDR sequence of the VH of SEQ ID NO: 64, and a VL comprising SEQ ID NO: 64 ID NO: 68 VL light chain CDR sequence.

[0252] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 64 and the amino acid sequence of SEQ ID NO: 68 The amino acid sequences of LCDR1, LCDR2 and LCDR3 of VL.

[0253] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 64 and the sequence of the VH of SEQ ID NO: 64 Backbone sequences have a backbone with at least 95%, 96%, 97%, 98% or 99% sequence identity. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 64 and a backbone having at least 95% sequence identity to the backbone sequence of the VH of SEQ ID NO: 64. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 64 and a backbone having at least 98% sequence identity to the backbone sequence of the VH of SEQ ID NO: 64.

[0254] In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises the light chain CDR sequence of the VL of SEQ ID NO: 68 and the sequence of the VL of SEQ ID NO: 68 Backbone sequences have a backbone with at least 95%, 96%, 97%, 98% or 99% sequence identity. In one aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 68 and a backbone having at least 95% sequence identity to the backbone sequence of the VL of SEQ ID NO: 68. In another aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 68 and a backbone having at least 98% sequence identity to the backbone sequence of the VL of SEQ ID NO: 68.

[0255] In some aspects, the second antigen is CD19, specifically human CD19.

[0256] In one aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region of SEQ ID NO: 75 ( HCDR) 1, HCDR 2 of SEQ ID NO: 76 and HCDR 3 of SEQ ID NO: 77; and a light chain variable region (VL) comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 79, LCDR 2 of SEQ ID NO: 80 and LCDR 3 of SEQ ID NO: 81.

[0257] In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is (derived from) a humanized antibody. In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is a humanized antigen binding domain (ie, the antigen binding domain of a humanized antibody). In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) are humanized variable regions.

[0258] In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more heavy chain framework sequences of the heavy chain variable region sequence of SEQ ID NO: 78 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 78. 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: 78. 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: 78. In certain aspects, a VH sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise The antibody retains the ability to bind to CD19. In certain aspects, in the amino acid sequence of SEQ ID NO: 78, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 78. VH optionally comprises the amino acid sequence of SEQ ID NO: 78, including post-translational modifications of that sequence. In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more light chain framework sequences of the light chain variable region sequence of SEQ ID NO: 82 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 82. 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: 82. 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: 82. In certain aspects, a VL sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise The antibody retains the ability to bind to CD19. In certain aspects, in the amino acid sequence of SEQ ID NO: 82, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 82. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 82, including post-translational modifications of that sequence.

[0261] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequence, and the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises at least about 95%, 96% of the amino acid sequence of SEQ ID NO: 82 %, 97%, 98% or 99% identical amino acid sequences. In one aspect, VH comprises the amino acid sequence of SEQ ID NO:78 and VL comprises the amino acid sequence of SEQ ID NO:82.

[0262] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: VH comprising the amino acid sequence of SEQ ID NO: 78; and VL comprising SEQ ID NO: The amino acid sequence of 82.

[0263] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises the VH sequence of SEQ ID NO: 78 and the VL sequence of SEQ ID NO: 82.

[0264] In another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises a VH comprising the heavy chain CDR sequence of the VH of SEQ ID NO: 78, and a VL comprising SEQ ID NO: 78 ID NO: 82 VL light chain CDR sequence.

[0265] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 78 and the amino acid sequence of SEQ ID NO: 82 The amino acid sequences of LCDR1, LCDR2 and LCDR3 of VL.

[0266] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 78 and the sequence of the VH of SEQ ID NO: 78 Backbone sequences have a backbone with at least 95%, 96%, 97%, 98% or 99% sequence identity. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 78 and a backbone having at least 95% sequence identity to the backbone sequence of the VH of SEQ ID NO: 78. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 78 and a backbone having at least 98% sequence identity to the backbone sequence of the VH of SEQ ID NO: 78.

[0267] In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises the light chain CDR sequence of the VL of SEQ ID NO: 82 and the sequence of the VL of SEQ ID NO: 82 Backbone sequences have a backbone with at least 95%, 96%, 97%, 98% or 99% sequence identity. In one aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 82 and a backbone having at least 95% sequence identity to the backbone sequence of the VL of SEQ ID NO: 82. In another aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 82 and a backbone with at least 98% sequence identity to the backbone sequence of the VL of SEQ ID NO: 82.

[0268] In another aspect, wherein the second antigen is CD19, specifically human CD19, the second antigen binding domain (and the third antigen binding domain when present) comprises: a heavy chain variable region (VH), It comprises the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 83, the HCDR 2 of SEQ ID NO: 84 and the HCDR 3 of SEQ ID NO: 85; and the light chain variable region (VL), which comprises SEQ ID Light chain complementarity determining region (LCDR) 1 of NO: 87, LCDR 2 of SEQ ID NO: 88 and LCDR 3 of SEQ ID NO: 89.

[0269] In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is (derived from) a humanized antibody. In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is a humanized antigen binding domain (ie, the antigen binding domain of a humanized antibody). In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) are humanized variable regions.

[0270] In one aspect, the VH and / or VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more heavy chain framework sequences of the heavy chain variable region sequence of SEQ ID NO: 86 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 86. 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: 86. 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: 86. In certain aspects, a VH sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise The antibody retains the ability to bind to CD19. In certain aspects, in the amino acid sequence of SEQ ID NO: 86, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 86. Optionally, the VH comprises the amino acid sequence of SEQ ID NO: 86, including post-translational modifications of that sequence. In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises one or more light chain framework sequences of the light chain variable region sequence of SEQ ID NO: 90 ( ie FR1, FR2, FR3 and / or FR4 sequences). 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: 90. 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: 90. 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: 90. In certain aspects, a VL sequence that is at least 95%, 96%, 97%, 98% or 99% identical comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but does not comprise The antibody retains the ability to bind to CD19. In certain aspects, in the amino acid sequence of SEQ ID NO: 90, a total of 1 to 10 amino acids are substituted, inserted and / or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (i.e., in FRs). In one aspect, the VL comprises the amino acid sequence of SEQ ID NO: 90. Optionally, the VL comprises the amino acid sequence of SEQ ID NO: 90, including post-translational modifications of that sequence.

[0273] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequence, and the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises at least about 95%, 96% of the amino acid sequence of SEQ ID NO: 90 %, 97%, 98% or 99% identical amino acid sequences. In one aspect, VH comprises the amino acid sequence of SEQ ID NO:86 and VL comprises the amino acid sequence of SEQ ID NO:90.

[0274] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: VH comprising the amino acid sequence of SEQ ID NO: 86; and VL comprising SEQ ID NO: Amino acid sequence of 90.

[0275] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises the VH sequence of SEQ ID NO: 86 and the VL sequence of SEQ ID NO: 90.

[0276] In another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises a VH comprising the heavy chain CDR sequence of the VH of SEQ ID NO: 86, and a VL comprising SEQ ID NO: 86 ID NO: 90 VL light chain CDR sequence.

[0277] In yet another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 86 and the amino acid sequence of SEQ ID NO : Amino acid sequences of LCDR1, LCDR2 and LCDR3 of VL of 90.

[0278] In one aspect, the VH of the second antigen binding domain (and, when present, the third antigen binding domain) comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 86 and the sequence of the VH of SEQ ID NO: 86 Backbone sequences have a backbone with at least 95%, 96%, 97%, 98% or 99% sequence identity. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 86 and a backbone having at least 95% sequence identity to the backbone sequence of the VH of SEQ ID NO: 86. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 86 and a backbone having at least 98% sequence identity to the backbone sequence of the VH of SEQ ID NO: 86.

[0279] In one aspect, the VL of the second antigen binding domain (and, when present, the third antigen binding domain) comprises the light chain CDR sequence of the VL of SEQ ID NO: 90 and the sequence of the VL of SEQ ID NO: 90 Backbone sequences have a backbone with at least 95%, 96%, 97%, 98% or 99% sequence identity. In one aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 90 and a backbone having at least 95% sequence identity to the backbone sequence of the VL of SEQ ID NO: 90. In another aspect, the VL comprises the light chain CDR sequence of the VL of SEQ ID NO: 90 and a backbone having at least 98% sequence identity to the backbone sequence of the VL of SEQ ID NO: 90. c) Charge modification

[0280] The (multispecific) antibodies of the invention may comprise amino acid substitutions in the Fab molecules contained therein which are particularly effective in reducing mismatches of light chains with mismatched heavy chains (Bence-Jones type by-products) , such mismatches may occur in the preparation of Fab-based multispecific antibodies in which a VH / VL exchange occurs in one (or more, if the molecule contains more than two antigen-binding Fab molecules) of its binding arms (otherwise See PCT Publication No. WO 2015 / 150447, particularly examples therein, the entire contents of which are incorporated herein by reference). The ratio of desired (multispecific) antibodies to undesired byproducts, in particular Bence Jones-type byproducts that occur in multispecific antibodies with a VH / VL domain swap in one of their binding arms, can be determined by Improvements are made by introducing oppositely charged amino acids at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modification").

[0281] Thus, in some aspects wherein the first and second antigen-binding domains (and, if present, the third antigen-binding domain) of a (multispecific) antibody are both Fab molecules, and In one of the binding domains (specifically the first antigen binding domain), the variable domains VL and VH of the Fab light chain and the Fab heavy chain replace each other, i) in the second antigen binding domain (and the third antigen binding domain when present) In the constant domain CL of the binding domain), the amino acid at position 124 is substituted by a positively charged amino acid (according to Kabat numbering), and wherein, in the second antigen-binding domain (and, when present, the third antigen-binding domain), 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 ii) in the first antigen-binding domain In the constant domain CL, 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, the amino acid at position 147 or The amino acid at position 213 was substituted with a negatively charged amino acid (numbering according to the Kabat EU index).

[0282] The (multispecific) antibody does not comprise the modifications mentioned under i) and ii). The constant domains CL and CH1 of the antigen-binding domain with VH / VL swapping were not replaced by each other (i.e. remained unswapped). In a more specific aspect, i) in the constant domain CL of the second antigen binding domain (and, when present, the third antigen binding domain), the amino acid at position 124 is independently via lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) are substituted, and in the constant domain CH1 of the second antigen-binding domain (and 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); or ii) in the first antigen binding domain In the constant domain CL of , the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and in the first antigen-binding domain The amino acid at position 147 or the amino acid at position 213 was independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index) in the constant domain CH1 of .

[0284] In one such aspect, in the constant domain CL of the second antigen binding domain (and, when present, the third antigen binding domain), the amino acid at position 124 is independently replaced by lysine (K) , arginine (R) or histidine (H) (according to Kabat numbering), and in the constant domain CH1 of the second antigen-binding domain (and third antigen-binding domain, if present), at position 147 The amino acid or the amino acid at position 213 was independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

[0285] In yet another aspect, in the constant domain CL of the second antigen binding domain (and, when present, the third antigen binding domain), the amino acid at position 124 is independently via lysine (K), Arginine (R) or histidine (H) substitution (according to Kabat numbering) and the amine at position 147 in the constant domain CH1 of the second (and third, if present) antigen-binding domain Amino acids were independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0286] In a preferred aspect, in the constant domain CL of the second antigen binding domain (and, when present, the third antigen binding domain), the amino acid at position 124 is independently replaced by lysine (K ), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) substituted and the amino acid at position 147 in the constant domain CH1 of the second (and third antigen-binding domain, if present) was independently replaced 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 with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index) replace.

[0287] In a more preferred aspect, in the constant domain CL of the second antigen binding domain (and, when present, the third antigen binding domain), the amino acid at position 124 is replaced by lysine (K) ( according to Kabat numbering) and the amino acid at position 123 is substituted with lysine (K) (according to Kabat numbering) and in the constant domain of the second antigen-binding domain (and third antigen-binding domain when present) In CH1, the amino acid at position 147 was substituted with glutamic acid (E) (numbered according to the Kabat EU index) and the amino acid at position 213 was substituted with glutamic acid (E) (numbered according to the Kabat EU index).

[0288] In an even more preferred aspect, in the constant domain CL of the second antigen binding domain (and, when present, the third antigen binding domain), the amino acid at position 124 is replaced by lysine (K) (according to Kabat numbering) and the amino acid at position 123 was 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 was substituted by Glutamic acid (E) (numbering according to Kabat EU index) was substituted and the amino acid at position 213 was substituted with glutamic acid (E) (numbering according to Kabat EU index).

[0289] In a preferred aspect, if the amino acid substitution according to the above aspects takes place in the constant domain CL and the constant domain CH1 of the second antigen binding domain (and, if present, the third antigen binding domain), then the second The constant domain CL of the second antigen-binding domain (and, when present, the third antigen-binding domain) is of the kappa isotype.

[0290] Alternatively, amino acid substitutions according to the above aspects may take place in the constant domain CL and the constant domain CH1 of the first antigen-binding domain, but not the second antigen-binding domain (and, if present, the third antigen-binding domain) in the constant domain CL and the constant domain CH1. In preferred such aspects, the constant domain CL of the first antigen binding domain is of the kappa isotype.

[0291] Thus, in one aspect, in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is independently modified by lysine (K), arginine (R) or histidine ( H) Substitution (according to Kabat numbering) and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 was independently modified by glutamic acid (E) or asparagine Acid (D) (number according to Kabat EU index) is substituted.

[0292] In yet another aspect, in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is independently modified by lysine (K), arginine (R) or histidine (H ) (according to Kabat numbering) and in the constant domain CH1 of the first antigen-binding domain, the amino acid at position 147 was independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index number) instead.

[0293] In yet another aspect, in the constant domain CL of the first antigen-binding domain, the amino acid at position 124 is independently modified by lysine (K), arginine (R) or histidine (H ) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and at the first In the constant domain CH1 of the antigen-binding domain, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index), and the amino acid at position 213 Independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to Kabat EU index).

[0294] In one aspect, in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is substituted with lysine (K) (according to Kabat numbering), and the amino acid at position 123 is substituted with lysine (K) (numbering according to Kabat) and in the constant domain CH1 of the first antigen-binding domain the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to the Kabat EU index), and The amino acid at position 213 was substituted with glutamic acid (E) (numbering according to Kabat EU index).

[0295] In another aspect, in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is substituted with lysine (K) (according to Kabat numbering), and the amino acid at position 123 Substituted by arginine (R) (numbering according to Kabat) and in the constant domain CH1 of the first antigen-binding domain the amino acid at position 147 was substituted by glutine (E) (numbering according to the Kabat EU index), And the amino acid at position 213 was substituted with glutamic acid (E) (numbering according to Kabat EU index).

[0296] In a preferred aspect, the (multispecific) antibody of the present invention comprises (A) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, wherein, the Fab light chain and the Fab The variable domains VL and VH of the heavy chain are substituted for each other and comprise (i) a heavy chain variable region (VH) selected from the group consisting of: (a) VH comprising the heavy chain of SEQ ID NO: 2 Chain complementarity determining region (HCDR) 1, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10, (b) VH comprising HCDR 1 of SEQ ID NO: 2, HCDR of SEQ ID NO: 4 2 and HCDR 3 of SEQ ID NO: 12, (c) VH, which comprises HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 5 and HCDR 3 of SEQ ID NO: 9, (d) VH, It comprises HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 6 and HCDR 3 of SEQ ID NO: 11, or (e) a VH comprising HCDR 1 of SEQ ID NO: 3, SEQ ID NO: HCDR 2 of 7 and HCDR 3 of SEQ ID NO: 13; and (ii) light chain variable region (VL), which comprises the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, SEQ ID NO: 21 LCDR 2 and LCDR 3 of SEQ ID NO: 22; and (B) a second antigen binding domain that binds to a second antigen and an optional third antigen binding domain; wherein, in the second antigen binding domain (and in In the constant domain CL of the third antigen-binding domain when present), the amino acid at position 124 is independently modified by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) substituted (in a preferred aspect, independently substituted with lysine (K) or arginine (R)), and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) substituted (in a preferred aspect, independently by lysine (K) or arginine (R)), and on the second antigen binding The amino acid at position 147 of the constant domain CH1 of the domain (and the third antigen-binding domain when present) is independently modified by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index) and the amino acid at position 213 was independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index). d) Multispecific antibody formats

[0297] The (multispecific) antibodies according to the invention may have various configurations. An exemplary configuration is shown in FIG. 1 .

[0298] In a preferred aspect, the antigen binding domain comprised in the (multispecific) antibody is a Fab molecule. In these aspects, the first antigen binding domain, second antigen binding domain, third antigen binding domain, etc. may be referred to herein as a first Fab molecule, a second Fab molecule, a third Fab molecule, etc., respectively.

[0299] In one aspect, the first and second antigen-binding domains of the (multispecific) antibody are fused to each other, optionally via a peptide linker. In preferred aspects, the first antigen binding domain and the second antigen binding domain are each Fab molecules. In one such aspect, 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. In another such aspect, 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. Alternatively, in which (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 at the C-terminus of the Fab heavy chain In the aspect where the Fab light chain of the first antigen binding domain is fused to the N-terminus of the Fab heavy chain of the first antigen binding domain, the Fab light chain of the first antigen binding domain and the Fab light chain of the second antigen binding domain may be fused to each other, optionally via a peptide linker .

[0300] (Multispecific) antibodies with a single antigen binding domain (e.g. a Fab molecule) capable of specifically binding to a second antigen (e.g. a target cell antigen such as TYRP-1, CEA, GPRC5D or CD19) can be used ( For example, as shown in Figure 1 A, D, G, H, K, L), particularly where internalization of the antigen is expected to occur after binding of the high affinity antigen binding domain. In such cases, the presence of more than one antigen binding domain directed against the second antigen may enhance internalization of the second antigen, thereby reducing its availability.

[0301] In other cases, however, there are (multispecific) antibodies (eg, as shown in Fig. 1B, FIG. 1C, FIG. 1E, FIG. 1F, FIG. 1I, FIG. 1J, FIG. 1M or FIG. 1N) would be advantageous, eg, to optimize targeting to a target or to cross-link target cell antigens.

[0302] Thus, in a preferred aspect, the (multispecific) antibody according to the invention comprises a third antigen binding domain.

[0303] In one aspect, the third antigen binding domain binds a second antigen (e.g., a target cell antigen such as TYRP-1, CEA, GPRC5D, or CD19). In one aspect, the third antigen binding domain is a Fab molecule.

[0304] In one aspect, the third antigenic domain is identical to the second antigen binding domain.

[0305] In some aspects, the third antigen binding domain and the second antigen binding domain are each Fab molecules, and the third antigen binding domain is the same as the second antigen binding domain. Thus, in these aspects, the second and third antigen binding domains comprise the same heavy and light chain amino acid sequences and have the same arrangement of domains (i.e. conventional or swapped). Also, in these aspects, the third antigen binding domain comprises the same amino acid substitutions, if any, as the second antigen binding domain. For example, an amino acid substitution described herein as "charge modification" would be made in the constant domain CL and the constant domain CH1 of each of the second and third antigen binding domains. Alternatively, such amino acid substitutions may be made in the constant domain CL and constant domain CH1 of the first antigen binding domain (which in a preferred aspect is also a Fab molecule), but not in the second antigen binding domain and the second antigen binding domain. The three antigen-binding domains are carried out in the constant domain CL and the constant domain CH1.

[0306] Similar to the second antigen binding domain, the third antigen binding domain is preferably a conventional Fab molecule. However, aspects are also conceivable in which the second and third antigen binding domains are swapped Fab molecules (and the first antigen binding domain is a conventional Fab molecule). Therefore, in a preferred aspect, the second antigen binding domain and the third antigen binding domain are each a conventional Fab molecule, and the first antigen binding domain is an exchanged Fab molecule as described herein, i.e., wherein the Fab heavy chain and light Fab molecules in which the variable domains VH and VL or the constant domains CL and CH1 of the chains are exchanged / replaced with each other. In other aspects, the second antigen binding domain and the third antigen binding domain are each swapped Fab molecules, and the first antigen binding domain is a conventional Fab molecule.

[0307] If a third antigen-binding domain is present, in a preferred aspect the first antigen-binding domain binds to CD3 and the second and third antigen-binding domains bind to the second antigen, in particular the target Cellular antigen (eg TYRP-1, CEA, GPRC5D or CD19) binding.

[0308] In a preferred aspect, the (multispecific) antibody of the invention comprises an Fc domain consisting of a first unit and a second unit. The first and second units of the Fc domain are capable of stable association.

[0309] The (multispecific) antibodies according to the invention may have different configurations, i.e. the first antigen-binding domain, the second antigen-binding domain (and optionally the third antigen-binding domain) may be fused to each other and in different way to fuse with the Fc domain. These components may be fused directly to each other or preferably via one or more suitable peptide linkers. In cases where the Fab molecule is fused to the N-terminus of the subunit of the Fc domain, it is usually fused through the immunoglobulin hinge region.

[0310] In some aspects, the first antigen-binding domain and the second antigen-binding domain are each a Fab molecule, and the first antigen-binding domain is at the C-terminus of the Fab heavy chain with the first or second unit of the Fc domain N-terminal fusion. In these aspects, the second antigen binding domain may be 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 or to the N-terminus of the other of the subunits of the Fc domain. In preferred such aspects, the second antigen binding domain is a conventional Fab molecule and the first antigen binding domain is an exchanged Fab molecule as described herein, i.e. wherein the variable domains VH and Fab molecules in which the VL or constant domains CL and CH1 are exchanged / replaced for each other. In other such aspects, the second antigen binding domain is an exchanged Fab molecule and the first antigen binding domain is a conventional Fab molecule.

[0311] In one aspect, the first antigen-binding domain and the second antigen-binding domain are each a Fab molecule, and the first antigen-binding domain is between the C-terminus of the Fab heavy chain and the first or second unit of the Fc domain The N-terminus is fused, 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 consists essentially of a first Fab molecule and a second Fab molecule, the Fc domain consists of a first unit and a second unit and optionally one or more peptide linkers constituting 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 first unit of the Fc domain or N-terminal fusion of the second unit. Such configurations are schematically depicted in Figure 1G and Figure 1K (in these examples, the first antigen binding domain is a VH / VL swapped Fab molecule). Additionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other, as appropriate.

[0312] 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 located at the C-terminus of the Fab heavy chain and next to the Fc domain. N-terminal fusion of one of the units. In a specific aspect, a (multispecific) antibody essentially consists of a first Fab molecule and a second Fab molecule, the Fc domain consisting of a first unit and a second unit and optionally one or more peptide linkers A composition 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. Such configurations are schematically depicted in Figures 1A and 1D (in these examples, the first antigen binding domain is a VH / VL swapped Fab molecule and the second 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 via a peptide linker. In a preferred aspect, the first Fab molecule and the second Fab molecule are each fused to an Fc domain via an immunoglobulin hinge region. In a specific aspect, the immunoglobulin hinge region is a human IgG1 hinge region, in particular wherein the Fc domain is an IgG1 Fc domain.

[0313] 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 at the C-terminus of the Fab heavy chain with the first or second unit of the Fc domain N-terminal fusion. In such aspects, the first antigen binding domain may be C-terminal to the Fab heavy chain and N-terminal to the other of the Fab heavy chain of the first antigen binding domain or (as described above) the subunit of the Fc domain fusion. In preferred such aspects, the second antigen binding domain is a conventional Fab molecule and the first antigen binding domain is an exchanged Fab molecule as described herein, i.e. wherein the variable domains VH of the Fab heavy and light chains and VL or constant domains CL and CH1 are exchanged / replaced Fab molecules with each other. In other such aspects, the second antigen binding domain is an exchanged Fab molecule and the first antigen binding domain is a conventional Fab molecule.

[0314] In one aspect, the first antigen-binding domain and the second antigen-binding domain are each a Fab molecule, and the second antigen-binding domain is between the C-terminus of the Fab heavy chain and the first or second unit of the Fc domain The N-terminus is fused, 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. In a specific aspect, the (multispecific) antibody consists essentially of a first Fab molecule and a second Fab molecule, the Fc domain consists of a first unit and a second unit and optionally one or more peptide linkers constituting wherein a first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the first unit of the Fc domain or N-terminal fusion of the second unit. Such configurations are schematically depicted in Figure 1H and Figure 1L (in these examples, the first antigen binding domain is a VH / VL swapped Fab molecule and the second antigen binding domain is a conventional Fab molecule). Additionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other, as appropriate.

[0315] In some aspects, a third antigen binding domain, in particular a third Fab molecule, is fused at the C-terminus of the Fab heavy chain to the N-terminus of either the first unit or the second unit of the Fc domain. In preferred such aspects, the second and third antigen binding domains are each conventional Fab molecules, and the first antigen binding domain is an exchanged Fab molecule as described herein, i.e., wherein the Fab heavy chain and Fab molecules in which the variable domains VH and VL or the constant domains CL and CH1 of the light chain are exchanged / replaced with each other. In other such aspects, the second and third antigen binding domains are each swapped Fab molecules and the first antigen binding domain is a conventional Fab molecule. In preferred such aspects, the first 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 the second antigen-binding The domain 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 a specific aspect, the (multispecific) antibody consists essentially of a first Fab molecule, a second Fab molecule and a third Fab molecule, the Fc domain consisting of a first unit and a second unit and optionally a or multiple 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 to the Fc domain at the C-terminus of the Fab heavy chain and wherein a third Fab molecule is fused to the N-terminus of the second unit of the Fc domain at the C-terminus of the Fab heavy chain. Figure 1B and Figure 1E (in these examples, the first antigen-binding domain is a VH / VL swapped Fab molecule, and the second and third antigen-binding domains are conventional Fab molecules) and Figure 1J and Figure 1N (in Such configurations are schematically depicted in these examples where the first antigen binding domain is a conventional Fab molecule and the second and third antigen binding domains are VH / VL swapped Fab molecules). The first Fab molecule and the third Fab molecule can be fused to the Fc domain directly or via a peptide linker. In a preferred aspect, the first Fab molecule and the third Fab molecule are each fused to an Fc domain via an immunoglobulin hinge region. In a specific aspect, the immunoglobulin hinge region is a human IgG1 hinge region, in particular wherein the Fc domain is an IgG1 Fc domain. Additionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other, as appropriate. In another such aspect, 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 the first antigen-binding domain Fusion at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain. In a specific aspect, the (multispecific) antibody consists essentially of a first Fab molecule, a second Fab molecule and a third Fab molecule, the Fc domain consisting of a first unit and a second unit and optionally a or multiple peptide linkers, wherein a first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a second Fab molecule, and the second Fab molecule is fused to the Fc domain at the C-terminus of the Fab heavy chain and wherein a third Fab molecule is fused to the N-terminus of the second unit of the Fc domain at the C-terminus of the Fab heavy chain. Figure 1C and Figure 1F (in these examples, the first antigen-binding domain is a VH / VL swapped Fab molecule, and the second and third antigen-binding domains are conventional Fab molecules) and Figure 1I and Figure 1M (in In these examples, the first antigen-binding domain is a conventional Fab molecule, and the second and third antigen-binding domains are VH / VL swapped Fab molecules) Such configurations are schematically depicted. The second Fab molecule and the third Fab molecule can be fused to the Fc domain directly or via a peptide linker. In a preferred aspect, the second Fab molecule and the third Fab molecule are each fused to an Fc domain via an immunoglobulin hinge region. In a specific aspect, the immunoglobulin hinge region is a human IgG1 hinge region, in particular wherein the Fc domain is an IgG1 Fc domain. Additionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be fused to each other, as appropriate.

[0318] In the configuration of a (multispecific) antibody in which the Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of each of the subunits of the Fc domain through an immunoglobulin hinge region, two The Fab molecule, hinge region and 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. In another aspect, the immunoglobulin is an IgG4 subclass immunoglobulin. In yet another preferred aspect, the immunoglobulin is a human immunoglobulin. In other aspects, the immunoglobulin is a chimeric or humanized immunoglobulin. In one aspect, the immunoglobulin comprises a human constant region, particularly a human Fc region.

[0319] In some (multispecific) antibodies of the 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 via a peptide linker. Depending on the configuration of the first Fab molecule and the second Fab molecule, the Fab light chain of the first Fab molecule can be fused to the N-terminus of the Fab light chain of the second Fab molecule at its C-terminus, or the Fab light chain of the second Fab molecule can The chain may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. Fusion of the Fab light chain of a first Fab molecule to a second Fab molecule further reduces mismatches of Fab heavy and light chains and also reduces the number of plastids required to express some (multispecific) antibodies of the invention.

[0320] The antigen binding domain may be fused directly to the Fc domain or to each other, or to the Fc or to each other via a peptide linker comprising one or more amino acids, usually about 2-20 amino acids sour. Peptide linkers are well known in the art and are described herein. Suitable non-immunizing peptide linkers include, for example, (G4S)n, (SG4)n, (G4S)n, G4(SG4)n or (G4S)nG5 peptide linkers. "N" is usually an integer from 1 to 10, especially 2 to 4. In one aspect, the peptide linker is at least 5 amino acids in length; in one aspect, 5 to 100 amino acids in length; in yet another aspect, 10 to 50 amino acids in length. In one aspect, the peptide linker is (GxS)n or (GxS)nGm, where G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=1, 2, 3, 4 or 5, and m=0, 1, 2, 3, 4 or 5); in one aspect, x =4 and n=2 or 3; In another aspect, x=4 and n=2; In yet another aspect, x=4, n=1, and m=5. In one aspect, the peptide link The child is (G4S)2. In another aspect, the peptide linker is G4SG5. 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 a first Fab fragment and a second Fab fragment comprises the sequence (D)-(G4S)2 (SEQ ID NOs 48 and 49). Another suitable such linker comprises the sequence (D)-G4SG5 (SEQ ID NO: 103 and SEQ ID NO: 104). Additionally, the linker may comprise (part of) an immunoglobulin hinge region. In particular, in cases where a Fab molecule is fused to the N-terminus of an Fc domain subunit, the fusion may be through an immunoglobulin hinge region or a portion thereof comprising or without an additional peptide linker .

[0321] In certain aspects, the (multispecific) antibody according to the invention comprises: a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares the carboxy-terminus with the Fab heavy chain constant region of the first Fab molecule Peptide bond (i.e., the first Fab molecule contains an exchanged Fab heavy chain in which the heavy chain variable region is replaced by the light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit (VL(1)-CH1 (1)-CH2-CH3(-CH4)); and polypeptides wherein the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH(2)-CH1(2)-CH2-CH3 (-CH4)). In some aspects, the (multispecific) antibody comprises in one step: a polypeptide, wherein the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH(1 )-CL(1)), and shares a carboxy-terminal peptide bond (VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule. In certain aspects, the polypeptides are covalently linked, eg, via disulfide bonds.

[0322] In certain aspects, the (multispecific) antibody according to the invention comprises: a polypeptide wherein the Fab heavy chain variable region of the first Fab molecule shares the carboxyl terminus with the Fab light chain constant region of the first Fab molecule Peptide bond (i.e., the first Fab molecule contains an exchanged Fab heavy chain in which the heavy chain constant region is replaced by the light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fc domain subunit (VH(1)-CL(1 )-CH2-CH3(-CH4)); and polypeptides in which the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH(2)-CH1(2)-CH2-CH3(- CH4)). In some aspects, the (multispecific) antibody further comprises: a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL(1) -CH1(1)), and share the carboxy-terminal peptide bond (VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule. In certain aspects, the polypeptides are covalently linked, eg, via disulfide bonds.

[0323] In some aspects, a (multispecific) antibody comprises a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e. the first A Fab molecule comprises an exchanged 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 second Fab molecule, which in turn shares the Fc domain subunit Carboxy-terminal peptide bond (VL(1)-CH1(1)-VH(2)-CH1(2)-CH2-CH3(-CH4)). In other aspects, a (multispecific) antibody comprises a polypeptide wherein the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, which in turn shares a peptide bond with the Fab light chain of the first Fab molecule. The Fab heavy chain constant region shares a carboxy-terminal peptide bond (i.e. the first Fab molecule contains an exchanged 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 with the Fc domain subunit Bond (VH(2)-CH1(2)-VL(1)-CH1(1)-CH2-CH3(-CH4)). In some of these aspects, the (multispecific) antibody further comprises: an exchanged Fab light chain polypeptide of the first Fab molecule, wherein the Fab heavy chain variable region of the first Fab molecule is separated from the Fab light chain of the first Fab molecule The chain constant regions share a carboxy-terminal peptide bond (VH(1)-CL(1)) and share a carboxy-terminal peptide bond (VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule. In still other of these aspects, the (multispecific) antibody further comprises: a polypeptide wherein the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule, It in turn shares a carboxy-terminal peptide bond (VH(1)-CL(1)-VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule; or a polypeptide in which the Fab of the second Fab molecule The light chain polypeptide 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 (VL(2)-CL(2)- VH(1)-CL(1)) (where appropriate). The (multispecific) antibody according to these aspects may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain of the third Fab molecule and the Fc domain The subunit shares a carboxy-terminal peptide bond (VH(3)-CH1(3)-CH2-CH3(-CH4)) and shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the third Fab molecule (VL(3)-CL (3)). In certain aspects, the polypeptides are covalently linked, eg, via disulfide bonds.

[0324] In some aspects, a (multispecific) antibody comprises a polypeptide wherein the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e. the first A Fab molecule comprises an exchanged 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 second Fab molecule, which in turn shares a carboxy-terminus with the Fc domain subunit Peptide bond (VH(1)-CL(1)-VH(2)-CH1(2)-CH2-CH3(-CH4)). In other aspects, the (multispecific) antibody comprises a polypeptide wherein 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 heavy chain variable region of the first Fab molecule. The Fab light chain constant region shares a carboxy-terminal peptide bond (i.e. the first Fab molecule comprises an exchanged 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 Fc domain subunit ( VH(2)-CH1(2)-VH(1)-CL(1)-CH2-CH3(-CH4)). In some of these aspects, the (multispecific) antibody further comprises: an exchanged Fab light chain polypeptide of the first Fab molecule, wherein the Fab light chain variable region of the first Fab molecule is the same as the Fab heavy chain of the first Fab molecule The chain constant regions share a carboxy-terminal peptide bond (VL(1)-CH1(1)) and share a carboxy-terminal peptide bond (VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule. In still other of these aspects, the (multispecific) antibody further comprises: a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule, It in turn shares a carboxy-terminal peptide bond (VL(1)-CH1(1)-VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule; or a polypeptide in which the Fab of the second Fab molecule The light chain polypeptide 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 (VL(2)-CL(2)- VH(1)-CL(1)) (where appropriate). The (multispecific) antibody according to these aspects may further comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavy chain of the third Fab molecule and the Fc domain The subunit shares a carboxy-terminal peptide bond (VH(3)-CH1(3)-CH2-CH3(-CH4)) and shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of the third Fab molecule (VL(3)-CL (3)). In certain aspects, the polypeptides are covalently linked, eg, via disulfide bonds.

[0325] In certain aspects, the (multispecific) antibody does not comprise an Fc domain. In preferred such aspects, the second antigen binding domain and (if present) the third antigen binding domain are each conventional Fab molecules and the first antigen binding domain is an exchanged Fab molecule as described herein, i.e. wherein, Fab A Fab molecule in which the variable domains VH and VL of the heavy and light chains or the constant domains CL and CH1 are exchanged / replaced for each other. In other such aspects, the second antigen binding domain and (if present) the third antigen binding domain are each an exchanged Fab molecule and the first antigen binding domain is a conventional Fab molecule.

[0326] In one such aspect, the (multispecific) antibody consists essentially of a first antigen-binding domain and a second antigen-binding domain, optionally comprising one or more peptide linkers, wherein the first antigen-binding Both the 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. Such configurations are schematically depicted in Figure 1O and Figure 1S (in these examples, the first antigen binding domain is a VH / VL swapped Fab molecule and the second antigen binding domain is a conventional Fab molecule).

[0327] In another such aspect, a (multispecific) antibody consists essentially of a first antigen-binding domain and a second antigen-binding domain, optionally comprising one or more peptide linkers, wherein the first antigen-binding Both the 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. Such configurations are schematically depicted in Figure 1P and Figure 1T (in these examples, the first antigen binding domain is a VH / VL swapped Fab molecule and the second antigen binding domain is a conventional Fab molecule).

[0328] In some aspects, 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 (multispecific) antibody further comprises a third antigen binding domain, specifically and 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 second Fab molecule. In certain of these aspects, the (multispecific) antibody consists essentially of a first Fab molecule, a second Fab molecule and a third Fab molecule, optionally comprising one or more peptide linkers, wherein the second Fab The 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 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 second Fab molecule. Figure 1Q and Figure 1U (in these examples, the first antigen-binding domain is a VH / VL swapped Fab molecule, and the second and third antigen-binding domains are conventional Fab molecules) or Figure 1X and Figure 1Z (in Such configurations are schematically depicted in these examples where the first antigen binding domain is a conventional Fab molecule and the second and third antigen binding domains are each VH / VL swapped Fab molecules).

[0329] 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, specifically and a third Fab molecule, wherein the third Fab molecule is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the second Fab molecule. In certain of these aspects, the (multispecific) antibody consists essentially of a first Fab molecule, a second Fab molecule and a third Fab molecule, optionally comprising one or more peptide linkers, wherein the first Fab The 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 N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the second Fab molecule. Figure 1R and Figure 1V (in these examples, the first antigen-binding domain is a VH / VL swapped Fab molecule, and the second and third antigen-binding domains are conventional Fab molecules) or Figure 1W and Figure 1Y (in Such configurations are schematically depicted in these examples where the first antigen binding domain is a conventional Fab molecule and the second and third antigen binding domains are each VH / VL swapped Fab molecules).

[0330] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide wherein the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, This 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 an exchanged Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region) (VH(2 )-CH1(2)-VL(1)-CH1(1)). In some aspects, the (multispecific) antibody comprises in one step: a polypeptide, wherein the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH(1 )-CL(1)), and shares a carboxy-terminal peptide bond (VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule.

[0331] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide with the Fab heavy chain constant region of the first Fab molecule linkage (i.e., the first Fab molecule comprises an exchanged 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 second Fab molecule (VL(1 )-CH1(1)-VH(2)-CH1(2)). In some aspects, the (multispecific) antibody comprises in one step: a polypeptide, wherein the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH(1 )-CL(1)), and shares a carboxy-terminal peptide bond (VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule. In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide wherein the Fab light chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, This 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 an exchanged Fab heavy chain in which the heavy chain constant region is replaced by the light chain constant region) (VH(2)- CH1(2)-VH(1)-CL(1)). In some aspects, the (multispecific) antibody further comprises: a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL(1) -CH1(1)), and share the carboxy-terminal peptide bond (VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule.

[0333] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide wherein the Fab heavy chain variable region of the first Fab molecule shares the carboxy-terminal peptide with the Fab light chain constant region of the first Fab molecule linkage (i.e. the first Fab molecule comprises an exchanged Fab heavy chain in which 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 of the second Fab molecule (VH(1)- CL(1)-VH(2)-CH1(2)). In some aspects, the (multispecific) antibody further comprises: a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL(1) -CH1(1)), and share the carboxy-terminal peptide bond (VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule.

[0334] In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide wherein the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule, which in turn shares a The Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond, 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 an exchanged Fab heavy chain, wherein, The heavy chain variable region was replaced by the light chain variable region) (VH(3)-CH1(3)-VH(2)-CH1(2)-VL(1)-CH1(1)). In some aspects, the (multispecific) antibody comprises in one step: a polypeptide, wherein the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH(1 )-CL(1)), and shares a carboxy-terminal peptide bond (VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule. In some aspects, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL(3)-CL(3)) of a third Fab molecule.

[0335] In certain aspects, a (multispecific) antibody according to the invention comprises a polypeptide wherein the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule, which in turn shares a The Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond, 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 an exchanged Fab heavy chain wherein, The heavy chain constant region was replaced by the light chain constant region) (VH(3)-CH1(3)-VH(2)-CH1(2)-VH(1)-CL(1)). In some aspects, the (multispecific) antibody further comprises: a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL(1) -CH1(1)), and share the carboxy-terminal peptide bond (VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule. In some aspects, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL(3)-CL(3)) of a third Fab molecule.

[0336] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide with the Fab heavy chain constant region of the first Fab molecule bond (i.e. the first Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain variable region is replaced by the light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule. The Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond (VL(1)-CH1(1)-VH(2)-CH1(2)-VH(3)-CH1(3)). In some aspects, the (multispecific) antibody comprises in one step: a polypeptide, wherein the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VH(1 )-CL(1)), and shares a carboxy-terminal peptide bond (VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule. In some aspects, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL(3)-CL(3)) of a third Fab molecule.

[0337] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide wherein the Fab heavy chain variable region of the first Fab molecule shares the carboxy-terminal peptide with the Fab light chain constant region of the first Fab molecule bond (i.e. the first Fab molecule comprises an exchanged Fab heavy chain in which 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 of the second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of the third Fab molecule. The Fab heavy chain of the Fab molecule shares a carboxy-terminal peptide bond (VH(1)-CL(1)-VH(2)-CH1(2)-VH(3)-CH1(3)). In some aspects, the (multispecific) antibody further comprises: a polypeptide wherein the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL(1) -CH1(1)), and share the carboxy-terminal peptide bond (VL(2)-CL(2)) with the Fab light chain polypeptide of the second Fab molecule. In some aspects, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL(3)-CL(3)) of a third Fab molecule. In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, This 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 an exchanged Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares The Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond, 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 an exchanged Fab heavy chain, wherein, The heavy chain variable region was replaced by the light chain variable region) (VH(1)-CH1(1)-VL(2)-CH1(2)-VL(3)-CH1(3)). In some aspects, the (multispecific) antibody comprises in one step: a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH(2) with the Fab light chain constant region of the second Fab molecule )-CL(2)), and share a carboxy-terminal peptide bond (VL(1)-CL(1)) with the Fab light chain polypeptide of the first Fab molecule. In some aspects, the (multispecific) antibody further comprises a polypeptide wherein 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 (VH(3)- CL(3)).

[0339] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide wherein the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, It 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 an exchanged 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 third Fab molecule. The Fab heavy chain variable region of the Fab molecule shares a carboxy-terminal peptide bond, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of a third Fab molecule (i.e. the third Fab molecule comprises an exchanged Fab heavy chain, wherein the heavy chain The constant region was replaced by the light chain constant region) (VH(1)-CH1(1)-VH(2)-CL(2)-VH(3)-CL(3)). In some aspects, the (multispecific) antibody comprises in one step: a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL(2) with the Fab heavy chain constant region of the second Fab molecule )-CH1(2)), and share the carboxy-terminal peptide bond (VL(1)-CL(1)) with the Fab light chain polypeptide of the first Fab molecule. In some aspects, the (multispecific) antibody further comprises a polypeptide wherein the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (VL(3)- CH1(3)).

[0340] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide wherein the Fab light chain variable region of the third Fab molecule shares the carboxy-terminal peptide with the Fab heavy chain constant region of the third Fab molecule linkage (i.e. the third Fab molecule comprises an exchanged Fab heavy chain in which the heavy chain variable region is replaced by the light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, This 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 an exchanged Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares The Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond (VL(3)-CH1(3)-VL(2)-CH1(2)-VH(1)-CH1(1)). In some aspects, the (multispecific) antibody comprises in one step: a polypeptide wherein the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VH(2) with the Fab light chain constant region of the second Fab molecule )-CL(2)), and share a carboxy-terminal peptide bond (VL(1)-CL(1)) with the Fab light chain polypeptide of the first Fab molecule. In some aspects, the (multispecific) antibody further comprises a polypeptide wherein 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 (VH(3)- CL(3)).

[0341] In certain aspects, the (multispecific) antibody according to the invention comprises a polypeptide wherein the Fab heavy chain variable region of the third Fab molecule shares the carboxy-terminal peptide with the Fab light chain constant region of the third Fab molecule bond (i.e. the third Fab molecule comprises an exchanged 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 variable region of the 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 an exchanged 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 first Fab molecule The Fab heavy chains share a carboxy-terminal peptide bond (VH(3)-CL(3)-VH(2)-CL(2)-VH(1)-CH1(1)). In some aspects, the (multispecific) antibody comprises in one step: a polypeptide wherein the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond (VL(2) with the Fab heavy chain constant region of the second Fab molecule )-CH1(2)), and share the carboxy-terminal peptide bond (VL(1)-CL(1)) with the Fab light chain polypeptide of the first Fab molecule. In some aspects, the (multispecific) antibody further comprises a polypeptide wherein the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (VL(3)- CH1(3)).

[0342] In one aspect, the invention provides a (multispecific) antibody comprising (A) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, wherein the Fab light chain and the variable domains VL and VH of the Fab heavy chain or the constant domains CL and CH1 are substituted for each other and comprise (i) a heavy chain variable region (VH) selected from the group consisting of (a) VH which Comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10, (b) a VH comprising HCDR 1 of SEQ ID NO: 2 , HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 12, (c) VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 5 and HCDR 3 of SEQ ID NO: 9 HCDR 3, (d) VH comprising HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 6 and HCDR 3 of SEQ ID NO: 11, or (e) VH comprising SEQ ID NO: 3 HCDR 1 of SEQ ID NO: 7 and HCDR 3 of SEQ ID NO: 13 (specifically VH, which includes HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4 and SEQ ID NO and (ii) a light chain variable region (VL) comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21 and SEQ ID NO : 22 of LCDR 3; (B) a second antigen binding domain that binds to a second antigen (specifically, a target cell antigen, more specifically TYRP-1, CEA, GPRC5D or CD19), wherein the second antigen binds The domain is a (conventional) Fab molecule; (C) an Fc domain consisting of a first unit and a second unit; wherein (i) the first antigen-binding domain described under (A) is in the Fab heavy chain The C-terminus of the Fab heavy chain described under (B) is fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain described under (B), and the second antigen-binding domain described under (B) is fused to the C-terminus of the Fab heavy chain in ( C) the N-terminal fusion of one of the subunits of the Fc domain described below, or (ii) the second antigen binding domain described under (B) at the C-terminus of the Fab heavy chain with the one described under (A) The N-terminus of the Fab heavy chain of the first antigen-binding domain described under (A) is fused at the C-terminus of the Fab heavy chain with the secondary of the Fc domain described under (C) N-terminal fusion of one of the units.

[0343] In a preferred aspect, the present invention provides a (multispecific) antibody comprising (A) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, wherein, Fab The variable domains VL and VH or the constant domains CL and CH1 of the light chain and the Fab heavy chain replace each other and comprise (i) a heavy chain variable region (VH) selected from the group consisting of: (a) VH , which comprises the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4, and HCDR 3 of SEQ ID NO: 10, (b) a VH comprising the HCDR of SEQ ID NO: 2 HCDR 1, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 12, (c) VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 5 and SEQ ID NO: HCDR 3 of 9, (d) VH comprising HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 6 and HCDR 3 of SEQ ID NO: 11, or (e) VH comprising SEQ ID NO HCDR 1 of : 3, HCDR 2 of SEQ ID NO: 7, and HCDR 3 of SEQ ID NO: 13 (specifically VH, which includes HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4, and SEQ ID NO: 2) ID NO: HCDR 3 of 10); and (ii) light chain variable region (VL), it comprises the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, the LCDR 2 of SEQ ID NO: 21 and SEQ ID NO: 21 LCDR 3 of ID NO: 22; (B) a second antigen-binding domain and a third antigen-binding domain that bind to (specifically, a target cell antigen, more specifically TYRP-1, CEA, GPRC5D or CD19), wherein the second antigen-binding domain and the third antigen-binding domain are each a (conventional) Fab molecule; and (C) an Fc domain consisting of a first unit and a second unit; wherein (i) in (A) The first antigen binding domain described below 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 described under (B), and the second antigen binding domain described under (B) The antigen binding domain and the third antigen binding domain described under (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 described under (C), or (ii ) the second antigen binding domain described under (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 described under (A), and under (A) The first antigen-binding domain and the third antigen-binding domain described under (B) are each at the C-terminus of the Fab heavy chain and at the N-terminus of one of the subunits of the Fc domain described under (C) fusion.

[0344] In another aspect, the invention provides a (multispecific) antibody comprising (A) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, wherein the Fab is lightly chain and the variable domains VL and VH of the Fab heavy chain or the constant domains CL and CH1 are substituted for each other and comprise (i) a heavy chain variable region (VH) selected from the group consisting of (a) VH, It comprises heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10, (b) VH comprising the HCDR of SEQ ID NO: 2 1. HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 12, (c) VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 5 and SEQ ID NO: 9 HCDR 3, (d) VH comprising HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 6 and HCDR 3 of SEQ ID NO: 11, or (e) VH comprising SEQ ID NO: HCDR 1 of 3, HCDR 2 of SEQ ID NO: 7, and HCDR 3 of SEQ ID NO: 13 (specifically VH, which includes HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4, and SEQ ID NO: HCDR 3 of 10); and (ii) light chain variable region (VL), it comprises the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, the LCDR 2 of SEQ ID NO: 21 and SEQ ID LCDR 3 of NO: 22; (B) a second antigen binding domain that binds to a second antigen (specifically, a target cell antigen, more specifically TYRP-1, CEA, GPRC5D or CD19), wherein the second antigen The binding domain is a (conventional) Fab molecule; (C) Fc domain, the Fc domain is composed of a first unit and a second unit; wherein (i) the first antigen binding domain described under (A) is the same as in ( B) The second antigen binding domains described below 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 described under (C).

[0345] In all the different configurations of the (multispecific) antibody according to the invention, the amino acid substitutions ("charge modifications") described herein, if present, may be in the second antigen-binding domain and (if present ) in the CH1 and CL domains of the third antigen binding domain / Fab molecule, or in the CH1 and CL domains of the first antigen binding domain / Fab molecule. Preferably, they are in the CH1 and CL domains of the second and, if present, third antigen binding domain / Fab molecule. According to the concept of the present invention, if the amino acid substitutions described herein are made in the second antigen binding domain (and, if present, the third antigen binding domain) / Fab molecule, then no Such amino acid substitutions exist. In contrast, if the amino acid substitutions described herein are made in the first antigen binding domain / Fab molecule, no such amines are present in the second antigen binding domain (and, if present, the third antigen binding domain) / Fab molecule. amino acid substitution. Amino acid substitutions are preferably made 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.

[0346] In a preferred aspect of the (multispecific) antibody according to the invention, in particular wherein the amino acid substitutions as described herein are in the second antigen binding domain (and if present) the third antigen binding domain) / Fab molecule, the constant domain CL of the second Fab molecule (and, if present, the third Fab molecule) is of the kappa isotype. In other aspects of the (multispecific) antibody according to the invention, in particular where the amino acid substitutions as described herein are made in the first antigen binding domain / Fab molecule, the first antigen binding domain The constant domain CL of the / Fab molecule is of the kappa isotype. In some aspects, the constant domain CL of the second antigen binding domain (and, if present, the third antigen binding domain) / Fab molecule and the constant domain CL of the first antigen binding domain / Fab molecule are of the kappa isotype.

[0347] In one aspect, the invention provides a (multispecific) antibody comprising (A) a first antigen binding domain that binds to CD3, wherein the first antigen binding domain is a Fab molecule, wherein the Fab light chain and the variable domains VL and VH of the Fab heavy chain are substituted for each other and comprise (i) a heavy chain variable region (VH) selected from the group consisting of: (a) VH comprising SEQ ID NO: 2 heavy chain complementarity determining region (HCDR) 1, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10, (b) VH comprising HCDR 1 of SEQ ID NO: 2, SEQ ID NO: 4 HCDR 2 of SEQ ID NO: 12 and HCDR 3, (c) VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 5 and HCDR 3 of SEQ ID NO: 9, (d) VH comprising HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 6 and HCDR 3 of SEQ ID NO: 11, or (e) VH comprising HCDR 1 of SEQ ID NO: 3, SEQ ID HCDR 2 of NO: 7 and HCDR 3 of SEQ ID NO: 13 (specifically VH, which includes HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10) and (ii) a light chain variable region (VL) comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21 and LCDR 3 of SEQ ID NO: 22; (B) A second antigen binding domain that binds to a second antigen (in particular, a target cell antigen, more specifically TYRP-1, CEA, GPRC5D or CD19), wherein the second antigen binding domain is a (conventional) Fab Molecule; (C) Fc domain, the Fc domain is composed of a first unit and a second unit; wherein, in the constant domain CL of the second antigen-binding domain described under (B), the amine group at position 124 The acid is substituted with lysine (K) (according to Kabat numbering) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (according to Kabat numbering) (optimally via arginine (R)) and wherein, in the constant domain CH1 of the second antigen-binding domain described under (B), 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); and wherein (i) the first antigen-binding domain described under (A) is at the C-terminus of the Fab heavy chain Fused to the N-terminus of the Fab heavy chain of the second antigen-binding domain described under (B), and the second antigen-binding domain described under (B) is at the C-terminus of the Fab heavy chain and described under (C) fused to the N-terminus of one of the subunits of the Fc domain, or (ii) the second antigen-binding domain described under (B) at the C-terminus of the Fab heavy chain with the second antigen-binding domain described under (A) An antigen binding domain is fused to the N-terminus of the Fab heavy chain, and the first antigen binding domain described under (A) is between the C-terminus of the Fab heavy chain and the subunit of the Fc domain described under (C) An N-terminal fusion.

[0348] In a preferred aspect, the present invention provides a (multispecific) antibody comprising (A) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, wherein the Fab The variable domains VL and VH of the light chain and the Fab heavy chain replace each other and comprise (i) a heavy chain variable region (VH) selected from the group consisting of: (a) VH comprising SEQ ID NO : 2 heavy chain complementarity determining region (HCDR) 1, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10, (b) VH comprising HCDR 1 of SEQ ID NO: 2, SEQ ID NO HCDR 2 of : 4 and HCDR 3 of SEQ ID NO: 12, (c) VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 5 and HCDR 3 of SEQ ID NO: 9, ( d) a VH comprising HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 6 and HCDR 3 of SEQ ID NO: 11, or (e) a VH comprising HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 7 and HCDR 3 of SEQ ID NO: 13 (specifically VH, which includes HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4 and HCDR of SEQ ID NO: 10 3); and (ii) a light chain variable region (VL) comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21 and LCDR of SEQ ID NO: 22 3; (B) a second antigen binding domain and a third antigen binding domain that bind to (specifically, a target cell antigen, more specifically TYRP-1, CEA, GPRC5D or CD19), wherein the second antigen binding domain and the third antigen binding domain are each a (conventional) Fab molecule; and (C) an Fc domain, the Fc domain is composed of a first unit and a second unit; wherein the second antigen binding described under (B) In domain and constant domain CL of the third antigen-binding domain described under (B), the amino acid at position 124 was substituted by lysine (K) (according to Kabat numbering), and the amino acid at position 123 was substituted by Lysine (K) or arginine (R) (according to Kabat numbering) (optimally substituted by arginine (R)), and wherein the second antigen binding domain described under (B) and ( B) In the constant domain CH1 of the third antigen-binding domain described below, the amino acid at position 147 was substituted with glutamic acid (E) (numbering according to the Kabat EU index) and the amino acid at position 213 was substituted with glutamic acid (E). amino acid (E) (according to Kabat EU index numbering); and wherein (i) the first antigen binding domain described under (A) is at the C-terminus of the Fab heavy chain with the second described under (B) The N-terminus of the Fab heavy chain of the antigen binding domain is fused, and the second antigen binding domain described under (B) and the third antigen binding domain described under (B) are each at the C-terminus of the Fab heavy chain and at the (C) an N-terminal fusion of one of the subunits of the Fc domain described below, or (ii) a second antigen-binding domain described under (B) at the C-terminus of the Fab heavy chain with that described under (A) The N-terminus of the Fab heavy chain of the first antigen binding domain is fused, and the first antigen binding domain described under (A) and the third antigen binding domain described under (B) are each at the Fab heavy chain fused to the N-terminus of one of the subunits of the Fc domain described under (C).

[0349] In another aspect, the invention provides a (multispecific) antibody comprising (A) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, wherein the Fab is chain and the variable domains VL and VH of the Fab heavy chain replace each other and comprise (i) a heavy chain variable region (VH) selected from the group consisting of: (a) VH comprising SEQ ID NO: Heavy chain complementarity determining region (HCDR) 1 of 2, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10, (b) VH comprising HCDR 1 of SEQ ID NO: 2, SEQ ID NO: HCDR 2 of 4 and HCDR 3 of SEQ ID NO: 12, (c) VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 5 and HCDR 3 of SEQ ID NO: 9, (d ) VH comprising HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 6 and HCDR 3 of SEQ ID NO: 11, or (e) VH comprising HCDR 1 of SEQ ID NO: 3, SEQ ID NO: 3 HCDR 2 of ID NO: 7 and HCDR 3 of SEQ ID NO: 13 (specifically VH, which includes HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10 ); and (ii) a light chain variable region (VL) comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21 and LCDR 3 of SEQ ID NO: 22 (B) a second antigen binding domain that binds to a second antigen (in particular, a target cell antigen, more specifically TYRP-1, CEA, GPRC5D or CD19), wherein the second antigen binding domain is (conventional) Fab molecule; (C) Fc domain, the Fc domain is composed of a first unit and a second unit; wherein, in the constant domain CL of the second antigen-binding domain described under (B), the amine at position 124 amino acid was substituted with lysine (K) (according to Kabat numbering) and the amino acid at position 123 was substituted with lysine (K) or arginine (R) (according to Kabat numbering) (optimally via spermine acid (R)) and wherein, in the constant domain CH1 of the second antigen binding domain described under (B), the amino acid at position 147 is replaced by glutamic acid (E) (numbering according to the Kabat EU index) substituted, and the amino acid at position 213 is substituted by glutamic acid (E) (according to Kabat EU index numbering); and wherein the first antigen binding domain described under (A) is the same as described under (B) Each of the second antigen binding domains is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described under (C).

[0350] According to any of the above aspects, the components of the (multispecific) antibody (e.g. Fab molecules, Fc domains) may be fused directly or via various linkers, in particular via Known peptide linkers comprising one or more amino acids (usually about 2-20 amino acids) are used for fusion. Suitable non-immune peptide linkers include, for example, (G4S)n, (SG4)n, (G4S)n, G4(SG4)n or (G4S)nG5 peptide linkers, wherein n is typically an integer from 1 to 10 , especially 2 to 4.

[0351] In one aspect, the invention provides a (multispecific) antibody comprising (A) a first antigen binding domain that binds CD3, wherein the first antigen binding domain is a Fab molecule, wherein the Fab light chain and The variable domains VL and VH of the Fab heavy chain replace each other and comprise (i) a heavy chain variable region (VH) selected from the group consisting of: (a) VH comprising the sequence of SEQ ID NO: 2 Heavy chain complementarity determining region (HCDR) 1, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10, (b) VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 3 of SEQ ID NO: 4 HCDR 2 and HCDR 3 of SEQ ID NO: 12, (c) VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 5 and HCDR 3 of SEQ ID NO: 9, (d) VH , which comprises the HCDR 1 of SEQ ID NO: 3, the HCDR 2 of SEQ ID NO: 6 and the HCDR 3 of SEQ ID NO: 11, or (e) VH, which comprises the HCDR 1 of SEQ ID NO: 3, the HCDR 1 of SEQ ID NO HCDR 2 of SEQ ID NO: 7 and HCDR 3 of SEQ ID NO: 13 (specifically VH, which includes HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10); And (ii) a light chain variable region (VL) comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21 and LCDR 3 of SEQ ID NO: 22; ( B) The second antigen-binding domain and the third antigen-binding domain that bind to TYRP-1, wherein each of the second antigen-binding domain and the third antigen-binding domain is a (conventional) Fab molecule, and includes: a heavy chain variable region ( VH ) comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 15, HCDR 2 of SEQ ID NO: 16 and HCDR 3 of SEQ ID NO: 17; and light chain variable region (VL), which Comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 19, LCDR 2 of SEQ ID NO: 20 and LCDR 3 of SEQ ID NO: 21; (C) Fc domain, the Fc domain is composed of the first unit and The second unit constitutes; wherein in the constant domain CL of the second antigen-binding domain and the third antigen-binding domain described under (B), the amino acid at position 124 is replaced 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) (optimally by arginine (R)), and wherein, in (B In the constant domain CH1 of the second antigen-binding domain and the third antigen-binding domain described under The amino acid is substituted by glutamic acid (E) (numbering according to the Kabat EU index); and wherein the second antigen binding domain otherwise described under (B) is at the C-terminus of the Fab heavy chain as described under (A) The N-terminus of the Fab heavy chain of the first antigen binding domain is fused, and the first antigen binding domain described under (A) and the third antigen binding domain described under (B) are each at the C of the Fab heavy chain fused to the N-terminus of one of the subunits of the Fc domain described under (C).

[0352] In yet another aspect, the invention provides a (multispecific) antibody comprising (A) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, wherein the Fab light chain and the variable domains VL and VH of the Fab heavy chain replace each other and comprise: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18 , SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19 (specifically, the amino acid sequence of SEQ ID NO: 16); and a light chain variable region comprising the sequence of SEQ ID NO: 11 Amino acid sequence; (B) the second antigen-binding domain and the third antigen-binding domain that bind to TYRP-1, wherein the second antigen-binding domain and the third antigen-binding domain are each a (conventional) Fab molecule and include: a heavy chain Variable region, it comprises the amino acid sequence of SEQ ID NO: 27; And light chain variable region, it comprises the amino acid sequence of SEQ ID NO: 31; (C) Fc domain, this Fc domain consists of the first unit and the second unit; wherein in the constant domain CL of the second antigen-binding domain and the third antigen-binding domain described under (B), the amino acid at position 124 is replaced 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) (optimally by arginine (R)), and wherein, in (B) In the constant domain CH1 of the second and third antigen-binding domains described below, the amino acid at position 147 was substituted with glutamic acid (E) (numbering according to the Kabat EU index), and position 213 where the amino acid is substituted by glutamic acid (E) (numbering according to the Kabat EU index); and wherein the second antigen-binding domain otherwise described under (B) is at the C-terminus of the Fab heavy chain and under (A) The N-terminus of the Fab heavy chain of the first antigen binding domain is fused, and the first antigen binding domain described under (A) and the third antigen binding domain described under (B) are each at the Fab heavy chain fused to the N-terminus of one of the subunits of the Fc domain described under (C).

[0353] In another aspect, the invention provides a (multispecific) antibody comprising (A) a first antigen binding domain that binds CD3, wherein the first antigen binding domain is a Fab molecule, wherein the Fab light chain and the variable domains VL and VH of the Fab heavy chain are substituted for each other and comprise (i) a heavy chain variable region (VH) selected from the group consisting of: (a) VH comprising SEQ ID NO: 2 heavy chain complementarity determining region (HCDR) 1, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10, (b) VH comprising HCDR 1 of SEQ ID NO: 2, SEQ ID NO: 4 HCDR 2 of SEQ ID NO: 12 and HCDR 3, (c) VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 5 and HCDR 3 of SEQ ID NO: 9, (d) VH comprising HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 6 and HCDR 3 of SEQ ID NO: 11, or (e) a VH comprising HCDR 1 of SEQ ID NO: 3, SEQ ID HCDR 2 of NO: 7 and HCDR 3 of SEQ ID NO: 13 (specifically VH, which includes HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10) and (ii) a light chain variable region (VL) comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21 and LCDR 3 of SEQ ID NO: 22; (B) A second antigen-binding domain and a third antigen-binding domain that bind to CEA, wherein each of the second and third antigen-binding domains is a (conventional) Fab molecule comprising: (i) a VH comprising HCDR 1 of SEQ ID NO: 53, HCDR 2 of SEQ ID NO: 54 and HCDR 3 of SEQ ID NO: 55, and VL comprising LCDR 1 of SEQ ID NO: 57, LCDR 2 of SEQ ID NO: 58 and LCDR 3 of SEQ ID NO: 59; (ii) VH comprising HCDR 1 of SEQ ID NO: 105, HCDR 2 of SEQ ID NO: 106 and HCDR 3 of SEQ ID NO: 107, and VL comprising SEQ ID NO: LCDR 1 of 109, LCDR 2 of SEQ ID NO: 110, and LCDR 3 of SEQ ID NO: 111; or (iii) VH comprising HCDR 1 of SEQ ID NO: 113, HCDR 2 of SEQ ID NO: 114 And the HCDR 3 of SEQ ID NO: 115, and VL, it comprises the LCDR 1 of SEQ ID NO: 117, the LCDR 2 of SEQ ID NO: 118 and the LCDR 3 of SEQ ID NO: 119; (C) Fc domain, the Fc The domain consists of a first unit and a second unit; wherein in the constant domain CL of the second and third antigen-binding domains described under (B), the amino acid at position 124 is replaced by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (numbering according to Kabat) (optimally with arginine (R)) , and wherein, in the constant domain CH1 of the second and third antigen-binding domains described under (B), the amino acid at position 147 is glutamic (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); and wherein the second antigen binding domain additionally described under (B) is at the C-terminus of the Fab heavy chain with N-terminal fusion of the Fab heavy chain of the first antigen-binding domain described under (A), and the first antigen-binding domain described under (A) and the third antigen-binding domain described under (B) Each is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain described under (C).

[0354] In yet another aspect, the invention provides a (multispecific) antibody comprising (A) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, wherein the Fab light chain and the variable domains VL and VH of the Fab heavy chain replace each other and comprise: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18 , SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19 (specifically, the amino acid sequence of SEQ ID NO: 16); and a light chain variable region comprising the sequence of SEQ ID NO: 11 Amino acid sequence; (B) a second antigen binding domain and a third antigen binding domain that binds to CEA, wherein the second antigen binding domain and the third antigen binding domain are each a (conventional) Fab molecule and comprise: (i) heavy Chain variable region, it comprises the amino acid sequence of SEQ ID NO: 56; And light chain variable region, it comprises the aminoacid sequence of SEQ ID NO: 60; (ii) heavy chain variable region, it comprises SEQ ID NO: the amino acid sequence of 108; and the light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 112; or (iii) the heavy chain variable region, which comprises the amino acid sequence of SEQ ID NO: 116 sequence; and the light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 120; (C) Fc domain, which is composed of the first unit and the second unit; wherein under (B) In the constant domain CL of the second antigen-binding domain and the third 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) amino acid (K) or arginine (R) (according to Kabat numbering) (optimally substituted by arginine (R)), and wherein the second antigen binding domain and the third antigen binding domain described under (B) In the constant domain CH1 of the antigen-binding domain, the amino acid at position 147 was substituted by glutamic acid (E) (numbering according to the Kabat EU index), and the amino acid at position 213 was replaced by glutamic acid (E) (according to Kabat EU index numbering). EU index number) is substituted; and wherein the second antigen-binding domain described under (B) is at the C-terminus of the Fab heavy chain with the N-terminus of the Fab heavy chain of the first antigen-binding domain described under (A) fused, and the first antigen binding domain described under (A) and the third antigen binding domain described under (B) are each at the C-terminus of the Fab heavy chain with the Fc domain described under (C) N-terminal fusion of one of the subunits.

[0355] In another aspect, the invention provides a (multispecific) antibody comprising (A) a first antigen binding domain that binds CD3, wherein the first antigen binding domain is a Fab molecule, wherein the Fab light chain and the variable domains VL and VH of the Fab heavy chain are substituted for each other and comprise (i) a heavy chain variable region (VH) selected from the group consisting of: (a) VH comprising SEQ ID NO: 2 heavy chain complementarity determining region (HCDR) 1, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10, (b) VH comprising HCDR 1 of SEQ ID NO: 2, SEQ ID NO: 4 HCDR 2 of SEQ ID NO: 12 and HCDR 3, (c) VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 5 and HCDR 3 of SEQ ID NO: 9, (d) VH comprising HCDR 1 of SEQ ID NO: 3, HCDR 2 of SEQ ID NO: 6 and HCDR 3 of SEQ ID NO: 11, or (e) VH comprising HCDR 1 of SEQ ID NO: 3, SEQ ID HCDR 2 of NO: 7 and HCDR 3 of SEQ ID NO: 13 (specifically VH, which includes HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10) and (ii) a light chain variable region (VL) comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21 and LCDR 3 of SEQ ID NO: 22; (B) Second and third antigen-binding domains that bind to GPRC5D, wherein each of the second and third antigen-binding domains is a (conventional) Fab molecule and comprises: VH comprising SEQ ID NO: 61 HCDR 1 of SEQ ID NO: 62 and HCDR 3 of SEQ ID NO: 63; and VL comprising LCDR 1 of SEQ ID NO: 65, LCDR 2 of SEQ ID NO: 66 and SEQ ID NO: 67 LCDR 3; (C) Fc domain, the Fc domain is composed of the first unit and the second unit; wherein in the constant domain CL of the second antigen-binding domain and the third antigen-binding domain described under (B) , the amino acid at position 124 was substituted with lysine (K) (according to Kabat numbering), and the amino acid at position 123 was substituted with lysine (K) or arginine (R) (according to Kabat numbering) ( optimally substituted with arginine (R), and wherein, in the constant domain CH1 of the second and third antigen-binding domains described under (B), the amino acid at position 147 is substituted by gluten Amino 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); and wherein additionally described under (B) 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 described under (A), and the first antigen binding domain described under (A) and the (B) The third antigen binding domains described below 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 described under (C).

[0356] In yet another aspect, the invention provides a (multispecific) antibody comprising (A) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, wherein the Fab light chain and the variable domains VL and VH of the Fab heavy chain replace each other and comprise: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18 , SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19 (specifically, the amino acid sequence of SEQ ID NO: 16); and a light chain variable region comprising the sequence of SEQ ID NO: 11 Amino acid sequence; (B) a second antigen-binding domain and a third antigen-binding domain that binds to GPRC5D, wherein the second antigen-binding domain and the third antigen-binding domain are each a (conventional) Fab molecule and comprise: (i) heavy chain variable region, which comprises the amino acid sequence of SEQ ID NO: 64; and light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 68; (C) Fc domain, which consists of the first Fc domain A subunit and a second subunit constitute; wherein in the constant domain CL of the second antigen-binding domain and the third antigen-binding domain described under (B), the amino acid at position 124 is via lysine (K) ( substituted according to Kabat numbering), and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (according to Kabat numbering), preferably with arginine (R), and wherein, In the constant domain CH1 of the second and third antigen-binding domains described under (B), the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to the Kabat EU index), and the position The amino acid at position 213 is substituted by glutamic acid (E) (numbering according to the Kabat EU index); and wherein the second antigen binding domain otherwise described under (B) is at the C-terminus of the Fab heavy chain and at the C-terminus of the Fab heavy chain at (A) The N-terminal of the Fab heavy chain of the first antigen binding domain described below is fused, and the first antigen binding domain described under (A) and the third antigen binding domain described under (B) are each at the Fab heavy chain The C-terminus of the chain is fused to the N-terminus of one of the subunits of the Fc domain described under (C).

[0357] In one aspect, the invention provides a (multispecific) antibody comprising (A) a first antigen binding domain that binds CD3, wherein the first antigen binding domain is a Fab molecule, wherein the Fab light chain and The variable domains VL and VH of the Fab heavy chain replace each other and comprise (i) a heavy chain variable region (VH) selected from the group consisting of: (a) VH comprising the sequence of SEQ ID NO: 2 Heavy chain complementarity determining region (HCDR) 1, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10, (b) VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 3 of SEQ ID NO: 4 HCDR 2 and HCDR 3 of SEQ ID NO: 12, (c) VH comprising HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 5 and HCDR 3 of SEQ ID NO: 9, (d) VH , which comprises the HCDR 1 of SEQ ID NO: 3, the HCDR 2 of SEQ ID NO: 6 and the HCDR 3 of SEQ ID NO: 11, or (e) VH, which comprises the HCDR 1 of SEQ ID NO: 3, the HCDR 1 of SEQ ID NO HCDR 2 of SEQ ID NO: 7 and HCDR 3 of SEQ ID NO: 13 (specifically VH, which includes HCDR 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 4 and HCDR 3 of SEQ ID NO: 10); And (ii) a light chain variable region (VL) comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, LCDR 2 of SEQ ID NO: 21 and LCDR 3 of SEQ ID NO: 22; ( B) A second and third antigen-binding domain that binds to CD19, wherein each of the second and third antigen-binding domains is a (conventional) Fab molecule and comprises: (i) a VH comprising SEQ HCDR 1 of ID NO: 75, HCDR 2 of SEQ ID NO: 76, and HCDR 3 of SEQ ID NO: 77, and VL comprising LCDR 1 of SEQ ID NO: 79, LCDR 2 of SEQ ID NO: 80, and SEQ ID NO: 79 LCDR 3 of ID NO: 81; or (ii) VH comprising HCDR 1 of SEQ ID NO: 83, HCDR 2 of SEQ ID NO: 84 and HCDR 3 of SEQ ID NO: 85, and VL comprising SEQ ID LCDR 1 of NO: 87, LCDR 2 of SEQ ID NO: 88, and LCDR 3 of SEQ ID NO: 89 (specifically, including: VH, which includes HCDR 1 of SEQ ID NO: 75, HCDR 1 of SEQ ID NO: 76 HCDR 2 and HCDR 3 of SEQ ID NO: 77, and VL comprising LCDR 1 of SEQ ID NO: 79, LCDR 2 of SEQ ID NO: 80 and LCDR 3 of SEQ ID NO: 81); (C) Fc domain , the Fc domain is composed of the first unit and the second unit; wherein in the constant domain CL of the second antigen-binding domain and the third antigen-binding domain described under (B), the amino acid at position 124 is passed Lysine (K) (numbering according to Kabat) was substituted, and the amino acid at position 123 was lysine (K) or arginine (R) (numbering according to Kabat) (optimally via arginine (R ) ) substituted, and wherein, in the constant domain CH1 of the second and third antigen-binding domains described under (B), the amino acid at position 147 is replaced by glutamic acid (E) (according to Kabat EU index number) and the amino acid at position 213 is substituted by glutamic acid (E) (according to Kabat EU index number); and wherein the second antigen binding domain otherwise described under (B) is in the Fab heavy chain The C-terminus is fused to the N-terminus of the Fab heavy chain of the first antigen-binding domain described under (A), and the first antigen-binding domain described under (A) and the third antigen-binding domain described under (B) The 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 described under (C).

[0358] In yet another aspect, the invention provides a (multispecific) antibody comprising (A) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, wherein the Fab light chain and the variable domains VL and VH of the Fab heavy chain replace each other and comprise: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18 , SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19 (specifically, the amino acid sequence of SEQ ID NO: 16); and a light chain variable region comprising the sequence of SEQ ID NO: 11 Amino acid sequence; (B) a second antigen-binding domain and a third antigen-binding domain binding to CD19, wherein the second antigen-binding domain and the third antigen-binding domain are each a (conventional) Fab molecule and comprise: (i) heavy Chain variable region, it comprises the amino acid sequence of SEQ ID NO: 78; And light chain variable region, it comprises the aminoacid sequence of SEQ ID NO: 82; Or (ii) heavy chain variable region, it comprises SEQ The amino acid sequence of ID NO: 86; And light chain variable region, it comprises the amino acid sequence of SEQ ID NO: 90 (particularly, comprises: Heavy chain variable region, it comprises the aminoacid sequence of SEQ ID NO: 78 Amino acid sequence; and light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 82); (C) Fc domain, the Fc domain is composed of the first unit and the second unit; wherein in ( B) In the constant domain CL of the second antigen-binding domain and the third antigen-binding domain described below, the amino acid at position 124 is substituted with lysine (K) (according to Kabat numbering), and the amine at position 123 amino acid is substituted by lysine (K) or arginine (R) (according to Kabat numbering), optimally by arginine (R), and wherein the second antigen binding described under (B) domain and the constant domain CH1 of the third antigen-binding domain, the amino acid at position 147 was substituted by glutamic acid (E) (numbering according to the Kabat EU index), and the amino acid at position 213 was replaced by glutamic acid (E ) (numbering according to the Kabat EU index); and wherein the second antigen-binding domain otherwise described under (B) is at the C-terminus of the Fab heavy chain with the Fab heavy chain of the first antigen-binding domain described under (A) The N-terminus of the chain is fused, and the first antigen-binding domain described under (A) and the third antigen-binding domain described under (B) are each at the C-terminus of the Fab heavy chain and described under (C) N-terminal fusion to one of the subunits of the Fc domain.

[0359] In one aspect according to these aspects of the invention, in the first unit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second unit of the Fc domain In subunits, 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 The leucine residue was replaced by an alanine residue (L368A) (numbering according to the Kabat EU index).

[0360] In yet another aspect according to these aspects of the invention, in the first unit of the Fc domain, the serine residue at position 354 is in turn replaced by a cysteine ​​residue (S354C) or the gluten residue at position 356. Amino acid residue is replaced by a cysteine ​​residue (E356C) (specifically the serine residue at position 354 is replaced by a cysteine ​​residue), and in the second unit of the Fc domain, position 349 The tyrosine residue was replaced by a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index).

[0361] In yet another aspect according to these aspects of the invention, in each of the first unit and the second unit of the Fc domain, the leucine residue at position 234 is replaced by an alanine residue (L234A ), the leucine residue at position 235 was replaced by an alanine residue (L235A), and the proline residue at position 329 was replaced by a glycine residue (P329G) (numbering according to the Kabat EU index).

[0362] In yet another aspect according to these aspects of the invention, the Fc domain is a human IgG1 Fc domain.

[0363] In a specific aspect, the (multispecific) antibody comprises: a polypeptide comprising a sequence at least about 95%, 96%, 97%, 98% or 99% identical to a sequence selected from the group consisting of Amino acid sequences of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 (specifically, the sequence of SEQ ID NO: 37); polypeptide (specifically, two polypeptides) comprising an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 34; a polypeptide comprising an amino acid sequence identical to that of SEQ ID NO: 34 An amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 33; and a polypeptide comprising at least about 95%, 96% identical to the sequence of SEQ ID NO: 32 %, 97%, 98% or 99% identical amino acid sequences. In another specific aspect, the (multispecific) antibody comprises: a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36. SEQ ID NO: 38 and SEQ ID NO: 40 (in particular, the amino acid sequence of SEQ ID NO: 37); a polypeptide (in particular, two polypeptides), the polypeptide comprising the amino acid sequence of SEQ ID NO: 34 an amino acid sequence; a polypeptide comprising the amino acid sequence of SEQ ID NO: 33; and a polypeptide comprising the amino acid sequence of SEQ ID NO: 32.

[0364] In one aspect, the invention provides a (multispecific) antibody that binds to CD3 and TYRP-1, the (multispecific) antibody comprising: a polypeptide comprising and selected from the group consisting of Amino acid sequences at least about 95%, 96%, 97%, 98% or 99% identical in sequence to: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 (specifically, the sequence of SEQ ID NO: 37); a polypeptide (specifically, two polypeptides), which comprises at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequences; polypeptides comprising amino acid sequences at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 33; and polypeptides , the polypeptide comprises an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 32. In one aspect, the invention provides a (multispecific) antibody that binds to CD3 and TYRP-1, the (multispecific) antibody comprising: a polypeptide comprising an amino acid sequence selected from the group consisting of : SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 (specifically, the amino acid sequence of SEQ ID NO: 37); polypeptide (specifically In terms of two polypeptides), the polypeptide comprises the amino acid sequence of SEQ ID NO: 34; the polypeptide comprises the amino acid sequence of SEQ ID NO: 33; and the polypeptide comprises the amino acid sequence of SEQ ID NO: 32 amino acid sequence.

[0365] In another specific aspect, the (multispecific) antibody comprises: a polypeptide comprising at least about 95%, 96%, 97%, 98% or 99% of a sequence selected from the group consisting of identical amino acid sequences: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 (specifically, the sequence of SEQ ID NO: 37); A polypeptide (in particular, two polypeptides) comprising an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 71; a polypeptide comprising An amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 69; and a polypeptide comprising at least about 95% of the sequence of SEQ ID NO: 70, 96%, 97%, 98% or 99% amino acid sequence identity. In another specific aspect, the (multispecific) antibody comprises: a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36. SEQ ID NO: 38 and SEQ ID NO: 40 (in particular, the amino acid sequence of SEQ ID NO: 37); a polypeptide (in particular, two polypeptides), the polypeptide comprising the amino acid sequence of SEQ ID NO: 71 an amino acid sequence; a polypeptide comprising the amino acid sequence of SEQ ID NO: 69; and a polypeptide comprising the amino acid sequence of SEQ ID NO: 70.

[0366] In one aspect, the invention provides a (multispecific) antibody that binds to CD3 and CEA, the (multispecific) antibody comprising: a polypeptide comprising a sequence selected from the group consisting of at least About 95%, 96%, 97%, 98% or 99% identical amino acid sequences: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 (in particular, the sequence of SEQ ID NO: 37); a polypeptide (in particular, two polypeptides) comprising at least about 95%, 96%, 97%, 98% of the sequence of SEQ ID NO: 71 Or a 99% identical amino acid sequence; a polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 69; and a polypeptide, the The polypeptide comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 70. In one aspect, the invention provides a (multispecific) antibody that binds to CD3 and CEA, the (multispecific) antibody comprising: a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 (in particular, the amino acid sequence of SEQ ID NO: 37); polypeptides (in particular , two polypeptides), the polypeptide comprises the amino acid sequence of SEQ ID NO: 71; the polypeptide, the polypeptide comprises the amino acid sequence of SEQ ID NO: 69; and the polypeptide, the polypeptide comprises the amino acid sequence of SEQ ID NO: 70 acid sequence.

[0367] In yet another specific aspect, the (multispecific) antibody comprises: a polypeptide comprising at least about 95%, 96%, 97%, 98% or 99% of a sequence selected from the group consisting of identical amino acid sequences: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 (specifically, the sequence of SEQ ID NO: 37); A polypeptide (in particular, two polypeptides) comprising an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 74; a polypeptide comprising An amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 72; and a polypeptide comprising at least about 95% of the sequence of SEQ ID NO: 73, 96%, 97%, 98% or 99% amino acid sequence identity. In another specific aspect, the (multispecific) antibody comprises: a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36. SEQ ID NO: 38 and SEQ ID NO: 40 (in particular, the amino acid sequence of SEQ ID NO: 37); a polypeptide (in particular, two polypeptides), the polypeptide comprising the amino acid sequence of SEQ ID NO: 74 an amino acid sequence; a polypeptide comprising the amino acid sequence of SEQ ID NO: 72; and a polypeptide comprising the amino acid sequence of SEQ ID NO: 73.

[0368] In one aspect, the present invention provides a (multispecific) antibody that binds to CD3 and GPRC5D, comprising: a polypeptide comprising and selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 39, SEQ ID NO: The sequence of the group consisting of ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 (in particular the sequence of SEQ ID NO: 37) is at least about 95%, 96%, 97%, 98%, or 99% % identical amino acid sequence; Polypeptide, this polypeptide comprises the sequence of SEQ ID NO:74 at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequence; Polypeptide, this polypeptide comprises and An amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 72; and a polypeptide comprising at least about 95%, 96% of the sequence of SEQ ID NO: 73 %, 97%, 98% or 99% identical amino acid sequences. In one aspect, the present invention provides a (multispecific) antibody that binds to CD3 and GPRC5D, comprising: a polypeptide comprising a polypeptide selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36 , the amino acid sequence of the group consisting of SEQ ID NO: 38 and SEQ ID NO: 40 (specifically the amino acid sequence of SEQ ID NO: 37); a polypeptide comprising the amine of SEQ ID NO: 74 an amino acid sequence; a polypeptide comprising the amino acid sequence of SEQ ID NO: 72; and a polypeptide comprising the amino acid sequence of SEQ ID NO: 73.

[0369] In another specific aspect, the (multispecific) antibody comprises: a polypeptide comprising at least about 95%, 96%, 97%, 98% or 99% of a sequence selected from the group consisting of identical amino acid sequences: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 (specifically, the sequence of SEQ ID NO: 37); A polypeptide (in particular, two polypeptides) comprising an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 94; a polypeptide comprising An amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 91 or SEQ ID NO: 92 (in particular, the sequence of SEQ ID NO: 91); and a polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 93. In another specific aspect, the (multispecific) antibody comprises: a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36. SEQ ID NO: 38 and SEQ ID NO: 40 (in particular, the amino acid sequence of SEQ ID NO: 37); a polypeptide (in particular, two polypeptides), the polypeptide comprising the amino acid sequence of SEQ ID NO: 94 Amino acid sequence; a polypeptide comprising the amino acid sequence of SEQ ID NO: 91 or SEQ ID NO: 92 (specifically, the sequence of SEQ ID NO: 91); and a polypeptide comprising SEQ ID NO: 93 amino acid sequence.

[0370] In one aspect, the invention provides a (multispecific) antibody binding to CD3 and CD19, the (multispecific) antibody comprising: a polypeptide comprising a sequence selected from the group consisting of at least About 95%, 96%, 97%, 98% or 99% identical amino acid sequences: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 (in particular, the sequence of SEQ ID NO: 37); a polypeptide (in particular, two polypeptides) comprising at least about 95%, 96%, 97%, 98% of the sequence of SEQ ID NO: 94 or 99% identical amino acid sequence; a polypeptide comprising at least about 95%, 96%, the sequence of SEQ ID NO: 91 or SEQ ID NO: 92 (specifically, the sequence of SEQ ID NO: 91), 97%, 98% or 99% identical amino acid sequences; and polypeptides comprising amino acids at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 93 sequence. In one aspect, the invention provides a (multispecific) antibody that binds to CD3 and CD19, the (multispecific) antibody comprising: a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 (in particular, the amino acid sequence of SEQ ID NO: 37); polypeptides (in particular , two polypeptides), the polypeptide comprises the amino acid sequence of SEQ ID NO: 94; the polypeptide, the polypeptide comprises the amino acid sequence of SEQ ID NO: 91 or SEQ ID NO: 92 (specifically, SEQ ID NO: 91); and a polypeptide comprising the amino acid sequence of SEQ ID NO: 93.

[0371] In another specific aspect, the (multispecific) antibody comprises: a polypeptide comprising and selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 38 and The sequences of the group consisting of SEQ ID NO: 40 (in particular the sequence of SEQ ID NO: 37) are at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequences; polypeptides, the A polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 98; a polypeptide comprising an amino acid sequence identical to that of SEQ ID NO: 95 or SEQ ID NO: 96 The sequence (specifically the sequence of SEQ ID NO: 95) is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence; and a polypeptide comprising the sequence of SEQ ID NO: 97 Amino acid sequences that are at least about 95%, 96%, 97%, 98% or 99% identical in sequence. In another specific aspect, the (multispecific) antibody comprises: a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36. SEQ ID NO: 38 and SEQ ID NO: 40 (in particular, the amino acid sequence of SEQ ID NO: 37); a polypeptide (in particular, two polypeptides), the polypeptide comprising SEQ ID NO: 98 Amino acid sequence; a polypeptide comprising the amino acid sequence of SEQ ID NO: 95 or SEQ ID NO: 96 (specifically, the sequence of SEQ ID NO: 95); and a polypeptide comprising SEQ ID NO: 97 amino acid sequence.

[0372] In one aspect, the present invention provides a (multispecific) antibody that binds to CD3 and CD19, comprising: a polypeptide comprising and selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 39, SEQ ID NO: The sequence of the group consisting of ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 (in particular the sequence of SEQ ID NO: 37) is at least about 95%, 96%, 97%, 98%, or 99% % identical amino acid sequence; a polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 98; a polypeptide (specifically two a polypeptide) comprising at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 95 or SEQ ID NO: 96 (specifically the sequence of SEQ ID NO: 95) and a polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 97. In one aspect, the invention provides a (multispecific) antibody that binds to CD3 and CD19, the (multispecific) antibody comprising: a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40 (in particular, the amino acid sequence of SEQ ID NO: 37); polypeptides (in particular , two polypeptides), the polypeptide comprises the amino acid sequence of SEQ ID NO: 98; the polypeptide, the polypeptide comprises the amino acid sequence of SEQ ID NO: 95 or SEQ ID NO: 96 (specifically, SEQ ID NO: 95); and a polypeptide comprising the amino acid sequence of SEQ ID NO: 97. 8. Fc domain variants

[0373] In a preferred aspect, the (multispecific) antibody of the invention comprises an Fc domain consisting of a first unit and a second unit.

[0374] The Fc domain of a (multispecific) antibody consists 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, with each subunit comprising CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stably associating with each other. In one aspect, the (multispecific) antibody of the invention comprises no more than one Fc domain.

[0375] In one aspect, the Fc domain of the (multispecific) antibody is an IgG Fc domain. In a preferred aspect, the Fc domain is an IgG1 Fc domain. In another aspect, the Fc domain is an IgG4 Fc domain. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (numbering according to the Kabat EU index), specifically the amino acid substitution S228P. This amino acid substitution reduces Fab arm exchange of IgG4 antibodies in vivo (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. An exemplary sequence of the human IgG1 Fc region is given in SEQ ID NO: 47. a) Fc domain modification to promote heterodimerization

[0376] The (multispecific) antibodies according to the invention comprise different antigen-binding domains which may be fused to one or the other of the two subunits of the Fc domain, so that the two subunits of the Fc domain are usually comprised in two in different polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. To improve the yield and purity of (multispecific) antibodies in recombinant production, it would be advantageous to introduce modifications in the Fc domain of (multispecific) antibodies that facilitate the desired polypeptide association.

[0377] Thus, in a preferred aspect, the Fc domain of the (multispecific) antibody according to the invention comprises modifications that facilitate the association of the first and second units 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 made in the CH3 domain of the Fc domain.

[0378] There are various methods of modifying the CH3 domain of an Fc domain to enhance heterodimerization, which are well described 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, WO 2013096291. Typically, in all of these approaches, the CH3 domain of the first unit of the Fc domain and the CH3 domain of the second unit of the Fc domain are engineered in a complementary manner such that each CH3 domain (or containing a CH3 domain heavy chain) is no longer able to homodimerize with itself, but is forced to heterodimerize with other CH3 domains that have been complementarily engineered (so that the first CH3 domain and the second CH3 domain heterodimerize and does not form homodimers between two first CH3 domains or two second CH3 domains). These different approaches for improving heavy chain heterodimerization are considered to be comparable to heavy chain-light chain modifications in (multispecific) antibodies (e.g., VH and VL swapping / replacement in one binding arm, and in CH1 / Different options for incorporation of substituents in the CL interface to introduce oppositely charged amino acids) which reduce heavy chain / light chain mismatches and Bence Jones-type by-products.

[0379] In a specific aspect, the modification that promotes the association of the first unit and the second unit of the Fc domain is a so-called "knob-into-hole" modification that includes "Knob" modification of one of the two subunits and "hole" modification of the other of the two subunits of the Fc domain.

[0380] The "punch and mortar" technique is described in, eg, 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 the first polypeptide and a corresponding cavity ("hole") at the interface of the second polypeptide, so that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. Protuberances are constructed by replacing smaller amino acid side chains on the interface of the first polypeptide with larger side chains (such as tyrosine or tryptophan). Complementary cavities of the same or similar size as the protrusions are created in the interface of the second polypeptide by replacing larger amino acid side chains with smaller ones (e.g., alanine or threonine) .

[0381] Thus, in a preferred aspect, in the CH3 domain of the first unit of the Fc domain of a (multispecific) antibody, the amino acid residue is replaced by an amino acid residue with a larger side chain volume Replacement, thereby creating a protrusion in the CH3 domain of the first unit, which can be positioned in the cavity in the CH3 domain of the second unit, and in the CH3 domain of the second unit of the Fc domain, the amino acid residue Replacement by amino acid residues with smaller side chain volumes creates a cavity within the CH3 domain of the second unit into which the protrusions within the CH3 domain of the second unit can be positioned.

[0382] 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 chroma amino acid (W).

[0383] 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 valerine Amino acid (V).

[0384] Protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide (eg, by site-specific mutations or by peptide synthesis).

[0385] In a specific aspect, in the first unit (the "knob" subunit) of the Fc domain (of the CH3 domain), the threonine residue at position 366 is replaced by a tryptophan residue (T366W), And in the second subunit ("hole" subunit) of the Fc domain (of the CH3 domain), the tyrosine residue at position 407 was replaced by a valine residue (Y407V). In one aspect, in the second unit of the Fc domain, the threonine residue at position 366 is again replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbered according to Kabat EU Index).

[0386] In yet another aspect, in the first unit of the Fc domain, the serine residue at position 354 is in turn replaced by a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is replaced by a half Cystine residue replacement (E356C) (specifically the serine residue at position 354 is replaced by a cysteine ​​residue) and, in the second unit of the Fc domain, the tyrosine residue at position 349 Replaced in turn by a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). Introduction of these two cysteine ​​residues leads to the formation of a disulfide bond between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0387] In a preferred aspect, the first unit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second unit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (according to Kabat EU index number).

[0388] In a preferred aspect, the antigen binding domain that binds to CD3 is fused (optionally via a second antigen binding domain that binds to a second antigen and / or a peptide linker) to the first unit of the Fc domain ( Contains the "pestle" modifier). Without wishing to be bound by theory, fusion of the CD3-binding antigen-binding domain to the knob-containing subunit of the Fc domain will (further) minimize the generation of antibodies comprising two CD3-binding antigen-binding domains (two knob-containing polypeptides space collision).

[0389] Other techniques of CH3 modification for forced heterodimerization can be envisaged as alternatives 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.

[0390] In one aspect, the heterodimerization method described in EP 1870459 may alternatively be used. The method is based on the introduction of oppositely charged amino acids at specific amino acid positions at the CH3 / CH3 domain interface between the two subunits of the Fc domain. A particular aspect of the (multispecific) antibody of the invention is the amino acid mutations R409D and K370E in one of the two CH3 domains (of the Fc domain); and the amino acid mutation in the other of the CH3 domains of the Fc domain D399K and E357K (numbered according to Kabat EU index).

[0391] In another aspect, the (multispecific) antibody of the invention comprises the amino acid mutation T366W in the CH3 domain of the first unit of the Fc domain and the amine in the CH3 domain of the second unit of the Fc domain Amino acid mutations T366S, L368A, Y407V, and amino acid mutations R409D, K370E in the CH3 domain of the first unit of the Fc domain, and amino acid mutations D399K, E357K in the CH3 domain of the second unit of the Fc domain ( Numbered according to Kabat EU index).

[0392] In another aspect, the (multispecific) antibody of the invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first unit of the Fc domain and in the CH3 domain of the second unit of the Fc domain Amino acid mutations Y349C, T366S, L368A, Y407V, or the (multispecific) antibody comprises amino acid mutations Y349C, T366W in the CH3 domain of the first unit of the Fc domain and of the second unit of the Fc domain Amino acid mutations in the CH3 domain S354C, T366S, L368A, Y407V, and amino acid mutations in the CH3 domain of the first unit of the Fc domain R409D, K370E and amines in the CH3 domain of the second unit of the Fc domain Acid mutations D399K, E357K (all numbered according to Kabat EU index).

[0393] In one aspect, the heterodimerization method described in WO 2013 / 157953 can be used instead. 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 yet another aspect, the first CH3 domain further comprises the amino acid mutation L351K. In another aspect, the second CH3 domain further comprises an amino acid mutation selected from Y349E, Y349D and L368E (particularly L368E) (numbering according to the Kabat EU index).

[0394] In one aspect, the heterodimerization method described in WO 2012 / 058768 can be used instead. 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 yet another aspect, the second CH3 domain further comprises an amino acid mutation at position T411, D399, S400, F405, N390 or K392 selected from, for example: a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W; b) D399R, D399W, D399Y, or D399K; c) S400E, S400D, S400R, or S400K; d) F405I, F405M, F405T, F405S, F405V, or F405W; e) N390R, N390K, or N390D; f) K392V, K392M, K392R, K392L, K392F or K392E (numbered according to Kabat EU index). In yet another aspect, the first CH3 domain comprises amino acid mutations L351Y, Y407A and the second CH3 domain comprises amino acid mutations T366V, K409F. In yet another aspect, the first CH3 domain comprises the amino acid mutation Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In yet another aspect, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to the Kabat EU index).

[0395] In one aspect, the heterodimerization method described in WO 2011 / 143545 may alternatively be used, e.g. at a position selected from the group consisting of 368 and 409 (numbered according to the Kabat EU index) Amino acid modification.

[0396] In one aspect, the heterodimerization method described in WO 2011 / 090762, which also uses the "knob and punch" technique described above, can be used instead. 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 (nu...

Claims

1. An antibody that binds to CD3, wherein the antibody comprises a first antigen-binding domain comprising: a VH sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19; and a VL sequence of SEQ ID NO:

23.

2. The antibody as requested in claim 1, wherein the first antigen-binding domain is a Fab molecule.

3. The antibody as claimed in claim 1 or 2, comprising an Fc domain consisting of a first unit and a second unit.

4. The antibody as claimed in claim 1 or 2, which includes a second antigen-binding domain that binds to a second antigen.

5. The antibody as requested in item 4, wherein the second antigen-binding domain is a Fab molecule.

6. An antibody as claimed in claim 1 or 2, wherein the first antigen-binding domain is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and Fab heavy chain are interchanged.

7. The antibody as claimed in claim 6, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are interchanged.

8. The antibody as requested in item 4, wherein the second antigen-binding domain is a conventional Fab molecule.

9. The antibody of claim 7, wherein the second antigen-binding domain is a Fab molecule, wherein in the constant domain CL, according to the Kabat number, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H), and according to the Kabat number, the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H), and in the constant domain CH1, according to the Kabat EU index number, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D), and according to the Kabat EU index number, the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D).

10. The antibody of claim 4, wherein the first antigen-binding domain and the second antigen-binding domain are fused together.

11. The antibody of claim 10, wherein the first antigen-binding domain and the second antigen-binding domain are fused to each other via a peptide linker.

12. The antibody of claim 4, wherein the first antigen-binding domain and the second antigen-binding domain are each Fab molecules, and (i) 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, or (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.

13. The antibody of claim 4, wherein the first antigen-binding domain and the second antigen-binding domain are each Fab molecules, and the antibody comprises an Fc domain consisting of a first unit and a second unit; and wherein (i) 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 unit of the Fc domain, or (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 antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first unit of the Fc domain.

14. The antibody as requested in item 3, wherein the Fc domain is an IgG Fc domain.

15. The antibody as requested in claim 3, wherein the Fc domain is the IgG1 Fc domain.

16. The antibody as requested in claim 3, wherein the Fc domain is a human Fc domain.

17. The antibody of claim 3, wherein the Fc includes a modification that promotes association between the primary unit and the secondary unit of the Fc domain.

18. The antibody of claim 3, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to and / or effector function of the Fc receptor.

19. The antibody as requested in item 4, wherein the second antigen is a target cell antigen.

20. The antibody as requested in claim 4, wherein the second antigen is a tumor cell antigen.

21. The antibody as requested in claim 4, wherein the second antigen is TYRP-1.

22. The antibody of claim 21, wherein the second antigen-binding domain comprises: VH, which comprises HCDR 1 of SEQ ID NO: 24, HCDR 2 of SEQ ID NO: 25 and HCDR 3 of SEQ ID NO: 26; and VL, which comprises LCDR 1 of SEQ ID NO: 28, LCDR 2 of SEQ ID NO: 29 and LCDR 3 of SEQ ID NO:

30.

23. The antibody of claim 22, wherein the second antigen-binding domain comprises: VH, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 27; and / or VL, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

31.

24. The antibody as requested in item 4, wherein the second antigen is CEA.

25. The antibody of claim 24, wherein the second antigen-binding domain comprises (i) VH, comprising HCDR 1 of SEQ ID NO: 53, HCDR 2 of SEQ ID NO: 54, and HCDR 3 of SEQ ID NO: 55, and VL, comprising LCDR 1 of SEQ ID NO: 57, LCDR 2 of SEQ ID NO: 58, and LCDR 3 of SEQ ID NO: 59; (ii) VH, comprising HCDR 1 of SEQ ID NO: 105, HCDR 2 of SEQ ID NO: 106, and HCDR 3 of SEQ ID NO: 107, and VL, comprising LCDR 1 of SEQ ID NO: 109, LCDR 2 of SEQ ID NO: 110, and LCDR 3 of SEQ ID NO: 111; or (iii) VH, comprising HCDR 1 of SEQ ID NO:

113.

1. HCDR 2 of SEQ ID NO: 114 and HCDR 3 of SEQ ID NO: 115, and VL, which include LCDR 1 of SEQ ID NO: 117, LCDR 2 of SEQ ID NO: 118 and LCDR 3 of SEQ ID NO:

119.

26. The antibody of claim 25, wherein the second antigen-binding domain comprises (i) VH, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 56, and / or VL, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 60; (ii) VH, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 108, and / or VL, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 112; or (iii) VH, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 60; or (iii) VH, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 60; or (iii) VH, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 60; or (iv) VL ... The amino acid sequence of NO: 116 is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 120, and / or VL contains 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:

120.

27. The antibody as requested in claim 4, wherein the second antigen is GPRC5D.

28. The antibody of claim 27, wherein the second antigen-binding domain comprises: VH, which comprises HCDR 1 of SEQ ID NO: 61, HCDR 2 of SEQ ID NO: 62 and HCDR 3 of SEQ ID NO: 63; and VL, which comprises LCDR 1 of SEQ ID NO: 65, LCDR 2 of SEQ ID NO: 66 and LCDR 3 of SEQ ID NO:

67.

29. The antibody of claim 28, wherein the second antigen-binding domain comprises: VH, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64; and / or VL, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

68.

30. The antibody as requested in claim 4, wherein the second antigen is CD19.

31. The antibody of claim 30, wherein the second antigen-binding domain comprises (i) VH, which comprises HCDR 1 of SEQ ID NO: 75, HCDR 2 of SEQ ID NO: 76 and HCDR 3 of SEQ ID NO: 77, and VL, which comprises LCDR 1 of SEQ ID NO: 79, LCDR 2 of SEQ ID NO: 80 and LCDR 3 of SEQ ID NO: 81; or (ii) VH, which comprises HCDR 1 of SEQ ID NO: 83, HCDR 2 of SEQ ID NO: 84 and HCDR 3 of SEQ ID NO: 85, and VL, which comprises LCDR 1 of SEQ ID NO: 87, LCDR 2 of SEQ ID NO: 88 and LCDR 3 of SEQ ID NO:

89.

32. The antibody of claim 31, wherein the second antigen-binding domain comprises (i) VH, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 78, and / or VL, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 82; or (ii) VH, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 86, and / or VL, which comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

90.

33. The antibody of claim 4, which includes a third antigen-binding domain that binds to a second antigen.

34. The antibody as claimed in claim 33, wherein the third antigen-binding domain is a Fab molecule.

35. The antibody of claim 33, wherein the third antigen binds to a conventional Fab molecule.

36. An antibody as claimed in claim 33, wherein the third antigen-binding domain is a Fab molecule, wherein in the constant domain CL, according to the Kabat number, the amino acid at position 124 is independently substituted with lysine (K), arginine (R), or histidine (H), and according to the Kabat number, the amino acid at position 123 is independently substituted with lysine (K), arginine (R), or histidine (H), and in the constant domain CH1, according to the Kabat EU index number, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D), and according to the Kabat EU index number, the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D).

37. The antibody of claim 33, wherein the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain are each Fab molecules, and the antibody comprises an Fc domain consisting of a first unit and a second unit; and wherein (i) 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 unit of the Fc domain, or (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 antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first unit 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 unit of the Fc domain.

38. The antibody of claim 33, wherein the third antigen-binding domain is identical to the second antigen-binding domain.

39. An isolated polynucleotide encoding an antibody as claimed in any one of claims 1-38.

40. A host cell comprising the isolated polynucleotide as claimed in claim 39.

41. A method for producing an antibody that binds to CD3, comprising the steps of: (a) culturing a host cell as claimed in claim 40 under conditions suitable for expressing the antibody.

42. The method of claim 41 further includes step (b) recovering the antibody.

43. A pharmaceutical composition comprising an antibody as claimed in any one of claims 1-38 and a pharmaceutically acceptable carrier.

44. Use of an antibody as claimed in any one of claims 1-38 or a pharmaceutical composition as claimed in claim 43 in the manufacture of a pharmaceutical preparation.

45. Use of an antibody of any one of claims 1-38 or a pharmaceutical composition of claim 43 in the manufacture of a medicament for treating a disease, wherein the disease is cancer or an autoimmune disease.

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