Antibodies that bind to CD3 and CD19

Abstract

The present invention relates generally to antibodies that bind to CD3 and CD19, for example to activate T cells. The invention further relates to polynucleotides encoding such antibodies, as well as vectors and host cells containing such polynucleotides. The invention further relates to methods of producing the antibodies and their use in the treatment of disease.

Description

[Technical Field]

[0001] The present invention generally relates to antibodies that bind to CD3 and CD19, for example, to activate T cells. In addition, the present invention relates to polynucleotides encoding such antibodies, as well as vectors and host cells containing such polynucleotides. The present invention further relates to methods for producing the antibodies and methods of using the antibodies to treat disease. [Background technology]

[0002] CD3 (cluster of differentiation 3) is a protein complex consisting of four subunits, the CD3γ chain, the CD3δ chain, and two CD3ε chains. CD3 associates with the T cell receptor and the ζ chain to generate activation signals in T lymphocytes.

[0003] CD3 has been extensively investigated as a drug target. Monoclonal antibodies targeting CD3 have been used as immunosuppressive therapy in the treatment of autoimmune diseases such as type 1 diabetes or transplant rejection. The CD3 antibody muromonab-CD3 (OKT3) was the first monoclonal antibody ever approved for clinical use in humans, in 1985.

[0004] A more recent application of CD3 antibodies is in the form of bispecific antibodies, which bind CD3 on one side and a target cell antigen such as CD19 on the other. The simultaneous binding of such antibodies to both targets strengthens the transient interaction between the target cell and T cells, leading to the activation of any cytotoxic T cells and subsequent lysis of the target cell. Bispecific antibodies that bind to both CD3 and CD19 are described, for example, in WO2017 / 055314.

[0005] For therapeutic purposes, an important requirement that antibodies must fulfill is that they are sufficiently stable both in vitro (for drug storage) and in vivo (after administration to the patient).

[0006] Modifications such as asparagine deamidation are typical degradations of recombinant antibodies and can affect both in vitro stability and in vivo biological function.

[0007] Given the tremendous 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

[0008] The present invention provides antibodies, including multispecific (e.g., bispecific) antibodies, that bind to CD3 and are resistant to degradation, e.g., by asparagine deamidation, and are therefore particularly stable for therapeutic purposes. The provided (multispecific) antibodies further combine excellent efficacy and productivity with low toxicity and favorable pharmacokinetic properties.

[0009] As shown herein, antibodies, including multispecific antibodies that bind to CD3, provided by the present invention retain greater than about 90% of their binding activity to CD3 after two weeks at pH 7.4 and 37°C compared to their binding activity after two weeks at pH 6 and -80°C, as determined by surface plasmon resonance (SPR).

[0010] In one aspect, the invention provides an antibody that binds to CD3 and CD19, wherein the antibody comprises (a) a first antigen-binding domain that binds to CD3, and (b) a second and optionally a third antigen-binding domain that binds CD19, the second and optionally a third antigen-binding domain comprising a heavy chain variable region (VH) that comprises 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: 5, and a light chain variable region (VL) that comprises a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 8, an LCDR2 of SEQ ID NO: 9, and an LCDR3 of SEQ ID NO: 10. 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: 7, 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: 11.

[0011] In a further aspect, the invention provides an antibody that binds to CD3 and CD19, wherein the antibody comprises (a) a first antigen-binding domain that binds to CD3 comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 11, and (b) a second and optionally a third antigen-binding domain that binds to CD19.

[0012] In one embodiment, the first, second and / or, if present, third antigen binding domain is a Fab molecule.

[0013] In one embodiment, the antibody comprises an Fc domain composed of a first and a second subunit.

[0014] 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 of the Fab light and heavy chains, in particular the variable domains VL and VH, replace each other.

[0015] In one aspect, the second antigen binding domain and, if present, the third antigen binding domain are conventional Fab molecules.

[0016] In one aspect, the second antigen-binding domain and, if present, the third antigen-binding domain are Fab molecules and in the constant domain CL, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (Kabat numbering), and 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).

[0017] In one aspect, the first and second antigen binding domains are fused to each other, optionally via a peptide linker.

[0018] 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.

[0019] In one aspect, the first antigen-binding domain, the second antigen-binding domain, and, if present, the third antigen-binding domain are each Fab molecules, and the antibody comprises an Fc domain consisting 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.

[0020] In one embodiment, the Fc domain is an IgG, particularly an IgG1 Fc domain. In one embodiment, the Fc domain is a human Fc domain. In one embodiment, the Fc domain 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 reduce effector function.

[0021] In one aspect, the second and, if present, third antigen-binding domain comprises (i) a VH comprising an HCDR1 of SEQ ID NO: 15, an HCDR2 of SEQ ID NO: 16, and an HCDR3 of SEQ ID NO: 17, and a VL comprising an LCDR1 of SEQ ID NO: 19, an LCDR2 of SEQ ID NO: 20, and an LCDR3 of SEQ ID NO: 21, or (ii) a VH comprising an HCDR1 of SEQ ID NO: 28, an HCDR2 of SEQ ID NO: 29, and an HCDR3 of SEQ ID NO: 30, and a VL comprising an LCDR1 of SEQ ID NO: 32, an LCDR2 of SEQ ID NO: 33, and an LCDR3 of SEQ ID NO: 34. In one aspect, the second, and, if present, the third, antigen-binding domain comprises (i) a VH comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18, and / or a VL comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22, or (ii) a VH comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31, and / or a VL comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35.

[0022] According to further aspects of the invention, there are provided isolated polynucleotides encoding the antibodies of the invention, and host cells comprising the isolated polynucleotides of the invention.

[0023] In another aspect, there is provided a method for producing an antibody that binds CD3 and CD19, comprising the steps of (a) incubating 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 CD19 produced by the methods of the invention.

[0024] The present invention further provides a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier.

[0025] The present invention also encompasses methods of using the antibodies and pharmaceutical compositions of the present invention. In one aspect, an antibody or pharmaceutical composition according to the present invention is provided for use as a medicament. In one aspect, an antibody or pharmaceutical composition according to the present invention is provided 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 certain aspects, the disease is cancer. In other aspects, the disease is an autoimmune disease. [Brief explanation of the drawings]

[0026] [Figure 1A-F] Exemplary configurations 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 in which the order of the Crossfab and Fab components is different ("flipped"). (C, F) Diagram of a "2+1 IgG Crossfab" molecule. [Figure 1G-N] Exemplary configurations of (multispecific) antibodies of the invention. (G, K) Diagrams of a "1+1 IgG Crossfab" molecule in which the order of the Crossfab and Fab components is different ("flipped"). (H, L) Diagrams of a "1+1 IgG Crossfab" molecule. (I, M) Diagrams of a "2+1 IgG Crossfab" molecule with two CrossFabs. (J, N) Diagrams of a "2+1 IgG Crossfab" molecule with two CrossFabs in which the order of the Crossfab and Fab components is different ("flipped"). [Figure 1O-V] Exemplary configurations of (multispecific) antibodies of the invention. (O, S) Diagram of a "Fab-Crossfab" molecule. (P, T) Diagram of a "Crossfab-Fab" molecule. (Q, U) Diagram of a "(Fab)2-Crossfab" molecule. (R, V) Diagram of a "Crossfab-(Fab)2" molecule. [Figure 1W-Z]Exemplary configurations of (multispecific) antibodies of the present invention. (W, Y) Diagrams of "Fab-(Crossfab)2" molecules. (X, Z) Diagrams of "(Crossfab)2-Fab" molecules. Black dots: optional modifications in the Fc domain that promote heterodimerization. ++, --: amino acids of opposite charge optionally introduced in the CH1 and CL domains. Crossfab molecules are shown as comprising swapped VH and VL domains, but alternatively comprise swapped CH1 and CL domains in embodiments where no charge modifications have been introduced in the CH1 and CL domains. [Figure 2] Relative binding activity of the original CD3 binder, CD3orig, and the optimized CD3 binder, CD3opt, to recombinant CD3 (IgG format) measured by SPR under non-stress conditions after 14 days at 40°C and pH 6 or after 14 days at 37°C and pH 7.4. [Figure 3] Binding of the original CD3 binder, CD3orig, and the optimized CD3 binder, CD3opt, to Jurkat NFAT cells (IgG format) as measured by flow cytometry. Antibody binding to Jurkat NFAT cells was detected using a fluorescently labeled anti-human Fc-specific secondary antibody. [Figure 4] Schematic diagram of the CD3 activation assay used in Example 3. [Figure 5] Jurkat NFAT cell activation by the original CD3 binder, CD3orig, and the optimized CD3 binder, CD3opt (IgG format). Jurkat NFAT reporter cells were co-incubated with anti-PGLALA-expressing CHO (CHO-PGLALA) cells in the presence of CD3orig IgG PGLALA or CD3opt IgG PGLALA, or CD3opt IgG wt as a negative control. CD3 activation was quantified after 24 hours by measuring luminescence. [Figure 6A-E](A) Schematic diagram of the T cell bispecific antibody (TCB) molecule used in the examples. All TCB antibodies tested were generated as "2+1 IgG CrossFab, 'inverted'" with charge modifications (VH / VL exchange in the CD3 binder, charge modifications in the target antigen binder, EE = 147E, 213E; RK = 123R, 124K). (B-E) Components for TCB assembly: light chain of an anti-TYRP1 Fab molecule with charge modifications in CH1 and CL (B), light chain of an anti-CD3 crossover Fab molecule (C), heavy chain with knob and PG-LALA mutations in the Fc region (D), and heavy chain with hole and PG-LALA mutations in the Fc region (E). [Figure 7A-C] Binding of CD19-TCB antibody to CD3-expressing Jurkat cells (A) and CD19-expressing Z-138 (B) and Nalm-6 (C) cells as measured by flow cytometry. [Figure 8A-B] Target-specific killing of CD19+ target cells induced by CD19-TCB antibody. (A) Z-138 target cells, (B) Nalm-6 target cells. [Figure 9A-F] T cell activation induced by CD19-TCB antibody after killing of Z-138 target cells. (A) CD25 expression on CD4 T cells, (B) CD69 expression on CD4 T cells, (C) CD107 expression on CD4 T cells, (D) CD25 expression on CD8 T cells, (E) CD69 expression on CD8 T cells, and (F) CD107 expression on CD8 T cells. [Figure 10A-F] T cell activation induced by CD19-TCB antibody after killing of Nalm-6 target cells. (A) CD25 expression on CD4 T cells, (B) CD69 expression on CD4 T cells, (C) CD107 expression on CD4 T cells, (D) CD25 expression on CD8 T cells, (E) CD69 expression on CD8 T cells, and (F) CD107 expression on CD8 T cells. [Figure 11] Example 10 In Vivo Study Design. [Figure 12] Body weight change with treatment with different doses of CD19-TCB or CD20-TCB in the study of Example 10, n=3 mice / group. Mean+ / -SEM. [Figure 13] Cytokine release in serum 4 hours after treatment with CD19-TCB or CD20-TCB with or without obinituzumab (Gazyva® pretreatment (GPT)) in the study of Example 10. (A) MIP-1β, (B) IL-6, (C) IFN-γ, (D) IL-5, (E) GM-CSF, (F) TNF-α, (G) IL-2, (H) IL-1β, (I) IL- 13, (J)MCP1, (K)IL-8, (L)IL-10, (M)G-CSF, (N)L-12p70, (O)IL-17. Panel bars from left to right: CD19-TCB 0.5mg / kg, CD19-TCB 0.15mg / kg, CD19-TCB 0.05mg / kg, GPT+CD19-TCB 0.5mg / kg, CD20-TCB 0.15mg / kg, GPT+CD20-TCB 0.15mg / kg. Mean+SEM. [Figure 14] B cell counts in blood at 4 hours, 24 hours, and 72 hours after treatment with CD19-TCB or CD20-TCB with or without obinituzumab (Gazyva®) pretreatment (GPT) in the study of Example 10. Mean + SEM. [Figure 15] Treatment schedule and experimental setup. Humanized NSG mice were subcutaneously implanted with lymphoma patient-derived xenografts (PDXs) (5 million cells). Tumor volume was calculated using caliper measurements. When mice reached 200 mm3, they were randomized into groups of 8 based on tumor size. Mice then received weekly intravenous injections of vehicle or 0.5 mg / kg CD19-TCB. [Figure 16] Effect of CD19-TCB treatment on tumor growth. Tumor volumes were calculated from caliper measurements taken twice (volume < 1000 mm3) or three times (volume ≥ 1000 mm3) per week for n = 7 mice in Group B and n = 8 mice in Group A, as shown in Figure 15. Mean + SD, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by Mann-Whitney test. Arrows indicate each of the four treatments with CD19-TCB or vehicle. DETAILED DESCRIPTION OF THE INVENTION

[0027] I. Definition Terms are used herein as commonly used in the art unless otherwise defined below.

[0028] As used herein, the terms "first," "second," or "third," with respect to antigen-binding domains, etc., are used for convenience in distinguishing between cases where more than one of each type of moiety is present. The use of these terms is not intended to confer a particular order or orientation of the moieties unless explicitly indicated as such.

[0029] The terms "anti-CD3 antibody" and "antibody that binds to CD3" refer to an antibody that is capable of binding to CD3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD3. In one embodiment, the binding of an anti-CD3 antibody to an unrelated, non-CD3 protein 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 to CD3 has an equilibrium dissociation constant (K) of ≦1 μM, ≦500 nM, ≦200 nM, or ≦100 nM. D For example, the antibody has a K of 1 μM or less as measured by SPR. D An 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.

[0030] Similarly, the terms "anti-CD19 antibody" and "antibody that binds to CD19" refer to an antibody that is capable of binding to CD19 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD19. In one aspect, the binding of an anti-CD19 antibody to an unrelated, non-CD19 protein is less than about 10% of the binding of the antibody to CD19, as measured, for example, by surface plasmon resonance (SPR). In certain aspects, an antibody that binds to CD19 has an equilibrium dissociation constant (K) of ≦1 μM, ≦500 nM, ≦200 nM, or ≦100 nM.D For example, the antibody has a K of 1 μM or less as measured by SPR. D An antibody is said to "specifically bind" to CD19 if it has the following structure: In certain embodiments, an anti-CD19 antibody binds to an epitope of CD19 that is conserved among CD19 from different species.

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

[0032] An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFab), single-domain antibodies, and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0033] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure.

[0034] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies that contain, for example, naturally occurring mutations or that arise during production of the monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an 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 containing all or part of the human immunoglobulin loci, such as those methods and other exemplary methods for producing monoclonal antibodies described herein.

[0035] 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 testing methods for antibody purification, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). In some embodiments, the antibody provided by the present invention is an isolated antibody.

[0036] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0037] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDRs and human FRs. In certain embodiments, 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 embodiments, 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 were 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 undergone humanization.

[0038] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell, or an antibody of non-human origin that utilizes human antibody-encoding sequences, such as the human antibody repertoire. This definition of human antibody specifically excludes humanized antibodies, which comprise non-human antigen-binding residues. In some aspects, a human antibody is derived from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In some 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 of the invention.

[0039] The term "antigen-binding domain" refers to a portion of an antibody comprising an 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).

[0040] 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 variable domains of the heavy and light chains (VH and VL, respectively) of a native antibody generally have similar structures, 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., W.H. Freeman & Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind to a specific antigen may be isolated by using the VH or VL domain of an antibody that binds the 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). When used herein in reference to variable region sequences, "Kabat numbering" refers to the numbering system described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0041] As used herein, amino acid positions in all heavy and light chain constant regions and domains are numbered according to the Kabat numbering system as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and is referred to herein as "Kabat numbering" or "Kabat numbering." Specifically, the Kabat numbering system (see pages 647-660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domains CL of 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), which is further clarified by reference to "Kabat EU index numbering" or "Kabat EU index numbering."

[0042] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain, e.g., "complementarity determining regions" (CDRs), that are hypervariable in sequence and determine antigen binding specificity. Generally, antibodies contain six CDRs, three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs 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 contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0043] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. Those skilled in the art will understand that CDR designations can be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0044] "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 generally appear in the following order in VH (or VL): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

[0045] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0046] 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 comprise amino acid sequence changes. In some aspects, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0047] 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 in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3.

[0048] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two light chains and two heavy chains disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light domain or light chain variable region, followed by a constant light domain (CL), also called a light chain constant region. Immunoglobulin heavy chains may be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains 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 connected via an immunoglobulin hinge region.

[0049] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by the antibody or immunoglobulin heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0050] A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain (a "Fab heavy chain") and the VL and CL domains of an immunoglobulin light chain (a "Fab light chain").

[0051] A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which the variable and constant domains of the Fab heavy and light chains have been exchanged (i.e., replaced with each other), i.e., the crossover Fab molecule comprises a peptide chain consisting of a light chain variable domain VL and a heavy chain constant domain 1 CH1 (VL-CH1, from N- to C-terminal), and a peptide chain consisting of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL, from N- to C-terminal). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule in which the constant domains of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0052] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native format, i.e., comprising a heavy chain (VH-CH1, N- to C-terminally) composed of a heavy chain variable domain and a constant domain, and a light chain (VL-CL, N- to C-terminally) composed of a light chain variable domain and a constant domain.

[0053] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a particular nucleic acid molecule encoding a full-length heavy chain, antibodies produced by host cells may comprise a full-length heavy chain or a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (Lys447), may or may not be present. The amino acid sequence of a 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, a 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, numbered according to the Kabat EU index). In one aspect, a 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, numbered according to the 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 referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also supra).A "subunit" of an Fc domain, as used herein, refers to one of the two polypeptides that form the dimeric Fc domain (i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain that is capable of stable self-association). For example, a subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

[0054] By "fused" is meant that the components (e.g., a Fab molecule and an Fc domain subunit) are joined either directly by a peptide bond or via one or more peptide linkers.

[0055] The term "multispecific" means that an antibody can specifically bind to at least two distinct antigenic determinants. A multispecific antibody can be, for example, a bispecific antibody. Typically, a bispecific antibody contains two antigen-binding sites, each specific for a different antigenic determinant. In some embodiments, a multispecific (e.g., bispecific) antibody can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two separate cells.

[0056] The term "valency," as used herein, refers to the presence of a specific number of antigen-binding sites within an antigen-binding molecule. In this context, the term "monovalent binding to an antigen" refers to the presence of one (and no more than one) antigen-binding site specific for an antigen within the antigen-binding molecule.

[0057] "Antigen-binding site" refers to the site of an antigen-binding molecule, i.e., one or more amino acid residues, that interacts with an antigen. For example, the antigen-binding site of an antibody comprises amino acid residues from the complementarity-determining region (CDR). A native immunoglobulin molecule typically contains two antigen-binding sites, and a Fab molecule typically has one antigen-binding site.

[0058] As used herein, the term "antigenic determinant" or "antigen" refers to the site on a polypeptide macromolecule to which an antigen-binding domain binds (e.g., a contiguous stretch of amino acids or a conformational configuration composed of different regions of non-contiguous amino acids), forming an antigen-binding domain-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virally infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). In a preferred embodiment, the antigen is a human protein.

[0059] Unless otherwise specified, "CD3" refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD3 and any form of CD3 resulting from intracellular processing. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, the CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is set forth in SEQ ID NO: 45 (without the signal peptide). See also UniProt (www.uniprot.org) accession number P07766 (version 209) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1.1. In another embodiment, the CD3 is cynomolgus monkey (Macaca fascicularis) CD3, particularly cynomolgus monkey CD3ε. The amino acid sequence of cynomolgus monkey CD3ε is set forth in SEQ ID NO: 46 (without the signal peptide). See also NCBI GenBank No. BAB71849.1. In some embodiments, the antibodies of the invention bind to an epitope of CD3 that is conserved among CD3 antigens from different species, particularly human CD3 and cynomolgus monkey CD3. In a preferred embodiment, the antibody binds to human CD3.

[0060] As used herein, "target cell antigen" refers to an antigenic determinant displayed on the surface of a target cell, e.g., a B cell. Preferably, the target cell antigen is not CD3 and / or is other than CD3 expressed on a different cell. According to the present invention, the target cell antigen is CD19, particularly human CD19.

[0061] "CD19" refers to cluster of differentiation 19 (also known as B-lymphocyte antigen CD19 or B-lymphocyte surface antigen B4) and, unless otherwise specified, refers to any native CD19 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD19 and any form of CD19 resulting from intracellular processing. The term also encompasses naturally occurring variants of CD19, such as splice variants or allelic variants. In one embodiment, CD19 is human CD19. For the human protein, see UniProt (www.uniprot.org) accession number P15391 (version 211) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_001761.3. In some embodiments, the antibodies of the invention bind to an epitope of CD3 that is conserved in CD19 antigens from different species, particularly human CD19 and cynomolgus monkey CD19. In preferred embodiments, the antibody binds to human CD19.

[0062] "Affinity" refers to the strength of the total 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 specified, as used herein, "binding affinity" refers to the specific binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined 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).

[0063] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more complementarity determining regions (CDRs) compared to a parent antibody that does not possess those alterations, such that the affinity of the antibody for antigen is improved by such alterations.

[0064] "Reduced binding," e.g., reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, e.g., as measured by SPR. For clarity, the term also includes a decrease in affinity to zero (or below the detection limit of the analytical method), i.e., a complete loss of interaction. Conversely, "increased binding" refers to an increase in binding affinity for a particular interaction.

[0065] 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.

[0066] A "modification that promotes association between a first subunit and a second subunit of an Fc domain" refers to manipulation of the peptide backbone or post-translational modification of an Fc domain subunit that reduces or prevents association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. As used herein, a modification that promotes association preferably includes separate alterations made to each of the two Fc domain subunits (i.e., the first and second Fc domain subunits) that are desired to associate, where the modifications are complementary to each other to promote association of the two Fc domain subunits. For example, a modification that promotes association may alter the structure or charge of one or both of the Fc domain subunits to achieve the desired steric or electrostatic association, respectively. Thus, (hetero)dimerization can occur between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, and the subunits may not be identical in the sense that the additional components (e.g., antigen-binding domains) fused to each of the subunits are not identical. In some embodiments, the modification that promotes association of the first and second subunits of the Fc domain comprises an amino acid mutation, specifically an amino acid substitution, within the Fc domain. In preferred embodiments, the modification that promotes association of the first and second subunits of the Fc domain comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.

[0067] The term "effector function" refers to a biological activity attributable to the Fc region of an antibody and varies 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, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0068] An "activating Fc receptor" is an Fc receptor that, upon engagement by the Fc domain of an antibody, triggers signaling events that stimulate the receptor-containing cell to exert effector function. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0069] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that causes immune effector cells to lyse antibody-coated target cells. Target cells are cells to which an antibody or its derivative, including an Fc region, specifically binds, typically via a protein portion 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 period by the ADCC mechanism defined above at a given concentration of antibody in the medium surrounding the target cells, and / or an increase in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time period by the ADCC mechanism. The reduction in ADCC is compared to the ADCC mediated by the same antibody produced by the same type of host cell using the same standard production, purification, formulation, and storage methods (known to those skilled in the art), but it is not engineered. For example, an amino acid substitution that reduces ADCC, which is a reduction in ADCC mediated by an antibody containing its Fc domain, is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, PCT Publication Nos. WO2006 / 082515 or 2012 / 130831).

[0070] As used herein, the terms "engineer, engineered, manipulating" are intended to include any manipulation or post-translational modification of the peptide backbone of a naturally occurring or recombinant polypeptide or fragment thereof. Manipulating includes modifying the amino acid sequence, modifying the glycosylation pattern, or modifying the side groups of individual amino acids, as well as combinations of these approaches.

[0071] As used herein, the term "amino acid mutation" is intended to encompass amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to arrive at a final construct, as long as the final construct has the desired characteristics, such as reduced binding to an Fc receptor or increased association with another peptide. Deletions and insertions in the amino acid sequence include deletions and insertions of amino and / or carboxy terminal amino acids. Preferred amino acid mutations are amino acid substitutions. For example, for the purpose of altering the binding characteristics of the Fc region, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid with different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include replacement with unnatural amino acids or replacement with natural 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, etc. It is contemplated that methods other than genetic engineering that alter the side chain group of an amino acid, such as chemical modification, may also be useful. Various names may be used herein to refer to the same amino acid mutation. For example, a proline to glycine substitution at position 329 of the Fc domain is referred to as 329G, G329, G 329 , P329G or Pro329Gly.

[0072] "Percent (%) amino acid sequence identity" 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, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, without considering any conservative substitutions as part of the sequence identity. 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 appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment across the entire 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 the source code is on file in the user documentation at the U.S. Copyright Office, Washington, DC, 20559, registered under U.S. Copyright Registration No. TXU510087, and described in WO2001 / 007611.

[0073] Unless otherwise specified, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program in the FASTA package version 36.3.8c, followed by the BLOSUM50 comparison matrix. The FASTA program package was created by W.R. Pearson and D.J. Lipman ("Improved Tools for Biological Sequence Analysis," PNAS 85(1988):2444-2448), W.R. Pearson ("Effective protein sequence comparison," Meth. Enzymol. 266(1996):227-258), and Pearson et. al. (Genomics 46(1997)24-36), and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using 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 alignment. The percent amino acid identity is given in the output alignment header.

[0074] The terms "polynucleotide" or "nucleic acid molecule" include any compound and / or substance comprising 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 their base sequence, where the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically presented 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 may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, and both single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides, including those containing derivatized sugar or phosphate backbone linkages or chemically modified residues, include modified nucleotide bases. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the present invention in vitro and / or in vivo, e.g., 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 enhance the stability of the RNA vector and / or expression of the encoded molecule, such that the mRNA can be injected into a subject to generate antibodies in vivo (see, e.g., Stadler et al. (2017) Nature Medicine 23:815-817, or EP 2 101 823 B1).

[0075] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid molecules include nucleic acid molecules contained within cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its native chromosomal location.

[0076] 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 molecule(s) in a single vector or in separate vectors, where such polynucleotide molecule(s) are present in one or more locations in a host cell.

[0077] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This 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."

[0078] The terms "host cell," "host cell line," and "host cell incubate" 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," and include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not have exactly the same nucleic acid content as the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included in the present invention. Host cells can be any type of cell line that can be used to produce antibodies of the present invention. Host cells include incubate cells, such as mammalian incubate 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 just a few, but also cells contained in transgenic animals, transgenic plants, or incubate plants, or animal tissues. In one aspect, the host cell of the invention is a eukaryotic cell, in particular a mammalian cell. In one aspect, the host cell is not a cell in the human body.

[0079] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredients contained in the preparation is effective, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition will be administered.

[0080] 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, vehicles, stabilizers, or preservatives.

[0081] As used herein, "treatment" (and its grammatical variants, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of disease in the individual being treated, and can be carried out prophylactically or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, remission or palliation of disease symptoms, and recovery or improved 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.

[0082] 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, the individual or subject is human.

[0083] An "effective amount" of an agent, eg, a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result. The term "package insert" is used to refer to instructions customarily included in the commercial packaging of a therapeutic product, which contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications and / or precautions regarding the use of such therapeutic product.

[0084] II. Compositions and Methods The present invention provides antibodies that bind to CD3 and CD19. The antibodies exhibit excellent stability in combination with other properties beneficial for therapeutic use, such as efficacy and safety, pharmacokinetics, and reproducibility. The antibodies of the present invention are useful, for example, in the treatment of diseases such as cancer.

[0085] A. Anti-CD3 / CD19 antibody In one aspect, the invention provides antibodies that bind to CD3 and CD19. In one aspect, isolated antibodies that bind to CD3 and CD19 are provided. In one aspect, the invention provides antibodies that specifically bind to CD3 and CD19. In certain aspects, the anti-CD3 / CD19 antibody retains greater than about 90% of its binding activity to CD3 after two weeks at pH 7.4 and 37°C compared to the binding activity after two weeks at pH 6 and -80°C, as determined by surface plasmon resonance (SPR).

[0086] In one aspect, the invention provides an antibody that binds to CD3 and CD19, wherein the antibody comprises: (a) a first antigen-binding domain comprising a heavy chain variable region (VH) that binds to CD3 and comprises 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: 5; and a light chain variable region (VL) that comprises a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 8, an LCDR2 of SEQ ID NO: 9, and an LCDR3 of SEQ ID NO: 10.

[0087] 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.

[0088] 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.

[0089] 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: 7. 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: 7. 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: 7. 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: 7. In certain aspects, a VH sequence with at least about 95%, about 96%, about 97%, about 98%, or about 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, although antibodies comprising that sequence retain the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 7. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In one aspect, the VH of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 7. Optionally, the VH of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 7, including post-translational modifications of that sequence.

[0090] In one aspect, the VL of the first antigen-binding domain comprises one or more light chain framework sequences (i.e., the FR1, FR2, FR3 and / or FR4 sequences) of the light chain variable region sequence of SEQ ID NO: 11. 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: 11. 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: 11. 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: 11. In certain aspects, a VL sequence with at least about 95%, about 96%, about 97%, about 98% or about 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions compared to the reference sequence, although antibodies comprising that sequence retain the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO: 11. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In one aspect, the VL of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 11. Optionally, the VL of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 11, including post-translational modifications of that sequence.

[0091] 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: 7, 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: 11. In one aspect, the VH of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 7, and the VL of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 11.

[0092] In a further aspect, the invention provides an antibody that binds to CD3 and CD19, wherein the antibody comprises a first antigen-binding domain that binds to CD3, the first antigen-binding domain comprising a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO:11.

[0093] In a further aspect, the invention provides an antibody that binds to CD3 and CD19, wherein the antibody comprises a first antigen-binding domain that binds to CD3 comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 11.

[0094] In a further aspect, the invention provides an antibody that binds to CD3 and CD19, wherein the antibody comprises a first antigen-binding domain that binds to CD3, the first antigen-binding domain comprising a VH comprising the heavy chain CDR sequence of the VH of SEQ ID NO: 7 and a VL comprising the light chain CDR sequence of the VL of SEQ ID NO: 11.

[0095] In a further aspect, the first antigen-binding domain comprises the VH HCDR1, HCDR2 and HCDR3 amino acid sequences of SEQ ID NO: 7 and the VL LCDR1, LCDR2 and LCDR3 amino acid sequences of SEQ ID NO:11.

[0096] In one embodiment, the VH of the first antigen-binding domain comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 7 and a framework with at least 95%, 96%, 97%, 98% or 99% sequence identity to the framework sequence of the VH of SEQ ID NO: 7. In one embodiment, the VH of the antigen-binding domain of the first antigen-binding domain comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 7 and a framework with at least 95% sequence identity to the framework sequence of the VH of SEQ ID NO: 7. In another embodiment, the VH of the first antigen-binding domain comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 7 and a framework with at least 98% sequence identity to the framework sequence of the VH of SEQ ID NO: 7.

[0097] In one embodiment, the VL of the second antigen binding domain comprises the light chain CDR sequence of the VL of SEQ ID NO: 11 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: 11. In one embodiment, the VL of the first antigen binding domain comprises the light chain CDR sequence of the VL of SEQ ID NO: 11 and a framework of at least 95% sequence identity to the framework sequence of the VL of SEQ ID NO: 11. In one embodiment, the VL of the first antigen binding domain comprises the light chain CDR sequence of the VL of SEQ ID NO: 11 and a framework of at least 98% sequence identity to the framework sequence of the VL of SEQ ID NO: 11.

[0098] In one aspect, the invention provides an antibody that binds to CD3 and CD19, wherein the antibody comprises a first antigen-binding domain that binds to CD3 comprising a VH sequence of any of the aspects provided above and a VL sequence of any of the aspects provided above.

[0099] In one embodiment, the antibody comprises a human constant region. In one embodiment, the antibody is an immunoglobulin molecule comprising a human constant region, particularly an IgG class immunoglobulin molecule comprising human CH1, CH2, CH3 and / or CL domains. Exemplary sequences of human constant domains are given in SEQ ID NOs: 52 and 53 (human kappa and lambda CL domains, respectively), and SEQ ID NO: 54 (human IgG1 heavy chain constant domain CH1-CH2-CH3). In one embodiment, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 53, particularly an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 52. In one embodiment, the antibody comprises a heavy chain constant region comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 54. In particular, the heavy chain constant region may comprise amino acid mutations in the Fc domain as described herein.

[0100] 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, particularly 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 at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 53, particularly the amino acid sequence of SEQ ID NO: 52. In particular, the light chain constant region may comprise amino acid mutations described herein in a "charge-modified" state and / or, in the case of a crossover Fab molecule, one or more (particularly two) N-terminal amino acid deletions or substitutions. In one embodiment, the first 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: 54. In particular, the heavy chain constant region (specifically the CH1 domain) may contain the amino acid mutations described herein under "charge modifications."

[0101] In one embodiment, the antibody is a monoclonal antibody.

[0102] In one embodiment the antibody is an IgG, particularly an IgG1 antibody. In one embodiment the antibody is a full-length antibody.

[0103] In another embodiment, the antibody is an antibody fragment, particularly a Fab molecule, selected from the group of Fv, scFv, Fab, and F(ab')2 molecules. In another embodiment, the antibody fragment is a diabody, triabody, or tetrabody.

[0104] In one embodiment, the first antigen-binding domain is a Fab molecule. In a preferred embodiment, the first antigen-binding moiety 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 Fab heavy chain are replaced by each other (i.e., the first antigen-binding domain is a crossover Fab molecule).

[0105] In further embodiments, antibodies according to any of the above embodiments can incorporate any of the features, alone or in combination, as described in Sections II.A.1.-8. below.

[0106] In a preferred embodiment, the antibody comprises an Fc domain, particularly an IgG Fc domain, more particularly an IgG1 Fc domain. In one embodiment, the Fc domain is a human Fc domain. In one embodiment, the Fc domain is a human IgG1 Fc domain. The Fc domain consists 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.).

[0107] According to the present invention, the antibody comprises a second and optionally a third antigen-binding domain that binds to CD19 (i.e., the antibody is a multispecific antibody, as described further herein below (Section II.A.7.)).

[0108] 1. Antibody fragment In certain aspects, the antibodies provided herein are antibody fragments.

[0109] In one embodiment, the antibody fragment is a Fab molecule, a Fab' molecule, a Fab'-SH molecule, or a F(ab')2 molecule, particularly a Fab molecule as described herein. A "Fab' molecule" differs from a Fab molecule only by the addition of residues at the carboxy terminus of the CH1 domain that contain one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' molecule in which the cysteine ​​residue(s) in the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 molecule that has two antigen-binding sites (two Fab molecules) and a portion of the Fc region.

[0110] In another embodiment, the antibody fragment is a diabody, triabody, or tetrabody. Diabodies are antibody fragments with two antigen-binding sites, which may 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).

[0111] In a further embodiment, the antibody fragment is a single-chain Fab molecule. A "single-chain Fab molecule" or "scFab" consists 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, with the antibody domains and linker arranged in one of the following orders from N- 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, the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. Single-chain Fab molecules are stabilized by a native disulfide bond between the CL and CH1 domains. In addition, these single-chain Fab molecules can be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (e.g., at position 44 of the variable heavy chain and position 100 of the variable light chain according to Kabat numbering).

[0112] 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 an antibody's heavy chain (VH) and light chain (VL) connected by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids, typically rich in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. This protein can retain 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, Plueckthun, 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. Patent Nos. 5,571,894 and 5,587,458.

[0113] In another embodiment, the antibody fragment is a single-domain antibody. A "single-domain antibody" is an antibody fragment that comprises 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 a particular embodiment, 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).

[0114] 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.

[0115] 2. Humanized antibodies In some embodiments, 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. Generally, 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 may also optionally comprise at least a portion of a human constant region. 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.

[0116] Humanized antibodies and methods for their production are reviewed by Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in 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"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection approach" for FR shuffling).

[0117] 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 human antibody consensus sequences of specific subtypes of heavy or light 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., See J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0118] 3. Glycosylation variants In certain aspects, the antibodies provided herein are altered to increase or decrease the extent of glycosylation of the antibody. 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.

[0119] If the antibody contains an Fc region, the oligosaccharides attached to the antibody may be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are commonly attached to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, e.g., 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, modifications of the oligosaccharides in the antibodies of the present invention may be made to generate antibody variants with specific improved properties.

[0120] In one embodiment, antibody variants are provided that have nonfucosylated oligosaccharides, i.e., oligosaccharide structures lacking fucose linkage (direct or indirect) to the Fc region. Such nonfucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are particularly N-linked oligosaccharides that lack the first GlcNAc-linked fucose residue at the stem of the biantennary oligosaccharide structure. In one embodiment, 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 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 the asparagine residue located at approximately position 297 of the Fc region (EU numbering of Fc region residues); however, due to minor antibody sequence variations, 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, particularly improved ADCC function. See, e.g., US 2003 / 0157108; US 2004 / 0093621.

[0121] 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); US2003 / 0157108; and WO2004 / 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., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107), or cells in which the activity of GDP-fucose synthesis or transporter proteins is reduced or abolished (see, e.g., US2004259150, US2005031613, US2004132140, US2004110282).

[0122] In a further embodiment, antibody variants are provided with 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); WO99 / 54342, WO2004 / 065540, and WO2003 / 011878.

[0123] Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in WO1997 / 30087; WO1998 / 58964; and WO1999 / 22764.

[0124] 4. Cysteine-engineered antibody variants In certain aspects, cysteine ​​engineered antibodies, e.g., THIOMAB, in which one or more residues of the antibody are substituted with cysteine ​​residues. TM In some cases, it may be desirable to generate cysteine-engineered antibodies. In preferred embodiments, the substituted residues occur at accessible sites on the antibody. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites on the antibody, which can be used to conjugate the antibody to other moieties, 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. Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO2016040856.

[0125] 5. Antibody derivatives In some aspects, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Suitable sites 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 during 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 molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0126] 6. Immunoconjugates The present invention also provides immunoconjugates comprising an anti-CD3 / CD19 antibody herein conjugated (chemically linked) 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.

[0127] In one aspect, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more of the aforementioned therapeutic agents. The antibody is typically connected to one or more of the therapeutic agents using a linker. An overview of ADC technology, including examples of therapeutic agents and drugs and linkers, is provided in Pharmacol Review 68:3-19 (2016).

[0128] In another aspect, the immunoconjugate comprises an antibody of the invention conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Jatropha curcas protein, dianthin protein, pokeweed protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0129] 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 production 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 radioconjugates are used for detection, examples include radioactive atoms for scintigraphy studies, e.g., Tc 99m Or I 123 , or nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), I 123 , I 131 , In 111 , F 19 , C 13 , N15 , O 17 Spin labels such as gadolinium, manganese, or iron may be included.

[0130] 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 (such as 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 conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker may also be a "cleavable linker" that facilitates the release of the cytotoxic drug within the cell. For example, an acid-labyrinth linker, a peptidase-sensitive linker, a photolabyrinth linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0131] The immunoconjugates or ADCs herein expressly contemplate conjugates prepared using cross-linker 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, Inc., Rockford, IL, USA).

[0132] 7. Multispecific antibodies The antibodies provided herein are multispecific antibodies, particularly bispecific antibodies. A multispecific antibody is a monoclonal antibody that has 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, a multispecific antibody has three or more binding specificities. According to the present invention, one of the binding specificities is for CD3 and the other is for CD19.

[0133] Multispecific antibodies can be prepared as full-length antibodies or antibody fragments. Techniques for producing multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knob-in-hole" engineering (see, e.g., U.S. Patent 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)); generating bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); using conventional light chain technology to circumvent the light chain mispairing problem (see, e.g., WO 98 / 50431); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and by the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and by the preparation of trispecific antibodies, for example, as described in Tutt et al. J. Immunol. 147:60 (1991).

[0134] Also included herein are engineered antibodies, or DVD-Igs, having three or more antigen-binding sites, including, for example, "Octopus antibodies" (see, e.g., WO2001 / 77342 and WO2008 / 024715). Other examples of multispecific antibodies having three or more antigen-binding sites can be found in WO2010 / 115589, WO2010 / 112193, WO2010 / 136172, WO2010 / 145792, and WO2013 / 026831. Multispecific antibodies or antigen-binding fragments thereof also include "dual-acting FAbs" or "DAFs" that contain antigen-binding sites that bind to CD3, as well as to another different antigen, or to two different epitopes on CD3 (see, e.g., US2008 / 0069820 and WO2015 / 095539).

[0135] Multispecific antibodies can also be provided in an asymmetric format with domain crossovers in one or more binding arms of the same antigen specificity (the so-called "CrossMab" technology), i.e., by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253), or complete Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299; see also Schaefer et al., PNAS, 108(2011)1187-1191 and Klein 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 direct correct Fab pairing. See, for example, WO2016 / 172485.

[0136] 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).

[0137] 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 B cell, and to an activation-invariant component of the T cell receptor (TCR) complex, such as CD3, to retarget T cells 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 CD19 as the target cell antigen.

[0138] 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., WO2004 / 106381, WO2005 / 061547, WO2007 / 042261 and WO2008 / 119567; Nagorsen and Baeuerle, Exp Cell Res 317, 1255-1260 (2011)), diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem diabodies ("TandAb"; Kipriyanov et al., J Mol Biol 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)), and so-called triomabs, which are all-hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Specific T cell bispecific antibody formats included herein are described in WO2013 / 026833, WO2013 / 026839, WO2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0139] Preferred embodiments of the antibody of the present invention are described below.

[0140] In one aspect, the invention provides an antibody that binds CD3 and CD19, comprising a first antigen-binding domain that binds CD3, and a second and optionally a third antigen-binding domain that binds CD19, as described herein.

[0141] According to a preferred embodiment of the present invention, the antigen-binding domain contained 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, second, and / or, if present, the third antigen-binding domain is a Fab molecule. In one embodiment, the Fab molecule is human. In a preferred embodiment, the Fab molecule is humanized. In another embodiment, the Fab molecule comprises human heavy and light chain constant domains.

[0142] Preferably, at least one of the antigen-binding domains is a crossover Fab molecule. Such a modification reduces mispairing of heavy and light chains of different Fab molecules, thereby improving the yield and purity of the (multispecific) antibodies of the present invention during recombinant production. In preferred crossover Fab molecules useful for the (multispecific) antibodies of the present invention, the variable domains of the Fab light chain and the Fab heavy chain (VL and VH, respectively) are exchanged. However, even with this domain exchange, the preparation of (multispecific) antibodies may contain certain side products due to so-called Bence-Jones type interactions between mispaired heavy and light chains (see Schaefer et al., PNAS, 108 (2011) 11187-11191). To further reduce mispairing of heavy and light chains from different Fab molecules and increase the purity and yield of the desired (multispecific) antibody, charged amino acids with opposite charges can be introduced into specific amino acid positions in the CH1 and CL domains of either the CD3-binding Fab molecule or the CD19-binding Fab molecule, as further described herein. The charge modifications can be performed in either the conventional Fab molecule(s) contained in the (multispecific) antibody (e.g., as shown in Figures 1A-1C, 1G-1J) or in the VH / VL crossover Fab molecule contained in the (multispecific) antibody (e.g., as shown in Figures 1D-1F, 1K-1N), but not both. In a preferred embodiment, the charge modifications are performed in the conventional Fab molecule contained in the (multispecific) antibody (which in a preferred embodiment binds to CD19).

[0143] In a preferred embodiment according to the present invention, the (multispecific) antibody is capable of simultaneously binding to CD3 and CD19. In one embodiment, the (multispecific) antibody is capable of cross-linking T cells and target cells by simultaneous binding to CD3 and CD19. In an even more preferred embodiment, such simultaneous binding results in lysis of target cells, particularly CD19-expressing target cells such as B cells. In one embodiment, such simultaneous binding results in activation of T cells. In another embodiment, such simultaneous binding results in a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, 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 CD19 does not result in activation of T cells.

[0144] In one embodiment, (multispecific) antibodies are able to redirect 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.

[0145] Preferably, the T cells according to any embodiment of the invention are cytotoxic T cells. In some embodiments, the T cells are CD4 + or CD8 + T cells, especially CD8 + T cells.

[0146] a) First antigen-binding domain The (multispecific) antibodies of the present invention comprise at least one antigen-binding domain (first antigen-binding domain) that binds to CD3. In preferred embodiments, the CD3 is human CD3 (SEQ ID NO: 45) or cynomolgus CD3 (SEQ ID NO: 46), particularly human CD3. In one embodiment, the first antigen-binding domain is cross-reactive with (i.e., specifically binds to) human and cynomolgus CD3. In some embodiments, it is a subunit of CD3 (CD3 epsilon).

[0147] In a preferred embodiment, the (multispecific) antibody comprises no more than one antigen-binding domain that binds to CD3. In one embodiment, the (multispecific) antibody provides monovalent binding to CD3.

[0148] In one embodiment, the antigen-binding domain that binds to CD3 is an antibody fragment selected from the group consisting of an Fv molecule, an scFv molecule, an Fab molecule, and an F(ab')2 molecule. In a preferred embodiment, the antigen-binding domain that binds to CD3 is a Fab molecule.

[0149] 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. In such an embodiment, the antigen-binding domain that binds to CD19 is preferably a conventional Fab molecule. In embodiments in which there are two or more antigen-binding domains that bind to CD19 contained in a (multispecific) antibody, particularly Fab molecules, the antigen-binding domain that binds to CD3 is preferably a crossover Fab molecule, and the antigen-binding domain that binds to CD19 is preferably a conventional Fab molecule.

[0150] In alternative embodiments, the antigen-binding domain that binds CD3 is a conventional Fab molecule. In such embodiments, the antigen-binding domain that binds CD19 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 are swapped / substituted for each other. In embodiments in which there are more than one antigen-binding domain that binds CD3 contained in a (multispecific) antibody, particularly Fab molecules, the antigen-binding domain that binds CD19 is preferably a crossover Fab molecule and the antigen-binding domain that binds CD3 is preferably a conventional Fab molecule.

[0151] 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 one such embodiment, the second (and, if present, the third) antigen-binding domain is a conventional Fab molecule.

[0152] In one embodiment, no more than one antigen-binding domain that binds to CD3 is present in the (multispecific) antibody (i.e., the antibody provides monovalent binding to CD3).

[0153] b) Second (and third) antigen-binding domains The (multispecific) antibody of the invention comprises at least one antigen-binding domain (second and optionally third antigen-binding domain), in particular a Fab molecule, that binds to CD19. The second antigen-binding domain can direct the (multispecific) antibody to a target site, for example a specific type of cell expressing CD19.

[0154] In one embodiment, the antigen-binding domain that binds to CD19 is an antibody fragment selected from the group consisting of an Fv molecule, an scFv molecule, a Fab molecule, and an F(ab')2 molecule. In a preferred embodiment, the antigen-binding domain that binds to CD19 is a Fab molecule.

[0155] In a particular embodiment, the (multispecific) antibody comprises two antigen-binding domains, in particular Fab molecules, that bind to CD19. In a preferred embodiment, all of these antigen-binding domains are 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 (if present) as described herein. In one embodiment, the (multispecific) antibody comprises no more than two antigen-binding domains, in particular Fab molecules, that bind to CD19.

[0156] In a preferred embodiment, the antigen-binding domain that binds to CD19 is a conventional Fab molecule. In such an 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 by each other.

[0157] In another embodiment, the antigen-binding domain that binds to CD19 is a crossover Fab molecule, i.e., a Fab molecule, as described herein, in which the variable domains VH and VL or constant domains CH1 and CL of the Fab heavy and light chains are swapped / substituted for each other. In such an embodiment, the antigen-binding domain that binds to CD3 is a conventional Fab molecule.

[0158] In one embodiment, the second (and, if present, the third) antigen-binding domain comprises a human constant region. In one embodiment, 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 given in SEQ ID NOs: 52 and 53 (human kappa and lambda CL domains, respectively), and SEQ ID NO: 54 (human IgG1 heavy chain constant domain CH1-CH2-CH3). 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: 52 or SEQ ID NO: 53, in particular the amino acid sequence of SEQ ID NO: 52. In particular, the light chain constant region may comprise the amino acid mutations described herein in a "charge-modified" state and / or, in the case of a crossover Fab molecule, one or more (in particular two) N-terminal amino acid deletions or substitutions. In some embodiments, the second (and third, if present) 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: 54. In particular, the heavy chain constant region (specifically the CH1 domain) may comprise the amino acid mutations described herein under "charge modifications."

[0159] In one aspect, the second (and third, if present) antigen binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 15, an HCDR2 of SEQ ID NO: 16, and an HCDR3 of SEQ ID NO: 17, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 19, an LCDR2 of SEQ ID NO: 20, and an LCDR3 of SEQ ID NO: 21.

[0160] In one embodiment, the second (and, if present, the third) antigen-binding domain is (derived from) a humanized antibody. In one embodiment, 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 embodiment, the VH and / or VL of the second (and, if present, the third) antigen-binding domain are humanized variable regions.

[0161] In one embodiment, the VH and / or VL of the second (and, if present, third) antigen binding domain comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0162] In one aspect, the VH of the second (and, if present, the third) antigen-binding domain comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3 and / or FR4 sequences) of SEQ ID NO: 18. 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: 18. 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 the amino acid sequence of SEQ ID NO: 18. 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 98% identical to the amino acid sequence of SEQ ID NO: 18. In certain embodiments, a VH sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising the sequence retains the ability to bind to CD19. 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: 18. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In one embodiment, the VH of the second (and, if present, the third) antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 18. Optionally, the VH of the second (and, if present, the third) antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 18, including post-translational modifications of that sequence.

[0163] In one embodiment, 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: 22. In one embodiment, 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: 22. In one embodiment, the VL of the second (and, if present, the third) antigen binding domain comprises an amino acid sequence which is at least about 95% identical to the amino acid sequence of SEQ ID NO: 22. In one embodiment, the VL of the second (and, if present, the third) antigen binding domain comprises an amino acid sequence which is at least about 98% identical to the amino acid sequence of SEQ ID NO: 22. In certain embodiments, a VL sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that sequence retains the ability to bind to CD19. 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: 22. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the 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: 22. Optionally, the VL of the second (and, if present, the third) antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 22, including post-translational modifications of that sequence.

[0164] In one aspect, the VH of the second (and if present, the third) 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: 18, and the VL of the second (and if present, the third) 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: 22. 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: 18, and the VL of the second (and if present, the third) antigen binding domain comprises the amino acid sequence of SEQ ID NO: 22.

[0165] In a further aspect, the second (and third, if present) antigen-binding domain comprises a VH comprising the sequence of SEQ ID NO: 18 and a VL comprising the sequence of SEQ ID NO: 22.

[0166] In a further aspect, the second (and third, if present) antigen-binding domain comprises the VH sequence of SEQ ID NO: 18 and the VL sequence of SEQ ID NO: 22.

[0167] In another aspect, the second (and third, if present) antigen-binding domain comprises a VH comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 18 and a VL comprising the light chain CDR sequences of the VL of SEQ ID NO: 22.

[0168] In a further aspect, the second (and third, if present) antigen-binding domain comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 18, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 22.

[0169] In one 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: 18 and a framework with at least 95%, 96%, 97%, 98% or 99% sequence identity to the framework sequences of the VH of SEQ ID NO: 18. In one 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: 18 and a framework with at least 95% sequence identity to the framework sequences of the VH of SEQ ID NO: 18. In one 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: 18 and a framework with at least 98% sequence identity to the framework sequences of the VH of SEQ ID NO: 18.

[0170] In one aspect, the VL of the second (and if present the third) antigen binding domain comprises the light chain CDR sequence of the VL of SEQ ID NO: 22 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: 22. In one aspect, the VL of the second (and if present the third) antigen binding domain comprises the light chain CDR sequence of the VL of SEQ ID NO: 22 and a framework of at least 95% sequence identity to the framework sequence of the VL of SEQ ID NO: 22. In another aspect, the VL of the second (and if present the third) antigen binding domain comprises the light chain CDR sequence of the VL of SEQ ID NO: 22 and a framework of at least 98% sequence identity to the framework sequence of the VL of SEQ ID NO: 22.

[0171] In another aspect, the second (and third, if present) antigen-binding domain comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 28, HCDR2 of SEQ ID NO: 29, and HCDR3 of SEQ ID NO: 30, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO: 32, LCDR2 of SEQ ID NO: 33, and LCDR3 of SEQ ID NO: 34.

[0172] In one embodiment, the second (and, if present, the third) antigen-binding domain is (derived from) a humanized antibody. In one embodiment, 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 embodiment, the VH and / or VL of the second (and, if present, the third) antigen-binding domain are humanized variable regions.

[0173] In one embodiment, the VH and / or VL of the second (and, if present, third) antigen binding domain comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0174] In one aspect, the VH of the second (and, if present, the third) antigen binding domain comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3 and / or FR4 sequences) of SEQ ID NO: 31. 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. 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 the amino acid sequence of SEQ ID NO: 31. 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 98% identical to the amino acid sequence of SEQ ID NO: 31. In certain embodiments, a VH sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising the sequence retains the ability to bind to CD19. 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. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In one embodiment, the VH of the second (and, if present, the third) antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 31. Optionally, the VH of the second (and, if present, the third) antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 31, including post-translational modifications of that sequence.

[0175] 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: 35. In one aspect, 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: 35. In one aspect, the VL of the second (and, if present, the third) antigen binding domain comprises an amino acid sequence which is at least about 95% identical to the amino acid sequence of SEQ ID NO: 35. In one aspect, the VL of the second (and, if present, the third) antigen binding domain comprises an amino acid sequence which is at least about 98% identical to the amino acid sequence of SEQ ID NO: 35. In certain embodiments, a VL sequence having at least about 95%, about 96%, about 97%, about 98%, or about 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an antibody comprising that sequence retains the ability to bind to CD19. 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: 35. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the 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: 35. Optionally, the VL of the second (and, if present, the third) antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 35, including post-translational modifications of that sequence.

[0176] In one aspect, the VH of the second (and if present, the third) 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: 31 and the VL of the second (and if present, the third) 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: 35. 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 and the VL of the second (and if present, the third) antigen binding domain comprises the amino acid sequence of SEQ ID NO: 35.

[0177] In a further aspect, the second (and third, if present) antigen-binding domain comprises a VH comprising the sequence of SEQ ID NO: 31 and a VL comprising the sequence of SEQ ID NO: 35.

[0178] In a further aspect, the second (and third, if present) antigen-binding domain comprises the VH sequence of SEQ ID NO: 31 and the VL sequence of SEQ ID NO: 35.

[0179] In another aspect, the second (and third, if present) antigen-binding domain comprises a VH comprising the heavy chain CDR sequences of the VH of SEQ ID NO: 31 and a VL comprising the light chain CDR sequences of the VL of SEQ ID NO: 35.

[0180] In a further aspect, the second (and third, if present) antigen-binding domain comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 31, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 35.

[0181] In one 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 and a framework with at least 95%, 96%, 97%, 98% or 99% sequence identity to the framework sequences of the VH of SEQ ID NO: 31. In one 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 and a framework with at least 95% sequence identity to the framework sequences of the VH of SEQ ID NO: 31. In one 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 and a framework with at least 98% sequence identity to the framework sequences of the VH of SEQ ID NO: 31.

[0182] In one aspect, the VL of the second (and if present the third) antigen binding domain comprises the light chain CDR sequence of the VL of SEQ ID NO: 35 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: 35. In one aspect, the VL of the second (and if present the third) antigen binding domain comprises the light chain CDR sequence of the VL of SEQ ID NO: 35 and a framework of at least 95% sequence identity to the framework sequence of the VL of SEQ ID NO: 35. In another aspect, the VL of the second (and if present the third) antigen binding domain comprises the light chain CDR sequence of the VL of SEQ ID NO: 35 and a framework of at least 98% sequence identity to the framework sequence of the VL of SEQ ID NO: 35.

[0183] c) Charge modification The (multispecific) antibodies of the present invention may contain, in the Fab molecules contained therein, amino acid substitutions that are particularly efficient in reducing mispairing of light chains with incompatible heavy chains (Bence-Jones by-products) that can occur in the generation of Fab-based multispecific antibodies with a VH / VL exchange in one (or more, in the case of molecules containing more than two antigen-binding Fab molecules) binding arm (see also WO2015 / 150447, in particular the Examples therein, which is incorporated herein by reference in its entirety). The ratio of the desired (multispecific) antibody to undesired by-products, in particular the Bence-Jones by-products that occur in multispecific antibodies with a VH / VL domain exchange in one of the binding arms, can be improved by introducing charged amino acids with an opposite charge to specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modifications").

[0184] Thus, in some aspects, the first and second (and, if present, 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 replace 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 positively charged 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 negatively charged 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 positively charged amino acid (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 substituted by a negatively charged amino acid (Kabat EU index numbering).

[0185] A (multispecific) antibody does not contain both of the modifications described in i) and ii): the constant domains CL and CH1 of the antigen-binding domain with a VH / VL exchange are not replaced by each other (i.e. remain unexchanged).

[0186] In a more specific aspect, 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 independently 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 substituted independently 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).

[0187] In one such 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 independently by lysine (K), arginine (R) or histidine (H) (Kabat numbering) and in the constant domain CHI 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 independently by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0188] In a further aspect, 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, 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).

[0189] 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 CHI 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).

[0190] 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) and 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).

[0191] 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 lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat); and in the constant domain 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).

[0192] In a preferred embodiment, when the amino acid substitutions according to the above aspects are made in the constant domain CL and 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.

[0193] Alternatively, the amino acid substitutions according to the above embodiments are made in the constant domains CL and CH1 of the first antigen-binding domain instead of the constant domains CL and CH1 of the second (and, if present, third) antigen-binding domain. In a preferred such embodiment, the constant domain CL of the first antigen-binding domain is of the kappa isotype.

[0194] Thus, in one aspect, in the constant domain CL of the first antigen-binding domain the amino acid at position 124 is substituted independently 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 substituted independently by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0195] In a further aspect, in the constant domain CL of said 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 said 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).

[0196] 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 CHI 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).

[0197] In one embodiment, in the constant domain CL of said 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 a lysine (K) (numbering according to Kabat), and in the constant domain CHI of said 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).

[0198] In another embodiment, in the constant domain CL of said first 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 CHI of said 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).

[0199] In a preferred embodiment, the (multispecific) antibody of the present invention (a) a first antigen-binding domain that binds to CD3, the first antigen-binding domain being a Fab molecule, wherein the Fab light chain and Fab heavy chain variable domains VL and VH are replaced with each other, and the first antigen-binding domain comprises 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: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, an LCDR2 of SEQ ID NO: 9, and an LCDR3 of SEQ ID NO: 10; (b) a second and optionally a third antigen-binding domain that binds to CD19; Including, In the constant domain CL of the second (and, if present, the third) antigen-binding domain, the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (in a preferred embodiment, independently by lysine (K) or arginine (R)) (Kabat numbering), and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (in a preferred embodiment, independently by lysine (K) or arginine (R)) (Kabat numbering); in the constant domain CHI of the second (and, if present, the third) antigen-binding domain, the amino acid at position 147 is substituted independently by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering), and the amino acid at position 213 is substituted independently by glutamic acid (E) or aspartic acid (D) (Kabat EU index numbering).

[0200] d) Multispecific antibody formats The (multispecific) antibodies according to the invention can have a variety of structures, exemplary configurations are shown in Figure 1.

[0201] In a preferred embodiment, the antigen-binding domains comprised in the (multispecific) antibody are Fab molecules. In such embodiments, the first, second, third, etc. antigen-binding domains may be referred to herein as the first, second, third, etc. Fab molecules, respectively.

[0202] 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 one such embodiment, the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain. In another such embodiment, the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain. In embodiments where (i) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain, or (ii) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, 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.

[0203] Multispecific antibodies with a single antigen-binding domain (such as a Fab molecule) capable of specifically binding to a second antigen, e.g., a target cell antigen such as CD19 (e.g., as shown in Figures 1A, 1D, and 1G), are particularly useful when internalization of the second antigen is expected following 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 enhance internalization of the second antigen, thereby reducing the availability of the second antigen.

[0204] 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 CD19 (see examples shown in Figure 1B, Figure 1C, Figure 1E, Figure 1F, Figure 1I, Figure 1J, Figure 1M or Figure 1N), e.g., to optimize targeting to the target site or to allow cross-linking of target cell antigens.

[0205] Therefore, in a preferred embodiment, the (multispecific) antibody according to the invention comprises a third antigen-binding domain.

[0206] In one embodiment, the third antigen-binding domain binds to CD 19. In one embodiment, the third antigen-binding domain is a Fab molecule.

[0207] In one embodiment, the third antigen-binding domain is identical to the second antigen-binding domain.

[0208] In some embodiments, the third and second antigen-binding domains are each a Fab molecule, 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, if present, comprises the same amino acid substitutions as the second antigen-binding domain. For example, the amino acid substitutions described herein as "charge-engineered" are made in the constant domains CL and CHI of the second and third antigen-binding domains, respectively. Alternatively, the amino acid substitutions can be made in the constant domains CL and CHI of the first antigen-binding domain (which in preferred embodiments is also a Fab molecule), but not in the constant domains CL and CHI of the second and third antigen-binding domains.

[0209] 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 have been 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.

[0210] 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 CD19.

[0211] 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.

[0212] The (multispecific) antibodies according to the invention can have different structures, i.e. the first, second (and optionally third) antigen-binding domains may be fused to each other and to the Fc domain in different ways. The components may be fused to each other directly or, preferably, via one or more suitable peptide linkers. When the fusion of a Fab molecule is to the N-terminus of a subunit of the Fc domain, it is typically via the immunoglobulin hinge region.

[0213] In some embodiments, the first and second antigen-binding domains are each a Fab molecule, 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 Fc domain subunit. In such embodiments, the second antigen-binding domain may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain or to the N-terminus of the other Fc domain subunit. 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.

[0214] 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 Fc domain subunit, 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 first and second Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, with the second Fab molecule 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 fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fc domain of the first or second subunit. Such structures are depicted schematically 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 may additionally be fused to each other.

[0215] 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 of the Fc domain subunits. In a specific embodiment, the (multispecific) antibody consists essentially of first and second Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, and the first and second Fab molecules are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits. Such structures are depicted schematically 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 second Fab molecules may be fused to the Fc domain directly or via a peptide linker. In a preferred embodiment, the first and second Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, particularly where the Fc domain is an IgG1 Fc domain.

[0216] In some embodiments, the first and second antigen-binding domains are each a Fab molecule, and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second Fc domain subunit. In such embodiments, the first antigen-binding domain may be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain or to the N-terminus of the other Fc domain subunit (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. 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.

[0217] In one embodiment, the first and second antigen-binding domains are each Fab molecules, with the second 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 first antigen-binding domain 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 first and second Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, with the first Fab molecule 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 fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fc domain of the first or second subunit. Such structures are depicted schematically 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 may additionally be fused to each other.

[0218] In some embodiments, the third antigen-binding domain, particularly the third Fab molecule, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second Fc domain subunit. 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. 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.

[0219] In preferred such embodiments, the first and third antigen-binding domains are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In a specific embodiment, the (multispecific) antibody consists essentially of first, second and third Fab molecules, 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 the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such structures are depicted schematically in Figures 1B and 1E (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 Figures 1J and 1N (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 first and third Fab molecules may be fused to an Fc domain directly or via a peptide linker. In a preferred embodiment, the first and third Fab molecules are each fused to an Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, particularly when the Fc domain is an IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.

[0220] 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 of the subunits 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 first, second and third Fab molecules, 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 its 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 its 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 its Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such structures are depicted schematically 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 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 specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, particularly when the Fc domain is an IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may additionally be fused to each other.

[0221] In the structure of a (multispecific) antibody in which the Fab molecules are fused via an immunoglobulin hinge region to the C-terminus of the Fab heavy chain and the N-terminus of each of the Fc domain subunits, 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 IgG class immunoglobulin. In an even more preferred form, the immunoglobulin is an IgG1 subclass immunoglobulin. In another embodiment, the immunoglobulin is an IgG4 subclass immunoglobulin. 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.

[0222] In some of the (multispecific) antibodies of the invention, the Fab light chain of a first Fab molecule and the Fab light chain of a 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 may be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. Fusing the Fab light chains of the first and second Fab molecules further reduces mispairing of incompatible Fab heavy and light chains and also reduces the number of plasmids required to express some of the (multispecific) antibodies of the invention.

[0223] The antigen-binding domains may be fused to the Fc domain or to each other 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 are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S) n , G4(SG4) n , or (G4S) nG5 peptide linkers are included. "n" is generally an integer between 1 and 10, typically between 2 and 4. In one embodiment, the peptide linker has a length of at least 5 amino acids, in one embodiment between 5 and 100 amino acids, and in a further embodiment between 10 and 50 amino acids. In one embodiment, the peptide linker is (GxS) n or (GxS) n G m wherein G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=1, 2, 3, 4 or 5, and m=0, 1, 2, 3, 4 or 5); in one embodiment, x=4 and n=2 or 3; in a further embodiment, x=4 and n=2; and in yet another embodiment, x=4, n=1, and m=5. In one embodiment, the peptide linker is (G4S)2. In another embodiment, the peptide linker is G4SG5. A peptide linker particularly suitable for fusing the Fab light chains of the first and second Fab molecules to each other is (G4S)2. An exemplary peptide linker suitable for connecting the Fab heavy chains of the first and second Fab fragments comprises the sequence (D)-(G4S)2 (SEQ ID NOs: 48 and 49). Another particularly suitable such linker comprises the sequence (D)-G4SG5 (SEQ ID NOs: 50 and 51). In addition, 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 the immunoglobulin hinge region or part thereof, with or without an additional peptide linker.

[0224] In certain embodiments, 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 replaces the light chain variable region), and a Fab heavy chain constant region of a first Fab molecule that shares a carboxy-terminal peptide bond with an Fc domain subunit (VL (1) -CH1 (1)-CH2-CH3(-CH4)) polypeptide, and the Fab heavy chain of the second Fab molecule share a carboxy-terminal peptide bond with the Fc domain subunit (VH (2) -CH1 (2) In some embodiments, the (multispecific) antibody comprises a polypeptide (VH) 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 (VL (2) -CL (2) In some embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0225] In certain embodiments, 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 replaces the light chain constant region), and a Fab light chain constant region of the first Fab molecule that shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (1) -CL (1) -CH2-CH3(-CH4)) polypeptide, and the Fab heavy chain of the second Fab molecule share a carboxy-terminal peptide bond with the Fc domain subunit (VH (2) -CH1 (2) In some embodiments, the (multispecific) antibody comprises a polypeptide (VL) 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 (VL (2) -CL (2) In some embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0226] In some embodiments, the (multispecific) antibody 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, where the heavy chain variable region replaces the light chain variable region), a Fab heavy chain constant region of the first Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule, and a Fab heavy chain of the second Fab molecule that 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 Fab heavy chain of the second Fab molecule that shares a carboxy-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, a Fab light chain variable region of the first Fab molecule that 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, with the heavy chain variable region replacing the light chain variable region), and a Fab heavy chain constant region of the first Fab molecule that 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 (VL (2) -CL (2)In other of these embodiments, where appropriate, the (multispecific) antibody further 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 the first Fab molecule, and a Fab light chain constant region of the first Fab molecule that shares a carboxy-terminal peptide bond with a Fab light chain polypeptide of a second Fab molecule (VH (1) -CL (1) -VL (2) -CL (2) ) polypeptide, or 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, and 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 (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 some embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0227] In some embodiments, the (multispecific) antibody 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, where the heavy chain constant region replaces the light chain constant region), a Fab light chain constant region of the first Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule, and a Fab heavy chain of the second Fab molecule that 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 Fab heavy chain of the second Fab molecule that shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, a Fab heavy chain variable region of the first Fab molecule that 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, where the heavy chain constant region replaces the light chain constant region), and a Fab light chain constant region of the first Fab molecule that 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 (VL (2) -CL (2) In other of these embodiments, where appropriate, the (multispecific) antibody further 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, and a Fab heavy chain constant region of a first Fab molecule that shares a carboxy-terminal peptide bond with a Fab light chain polypeptide of a second Fab molecule (VL (1) -CH1 (1) -VL (2) -CL (2) ) polypeptide, or 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, and 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 (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 some embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0228] In some embodiments, the (multispecific) antibody does not comprise an Fc domain. In preferred such embodiments, the 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 swapped / replaced. In other such embodiments, the second and, if present, third antigen-binding domains, respectively, are crossover Fab molecules, and the first antigen-binding domain is a conventional Fab molecule.

[0229] In one such embodiment, the (multispecific) antibody consists essentially of a first and a second antigen-binding domain and optionally one or more peptide linkers, wherein 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 a structure is depicted schematically 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).

[0230] In another such embodiment, the (multispecific) antibody consists essentially of first and second antigen-binding domains and, optionally, one or more peptide linkers, where the first and second antigen-binding domains 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 structures are depicted schematically 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).

[0231] 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 consists essentially of first, second and third Fab molecules 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 depicted schematically 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).

[0232] 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 consists essentially of first, second and third Fab molecules 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 depicted schematically 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).

[0233] In certain embodiments, the (multispecific) antibody according to the invention comprises a Fab heavy chain of the second Fab molecule that shares a carboxy-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, and a Fab light chain variable region of the first Fab molecule that 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 the light chain variable region) (VH (2) -CH1 (2) -VL (1) -CH1 (1) In some embodiments, the (multispecific) antibody 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 the first Fab molecule. (1) -CL (1) ) and the Fab light chain polypeptide (VL (2) -CL (2) ) further includes.

[0234] In certain embodiments, the (multispecific) antibody according to 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), and a Fab heavy chain constant region of a first Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule (VL (1) -CH1 (1) -VH (2) -CH1 (2) In some embodiments, the (multispecific) antibody 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 the first Fab molecule. (1) -CL (1) ) and the Fab light chain polypeptide (VL (2) -CL (2) ) further includes.

[0235] In certain embodiments, the (multispecific) antibody according to the invention comprises a Fab heavy chain of the second Fab molecule that shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, and a Fab heavy chain variable region of the first Fab molecule that 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) (VH (2) -CH1 (2) -VH (1) -CL (1) In some embodiments, the (multispecific) antibody 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 the first Fab molecule. (1) -CH1 (1) ) and the Fab light chain polypeptide (VL (2) -CL (2) ) further includes.

[0236] In certain embodiments, the (multispecific) antibody according to 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), and a Fab light chain constant region of the first Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule (VH (1) -CL (1) -VH (2) -CH1 (2) In some embodiments, the (multispecific) antibody 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 the first Fab molecule. (1) -CH1 (1) ) and the Fab light chain polypeptide (VL (2) -CL (2) ) further includes.

[0237] In certain embodiments, the (multispecific) antibody according to the invention comprises a Fab heavy chain of a third Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule, a Fab heavy chain of a second Fab molecule that shares a carboxy-terminal peptide bond with a Fab light chain variable region of a first Fab molecule, and a Fab light chain variable region of the first Fab molecule that shares a carboxy-terminal peptide bond with a 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) (VH (3) -CH1 (3) -VH (2) -CH1 (2) -VL (1) -CH1 (1) In some embodiments, the (multispecific) antibody 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 the first Fab molecule. (1) -CL (1)) and the Fab light chain polypeptide (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) ) further includes.

[0238] In certain embodiments, the (multispecific) antibody according to the invention comprises a Fab heavy chain of a third Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule, a Fab heavy chain of a second Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain variable region of a first Fab molecule, and a Fab heavy chain variable region of the first Fab molecule that shares a carboxy-terminal peptide bond with a 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) (VH (3) -CH1 (3) -VH (2) -CH1 (2) -VH (1) -CL (1) In some embodiments, the (multispecific) antibody 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 the first Fab molecule. (1) -CH1 (1) ) and the Fab light chain polypeptide (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) ) further includes.

[0239] In certain embodiments, the (multispecific) antibody according to 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 replaces the light chain variable region), a Fab heavy chain constant region of the first Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule, and a Fab heavy chain of the second Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain of a third Fab molecule (VL (1) -CH1 (1) -VH (2) -CH1 (2) -VH (3) -CH1 (3) In some embodiments, the (multispecific) antibody 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 the first Fab molecule. (1) -CL (1) ) and the Fab light chain polypeptide (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) ) further includes.

[0240] In certain embodiments, the (multispecific) antibody according to 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, where the heavy chain constant region replaces the light chain constant region), a Fab light chain constant region of the first Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain of a second Fab molecule, and a Fab heavy chain of the second Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain of a third Fab molecule (VH (1) -CL (1) -VH (2) -CH1 (2) -VH (3) -CH1 (3)In some embodiments, the (multispecific) antibody 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 the first Fab molecule. (1) -CH1 (1) ) and the Fab light chain polypeptide (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) ) further includes.

[0241] In certain embodiments, the (multispecific) antibody according to the invention comprises a Fab heavy chain of a first Fab molecule that shares a carboxy-terminal peptide bond with a Fab light chain variable region of a second Fab molecule, a Fab light chain variable region of the second Fab molecule that 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, where the heavy chain variable region replaces the light chain variable region), a Fab heavy chain constant region of the second Fab molecule that shares a carboxy-terminal peptide bond with a Fab light chain variable region of a third Fab molecule, and a Fab light chain variable region of the third Fab molecule that 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, where the heavy chain variable region replaces the light chain variable region) (VH (1) -CH1 (1) -VL (2) -CH1 (2) -VL (3) -CH1 (3) In some embodiments, the (multispecific) antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL (1) -CL (1)In some embodiments, the (multispecific) antibody further comprises a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule. (3) -CL (3) ) further includes.

[0242] In certain embodiments, the (multispecific) antibody according to the invention comprises a Fab heavy chain of a first Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain variable region of a second Fab molecule, a Fab heavy chain variable region of the second Fab molecule that shares a carboxy-terminal peptide bond with a Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, where the heavy chain constant region is replaced by a light chain constant region), a Fab light chain constant region of the second Fab molecule that shares a carboxy-terminal peptide bond with a Fab heavy chain variable region of a third Fab molecule, and a Fab heavy chain variable region of the third Fab molecule that 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, where the heavy chain constant region is replaced by a light chain constant region) (VH (1) -CH1 (1) -VH (2) -CL (2) -VH (3) -CL (3) In some embodiments, the (multispecific) antibody comprises a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule. (2) -CH1 (2) ) and the Fab light chain polypeptide (VL (1) -CL (1) In some embodiments, the (multispecific) antibody further comprises a polypeptide (VL) in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule. (3) -CH1 (3) ) further includes.

[0243] In certain embodiments, the (multispecific) antibody according to the invention comprises a Fab light chain variable region of a third Fab molecule that 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, where the heavy chain variable region is replaced by a light chain variable region), a Fab heavy chain constant region of a third Fab molecule that shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, a Fab light chain variable region of a second Fab molecule that shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, where the heavy chain variable region is replaced by a light chain variable region), and a Fab heavy chain constant region of a second Fab molecule that shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule (VL (3) -CH1 (3) -VL (2) -CH1 (2) -VH (1) -CH1 (1) In some embodiments, the (multispecific) antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL (1) -CL (1) In some embodiments, the (multispecific) antibody further comprises a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule. (3) -CL (3) ) further includes.

[0244] In certain embodiments, the (multispecific) antibody according to the invention comprises a Fab heavy chain variable region of a third Fab molecule that 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, where the heavy chain constant region is replaced by a light chain constant region), a Fab light chain constant region of the third Fab molecule that shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, a Fab heavy chain variable region of the second Fab molecule that shares a carboxy-terminal peptide bond with the Fab light chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, where the heavy chain constant region is replaced by a light chain constant region), and a Fab light chain constant region of the second Fab molecule that shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule (VH (3) -CL (3) -VH (2) -CL (2) -VH (1) -CH1 (1) In some embodiments, the (multispecific) antibody comprises a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule. (2) -CH1 (2) ) and the Fab light chain polypeptide (VL (1) -CL (1) In some embodiments, the (multispecific) antibody further comprises a polypeptide (VL) in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule. (3) -CH1 (3) ) further includes.

[0245] In one aspect, the present invention provides a method for producing a pharmaceutical composition 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 or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain 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: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, an LCDR2 of SEQ ID NO: 9, and an LCDR3 of SEQ ID NO: 10; b) a second antigen-binding domain that binds to CD19, the second antigen-binding domain being a (conventional) Fab molecule; c) an Fc domain composed of the first and second subunits; Including, (i) the first antigen-binding domain under 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 under b), and the second antigen-binding domain under 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 under c); or (ii) The second antigen-binding domain under b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain under a), and the first antigen-binding domain under a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under c).

[0246] In a preferred embodiment, the present invention provides a (multispecific) antibody comprising: a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, and the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and Fab heavy chain are substituted for each other, and the first antigen-binding domain comprises 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: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, an LCDR2 of SEQ ID NO: 9, and an LCDR3 of SEQ ID NO: 10; b) a second and a third antigen-binding domain that binds to CD19, wherein the second and third antigen-binding domains are each (conventional) Fab molecules; c) an Fc domain composed of the first and second subunits; Including, (i) the first antigen-binding domain under 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 under b), and the second antigen-binding domain under b) and the third antigen-binding domain under b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under c); or (ii) The second antigen-binding domain under b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain under a), and the first antigen-binding domain under a) and the third antigen-binding domain under b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under c).

[0247] In another aspect, the present invention provides a (multispecific) antibody 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 or the constant domains CL and CH1 of the Fab light chain and the Fab heavy chain 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: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, an LCDR2 of SEQ ID NO: 9, and an LCDR3 of SEQ ID NO: 10; b) a second antigen-binding domain that binds to CD19, the second antigen-binding domain being a (conventional) Fab molecule; c) an Fc domain consisting of a first and a second subunit; and a bispecific antibody comprising: (i) A (multispecific) antibody is provided in which a) a first antigen-binding domain and b) a second antigen-binding domain are fused at the C-terminus of the Fab heavy chain and to the N-terminus of one of the Fc domain subunits of c).

[0248] In all the different structures of the (multispecific) antibodies according to the invention, the amino acid substitutions ("charge modifications") described herein may, if present, be in either the CH1 and CL domains of the second and (if present) third antigen-binding domain / Fab molecule, or in the CH1 or CL domain 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. In accordance with the concept of the present invention, when an amino acid substitution described herein is made in the second (and, if present, third) antigen-binding domain / Fab molecule, such an amino acid substitution is not made in the first antigen-binding domain / Fab molecule. Conversely, when an amino acid substitution described herein is made in the first antigen-binding domain / Fab molecule, such an amino acid substitution is 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.

[0249] In preferred embodiments of the (multispecific) antibodies according to the invention, particularly 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 other embodiments of the (multispecific) antibodies according to the invention, particularly 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 and the constant domain CL of the first antigen-binding domain / Fab molecule are of the kappa isotype.

[0250] In one aspect the invention provides a (multispecific) antibody comprising: a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, and the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted with each other, and the first antigen-binding domain comprises 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: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, an LCDR2 of SEQ ID NO: 9, and an LCDR3 of SEQ ID NO: 10; b) a second antigen-binding domain that binds to CD19, the second antigen-binding domain being a (conventional) Fab molecule; c) an Fc domain consisting of a first and a second subunit; Including, in the constant domain CL of said 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 said 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 under 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 under b), and the second antigen-binding domain under 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 under c); or (ii) The second antigen-binding domain under b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain under a), and the first antigen-binding domain under a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under c). In a preferred embodiment, the present invention provides a (multispecific) antibody comprising:

[0251] a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, and the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted with each other, and the first antigen-binding domain comprises 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: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, an LCDR2 of SEQ ID NO: 9, and an LCDR3 of SEQ ID NO: 10; b) a second and a third antigen-binding domain that binds to CD19, wherein the second and third antigen-binding domains are each (conventional) Fab molecules; c) an Fc domain consisting of a first and a second subunit; Including, in the constant domain CL of the second antigen-binding domain of b) and 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); in the constant domain CHI of the second antigen-binding domain of b) and 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 under 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 under b), and the second antigen-binding domain under b) and the third antigen-binding domain under b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under c); or (ii) The second antigen-binding domain under b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain under a), and the first antigen-binding domain under a) and the third antigen-binding domain under b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain under c).

[0252] In another aspect, the present invention provides a (multispecific) antibody 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, an HCDR2 of SEQ ID NO: 3, and an HCDR3 of SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, an LCDR2 of SEQ ID NO: 9, and an LCDR3 of SEQ ID NO: 10; b) a second antigen-binding domain that binds to CD19, the second antigen-binding domain being a (conventional) Fab molecule; c) an Fc domain consisting of a first and a second subunit; Including, in the constant domain CL of said 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 said 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 fused at the C-terminus of the Fab heavy chain and to the N-terminus of one of the Fc domain subunits of c).

[0253] According to any of the above embodiments, the components of the (multispecific) antibody (e.g., Fab molecules, Fc domains) may be fused directly or via various linkers, in particular via 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, (G4S) n , (SG4) n, (G4S) n or G4 (SG4) n A peptide linker is included, and "n" is usually an integer from 1 to 10, typically from 2 to 4.

[0254] In a preferred embodiment, the present invention provides a (multispecific) antibody 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, an HCDR2 of SEQ ID NO: 3, and an HCDR3 of SEQ ID NO: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, an LCDR2 of SEQ ID NO: 9, and an LCDR3 of SEQ ID NO: 10; b) second and third antigen-binding domains that bind to CD19, each of the second and third antigen-binding domains being a (conventional) Fab molecule and comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 15, an HCDR2 of SEQ ID NO: 16 and an HCDR3 of SEQ ID NO: 17, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 19, an LCDR2 of SEQ ID NO: 20 and an LCDR3 of SEQ ID NO: 21; c) an Fc domain consisting of a first and a second subunit; in the constant domain CL of the second and third antigen-binding domains 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 domains 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); and further, the second antigen-binding domain of b) is fused to the N-terminus of the Fab heavy chain of the first antigen-binding domain of a), at the C-terminus of the Fab heavy chain, and the first antigen-binding domain of a) and the third antigen-binding domain of b) are each fused to the N-terminus of one of the subunits of the Fc domain of c), at the C-terminus of the Fab heavy chain.

[0255] In a further preferred embodiment, the present invention provides 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: 11; b) second and third antigen-binding domains that bind to CD19, each of which is a (conventional) Fab molecule and comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; and c) an Fc domain consisting of a first and a second subunit; in the constant domain CL of the second and third antigen-binding domains 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 domains 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); And further, the second antigen-binding domain of b) is fused to the N-terminus of the Fab heavy chain of the first antigen-binding domain of a), at the C-terminus of the Fab heavy chain, and the first antigen-binding domain of a) and the third antigen-binding domain of b) are each fused to the N-terminus of one of the subunits of the Fc domain of c), at the C-terminus of the Fab heavy chain.

[0256] In a further preferred embodiment, the present invention provides 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: 5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, an LCDR2 of SEQ ID NO: 9, and an LCDR3 of SEQ ID NO: 10; b) second and third antigen-binding domains that bind to CD19, each of the second and third antigen-binding domains being a (conventional) Fab molecule and comprising a heavy chain variable region (VH) comprising 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, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 32, an LCDR2 of SEQ ID NO: 33 and an LCDR3 of SEQ ID NO: 34; c) an Fc domain consisting of a first and a second subunit; in the constant domain CL of the second and third antigen-binding domains 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 domains 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); And further, the second antigen-binding domain of b) is fused to the N-terminus of the Fab heavy chain of the first antigen-binding domain of a), at the C-terminus of the Fab heavy chain, and the first antigen-binding domain of a) and the third antigen-binding domain of b) are each fused to the N-terminus of one of the subunits of the Fc domain of c), at the C-terminus of the Fab heavy chain.

[0257] In a further preferred embodiment, the present invention provides a (multispecific) antibody 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: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; b) second and third antigen-binding domains that bind to CD19, each of which is a (conventional) Fab molecule and comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 35; c) an Fc domain consisting of a first and a second subunit; in the constant domain CL of the second and third antigen-binding domains 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 domains 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); And further, the second antigen-binding domain of b) is fused to the N-terminus of the Fab heavy chain of the first antigen-binding domain of a), at the C-terminus of the Fab heavy chain, and the first antigen-binding domain of a) and the third antigen-binding domain of b) are each fused to the N-terminus of one of the subunits of the Fc domain of c), at the C-terminus of the Fab heavy chain.

[0258] 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).

[0259] In a further embodiment according to these aspects of the invention, the first subunit of the Fc domain additionally has the serine residue at position 354 replaced by a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 replaced by a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 replaced by a cysteine ​​residue), and the second subunit of the Fc domain additionally has the tyrosine residue at position 349 replaced by a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index).

[0260] 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).

[0261] In a further embodiment according to these aspects of the invention, the Fc domain is a human IgG1 Fc domain.

[0262] In preferred specific embodiments, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence which is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 23 or SEQ ID NO: 39 (in particular SEQ ID NO: 39), a polypeptide comprising an amino acid sequence which is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 24, a polypeptide (in particular two polypeptides) comprising an amino acid sequence which is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 25, and a polypeptide comprising an amino acid sequence which is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 27. In further preferred specific embodiments, the (multispecific) antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 39 (in particular SEQ ID NO: 39), a polypeptide comprising the amino acid sequence of SEQ ID NO: 24, a polypeptide (in particular two polypeptides) comprising the amino acid sequence of SEQ ID NO: 25, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.

[0263] In one aspect, the invention provides (multispecific) antibodies that bind to CD3 and CD19, 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: 23 or SEQ ID NO: 39 (in particular SEQ ID NO: 39), 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: 24, a polypeptide (in particular two polypeptides) that comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 25, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 27. In one aspect, the present invention provides (multispecific) antibodies that bind to CD3 and CD19, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 39 (particularly SEQ ID NO: 39), a polypeptide comprising the amino acid sequence of SEQ ID NO: 24, a polypeptide (particularly two polypeptides) comprising the amino acid sequence of SEQ ID NO: 25, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.

[0264] In preferred specific embodiments, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence which is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 36 or SEQ ID NO: 40 (particularly SEQ ID NO: 36), a polypeptide comprising an amino acid sequence which is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 37, a polypeptide (particularly two polypeptides) comprising an amino acid sequence which is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 38, and a polypeptide comprising an amino acid sequence which is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 27. In further preferred specific embodiments, the (multispecific) antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 40 (particularly SEQ ID NO: 36), a polypeptide comprising the amino acid sequence of SEQ ID NO: 37, a polypeptide (particularly two polypeptides) comprising the amino acid sequence of SEQ ID NO: 38, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.

[0265] In one aspect, the present invention provides (multispecific) antibodies that bind to CD3 and CD19, 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: 36 or SEQ ID NO: 40 (particularly SEQ ID NO: 36), 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: 37, a polypeptide (particularly two polypeptides) that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 38, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 27. In one aspect, the present invention provides (multispecific) antibodies that bind to CD3 and CD19, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 40 (particularly SEQ ID NO: 36), a polypeptide comprising the amino acid sequence of SEQ ID NO: 37, a polypeptide (particularly two polypeptides) that comprises the amino acid sequence of SEQ ID NO: 38, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 27.

[0266] 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.

[0267] 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, with each subunit comprising 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 present invention does not comprise more than one Fc domain.

[0268] In one embodiment, the Fc domain of the (multispecific) antibody is an IgG Fc domain. In a preferred embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228, in particular the amino acid substitution S228P (Kabat EU index numbering). This amino acid substitution reduces Fab arm exchange of IgG4 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 IgG1 Fc domain. An exemplary sequence of a human IgG1 Fc region is shown in SEQ ID NO: 47.

[0269] a) Fc domain modifications that promote heterodimerization The (multispecific) antibodies according to the invention comprise different antigen-binding domains that can be fused to one or the other of the two subunits of the Fc domain, which are therefore 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. To improve the yield and purity of (multispecific) antibodies in recombinant production, it is advantageous to introduce modifications in the Fc domain of the (multispecific) antibody that promote the association of the desired polypeptides.

[0270] 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 longest site of protein-protein interaction between the two subunits of a human IgG Fc domain is within the CH3 domain of the Fc domain. Thus, in one embodiment, said modification is in the CH3 domain of the Fc domain.

[0271] Several approaches exist for modifications in the CH3 domain of the Fc domain to enhance heterodimerization, and are fully described, for example, in WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012058768, WO2013157954, WO2013096291. Typically, in all such approaches, the CH3 domains of the first Fc subunit and the second Fc subunit are designed to be complementary to each other, such that each CH3 domain (or its constituent heavy chain) is unable to homodimerize with itself but is forced to heterodimerize with the complementary engineered CH3 domain (i.e., the first and second CH3 domains heterodimerize, preventing homodimerization between the two first or second CH3 domains). These different approaches to improved heavy chain heterodimerization are considered as different alternatives in combination with heavy-light chain modifications in (multispecific) antibodies to reduce heavy / light chain mispairing and Bence-Jones side effects (e.g., swapping / replacing VH and VL in one binding arm and introducing oppositely charged amino acid substitutions at the CH1 / CL interface).

[0272] 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.

[0273] 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). Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine).

[0274] Thus, in a preferred embodiment, in the CH3 domain of a first subunit of the 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 positioned in a cavity in the CH3 domain of a 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 into which the protrusion in the CH3 domain of the first subunit can be positioned.

[0275] Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

[0276] Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V).

[0277] The protuberances and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0278] In a specific embodiment, in the (CH3 domain of) the first subunit ("knob" subunit) of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the (CH3 domain of) the second subunit ("hole" subunit) of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, additionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0279] In a further embodiment, the first subunit of the Fc domain additionally 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 additionally 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 results in the formation of disulfide bridges between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0280] 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).

[0281] In a preferred embodiment, an antigen-binding domain that binds CD3 is fused (optionally via a second antigen-binding domain that binds CD19 and / or a peptide linker) to a first subunit of an Fc domain (comprising a "knob" modification). Without being bound by theory, the fusion of an 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).

[0282] Other techniques for CH3 modifications that enhance heterodimerization are contemplated as alternatives of the present invention and are described, for example, in WO96 / 27011, WO98 / 050431, EP1870459, WO2007 / 110205, WO2007 / 147901, WO2009 / 089004, WO2010 / 129304, WO2011 / 90754, WO2011 / 143545, WO2012 / 058768, WO2013 / 157954, WO2013 / 096291.

[0283] In one embodiment, the heterodimerization technique described in EP 1 870 459 is used instead. This technique is based on the introduction of oppositely charged amino acids 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 comprises the amino acid mutations R409D, K370E in one of the two CH3 domains (of the Fc domain) and D399K, E357K in the other CH3 domain of the Fc domain (numbering according to the Kabat EU index).

[0284] In another form, 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 in the CH3 domain of the second subunit of the Fc domain (numbering according to Kabat EU index).

[0285] 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).

[0286] In one embodiment, the heterodimerization technique described in WO2013 / 157953 is alternatively used. 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 additional 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).

[0287] In one embodiment, the heterodimerization approach described in WO2012 / 058768 is alternatively used. 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 an additional amino acid mutation at position T411, D399, S400, F405, N390 or K392, for example selected from a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (Kabat In a further embodiment, 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 embodiment, the first CH3 domain comprises the amino acid mutation 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 EU index, Kabat).

[0288] In one aspect, the heterodimerization approach described in WO2011 / 143545 is alternatively used, e.g., with an amino acid modification at position selected from the group consisting of 368 and 409 (numbering according to the Kabat EU index).

[0289] In one embodiment, the heterodimerization approach described in WO 2011 / 090762, which also employs the knob-into-hole technique described above, is alternatively used. 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).

[0290] In one embodiment, the (multispecific) antibody or its Fc domain is of the IgG2 subclass and the heterodimerization approach described in WO2010 / 129304 is alternatively used.

[0291] In an alternative embodiment, the modification that promotes association of the first and second Fc domain subunits comprises a modification that mediates an electrostatic steering effect, e.g., as described in PCT Publication WO 2009 / 089004. Generally, this method involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues such that homodimer formation is electrostatically unfavorable, but 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., with 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., with 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., with glutamic acid (E) or aspartic acid (D), particularly K409D or R409D). In a further embodiment, 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 the Kabat EU index).

[0292] In a further embodiment, the heterodimerization approach described in WO2007 / 147901 is alternatively used. 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).

[0293] In yet another embodiment, the heterodimerization approach described in WO2007 / 110205 can alternatively be used.

[0294] 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).

[0295] b) Fc domain modifications that reduce Fc receptor binding and / or effector function The Fc domain confers favorable pharmacokinetic properties and favorable tissue-blood distribution ratios to (multispecific) antibodies, including a long serum half-life that contributes to favorable accumulation in target tissues. However, it can also result in undesirable targeting of (multispecific) antibodies to cells expressing Fc receptors rather than to preferred antigen-bearing cells. Furthermore, simultaneous activation of Fc receptor signaling pathways can result in cytokine release, which, combined with the T cell-activating properties and long half-life of (multispecific) antibodies, can result in excessive cytokine receptor activation and severe side effects upon systemic administration. Activation of (Fc receptor-bearing) immune cells other than T cells can even reduce the effectiveness of (multispecific) antibodies, for example, due to the potential for T cell destruction by NK cells.

[0296] Thus, in a preferred embodiment, the Fc domain of the (multispecific) antibody according to the invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain. In one such embodiment, the Fc domain (or a (multispecific) antibody comprising said Fc domain) exhibits less than 50%, in particular less than 20%, even more particularly less than 10%, and most particularly less than 5% of the binding affinity to Fc receptors compared to a native IgG1 Fc domain (or a (multispecific) antibody comprising a native IgG1 Fc domain), and / or exhibits less than 50%, in particular less than 20%, even more particularly less than 10%, and most particularly less than 5% of the effector function compared to a native IgG1 Fc domain (or a (multispecific) antibody comprising a native IgG1 Fc domain). In one embodiment, the Fc domain (or a (multispecific) antibody comprising said Fc domain) does not substantially bind to and / or induce effector function on Fc receptors. 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, and most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In a preferred embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to a native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or a (multispecific) antibody comprising said Fc domain) exhibits more than about 70%, particularly more than about 80%, and even more particularly more than about 90% of the binding affinity for FcRn of a native IgG1 Fc domain (or a (multispecific) antibody comprising a native IgG1 Fc domain).

[0297] In some embodiments, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function compared to a non-engineered Fc domain. In preferred embodiments, the Fc domain of a (multispecific) antibody comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor and / or the effector function. 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 an Fc receptor. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where more than one amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor is present, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, a (multispecific) antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, and 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 several embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, and most particularly human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complement components (particularly binding affinity to C1q) is also reduced. In one embodiment, binding affinity to 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 for FcRn of a non-engineered form of the Fc domain (or a (multispecific) antibody comprising a non-engineered form of said Fc domain).The Fc domain or (multispecific) antibodies of the present invention comprising said Fc domain may exhibit more than about 80%, or even more than about 90%, of such affinity. In some embodiments, 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 consisting 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 reduced 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). In one embodiment, the amino acid mutation that reduces the binding affinity to an Fc receptor and / or effector function of the Fc domain 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 one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 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, particularly P329G (numbering according to Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbering according to Kabat EU index). In a more specific embodiment, the further amino acid substitution is 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 with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) in each of the first and second subunits of the Fc domain (Kabat EU index numbering).

[0298] In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor (and similarly, complement) binding of the human IgG1 Fc domain, as described in PCT publication WO2012 / 130831, which is incorporated herein by reference in its entirety. WO2012 / 130831 also describes methods for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector function.

[0299] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions compared to IgG1 antibodies. Thus, in some embodiments, the Fc domain of the (multispecific) antibody of the present invention is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P (numbering according to the Kabat EU index). To further reduce binding affinity to Fc receptors and / or effector functions, in one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numbering according to the Kabat EU index). In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numbering according to the Kabat EU index). In a preferred embodiment, the IgG4 Fc domain comprises 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). Such IgG4 Fc domain mutants and their Fcγ receptor binding properties are described in PCT Publication WO2012 / 130831, which is incorporated herein by reference in its entirety.

[0300] In a preferred embodiment, the Fc domain which exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain is a human IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numbering according to the Kabat EU index).

[0301] In some embodiments, N-glycosylation of the Fc domain is eliminated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, specifically replacing asparagine with alanine (N297A) or aspartic acid (N297D) (numbering according to the Kabat EU index).

[0302] In addition to the Fc domains described herein above and in PCT Publication WO2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also include those with one or more substitutions at Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (Kabat EU index numbering). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0303] 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 may include site-directed mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. Correct nucleotide changes can be confirmed, for example, by sequencing.

[0304] Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (GE Healthcare), and Fc receptors can be obtained by recombinant expression. Alternatively, the binding affinity of an Fc domain or a (multispecific) antibody comprising an Fc domain for an Fc receptor can be assessed using a cell line known to express a particular Fc receptor, for example, human NK cells expressing the FcγIIIa receptor.

[0305] The effector function of an Fc domain or an Fc domain-containing (multispecific) antibody can be measured by methods known in the art. Examples of in vitro assays to assess 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 (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox96® 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).

[0306] In some embodiments, binding of the Fc domain to complement components, particularly C1q, is reduced. Thus, in some forms in which the Fc domain is engineered to have reduced effector function, the reduced effector function includes reduced CDC. C1q binding assays can be performed to determine whether an Fc domain or a (multispecific) antibody comprising an Fc domain can bind C1q and therefore has CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may 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)).

[0307] 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); WO2013 / 120929).

[0308] B. Polynucleotides The present invention further provides isolated polynucleotides encoding the antibodies of the present invention. The isolated polynucleotides may be a single polynucleotide or multiple polynucleotides.

[0309] Polynucleotides encoding the (multispecific) antibodies of the present invention may be expressed as a single polynucleotide encoding the entire antibody, or as multiple (e.g., two or more) co-expressed polynucleotides. Polypeptides encoded by the co-expressed polynucleotides may associate, for example, via 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 derived from a portion of the antibody comprising the antibody heavy chain. When co-expressed, the heavy chain polypeptides associate with the light chain polypeptides to form the antibody. In another example, a portion of an antibody comprising one of two Fc domain subunits and, optionally, one or more Fab molecules (parts) may be encoded by a separate polynucleotide derived from a portion of the antibody comprising the other of the two Fc domain subunits and, optionally, one or more Fab molecules (parts). When co-expressed, the Fc domain subunits associate to form the Fc domain.

[0310] In some aspects, the isolated polynucleotide encodes an entire antibody molecule according to the invention as described herein, hi other aspects, the isolated polynucleotide encodes a polypeptide comprised in an antibody according to the invention as described herein.

[0311] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, e.g., in the form of messenger RNA (mRNA). The RNA of the present invention may be single-stranded or double-stranded.

[0312] C. Recombinant Methods Antibodies of the present invention may be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the antibody, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such polynucleotides may be readily isolated and sequenced using conventional procedures. In one aspect, vectors, particularly expression vectors, containing polynucleotides of the present invention (e.g., a single polynucleotide or multiple polynucleotides) are provided. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences along 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 may be part of a plasmid, virus, or nucleic acid fragment. An expression vector comprises an expression cassette into which a polynucleotide encoding an antibody (i.e., 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), although not translated into amino acids, is considered part of the coding region when present; however, any adjacent 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 two or more coding regions; for example, a vector of the present invention may encode one or more polypeptides, which are separated into final proteins by post- or co-translational proteolytic cleavage. Furthermore, a vector, polynucleotide, or nucleic acid of the present invention may encode a heterologous coding region, either fused or unfused to a polynucleotide encoding an antibody of the present invention, or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable association refers to the association of a coding region for a gene product (e.g., a polypeptide) with one or more regulatory sequences in such a manner that expression of the gene product is under the influence or control of the regulatory sequence(s). Two DNA segments (such as a polypeptide coding region and its associated promoter) are "operably linked" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA segments does not interfere with the ability of the expression control sequences to direct expression of the gene product or the ability of the DNA template to be transcribed. Thus, a promoter region can be operably associated with a polypeptide-encoding nucleic acid if the promoter is capable of effecting transcription of the nucleic acid. The promoter can also be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements besides a promoter, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with a polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art.These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., the immediate-early promoter, in combination with intron-A), Simian Virus 40 (e.g., the early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Further suitable transcriptional control regions include tissue-specific promoters and enhancers, and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translational control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translational start and stop codons, and elements derived from viral systems (particularly internal ribosome entry sites, or IRES, also called CITE sequences). The expression cassette may also contain other features such as, for example, an origin of replication and / or chromosomal integration elements, e.g., retroviral long terminal repeats (LTRs) or adeno-associated viral (AAV) inverted terminal repeats (ITRs).

[0313] Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding secretory or signal peptides to 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 a nucleic acid encoding an 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 upon initiation of transport of the growing protein chain across the rough endoplasmic reticulum. Those skilled in the art know that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the translated polypeptide to produce the secretory or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy or light chain signal peptide, is used, or a functional derivative of that sequence that retains the ability to direct the secretion of an operably associated polypeptide is used. 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.

[0314] DNA encoding short protein sequences that can be used to facilitate later purification (e.g., a histidine tag) or to serve to label the antibody may be included within or at the end of the antibody (fragment) encoding polynucleotide.

[0315] In a further aspect, a host cell is provided that comprises a polynucleotide of the invention (i.e., a single polynucleotide or multiple polynucleotides). In a particular aspect, a host cell is provided that comprises a vector of the invention. The polynucleotide and vector may incorporate any of the features described herein in connection with the polynucleotide and vector, respectively, 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 (portions of) an antibody of the invention. As used herein, the term "host cell" refers to any type of cell line that can be engineered to produce an antibody of the invention or a fragment thereof. Suitable host cells for replicating and supporting the expression of antibodies are well known in the art. Such cells can be transfected or transduced, where appropriate, with a particular expression vector, and large quantities of the vector-containing cells can be grown to inoculate large-scale fermenters, thereby obtaining sufficient quantities of antibody for clinical use. Suitable host cells include prokaryotic microorganisms such as Escherichia coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, polypeptides can be produced in bacteria, especially if glycosylation is not required. After expression, the polypeptides can be isolated from the bacterial cell paste in appropriate fractions and further purified. In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" to produce 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). Suitable host cells 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 can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used 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 can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension can be useful. Other examples of useful mammalian host cell lines include SV40 (COS-7) transformed monkey kidney CV1 cells, human embryonic kidney cells (e.g., 293 or 293T cells as described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as 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 include dhfr. -These include 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 specific mammalian host cells suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, to name just a few, but also cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues. In one embodiment, the host cell is a eukaryotic cell, particularly 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, or Sp20 cell). In one embodiment, the host cell is not a cell in the human body.

[0316] Standard techniques for expressing foreign genes in these systems are known in the art. Cells that express a polypeptide containing the heavy or light chain of an antigen-binding domain, such as an antibody, can be engineered to also express the other antibody chain, such that the expressed product is an antibody having both a heavy and a light chain.

[0317] In one aspect, a method of producing an antibody according to the invention is provided, the method comprising incubating 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).

[0318] The components of the (multispecific) antibody of the present invention can be genetically fused to each other. The (multispecific) antibody can be designed so that its components are fused to each other directly or indirectly through a linker sequence. The composition and length of the linker can be determined according to methods well known in the art and tested for effectiveness. Examples of linker sequences between different components of the (multispecific) antibody are provided herein. If necessary, additional sequences, such as endopeptidase recognition sequences, can also be included to incorporate cleavage sites for separating the individual components of the fusion.

[0319] 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, and size-exclusion chromatography. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled 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 antibodies of the present 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 antibodies essentially as described in the Examples. Antibody purity can be determined by any of a variety of well-known analytical methods, including gel electrophoresis and high-pressure liquid chromatography.

[0320] D. Assay The antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0321] 1. Binding assay Antibody binding (affinity) 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 receptor or target proteins available, for example, by recombinant expression. Alternatively, antibody binding to different receptors or target antigens can be assessed, for example, by flow cytometry (FACS), using cell lines expressing the particular receptor or target antigen. Specific illustrative and exemplary embodiments for measuring binding activity to CD3 are described below.

[0322] In one embodiment, binding activity to CD3 is determined by SPR as follows. SPR was performed on a Biacore T200 instrument (GE Healthcare). Anti-Fab capture antibodies (GE Healthcare, #28958325) were 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) was used as the running and dilution buffer. CD3 antibody at a concentration of 2 μg / ml (in 20 mM His, 140 mM NaCl, pH 6.0) was injected at a flow rate of 5 μl / min for approximately 60 seconds. The CD3 antigen used was a heterodimer of CD3 delta and CD3 epsilon ectodomains fused to a human Fc domain with a knob-into-hole modification and a C-terminal Avi-tag (see SEQ ID NOs: 41 and 42). CD3 antigen is injected at a concentration of 10 μg / ml for 120 seconds, and dissociation is monitored for approximately 120 seconds at a flow rate of 5 μl / min. The chip surface is regenerated by two consecutive injections of 10 mM glycine, pH 2.1, each for approximately 60 seconds. Bulk refractive index differences are corrected by subtracting a blank injection and the response obtained from a blank control flow cell. To normalize the binding signal, CD3 binding is divided by the anti-Fab response (the signal (RU) obtained upon capture of CD3 antibody on immobilized anti-Fab antibody). The binding activity of an antibody to CD3 after a specific treatment, relative to the binding activity of an antibody to CD3 after a different treatment (also called the relative activity concentration (RAC)), is calculated by referencing the binding activity of the antibody sample after a specific treatment against the binding activity of the corresponding antibody sample after a different treatment.

[0323] 2. Activity Assay The biological activity of the (multispecific) antibodies of the invention can be measured by various assays, as described in the Examples. Biological activity may include, for example, inducing T cell proliferation, 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 B cells, and inducing tumor regression and / or improving survival.

[0324] E. Composition, Formulation and Route of Administration In further aspects, the invention provides pharmaceutical compositions comprising any of the antibodies provided herein, e.g., for use in any of the following therapeutic methods. Additionally, methods are provided for producing antibodies of the invention in a form suitable for in vivo administration, comprising (a) obtaining an antibody according to the invention, and (b) combining the antibody with at least one pharmaceutically acceptable carrier, thereby formulating a formulation of the antibody for in vivo administration. In another aspect, a pharmaceutical composition comprises an antibody according to the invention and at least one additional therapeutic agent, e.g., as described below.

[0325] Additionally 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) combining the antibody with at least one pharmaceutically acceptable carrier, thereby formulating a formulation of the antibody for in vivo administration.

[0326] Pharmaceutical compositions of the present invention comprise an effective amount of an antibody dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., that do not elicit adverse, allergic, or other untoward reactions when administered to animals, such as humans, as appropriate. The preparation of pharmaceutical compositions comprising an antibody and, optionally, additional active ingredients, is known to those skilled in the art in view of the present disclosure, as exemplified in Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, incorporated herein by reference. Furthermore, it will be understood that for animal (e.g., human) administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards or corresponding national authorities. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carriers" include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, vehicles, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such similar materials, and combinations thereof, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 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.

[0327] 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 localized treatment is desired. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

[0328] Parenteral compositions include those designed to be administered by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection. For injection, the antibodies of the present invention can be formulated in aqueous solutions, particularly physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. Solutions may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the antibodies may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. Inorganic injectable solutions are prepared by incorporating the antibodies of the present invention in the required amount in an appropriate solvent, with various other ingredients, as optionally listed below. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes. 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 techniques, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered liquid medium. The liquid medium should be suitably buffered, if necessary, and the liquid diluent should first be 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 minimized to a safe level, e.g., less than 0.5 ng / mg protein.Suitable pharmaceutically acceptable carriers include, but are not limited to, buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; tannins, etc. Proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound 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 cleat or triglycerides, or liposomes.

[0329] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations 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 injectable compositions can be achieved by using agents delaying absorption, such as aluminum monostearate, gelatin, or combinations thereof, in the compositions.

[0330] In addition to the aforementioned compositions, antibodies can also be formulated as depot preparations. Such long-acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the antibodies can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0331] Pharmaceutical compositions containing the antibodies of the present invention may be produced by conventional mixing, dissolving, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, vehicles, or adjuvants that facilitate processing of the protein into a pharmaceutically usable preparation. Appropriate formulations vary depending on the chosen route of administration.

[0332] Antibodies may be formulated in the compositions in free acid or free base, neutral, or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or free base. These include acid addition salts, for example, those formed with free amino groups of the protein composition, or those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, or from organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0333] F. Therapeutic Methods and Compositions Any of the antibodies provided herein can be used in therapeutic methods. The antibodies of the invention can be used as immunotherapeutic agents, for example, in the treatment of cancer or autoimmune diseases.

[0334] For use in therapeutic methods, the antibodies of the invention will be formulated, dosed, and administered in a manner consistent with good medical practice, with factors to consider in this regard including 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 drug delivery, the method of administration, the administration schedule, and other factors known to medical practitioners.

[0335] In one aspect, an antibody of the present invention is provided for use as a pharmaceutical. In a further aspect, an antibody of the present invention is provided for use in the treatment of a disease. In a particular aspect, an antibody of the present invention is provided for use in a method of treatment. In one aspect, the present invention provides an antibody of the present invention for use in the treatment of a disease in an individual in need thereof. In a particular aspect, the present invention provides an 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, particularly a CD19-expressing cancer. In a specific aspect, the cancer is a B-cell cancer. In one aspect, the B-cell cancer is a B-cell lymphoma or a B-cell leukemia. In one aspect, the B-cell cancer is non-Hodgkin's lymphoma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia. In another aspect, the disease is an autoimmune disease. In a particular aspect, the disease is lupus, particularly systemic lupus erythematosus (SLE) or lupus nephritis (LN).

[0336] In certain aspects, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, 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 B cells. In certain aspects, the invention provides an antibody of the invention for use in a method of inducing lysis of target cells, particularly B cells, in an individual, comprising administering to the individual an amount of the antibody effective to induce lysis of the target cells. The "individual" in any of the above aspects is a mammal, preferably a human.

[0337] In a further aspect, the invention provides use of an antibody of the invention in the manufacture or preparation of a medicament. In one aspect, the medicament is for treating 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, particularly a CD19-expressing cancer. In a specific aspect, the cancer is a B-cell cancer. In one aspect, the B-cell cancer is a B-cell lymphoma or a B-cell leukemia. In one aspect, the B-cell cancer is non-Hodgkin's lymphoma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia. In another aspect, the disease is an autoimmune disease. In a particular aspect, the disease is lupus, particularly systemic lupus erythematosus (SLE) or lupus nephritis (LN). In one aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, 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 medicament is for inducing lysis of target cells, particularly B cells. In a still further aspect, the medicament is for use in a method of inducing lysis of target cells, particularly B cells, in an individual, comprising administering to the individual an effective amount of the medicament to induce lysis of the target cells. The "individual" according to any of the above aspects may be a mammal, preferably a human.

[0338] In a further aspect, the present invention provides a method for treating a disease. In one aspect, the method comprises administering an effective amount of an antibody of the present invention to an individual having such a disease. In one aspect, a composition is administered to the individual and comprises an antibody of the present invention in a pharmaceutically acceptable form. In a particular aspect, the disease is a proliferative disease. In a particular aspect, the disease is cancer, particularly a CD19-expressing cancer. In a specific aspect, the cancer is a B-cell cancer. In one aspect, the B-cell cancer is a B-cell lymphoma or a B-cell leukemia. In one aspect, the B-cell cancer is non-Hodgkin's lymphoma or acute lymphoblastic leukemia or chronic lymphocytic leukemia. In another aspect, the disease is an autoimmune disease. In a particular aspect, the disease is lupus, particularly systemic lupus erythematosus (SLE) or lupus nephritis (LN). In certain embodiments, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, 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 embodiments may be a mammal, preferably a human.

[0339] In a further aspect, the invention provides a method for inducing lysis of target cells, particularly CD19-expressing cells such as B cells. In one aspect, the method comprises contacting the target cells with an antibody of the invention in the presence of T cells, particularly cytotoxic T cells. In a further aspect, a method is provided for inducing lysis of target cells, particularly CD19-expressing cells such as B cells, in an individual. In one such aspect, the method comprises administering to the individual an effective amount of an antibody of the invention to induce lysis of the target cells. In one aspect, the "individual" is a human.

[0340] Those skilled in the art will readily recognize that in many cases, an antibody may provide only a partial benefit without providing a cure. In some aspects, a physiological change that has some effect is also considered therapeutically beneficial. Thus, in some aspects, the amount of antibody that effects a physiological change is considered an "effective amount." The subject, patient, or individual in need of treatment is typically a mammal, and more particularly, a human.

[0341] In some embodiments, an effective amount of an antibody of the invention is administered to an individual to treat a disease.

[0342] The appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease 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 practitioner 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 or multiple doses over various time periods, bolus administration, and pulse infusion.

[0343] The antibodies of the invention are preferably administered to a patient at one time or over a series of treatments. Depending on the type and severity of the disease, an initial candidate dosage of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of antibody can be administered to a patient, whether by one or more separate administrations or by continuous infusion, for example. A typical daily dosage might range from about 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on symptoms, treatment is usually continued until a desired suppression of disease symptoms occurs. 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, dosages may also include about 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 100 μg / kg / body weight, about 200 μg / kg / body weight, about 350 μg / kg / body weight, about 500 μg / kg / body weight, about 1 mg / kg / body weight, about 5 mg / kg / body weight, about 10 mg / kg / body weight, about 50 mg / kg / body weight, about 100 mg / kg / body weight, about 200 mg / kg / body weight, about 350 mg / kg / body weight, about 500 mg / kg / body weight, up to about 1000 mg / kg / body weight, or even more per administration, and any range derivable therein. Non-limiting examples of ranges derivable from the numbers recited herein include about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc., which 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) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., so that the patient receives about two to about 20, or, for example, about six, doses of antibody). An initial larger dose, followed by one or more smaller doses, may be administered. However, other dosing regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0344] The antibodies of the present 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 present invention, or pharmaceutical compositions thereof, are administered or applied in an effective amount.

[0345] For systemic administration, the effective dose can be estimated initially from in vitro assays, such as cell incubation assays. The IC 50 A dose may also be formulated in animal models to achieve a circulating concentration range including 100 mg / kg / day. Such information can be used to more accurately determine useful doses in humans.

[0346] Initial dosages can also be estimated from in vivo data, eg, from animal models, using techniques well known in the art.

[0347] Dosage and dosing intervals can be adjusted individually to provide plasma concentrations of antibody sufficient to maintain therapeutic effect. Typical patient doses useful for administration by injection range from about 0.1 to 50 mg / kg / day, typically about 0.1 to 1 mg / kg / day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Plasma levels may be measured, for example, by HPLC.

[0348] An effective dose of the antibodies of the present invention generally provides therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of antibodies can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Using cell culture assays and animal experiments, LD 50 (the dose that causes death in 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 / ED 50Antibodies that exhibit a large therapeutic index are preferred. In one aspect, the antibodies according to the invention exhibit a high therapeutic index. Data obtained from cell incubation assays and animal studies can be used in formulating a dosage range suitable for human use. Dosages are preferably administered at or above the ED with little or no toxicity. 50 The blood concentration range includes: (a) a concentration of 0.05 mg / kg / day or more ...

[0349] The attending physician of a patient treated with an antibody of the invention will know how and when to discontinue, interrupt, or adjust administration due to toxicity, organ failure, etc. Conversely, the attending physician will also know to adjust treatment levels upward (preemptively precluding toxicity) if the clinical response is inadequate. The magnitude of an administered dose in the management of the disorder of interest will vary depending on the severity of the condition being treated, the route of administration, etc. The severity of the condition may, for example, be assessed, in part, by standard prognostic evaluation methods. Furthermore, the dose, and perhaps dosing frequency, will also vary according to the age, weight, and response of the individual patient.

[0350] The antibodies of the present invention may be administered in combination with one or more other agents in a therapeutic setting. For example, the antibodies 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 ingredients appropriate for the particular disease being treated, preferably those with complementary activities that do not adversely affect each other. In some embodiments, the additional therapeutic agent is an immunosuppressant, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that sensitizes cells to apoptosis-inducing factors. 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, 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.

[0351] Such other agents are suitably present in the combination in amounts effective for the intended purpose. The effective amount of such other agents will depend on the amount of antibody used, the type of disorder or treatment, and other factors discussed above. Antibodies are typically used in the same dosages and by the routes of administration described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0352] Such combination therapy as described above encompasses combined administration (wherein two or more therapeutic agents are contained in the same or separate compositions) and separate administration, where administration of an antibody of the invention may occur before, simultaneously with, and / or after administration of an additional therapeutic agent and / or adjuvant. Antibodies of the invention may also be used in combination with radiation therapy.

[0353] G. Manufactured products In another aspect of the present invention, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert affixed to or associated with the container. Suitable containers include, by way of example, bottles, vials, syringes, IV infusion bags, etc. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition to be used alone or in combination with another composition effective in 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 a vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat a selected condition. Further, the article of manufacture may include (a) a first container containing a composition, the composition comprising an antibody of the present invention, and (b) a second container containing a composition, the composition further comprising a cytotoxic or other therapeutic agent. 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 additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0354] H. Methods and Compositions for Diagnostics and Detection In certain embodiments, any of the antibodies provided herein is useful for detecting the presence of its target (e.g., CD3 or CD19) in a biological sample. As used herein, the term "detect" encompasses quantitative or qualitative detection. In some embodiments, the biological sample comprises cells or tissue, such as prostate tissue.

[0355] In one embodiment, an antibody according to the present invention is provided for use in a method of diagnosis or detection. In a further embodiment, a method for detecting the presence of CD3 or CD19 in a biological sample is provided. In a particular embodiment, the method comprises contacting the biological sample with an antibody of the present invention under conditions that allow binding of the antibody to CD3 or CD19, and detecting whether a complex is formed between the antibody and CD3 or CD19. Such a method may be an in vitro or in vivo method. In one embodiment, the antibody of the present invention is used to select subjects eligible for treatment with an antibody that binds to CD3 and / or CD19, for example, when CD3 and / or CD19 are biomarkers for patient selection.

[0356] Exemplary disorders that may be diagnosed using the antibodies of the invention include cancer, particularly B-cell cancers.

[0357] In some embodiments, antibodies according to the present invention are provided that are labeled. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels) and indirectly detectable moieties (e.g., enzymes or ligands), for example, by enzymatic reaction or molecular interaction. Exemplary labels include radioisotopes, 32 P, 14 C. 125 I, 3 H, and 131These 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, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase conjugated to enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0358] TIFF0007741111000001.tif249170TIFF0007741111000002.tif248170TIFF0007741111000003.tif254170TIFF0007741111000004.tif123170 [Example]

[0359] 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.

[0360] Example 1 - Generation of optimized CD3 binder As previously described (see, e.g., WO2014 / 131712, which is incorporated herein by reference), "CD3 origStarting with a CD3 binder called " and comprising the VH and VL sequences of SEQ ID NOs: 6 and 11, respectively, we aimed to optimize the properties of this binder by removing the two asparagine deamidation sequence motifs at Kabat positions 97 and 100 of the heavy chain CDR3.

[0361] To this end, we generated an antibody library suitable for phage display in which the heavy chains of Kabat asparagines at both positions 97 and 100 were deleted, and further randomized CDRs H1, H2, and H3 to compensate for the loss of affinity caused by replacing Asn97 and Asn100 through an affinity maturation process.

[0362] This library was placed on filamentous phage via fusion to the minor coat protein p3 (Marks et al. (1991) J Mol Biol 222, 581-597) and selected for binding to recombinant CD3ε.

[0363] Ten candidate clones were identified in the initial screen and showed acceptable binding on the recombinant antigen as measured by SPR as Fab fragments (produced in E. coli).

[0364] However, only one of these clones exhibited acceptable binding activity to CD3-expressing cells as measured by flow cytometry after conversion to an IgG format.

[0365] As used herein, "CD3 opt The selected clone, designated "," and containing the VH and VL sequences of SEQ ID NOs: 7 and 11, respectively, was further evaluated and converted to a bispecific format, as described below.

[0366] Example 2 - Binding of optimized CD3 binders to CD3 Binding to recombinant CD3 and an optimized CD3 binder, "CD3 opt " and the original CD3 binder "CD3 orig " (SEQ ID NOs: 12 and 14 (CD3 orig ) and SEQ ID NOs: 13 and 14 (CD3 opt )), binding to recombinant CD3 was determined by surface plasmon resonance (SPR).

[0367] To assess the effect of deamidation site removal and its effect on antibody stability, the binding of the original optimized CD3 binder to recombinant CD3 was tested after 14 days of temperature stress at 37°C or 40°C. Samples stored at -80°C were used as references. The reference sample and the 40°C-stressed sample were in 20 mM His, 140 mM NaCl, pH 6.0, while the 37°C-stressed sample was in PBS, pH 7.4, all at concentrations of 1.2–1.3 mg / ml. After the stress period (14 days), the samples were dialyzed back into PBS, 20 mM His, 140 mM NaCl, pH 6.0, for further analysis.

[0368] The relative activity concentration (RAC) of the samples was determined by SPR as follows.

[0369] SPR was performed on a Biacore T200 instrument (GE Healthcare). Anti-Fab capture antibodies (GE Healthcare, #28958325) were immobilized on a Series S Sensor Chip CM5 (GE Healthcare) using standard amine coupling chemistry, resulting in a surface density of 4000–6000 resonance units (RU). HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) was used as the running and dilution buffer. CD3 antibody at a concentration of 2 μg / ml was injected for 60 seconds at a flow rate of 5 μl / min. CD3 antigen (see below) was injected at a concentration of 10 μg / ml for 120 seconds, and dissociation was monitored for 120 seconds at a flow rate of 5 μl / min. The chip surface was regenerated by two consecutive injections of 10 mM glycine pH 2.1 for 60 seconds each. Bulk refractive index differences were corrected by subtracting a blank injection and by subtracting the response obtained from a blank control flow cell. For evaluation, binding reactions were taken 5 seconds after the end of the injection. To normalize the binding signal, CD3 binding was divided by the anti-Fab response (signal (RU) obtained upon capture of CD3 antibodies on immobilized anti-Fab antibodies). Relative activity concentrations were calculated by referencing each temperature-stressed sample to the corresponding unstressed sample.

[0370] The antigen used was a heterodimer of CD3 delta and CD3 epsilon ectodomains fused to a human Fc domain with a knob-into-hole modification and a C-terminal Avi-tag (see SEQ ID NOs: 41 and 42).

[0371] The results of this experiment are shown in Figure 2. As can be seen, the optimized CD3 binder, CD3 opt is the original CD3 binder, CD3 origshowed strongly improved binding to CD3 after temperature stress (37°C, pH 7.4 for 2 weeks) compared to . This result indicates that the removal of the deamidation site was successful and resulted in an antibody with superior stability characteristics in relation to the in vivo half-life and formulation of the antibody at neutral pH.

[0372] Binding to CD3 on Jurkat cells and an optimized CD3 binder, "CD3 opt " and the original CD3 binder "CD3 orig " (SEQ ID NOs: 12 and 14 (CD3 orig ) and SEQ ID NOs: 13 and 14 (CD3 opt )), binding to CD3 on the human reporter T cell line Jurkat NFAT was determined by FACS.

[0373] Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega #CS176501) are a human acute lymphoblastic leukemia reporter cell line with the NFAT promoter and expressing human CD3. Cells were incubated at 0.1–0.5 mio cells per ml in RPMI 1640, 2 g / L glucose, 2 g / L NaHCO3, 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1× NEAA, and 1× sodium pyruvate. A final concentration of 200 μg of hygromycin B per ml was added whenever cells were passaged.

[0374] For the binding assay, Jurkat NFAT cells were harvested, washed with PBS, and resuspended in FACS buffer. Antibody staining was performed in a 96-well round-bottom plate. 100,000–200,000 cells were seeded per well. The plate was centrifuged at 400 × g for 4 minutes, and the supernatant was removed. Test antibodies were diluted in FACS buffer, and 20 μl of antibody solution was added to the cells for 30 minutes at 4°C. To remove unbound antibody, the cells were washed twice with FACS buffer, followed by the addition of diluted secondary antibody (PE-conjugated AffiniPure F(ab')2 Fragment Goat Anti-Human IgG Fcg Fragment Specific; Jackson ImmunoResearch #109-116-170). After a 30-minute incubation at 4°C, unbound secondary antibody was washed away. Before measurement, cells were resuspended in 200 μl FACS buffer and then analyzed by flow cytometry using a BD Canto II instrument.

[0375] As shown in Figure 3, the optimized CD3 binder "CD3 opt " and the original CD3 binder "CD3 orig " bound relatively well to CD3 on Jurkat cells.

[0376] Example 3 - Functional activity of optimized CD3 binders Optimized CD3 binder "CD3 opt The functional activity of " was tested in a Jurkat reporter cell assay and was significantly higher than that of the original CD3 binder "CD3 orig To test the functional activity of IgG, the activity of CD3 opt Human IgG1PGLALA or CD3 orig Anti-PGLALA-expressing CHO cells were co-incubated with Jurkat NFAT reporter cells in the presence of increasing concentrations of human IgG1PGLALA. Activation of CD3 on Jurkat NFAT reporter cells by cross-linking T cells induces the production of luciferase, and luminescence can be measured as a marker of activation. origHuman IgG1wt was included as a negative control, which is unable to bind to anti-PGLALA-expressing CHO cells and therefore unable to crosslink to Jurkat NFAT cells. A schematic diagram of the assay is shown in Figure 4.

[0377] Anti-PGLALA-expressing CHO cells were CHO-K1 cells engineered to express an antibody that specifically binds to human IgG1Fc (PGLALA) on their surface (see WO2017 / 072210, incorporated herein by reference). These cells were incubated in DMEM / F12 medium containing 5% FCS + 1% GluMax. Jurkat NFAT reporter cells were as described in Example 2.

[0378] When CD3 huIgG1PGLALA simultaneously binds to anti-PGLALA expressed on CHO cells and CD3 expressed on Jurkat NFAT reporter cells, the NFAT promoter is activated, leading to the expression of active firefly luciferase. The intensity of the luminescent signal (obtained upon addition of luciferase substrate) is proportional to the intensity of CD3 activation and signaling. Jurkat-NFAT reporter cells were grown in suspension and incubated in RPMI 1640, 2 g / L glucose, 2 g / L NaHCO3, 10% FCS, 25 mM HEPES, 2 mM L-glutamin, 1x NEAA, 1x sodium pyruvate, with 0.1–0.5 mio cells per ml and 200 μg hygromycin per ml. For the assay, CHO cells were harvested and viability was determined using a ViCell™ system. In a flat-bottom, white-walled 96-well plate (Greiner Bio-One #655098), 30,000 target cells / well were plated in 100 μl of medium, and 50 μl / well of diluted antibody or medium (for control) was added to the CHO cells. Jurkat-NFAT reporter cells were then harvested and viability assessed using ViCell. Cells were resuspended at 1.2 mio cells / ml in cell culture medium without hygromycin B and added to the CHO cells at 60,000 cells / well (50 μl / well), yielding a final effector-to-target (E:T) ratio of 2:1 and a final volume of 200 μl per well. Four μl of GloSensor (Promega #E1291) was then added to each well (2% of the final volume). The cells were incubated at 37°C in a humidified incubator for 24 hours. At the end of the incubation period, luminescence was detected using a TECAN Spark 10M.

[0379] As shown in Figure 5, the optimized CD3 binder, CD3 opt on Jurkat NFAT cells, CD3 orig had similar activity when crosslinked as

[0380] Example 4 - Generation of T cell bispecific antibodies containing optimized CD3 binders The optimized CD3 binder identified in Example 1 ("CD3 opt ", SEQ ID NOS: 7 (VH) and 11 (VL)) to generate T cell bispecific antibodies (TCBs) ("CD19-TCBs") that target CD3 and CD19 and use anti-CD19 antibodies 2B11 or 018 as the CD19-binding moiety (SEQ ID NOS: 15-22 or 28-35, respectively).

[0381] A schematic representation of the TCB molecules is shown in Figure 6A, and their full sequences are shown in SEQ ID NOs: 39, 24, 25 and 27 (2B11), and SEQ ID NOs: 36, 37, 38 and 27 (018).

[0382] any of the above anti-CD19 antibodies as the target cell antigen-binding portion, and CD3 as the CD3 binder. orig Corresponding molecules containing the following were also prepared (SEQ ID NOs: 39, 24, 25 and 26 (2B11), and SEQ ID NOs: 40, 37, 38 and 26 (018)).

[0383] As shown in Figures 6B-6E, the variable regions of the heavy and light chain DNA sequences were subcloned in frame with either the constant heavy or constant light chain previously inserted into the respective recipient mammalian expression vectors.

[0384] To improve correct pairing of the light chain with the corresponding heavy chain, mutations were introduced into the human CL (E123R, Q124K) and human CH1 (K147E, K213E) of the CD19-binding F...

Claims

1. An antibody that binds to CD3 and CD19, (a) a first antigen-binding domain that binds to CD3, comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 11; (b) a second antigen-binding domain that binds to CD19; and Including, An antibody in which the first and second antigen-binding domains are each Fab molecules, and (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain, or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain.

2. The antibody of claim 1, comprising a third antigen-binding domain that binds to CD19.

3. The antibody of claim 2 , wherein the third antigen-binding domain is a Fab molecule.

4. An antibody according to any one of claims 1 to 3, comprising an Fc domain composed of a first and a second subunit.

5. 5. The antibody of claim 1, wherein the first antigen-binding domain is a Fab molecule, and the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and Fab heavy chain are replaced by each other.

6. 6. The antibody of claim 5, wherein the first antigen-binding domain is a Fab molecule, and the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted for each other.

7. 7. The antibody of claim 1, wherein the second and, if present, the third antigen-binding domain is a conventional Fab molecule.

8. 8. The antibody of claim 1 , wherein the second and, if present, the third antigen-binding domain is a Fab molecule, 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. 9. The antibody of claim 1, wherein the first and second antigen-binding domains are fused to each other via a peptide linker.

10. 10. The antibody of claim 1, wherein the first, second, and, if present, third antigen-binding domains are each Fab molecules, and the antibody comprises an Fc domain composed of first and second subunits, and (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain 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 the third antigen-binding domain, if present, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

11. 11. The antibody of claim 10, comprising first, second and third antigen-binding domains, an Fc domain and optionally one or more peptide linkers, wherein 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, the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain.

12. 12. The antibody of claim 4, comprising an Fc domain, wherein the domain is an IgG Fc domain.

13. Fc domain is IgG １ The antibody of claim 12, which is an Fc domain.

14. An antibody described in any one of claims 4 to 13, which comprises an Fc domain, and the domain is a human Fc domain.

15. Fc domain, and the domain is human IgG １ An antibody described in any one of claims 4 to 14, which is an Fc domain.

16. An antibody described in any one of claims 4 to 15, comprising an Fc domain, wherein the Fc comprises a modification that promotes association of the first and second subunits of the Fc domain.

17. An antibody described in any one of claims 4 to 16, comprising an Fc domain, wherein in the CH3 domain of a first subunit of the Fc domain, 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 positioned in a cavity in the CH3 domain of the second subunit, and wherein 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, into which the protrusion in the CH3 domain of the first subunit can be positioned.

18. An antibody according to any one of claims 4 to 17, comprising an Fc domain, wherein in a first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in a second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V) (numbering according to the Kabat EU index). (a) in the second subunit of the Fc domain, additionally, the threonine residue at position 366 is replaced by a serine residue (T366S) and the leucine residue at position 368 is replaced by an alanine residue (L368A); and / or (b) the antibody of claim 18, wherein in the first subunit of the Fc domain, the serine residue at position 354 is additionally replaced with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is additionally replaced with a cysteine ​​residue (E356C), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is additionally replaced with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index).

20. 20. The antibody of any one of claims 4 to 19, comprising an Fc domain, wherein the Fc comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

21. 21. The antibody of any one of claims 4 to 20, comprising an Fc domain, wherein the Fc comprises an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331 and P329 (numbering according to the Kabat EU index).

22. An antibody described in any one of claims 4 to 21, comprising an Fc domain, wherein each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).

23. the second and, if present, third antigen-binding domains that bind to CD19 (i) a VH comprising an HCDR1 of SEQ ID NO: 15, an HCDR2 of SEQ ID NO: 16, and an HCDR3 of SEQ ID NO: 17, and a VL comprising an LCDR1 of SEQ ID NO: 19, an LCDR2 of SEQ ID NO: 20, and an LCDR3 of SEQ ID NO: 21; or (ii) An antibody described in any one of claims 1 to 22, comprising a VH comprising an HCDR1 of SEQ ID NO: 28, an HCDR2 of SEQ ID NO: 29, and an HCDR3 of SEQ ID NO: 30, and a VL comprising an LCDR1 of SEQ ID NO: 32, an LCDR2 of SEQ ID NO: 33, and an LCDR3 of SEQ ID NO:

34.

24. the second and, if present, third antigen-binding domains that bind to CD19 (i) a VH comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 18, and / or a VL comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22; or (ii) the antibody of claim 23, comprising a VH comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 31, and / or a VL comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

35.

25. 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 Fab heavy chain 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: 11; b) second and third antigen-binding domains that bind to CD19, each of which is a (conventional) Fab molecule and comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; and c) an Fc domain consisting of a first and a second subunit; where: in the constant domain CL of the second and third antigen-binding domains of b) the amino acid at position 124 is substituted by lysine (K) (Kabat numbering) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (Kabat numbering); and in the constant domain CHI of the second and third antigen-binding domains of b) the amino acid at position 147 is substituted by glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is substituted by glutamic acid (E) (Kabat EU index numbering); And further, 25. The antibody of any one of claims 1 to 24, wherein 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).

26. Fc domain is human IgG １ The Fc domain is an Fc domain, in which in the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and the serine residue at position 354 is replaced with a cysteine ​​residue (S354C). In the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), the threonine residue at position 366 is replaced with a serine residue (T366S), the leucine residue at position 368 is replaced with an alanine residue (L368A), and the tyrosine residue at position 349 is replaced with a cysteine ​​residue (Y349C) (Kabat et al., 2004).

26. The antibody of claim 25, further comprising, in each of the first and second subunits of the Fc domain, a leucine residue at position 234 replaced with an alanine residue (L234A), a leucine residue at position 235 replaced with an alanine residue (L235A), and a proline residue at position 329 replaced with a glycine residue (P329G) (Kabat EU index numbering).

27. 27. The antibody of any one of claims 1 to 26, comprising a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 39, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 24, two polypeptides comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 25, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:

27.

28. 28. An isolated polynucleotide encoding the antibody of any one of claims 1 to 27.

29. 29. A host cell comprising the isolated polynucleotide of claim 28.

30. 30. A method for producing an antibody that binds to CD3 and CD19, comprising the step of: (a) culturing the host cell of claim 29 under conditions suitable for expression of the antibody.

31. 31. The method of claim 30, further comprising the step of (b) recovering the antibody.

32. An antibody that binds to CD3 and CD19, produced by the method of claim 30 or 31.

33. 33. A pharmaceutical composition comprising the antibody of any one of claims 1 to 27 or 32 and a pharmaceutically acceptable carrier.

34. 34. An antibody according to any one of claims 1 to 27 or 32 or a pharmaceutical composition according to claim 33 for use as a medicament.

35. 34. An antibody according to any one of claims 1 to 27 or 32 or a pharmaceutical composition according to claim 33 for use in the treatment of a disease.

36. 36. The antibody or pharmaceutical composition for use according to claim 35, wherein the disease is cancer or an autoimmune disease.

37. 37. The antibody or pharmaceutical composition for use according to claim 36, wherein the cancer is (i) a CD19-expressing cancer, and / or (ii) a B-cell cancer.

38. 38. The antibody or pharmaceutical composition for use according to claim 36 or 37, wherein the cancer is a B-cell lymphoma or a B-cell leukemia.

39. 39. The antibody or pharmaceutical composition for use according to any one of claims 36 to 38, wherein the cancer is non-Hodgkin's lymphoma or acute lymphoblastic leukemia or chronic lymphocytic leukemia.

40. 37. The antibody or pharmaceutical composition for use according to claim 36, wherein the autoimmune disease is lupus.

41. 41. The antibody or pharmaceutical composition for use according to claim 36 or 40, wherein the autoimmune disease is systemic lupus erythematosus (SLE) or lupus nephritis (LN).

42. 34. Use of an antibody according to any one of claims 1 to 27 or 32 or a pharmaceutical composition according to claim 33 in the manufacture of a medicament.

43. 34. Use of an antibody according to any one of claims 1 to 27 or 32 or a pharmaceutical composition according to claim 33 in the manufacture of a medicament for the treatment of a disease.

44. 44. The use according to claim 43, wherein the disease is cancer or an autoimmune disease.

45. 45. The use of claim 44, wherein the cancer is (i) a CD19-expressing cancer, and / or (ii) a B-cell cancer.

46. 46. ​​The use of claim 44 or 45, wherein the cancer is B-cell lymphoma or B-cell leukemia.

47. 47. The use of any one of claims 44 to 46, wherein the cancer is non-Hodgkin's lymphoma or acute lymphoblastic leukemia or chronic lymphocytic leukemia.

48. 45. The use of claim 44, wherein the autoimmune disease is lupus.

49. 49. The use according to claim 44 or 48, wherein the autoimmune disease is systemic lupus erythematosus (SLE) or lupus nephritis (LN).

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