Antibodies that bind to CD3 and PLAP

Abstract

The present invention relates generally to antibodies that bind to CD3 and PLAP, for example to activate T cells. Additionally, the present invention relates to polynucleotides encoding such antibodies, as well as vectors and host cells comprising such polynucleotides. The present invention further relates to methods of producing the antibodies and methods of using them in the treatment of disease.

Description

[Technical field]

[0001] The present invention relates generally to antibodies that bind to CD3 and PLAP, for example to activate T cells. Additionally, the present invention relates to polynucleotides encoding such antibodies, as well as vectors and host cells comprising such polynucleotides. The present invention further relates to methods of producing the antibodies and using them in the treatment of disease. [Background technology]

[0002] CD3 (cluster of differentiation 3) is a protein complex composed of four subunits, the CD3 gamma chain, the CD3 delta chain, and two CD3 epsilon chains. CD3 associates with the T cell receptor and the zeta chain to generate activation signals in T lymphocytes.

[0003] CD3 has been widely studied as a drug target. Monoclonal antibodies targeting CD3 have been used as immunosuppressive therapy in the treatment of autoimmune diseases such as type I diabetes or transplant rejection. The CD3 antibody muromonab-CD3 (OKT3) was the first monoclonal antibody approved for clinical use in humans in 1985.

[0004] A more recent application of CD3 antibodies is in the form of bispecific antibodies that bind CD3 on the one hand and target cell antigens such as PLAP on the other hand. PLAP (placental alkaline phosphatase) is a dimeric enzyme anchored to the cell membrane by glycosylphosphatidylinositol and is highly expressed in the placenta but has limited or no expression in healthy tissues. Ectopic expression of PLAP has been found to be associated with cancers of the ovary, testis, lung, and gastrointestinal tract (Fishman and Singer, Cancer Res 36 (1976) 4256-4261), making it an interesting target for tumor-targeted immunotherapy using CD3 bispecific antibodies. Binders against PLAP are described, for example, in WO 2019 / 240934.

[0005] Simultaneous binding of such antibodies to both targets results in a transient interaction between the target cell and the T cell, leading to activation of cytotoxic T cells and subsequent lysis of the target cell. Bispecific antibodies that bind to CD3 and PLAP are described, for example, in WO 2021 / 154534.

[0006] An important requirement that antibodies must fulfill for therapeutic purposes is sufficient stability both in vitro (for drug storage) and in vivo (after administration to the patient).

[0007] Modifications such as asparagine deamidation are typical degradation events in recombinant antibodies and can affect both in vitro stability and in vivo biological function.

[0008] Given the enormous therapeutic potential of antibodies, particularly bispecific antibodies for T cell activation, there is a need for CD3 antibodies, including multispecific antibodies, with optimized properties. Summary of the Invention

[0009] The present invention provides antibodies, particularly multispecific (e.g. bispecific) antibodies, that bind to CD3 and are resistant to degradation, e.g. by asparagine deamidation, and thus are particularly stable when required for therapeutic purposes. The antibodies also have excellent thermal stability and binding affinity. The provided (multispecific) antibodies further combine excellent efficacy and productivity with low toxicity and favorable pharmacokinetic properties.

[0010] As indicated herein, the CD3-binding antibodies provided by the invention, including multispecific antibodies, retain about 90% or more of their binding activity to CD3 after 2 weeks at pH 7.4, 37° C., as determined by surface plasmon resonance (SPR), compared to the binding activity after 2 weeks at pH 6, −80° C. As further indicated herein, the CD3-binding antibodies, including multispecific antibodies, provided by the invention, have aggregation temperatures of 55° C. or higher, as determined by dynamic light scattering (DLS), and KD values ​​for monovalent binding to human and cynomolgus CD3 in the picomolar range, as determined by SPR.

[0011] In one aspect, the invention provides an antibody that binds to CD3 and placental alkaline phosphatase (PLAP), wherein the antibody comprises (a) a first antigen-binding domain that binds to CD3 comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3, and an HCDR3 of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, an LCDR2 of SEQ ID NO: 11, and an LCDR3 of SEQ ID NO: 12; and (b) a second antigen-binding domain that binds PLAP, and optionally a third antigen-binding domain. In one aspect, the VH of the first antigen-binding domain 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: 9, and / or the VL of the first antigen-binding domain 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: 13.

[0012] In a further aspect, the invention provides an antibody that binds to CD3 and PLAP, wherein the antibody comprises (a) a first antigen-binding domain that binds to CD3 comprising a VH sequence of SEQ ID NO: 9 and a VL sequence of SEQ ID NO: 13; and (b) a second antigen-binding domain that binds to PLAP, and optionally a third antigen-binding domain.

[0013] In one aspect, the first antigen binding domain, the second antigen binding domain and / or, if present, the third antigen binding domain are Fab molecules.

[0014] In one embodiment, the antibody comprises an Fc domain composed of a first and a second subunit.

[0015] In one embodiment the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH, of a Fab light chain and a Fab heavy chain are replaced by each other.

[0016] In one aspect the second antigen binding domain and, if present, the third antigen binding domain are conventional Fab molecules.

[0017] In one aspect, the second antigen binding domain and, if present, the third antigen binding domain are Fab molecules, wherein in the constant domain CL the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and in the constant domain CH1 the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering) and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0018] In one aspect, the first and second antigen binding domains are fused to each other, optionally via a peptide linker.

[0019] In one aspect, the first and second antigen binding domains are each Fab molecules, and either (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.

[0020] In one aspect, the first antigen binding domain, the second antigen binding domain and, if present, the third antigen binding domain are each a Fab molecule and the antibody comprises an Fc domain comprised of a first and a second subunit, and either (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 subunit 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 subunit of the Fc domain, and, if present, the third antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

[0021] In one embodiment the Fc domain is an IgG Fc domain, particularly an IgG 1 The Fc domain is an Fc domain. In one embodiment, the Fc domain is a human Fc domain. In one embodiment, the Fc comprises a modification that promotes association of the first and second subunits of the Fc domain. In one embodiment, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

[0022] In one aspect, the second antigen binding domain and, if present, the third antigen binding domain comprises (i) an HCDR1 of SEQ ID NO: 28, an HCDR2 of SEQ ID NO: 29, and an HCDR3 of SEQ ID NO: 30, (ii) an HCDR1 of SEQ ID NO: 32, an HCDR2 of SEQ ID NO: 33, and an HCDR3 of SEQ ID NO: 34, (iii) an HCDR1 of SEQ ID NO: 36, an HCDR2 of SEQ ID NO: 37, and an HCDR3 of SEQ ID NO: 38, (iv) an HCDR1 of SEQ ID NO: 40, an HCDR2 of SEQ ID NO: 41, and an HCDR3 of SEQ ID NO: 42, or (v) a VH comprising an HCDR1 of SEQ ID NO: 44, an HCDR2 of SEQ ID NO: 45, and an HCDR3 of SEQ ID NO: 46, and a VL comprising an LCDR1 of SEQ ID NO: 48, an LCDR2 of SEQ ID NO: 49, and an LCDR3 of SEQ ID NO: 50. In one aspect, the second antigen binding domain and, if present, the third antigen binding domain comprises a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43 or SEQ ID NO:47 and / or a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:51.

[0023] In a particular aspect, the second antigen binding domain and, if present, the third antigen binding domain comprises a VH comprising an HCDR1 of SEQ ID NO: 36, an HCDR2 of SEQ ID NO: 37, and an HCDR3 of SEQ ID NO: 38, and a VL comprising an LCDR1 of SEQ ID NO: 48, an LCDR2 of SEQ ID NO: 49, and an LCDR3 of SEQ ID NO: 50. In one aspect, the second antigen binding domain and, if present, the third antigen binding domain comprises a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39 and / or a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 51.

[0024] In a further aspect, the second antigen binding domain, and, if present, the third antigen binding domain, comprises a VH comprising an HCDR1 of SEQ ID NO: 44, an HCDR2 of SEQ ID NO: 45, and an HCDR3 of SEQ ID NO: 46, and a VL comprising an LCDR1 of SEQ ID NO: 48, an LCDR2 of SEQ ID NO: 49, and an LCDR3 of SEQ ID NO: 50. In one aspect, the second antigen binding domain, and, if present, the third antigen binding domain, comprises a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47 and / or a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 51.

[0025] According to a further aspect of the invention there are provided isolated polynucleotides encoding the antibodies of the invention, and host cells comprising the isolated polynucleotides of the invention.

[0026] In another aspect, there is provided a method for producing an antibody that binds CD3 and PLAP, comprising (a) culturing a host cell of the invention under conditions suitable for expression of the antibody, and, optionally, (b) recovering the antibody. The invention also encompasses antibodies that bind CD3 and PLAP produced by the methods of the invention.

[0027] The present invention further provides a pharmaceutical composition comprising an antibody of the invention and a pharma- ceutically acceptable carrier.

[0028] The present invention also encompasses methods of using the antibodies and pharmaceutical compositions of the present invention. In one aspect, the present invention provides an antibody or pharmaceutical composition according to the present invention for use as a medicament. In one aspect, the present invention provides an antibody or pharmaceutical composition according to the present invention for use in treating a disease. Also provided is the use of an antibody or pharmaceutical composition according to the present invention in the manufacture of a medicament, and the use of an antibody or pharmaceutical composition according to the present invention in the manufacture of a medicament for treating a disease. The present invention also provides a method of treating a disease in an individual, comprising administering to the individual an effective amount of an antibody or pharmaceutical composition according to the present invention. In a particular aspect, the disease is cancer. In another aspect, the disease is an autoimmune disease. [Brief description of the drawings]

[0029] [Figure 1A-F] Exemplary structures of (multispecific) antibodies of the invention. (A,D) Diagram of a "1+1 CrossMab" molecule. (B,E) Diagram of a "2+1 IgG Crossfab" molecule with Crossfab and Fab components in an alternative order ("flipped"). (C,F) Diagram of a "2+1 IgG Crossfab" molecule. Black circles: optional modifications in the Fc domain to promote heterodimerization. ++, --: amino acids of opposite charge optionally introduced in the CH1 and CL domains. Although Crossfab molecules are depicted as including swaps of VH and VL regions, in embodiments where no charge modifications are introduced in the CH1 and CL domains, they may alternatively include swaps of the CH1 and CL domains. [Figure 1G-N]Exemplary structures of (multispecific) antibodies of the invention. (G,K) Diagrams of "1+1 IgG Crossfab" molecules with Crossfab and Fab components in an alternative order ("flipped"). (H,L) Diagrams of "1+1 IgG Crossfab" molecules. (I,M) Diagrams of "2+1 IgG Crossfab" molecules with two CrossFabs. (J,N) Diagrams of "2+1 IgG Crossfab" molecules with two CrossFabs and Crossfab and Fab components in an alternative order ("flipped"). Black circles: optional modifications in the Fc domain to promote heterodimerization. ++, --: amino acids of opposite charge optionally introduced in the CH1 and CL domains. Although Crossfab molecules are depicted as including swaps of VH and VL regions, in embodiments where no charge modifications are introduced in the CH1 and CL domains, they may alternatively include swaps of the CH1 and CL domains. [Figure 1O-V] Exemplary structures of (multispecific) antibodies of the invention. (O,S) Diagrams of "Fab-Crossfab" molecules. (P,T) Diagrams of "Crossfab-Fab" molecules. (Q,U) Diagrams of "(Fab)2-Crossfab" molecules. (R,V) Diagrams of "Crossfab-(Fab)2" molecules. Black circles: optional modifications in the Fc domains that promote heterodimerization. ++, --: amino acids of opposite charge optionally introduced in the CH1 and CL domains. Although Crossfab molecules are depicted as including swapping of VH and VL regions, in embodiments where no charge modifications are introduced in the CH1 and CL domains, they may alternatively include swapping of the CH1 and CL domains. [Figure 1W-Z]Exemplary structures of (multispecific) antibodies of the invention. (W, Y) Diagram of "Fab-(Crossfab)2" molecule. (X, Z) Diagram of "(Crossfab)2-Fab" molecule. Black circle: optional modification in Fc domain to promote heterodimerization. ++, --: amino acids of opposite charge optionally introduced in CH1 and CL domains. Crossfab molecules are depicted as including swapping of VH and VL domains, but in embodiments where no charge modifications are introduced in CH1 and CL domains, they may alternatively include swapping of CH1 and CL domains. [Diagram 2] (A) Schematic diagram of T cell bispecific antibody (TCB) molecules used in the examples. All tested TCB antibody molecules were produced as "2+1 IgG CrossFab, inverted" with charge modifications (VH / VL exchange in CD3 binder, charge modifications in target cell antigen binder, EE=147E, 213E; RK=123R, 124K). (B-E) Building blocks for the assembly of TCB: light chain of anti-TYRP1 Fab molecule with charge modifications in CH1 and CL (B), light chain of anti-CD3 crossover Fab molecule (C), heavy chain with knob and PG LALA mutations in the Fc region (D), heavy chain with hole and PG LALA mutations in the Fc region (E). [Diagram 3] Schematic diagram of the surface plasmon resonance (SPR) setup used in Example 3. Anti-PG antibodies bound to a C1 sensor chip. Human and cynomolgus CD3 (fused to the Fc region) are passed over the surface to analyze the interaction of the anti-CD3 antibodies with CD3 in the TCB. [Figure 4A-B]Tumor cell killing and T cell activation by TCBs containing different CD3 binders. Killing of the melanoma cell line M150543 by PBMCs of healthy donors upon treatment with TYRP1 TCBs containing P035.093, CD3orig or CD3opt CD3 binders was determined by LDH release after 24 hours (A) and 48 hours (B). In parallel, upregulation of CD25 and CD69 in CD8 and CD4 T cells in PBMCs was measured by flow cytometry as a marker of T cell activation after 48 hours. CD4 CD25 (C), CD4 CD69 (D), CD8 CD25 (E), CD8 CD69 (F). [Figure 4C-F] Tumor cell killing and T cell activation by TCBs containing different CD3 binders. Killing of the melanoma cell line M150543 by PBMCs of healthy donors upon treatment with TYRP1 TCBs containing P035.093, CD3orig or CD3opt CD3 binders was determined by LDH release after 24 hours (A) and 48 hours (B). In parallel, upregulation of CD25 and CD69 in CD8 and CD4 T cells in PBMCs was measured by flow cytometry as a marker of T cell activation after 48 hours. CD4 CD25 (C), CD4 CD69 (D), CD8 CD25 (E), CD8 CD69 (F). [Diagram 5] (A) Schematic diagram of the monovalent IgG molecule generated in Example 6. The monovalent IgG molecule was produced as a human IgG1 with a VH / VL exchange in the CD3 binder. (B-E) Components for the assembly of the monovalent IgG: light chain of an anti-CD3 crossover Fab molecule (B), heavy chain with a knob and a PG LALA mutation in the Fc region (C), heavy chain with a hole and a PG LALA mutation in the Fc region (D). [Figure 6A-F]PLAPTCB containing the optimized anti-CD3 binder P035.093 was tested in a Jurkat-NFAT reporter assay using an engineered CHO-K1 pool as target cells. Activation of Jurkat NFAT reporter cells was determined by measuring luminescence 6 hours after treatment. Each panel represents a different target cell line as follows: (A) no target cells, (B) CHO-K1 parental, (C) CHO-K1 human ALPP, (D) CHO-K1 human ALPG, (E) CHO-K1 human ALPI, (F) CHO-K1 human ALPL. [Figure 6G-I] PLAPTCB containing the optimized anti-CD3 binder P035.093 was tested in a Jurkat-NFAT reporter assay using an engineered CHO-K1 pool as target cells. Activation of Jurkat NFAT reporter cells was determined by measuring luminescence 6 hours after treatment. Each panel represents a different target cell line as follows: (G) CHO-K1 mouse ALPG, (H) CHO-K1 mouse ALPI, (I) LoVo. [Figure 7] PLAP TCB containing the optimized anti-CD3 binder P035.093 was tested in a Jurkat-NFAT reporter assay using engineered CHO-K1 pools as target cells. Activation of Jurkat NFAT reporter cells was determined by measuring luminescence 6 hours after treatment. Each panel represents a different target cell line: (A) CHO-K1 human ALPP, (B) CHO-K1 cynomolgus ALPP. [Figure 8] Tumor cell killing of the colorectal adenocarcinoma cell line LoVo by PBMCs from healthy donors was assessed when treated with PLAP TCB. Tumor cell killing was measured in the absence (A, B) or presence (C, D) of target cells by quantifying cell death after 48 h (A, C) and 72 h (B, D) using the CytotoxGlo kit (Promega). [Figure 9]Upregulation of CD25 in CD4 (A, C) and CD8 (B, D) T cells was analyzed in PBMCs from healthy donors treated with PLAP TCB in the presence (C, D) or absence (A, B) of LoVo cell line as target cells. Analysis was performed by flow cytometry after 72 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] I. Definition Unless otherwise defined below, terms are used herein as commonly used in the art.

[0031] As used herein, the terms "first," "second," or "third," with respect to antigen-binding domains and the like, are used for convenience to distinguish when there is more than one of each type of moiety. The use of these terms is not intended to confer a particular order or orientation of the moieties unless explicitly stated.

[0032] The terms "anti-CD3 antibody" and "antibody that binds CD3" refer to an antibody that can bind CD3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent that targets CD3. In one embodiment, the extent of binding of an anti-CD3 antibody to unrelated, non-CD3 proteins is less than about 10% of the binding of the antibody to CD3, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds CD3 has a dissociation constant (K D The antibody has a K of 1 μM or less, as measured, for example, by SPR. D An anti-CD3 antibody is said to "specifically bind" to CD3 if it has the following structure: In certain embodiments, an anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3 from different species.

[0033] Similarly, the terms "anti-PLAP antibody" and "antibody that binds to PLAP" refer to an antibody that can bind to PLAP with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent that targets PLAP. In one embodiment, the extent of binding of an anti-PLAP antibody to unrelated, non-PLAP proteins is less than about 10% of the binding of the antibody to PLAP, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to PLAP has a dissociation constant (K D The antibody has a K of 1 μM or less, as measured, for example, by SPR. D An anti-PLAP antibody is said to "specifically bind" to PLAP if it has the following structure: In certain embodiments, an anti-PLAP antibody binds to an epitope of PLAP that is conserved among PLAP from different species.

[0034] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0035] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab') 2 Antibody fragments include, but are not limited to, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFab), single domain antibodies, and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0036] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure.

[0037] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies that make up the population are identical and / or bind to the same epitope, except for possible variant antibodies that are normally present in minor amounts, such as those that contain naturally occurring mutations or that arise during the manufacture of the monoclonal antibody preparation. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals that contain all or part of the human immunoglobulin loci, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0038] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC, affinity chromatography, size exclusion chromatography) methods. For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007). In some embodiments, the antibody provided by the present invention is an isolated antibody.

[0039] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remaining portions of the heavy and / or light chain are derived from a different source or species.

[0040] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDRs and human FRs. In certain aspects, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDRs corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. Such variable domains are referred to herein as "humanized variable regions." A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. In some aspects, some FR residues of a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been subjected to humanization.

[0041] A "human antibody" is one having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. In certain aspects, a human antibody is derived from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In certain aspects, a human antibody is derived from a hybridoma cell line. Antibodies or antibody fragments isolated from a human antibody library are also considered human antibodies or human antibody fragments herein.

[0042] The term "antigen-binding domain" refers to the part of an antibody that comprises the area 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 called antibody variable regions). In a preferred embodiment, the antigen-binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0043] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies usually have a similar structure, and each domain contains four conserved framework regions (FR) and complementarity determining regions (CDR). See, for example, Kindt et al., Kuby Immunology, 6 th ed., WH Freeman & Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated using a VH or VL domain from an antibody that binds that antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). As used herein with respect to variable region sequences, "Kabat numbering" refers to the numbering system established by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0044] As used herein, amino acid positions of all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system, referred to herein as "Kabat numbering" or "Kabat numbering", as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Specifically, the Kabat numbering system (see pages 647-660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domains CL of the kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3). The Kabat EU index numbering system is further clarified herein by referring to it as "Kabat EU index numbering" or "Kabat EU index numbering."

[0045] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence and determines antigen binding specificity, e.g., a "complementarity determining region" ("CDR"). Typically, antibodies contain six CDRs; three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include the following: (a) hypervariable loops located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contact sites located 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)).

[0046] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the designations of CDRs can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0047] "Framework" or "FR" refers to variable domain residues other than the complementarity determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, the HVR and FR sequences are generally represented in the following order in VH (or VL): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

[0048] Unless otherwise indicated, CDR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0049] For purposes herein, an "acceptor human framework" is a framework that comprises the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In some embodiments, 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 less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0050] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as found in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

[0051] The term "immunoglobulin molecule" herein refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons composed of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called the variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called the variable light domain or light chain variable region, followed by a constant light (CL) domain, also called the light chain constant region. The heavy chains of immunoglobulins can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which are subtypes, e.g., γ (IgG), ... 1 (IgG 1 ), γ 2 (IgG 2 ), γ 3 (IgG 3 ), γ 4 (IgG 4 ), α1(IgA 1 ) and α 2 (IgA 2 The light chains of immunoglobulins may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain. Immunoglobulins essentially consist of two Fab molecules and an Fc domain linked via an immunoglobulin hinge region.

[0052] The "class" of an antibody or immunoglobulin refers to the type of constant domain or region possessed by the antibody or immunoglobulin heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which have subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0053] A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain (a "Fab heavy chain") and the VL and CL domains of a light chain (a "Fab light chain").

[0054] By "crossover" Fab molecule (also referred to as "Crossfab") is meant a Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (i.e., replaced by one another), i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CH1 (VL-CH1 in the N-terminal to C-terminal direction) and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL in the N-terminal to C-terminal direction). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule in which the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0055] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native format, i.e., a Fab molecule comprising a heavy chain (VH-CH1, from the N to C terminus) composed of a heavy chain variable domain and a constant domain, and a light chain (VL-CL, from the N to C terminus) composed of a light chain variable domain and a constant domain.

[0056] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, an antibody produced by a host cell may undergo post-translational truncation of one or more, in particular one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain or a truncated variant of the full-length heavy chain. This is the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. The amino acid sequence of the heavy chain comprising an Fc region (or a subunit of an Fc domain as defined herein) is shown herein without the C-terminal glycine-lysine dipeptide, unless otherwise indicated. In one aspect, the heavy chain comprising an Fc region (subunit) as specified herein comprised in an antibody according to the invention comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to Kabat EU index). In one aspect, the heavy chain comprising an Fc region (subunit) as specified herein comprised in an antibody according to the invention comprises an additional C-terminal glycine residue (G446, numbering according to Kabat EU index). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above).As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that comprises the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. For example, a subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

[0057] By "fused" it is meant that the components (eg, a Fab molecule and an Fc domain subunit) are linked by a peptide bond, either directly or via one or more peptide linkers.

[0058] The term "multispecific" means that an antibody can specifically bind to at least two different antigenic determinants. A multispecific antibody can be, for example, a bispecific antibody. Typically, a bispecific antibody comprises two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, a multispecific (e.g., bispecific) antibody can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different cells.

[0059] The term "valency" as used herein indicates that a particular number of antigen-binding sites are present within an antigen-binding molecule. Thus, the term "monovalent binding to an antigen" indicates that one (and not more than one) antigen-binding site specific for that antigen is present within the antigen-binding molecule.

[0060] "Antigen-binding site" refers to the site of an antigen-binding molecule, i.e., one or more amino acid residues, that provides interaction with an antigen. For example, the antigen-binding site of an antibody comprises amino acid residues of the complementarity determining regions (CDRs). A naturally occurring immunoglobulin molecule typically has two antigen-binding sites, and a Fab molecule typically has a single antigen-binding site.

[0061] As used herein, the term "antigenic determinant" or "antigen" refers to a site on a polypeptide macromolecule (e.g., a conformational configuration made up of a contiguous stretch of amino acids or distinct regions of non-contiguous amino acids) to which an antigen binding domain binds, forming an antigen binding domain-antigen complex. Useful antigenic determinants can be found, for example, on tumor cell surfaces, on virally infected cell surfaces, on other diseased cell surfaces, on immune cell surfaces, free in serum, and / or within the extracellular matrix (ECM). In a preferred embodiment, the antigen is a human protein.

[0062] "CD3" refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length", unprocessed CD3, as well as any form of CD3 resulting from processing within a cell. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, the CD3 is human CD3, in particular the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in SEQ ID NO: 63 (without signal peptide). See also (www.uniprot.org) Accession No. P07766 (Version 209), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. In another embodiment, the CD3 is Macaca fascicularis CD3, in particular Macaca fascicularis CD3ε. The amino acid sequence of Macaca fascicularis CD3ε is shown in SEQ ID NO: 64 (without signal peptide). See also NCBI GenBank no. BAB71849.1. In a particular embodiment, the antibody of the invention binds to an epitope of CD3 that is conserved between CD3 antigens from different species, in particular human and Macaca fascicularis CD3. In a preferred embodiment, the antibody binds to human CD3.

[0063] As used herein, "target cell antigen" refers to an antigenic determinant presented on the surface of a target cell, such as a cancer cell. Preferably, the target cell antigen is not CD3 and / or is expressed on a different cell than CD3. According to the present invention, the target cell antigen is PLAP, in particular human PLAP.

[0064] "PLAP" stands for placental alkaline phosphatase (Enzyme Commission (EC) number 3.1.3.1). Human PLAP is a glycosylated protein of 535 amino acids encoded by the ALPP gene (hence sometimes referred to as "ALPP"). There are four distinct human alkaline phosphatases: (1) placental-type ALP (PLAP, ALPP gene) and (2) germline-type ALP (PLAP-like, ALPG gene), which share 98% homology; (3) intestinal-type ALP (ALPI gene), and (4) non-specific tissue ALP or liver / bone / kidney ALP (ALPL gene), which share 88% and 56% homology with PLAP, respectively (Moss, Clinical Chemistry 38 (1992) 2486-2492; Harris, Clin Chim Acta 186 (1990) 133-150). As used herein, "PLAP" refers to any native PLAP from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length", unprocessed PLAP, as well as any form of PLAP resulting from processing within a cell. The term also encompasses naturally occurring variants of PLAP, such as splice variants or allelic variants. In one embodiment, the PLAP is human PLAP. See UniProt (www.uniprot.org) accession number P05187 (entry version 224) for the human protein. The amino acid sequence of human PLAP is also shown in SEQ ID NO: 73 (without signal peptide). The amino acid sequence of cynomolgus monkey PLAP is shown in SEQ ID NO: 74 (with signal peptide). In a particular embodiment, the antibody of the present invention binds to an epitope of PLAP that is conserved between different species of PLAP antigens, particularly human and cynomolgus monkey PLAP. In a preferred embodiment, the antibody binds to human PLAP.

[0065] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is usually measured by the dissociation constant (K D Affinity can be measured by well-established methods known in the art, including those described herein. A preferred method for measuring affinity is surface plasmon resonance (SPR).

[0066] An "affinity matured" antibody refers to an antibody that has one or more modifications in one or more complementarity determining regions (CDRs) that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess such modifications.

[0067] "Reduced binding", e.g., reduced binding to Fc receptors, refers to a reduced affinity for the respective interaction, e.g., as measured by SPR. For clarity, the term also includes a reduction in affinity to zero (or below the detection limit of the analytical method), i.e., a complete loss of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0068] As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and described herein.

[0069] A "modification that promotes the association of a first and a second subunit of an Fc domain" is a peptide backbone manipulation or post-translational modification of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising an Fc domain subunit with an identical polypeptide to form a homodimer. As used herein, a modification that promotes association preferably includes separate modifications made to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to be associated, which are complementary to each other to promote the association of the two Fc domain subunits. For example, a modification that promotes association can change the structure or charge of one or both of the Fc domain subunits to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may not be identical in the sense that the additional components (e.g., antigen binding domains) fused to each of the subunits are not the same. In some embodiments, the modification that promotes the association of the first and second subunits of the Fc domain comprises an amino acid mutation, specifically an amino acid substitution, in the Fc domain. In a preferred embodiment, the modification that promotes the association of the first and second subunits of the Fc domain comprises a distinct amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.

[0070] The term "effector function" refers to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); cytokine secretion; immune complex-mediated antigen uptake by antigen-presenting cells; downregulation of cell surface receptors (e.g., B cell receptor); and activation of B cells.

[0071] An "activating Fc receptor" is an Fc receptor that, following binding of the Fc domain of an antibody, triggers signaling events that stimulate a receptor-bearing cell to carry out an effector function. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32) and FcαRI (CD89).

[0072] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that causes lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which an antibody or its derivatives, including an Fc region, specifically binds, typically via a protein portion that is N-terminal to the Fc region. As used herein, the term "reduced ADCC" is defined as either a reduction in the number of target cells lysed in a given time at a given antibody concentration in the medium surrounding the target cells by the mechanism of ADCC as defined above, 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 by the mechanism of ADCC. The reduced ADCC is compared to the ADCC mediated by the same antibody, but not engineered, produced by the same type of host cell, using the same standard production, purification, formulation and storage methods, which are known to those skilled in the art. For example, the reduced ADCC mediated by an antibody that includes an amino acid substitution in its Fc domain that reduces ADCC is compared 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, for example, PCT Publication No. WO2006 / 082515 or PCT Publication No. WO2012 / 130831).

[0073] As used herein, the terms "engineer, engineered, engineering" are intended to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modification of the amino acid sequence, modification of the glycosylation pattern or modification of the side groups of individual amino acids, and combinations of these techniques.

[0074] The term "amino acid mutation" as used herein is meant to encompass amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct has the desired characteristics (e.g., reduced binding to Fc receptors or increased association with another peptide). Deletions and insertions in the amino acid sequence include amino- and / or carboxy-terminal deletions and insertions of amino acids. Preferred amino acid mutations are amino acid substitutions. Non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred, e.g., to alter the binding properties of the Fc region. Amino acid substitutions include replacement with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). 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 of modifying the side chain groups of amino acids other than genetic engineering, such as chemical modification, may also be useful. Various names may be used herein to indicate the same amino acid mutation. For example, a proline to glycine substitution at position 329 of the Fc domain is represented by 329G, G329, G 329 , P329G or Pro329Gly.

[0075] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine the appropriate parameters for performing sequence alignment, including any algorithms required to achieve maximum 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 written by Genentech, Inc., and its source code together with user documentation has been submitted to the U.S. Copyright Office, Washington, DC, 20559, where it is registered under U.S. Copyright Registration No. TXU510087, and is described in WO 2001 / 007611.

[0076] Unless otherwise indicated, for purposes of this specification, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c or later with the BLOSUM50 comparison matrix. The FASTA program package was written by WR Pearson and DJ Lipman ("Improved Tools for Biological Sequence Analysis", PNAS 85 (1988) 2444-2448), WR Pearson ("Effective protein sequence comparison" Meth. Enzymol. 266 (1996) 227-258) and Pearson et.al. (Genomics 46 (1997) 24-36) and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch(global protein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2), ensuring global rather than local alignments. Percent amino acid identity is given in the output alignment header.

[0077] The term "polynucleotide" or "nucleic acid molecule" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, which represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, and both single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein can include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and / or in vivo, for example in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to improve the stability of the RNA vector and / or the expression of the encoded molecule, allowing the mRNA to be injected into a subject to generate antibodies in vivo (see, for example, Stadler et al. (2017) Nature Medicine 23:815-817, or EP 2101823 B1).

[0078] An "isolated" nucleic acid molecule refers to a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid molecules include nucleic acid molecules contained within a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0079] An "isolated polynucleotide (or nucleic acid) encoding an antibody" refers to one or more polynucleotide molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such polynucleotide molecules in a single vector or separate vectors, and present in one or more locations in a host cell.

[0080] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which the vector is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0081] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," including the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell are included herein. A host cell is any type of cell line that can be used to generate the antibodies of the invention. Host cells include cultured cells, such as cultured mammalian cells, such as HEK cells, CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, to name a few, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues. In one embodiment, the host cell of the invention is a eukaryotic cell, in particular a mammalian cell. In one embodiment, the host cell is not a cell within the human body.

[0082] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form that is effective for the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that are unacceptably toxic to a subject to which the composition will be administered.

[0083] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, additives, stabilizers, or preservatives.

[0084] As used herein, "treatment" (and its grammatical variants, "treat" or "treating") refers to a clinical intervention to alter the natural course of a disease in the subject being treated, and can be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or remitigating the condition, and achieving remission or improving prognosis. In some aspects, the antibodies of the invention are used to delay the onset of disease or to slow the progression of the disease.

[0085] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, an individual or subject is a human.

[0086] An "effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0087] The term "package insert" is used to refer to instructions customarily included in commercial packaging of a therapeutic product that contain information regarding the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings regarding the use of such therapeutic product.

[0088] II. Compositions and Methods The present invention provides antibodies that bind to CD3 and PLAP. The antibodies exhibit excellent stability, e.g., in terms of efficacy and safety, pharmacokinetics, and manufacturability, combined with other properties that are advantageous for therapeutic applications. The antibodies of the present invention are useful, e.g., for the treatment of diseases such as cancer or autoimmune diseases.

[0089] A. Anti-CD3 / PLAP antibody In one aspect, the invention provides antibodies that bind to CD3 and PLAP. In one aspect, isolated antibodies that bind to CD3 and PLAP are provided. In one aspect, the invention provides antibodies that specifically bind to CD3 and PLAP. In a particular aspect, the anti-CD3 anti-PLAP antibody retains greater than about 90% binding activity to CD3 after 2 weeks at pH 7.4 and 37°C relative to the binding activity after 2 weeks at pH 6 and -80°C as determined by surface plasmon resonance (SPR).

[0090] In one aspect, the invention provides a bispecific antibody that binds to CD3 and FolR1, wherein the antibody comprises (a) a first antigen-binding domain that binds to CD3 comprising a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 3, and HCDR3 of SEQ ID NO: 6, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12.

[0091] In one embodiment, the antibody is a humanized antibody. In one embodiment, the first antigen-binding domain is a humanized antigen-binding domain (i.e., the antigen-binding domain of a humanized antibody). In one embodiment, the VH and / or VL of the first antigen-binding domain are humanized variable regions.

[0092] In one aspect, the VH and / or VL of the first antigen binding domain comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0093] In one aspect, the VH of the first antigen binding domain comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of the heavy chain variable region sequence of SEQ ID NO: 9. In one aspect, the VH of the first antigen binding domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 9. In one aspect, the VH of the first antigen binding domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 9. In one aspect, the VH of the first antigen binding domain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 9. In certain aspects, a VH sequence with at least 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody comprising the sequence retains the ability to bind to CD3. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the substitutions, insertions or deletions occur in the regions outside the CDRs (i.e., FRs). In one embodiment, the VH of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 9. Optionally, the VH of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 9, including post-translational modifications of that sequence.

[0094] In one aspect, the VL of the first antigen binding domain 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: 13. In one aspect, the VL of the first antigen binding domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13. In one aspect, the VL of the first antigen binding domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 13. In one aspect, the VL of the first antigen binding domain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 13. In a particular aspect, a VL sequence with at least 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody comprising the sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in the amino acid sequence of SEQ ID NO: 13. In certain aspects, the substitutions, insertions or deletions occur in the regions outside the CDRs (i.e., FRs). In one aspect, the VL of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 13. Optionally, the VL of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 13, including post-translational modifications of that sequence.

[0095] In one aspect, the VH of the first antigen binding domain comprises an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9 and the VL of the first antigen binding domain comprises an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13. In one aspect, the VH of the first antigen binding domain comprises the amino acid sequence of SEQ ID NO: 9 and the VL of the first antigen binding domain comprises the amino acid sequence of SEQ ID NO: 13.

[0096] In a further aspect, the invention provides an antibody that binds to CD3 and PLAP, wherein the antibody comprises a first antigen-binding domain that binds to CD3, comprising a VH comprising the amino acid sequence of SEQ ID NO:9 and a VL comprising the amino acid sequence of SEQ ID NO:13.

[0097] In a further aspect, the invention provides an antibody that binds to CD3 and PLAP, wherein the antibody comprises a first antigen-binding domain that binds to CD3 comprising the VH sequence of SEQ ID NO: 9 and the VL sequence of SEQ ID NO: 13.

[0098] In another aspect, the invention provides an antibody that binds to CD3 and PLAP, wherein the antibody comprises a first antigen-binding domain that binds to CD3, comprising a VH comprising the heavy chain CDR sequence of VH of SEQ ID NO: 9 and a VL comprising the light chain CDR sequence of VL of SEQ ID NO: 13.

[0099] In a further aspect, the first antigen-binding domain comprises the VH HCDR1, HCDR2 and HCDR3 amino acid sequence of SEQ ID NO: 9 and the VL LCDR1, LCDR2 and LCDR3 amino acid sequence of SEQ ID NO:13.

[0100] In one aspect the VH of the first antigen binding domain comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 9 and a framework of at least 95%, 96%, 97%, 98% or 99% sequence identity to the framework sequence of the VH of SEQ ID NO: 9. In one aspect the VH of the first antigen binding domain comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 9 and a framework of at least 95% sequence identity to the framework sequence of the VH of SEQ ID NO: 9. In another aspect the VH of the first antigen binding domain comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 9 and a framework of at least 98% sequence identity to the framework sequence of the VH of SEQ ID NO: 9.

[0101] In one aspect the VL of the first antigen binding domain comprises the light chain CDR sequence of the VL of SEQ ID NO: 13 and a framework of at least 95%, 96%, 97%, 98% or 99% sequence identity to the framework sequence of the VL of SEQ ID NO: 13. In one aspect the VL of the first antigen binding domain comprises the light chain CDR sequence of the VL of SEQ ID NO: 13 and a framework of at least 95% sequence identity to the framework sequence of the VL of SEQ ID NO: 13. In another aspect the VL of the first antigen binding domain comprises the light chain CDR sequence of the VL of SEQ ID NO: 13 and a framework of at least 98% sequence identity to the framework sequence of the VL of SEQ ID NO: 13.

[0102] In one aspect, the invention provides an antibody that binds to CD3 and PLAP, wherein the antibody comprises a first antigen-binding domain that binds to CD3 comprising a VH sequence as in any of the aspects provided above and a VL sequence as in any of the aspects provided above.

[0103] In one embodiment, the antibody comprises a human constant region. In one embodiment, 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 SEQ ID NOs: 70 and 71 (human kappa and lambda CL domains, respectively), and SEQ ID NO: 72 (human IgG 1 The heavy chain constant domains are given by CH1-CH2-CH3). In one embodiment, the antibody comprises a light chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 71, in particular the amino acid sequence of SEQ ID NO: 70. In one embodiment, the antibody comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 72. In particular, the heavy chain constant region may comprise amino acid mutations in the Fc domain as described herein.

[0104] In one embodiment, the first antigen binding domain comprises a human constant region. In one embodiment, 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 embodiment, the first antigen binding domain comprises a light chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 71, in particular the amino acid sequence of SEQ ID NO: 70. In particular, the light chain constant region may comprise an amino acid mutation as described herein under "charge modification" and / or, in the case of a crossover Fab molecule, a deletion or substitution of one or more (in particular two) N-terminal amino acids. In some embodiments, the first antigen binding domain comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 72. In particular, the heavy chain constant region (specifically the CH1 domain) may contain the amino acid mutations described under "charge modifications" herein.

[0105] In one embodiment, the antibody is a monoclonal antibody.

[0106] In one embodiment the antibody is an IgG antibody, particularly an IgG 1 An antibody. In one aspect, the antibody is a full-length antibody.

[0107] In another embodiment, the antibody is an Fv molecule, an scFv molecule, an Fab molecule, or an F(ab') molecule. 2 In another embodiment, the antibody fragment is a diabody, a triabody or a tetrabody.

[0108] In one embodiment, the first antigen-binding domain is a Fab molecule. In a preferred embodiment, the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are replaced by each other (i.e. the first antigen-binding domain is a crossover Fab molecule).

[0109] In further aspects, an antibody according to any of the above aspects may incorporate any of the features described in Sections II.A.1.-8. below, either alone or in combination.

[0110] In a preferred embodiment, the antibody comprises an Fc domain, in particular an IgG Fc domain, more particularly an IgG 1 In one embodiment, the Fc domain is a human Fc domain. In one embodiment, the Fc domain is a human IgG 1 An Fc domain is composed of a first and a second subunit and may incorporate, alone or in combination, any of the features described herein below in relation to Fc domain variants (section II.A.8.).

[0111] According to the present invention, the antibody comprises a second antigen-binding domain that binds to PLAP, and optionally a third antigen-binding domain (i.e., the antibody is a multispecific antibody, as further described herein below (Section II.A.7.)).

[0112] 1. Antibody fragment In certain aspects, the antibodies provided herein are antibody fragments.

[0113] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab') 2 "Fab' molecules" differ from Fab molecules by the addition of residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' molecule in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. Pepsin treatment produces an F(ab') molecule that has two antigen-binding sites (two Fab molecules) and a portion of the Fc region. 2 The molecule is obtained.

[0114] In another embodiment, the antibody fragment is a diabody, triabody, or tetrabody. Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. See, e.g., EP 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0115] In a further embodiment, the antibody fragment is a single chain Fab molecule. A "single chain Fab molecule" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, said antibody domains and said linker having one of the following orders from N-terminus to C-terminus: 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, said linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single chain Fab molecule is stabilized by a natural disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules may be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., position 44 of the variable heavy chain and position 100 of the variable light chain according to the Kabat numbering).

[0116] In another embodiment, 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, the linker is a short polypeptide of 10-25 amino acids, usually rich in glycine for flexibility and rich in serine or threonine for solubility, which can link the N-terminus of VH to the C-terminus of VL, or vice versa. The protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. For a review of scFv fragments, see, for example, Pluckthun, 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 U.S. Pat. Nos. 5,571,894 and 5,587,458.

[0117] In another embodiment, the antibody fragment is a single domain antibody. A "single domain antibody" is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0118] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and recombinant production in recombinant host cells (e.g., E. coli), as described herein.

[0119] 2. Humanized antibodies 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 parent non-human antibody. In general, 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 comprises at least a portion of a human constant region. In some aspects, some FR residues of a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0120] Humanized antibodies and methods for producing them are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, for example, Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting), Padlan, Mol. Immunol. 28:489-498 (1991) (describing resurfacing), Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"), as well as Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0121] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0122] 3. Glycosylation variants In certain aspects, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0123] If the antibody comprises an Fc region, the oligosaccharides attached thereto may be modified. Natural antibodies produced by mammalian cells usually contain branched, biantennary oligosaccharides attached by N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibodies of the present invention may be performed to generate antibody variants with specific improved properties.

[0124] In one aspect, antibody variants are provided that have nonfucosylated oligosaccharides, i.e. oligosaccharide structures that lack fucose attached (directly or indirectly) to the Fc region. Such nonfucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are in particular N-linked oligosaccharides that lack a fucose residue attached to the first GlcNAc at the stem of the biantennary oligosaccharide structure. In one aspect, antibody variants are provided that have an increased proportion of nonfucosylated oligosaccharides in the Fc region compared to the native or parent antibody. For example, the proportion of nonfucosylated oligosaccharides may 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 nonfucosylated oligosaccharides is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g. complex, hybrid and high mannose structures) attached to Asn297, as determined, for example, by MALDI-TOF mass spectrometry as described in WO 2006 / 082515. Asn297 refers to an asparagine residue located at approximately position 297 of the Fc region (EU numbering of Fc region residues). However, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300. Such antibodies with an increased percentage of nonfucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, in particular improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 and 2004 / 0093621.

[0125] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108; and WO 2004 / 056312, especially Example 11), and knockout cell lines, such as FUT8 knockout CHO cells of the alpha-1,6-fucosyltransferase gene (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al. al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107), or cells with reduced or abolished activity of GDP-fucose synthesis or transporter proteins (see, e.g., U.S. Patent Application Publication Nos. 2004259150, 2005031613, 2004132140, and 2004110282).

[0126] In a further aspect, antibody variants are provided that have bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, 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.

[0127] Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087, WO 1998 / 58964 and WO 1999 / 22764.

[0128] 4. Cysteine ​​Engineered Antibody Variants In certain aspects, it may be desirable to generate cysteine ​​engineered antibodies, such as THIOMAB™ antibodies, in which one or more residues of an antibody are replaced with cysteine ​​residues. In preferred aspects, the replaced residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which can be used to conjugate the antibody to other sites, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541, U.S. Pat. No. 8,30,930, U.S. Pat. No. 7,855,275, U.S. Pat. No. 9,000,130, or WO2016040856.

[0129] 5. Antibody derivatives In certain aspects, the antibodies provided herein may be further modified to include additional non-protein moieties known in the art and readily available. 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-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when multiple polymers are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0130] 6. Immunoconjugates The invention also provides immunoconjugates comprising an anti-CD3 / PLAP antibody herein conjugated to one or more therapeutic agents, such as a cytotoxic agent, a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof) or a radioactive isotope.

[0131] In one aspect, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more of the above-mentioned therapeutic agents. The antibody is typically linked to one or more therapeutic agents using a linker. A review of ADC technology, including examples of therapeutic agents and therapeutic agents and linkers, is provided in Pharmacol Review 68:3-19 (2016).

[0132] In another aspect, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Abrasiongum protein, dianthin protein, pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, curcin, crotin, saponaria inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0133] In another embodiment, the immunoconjugate comprises an antibody of the invention conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the generation of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 When used for detection, the radioconjugates include radioactive atoms for scintigraphic examinations, such as Tc 99m Or I 123 or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as I23 , I 131 , In 111 , F 19 , C 13 , N 15 , O 17 , gadolinium, manganese or iron.

[0134] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and diactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies. See WO 94 / 11026. The linker may be a "cleavable linker" that facilitates the release of the cytotoxic drug inside the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.

[0135] Immunoconjugates or ADCs herein expressly contemplate conjugates prepared using cross-linking reagents including, but not limited to, 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), which are commercially available (e.g., from Pierce Biotechnology, Rockford, Ill., USA).

[0136] 7. Multispecific antibodies The antibodies provided herein are multispecific antibodies, particularly bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigenic determinants (e.g., two different proteins, or two different epitopes on the same protein). In certain embodiments, multispecific antibodies have three or more binding specificities. According to the present invention, one of the binding specificities is for CD3 and another is for PLAP.

[0137] Multispecific antibodies can be prepared as full-length antibodies or antibody fragments. Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knobs-in-holes" engineering (see, e.g., U.S. Patent No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (see, e.g., WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); using general light chain technology to avoid light chain mispairing problems (see, e.g., WO 98 / 50431); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605). 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 by preparation of trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0138] Also included herein are engineered antibodies with three or more antigen binding sites, including, for example, "Octopus antibodies," or DVD-Igs (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. Multispecific antibodies or antigen-binding fragments thereof also include "dual acting FAbs" or "DAFs" that contain antigen-binding sites that bind to CD3 and another different antigen, or to two different epitopes of CD3 (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820 and WO 2015 / 095539).

[0139] Multispecific antibodies can also be provided in an asymmetric form (so-called "CrossMab" technology) involving domain crossing in one or more binding arms with the same antigen specificity, i.e. by swapping VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253) or complete Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299; see also Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to induce correct Fab pairing. See, for example, WO 2016 / 172485.

[0140] A variety of additional molecular formats of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

[0141] A particular type of multispecific antibody is a bispecific antibody designed to simultaneously bind to a surface antigen on a target cell, e.g., a cancer cell, and to an activation-invariant component of the T cell receptor (TCR) complex, e.g., CD3, to retarget the T cell to kill the target cell. Thus, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies, in which one of the binding specificities is for CD3 and the other is for PLAP as the target cell antigen.

[0142] Examples of bispecific antibody formats that may be useful for this purpose include so-called "BiTE" (bispecific T cell engager) molecules, in which two scFv molecules are fused by a flexible linker (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261 and WO 2008 / 119567; Nagorsen and Baeuerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies ("TandAbs"; Kipriyanov et al., J Mol Biol 1999, 2010 ... 293, 41-56 (1999); "DART" (dual affinity retargeting) molecules based on the diabody format but featuring a C-terminal disulfide bridge for stabilizing attachment (Johnson et al., J Mol Biol 399, 436-449 (2010)), as well as the so-called triomabs, which are all-hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Included herein are certain T cell bispecific antibody formats described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0143] Preferred embodiments of the antibodies of the present invention are described below.

[0144] In one aspect, the invention provides an antibody that binds CD3 and PLAP, comprising a first antigen binding domain that binds CD3, a second antigen binding domain that binds PLAP, and optionally a third antigen binding domain, as described herein.

[0145] According to a preferred embodiment of the invention, the antigen-binding domain comprised in the antibody is a Fab molecule (i.e., an antigen-binding domain composed of a heavy chain and a light chain, each of which comprises a variable domain and a constant domain). In one embodiment, the first antigen-binding domain, the second antigen-binding domain and / or, if present, the third antigen-binding domain are Fab molecules. In one embodiment, the Fab molecule is human. In a preferred embodiment, the Fab molecule is humanized. In yet another embodiment, the Fab molecule comprises human heavy and light chain constant domains.

[0146] Preferably, at least one of the antigen-binding domains is a crossover Fab molecule. Such a modification reduces mispairing of the heavy and light chains of different Fab molecules, thereby improving the yield and purity of the (multispecific) antibodies of the invention in recombinant production. In preferred crossover Fab molecules useful for the (multispecific) antibodies of the invention, the variable domains of the Fab light chain and the Fab heavy chain (VL, VH, respectively) are exchanged. However, even with this domain exchange, the preparation of (multispecific) antibodies may contain certain by-products due to so-called Bence-Jones type interactions between mispaired heavy and light chains (see Schaefer et al, PNAS, 108 (2011) 11187-11191). In order to further reduce the mispairing of the heavy and light chains of different Fab molecules and thereby improve the purity and yield of the desired (multispecific) antibodies, charged amino acids with opposite charges can be introduced at specific amino acid positions in the CH1 and CL domains of either the Fab molecule binding to CD3 or the Fab molecule(s) binding to PLAP, as described below. Charge modifications are made either in the conventional Fab molecule(s) comprised in the (multispecific) antibody (e.g. as shown in Figures 1A-C, G-J) or in the VH / VL crossover Fab molecule(s) comprised in the multispecific antibody (e.g. as shown in Figures 1D-F, K-N), but not in both. In a preferred embodiment, charge modifications are made within a conventional Fab molecule comprised in the (multispecific) antibody (which in a preferred embodiment binds PLAP).

[0147] In a preferred embodiment according to the invention, the (multispecific) antibody is capable of binding simultaneously to CD3 and PLAP. In one embodiment, the (multispecific) antibody is capable of cross-linking T cells and target cells by simultaneous binding to CD3 and PLAP. In an even more preferred embodiment, such simultaneous binding results in lysis of the target cell, in particular a PLAP-expressing target cell. In one embodiment, such simultaneous binding causes activation of the T cell. In another embodiment, such simultaneous binding results in a cellular response of the T lymphocyte, in particular a cytotoxic T lymphocyte, selected from the group of proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. In one embodiment, binding of the (multispecific) antibody to CD3 without simultaneous binding to PLAP does not result in T cell activation.

[0148] In one embodiment, the (multispecific) antibody is capable of redirecting the cytotoxic activity of T cells to target cells. In a preferred embodiment, said redirection is independent of MHC-mediated peptide antigen presentation by the target cells and / or the specificity of the T cells.

[0149] Preferably, the T cells according to any of the embodiments of the present invention are cytotoxic T cells. In some embodiments, the T cells are CD4 + or CD8 + T cells, especially CD8 + T cells.

[0150] a) A first antigen-binding domain The (multispecific) antibody of the invention comprises at least one antigen-binding domain (first antigen-binding domain) that binds to CD3. In a preferred embodiment, the CD3 is human CD3 (SEQ ID NO: 63) or cynomolgus CD3 (SEQ ID NO: 64), most particularly human CD3. In one embodiment, the first antigen-binding domain is cross-reactive (i.e. specifically binds) to human and cynomolgus CD3. In some embodiments, the CD3 is the epsilon subunit of CD3 (CD3 epsilon).

[0151] In a preferred embodiment, the (multispecific) antibody comprises not more than one antigen-binding domain that binds to CD3. In one embodiment, the (multispecific) antibody provides monovalent binding to CD3.

[0152] In one embodiment, the antigen-binding domain that binds to CD3 is an Fv molecule, an scFv molecule, an Fab molecule, and an F(ab') 2 In a preferred embodiment, the antigen-binding domain that binds to CD3 is a Fab molecule.

[0153] In a preferred embodiment, the antigen-binding domain that binds to CD3 is a crossover Fab molecule as described herein, i.e. a Fab molecule in which the variable domains VH and VL, or the constant domains CH1 and CL, of the Fab heavy and light chains, have been swapped / replaced with each other. In such an embodiment, the antigen-binding domain that binds to PLAP is preferably a conventional Fab molecule. In an embodiment in which there is more than one antigen-binding domain, in particular a Fab molecule, that binds to PLAP contained in a multispecific antibody, the antigen-binding domain that binds to CD3 is preferably a crossover Fab molecule and the antigen-binding domain that binds to PLAP is a conventional Fab molecule.

[0154] In an alternative embodiment, the antigen-binding domain that binds CD3 is a conventional Fab molecule. In such an embodiment, the antigen-binding domain(s) that binds PLAP are crossover Fab molecules as described herein, i.e. Fab molecules in which the variable domains VH and VL, or the constant domains CH1 and CL, of the Fab heavy and Fab light chains, have been swapped / replaced with each other. In an embodiment in which there are more than one antigen-binding domain that binds CD3, in particular Fab molecules, contained in a (multispecific) antibody, the antigen-binding domain that binds PLAP is preferably a crossover Fab molecule and the antigen-binding domain that binds CD3 is a conventional Fab molecule.

[0155] In a preferred embodiment, the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain, in particular the variable domains VL and VH, are replaced by each other (i.e. according to such embodiment, the first antigen-binding domain is a crossover Fab molecule in which the variable or constant domains of the Fab light chain and the Fab heavy chain are exchanged). In such embodiment, the second (and, if present, the third) antigen-binding domain is a conventional Fab molecule.

[0156] In one embodiment, 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).

[0157] b) The second (and third) antigen-binding domain The (multispecific) antibody of the invention comprises at least one antigen-binding domain (a second antigen-binding domain and optionally a third antigen-binding domain), in particular a Fab molecule, that binds to PLAP. The second antigen-binding domain can direct the (multispecific) antibody to a target site, for example a specific type of tumor cell expressing PLAP.

[0158] In one embodiment, the antigen-binding domain that binds to PLAP is an Fv molecule, an scFv molecule, an Fab molecule, and an F(ab') 2 In a preferred embodiment, the antigen-binding domain that binds to PLAP is a Fab molecule.

[0159] In a particular embodiment, the (multispecific) antibody comprises two antigen-binding domains, in particular Fab molecules, that bind to PLAP. In a preferred embodiment, these antigen-binding domains are all identical, i.e. have the same molecular format (e.g. conventional or crossover Fab molecules) and comprise the same amino acid sequence, including the same amino acid substitutions in the CH1 and CL domains as described herein (if present). In one embodiment, the (multispecific) antibody comprises not more than two antigen-binding domains, in particular Fab molecules, that bind to PLAP.

[0160] In a preferred embodiment, the antigen binding domain(s) that bind to PLAP are conventional Fab molecules. In such an embodiment, the antigen binding domain(s) that bind to CD3 are crossover Fab molecules as described herein, i.e., Fab molecules in which the variable domains VH and VL, or the constant domains CH1 and CL, of the Fab heavy and Fab light chains are exchanged / replaced by each other.

[0161] In an alternative embodiment, the antigen binding domain(s) that bind PLAP are crossover Fab molecules as described herein, i.e., Fab molecules in which the variable domains VH and VL, or the constant domains CH1 and CL, of the Fab heavy and Fab light chains have been swapped / replaced with each other. In such an embodiment, the antigen binding domain(s) that bind CD3 are conventional Fab molecules.

[0162] In one aspect, the second (and, if present, the third) antigen binding domain comprises a human constant region. In one aspect, the second (and, if present, the third) antigen binding domain 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 SEQ ID NOs: 70 and 71 (human kappa and lambda CL domains, respectively), and SEQ ID NO: 72 (human IgG 1The heavy chain constant domains CH1-CH2-CH3) are given in the amino acid sequence of SEQ ID NO: 72. In one embodiment, the second (and, if present, the third) antigen binding domain comprises a light 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: 70 or SEQ ID NO: 71, in particular the amino acid sequence of SEQ ID NO: 70. In particular, the light chain constant region may comprise an amino acid mutation described herein under "charge modification" and / or in the case of crossover Fab molecules, one or more (in particular two) N-terminal amino acid deletions or substitutions. In some embodiments, the second (and, if present, the third) antigen binding domain comprises a heavy chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 72. In particular, the heavy chain constant region (specifically the CH1 domain) may comprise an amino acid mutation described herein under "charge modification".

[0163] In one aspect, the second (and, if present, the third) antigen binding domain comprises a VH comprising: (i) a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 28, a HCDR2 of SEQ ID NO: 29, and a HCDR3 of SEQ ID NO: 30; (ii) a HCDR1 of SEQ ID NO: 32, a HCDR2 of SEQ ID NO: 33, and a HCDR3 of SEQ ID NO: 34; (iii) a HCDR1 of SEQ ID NO: 36, a HCDR2 of SEQ ID NO: 37, and a HCDR3 of SEQ ID NO: 38; (iv) a HCDR1 of SEQ ID NO: 40, a HCDR2 of SEQ ID NO: 41, and a HCDR3 of SEQ ID NO: 42; or (v) a VH comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 48, a LCDR2 of SEQ ID NO: 49, and a LCDR3 of SEQ ID NO: 50.

[0164] In one aspect, the second (and, if present, the third) antigen binding domain is (derived from) a humanized antibody. In one aspect, the second (and, if present, the third) antigen binding domain is a humanized antigen binding domain (i.e. the antigen binding domain of a humanized antibody). In one aspect, the VH and / or VL of the second (and, if present, the third) antigen binding domain are humanized variable regions.

[0165] In one aspect the VH and / or VL of the second (and, if present, the third) antigen binding domain comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0166] In one aspect, the VH of the second (and, if present, the third) antigen binding domain comprises one or more of the heavy chain framework sequences (i.e. FR1, FR2, FR3 and / or FR4 sequences) of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, or SEQ ID NO:47. In one aspect, the VH of the second (and, if present, the third) antigen binding domain comprises an amino acid sequence which is at least about 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, or SEQ ID NO:47. In one aspect, the VH of the second (and, if present, the third) antigen binding domain comprises an amino acid sequence which is at least about 95% identical to an amino acid sequence of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, or SEQ ID NO:47. In one embodiment, the VH of the second (and, if present, the third) antigen-binding domain comprises an amino acid sequence that is at least about 98 percent identical to the amino acid sequence of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, or SEQ ID NO:47. In certain embodiments, a VH sequence with at least 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody comprising the sequence retains the ability to bind to PLAP. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43, or SEQ ID NO:47. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the CDRs (i.e., FRs). In one aspect, the VH of the second (and, if present, the third) antigen binding domain comprises the amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, or SEQ ID NO: 47. Optionally, the VH of the second (and, if present, the third) antigen binding domain comprises the amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, or SEQ ID NO: 47, including post-translational modifications of said sequences.

[0167] In one aspect, the VL of the second (and, if present, the third) antigen binding domain comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3 and / or FR4 sequences) of SEQ ID NO: 51. In one aspect, the VL of the second (and, if present, the third) antigen binding domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 51. In one aspect, the VL of the second (and, if present, the third) antigen binding domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 51. In one aspect, the VL of the second (and, if present, the third) antigen binding domain comprises an amino acid sequence that is at least about 98 percent identical to the amino acid sequence of SEQ ID NO: 51. In certain embodiments, a VL sequence having at least 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but an antibody comprising the sequence retains the ability to bind to PLAP. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 51. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the CDRs (i.e., FRs). In one embodiment, the VL of the second (and, if present, the third) antigen binding domain comprises the amino acid sequence of SEQ ID NO: 51. Optionally, the VL of the second (and, if present, the third) antigen binding domain comprises the amino acid sequence of SEQ ID NO: 51, including post-translational modifications of that sequence.

[0168] In one aspect, the VH of the second (and, if present, the third) antigen binding domain comprises an amino acid sequence which is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43 or SEQ ID NO:47 and the VL of the second (and, if present, the third) antigen binding domain comprises an amino acid sequence which is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:51. In one aspect, the VH of the second (and, if present, the third) antigen binding domain comprises the amino acid sequence of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43 or SEQ ID NO:47 and the VL of the second (and, if present, the third) antigen binding domain comprises the amino acid sequence of SEQ ID NO:51.

[0169] In a further aspect, the second (and, if present, the third) antigen binding domain comprises a VH comprising the sequence of SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43 or SEQ ID NO: 47, and a VL comprising the sequence of SEQ ID NO: 51.

[0170] In a further aspect, the second (and, if present, the third) antigen-binding domain comprises the VH sequence of SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43 or SEQ ID NO: 47 and the VL sequence of SEQ ID NO: 51.

[0171] In another aspect, the second (and, if present, the third) antigen binding domain comprises a VH comprising the heavy chain CDR sequence of the VH of SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43 or SEQ ID NO: 47, and a VL comprising the light chain CDR sequence of the VL of SEQ ID NO: 51.

[0172] In a further aspect, the second (and, if present, the third) antigen binding domain comprises the VH HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43 or SEQ ID NO: 47 and the VL LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO: 51.

[0173] In one aspect the VH of the second (and, if present, the third) antigen binding domain comprises the heavy chain CDR sequence of the VH of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43 or SEQ ID NO:47 and a framework of at least 95%, 96%, 97%, 98% or 99% sequence identity to the framework sequence of the VH of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43 or SEQ ID NO:47, respectively. In one aspect the VH of the second (and, if present, the third) antigen binding domain comprises the heavy chain CDR sequence of the VH of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43 or SEQ ID NO:47 and a framework of at least 95% sequence identity to the framework sequence of the VH of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43 or SEQ ID NO:47, respectively. In another aspect, the VH of the second (and, if present, the third) antigen binding domain comprises the heavy chain CDR sequences of the VH of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43 or SEQ ID NO:47 and a framework having at least 98% sequence identity to the framework sequence of the VH of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43 or SEQ ID NO:47, respectively.

[0174] In one aspect, the VL of the second (and, if present, the third) antigen binding domain comprises a light chain CDR sequence of the VL of SEQ ID NO: 51 and a framework of at least 95%, 96%, 97%, 98% or 99% sequence identity to the framework sequence of the VL of SEQ ID NO: 51. In one aspect, the VL of the second (and, if present, the third) antigen binding domain comprises a light chain CDR sequence of the VL of SEQ ID NO: 51 and a framework of at least 95% sequence identity to the framework sequence of the VL of SEQ ID NO: 51. In another aspect, the VL of the second (and, if present, the third) antigen binding domain comprises a light chain CDR sequence of the VL of SEQ ID NO: 51 and a framework of at least 98% sequence identity to the framework sequence of the VL of SEQ ID NO: 51.

[0175] c) Charge modification The (multispecific) antibodies of the invention may contain, in the Fab molecules contained therein, amino acid substitutions that are particularly effective in reducing mispairing of light chains with mismatched heavy chains (Bence Jones type by-products) that may occur during the preparation of Fab-based multispecific antibodies with a VH / VL exchange in one (or in the case of molecules containing more than two antigen-binding Fab molecules) binding arm (see also WO 2015 / 150447, which is incorporated herein by reference in its entirety, in particular the Examples therein). The ratio of desirable (multispecific) antibodies to undesirable by-products, in particular the Bence Jones type by-products that arise in multispecific antibodies with a VH / VL domain exchange in one binding arm, may be improved by introducing charged amino acids with opposite charges at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modification").

[0176] Thus, in some embodiments, where the first and the second (and, if present, the third) antigen-binding domains of the (multispecific) antibody are both Fab molecules and in one of the antigen-binding domains (particularly the first antigen-binding domain), the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other: i) in the constant domain CL of the second (and, if present, the third) antigen-binding domain the amino acid at position 124 is substituted by a positive amino acid (Kabat numbering) and in the constant domain CH1 of the second (and, if present, the third) antigen-binding domain the amino acid at position 147 or the amino acid at position 213 is substituted by a negative amino acid (Kabat EU index numbering); or ii) in the constant domain CL of the first antigen-binding domain the amino acid at position 124 is substituted by a positive amino acid (numbering according to Kabat) 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 is substituted by a negative amino acid (numbering according to Kabat EU index).

[0177] A (multispecific) antibody does not comprise both modifications described under i) and ii): the constant domains CL and CH1 of an antigen-binding domain having a VH / VL exchange are not replaced (i.e. not exchanged) by each other.

[0178] In a more specific embodiment, i) in the constant domain CL of the second (and, if present, the third) antigen binding domain the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering) and in the constant domain CH1 of the second (and, if present, the third) antigen binding domain the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering); or ii) in the constant domain CL of the first antigen-binding domain the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (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 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0179] In such embodiments, in the constant domain CL of the second (and, if present, the third) antigen binding domain the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering) and in the constant domain CH1 of the second (and, if present, the third) antigen binding domain the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0180] In a further aspect, in the constant domain CL of the second (and, if present, the third) antigen binding domain the amino acid at position 124 is independently substituted by a lysine (K), an arginine (R) or a histidine (H) (Kabat numbering) and in the constant domain CH1 of the second (and, if present, the third) antigen binding domain the amino acid at position 147 is independently substituted by a glutamic acid (E) or an aspartic acid (D) (Kabat EU index numbering).

[0181] In a preferred embodiment, in the constant domain CL of the second (and, if present, the third) antigen binding domain the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and in the constant domain CH1 of the second (and, if present, the third) antigen binding domain the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering) and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0182] In a more preferred embodiment, in the constant domain CL of the second (and, if present, the third) antigen binding domain the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat), and in the constant domain CHI of the second (and, if present, the third) antigen binding domain the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0183] In an even more preferred embodiment, in the constant domain CL of the second (and, if present, the third) antigen binding domain the amino acid at position 124 is substituted by a lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by an arginine (R) (numbering according to Kabat) and in the constant domain CH1 of the second (and, if present, the third) antigen binding domain the amino acid at position 147 is substituted by a glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by a glutamic acid (E) (numbering according to Kabat EU index).

[0184] In a preferred embodiment, when the amino acid substitutions according to the above embodiments are made in the constant domain CL and in the constant domain CH1 of the second (and, if present, the third) antigen binding domain, the constant domain CL of the second (and, if present, the third) antigen binding domain is of the kappa isotype.

[0185] Alternatively, the amino acid substitutions according to the above embodiments may be made in the constant domain CL and the constant domain CH1 of the first antigen-binding domain instead of the constant domain CL and the constant domain CH1 of the second (and, if present, the third) antigen-binding domain. In a preferred such embodiment, the constant domain CL of the first antigen-binding domain is of the kappa isotype.

[0186] Thus, in one embodiment in the constant domain CL of the first antigen-binding domain the amino acid at position 124 is independently substituted by a lysine (K), an arginine (R) or a histidine (H) (numbering according to Kabat) 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 is independently substituted by a glutamic acid (E) or an aspartic acid (D) (numbering according to Kabat EU index).

[0187] In a further embodiment, in the constant domain CL of the first antigen-binding domain the amino acid at position 124 is independently substituted by a lysine (K), an arginine (R) or a histidine (H) (numbering according to Kabat) and in the constant domain CH1 of the first antigen-binding domain the amino acid at position 147 is independently substituted by a glutamic acid (E) or an aspartic acid (D) (numbering according to Kabat EU index).

[0188] In yet another embodiment, in the constant domain CL of the first antigen-binding domain the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and in the constant domain CH1 of the first antigen-binding domain the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering) and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0189] In one embodiment, in the constant domain CL of the first antigen binding domain the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by lysine (K) (numbering according to Kabat), and in the constant domain CH1 of the first antigen binding domain the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0190] In another embodiment, in the constant domain CL of the first antigen-binding domain the amino acid at position 124 is substituted by a lysine (K) (numbering according to Kabat), the amino acid at position 123 is substituted by an arginine (R) (numbering according to Kabat), and in the constant domain CH1 of the first antigen-binding domain the amino acid at position 147 is substituted by a glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by a glutamic acid (E) (numbering according to Kabat EU index).

[0191] In a preferred embodiment, the (multispecific) antibody of the invention comprises (a) a first antigen-binding domain that binds to CD3, the first antigen-binding domain being a Fab molecule in which the Fab light chain and Fab heavy chain variable domains VL and VH are replaced with each other, the first antigen-binding domain comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3 and an HCDR3 of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, an LCDR2 of SEQ ID NO: 11 and an LCDR3 of SEQ ID NO: 12; (b) a second antigen-binding domain that binds to PLAP, and optionally a third antigen-binding domain, In the constant domain CL of the second (and, if present, the third) antigen binding domain, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering) (in a preferred embodiment, independently by lysine (K) or arginine (R)), the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering) (in a preferred embodiment, independently by lysine (K) or arginine (R)), and in the constant domain CH1 of the second (and, if present, the third) antigen binding domain, the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering) and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering). (numbered according to the EU index), a second antigen-binding domain that binds to PLAP, and optionally a third antigen-binding domain.

[0192] d) Multispecific antibody formats The (multispecific) antibodies according to the invention can have a variety of structures, exemplary structures are shown in FIG.

[0193] In a preferred embodiment, the antigen-binding domains comprised in the (multispecific) antibody are Fab molecules. In such an embodiment, the first, second, third etc. antigen-binding domains may be referred to herein as first, second, third etc. Fab molecules, respectively.

[0194] In one embodiment, the first and second antigen binding domains of the (multispecific) antibody are fused to each other, optionally via a peptide linker. In a preferred embodiment, the first and second antigen binding domains are each Fab molecules. In such an embodiment, the first antigen binding domain is fused to the N-terminus of the Fab heavy chain of the second antigen binding domain at the C-terminus of the Fab heavy chain. In another such embodiment, the second antigen binding domain is fused to the N-terminus of the Fab heavy chain of the first antigen binding domain at the C-terminus of the Fab heavy chain. In an embodiment in which (i) 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 or (ii) the second antigen binding domain is fused to the N-terminus of the Fab heavy chain of the first antigen binding domain at the C-terminus of the Fab heavy chain, the Fab light chain of the first antigen binding domain and the Fab light chain of the second antigen binding domain may additionally be fused to each other, optionally via a peptide linker.

[0195] A (multispecific) antibody having a single antigen-binding domain (e.g., a Fab molecule) capable of specifically binding a second antigen, e.g., a target cell antigen, e.g., PLAP (e.g., as shown in Figure 1A, D, G, H, K, L) is useful when internalization of the second antigen is expected after binding of the high affinity antigen-binding domain. In such cases, the presence of more than one antigen-binding domain specific for the second antigen may promote internalization of the second antigen, thereby reducing its availability.

[0196] However, in other cases, it may be advantageous to have a (multispecific) antibody that comprises two or more antigen binding domains (such as Fab molecules) specific for a second antigen, e.g., a target cell antigen such as PLAP (see, e.g., those shown in Figures 1B, 1C, 1E, 1F, 1I, 1J, 1M or 1N), e.g. to optimize targeting to a target site or to allow cross-linking of target cell antigens.

[0197] Thus, in a preferred embodiment, the (multispecific) antibody according to the invention comprises a third antigen-binding domain.

[0198] In one embodiment, the third antigen binding domain binds to PLAP. In one embodiment, the third antigen binding domain is a Fab molecule.

[0199] In one embodiment, the third antigen binding domain is identical to the second antigen binding domain.

[0200] In some embodiments, the third and second antigen binding domains are each Fab molecules, and the third antigen binding domain is identical to the second antigen binding domain. Thus, in these embodiments, the second and third antigen binding domains comprise the same heavy and light chain amino acid sequences and have the same domain configuration (i.e., conventional or crossover). Furthermore, in these embodiments, the third antigen binding domain comprises the same amino acid substitutions, if any, as the second antigen binding domain. For example, the amino acid substitutions described herein as "charge-modified" are made in the constant domains CL and CH1 of the second and third antigen binding domains, respectively. Alternatively, said amino acid substitutions may be made in the constant domains CL and CH1 of the first antigen binding domain (which in a preferred embodiment is also a Fab molecule), but not in the constant domains CL and CH1 of the second and third antigen binding domains.

[0201] Like the second antigen-binding domain, the third antigen-binding domain is preferably a conventional Fab molecule. However, embodiments in which the second and third antigen-binding domains are crossover Fab molecules (and the first antigen-binding domain is a conventional Fab molecule) are also contemplated. Thus, in a preferred embodiment, the second and third antigen-binding domains are each conventional Fab molecules and the first antigen-binding domain is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are swapped / replaced with each other. In other embodiments, the second and third antigen-binding domains are each crossover Fab molecules and the first antigen-binding domain is a conventional Fab molecule.

[0202] When a third antigen binding domain is present, in a preferred embodiment, the first antigen binding domain binds to CD3 and the second and third antigen binding domains bind to PLAP.

[0203] In a preferred embodiment, the (multispecific) antibody of the invention comprises an Fc domain composed of a first and a second subunit, the first and second subunits of the Fc domain being capable of stable association.

[0204] The (multispecific) antibodies according to the invention can have different structures, i.e. the first, second (and optionally third) antigen-binding domains can be fused in different ways to each other and to the Fc domain. The components can be fused to each other directly or, preferably, via one or more suitable peptide linkers. If the fusion of the Fab molecule is to the N-terminus of the Fc domain subunit, it is typically via the immunoglobulin hinge region.

[0205] In some embodiments, the first and second antigen-binding domains are each 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 first or second subunit of the Fc domain. In such embodiments, the second antigen-binding domain can 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 can be fused to the N-terminus of the other subunit of the Fc domain. In preferred such embodiments, the second antigen-binding domain is a conventional Fab molecule and the first antigen-binding domain is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are swapped / replaced with each other. In other such embodiments, the second antigen-binding domain is a crossover Fab molecule and the first antigen-binding domain is a conventional Fab molecule.

[0206] In one embodiment, the first and second antigen binding domains are each Fab molecules, with the first antigen binding domain fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the second antigen binding domain 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 embodiment, the (multispecific) antibody consists essentially of a first and a second Fab molecule, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, where the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Such structures are shown diagrammatically in Figures 1G and 1K (the first antigen binding domain in these examples is a VH / VL crossover Fab molecule). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can further be fused to each other.

[0207] In another embodiment, the first and second antigen-binding domains are each Fab molecules, and the first and second antigen-binding domains are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one subunit of the Fc domain. In a specific embodiment, the (multispecific) antibody consists essentially of a first and a second Fab molecule, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, and the first and the second Fab molecule are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one subunit of the Fc domain. Such a structure is shown diagrammatically in Figures 1A and 1D (in these examples, the first antigen-binding domain is a VH / VL crossover Fab molecule and the second antigen-binding domain is a conventional Fab molecule). The first and the second Fab molecule may be fused to the Fc domain directly or via a peptide linker. In a preferred embodiment, the first and the second Fab molecule are each fused to the Fc domain via an immunoglobulin hinge region. In certain embodiments, the immunoglobulin hinge region is an IgG Fc domain. 1In the case of an Fc domain, human IgG 1 This is the hinge region.

[0208] In some embodiments, the first and second antigen-binding domains are each 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 first or second subunit of the Fc domain. In such embodiments, the first antigen-binding domain can 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 can be fused to the N-terminus of the other subunit of the Fc domain (as described above). In preferred such embodiments, the second antigen-binding domain is a conventional Fab molecule, and the first antigen-binding domain is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are swapped / replaced with each other. In other such embodiments, the second antigen-binding domain is a crossover Fab molecule, and the first antigen-binding domain is a conventional Fab molecule.

[0209] In one embodiment, the first and second antigen binding domains are each Fab molecules, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, 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 embodiment, the (multispecific) antibody consists essentially of a first and a second Fab molecule, an Fc domain composed of a first and a second subunit, and optionally one or more peptide linkers, 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 second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Such structures are shown diagrammatically in Figures 1H and 1L (in these examples, the first antigen binding domain is a VH / VL crossover Fab molecule and the second antigen binding domain is a conventional Fab molecule). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can further be fused to each other.

[0210] In some embodiments, the third antigen-binding domain, in particular the third Fab molecule, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In preferred such embodiments, the second and third antigen-binding domains are each conventional Fab molecules and the first antigen-binding domain is a crossover Fab molecule as described herein, i.e. a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains have been swapped / replaced with each other. In other such embodiments, the second and third antigen-binding domains are each crossover Fab molecules and the first antigen-binding domain is a conventional Fab molecule.

[0211] In a preferred such embodiment, the first and third antigen-binding domains are each fused at their C-terminus of the Fab heavy chain to the N-terminus of one subunit of the Fc domain, and the second antigen-binding domain is fused at its C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In a specific embodiment, the (multispecific) antibody essentially consists of a first Fab molecule, a second Fab molecule and a third Fab molecule, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at its C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, the first Fab molecule is fused at its C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at its C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such structures are shown diagrammatically in Figures 1B and 1E (in which the first antigen binding domain is a VH / VL crossover Fab molecule and the second and third antigen binding domains are conventional Fab molecules) and in Figures 1J and 1N (in which the first antigen binding domain is a conventional Fab molecule and the second and third antigen binding domains are VH / VL crossover Fab molecules). The first and third Fab molecules may be fused to the Fc domain directly or via a peptide linker. In a preferred embodiment, the first and third Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In a particular embodiment, the immunoglobulin hinge region is preferably a Fab molecule, particularly when the Fc domain is an IgG 1 In the case of an Fc domain, human IgG 1 Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can further be fused to each other.

[0212] In another such embodiment, 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 subunit of the Fc domain, 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 embodiment, the (multispecific) antibody consists essentially of a first Fab molecule, a second Fab molecule and a third Fab molecule, an Fc domain composed of the first and second subunits and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such structures are shown diagrammatically in Figures 1C and 1F (in these examples, the first antigen binding domain is a VH / VL crossover Fab molecule and the second and third antigen binding domains are conventional Fab molecules) and in Figures 1I and 1M (in these examples, the first antigen binding domain is a conventional Fab molecule and the second and third antigen binding domains are VH / VL crossover Fab molecules). The second and third Fab molecules may be fused to the Fc domain directly or via a peptide linker. In a preferred embodiment, the second and third Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In a particular embodiment, the immunoglobulin hinge region is preferably a Fab region, particularly when the Fc domain is an IgG 1 In the case of an Fc domain, human IgG 1 Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can further be fused to each other.

[0213] In the structure of a (multispecific) antibody in which a Fab molecule is fused at the C-terminus of the Fab heavy chain via an immunoglobulin hinge region to the N-terminus of each subunit of the Fc domain, the two Fab molecules, the hinge region and the Fc domain essentially form an immunoglobulin molecule. In a preferred embodiment, the immunoglobulin molecule is an immunoglobulin of the IgG class. In an even more preferred embodiment, the immunoglobulin is an IgG 1 In another embodiment, the immunoglobulin is an IgG 4 In a further preferred embodiment, the immunoglobulin is a human immunoglobulin. In another embodiment, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one embodiment, the immunoglobulin comprises a human constant region, in particular a human Fc region.

[0214] In some of the (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 structure of the first and second Fab molecules, the Fab light chain of the first Fab molecule can be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule can be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. The fusion of the Fab light chains of the first and second Fab molecules further reduces mispairing of mismatched Fab heavy and light chains and also reduces the number of plasmids required for the expression of some of the (multispecific) antibodies of the invention.

[0215] The antigen-binding domains may be fused to the Fc domain or to each other, either directly or via a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, (G 4 S) n , (S.G. 4 ) n , (G 4 S) n , G 4 (SG4 ) n Or (G 4 S) n G 5 Peptide linkers are included. "n" is usually an integer from 1 to 10, typically from 2 to 4. In one embodiment, the peptide linker has a length of at least 5 amino acids, in one embodiment from 5 to 100 amino acids, and in a further embodiment from 10 to 50 amino acids. In one embodiment, the peptide linker has a length of (GxS) n or (GxS) n G m wherein G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, m=0, 1, 2 or 3) or (x=4, n=1, 2, 3, 4 or 5, m=0, 1, 2, 3, 4 or 5), in one embodiment x=4 and n=2 or 3, and in a further embodiment x=4, n=1 and m=5. In one embodiment the peptide linker is (G 4 S) 2 In another embodiment, the peptide linker is G 4 SG 5 A particularly suitable peptide linker for fusing the Fab light chains of the first and second Fab molecules to one another is (G 4 S) 2 An exemplary peptide linker suitable for linking the Fab heavy chains of the first and second Fab fragments has the sequence (D)-(G 4 S) 2 (SEQ ID NOs: 66 and 67). Another suitable linker includes the sequence (D)-G 4 SG 5 (SEQ ID NOs: 68 and 69). In a particular embodiment, the linker comprises the sequence of SEQ ID NO: 67. Additionally, the linker may comprise (part of) an immunoglobulin hinge region. In particular, when a Fab molecule is fused to the N-terminus of an Fc domain subunit, the fusion may be via an immunoglobulin hinge region or part thereof, with or without an additional peptide linker.

[0216] In a particular embodiment, the (multispecific) antibody of the invention comprises a Fab light chain variable region of a first Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of a first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn is a polypeptide that shares a carboxy-terminal peptide bond with an Fc domain subunit (VL (1) -CH1 (1) -CH2-CH3(-CH4)) and a polypeptide in which the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2) In some embodiments, the (multispecific) antibody comprises a polypeptide in which 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 the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) In certain embodiments, the polypeptides are covalently linked, for example by a disulfide bond.

[0217] In a particular embodiment, the (multispecific) antibody of the invention comprises a Fab heavy chain variable region of a first Fab molecule that shares a carboxy-terminal peptide bond with a Fab light chain constant region of a first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn is a polypeptide that shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (1) -CL (1) -CH2-CH3(-CH4)) and a polypeptide in which the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2)In some embodiments, the (multispecific) antibody comprises a polypeptide in which 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 the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) In certain embodiments, the polypeptides are covalently linked, for example by a disulfide bond.

[0218] In some embodiments, a (multispecific) antibody comprises a polypeptide in which the Fab light chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of a first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VL (1) -CH1 (1) -VH (2) -CH1 (2) In other embodiments, the (multispecific) antibody comprises a polypeptide in which 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 carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced with a light chain variable region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2) -VL (1) -CH1 (1) In some of these embodiments, the (multispecific) antibody comprises a crossover Fab light chain polypeptide (VH) of a first Fab molecule in which 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.(1) -CL (1) ) and the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) In other of these embodiments, the (multispecific) antibody optionally further comprises a polypeptide (VH) in which the Fab heavy chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab light chain constant region of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain polypeptide of a second Fab molecule. (1) -CL (1) -VL (2) -CL (2) ), or a polypeptide in which the Fab light chain polypeptide of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VL (2) -CL (2) -VH (1) -CL (1) The (multispecific) antibody according to these embodiments further comprises (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide of the third Fab molecule (VL (3) -CL (3) In certain embodiments, the polypeptides are covalently linked, for example by a disulfide bond.

[0219] In some embodiments, a (multispecific) antibody comprises a polypeptide in which the Fab heavy chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced with 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-terminal peptide bond with an Fc domain subunit (VH (1) -CL (1) -VH (2) -CH1 (2) In other embodiments, the (multispecific) antibody comprises a polypeptide in which the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2) -VH (1) -CL (1) In some of these embodiments, the (multispecific) antibody comprises a crossover Fab light chain polypeptide (VL) of a first Fab molecule in which 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. (1) -CH1 (1) ) and the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) In other of these embodiments, the (multispecific) antibody optionally further comprises a polypeptide (VL) in which the Fab light chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain polypeptide of a second Fab molecule. (1) -CH1 (1) -VL (2) -CL (2)), or a polypeptide in which the Fab light chain polypeptide of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VL (2) -CL (2) -VH (1) -CL (1) The (multispecific) antibody according to these embodiments further comprises (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and the Fab light chain polypeptide of the third Fab molecule (VL (3) -CL (3) In certain embodiments, the polypeptides are covalently linked, for example by a disulfide bond.

[0220] In certain embodiments, the (multispecific) antibody does not comprise an Fc domain. In preferred such embodiments, said second and, if present, third antigen-binding domains, respectively, are conventional Fab molecules and the first antigen-binding domain is a crossover Fab molecule as described herein, i.e. a Fab molecule in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains have been exchanged / replaced with each other. In other such embodiments, said second and, if present, third antigen-binding domains, respectively, are crossover Fab molecules and the first antigen-binding domain is a conventional Fab molecule.

[0221] In such an embodiment, the (multispecific) antibody consists essentially of a first and a second antigen-binding domain and optionally one or more peptide linkers, where the first and second antigen-binding domains are both 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 structures are shown diagrammatically in Figures 1O and 1S (in these examples, the first antigen-binding domain is a VH / VL crossover Fab molecule and the second antigen-binding domain is a conventional Fab molecule).

[0222] In another such embodiment, the (multispecific) antibody consists essentially of a first and a second antigen-binding domain and optionally one or more peptide linkers, where the first and the second antigen-binding domain are both 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 a structure is shown diagrammatically in Figures 1P and 1T (in these examples the first antigen-binding domain is a VH / VL crossover Fab molecule and the second antigen-binding domain is a conventional Fab molecule).

[0223] In some embodiments, the second Fab molecule is fused at its C-terminus 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, in particular a third Fab molecule, said third Fab molecule being fused at its C-terminus to the N-terminus of the Fab heavy chain of the second Fab molecule. In certain such embodiments, the (multispecific) antibody essentially consists of a first Fab molecule, a second Fab molecule, and a third Fab molecule, and optionally one or more peptide linkers, and the second Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the first Fab molecule, and the third Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the second Fab molecule. Such structures are shown diagrammatically in Figures 1Q and 1U (in these examples, the first antigen-binding domain is a VH / VL crossover Fab molecule and the second and third antigen-binding domains are each conventional Fab molecules) or in Figures 1X and 1Z (in these examples, the first antigen-binding domain is a conventional Fab molecule and the second and third antigen-binding domains are each VH / VL crossover Fab molecules).

[0224] In some embodiments, the first Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the second Fab molecule, and the (multispecific) antibody further comprises a third antigen-binding domain, in particular a third Fab molecule, said third Fab molecule being fused at its N-terminus to the C-terminus of the Fab heavy chain of the second Fab molecule. In certain such embodiments, the (multispecific) antibody essentially consists of a first Fab molecule, a second Fab molecule and a third Fab molecule and optionally one or more peptide linkers, and the first Fab molecule is fused at its C-terminus to the N-terminus of the Fab heavy chain of the second Fab molecule, and the third Fab molecule is fused at its N-terminus to the C-terminus of the Fab heavy chain of the second Fab molecule. Such structures are shown diagrammatically in Figures 1R and 1V (in these examples, the first antigen-binding domain is a VH / VL crossover Fab molecule and the second and third antigen-binding domains are each conventional Fab molecules) or in Figures 1W and 1Y (in these examples, the first antigen-binding domain is a conventional Fab molecule and the second and third antigen-binding domains are each VH / VL crossover Fab molecules).

[0225] In a particular embodiment, the (multispecific) antibody according to the invention comprises a polypeptide (VH) in which 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 carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e. the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region has been replaced by a light chain variable region). (2) -CH1 (2) -VL (1) -CH1 (1) In some embodiments, the (multispecific) antibody comprises a polypeptide in which 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 the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) )

[0226] In a particular embodiment, the (multispecific) antibody according to the invention comprises a polypeptide (VL) in which the Fab light chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of a first Fab molecule (i.e. the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule. (1) -CH1 (1) -VH (2) -CH1 (2) In some embodiments, the (multispecific) antibody comprises a polypeptide in which 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 the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) )

[0227] In a particular embodiment, the (multispecific) antibody according to the invention comprises a polypeptide (VH) in which the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e. the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region has been replaced by a light chain constant region). (2) -CH1 (2) -VH (1) -CL (1) In some embodiments, the (multispecific) antibody comprises a polypeptide in which 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 the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) )

[0228] In a particular embodiment, the (multispecific) antibody according to the invention comprises a polypeptide (VH) in which the Fab heavy chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab light chain constant region of a first Fab molecule (i.e. the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule. (1) -CL (1) -VH (2) -CH1 (2) In some embodiments, the (multispecific) antibody comprises a polypeptide in which 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 the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) )

[0229] In a particular embodiment, the (multispecific) antibody according to the invention comprises a polypeptide (VH) in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e. the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region has been replaced with a light chain variable region). (3) -CH1 (3) -VH (2) -CH1 (2) -VL (1) -CH1 (1) In some embodiments, the (multispecific) antibody comprises a polypeptide in which 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 the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2)In some embodiments, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL (3) -CL (3) ).

[0230] In a particular embodiment, the (multispecific) antibody according to the invention comprises a polypeptide (VH) in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a first Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e. the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region has been replaced by a light chain constant region). (3) -CH1 (3) -VH (2) -CH1 (2) -VH (1) -CL (1) In some embodiments, the (multispecific) antibody comprises a polypeptide in which 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 the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) In some embodiments, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL (3) -CL (3) ).

[0231] In a particular embodiment, the (multispecific) antibody according to the invention comprises a polypeptide (VL) in which the Fab light chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a first Fab molecule (i.e. the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region is replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule. (1) -CH1(1) -VH (2) -CH1 (2) -VH (3) -CH1 (3) In some embodiments, the (multispecific) antibody comprises a polypeptide in which 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 the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) In some embodiments, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ).

[0232] In a particular embodiment, the (multispecific) antibody according to the invention comprises a polypeptide (VH) in which the Fab heavy chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a first Fab molecule (i.e. the first Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule. (1) -CL (1) -VH (2) -CH1 (2) -VH (3) -CH1 (3) In some embodiments, the (multispecific) antibody comprises a polypeptide in which 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 the Fab light chain polypeptide of the second Fab molecule (VL (2) -CL (2) In some embodiments, the (multispecific) antibody further comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ).

[0233] In a particular embodiment, the (multispecific) antibody according to the invention comprises a polypeptide (VH) in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab light chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of a second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region has been replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with a Fab light chain variable region of a third Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain constant region of a third Fab molecule (i.e. the third Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region has been replaced by a light chain variable region). (1) -CH1 (1) -VL (2) -CH1 (2) -VL (3) -CH1 (3) In some embodiments, the (multispecific) antibody comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ) and the Fab light chain polypeptide (VL (1) -CL (1) In some embodiments, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (VH (3) -CL (3) ).

[0234] In a particular embodiment, the (multispecific) antibody according to the invention comprises a polypeptide (VH) in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with a Fab heavy chain variable region of a second Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain constant region of a second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region has been replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with a Fab heavy chain variable region of a third Fab molecule, which in turn shares a carboxy-terminal peptide bond with a Fab light chain constant region of a third Fab molecule (i.e. the third Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region has been replaced by a light chain constant region). (1) -CH1 (1) -VH (2) -CL (2) -VH (3) -CL (3) In some embodiments, the (multispecific) antibody comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) ) and the Fab light chain polypeptide (VL (1) -CL (1) In some embodiments, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (VL (3) -CH1 (3) ).

[0235] In a particular embodiment, the (multispecific) antibody according to the invention comprises a polypeptide (VL) in which the Fab light chain variable region of a third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a third Fab molecule (i.e. the third Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region has been replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain variable region has been replaced by a light chain variable region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a first Fab molecule. (3) -CH1 (3) -VL (2) -CH1 (2) -VH (1) -CH1 (1) In some embodiments, the (multispecific) antibody comprises a polypeptide in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (VH (2) -CL (2) ) and the Fab light chain polypeptide (VL (1) -CL (1) In some embodiments, the (multispecific) antibody further comprises a polypeptide in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (VH (3) -CL (3) ).

[0236] In a particular embodiment, the (multispecific) antibody according to the invention comprises a polypeptide (VH) in which the Fab heavy chain variable region of a third Fab molecule 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 a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a second Fab molecule (i.e. the second Fab molecule comprises a crossover Fab heavy chain in which the heavy chain constant region is replaced by a light chain constant region), which in turn shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule. (3) -CL (3) -VH (2) -CL (2) -VH (1) -CH1 (1) In some embodiments, the (multispecific) antibody comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) ) and the Fab light chain polypeptide (VL (1) -CL (1) In some embodiments, the (multispecific) antibody further comprises a polypeptide in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (VL (3) -CH1 (3) ).

[0237] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a first antigen-binding domain that binds to CD3, the first antigen-binding domain being a Fab molecule in which the variable domains VL and VH of the Fab light chain and the constant domains CL and CH1 are replaced with each other, the first antigen-binding domain comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3 and an HCDR3 of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, an LCDR2 of SEQ ID NO: 11 and an LCDR3 of SEQ ID NO: 12; b) a second antigen-binding domain that binds to PLAP, the second antigen-binding domain being a (conventional) Fab molecule; c) an Fc domain composed of a first and a second subunit; and (i) the first antigen-binding domain of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain of b) and the second antigen-binding domain of b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c); or (ii) a (multispecific) antibody, in which the second antigen-binding domain of b) is fused at its C-terminus to the N-terminus of the Fab heavy chain of the first antigen-binding domain of a) and the first antigen-binding domain of a) is fused at its C-terminus to the N-terminus of one of the subunits of the Fc domain of c).

[0238] In a preferred embodiment, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: a) a first antigen-binding domain that binds to CD3, the first antigen-binding domain being a Fab molecule in which the variable domains VL and VH of the Fab light chain and the constant domains CL and CH1 are replaced with each other, the first antigen-binding domain comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3 and an HCDR3 of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, an LCDR2 of SEQ ID NO: 11 and an LCDR3 of SEQ ID NO: 12; b) a second and a third antigen-binding domain that binds to PLAP, the second and third antigen-binding domains being each a (conventional) Fab molecule; c) an Fc domain composed of a first and a second subunit; and (i) the first antigen-binding domain of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain of b) and the second antigen-binding domain of b) and the third antigen-binding domain of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c); or (ii) the second antigen-binding domain of 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 of a), and the first antigen-binding domain of a) and the third antigen-binding domain of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

[0239] In a preferred embodiment, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: a) a first antigen-binding domain that binds to CD3, the first antigen-binding domain being a Fab molecule in which the variable domains VL and VH of the Fab light chain and the constant domains CL and CH1 are replaced with each other, the first antigen-binding domain comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, an HCDR2 of SEQ ID NO: 3 and an HCDR3 of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, an LCDR2 of SEQ ID NO: 11 and an LCDR3 of SEQ ID NO: 12; b) a second antigen-binding domain that binds to PLAP, the second antigen-binding domain being a (conventional) Fab molecule; c) an Fc domain composed of a first and a second subunit; and (i) a first antigen-binding domain of a) and a second antigen-binding domain of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

[0240] In all the different structures of the (multispecific) antibodies according to the invention, the amino acid substitutions ("charge modifications") described herein may be either in the CH1 and CL domains of the second and (if present) 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 invention, when the amino acid substitutions described herein are made in the second (and, if present, third) antigen binding domain / Fab molecule, such amino acid substitutions are not made in the first antigen binding domain / Fab molecule. Conversely, when the amino acid substitutions described herein are made in the first antigen binding domain / Fab molecule, such amino acid substitutions are not made in the second (and, if present, third) antigen binding domain / Fab molecule. The 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 are replaced by each other.

[0241] In a preferred embodiment of the (multispecific) antibody according to the invention, in particular when the amino acid substitutions described herein are made in the second (and, if present, the third) antigen binding domain / Fab molecule, the constant domain CL of the second (and, if present, the third) Fab molecule is of the kappa isotype. In another embodiment of the (multispecific) antibody according to the invention, in particular when the amino acid substitutions described herein are made in the first antigen binding domain / Fab molecule, the constant domain CL of the first antigen binding domain / Fab molecule is of the kappa isotype. In some embodiments, the constant domain CL of the second (and, if present, the third) antigen binding domain / Fab molecule as well as the constant domain CL of the first antigen binding domain / Fab molecule are of the kappa isotype.

[0242] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: a) a first antigen-binding domain that binds to CD3, the first antigen-binding domain being a Fab molecule in which the Fab light chain and Fab heavy chain variable domains VL and VH are replaced with each other, the first antigen-binding domain comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, a HCDR2 of SEQ ID NO: 3 and a HCDR3 of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11 and a LCDR3 of SEQ ID NO: 12; b) a second antigen-binding domain that binds to PLAP, the second antigen-binding domain being a (conventional) Fab molecule; c) an Fc domain composed of a first and a second subunit; and in which in the constant domain CL of the second antigen-binding domain of b) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), most preferably by arginine (R) (numbering according to Kabat); and in the constant domain CHI of the second antigen-binding domain of b) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); (i) the first antigen-binding domain of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain of b) and the second antigen-binding domain of b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c); or (ii) a (multispecific) antibody, in which the second antigen-binding domain of b) is fused at its C-terminus to the N-terminus of the Fab heavy chain of the first antigen-binding domain of a) and the first antigen-binding domain of a) is fused at its C-terminus to the N-terminus of one of the subunits of the Fc domain of c).

[0243] In a preferred embodiment, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: a) a first antigen-binding domain that binds to CD3, the first antigen-binding domain being a Fab molecule in which the Fab light chain and Fab heavy chain variable domains VL and VH are replaced with each other, the first antigen-binding domain comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, a HCDR2 of SEQ ID NO: 3 and a HCDR3 of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11 and a LCDR3 of SEQ ID NO: 12; b) a second and a third antigen-binding domain that binds to PLAP, the second and third antigen-binding domains being each a (conventional) Fab molecule; c) an Fc domain composed of a first and a second subunit; and in which in the constant domain CL of the second antigen-binding domain of b) and of the third antigen-binding domain of b) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), most preferably by arginine (R) (numbering according to Kabat); and in the constant domain CHI of the second antigen-binding domain of b) and of the third antigen-binding domain of b) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); (i) the first antigen-binding domain of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain of b) and the second antigen-binding domain of b) and the third antigen-binding domain of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c); or (ii) the second antigen-binding domain of 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 of a), and the first antigen-binding domain of a) and the third antigen-binding domain of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

[0244] In another aspect, the present invention provides a method for producing a composition comprising: a) a first antigen-binding domain that binds to CD3, the first antigen-binding domain being a Fab molecule in which the Fab light chain and Fab heavy chain variable domains VL and VH are replaced with each other, the first antigen-binding domain comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, a HCDR2 of SEQ ID NO: 3 and a HCDR3 of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11 and a LCDR3 of SEQ ID NO: 12; b) a second antigen-binding domain that binds to PLAP, the second antigen-binding domain being a (conventional) Fab molecule; c) an Fc domain composed of a first and a second subunit; and in which in the constant domain CL of the second antigen-binding domain of b) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), most preferably by arginine (R) (numbering according to Kabat); and in the constant domain CHI of the second antigen-binding domain of b) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); The present invention provides a (multispecific) antibody, in which a) a first antigen-binding domain and b) a second antigen-binding domain are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

[0245] According to any of the above aspects, the components of the (multispecific) antibody (e.g., Fab molecules, Fc domains) can be fused directly or via various linkers, in particular peptide linkers comprising one or more amino acids, typically about 2 to 20 amino acids, as described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G 4 S) n , (S.G. 4 ) n , (G 4 S) n , G 4 (SG 4 ) n Or (G 4 S) n G 5 A peptide linker is included, where n is usually an integer from 1 to 10, typically from 2 to 4.

[0246] In a preferred embodiment, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: a) a first antigen-binding domain that binds to CD3, the first antigen-binding domain being a Fab molecule in which the Fab light chain and Fab heavy chain variable domains VL and VH are replaced with each other, the first antigen-binding domain comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, a HCDR2 of SEQ ID NO: 3 and a HCDR3 of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11 and a LCDR3 of SEQ ID NO: 12; b) a second and a third antigen-binding domain that binds to PLAP, the second and the third antigen-binding domains being each a (conventional) Fab molecule and comprising: (i) a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:28, an HCDR2 of SEQ ID NO:29, and an HCDR3 of SEQ ID NO:30; (ii) an HCDR1 of SEQ ID NO:32, an HCDR2 of SEQ ID NO:33, and an HCDR3 of SEQ ID NO:34; (iii) an HCDR1 of SEQ ID NO:36, an HCDR2 of SEQ ID NO:37, and an HCDR3 of SEQ ID NO:38; (iv) an HCDR1 of SEQ ID NO:40, an HCDR2 of SEQ ID NO:41, and an HCDR3 of SEQ ID NO:42; or (v) a VH comprising an HCDR1 of SEQ ID NO:44, an HCDR2 of SEQ ID NO:45, and an HCDR3 of SEQ ID NO:46, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO:48, an LCDR2 of SEQ ID NO:49, and an LCDR3 of SEQ ID NO:50; c) an Fc domain composed of a first and a second subunit; and in which in the constant domain CL of the second and third antigen-binding domain of b) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), most preferably by arginine (R) (numbering according to Kabat); and in the constant domain CHI of the second and third antigen-binding domain of b) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); Further provided is a (multispecific) antibody, in which the second antigen-binding domain of 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 of a), and the first antigen-binding domain of a) and the third antigen-binding domain of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

[0247] In a preferred embodiment, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: a) a first antigen-binding domain that binds to CD3, the first antigen-binding domain being a Fab molecule in which the Fab light chain and Fab heavy chain variable domains VL and VH are replaced with each other, the first antigen-binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13; b) a second and a third antigen-binding domain that binds to PLAP, each of the second and third antigen-binding domains being a (conventional) Fab molecule and comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43 or SEQ ID NO:47 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:51; c) an Fc domain composed of a first and a second subunit; and in which in the constant domain CL of the second and third antigen-binding domain of b) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), most preferably by arginine (R) (numbering according to Kabat); and in the constant domain CHI of the second and third antigen-binding domain of b) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); Further provided is a (multispecific) antibody, in which the second antigen-binding domain of 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 of a), and the first antigen-binding domain of a) and the third antigen-binding domain of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

[0248] In a further preferred embodiment, the present invention provides a method for producing a method for the treatment of a pulmonary arthritis, comprising: a) a first antigen-binding domain that binds to CD3, the first antigen-binding domain being a Fab molecule in which the Fab light chain and Fab heavy chain variable domains VL and VH are replaced with each other, the first antigen-binding domain comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, a HCDR2 of SEQ ID NO: 3 and a HCDR3 of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11 and a LCDR3 of SEQ ID NO: 12; b) a second and a third antigen-binding domain that binds to PLAP, the second and the third antigen-binding domains being each a (conventional) Fab molecule and comprising: (i) a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:28, an HCDR2 of SEQ ID NO:29, and an HCDR3 of SEQ ID NO:30; (ii) an HCDR1 of SEQ ID NO:32, an HCDR2 of SEQ ID NO:33, and an HCDR3 of SEQ ID NO:34; (iii) an HCDR1 of SEQ ID NO:36, an HCDR2 of SEQ ID NO:37, and an HCDR3 of SEQ ID NO:38; (iv) an HCDR1 of SEQ ID NO:40, an HCDR2 of SEQ ID NO:41, and an HCDR3 of SEQ ID NO:42; or (v) a VH comprising an HCDR1 of SEQ ID NO:44, an HCDR2 of SEQ ID NO:45, and an HCDR3 of SEQ ID NO:46, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO:48, an LCDR2 of SEQ ID NO:49, and an LCDR3 of SEQ ID NO:50; c) an Fc domain composed of a first and a second subunit; and in which in the constant domain CL of the second and third antigen-binding domain of b) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), most preferably by arginine (R) (numbering according to Kabat); and in the constant domain CHI of the second and third antigen-binding domain of b) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); Further provided is a (multispecific) antibody, in which the second antigen-binding domain of 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 of a), and the first antigen-binding domain of a) and the third antigen-binding domain of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

[0249] In a further preferred embodiment, the present invention provides a method for producing a method for the treatment of a pulmonary arthritis, comprising: a) a first antigen-binding domain that binds to CD3, the first antigen-binding domain being a Fab molecule in which the Fab light chain and Fab heavy chain variable domains VL and VH are replaced with each other, the first antigen-binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13; b) a second and a third antigen-binding domain that binds to PLAP, each of the second and third antigen-binding domains being a (conventional) Fab molecule and comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43 or SEQ ID NO:47 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:51; c) an Fc domain composed of a first and a second subunit; and in which in the constant domain CL of the second and third antigen-binding domain of b) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), most preferably by arginine (R) (numbering according to Kabat); and in the constant domain CHI of the second and third antigen-binding domain of b) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); Further provided is a (multispecific) antibody, in which the second antigen-binding domain of 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 of a), and the first antigen-binding domain of a) and the third antigen-binding domain of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

[0250] In one embodiment according to these aspects of the invention, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), optionally the threonine residue at position 366 is replaced by a serine residue (T366S) and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbering according to the Kabat EU index).

[0251] In a further embodiment according to these aspects of the invention, in the first subunit of the Fc domain, additionally the serine residue at position 354 is replaced by a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C) (in particular the serine residue at position 354 is replaced by a cysteine ​​residue) and in the second subunit of the Fc domain, additionally the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index).

[0252] In a further embodiment according to these aspects of the invention, in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A) and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to the Kabat EU index).

[0253] In a further embodiment according to these aspects of the invention, the Fc domain is human IgG 1 The Fc domain.

[0254] In a specific embodiment, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 52, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 53, and a polypeptide (in particular two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 62. In a further specific embodiment, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence of SEQ ID NO: 52, a polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and a polypeptide (in particular two polypeptides) comprising the amino acid sequence of SEQ ID NO: 62.

[0255] In one aspect, the invention provides (multispecific) antibodies that bind to CD3 and PLAP, comprising a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 52, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 53, and a polypeptide (particularly two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 62. In one aspect, the invention provides (multispecific) antibodies that bind to CD3 and PLAP, comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence of SEQ ID NO: 52, a polypeptide comprising an amino acid sequence of SEQ ID NO: 53, and a polypeptide (particularly two polypeptides) comprising the amino acid sequence of SEQ ID NO: 62.

[0256] In another particular embodiment, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 54, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 55, and a polypeptide (in particular two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 62. In a further particular embodiment, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence of SEQ ID NO: 54, a polypeptide comprising an amino acid sequence of SEQ ID NO: 55, and a polypeptide (in particular two polypeptides) comprising the amino acid sequence of SEQ ID NO: 62.

[0257] In one aspect, the invention provides (multispecific) antibodies that bind to CD3 and PLAP, comprising a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 54, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 55, and a polypeptide (particularly two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 62. In one aspect, the invention provides (multispecific) antibodies that bind to CD3 and PLAP, comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence of SEQ ID NO: 54, a polypeptide comprising an amino acid sequence of SEQ ID NO: 55, and a polypeptide (particularly two polypeptides) comprising the amino acid sequence of SEQ ID NO: 62.

[0258] In a particularly specific embodiment, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 56, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 57, and a polypeptide (in particular two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 62. In a further specific embodiment, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence of SEQ ID NO: 56, a polypeptide comprising an amino acid sequence of SEQ ID NO: 57, and a polypeptide (in particular two polypeptides) comprising the amino acid sequence of SEQ ID NO: 62.

[0259] In one aspect, the invention provides (multispecific) antibodies that bind to CD3 and PLAP, comprising a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 56, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 57, and a polypeptide (particularly two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 62. In one aspect, the invention provides (multispecific) antibodies that bind to CD3 and PLAP, comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence of SEQ ID NO: 56, a polypeptide comprising an amino acid sequence of SEQ ID NO: 57, and a polypeptide (particularly two polypeptides) comprising the amino acid sequence of SEQ ID NO: 62.

[0260] In another particular embodiment, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 58, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 59, and a polypeptide (in particular two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 62. In a further particular embodiment, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence of SEQ ID NO: 58, a polypeptide comprising an amino acid sequence of SEQ ID NO: 59, and a polypeptide (in particular two polypeptides) comprising the amino acid sequence of SEQ ID NO: 62.

[0261] In one aspect, the invention provides (multispecific) antibodies that bind to CD3 and PLAP, comprising a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 58, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 59, and a polypeptide (particularly two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 62. In one aspect, the invention provides (multispecific) antibodies that bind to CD3 and PLAP, comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence of SEQ ID NO: 58, a polypeptide comprising an amino acid sequence of SEQ ID NO: 59, and a polypeptide (particularly two polypeptides) comprising the amino acid sequence of SEQ ID NO: 62.

[0262] In another particular embodiment, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 60, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 61, and a polypeptide (in particular two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 62. In a further particular embodiment, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence of SEQ ID NO: 60, a polypeptide comprising an amino acid sequence of SEQ ID NO: 61, and a polypeptide (in particular two polypeptides) comprising the amino acid sequence of SEQ ID NO: 62.

[0263] In one aspect, the invention provides (multispecific) antibodies that bind to CD3 and PLAP, comprising a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 60, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 61, and a polypeptide (particularly two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 62. In one aspect, the invention provides (multispecific) antibodies that bind to CD3 and PLAP, comprising a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, a polypeptide comprising an amino acid sequence of SEQ ID NO: 60, a polypeptide comprising an amino acid sequence of SEQ ID NO: 61, and a polypeptide (particularly two polypeptides) comprising the amino acid sequence of SEQ ID NO: 62.

[0264] 8. Fc domain variants In a preferred embodiment, the (multispecific) antibody of the invention comprises an Fc domain composed of a first and a second subunit.

[0265] The Fc domain of a (multispecific) antibody consists of a pair of polypeptide chains comprising the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment, the (multispecific) antibody of the invention comprises not more than one Fc domain.

[0266] In one embodiment, the Fc domain of the (multispecific) antibody is an IgG Fc domain. In a preferred embodiment, the Fc domain is an IgG 1 In another embodiment, the Fc domain is an IgG 4 In a more specific embodiment, the Fc domain is an IgG1 Fc domain that comprises an amino acid substitution at position S228 (Kabat EU index numbering), in particular the amino acid substitution S228P. 4 Fc domain. This amino acid substitution is 4 It reduces Fab arm exchange of antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further preferred embodiment, the Fc domain is a human Fc domain. In a more preferred embodiment, the Fc domain is a human IgG 1 Fc domain. Human IgG 1 An exemplary sequence of the Fc region is shown in SEQ ID NO:65.

[0267] a) Fc domain modifications that promote heterodimerization The (multispecific) antibody according to the invention comprises different antigen-binding domains that can be fused to one or the other of the two subunits of the Fc domain, which are thus typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization results in several possible combinations of the two polypeptides. Thus, to improve the yield and purity of the (multispecific) antibody in recombinant production, it would be advantageous to introduce modifications in the Fc domain of the (multispecific) antibody that promote the association of the desired polypeptides.

[0268] Thus, in a preferred embodiment, the Fc domain of the (multispecific) antibody according to the invention comprises a modification that promotes the association of the first and second subunits of the Fc domain. The site of greatest protein-protein interaction between the two subunits of a human IgG Fc domain is the CH3 domain of the Fc domain. Thus, in one embodiment, said modification is within the CH3 domain of the Fc domain.

[0269] Several approaches exist for modifying the CH3 domain of an Fc domain to force heterodimerization and are fully described, for example, in WO 96 / 27011, WO 98 / 050431, EP1870459, 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 such approaches, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both designed to be complementary, so that each CH3 domain (or the heavy chain that constitutes it) cannot homodimerize with itself, but is forced to heterodimerize with another CH3 domain that is complementarily engineered (so that the first and second CH3 domains heterodimerize and no homodimers are formed between the two first or two second CH3 domains). These different approaches to improve heavy chain heterodimerization are intended as different options to reduce heavy / light chain mispairing and Bence-Jones type by-products in combination with heavy-light chain modifications in (multispecific) antibodies (e.g., swapping / substitution of VH and VL in one binding arm or introduction of substitutions of oppositely charged amino acids at the CH1 / CL interface).

[0270] In a specific embodiment, the modification that promotes association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which comprises a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain.

[0271] Knob-into-hole technology is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001). In general, the method involves introducing a protrusion ("knob") into the interface of a first polypeptide and a corresponding cavity ("hole") into the interface of a second polypeptide, such that the protrusion is positioned within the cavity, thereby promoting heterodimer formation and preventing homodimer formation. The protrusion is constructed by replacing a small amino acid side chain at the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of identical or similar size to the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a small amino acid side chain (e.g., alanine or threonine).

[0272] Thus, in a preferred embodiment, in the CH3 domain of a first subunit of an Fc domain of a (multispecific) antibody, amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be accommodated in a cavity in the CH3 domain of the second subunit, and in the CH3 domain of a second subunit of the Fc domain, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit that can accommodate the protrusion in the CH3 domain of the first subunit.

[0273] Preferably, said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0274] Preferably, said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0275] The protrusions and cavities can be created by modifying the nucleic acid encoding the polypeptide, for example by site-directed mutagenesis or by peptide synthesis.

[0276] In a particular embodiment, in the CH3 domain of the first subunit of the Fc domain (the "knob" subunit), the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain (the "hole" subunit), the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0277] In yet a further embodiment, the first subunit of the Fc domain furthermore replaces the serine residue at position 354 with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine ​​residue), and the second subunit of the Fc domain furthermore replaces the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). The introduction of these two cysteine ​​residues creates a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0278] In a preferred embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the Kabat EU index).

[0279] In a preferred embodiment, the antigen binding domain that binds CD3 is fused (optionally via a second antigen binding domain that binds PLAP and / or a peptide linker) to a first subunit of an Fc domain (comprising the "knob" modification). Without wishing to be bound by theory, fusing the antigen binding domain that binds CD3 to a knob-containing subunit of an Fc domain (further) minimizes the generation of antibodies comprising two antigen binding domains that bind CD3 (steric clash of the two knob-containing polypeptides).

[0280] Other techniques of CH3 modification to effect heterodimerization are contemplated as alternatives according to the 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.

[0281] In one embodiment, the heterodimerization approach described in EP1870459 is used instead. This approach is based on introducing charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. A particular embodiment of the (multispecific) antibody of the invention is the amino acid mutation R409D;K370E in one of the two CH3 domains (of the Fc domain) and the amino acid mutation D399K;E357K in the other CH3 domain of the Fc domain (numbering according to the Kabat EU index).

[0282] In another embodiment the (multispecific) antibody of the invention comprises the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally the amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K, E357K (numbering according to Kabat EU index) in the CH3 domain of the second subunit of the Fc domain.

[0283] In another embodiment the (multispecific) antibody of the invention comprises the amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or said (multispecific) antibody comprises the amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain and additionally the amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K, E357K in the CH3 domain of the second subunit of the Fc domain (all numbered according to Kabat EU index).

[0284] In one embodiment, the heterodimerization approach described in WO 2013 / 157953 is used instead. In one embodiment, 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 a further embodiment, the first CH3 domain comprises the further amino acid mutation L351K. In a further embodiment, 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).

[0285] In one embodiment, the heterodimerization approach described in WO2012 / 058768 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain comprises further amino acid mutations at positions T411, D399, S400, F405, N390 or K392, such as a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y or In a further aspect, the first CH3 domain comprises the amino acid mutations L351Y, Y407A and the second CH3 domain comprises the amino acid mutations T366V, K409F. In a further aspect, the first CH3 domain comprises the amino acid mutations Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to the Kabat EU index).

[0286] In one aspect, the heterodimerization approach described in WO 2011 / 143545 is alternatively used, e.g., with an amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to the Kabat EU index).

[0287] In one embodiment, the heterodimerization approach described in WO 2011 / 090762, which also uses the knobs-into-holes technique described above, is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutation T366W and the second CH3 domain comprises the amino acid mutation Y407A. In one embodiment, the first CH3 domain comprises the amino acid mutation T366Y and the second CH3 domain comprises the amino acid mutation Y407T (numbering according to the Kabat EU index).

[0288] In one embodiment the (multispecific) antibody or Fc domain thereof is 2 Of the subclass, the heterodimerization approach described in WO 2010 / 129304 is alternatively used.

[0289] In an alternative embodiment, the modification that promotes the association of the first and second subunits of the Fc domain comprises a modification that mediates an electrostatic steering effect, for example as described in WO 2009 / 089004. Typically, this method involves the replacement of one or more amino acid residues at the interface of the two Fc domain subunits with a charged amino acid residue, such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. In one such embodiment, the first CH3 domain comprises an amino acid substitution of K392 or N392 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), particularly K392D or N392D) and the second CH3 domain comprises an amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g., lysine (K) or arginine (R), particularly D399K, E356K, D356K, or E357K, more particularly D399K and E356K). In a further embodiment, the first CH3 domain further comprises an amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), particularly K409D or R409D). In a further aspect, the first CH3 domain additionally or alternatively comprises an amino acid substitution at K439 and / or K370 with a negatively charged amino acid (e.g., glutamic acid (E), or aspartic acid (D)) (all numbering according to Kabat EU index).

[0290] In a further embodiment, the heterodimerization approach described in WO 2007 / 147901 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutations K253E, D282K, and K322D, and the second CH3 domain comprises the amino acid mutations D239K, E240K, and K292D (numbering according to the Kabat EU index).

[0291] In yet another embodiment, the heterodimerization approach described in WO 2007 / 110205 can alternatively be used.

[0292] In one embodiment, the first subunit of the Fc domain comprises the amino acid substitutions K392D and K409D and the second subunit of the Fc domain comprises the amino acid substitutions D356K and D399K (numbering according to the Kabat EU index).

[0293] b) Fc domain modifications that reduce Fc receptor binding and / or effector function The Fc domain confers favorable pharmacokinetic properties to the (multispecific) antibody, including good accumulation in target tissues and a long serum half-life that contributes to favorable tissue-blood distribution ratios. However, it may at the same time result in undesirable targeting of the (multispecific) antibody to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. Furthermore, coactivation of Fc receptor signaling pathways may result in cytokine release, which, in combination with the T cell activating properties and long half-life of the (multispecific) antibody, may result in excessive activation of cytokine receptors and severe side effects upon systemic administration. Activation of (Fc receptor-bearing) immune cells other than T cells may even reduce the efficacy of the (multispecific) antibody, for example due to the possible destruction of T cells by NK cells.

[0294] Thus, in a preferred embodiment, the Fc domain of the (multispecific) antibody according to the invention is a natural IgG 1 In one such embodiment, the Fc domain (or a (multispecific) antibody comprising said Fc domain) exhibits reduced binding affinity to an Fc receptor and / or reduced effector function compared to a naturally occurring IgG. 1 Fc domain (or natural IgG 1 50%, in particular less than 20%, more particularly less than 10%, and most particularly less than 5% of the binding affinity to Fc receptors and / or to native IgG (as compared to (multispecific) antibodies comprising an Fc domain). 1 Fc domain (or natural IgG 1In one embodiment, the Fc domain (or the (multispecific) antibody comprising said Fc domain) does not substantially bind to an Fc receptor and / or does not induce effector function. In a preferred embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group of CDC, ADCC, ADCP and cytokine secretion. In a preferred embodiment, the effector function is ADCC. In one embodiment, the Fc domain is a natural IgG 1 The Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to native IgG. Substantially similar binding to FcRn indicates that the Fc domain (or a (multispecific) antibody comprising said Fc domain) exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to native IgG. 1 Fc domain (or natural IgG 1 This is achieved when the antibody exhibits a binding affinity for FcRn of more than about 70%, particularly more than about 80%, and even more particularly more than about 90%, of the antibodies (including an Fc domain) that are capable of binding to FcRn.

[0295] In certain embodiments, the Fc domain is engineered to reduce the binding affinity to the Fc receptor and / or the effector function compared to a non-engineered Fc domain. In a preferred embodiment, the Fc domain of the (multispecific) antibody comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to the Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, a combination of these amino acid mutations may reduce the binding affinity of the Fc domain to the Fc receptor by at least 10-fold, at least 20-fold or even at least 50-fold. In one embodiment, the (multispecific) antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor compared to a (multispecific) antibody comprising a non-engineered Fc domain. In a preferred embodiment, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In a particular embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI or FcγRIIa, most particularly human FcγRIIIa. Preferably, the binding to each of these receptors is reduced. In some embodiments, the binding affinity to complement components (particularly the binding affinity to C1q) is also reduced. In one embodiment, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or a (multispecific) antibody comprising said Fc domain) exhibits more than about 70% of the binding affinity to FcRn of a non-engineered Fc domain (or a (multispecific) antibody comprising said non-engineered Fc domain).The Fc domain or (multispecific) antibodies of the invention comprising said Fc domain may exhibit more than about 80% or even more than about 90% of such affinity. In a particular embodiment, the Fc domain of the (multispecific) antibody is engineered to have reduced effector function compared to a non-engineered Fc domain. Reduced effector function may include, but is not limited to, one or more of: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced cross-linking with target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a preferred embodiment, the reduced effector function is reduced ADCC. In one embodiment, the reduction in ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a (multispecific) antibody comprising a non-engineered Fc domain).

[0296] In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or the effector function is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numbering according to Kabat EU index). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numbering according to Kabat EU index). In some embodiments, the Fc domain comprises the amino acid substitutions L234A and L235A (numbering according to Kabat EU index). In such embodiments, the Fc domain comprises an IgG 1 Fc domain, especially human IgG 1It is an Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, in particular P329G (numbering according to Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297 and P331 (numbering according to Kabat EU index). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a preferred embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numbering according to Kabat EU index). In a more preferred embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG"). Specifically, in a preferred embodiment, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e., the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A) and the proline residue at position 329 is replaced by a glycine residue (P329G) in each of the first and second subunits of the Fc domain (Kabat EU index numbering).

[0297] In such embodiments, the Fc domain is 1 Fc domain, especially human IgG 1 The "P329G LALA" combination of amino acid substitutions is an Fc domain of human IgG1 as described in WO 2012 / 130831, which is incorporated by reference in its entirety. 1 The Fc domain almost completely abolishes Fcγ receptor (as well as complement) binding. WO 2012 / 130831 also describes methods for preparing such mutant Fc domains and determining their properties (such as Fc receptor binding or effector function).

[0298] IgG 4 The antibody is IgG 1 In some embodiments, the Fc domain of the (multispecific) antibody of the invention exhibits reduced binding affinity to Fc receptors and reduced effector function compared to IgG 4 Fc domain, especially human IgG 4 In one embodiment, the IgG 4 The Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P (numbering according to the Kabat EU index). In order to further reduce its binding affinity to the Fc receptor and / or its effector function, in one embodiment, the IgG 4 The Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numbering according to the Kabat EU index). 4 The Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numbering according to the Kabat EU index). 4 The Fc domain contains amino acid substitutions at positions S228, L235 and P329, specifically the amino acid substitutions S228P, L235E and P329G (numbering according to the Kabat EU index). 4 Fc domain variants and their Fcγ receptor binding properties are described in WO 2012 / 130831, which is incorporated by reference in its entirety.

[0299] In a preferred embodiment, native IgG 1 The Fc domain, which exhibits reduced binding affinity to an Fc receptor and / or reduced effector function compared to the Fc domain, is a human IgG1 domain comprising the amino acid substitutions L234A, L235A, and optionally P329G. 1 A human IgG comprising an Fc domain or the amino acid substitutions S228P, L235E, and optionally P329G (numbering according to the Kabat EU index). 4 The Fc domain.

[0300] In certain embodiments, N-glycosylation of the Fc domain is eliminated, hi one such embodiment, the Fc domain comprises an amino acid mutation at position N297, specifically an amino acid substitution replacing asparagine with alanine (N297A) or aspartic acid (N297D) (numbering according to the Kabat EU index).

[0301] In addition to the Fc domains described herein above and in WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also include those with one or more substitutions of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (US Pat. No. 6,737,056) (Kabat EU index numbering). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variants with substitutions of residues 265 and 297 to alanine (US Pat. No. 7,332,581).

[0302] Variant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The exact nucleotide changes can be confirmed, for example, by sequencing.

[0303] Binding to an Fc receptor can be easily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment, such as a BIAcore instrument (GE Healthcare) and the Fc receptor, which can be obtained, for example, by recombinant expression. Alternatively, the binding affinity of an Fc domain or an Fc domain-containing (multispecific) antibody to an Fc receptor can be assessed using a cell line known to express a particular Fc receptor, such as human NK cells expressing the FcγIIIa receptor.

[0304] The effector function of Fc domain or (multispecific) antibody containing Fc domain can be measured by methods known in the art. Examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays may be used (e.g., the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0305] In some embodiments, the binding of the Fc domain to complement components, specifically to C1q, is reduced. Thus, in some embodiments where the Fc domain is designed to have reduced effector function, said reduced effector function includes reduced CDC. A C1q binding assay can be performed to determine whether an Fc domain or a (multispecific) antibody comprising an Fc domain is capable of binding to C1q and thus has CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, et al., Blood 101:1045-1052 (2003); and Cragg, and Glennie, Blood 103:2738-2743 (2004)).

[0306] Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929).

[0307] B. Polynucleotides The present invention further provides an isolated polynucleotide encoding an antibody of the present invention. The isolated polynucleotide may be a single polynucleotide or multiple polynucleotides.

[0308] The polynucleotides encoding the (multispecific) antibodies of the invention may be expressed as a single polynucleotide encoding the entire antibody or as multiple (e.g., two or more) polynucleotides that are coexpressed. The polypeptides encoded by the coexpressed polynucleotides can associate, for example, by disulfide bonds or other means, to form a functional antibody. For example, the light chain portion of an antibody may be encoded by a separate polynucleotide from the portion of the antibody that comprises the heavy chain of the antibody. When coexpressed, the heavy chain polypeptides associate with the light chain polypeptides to form the antibody. In another example, the portion of an antibody that comprises one of the two Fc domain subunits and optionally (a portion of) one or more Fab molecules can be encoded by a separate polynucleotide from the portion of the antibody that comprises the other of the two Fc domain subunits and optionally (a portion of) a Fab molecule. When coexpressed, the Fc domain subunits associate to form the Fc domain.

[0309] In some aspects, the isolated polynucleotide encodes an entire antibody molecule according to the invention as described herein, while in other aspects, the isolated polynucleotide encodes a polypeptide comprised in an antibody according to the invention as described herein.

[0310] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the invention is RNA, for example in the form of messenger RNA (mRNA). The RNA of the invention may be single-stranded or double-stranded.

[0311] C. Recombinant Methods The antibodies of the present invention can be obtained, for example, by solid state peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production. In the case of recombinant production, one or more polynucleotides encoding the antibody are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell, for example as described above. Such polynucleotides can be easily isolated and sequenced using conventional procedures. In one aspect, vectors, particularly expression vectors, are provided that contain the polynucleotides of the present invention (i.e., a single polynucleotide or multiple polynucleotides). Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the antibody, together with appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY (1989). An expression vector can be part of a plasmid, a virus, or can be a nucleic acid fragment. An expression vector contains an expression cassette into which a polynucleotide encoding an antibody (i.e., a coding region) is cloned in operative association with a promoter and / or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids. A "stop codon" (TAG, TGA, or TAA) is not translated into amino acids, but is considered to be part of the coding region when present. However, any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc., are not part of the coding region.Two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector) or in separate polynucleotide constructs (e.g., on separate (different) vectors). Furthermore, any vector may contain a single coding region or may contain two or more coding regions, e.g., a vector of the invention may encode one or more polypeptides that are post-translationally or co-translationally separated into the final protein by proteolytic cleavage. Furthermore, a vector, polynucleotide, or nucleic acid of the invention may encode a heterologous coding region, either fused or unfused to the polynucleotide encoding the antibody or variant or derivative thereof of the invention. Heterologous coding regions include, but are not limited to, specialized elements or motifs such as secretory signal peptides or heterologous functional domains. An operably associated is when the coding region of a gene product, such as a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and its associated promoter) are "operably associated" if induction of promoter function results in transcription of an mRNA encoding a desired gene product, and if the nature of the binding between the two DNA fragments does not interfere with the ability of the expression control sequences to direct expression of the gene product or does not interfere with the ability of the DNA template to transcribe. Thus, if the promoter is capable of effecting transcription of a nucleic acid encoding a polypeptide, the promoter region will be operably associated with the nucleic acid encoding the polypeptide. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in a given cell. Other transcriptional regulatory elements other than the promoter, such as enhancers, operators, repressors, and transcription termination signals, may be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional regulatory regions are disclosed herein. A variety of transcriptional regulatory regions are known to those skilled in the art.These include, but are not limited to, transcriptional regulatory regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., immediate early promoter and intron A), Simian Virus 40 (e.g., immediate early promoter), and retroviruses (e.g., Rous sarcoma virus, etc.). Other transcriptional regulatory regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, and other sequences capable of controlling gene expression in eukaryotic cells. Further suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., promoters inducible by tetracycline). Similarly, various translational control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (in particular, internal ribosome entry sites or IRES, also called CITE sequences). The expression cassette may also include other features such as an origin of replication and / or chromosomal integration elements, such as the retroviral long terminal repeat (LTR) or the adeno-associated viral (AAV) inverted terminal repeat (ITR).

[0312] The polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. For example, if secretion of an antibody is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding the antibody or fragment thereof of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once transport of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those skilled in the art are aware that polypeptides secreted by vertebrate cells usually have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to generate the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide is used, such as an immunoglobulin heavy or light chain signal peptide, or a functional derivative of that sequence that retains the ability to direct the secretion of a polypeptide with which it is operably associated. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0313] DNA encoding short protein sequences (e.g., histidine tags) that can be used to facilitate subsequent purification or to serve to label the antibody may be included within or at the end of the antibody (fragment) encoding polynucleotide.

[0314] In a further aspect, a host cell is provided that comprises a polynucleotide (i.e., a single polynucleotide or multiple polynucleotides) of the invention. In a particular aspect, a host cell is provided that comprises a vector of the invention. The polynucleotide and vector can incorporate any of the features described herein in connection with the polynucleotide and vector, respectively, either alone or in combination. In one such aspect, the host cell comprises (e.g., is transformed or transfected with) one or more vectors that comprise one or more polynucleotides encoding (a portion of) an antibody of the invention. As used herein, the term "host cell" refers to any type of cell system that can be engineered to produce an antibody of the invention or a fragment thereof. Host cells suitable for replicating and supporting the expression of antibodies are well known in the art. Such cells can be suitably transfected or transduced with a particular expression vector, and large amounts of vector-containing cells can be grown to inoculate large-scale fermenters to obtain sufficient amounts of antibody for clinical applications. Suitable host cells include prokaryotic microorganisms such as E. coli, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, polypeptides can be produced in bacteria, especially when glycosylation is not required. After expression, the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding polypeptides, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of polypeptides with partially or completely human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006). Host cells suitable for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells.Numerous baculovirus strains have been identified and may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures may also be utilized as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (e.g., 293 cells or 293T cells described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (e.g., Mather et al., Annals NY Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines are dhfr. -Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), p. 255-268 (2003). Host cells include cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacterial cells, and plant cells, to name a few, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues. In one embodiment, the host cell is a eukaryotic cell, in particular a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) cell or a lymphocytic cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, the host cell is not a cell within the human body.

[0315] Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing a polypeptide containing either the heavy or light chain of an antigen-binding domain, such as an antibody, may also be engineered to express the other antibody chain, such that the expression product is an antibody having both a heavy and a light chain.

[0316] In one aspect, a method of producing an antibody according to the invention is provided, the method comprising culturing a host cell comprising a polynucleotide encoding an antibody provided herein under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell medium).

[0317] The components of the (multispecific) antibody of the present invention can be genetically fused to each other. The (multispecific) antibody can be designed such that its components are fused to each other directly or indirectly via a linker sequence. The composition and length of the linker can be determined according to methods known in the art and tested for effectiveness. Examples of linker sequences between different components of the (multispecific) antibody are provided herein. Additional sequences, such as endopeptidase recognition sequences, can also be included as necessary to incorporate cleavage sites for separating the individual components of the fusion.

[0318] The antibodies prepared as described herein can be purified by techniques known in the art, such as, for example, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to one of skill in the art. For affinity chromatography purification, the antibody, ligand, receptor, or antigen to which the antibody binds can be used. For example, for affinity chromatography purification of the antibodies of the invention, a matrix containing protein A or protein G can be used. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to isolate the antibody, essentially as described in the Examples. The purity of the antibody can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like.

[0319] D. Assay The antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0320] 1. Binding Assay The binding (affinity) of an antibody to an Fc receptor or target antigen can be determined, for example, by surface plasmon resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare) and a receptor or target protein that can be obtained by recombinant expression. Alternatively, the binding of an antibody to different receptors or target antigens can be assessed, for example, by flow cytometry (FACS) using a cell line expressing a particular receptor or target antigen. Specific illustrative and exemplary embodiments for measuring the binding activity to CD3 are described below.

[0321] In one embodiment, the binding activity to CD3 is determined by SPR as follows: SPR is performed on a Biacore T200 instrument (GE Healthcare). Anti-Fab capture antibodies (GE Healthcare, #28958325) are immobilized on a Series S Sensor Chip CM5 (GE Healthcare) at a surface density of 4000-6000 resonance units (RU) using standard amine coupling chemistry. HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) is used as running and dilution buffer. CD3 antibody (in 20 mM His, 140 mM NaCl, pH 6.0) at a concentration of 2 μg / ml is injected at a flow rate of 5 μl / min for approximately 60 seconds. The CD3 antigen used is a heterodimer of CD3 delta and CD3 epsilon ectodomain fused to a human Fc domain with knob-into-hole modification and a C-terminal Avi tag (see SEQ ID NOs: 24 and 25). CD3 antigen is injected for 120 seconds at a concentration of 10 μg / ml and dissociation is monitored for approximately 120 seconds at a flow rate of 5 μl / min. The chip surface is regenerated by two successive injections of 10 mM glycine pH 2.1 for approximately 60 seconds each. Bulk refractive index differences are corrected by subtracting a blank injection and by subtracting the response obtained from a blank control flow cell. For evaluation, the binding response is obtained 5 seconds after the end of the injection. To normalize the binding signal, the CD3 binding is divided by the anti-Fab response (signal (RU) obtained upon capture of the CD3 antibody on the immobilized anti-Fab antibody). The binding activity of the antibody to CD3 after one treatment relative to the binding activity of the antibody after a different treatment (also called relative activity concentration (RAC)) is calculated by referencing the binding activity of an antibody sample after one treatment to the binding activity of the corresponding antibody sample after another treatment.

[0322] 2. Activity Assay The biological activity of the (multispecific) antibodies of the invention can be measured by various assays, which are described in the Examples. Biological activity may include, for example, inducing proliferation of T cells, inducing signal transduction in T cells, inducing expression of activation markers in T cells, inducing cytokine secretion by T cells, inducing lysis of target cells, such as cancer cells, and inducing tumor regression and / or improving survival.

[0323] E. Compositions, Formulations, and Routes of Administration In a further aspect, the invention provides a pharmaceutical composition comprising any of the antibodies provided herein, e.g., a pharmaceutical composition for use in any of the therapeutic methods described below. In one aspect, the pharmaceutical composition comprises an antibody according to the invention and a pharma- ceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises an antibody according to the invention and at least one additional therapeutic agent, e.g., as described below.

[0324] Further provided is a method of producing an antibody of the invention in a form suitable for in vivo administration, the method comprising: (a) obtaining an antibody according to the invention; and (b) formulating the antibody with at least one pharma- ceutically acceptable carrier, whereby the antibody preparation is formulated for in vivo administration.

[0325] The pharmaceutical composition of the present invention comprises an effective amount of the antibody dissolved or dispersed in a pharma- ceutically acceptable carrier. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce side effects, allergic reactions or other untoward reactions when administered as required to animals, e.g., humans. The preparation of pharmaceutical compositions containing antibodies and optionally additional active ingredients will be known to those of skill in the art in light of the present disclosure, as set forth in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, which is incorporated herein by reference. Furthermore, it will be understood that for administration to animals (e.g., humans), the formulation should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards or the corresponding agencies of other countries. A preferred composition is a lyophilized formulation or an aqueous solution. As used herein, "pharmaceutical acceptable carrier" includes any solvent, buffer, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal), isotonicity agent, absorption retardant, salt, preservative, antioxidant, protein, drug, drug stabilizer, polymer, gel, binder, excipient, disintegrant, lubricant, sweetener, flavoring agent, dye, and the like, and combinations thereof, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pages 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0326] The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional administration if desired for localized treatment. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

[0327] Parenteral compositions include those designed for administration by injection, e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. For injection, the antibody of the present invention may be formulated in aqueous solutions, particularly in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. The solutions may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the antibody may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the antibody of the present invention in the required amount in an appropriate solvent, with various other ingredients as listed below, as required. Sterilization may be readily accomplished, for example, by filtration through a sterile filtration membrane. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and / or other ingredients. In the case of sterile powders for preparing sterile injectable solutions, suspensions or emulsions, the preferred preparation method is vacuum drying or freeze-drying technology, which yields a powder of the active ingredient and any additional desired ingredients from a previously sterile filtered liquid medium. The liquid medium should be appropriately buffered if necessary, and the liquid diluent is first rendered isotonic with sufficient saline or glucose prior to injection. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept to a minimum safe level, e.g., less than 0.5ng / mg protein.Suitable pharma- ceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins (such as serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine); glucose, mannose, or the like. or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension (e.g., sodium carboxymethylcellulose, sorbitol, dextran, etc.). Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.

[0328] The active ingredient may also be incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), for example, by microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may also be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, for example, films or microcapsules. In certain embodiments, sustained absorption of the injectable composition can be brought about by the use in the composition of agents that delay absorption, such as aluminum monostearate, gelatin, or a combination thereof.

[0329] In addition to the above compositions, the antibody can also be formulated as a depot preparation. Such sustained release preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the antibody can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as a poorly soluble derivative, for example, as a poorly soluble salt.

[0330] Pharmaceutical compositions containing the antibody of the present invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, encapsulating or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, additives or auxiliary agents that facilitate the processing of proteins into medicaments that can be used as pharmaceuticals. The appropriate formulation depends on the selected route of administration.

[0331] Antibodies can be formulated in compositions in free acid or free base, neutral or salt form. Pharmaceutically acceptable salts are those that substantially retain the biological activity of the free acid or base. These include acid addition salts, such as those formed with free amino groups of proteinaceous compositions, or those formed with organic acids, such as hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxide; or organic bases, such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0332] F. Treatment Methods and Compositions Any of the antibodies provided herein can be used in methods of treatment. The antibodies of the invention can be used as immunotherapeutic agents, for example, in the treatment of cancer or autoimmune diseases.

[0333] For use in therapy, the antibodies of the invention will be formulated, dosed and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to medical practitioners.

[0334] In one aspect, the antibody of the invention is provided for use as a medicament. In a further aspect, the antibody of the invention is provided for use in the treatment of a disease. In a particular aspect, the antibody of the invention is provided for use in a method of therapy. In one aspect, the invention provides the antibody of the invention for use in the treatment of a disease in an individual in need of such treatment. In a particular aspect, the invention provides the antibody for use in a method of treating an individual having a disease, comprising administering to the individual an effective amount of the antibody. In a particular aspect, the disease is a proliferative disease. In a particular aspect, the disease is cancer, in particular a PLAP-expressing cancer. In a particular aspect, the cancer is ovarian cancer, lung cancer, or cancer of the digestive tract (e.g., gastric, colonic, pancreatic, or esophageal cancer). In a further particular aspect, the cancer is a cancer selected from the group consisting of ovarian cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, and esophageal cancer. In a particular aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., an anti-cancer agent if the disease being treated is cancer, or an immunosuppressant if the disease being treated is an autoimmune disease). In a further aspect, the invention provides an antibody of the invention for use in inducing lysis of target cells, particularly cancer cells. In a particular aspect, the invention provides an antibody of the invention for use in a method of inducing lysis of target cells, particularly cancer cells, in an individual comprising administering to the individual an effective amount of the antibody to induce lysis of the target cells. An "individual" according to any of the above aspects is a mammal, preferably a human.

[0335] In a further aspect, the invention provides the use of an antibody of the invention in the manufacture or preparation of a medicament. In one aspect, the medicament is for the treatment of a disease in an individual in need thereof. In a further aspect, the medicament is for use in a method of treating a disease comprising administering an effective amount of the medicament to an individual having the disease. In a particular aspect, the disease is a proliferative disease. In a particular aspect, the disease is cancer, in particular a PLAP-expressing cancer. In a particular aspect, the cancer is cancer of the ovary, cancer of the lung, or cancer of the digestive tract (e.g., gastric, colonic, pancreatic, or esophageal cancer). In a further aspect, the cancer is a cancer selected from the group consisting of ovarian cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, and esophageal cancer. In one aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., an anti-cancer agent if the disease being treated is cancer, or an immunosuppressant agent if the disease being treated is an autoimmune disease). In a further aspect, the medicament is for inducing lysis of target cells, in particular cancer cells. In a still further aspect, the medicament is for use in a method of inducing lysis of target cells, particularly cancer cells, in an individual comprising administering to the individual an effective amount of the medicament to induce lysis of the target cells. An "individual" according to any of the above aspects may be a mammal, preferably a human.

[0336] In a further aspect, the invention provides a method for treating a disease. In one aspect, the method comprises administering to an individual having such a disease an effective amount of an antibody of the invention. In one aspect, a composition comprising an antibody of the invention in a pharma- ceutically acceptable form is administered to said individual. In a particular aspect, the disease is a proliferative disease. In a particular aspect, the disease is cancer, in particular a PLAP-expressing cancer. In a particular aspect, the cancer is cancer of the ovary, cancer of the lung, or cancer of the digestive tract (e.g., gastric, colonic, pancreatic, or esophageal cancer). In a further particular aspect, the cancer is a cancer selected from the group consisting of ovarian cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, and esophageal cancer. In a particular aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., an anti-cancer agent if the disease being treated is cancer, or an immunosuppressant agent if the disease being treated is an autoimmune disease). An "individual" according to any of the above aspects may be a mammal, preferably a human.

[0337] In a further aspect, the present invention provides a method for inducing lysis of a target cell, particularly a PLAP-expressing cell. In one aspect, the method comprises contacting a target cell with an antibody of the present invention in the presence of a T cell, particularly a cytotoxic T cell. In a further aspect, a method for inducing lysis of a target cell, particularly a PLAP-expressing cell, in an individual is provided. In one such aspect, the method comprises administering to the individual an effective amount of an antibody of the present invention to induce lysis of the target cell. In one aspect, the "individual" is a human.

[0338] Those skilled in the art can easily recognize that in many cases, an antibody may not provide a cure, but may only provide a partial benefit. In some aspects, any physiological change that provides some benefit is also considered to be therapeutically beneficial. Thus, in some aspects, the amount of antibody that provides a physiological change is considered an "effective amount." The subject, patient, or individual in need of treatment is typically a mammal, and in particular a human.

[0339] In some aspects, an effective amount of an antibody of the invention is administered to an individual for the treatment of a disease.

[0340] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the route of administration, the patient's weight, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous or current therapeutic interventions, the patient's medical history and response to the antibody, and the discretion of the attending physician. The physician responsible for administration will, in any event, determine the concentration of active ingredient in the composition and the appropriate dose for the individual subject. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0341] The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, whether by single or multiple individual administrations or by continuous infusion, about 1 μg / kg-15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of antibody may be an initial candidate dose for administration to the patient. Depending on the factors mentioned above, a typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more. In the case of repeated administration over several days or more, treatment is usually continued until a desired suppression of disease symptoms occurs, depending on the condition. One exemplary dosage of the antibody would be in the range of about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, the dose is about 1 microgram / kg body weight, about 5 microgram / kg body weight, about 10 microgram / kg body weight, about 50 microgram / kg body weight, about 100 microgram / kg body weight, about 200 microgram / kg body weight, about 350 microgram / kg body weight, about 500 microgram / kg body weight, about 1 milligram / kg body weight, about 5 milligram / kg body weight, about 10 milligram / kg body weight, about 50 milligram / kg body weight, about 100 milligram / kg body weight, about 200 milligram / kg body weight, about 350 milligram / kg body weight, about 500 milligram / kg body weight, about 1000 mg / kg body weight or more per administration, and any range derivable therein. In non-limiting examples of ranges derived from the numbers recited herein, ranges such as about 5 mg / kg body weight to about 100 mg / kg body weight, about 5 microgram / kg body weight to about 500 milligram / kg body weight, etc. may be administered based on the above numbers. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses may be administered intermittently, for example every week or every three weeks (e.g., so that the patient receives from about 2 to about 20, or for example about 6 doses of the antibody). An initial higher loading dose, followed by one or more lower doses, can be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0342] The antibodies of the invention are typically used in an amount effective to achieve their intended purpose. For use in treating or preventing a disease state, the antibodies of the invention or pharmaceutical compositions thereof are administered or applied in an effective amount.

[0343] For systemic administration, the effective dose can be estimated initially from in vitro assays, such as cell culture assays. The IC 50 A dose can also be formulated in animal models to achieve a circulating concentration range including, but not limited to, 100 mg / kg / day. Such information can be used to more accurately determine useful doses in humans.

[0344] Initial dosages can also be estimated from in vivo data, eg, from animal models, using techniques well known in the art.

[0345] Dosage and administration intervals can be adjusted individually to obtain plasma concentrations of the antibody sufficient to maintain therapeutic efficacy. Usual patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.

[0346] An effective dose of the antibody of the invention will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of the antibody can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Using cell culture assays and animal studies, LD 50 (lethal dose for 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50The therapeutic index can be expressed as a ratio. Antibodies that exhibit large therapeutic indices are preferred. In one aspect, the antibodies according to the invention exhibit a high therapeutic index. The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages suitable for use in humans. The dosage is preferably within the ED 50 The dosage may vary within this range depending on various factors, such as the dosage form employed, the route of administration utilized, the condition of the subject, etc. The exact formulation, route of administration and dosage can be selected by the individual physician in view of the patient's condition (see, for example, Fingl et al., 1975, The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, which is incorporated herein by reference in its entirety).

[0347] The attending physician of a patient treated with an antibody of the invention will know how and when to terminate, interrupt or adjust administration due to toxicity, organ failure, and the like. Conversely, the attending physician will also know to adjust treatment to higher levels if the clinical response is not adequate (barring toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition being treated, the route of administration, and the like. The severity of the condition can, for example, be assessed in part by standard prognostic evaluation methods. Furthermore, the dose, and perhaps frequency of administration, will also vary according to the age, weight, and response of the individual patient.

[0348] The antibody of the present invention may be administered in combination with one or more other agents in a treatment. For example, the antibody of the present invention may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a condition or disease in an individual in need of such treatment. Such additional therapeutic agents may include any active ingredient suitable for the particular disease being treated, preferably active ingredients with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptosis inducers. In certain embodiments, the additional therapeutic agent is an anti-cancer agent, such as a microtubule disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent. In other embodiments, the additional therapeutic agent is an immunosuppressant. In certain embodiments, the additional therapeutic agent is one or more selected from the group of corticosteroids, hydroxychloroquine, mycophenolate mofetil, mycophenolic acid, methotrexate, azathioprine, cyclophosphamide, calcineurin inhibitors, belimumab, rituximab, and obinutuzumab.

[0349] Such other agents are suitably present in combination in amounts effective for the intended purpose. The effective amount of such other agents depends on the amount of antibody used, the type of disorder or treatment, and other factors discussed above. The antibodies are generally used in the same dosages and routes of administration as described herein, or about 1-99% of the dosages described herein, or any dosage and any route determined empirically / clinically appropriate.

[0350] Such combination therapy as described above includes combined administration (two or more therapeutic agents in the same or separate compositions) and separate administration, where administration of the antibody of the invention can occur prior to, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant. The antibody of the invention can also be used in combination with radiation therapy.

[0351] G. Manufactured products In another aspect of the invention, an article of manufacture is provided that includes materials useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds the composition by itself or in combination with another composition effective for treating, preventing, and / or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat a selected condition. Additionally, the article of manufacture may include (a) a first container in which a composition comprising the antibody of the invention is contained; and (b) a second container in which a composition comprising an additional cytotoxic or other therapeutic agent is contained. The article of manufacture in this aspect of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0352] H. Methods and Compositions for Diagnosis and Detection In certain embodiments, any of the antibodies provided herein is useful for detecting the presence of its target (e.g., CD3 or PLAP) in a biological sample. As used herein, the term "detect" includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises a cell or tissue, such as prostate tissue.

[0353] In one aspect, an antibody according to the invention is provided for use in a method of diagnosis or detection. In a further aspect, a method is provided for detecting the presence of CD3 and PLAP in a biological sample. In a particular aspect, the method comprises contacting a biological sample with an antibody of the invention under conditions that allow the antibody of the invention to bind to CD3 or PLAP, and detecting whether a complex is formed between the antibody and CD3 or PLAP. Such a method may be an in vitro method or an in vivo method. In one aspect, the antibody of the invention is used to select subjects eligible for treatment with an antibody that binds to CD3 or PLAP, e.g., CD3 or PLAP is a biomarker for patient selection.

[0354] Exemplary disorders that may be diagnosed using the antibodies of the invention include cancer, particularly PLAP-expressing cancers.

[0355] In certain aspects, an antibody according to the invention is provided, wherein the antibody is labeled. Labels include, but are not limited to, labels or moieties that are directly detected (such as fluorescent, chromophore, electron dense, chemiluminescent, radioactive labels, etc.), as well as moieties such as enzymes or ligands that are indirectly detected, for example, through enzymatic reactions or molecular interactions. Exemplary labels include radioisotopes. 32 P, 14 C. 125 I, 3 H, and 131Examples of suitable oxidases include, but are not limited to, I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase conjugated with enzymes that use hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0356] III. Sequence TIFF2025503399000001.tif254170TIFF2025503399000002.tif255169TIFF2025503399000003.t if255169TIFF2025503399000004.tif255169TIFF2025503399000005.tif250170TIFF20255033990 00006.tif255169TIFF2025503399000007.tif252170TIFF2025503399000008.tif255170TIFF202 5503399000009.tif255169TIFF2025503399000010.tif255169TIFF2025503399000011.tif194170 EXAMPLES

[0357] IV. Working Examples The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that various other embodiments may be practiced.

[0358] Example 1 – Preparation of an optimized anti-CD3 (multispecific) antibody The optimized anti-CD3 antibody "P035.093" was generated by a phage display selection campaign using a library derived from a previously described (see, e.g., WO 2014 / 131712, incorporated herein by reference) CD3 binder (also referred to herein as "CD3orig"), comprising VH and VL of SEQ ID NOs: 7 and 13, respectively. In these libraries, positions N97 and N100 (Kabat numbering) in the CDR3 region of the heavy chain were silenced or removed. As shown in FIG. 2A, P035.093 was converted to a T cell bispecific antibody (TCB) format using an anti-TYRP1 antibody as an exemplary target cell antigen binding moiety (SEQ ID NOs: 14 and 15). CD3 as a CD3 binder orig or the previously described CD3 binder ("CD 3opt ", see, for example, WO 2020 / 127619, were also prepared.

[0359] As shown in Figures 2B-E, the variable regions of the heavy and light chain DNA sequences were subcloned in frame with the constant heavy or constant light chain previously inserted into the respective recipient mammalian expression vector.

[0360] The sequences of the anti-CD3 antibodies tested are shown in the SEQ ID NOs: in Table 1.

[0361] Table 1. Optimized anti-CD3 antibody (P035.093) and comparative anti-CD3 antibody (CD3 orig , CD3 opt ) array. TIFF2025503399000012.tif19170

[0362] To further improve precise pairing of the light chain with the corresponding heavy chain, mutations were introduced into human CL (E123R, Q124K) and human CH1 (K147E, K213E) of the TYRP1-binding Fab molecule.

[0363] To ensure precise pairing of heavy chains (to form heterodimeric molecules), knob-into-hole mutations were introduced into the constant region of each antibody heavy chain (T366W / S354C, T366S / L368A / Y407V / Y349C, respectively).

[0364] Furthermore, P329G, L234A and L235A mutations were introduced into the constant region of the antibody heavy chain to abolish binding to Fcγ receptors.

[0365] The complete sequences of the prepared TCB molecules are SEQ ID NOs: 16, 19, 20 and 21 (P035.093), SEQ ID NOs: 18, 19, 20 and 21 (CD3 opt ), SEQ ID NOs: 17, 19, 20 and 21 (CD3 orig ) as shown in

[0366] TCB was prepared by Evitria (Switzerland) using a proprietary vector system and conventional (non-PCR-based) cloning techniques in suspension-adapted CHO K1 cells (originally acquired from ATCC and adapted by Evitria for serum-free culture in suspension culture). For production, Evitria used proprietary animal-component-free serum-free media (eviGrow and eviMake2) and a proprietary transfection reagent (eviFect). These cells were transfected with the corresponding expression vectors in a 1:1:2:1 ratio ("vector knob heavy chain":"vector hole heavy chain":"vector TYRP-1 light chain":"vector CD3 light chain"). The supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter).

[0367] As an alternative to the Evitria preparation, TCB molecules were prepared in-house by transient transfection of HEK293 EBNA cells. Cells were centrifuged and the medium replaced with pre-warmed CD CHO medium (Thermo Fisher, #10743029). Expression vectors were mixed in CD CHO medium, polyethylenimine (PEI; Polysciences, #23966-1) was added, the solution was vortexed and incubated at room temperature for 10 min. Cells (2 mio / mL) were then mixed with the vector / PEI solution and transferred to a flask and incubated in 5% CO. 2 The cells were incubated at 37°C for 3 hours in a shaking incubator at ambient temperature. After incubation, Excel medium containing supplements (80% of the total volume) was added (W. Zhou and A. Kantardjieff, Mammalian Cell Cultures for Biologics Manufacturing, DOI:10.1007 / 978-3-642-54050-9;2014). One day after transfection, supplements (Feed, 12% of the total volume) were added. Cell supernatants were harvested after 7 days by centrifugation and subsequent filtration (0.2 μm filter).

[0368] Proteins were purified from harvested supernatants by standard methods. Briefly, Fc-containing proteins were purified from filtered cell culture supernatants by Protein A affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0, followed immediately by neutralization of the pH of the sample. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15, #UFC903096) and aggregated proteins were separated from monomeric proteins by size-exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[0369] The concentration of purified proteins was determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423. Protein purity and molecular weight were analyzed by CE-SDS in the presence and absence of reducing agents using a LabChip GXII (Perkin Elmer). Aggregate content was measured by equilibrating analytical size exclusion columns (TSKgel G3000 SW XL or UP-SW3000) with running buffer (25 mM K, respectively). 2 HPO 4 , 125 mM NaCl, 200 mM L-arginine monohydrochloride, pH 6.7 or 200 mM KH 2 PO 4 , 250 mM KCl, pH 6.2) at 25°C by HPLC chromatography.

[0370] The results of biochemical and biophysical analysis of the prepared TCB molecules are shown in Table 2.

[0371] All the TCB molecules could be produced with good quality.

[0372] Table 2. Biochemical and biophysical characterization of anti-CD3 antibodies in TCB format. TIFF2025503399000013.tif27170

[0373] Example 2 – Determination of the thermal stability of an optimized anti-CD3 (multispecific) antibody The thermal stability of the anti-CD3 antibody (TCB format) prepared in Example 1 was monitored by dynamic light scattering (DLS) using an Optim2 instrument (Avacta Analytical, UK) and by monitoring the temperature-dependent intrinsic protein fluorescence by applying a temperature ramp.

[0374] 10 μg of filtered protein sample with a protein concentration of 1 mg / ml was run in duplicate on the Optim2. The temperature was ramped from 25° C. to 85° C. at 0.1° C. / min and the ratio of fluorescence intensity at 350 nm / 330 nm and scattering intensity at 266 nm were collected.

[0375] The results are shown in Table 3. The aggregation temperature (T agg ) and the midpoint of the observed temperature-in...

Claims

1. An antibody that binds to CD3 and PLAP, wherein the antibody comprises: (a) a first antigen-binding domain that binds to CD3, comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, a HCDR2 of SEQ ID NO: 3, and a HCDR3 of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 10, a LCDR2 of SEQ ID NO: 11, and a LCDR3 of SEQ ID NO: 12; and (b) a second antigen-binding domain that binds to PLAP, and optionally a third antigen-binding domain. An antibody that binds to CD3 and PLAP, comprising:

2. 2. The antibody of claim 1, wherein the VH of the first antigen-binding domain comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 9, and / or the VL of the first antigen-binding domain comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

13.

3. An antibody that binds to CD3 and PLAP, wherein the antibody comprises (a) a first antigen-binding domain that binds to CD3, comprising the VH sequence of SEQ ID NO: 9 and the VL sequence of SEQ ID NO: 13; and (b) a second antigen-binding domain that binds to PLAP, and optionally a third antigen-binding domain. An antibody that binds to CD3 and PLAP, comprising:

4. 4. The antibody of claim 1, wherein the first antigen-binding domain, the second antigen-binding domain and / or, if present, the third antigen-binding domain is a Fab molecule.

5. The antibody according to any one of claims 1 to 4, comprising an Fc domain composed of a first and a second subunit.

6. The antibody according to any one of claims 1 to 5, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are replaced by each other.

7. The antibody of any one of claims 1 to 6, wherein the second antigen-binding domain and, if present, the third antigen-binding domain are conventional Fab molecules.

8. 8. The antibody of claim 1 , wherein the second antigen-binding domain and, if present, the third antigen-binding domain are Fab molecules and wherein in the constant domain CL the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and in the constant domain CHI the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

9. The antibody of any one of claims 1 to 8, wherein the first and second antigen-binding domains are fused to each other, optionally via a peptide linker.

10. 10. The antibody of any one of claims 1 to 9, wherein the first and second antigen-binding domains are each Fab molecules and either (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.

11. 11. The antibody of claim 1, wherein the first antigen-binding domain, the second antigen-binding domain, and, if present, the third antigen-binding domain are each Fab molecules, the antibody comprising an Fc domain composed of first and second subunits, 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 subunit of the Fc domain.

11. The antibody of any one of claims 1 to 10, wherein either (i) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and, if present, the third antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

12. Fc domain is IgG Fc domain, especially IgG １ An antibody according to any one of claims 5 to 11, which is an Fc domain.

13. The antibody of any one of claims 5 to 12, wherein the Fc domain is a human Fc domain.

14. The antibody of any one of claims 5 to 13, wherein the Fc comprises a modification that promotes association of the first and second subunits of the Fc domain.

15. The antibody of any one of claims 5 to 14, wherein the Fc domain comprises one or more amino acid substitutions that reduce Fc receptor binding and / or effector function.

16. 16. The antibody of any one of claims 1 to 15, wherein the second antigen-binding domain and, if present, the third antigen-binding domain, comprises: (i) a HCDR1 of SEQ ID NO:28, a HCDR2 of SEQ ID NO:29, and a HCDR3 of SEQ ID NO:30; (ii) a HCDR1 of SEQ ID NO:32, a HCDR2 of SEQ ID NO:33, and a HCDR3 of SEQ ID NO:34; (iii) a HCDR1 of SEQ ID NO:36, a HCDR2 of SEQ ID NO:37, and a HCDR3 of SEQ ID NO:38; (iv) a HCDR1 of SEQ ID NO:40, a HCDR2 of SEQ ID NO:41, and a HCDR3 of SEQ ID NO:42; or (v) a VH comprising a HCDR1 of SEQ ID NO:44, a HCDR2 of SEQ ID NO:45, and a HCDR3 of SEQ ID NO:46, and a VL comprising a LCDR1 of SEQ ID NO:48, a LCDR2 of SEQ ID NO:49, and a LCDR3 of SEQ ID NO:

50.

17. The antibody of any one of claims 1 to 16, wherein the second antigen-binding domain and, if present, the third antigen-binding domain comprises a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:43 or SEQ ID NO:47 and / or a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

51.

18. An isolated polynucleotide encoding the antibody of any one of claims 1 to 17.

19. 20. A host cell comprising the isolated polynucleotide of claim 18.

20. 20. A method for producing an antibody that binds to CD3 and PLAP, comprising: (a) culturing the host cell of claim 19 under conditions suitable for expression of the antibody; and, optionally, (b) recovering the antibody.

21. 21. An antibody that binds to CD3 and PLAP produced by the method of claim 20.

22. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 17 or 21 and a pharma- ceutically acceptable carrier.

23. An antibody according to any one of claims 1 to 17 or 21, or a pharmaceutical composition according to claim 22, for use as a medicament.

24. An antibody according to any one of claims 1 to 17 or 21, or a pharmaceutical composition according to claim 22, for use in the treatment of a disease.

25. The antibody of claim 24, wherein the disease is cancer or an autoimmune disease.

26. Use of an antibody according to any one of claims 1 to 17 or 21, or a pharmaceutical composition according to claim 22, in the manufacture of a medicament.

27. Use of an antibody according to any one of claims 1 to 17 or 21, or a pharmaceutical composition according to claim 22, in the manufacture of a medicament for the treatment of a disease.

28. 28. The use according to claim 27, wherein the disease is cancer or an autoimmune disease.

29. A method of treating a disease in an individual, comprising administering to said individual an effective amount of an antibody according to any one of claims 1 to 17 or 21 or a pharmaceutical composition according to claim 22.

30. 30. The method of claim 29, wherein the disease is cancer.

31. 10. The invention as hereinbefore described.

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