RU2844750C1 - Antibodies binding to cd3 and cd19 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to biotechnology, namely to an antibody that binds to CD3 and CD19, a method for preparing it, as well as to a composition containing it. Also disclosed is a recovered polynucleotide coding an antibody that binds to CD3 and CD19, as well as a host cell containing same.

EFFECT: invention is effective for treating cancer and autoimmune disease.

35 cl, 16 dwg, 11 tbl, 11 ex

Description

AREA OF TECHNOLOGY

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

LEVEL OF TECHNOLOGY

CD3 (cluster of differentiation 3) is a protein complex consisting of four subunits - a CD3γ chain, a CD3δ chain, and two CD3ε chains. CD3 binds to the T-cell receptor and the ζ chain, generating an activation signal in T lymphocytes.

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

A more recent application of CD3 antibodies is in the form of bispecific antibodies that bind CD3 on one side and a target cell antigen, such as CD19, on the other side. The simultaneous binding of such an antibody to both of its targets will promote a transient interaction between the target cell and the T cell, leading to the activation of any cytotoxic T cell and subsequent lysis of the target cell. Bispecific antibodies binding to CD3 and CD19 are described, for example, in WO 2017/055314.

For therapeutic purposes, an important requirement that antibodies must satisfy is sufficient stability both in vitro (for storing the drug) and in vivo (after administration to the patient).

Modifications such as asparagine deamidation are typical degradation pathways for recombinant antibodies and can affect both in vitro stability and in vivo biological functions.

Given the enormous therapeutic potential of antibodies, in particular bispecific antibodies for T cell activation, there is a need for CD3 antibodies, including multispecific antibodies, with optimized properties.

ESSENCE OF THE INVENTION

The present invention provides antibodies, including multispecific (e.g. bispecific) antibodies, that bind to CD3 and are resistant to degradation by asparagine deamidation and thus exceptionally stable, which is required for therapeutic purposes. The presented (multispecific) antibodies additionally combine good efficacy and manufacturability with low toxicity and favorable pharmacokinetic properties.

As shown herein, antibodies, such as the multispecific antibodies that bind to CD3, provided herein, retain greater than about 90% of the binding activity to CD3 after 2 weeks at pH 7.4, 37°C, compared to the binding activity after 2 weeks at pH 6, -80°C, as determined by surface plasmon resonance (SPR).

In one aspect, the present 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 a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:2, HCDR 2 of SEQ ID NO:3 and HCDR 3 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, LCDR 2 of SEQ ID NO:9 and LCDR 3 of SEQ ID NO:10; and (b) a second and optionally third antigen-binding domain that binds to CD19. 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.

In a further aspect, the present 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 third antigen-binding domain that binds to CD19.

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

In one aspect, the antibody comprises an Fc domain consisting of a first and a second subunit.

In one aspect, the first antigen-binding domain is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are substituted for each other.

In one aspect, the second and, if present, the third antigen-binding domain is a standard Fab molecule.

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

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

In one aspect, each of the first and second antigen-binding domains is a Fab molecule 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.

In one aspect, each of the first, second and, if present, third antigen-binding domains is a Fab molecule, and the antibody comprises an Fc domain consisting of a first and a second subunit; and wherein 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 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.

In one aspect, the Fc domain is an IgG Fc domain, particularly IgG ₁ . In one aspect, the Fc domain is a human Fc domain. In one aspect, the Fc comprises a modification that facilitates association of the first and second subunits of the Fc domain. In one aspect, the Fc domain comprises one or more amino acid substitutions that reduce Fc receptor binding and/or effector function.

In one aspect, the second and, if present, the third antigen-binding domain comprises: (i) a VH comprising HCDR 1 of SEQ ID NO: 15, HCDR 2 of SEQ ID NO: 16 and HCDR 3 of SEQ ID NO: 17, and a VL comprising LCDR 1 of SEQ ID NO: 19, LCDR 2 of SEQ ID NO: 20 and LCDR 3 of SEQ ID NO: 21; or (ii) a VH comprising HCDR 1 of SEQ ID NO: 28, HCDR 2 of SEQ ID NO: 29 and HCDR 3 of SEQ ID NO: 30 and a VL comprising LCDR 1 of SEQ ID NO: 32, LCDR 2 of SEQ ID NO: 33 and LCDR 3 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 that is 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 that is 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 that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:31, and/or a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:35.

According to a further aspect of the invention, there are provided an isolated polynucleotide encoding an antibody of the invention and a host cell comprising the isolated polynucleotide of the invention.

In another aspect, a method for producing an antibody that binds to CD3 and CD19 is provided, comprising the steps of: (a) culturing a host cell of the invention under conditions suitable for expressing the antibody, and optionally (b) isolating the antibody. The present invention also includes an antibody that binds to CD3 and CD19, obtained by the method of the present invention.

The invention further provides a pharmaceutical composition comprising an antibody according to the invention and a pharmaceutically acceptable carrier.

Also included in the invention are methods of using an antibody and a pharmaceutical composition of the invention. In one aspect, the present invention provides an antibody or a pharmaceutical composition according to the invention for use as a medicine. In one aspect, there are provided an antibody or a pharmaceutical composition according to the invention for use in the treatment of a disease. Also provided is the use of an antibody or a pharmaceutical composition according to the invention in the manufacture of a medicine and the use of an antibody or a pharmaceutical composition according to the invention in the manufacture of a medicine for the treatment of a disease. The invention also provides a method of treating a disease in an individual, comprising administering to said individual an effective amount of an antibody or a pharmaceutical composition according to the invention. In some aspects, the disease is cancer. In other aspects, the disease is an autoimmune disease.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1. Exemplary configurations of (multispecific) antibodies according to the invention. (A, G) Illustration of a “1+1 CrossMab” molecule. (B, E) Illustration of a “2+1 IgG Crossfab” molecule with an alternative order of the Crossfab and Fab components (“inverted molecule”). (C, E) Illustration of a “2+1 IgG Crossfab” molecule. (G, M) Illustration of a “1+1 IgG Crossfab” molecule with an alternative order of the Crossfab and Fab components (“inverted molecule”). (I, H) Illustration of a “1+1 IgG Crossfab” molecule. (J, P) Illustration of a “2+1 IgG Crossfab” molecule with two CrossFabs. (K, R) Illustration of a “2+1 IgG Crossfab” molecule with two CrossFabs and an alternative order of the Crossfab and Fab components (“inverted molecule”). (C, X) Illustration of a “Fab-Crossfab” molecule. (T, C) Illustration of the Crossfab-Fab molecule. (U, W) Illustration of the (Fab) ₂ -Crossfab molecule. (F, Щ) Illustration of the Crossfab-(Fab)2 molecule. (E, Z) Illustration of the Fab-(Crossfab) ₂ molecule. (U, AA) Illustration of the (Crossfab)2-Fab molecule. Black dot: optional modification in the Fc domain that promotes heterodimerization. ++, --: amino acids of opposite charges optionally introduced into the CH1 and CL domains. The Crossfab molecules in the image contain an exchange of the VH and VL regions, but may, in aspects in which charge modifications are introduced into the CH1 and CL domains, alternatively contain an exchange of the CH1 and CL domains.

Fig. 2. Relative binding activity of original and optimized CD3 binding fragments CD3 _orig and CD3 _opt to recombinant CD3 as measured by SPR under stress-free conditions after 14 days at 40°C, pH 6, or after 14 days at 37°C, pH 7.4 (IgG format).

Fig. 3. Binding of original and optimized CD3 binding fragments CD3 _orig and CD3 _opt to Jurkat NFAT cells as measured by flow cytometry (IgG format). Detection of antibodies bound to Jurkat NFAT cells was performed using a fluorescently labeled Fc-specific secondary antibody to human antibody.

Fig. 4. Schematic illustration of the CD3 activation assay used in Example 3.

Fig. 5. Activation of Jurkat NFAT by original and optimized CD3 binding fragments CD3 _orig and CD3 _opt (IgG format). Jurkat NFAT reporter cells were co-incubated with CHO cells expressing the PGLALA antibody (CHO-PGLALA) in the presence of CD3 _orig or CD3 _opt IgG PGLALA or CD3 _opt IgG wt as a negative control. Quantification of CD3 activation was performed by measuring luminescence after 24 h.

Fig. 6. (A) Schematic illustration of the bispecific T-cell antibody (TCB) molecules used in the examples. All tested TCB antibody molecules were prepared as “2+1 IgG CrossFab inverted” with charge modifications (VH/VL interchange in the CD3 binding molecule, charge modifications in the target antigen binding molecules, EE=147E, 213E; RK=123R, 124K). (B-D) Components for TCB assembly: light chain of the Fab molecule to TYRP1 with charge modifications at CH1 and CL (B), light chain of the Fab molecule to CD3 crossover (C), heavy chain with knob mutations and PG LALA in the Fc region (D), heavy chain with hole mutations and PG LALA in the Fc region (E).

Fig. 7. Binding of TSV antibodies to CD19 to CD3-expressing Jurkat cells (A) and to CD19-expressing Z-138 (B) and Nalm-6 (C) cells as measured by flow cytometry.

Fig. 8. Target-specific killing of CD19+ target cells induced by TCB antibodies to CD19. (A) Z-138 target cells, (B) Nalm-6 target cells.

Fig. 9. T cell activation induced by TCB antibodies to CD19 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, (E) CD107 expression on CD8 T cells.

Fig. 10. T cell activation induced by TCB antibodies to CD19 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, (E) CD107 expression on CD8 T cells.

Fig. 11. Scheme of in vivo study of Example 10.

Fig. 12. Change in body weight upon treatment with different doses of CD19-TCB or CD20-TCB, in Study Example 10. n=3 mice per group. Mean+/- sem.

Fig. 13. Cytokine release in serum 4 hours after treatment with CD19-TCB or CD20-TCB, with or without pretreatment with obinituzumab (Gazyva(R)) (GPT), in the Example 10 study. (A) MIP-1β, (B) IL-6, (C) IFN-γ, (D) IL-5, (E) GM-CSF, (F) TNF-α, (G) IL-2, (I) IL-1β, (J) IL-13, (K) MCP1, (M) IL-8, (H) IL-10, (R) GM-CSF, (R) IL-12p70, (C) IL-17. Bands in each panel from left to right: CD19-TCB 0.5 mg/kg, CD19-TCB 0.15 mg/kg, CD19-TCB 0.05 mg/kg, GPT+CD19-TCB 0.5 mg/kg, CD20-TCB 0.15 mg/kg, GPT+CD20-TCB 0.15 mg/kg. Mean+SPS.

Fig. 14. Blood B cell counts at 4 hours, 24 hours, and 72 hours after treatment with CD19-TCB or CD20-TCB, with or without pretreatment with obinituzumab (Gazyva®) (GPT), in the Example 10 study. Mean+SDS.

Fig. 15. Treatment scheme and experimental setup. Humanized NSG mice were inoculated subcutaneously with patient-derived lymphoma xenograft (PDX) (5 million cells). Tumor volumes were calculated from capillary measurements. When they reached 200 mm, mice were randomized into groups of 8 based on their tumor size. Mice were then injected weekly (i.v.) with vehicle or 0.5 mg/kg CD19-TCB.

Fig. 16. Effect of CD19-TCB treatment on tumor growth. Tumor volumes were calculated from capillary measurements two (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 described in Fig. 15. Mean+SD with *p≤0.05, **p≤0.01, ***p≤0.001 by Mann-Whitney test. Arrows point to each of the 4 treatments with CD19-TCB or vehicle.

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

I. DEFINITIONS

In this document, terms are used as generally accepted in the art unless otherwise defined below.

In the context of this document, the terms "first", "second" or "third" with respect to antigen-binding domains, etc. are used to facilitate distinction when more than one of each type of fragment is present. The use of these terms does not imply a particular order or orientation of the fragment unless explicitly stated otherwise.

The terms "anti-CD3 antibody" and "antibody that binds to CD3" refer to an antibody that is capable of binding CD3 with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent for targeting CD3. In one aspect, the extent of binding of an anti-CD3 antibody to an unrelated protein other than CD3 is less than about 10% of the binding of the antibody to CD3, as measured, for example, by surface plasmon resonance (SPR). In certain aspects, an antibody that binds to CD3 has a dissociation constant (K _D ) of ≤1 μM, ≤500 nM, ≤200 nM, or ≤100 nM. An antibody is said to "specifically bind" to CD3 if the antibody has a K _D of 1 μM or less, as measured, for example, by SPR. In certain aspects, an anti-CD3 antibody binds to an epitope of CD3 that is conserved between CD3 from different species.

Similarly, the terms "anti-CD19 antibody" and "antibody that binds to CD19" refer to an antibody that is capable of binding CD19 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting CD19. In one aspect, the extent of binding of the anti-CD19 antibody to an unrelated protein other than CD19 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, the antibody that binds to CD19 has a dissociation constant (K _D ) of ≤1 μM, ≤500 nM, ≤200 nM, or ≤100 nM. An antibody is said to "specifically bind" to CD19 if the antibody has a K _D of 1 μM or less as measured by a measurement such as SPR. In certain aspects, an anti-CD19 antibody binds to an epitope of CD19 that is conserved across CD19 from different species.

The term "antibody" as used herein is used in the broadest sense and includes various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the necessary antigen-binding activity.

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

In the context of this document, the terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably to refer to an antibody having a structure substantially similar to the structure of a native antibody.

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 the same epitope, with the exception of possible variant antibodies, such as those containing natural mutations or arising during the production of the monoclonal antibody preparation, such variants generally being present in small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the characteristic of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies can be produced by a variety of methods, including, but not limited to, hybridoma techniques, recombinant DNA techniques, phage display techniques, and methods using transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for producing monoclonal antibodies are described herein.

An "isolated" antibody is an antibody that has been separated from components of its natural environment. In some aspects, the antibody is purified to greater than 95% or 99% purity by determination, such as by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reversed phase HPLC, affinity chromatography, size exclusion chromatography) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). In some aspects, the antibodies provided herein are isolated antibodies.

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.

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

"A human antibody" is an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or obtained from a source other than a human that utilizes human antibody repertoires or other human antibody encoding sequences. This definition of a human antibody explicitly excludes a humanized antibody that contains non-human antigen-binding residues. In certain aspects, a human antibody is obtained from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In certain aspects, a human antibody is obtained from a hybridoma cell line. Antibodies or antibody fragments isolated from human antibody libraries are also considered human antibodies or human antibody fragments herein.

The term "antigen-binding domain" refers to a portion of an antibody that contains a region that binds to and is complementary to a portion of an antigen or to the entire antigen. The antigen-binding domain can be formed, for example, by one or more variable domains of an antibody (also called variable regions of an antibody). In preferred aspects, the antigen-binding domain comprises a variable domain of an antibody light chain (VL) and a variable domain of an antibody heavy chain (VH).

The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that participates in the binding of the antibody to an antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain containing four conserved framework regions (FRs) and complementarity determining regions (CDRs). See, e.g., Kindt et al., Kuby Immunology, ^6th ed., W H Freeman & Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated by using the VH or VL domain from 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). In the context of this document, in connection with variable region sequences, “Kabat numbering” refers to the numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

For the purposes of this document, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), referred to herein as "Kabat-consistent numbering" or "Kabat numbering." In particular, the Kabat numbering system (see pages 647-660 in 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 domain CL of the kappa or lambda isotype, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge region, CH2, and CH3), which is then further emphasized in this document by the name "EU Kabat numbering" or "EU Kabat index numbering".

As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of the variable domain of an antibody that are hypervariable in sequence and that determine antigen-binding specificity, such as the "complementarity determining regions" ("CDRs"). Antibodies generally contain six CDRs: three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs as used herein include:

(a) hypervariable loops located at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

(c) antigenic contacts located at amino acid residues 27–36 (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)).

Unless otherwise noted, CDRs are defined in accordance with Kabat et al., supra. One skilled in the art will appreciate that assignment of CDRs may also be defined in accordance with Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

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

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

For the purposes of this document, a "human acceptor framework" is a framework comprising an amino acid sequence of a variable light chain (VL) framework or a variable heavy chain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. A human acceptor framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may comprise changes in the amino acid sequence. 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, a human VL acceptor framework is identical in sequence to a human immunoglobulin VL framework sequence or a human consensus framework sequence.

A "human framework consensus sequence" is a framework region that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, a set of human immunoglobulin VL or VH sequences refers to a subset of variable domain sequences. In general, the subset of sequences is the subset according to Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

As used herein, the term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 Daltons, consisting of two light chains and two heavy chains that are linked by disulfide bonds. In the direction from the N-terminus to the C-terminus, each heavy chain contains a variable domain (VH), also called the heavy chain variable domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant region. Similarly, in the direction from the N-terminus to the C-terminus, each light chain contains a variable domain (VL), also called the light chain variable domain or light chain variable region, followed by a light chain constant domain (CL), also called the light chain constant region. The immunoglobulin heavy chain can be classified into 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, such as γ ₁ (IgGi), γ ₂ (IgG ₂ ), γ ₃ (IgG ₃ ), γ ₄ (IgG ₄ ), α ₁ (IgA ₁ ), and α ₂ (IgA ₂ ). The immunoglobulin light chain can be classified into one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. Immunoglobulin consists primarily of two Fab molecules and an Fc domain linked by the immunoglobulin hinge region.

The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region contained in its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some can be further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . The heavy chain constant domains corresponding to the different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively.

"Fab molecule" refers to a protein consisting of the VH and CH1 domain of the heavy chain ("Fab heavy chain") and the VL and CL domain of the light chain ("Fab light chain") of an immunoglobulin.

By "crossover" Fab molecule (also called "Crossfab") is meant a Fab molecule in which the variable domains or constant domains of the heavy and light chains of the Fab are exchanged (i.e. substituted for each other), i.e. a crossover Fab molecule comprises a peptide chain consisting of the light chain variable domain VL and the heavy chain constant domain 1 CH1 (VL-CH1, in the N- to C-terminal direction), and a peptide chain consisting of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in the N- to C-terminal direction). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain containing the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. At the same time, in a crossover Fab molecule in which the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain containing the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

In contrast, a "standard" Fab molecule means that the Fab molecule has its natural format, i.e. it contains a heavy chain consisting of the variable and constant domains of the heavy chain (VH-CH1, in the direction from N- to C-terminus), and a light chain consisting of the variable and constant domains of the light chain (VL-CL, in the direction from N- to C-terminus).

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

By "fused" is meant that the components (e.g., the Fab molecule and the Fc domain subunit) are linked by peptide bonds, either directly or through one or more peptide linkers.

The term "multispecific" means that the antibody is capable of specifically binding to at least two different antigenic determinants. A multispecific antibody may, for example, be a bispecific antibody. Typically, a bispecific antibody comprises two antigen-binding sites, each of which is specific for a separate antigenic determinant. In certain aspects, a multispecific (e.g., bispecific) antibody is capable of simultaneously binding two antigenic determinants, in particular two antigenic determinants expressed on two different cells.

In the context of this document, the term "valent" refers to the presence of a specified number of antigen-binding sites in an antigen-binding molecule. Therefore, the term "monovalent antigen binding" refers to the presence of one (and no more than one) antigen-binding site specific for an antigen in an antigen-binding molecule.

"Antigen-binding site" refers to the site, i.e., one or more amino acid residues, of an antigen-binding molecule that mediates interaction with an antigen. For example, the antigen-binding site of an antibody contains amino acid residues from the complementarity-determining regions (CDRs). A native immunoglobulin molecule typically has two antigen-binding sites; a Fab molecule typically has one antigen-binding site.

As used herein, the term "antigenic determinant" or "antigen" refers to a site (e.g., a continuous sequence of amino acids or a conformational configuration consisting of different regions of non-contiguous amino acids) in a polypeptide macromolecule to which an antigen-binding domain binds to form an antigen-binding domain-antigen complex. Suitable antigenic determinants may be found, for example, on the surface of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, in free serum, and/or in the extracellular matrix (ECM). In a preferred aspect, the antigen is a human protein.

"CD3" refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed CD3 as well as any form of CD3 produced by cellular processing. The term also includes naturally occurring CD3 variants, such as splice variants or allelic variants. In one aspect, the CD3 is human CD3, in particular human CD3 epsilon (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 No. P07766 (version 209) or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1. In another aspect, 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 signal peptide). See also NCBI GenBank No. BAB71849.1. In certain aspects, an antibody of the invention binds to an epitope of CD3 that is conserved between CD3 antigens from different species, particularly human and cynomolgus monkey CD3. In preferred aspects, the antibody binds to human CD3.

In the context of the present document, the term "target cell antigen" refers to an antigenic determinant presented on the surface of a target cell, for example a B cell. Preferably, the target cell antigen is not CD3 and/or is expressed on a cell other than CD3. According to the present invention, the target cell antigen is CD19, in particular human CD19.

The term "CD19" refers to cluster of differentiation 19 (also known as B-lymphocyte antigen CD19 or B-lymphocyte surface antigen B4) and 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), unless otherwise specified. The term encompasses "full-length" unprocessed CD19 as well as any form of CD19 produced by cellular processing. The term also includes naturally occurring CD19 variants, such as splice variants or allelic variants. In one aspect, the CD19 is human CD19. See UniProt (www.uniprot.org) human protein accession number P15391 (version 211) or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP 001761.3. In certain aspects, an antibody of the invention binds to an epitope of CD19 that is conserved between CD19 antigens from different species, in particular human and cynomolgus monkey CD19. In preferred aspects, the antibody binds to human CD19.

"Affinity" refers to the strength of the net non-covalent interactions between one 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 intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K _D ). Affinity can be measured by well-established methods known in the art, including the methods described herein. A preferred method for measuring affinity is surface plasmon resonance (SPR).

An "affinity matured" antibody refers to an antibody with one or more alterations in one or more complementarity determining regions (CDRs) compared to a parent antibody that does not contain such alterations, wherein such alterations result in an improvement in the affinity of the antibody for the antigen.

"Decreased binding", such as decreased binding to an Fc receptor, refers to a decrease in the affinity for the relevant interaction as defined by, for example, the SPR. For clarity, this term also includes a decrease in affinity to zero (or below the detection limit of the assay), i.e., complete elimination of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for the relevant interaction.

In the context of this document, "T cell activation" refers to one or more cellular responses of a T lymphocyte, in particular a cytotoxic T lymphocyte, selected from: proliferation, differentiation, secretion of cytokines, release of cytotoxic effector molecules, cytotoxic activity and expression of activation markers. Suitable assays for determining T cell activation are known in the art and are described herein.

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

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

An "activating Fc receptor" is an Fc receptor that, upon interaction with the Fc domain of an antibody, triggers signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which antibodies or their derivatives containing an Fc region specifically bind, generally via the protein portion N-terminal to the Fc region. As used herein, the term "reduced ADCC" is defined as a decrease in the number of target cells lysed in a given time at a given concentration of antibody in the medium surrounding the target cells via the ADCC mechanism defined above, and/or as an increase in the concentration of antibody in the medium surrounding the target cells required to ensure lysis of a given number of target cells in a given time via the ADCC mechanism. The reduction in ADCC is determined relative to the ADCC mediated by the same antibody produced in the same host cell type using the same standard production, purification, formulation, and storage methods (which are well known to those skilled in the art), but which has not been engineered. For example, the reduction in ADCC mediated by an antibody containing an amino acid substitution in the Fc domain that reduces ADCC is determined relative to the ADCC mediated by the same antibody without the amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, PCT Publication No. WO 2006/082515 or PCT Publication No. WO 2012/130831).

In the context of this document, the terms "design, designed, designing" are considered to include any manipulation of the peptide backbone or post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof.

Design involves modifications of the amino acid sequence, glycosylation profile, or side chain groups of individual amino acids, as well as combinations of these approaches.

In the context of this document, the term "amino acid mutation" is intended to include amino acid substitutions, deletions, insertions and modifications. Any combination of substitution, deletion, insertion and modification may be made to obtain the final construct, provided that 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 amino- and/or carboxy-terminal deletions and insertions of amino acids. Preferred amino acid mutations are amino acid substitutions. In order to alter, for example, the binding characteristics of the Fc region, non-conservative amino acid substitutions, i.e., substitution of one amino acid by another amino acid having different structural and/or chemical properties, are particularly preferred. Amino acid substitutions include substitution with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be created using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is understood that methods of altering amino acid side chain groups by methods other than genetic engineering, such as chemical modification, may also be applicable. Different designations may be used herein to refer to the same amino acid mutation. For example, a substitution of proline at position 329 of the Fc domain with glycine may be designated as 329G, G329, G ₃₂₉ , P329G, or Pro329Gly.

"Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways that are known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA software package. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. Alternatively, percent identity values can be obtained using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc., and the source code was filed with user documentation in the United States Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration Number TXU510087, and is described in WO 2001/007611.

Unless otherwise stated, for the purposes of this document % amino acid sequence identity values are obtained using the ggsearch program from the FASTA package version 36.3.8c or later with the BLOSUM50 comparison matrix. The FASTA package was written 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 freely available at www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or www.ebi.ac.uk/Tools/sss/fasta. Alternatively, the publicly available server at fasta.bioch.virginia.edu/fasta_www2/index.cgi can be used to compare sequences, using the ggsearch program (global protein:protein) and default parameters (BLOSUM50; open: -10; ext: -2; Ktup=2) to ensure that a global rather than local alignment is performed. Percent amino acid identity is reported in the header of the alignment output.

The term "polynucleotide" or "nucleic acid molecule" includes any compound and/or substance that comprises a polymer of nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, a nucleic acid molecule is described by the sequence of bases, wherein said bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically presented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA and RNA, and mixed polymers containing two or more of these molecules. A nucleic acid molecule may be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. In addition, a nucleic acid molecule described herein may comprise nucleotides of natural and non-natural origin. Examples of nucleotides of non-natural origin include modified nucleotide bases with derivatized sugars or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also include DNA and RNA molecules suitable as a vector for directing expression of an antibody of the invention in vitro and/or in vivo, such as in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule so that the mRNA can be administered to 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).

An "isolated" nucleic acid molecule refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid molecule includes a nucleic acid molecule that is contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

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

As used herein, the term "vector" refers to a nucleic acid molecule capable of increasing the copy number of another nucleic acid to which it is linked. This term includes a vector in the form of a self-replicating nucleic acid structure, as well as a vector incorporated into the genome of a host cell into which it is introduced. Some vectors are capable of directing the expression of nucleic acids to which they are functionally linked. Such vectors are referred to herein as "expression vectors."

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include initially transformed cells and progeny derived therefrom, regardless of passage number. Progeny may not be completely identical to the parent cell in nucleic acid content and may contain mutations. Included herein are mutant progeny that have the same function or biological activity for which the initially transformed cells are screened or selected. A host cell is any type of cellular system that can be used to generate the antibodies of the present invention. Host cells include cultured cells, such as cultured mammalian cells such as HEK cells, CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells and plant cells, to name just a few, but also cells present in the body of a transgenic animal, a transgenic plant or a cultured plant or animal tissue. 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.

The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form that provides the effectiveness of the biological activity of the active ingredient it contains and that does not contain additional components that are unacceptably toxic to the subject to which the composition is to be administered.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or pharmaceutical formulation other than the active ingredient that is nontoxic to the subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, the term "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to a clinical intervention to alter the natural course of a disease in a subject to be treated, and may be performed either prophylactically or during clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, reducing the intensity or transient relief of a painful condition, and remission or improvement of prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated 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 respects, an individual or subject is a human.

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

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

II. COMPOSITIONS AND METHODS

The present invention provides antibodies that bind to CD3 and CD19. These antibodies exhibit excellent stability in combination with other favorable properties for therapeutic use, such as in terms of efficacy and safety, pharmacokinetics, and manufacturing capability. The antibodies of the invention are useful, for example, for the treatment of diseases such as cancer or an autoimmune disease.

A. Antibodies to CD3/CD19

In one aspect, the present invention provides antibodies that bind to CD3 and CD19. In one aspect, the present invention provides isolated antibodies that bind to CD3 and CD19. In one aspect, the present invention provides antibodies that specifically bind to CD3 and CD19. In certain aspects, the CD3/CD19 antibodies retain greater than about 90% of the binding activity to CD3 after 2 weeks at pH 7.4, 37°C, compared to the binding activity after 2 weeks at pH 6, -80°C, as determined by surface plasmon resonance (SPR).

In one aspect, the present 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 a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:2, HCDR 2 of SEQ ID NO:3 and HCDR 3 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, LCDR 2 of SEQ ID NO:9 and LCDR 3 of SEQ ID NO:10.

In one aspect, the antibody is a humanized antibody. In one aspect, the first antigen-binding domain is a humanized antigen-binding domain (i.e., an antigen-binding domain of a humanized antibody). In one aspect, the VH and/or VL of the first antigen-binding domain is a humanized variable region.

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

In one aspect, the VH of the first antigen binding domain comprises one or more heavy chain framework sequences (i.e., a FR1, FR2, FR3 and/or FR4 sequence) 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 a sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO:7. In one aspect, the VH of the first antigen binding domain comprises a sequence that is at least approximately 98% identical to the amino acid sequence of SEQ ID NO:7. In certain aspects, a VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but an antibody comprising the sequence retains the ability to bind to CD3. In certain aspects, a total of 1-10 amino acids in the amino acid sequence of SEQ ID NO:7 have been substituted, inserted, and/or deleted. In some aspects, the substitutions, insertions, or deletions are in regions outside of the CDRs (i.e., in the FRs). In one aspect, the VH domain 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 this sequence.

In one aspect, the VL of the first antigen binding domain comprises one or more light chain framework sequences (i.e., a FR1, FR2, FR3 and/or FR4 sequence) 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 a sequence that is at least approximately 95% identical to the amino acid sequence of SEQ ID NO:11. In one aspect, the VL of the first antigen binding domain comprises a sequence that is at least approximately 98% identical to the amino acid sequence of SEQ ID NO:11. In certain aspects, a VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but an antibody comprising the sequence retains the ability to bind to CD3. In certain aspects, a total of 1-10 amino acids in the amino acid sequence of SEQ ID NO:11 have been substituted, inserted, and/or deleted. In some aspects, the substitutions, insertions, or deletions are in regions outside of the CDRs (i.e., in the FRs). In one aspect, the VL domain 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 this sequence.

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, and 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 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.

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 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.

In a further aspect, the present 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 SEQ ID NO:7 and a VL sequence of SEQ ID NO:11.

In another aspect, the present 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 comprising the VH heavy chain CDR sequences of SEQ ID NO:7, and a VL comprising the VL light chain CDR sequences of SEQ ID NO:11.

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

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

In one aspect, the VL of the first antigen-binding domain comprises the VL light chain CDR sequences of SEQ ID NO: 11 and a framework region with at least 95%, 96%, 97%, 98% or 99% sequence identity to the VL framework sequence of SEQ ID NO: 11. In one aspect, the VL of the first antigen-binding domain comprises the VL light chain CDR sequences of SEQ ID NO: 11 and a framework region with at least 95% sequence identity to the VL framework sequence of SEQ ID NO: 11. In another aspect, the VL of the first antigen-binding domain comprises the VL light chain CDR sequences of SEQ ID NO: 11 and a framework region with at least 98% sequence identity to the VL framework sequence of SEQ ID NO: 11.

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 corresponding to any of the above aspects, and a VL sequence corresponding to any of the above aspects.

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

In one aspect, the first antigen-binding domain comprises a human constant region. In one aspect, the first antigen-binding fragment is a Fab molecule comprising a human constant region, in particular a human CH1 and/or CL domain. In one aspect, the first antigen-binding domain comprises a light chain constant region comprising 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: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 amino acid mutations described herein as "charge modifications" and/or may comprise a deletion or substitution of one or more (in particular two) N-terminal amino acids if it belongs to a crossover Fab molecule. In some aspects, the first antigen-binding domain comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of the CH1 domain contained in the amino acid sequence of SEQ ID NO:54. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise amino acid mutations described herein as "charge modifications."

In one aspect, the antibody is a monoclonal antibody.

In one aspect, the antibody is an IgG, in particular an IgG ₁ antibody. In one aspect, the antibody is a full-length antibody.

In another aspect, the antibody 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 particular, a Fab molecule. In another aspect, the antibody fragment is a diabody, triabody, or tetrabody.

In one aspect, the first antigen-binding domain is a Fab molecule. In a preferred aspect, the first antigen-binding domain is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are substituted for each other (i.e., the first antigen-binding domain is a crossover Fab molecule).

In a further aspect, the antibody of any of the above aspects may have any of the features, individually or in combination, described in Sections II.A.1.-8. below.

In a preferred aspect, the antibody comprises an Fc domain, in particular an IgG Fc domain, more particularly an IgG ₁ Fc domain. In one aspect, the Fc domain is a human Fc domain. In one aspect, the Fc domain is a human IgG ₁ Fc domain. The Fc domain is comprised of a first and a second subunit and may have any of the features, individually or in combination, described below herein with respect to variants of Fc domains (Section II.A.8.).

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 further described below herein (Section II.A. 7.).

1. Antibody fragments

In certain aspects, the antibody provided herein is an antibody fragment.

In one aspect, the antibody fragment is a Fab, Fab', Fab'-SH or F(ab') ₂ molecule, in particular a Fab molecule as described herein. A "Fab'molecule" differs from a Fab molecule by the addition of residues at the carboxy terminus of the CH1 domain, including one or more cysteine residues from the hinge region of the antibody. Fab'-SH are Fab' molecules in which the cysteine residue(s) of the constant domains bear a free thiol group. Treatment with pepsin produces an F(ab') ₂ molecule that contains two antigen-binding sites (two Fab molecules) and a portion of the Fc region.

In another aspect, 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 404097; 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).

In a further aspect, the antibody fragment is a single-chain Fab molecule. A "single-chain Fab molecule" or "scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein said antibody domains and said linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In particular, said linker is a polypeptide of at least 30 amino acids, preferably from 32 to 50 amino acids. Said single-chain Fab molecules are stabilized by a natural disulfide bond between the CL domain and the CH1 domain. In addition, the single-chain Fab molecules can be further stabilized by creating interchain disulfide bonds by inserting cysteine residues (e.g. at position 44 of the variable domain of the heavy chain and position 100 of the variable domain of the light chain according to Kabat numbering).

In another aspect, the antibody fragment is a single-chain variable fragment (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy (VH) and light chains (VL) of an antibody connected by a linker. In particular, the linker is a short polypeptide of 10-25 amino acids and is typically enriched 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. The protein retains the specificity of the original antibody despite the removal of the constant regions and the introduction of a linker. For a review of scFv fragments, see, e.g., Pluckthun, in 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.

In another aspect, the antibody fragment is a single-domain antibody. "Single-domain antibodies" are antibody fragments that comprise all or part of a heavy chain variable domain or all or part of a light chain variable domain of an antibody. In certain aspects, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

Antibody fragments can be produced by a variety of methods, including, but not limited to, proteolytic cleavage of an intact antibody, as well as recombinant production by recombinant host cells (e.g., E. coli), as described herein.

2. Humanized antibodies

In certain aspects, the antibody provided herein is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining 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 an antibody of non-human origin and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody optionally also comprises at least a portion of a human constant region. In some aspects, some FR residues in the humanized antibody are replaced with corresponding residues of a non-human antibody (e.g., the antibody from which the CDR residues are derived), such as to restore or improve the specificity or affinity of the antibody.

Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619–1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323–329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029–10033 (1989); U.S. Patents Ms 5821337, 7527791, 6982321, and 7087409; Kashmiri et al, Methods 36:25–34 (2005) (describing grafting of a specificity determining region (SDR)); Padlan, Mol. Immunol. 28:489–498 (1991) (describing "surface modification"); Dall'Acqua et al., Methods 36:43-60 (2005) (described "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (described a "directed selection" approach to FR shuffling).

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

3. Glycosylation variants

In some aspects, the antibody provided herein is altered to increase or decrease the degree of glycosylation of the antibody. Addition or removal of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

If the antibody comprises an Fc region, the oligosaccharide attached thereto can be altered. Native antibodies produced by mammalian cells typically comprise a branched biantennary oligosaccharide that is typically attached via an N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide can 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 aspects, modifications to the oligosaccharide in an antibody of the present invention can be made to create antibody variants with certain improved properties.

In one aspect, antibody variants are provided that comprise a non-fucosylated oligosaccharide, i.e., an oligosaccharide structure lacking fucose attached (directly or indirectly) to the Fc region. Such a non-fucosylated oligosaccharide (also referred to as an "afucosylated" oligosaccharide) is, in particular, an N-linked oligosaccharide lacking a fucose residue attached to the first GlcNAc in the backbone of the biantennary oligosaccharide structure. In one aspect, antibody variants are provided that comprise an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to a native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides 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). The percentage of non-fucosylated oligosaccharides represents the (average) amount of oligosaccharides without fucose residues relative to the sum of all oligosaccharides attached to Asn 297 (e.g., complex, hybrid, and high-mannose structures), as measured by TOF-MALDI mass spectrometry, e.g., as described in WO 2006/082515. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); wherein Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e. between positions 294 and 300, due to minor sequence variations in the antibodies. Such antibodies containing an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved binding to the FcγRIIIa receptor and/or improved effector function, in particular improved ADCC function. See, for example, US 2003/0157108; US 2004/0093621.

Examples of cell lines capable of producing antibodies with reduced fucosylation include protein fucosylation-deficient CHO Lec13 cells (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108 and WO 2004/056312, particularly Example 11), as well as knockout cell lines such as FUT8 alpha-1,6-fucosyltransferase gene knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107), or cells with reduced or eliminated activity of GDP-fucose synthesis or transport protein (see, for example, US2004259150, US2005031613, US2004132140, US2004110282).

In a further aspect, antibody variants with bisected oligosaccharides are provided, for example, in which a biantennary oligosaccharide attached to the Fc region of the antibody is 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 in, for example, Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99/54342; WO 2004/065540, WO 2003/011878.

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

4. Cysteine-engineered antibody variants

In certain aspects, it may be desirable to create cysteine engineered antibodies, such as THIOMAB™ antibodies, in which one or more residues are substituted with cysteine residues. In preferred aspects, the substituted residues are at accessible sites on the antibody. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites on the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as further described herein. Cysteine engineered antibodies can be prepared as described, for example, in U.S. Patent Nos. 7,521,541, 830,930, 7,855,275, 9000,130, or WO 2016040856.

5. Antibody derivatives

In certain aspects, the antibody provided herein can be further modified to contain additional non-protein moieties that are known in the art and readily available. Moieties suitable for derivatization of the antibody 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers, propropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerin), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymer molecules attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations that include, but are not limited to, the specific properties or functions of the antibody that are to be improved, whether the antibody derivative will be used in therapy under certain conditions, etc.

6. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-CD3/CD19 antibody according to this document conjugated (chemically linked) to one or more therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g. protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin or fragments thereof) or radioactive isotopes.

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

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 toxin A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.

In another aspect, an immunoconjugate comprises an antibody of the invention conjugated to a radioactive atom to form a radioconjugate. A number of radioactive isotopes are available for the preparation of radioconjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioactive isotopes of Lu. If the radioconjugate is used for detection purposes, it may contain a radioactive atom for scintigraphic studies, such as Tc ^99m or I ¹²³ , or a spin label for nuclear magnetic resonance (NMR) (also known as magnetic resonance imaging, MRI) such as I ¹²³ , I ¹³¹ , In ¹¹¹ , F ¹⁹ , C ¹³ , N ¹⁵ , O ¹⁷ , gadolinium, manganese or iron.

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

As used herein, immunoconjugates or ADCs expressly include, but are not limited to, conjugates prepared using cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl (4-vinyl sulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

7. Multispecific antibodies

The antibody provided herein is a multispecific antibody, in particular a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificity for at least two antigenic determinants (e.g., two different proteins or two different epitopes on a single protein). In some aspects, a multispecific antibody has three or more binding specificities. According to the present invention, one of the binding specificities is directed to CD3 and the other to CD19.

Multispecific antibodies can be produced as full-length antibodies or antibody fragments. Techniques for generating multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), and knob-into-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 generated by engineering using the electrostatic interaction effect to create Fc-heterodimeric antibody molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992) and WO 2011/034605); using common light chain technology to overcome the problem of light chain mispairing (see, e.g., WO 98/50431); using diabody technology to produce bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and the production of trispecific antibodies, as described, e.g., in Tutt et al. J. Immunol. 147:60 (1991).

Engineered antibodies with three or more antigen-binding sites, including, for example, "octopus antibodies" or DVD-Ig, are also included herein (see, for example, WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies with three or more antigen-binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792 and WO 2013/026831. Multispecific antibodies or antigen-binding fragments thereof also include “dual-acting FAbs” or “DAFs” that contain an antigen-binding site that binds to CD3 as well as another different antigen or to two different epitopes of CD3 (see, e.g., US 2008/0069820 and WO 2015/095539).

Multispecific antibodies may be provided in an asymmetric form with a crossover of domains in one or more binding arms with 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 into the domain interfaces to ensure proper Fab pairing. See, for example, WO 2016/172485.

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

A particular type of multispecific antibodies are bispecific antibodies, which are designed to simultaneously bind to a surface antigen on a target cell, such as a B cell, and to an activating invariant component of the T cell receptor (TCR) complex, such as CD3, to redirect T cells to kill the target cells. Therefore, the antibody provided herein is a multispecific antibody, in particular a bispecific antibody, wherein one of the binding specificities is directed to CD3 and the other to CD19 as a target cell antigen.

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

Preferred aspects of the antibody of the present invention are described below.

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

According to preferred aspects of the invention, the antigen-binding domains contained in the antibody are Fab molecules (i.e. antigen-binding domains consisting of a heavy and a light chain, each of which comprises a variable and a constant domain). In one aspect, the first, second and/or, if present, the third antigen-binding domain is a Fab molecule. In one aspect, said Fab molecule is human. In a preferred aspect, said Fab molecule is humanized. In another aspect, said Fab molecule comprises constant domains of a human heavy and light chain.

Preferably, at least one of the antigen-binding domains is a crossover Fab molecule. Such a modification reduces mispairing of heavy and light chains from different Fab molecules, thereby improving the yield and purity of the (multispecific) antibody of the invention when produced recombinantly. In a preferred crossover Fab molecule useful for the (multispecific) antibody of the invention, the variable domains of the Fab light chain and the Fab heavy chain (VL and VH, respectively) are exchanged. However, even with such an exchange of domains, the (multispecific) antibody preparation may contain some by-products due to the so-called Bence-Jones interaction between the 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 thus improve the purity and yield of the desired (multispecific) antibody, charged amino acids of opposite charges can be introduced at specific amino acid positions in the CH1 and CL domains of either the CD3-binding Fab molecule or the CD19-binding Fab molecule(s), as further described herein. Charge modifications are performed on standard Fab molecules contained in the (multispecific) antibody (as shown, for example, in Fig. 1A-C, G-L) or on VH/VL crossover Fab molecules contained in the (multispecific) antibody (as shown, for example, in Fig. 1D-E, M-P) (but not on both). In preferred aspects, charge modifications are made to standard Fab molecules contained in a (multispecific) antibody (which in preferred aspects binds to CD19).

In a preferred aspect, according to the present invention (multispecific), the antibody is capable of simultaneously binding to CD3 and CD19. In one aspect, the (multispecific) antibody is capable of cross-linking a T cell and a target cell by simultaneously binding to CD3 and CD19. In an even more preferred aspect, such simultaneous binding results in lysis of the target cell, in particular a CD19 expressing target cell, such as a B cell. In one aspect, such simultaneous binding results in activation of T cells. In other aspects, such simultaneous binding results in a cellular response of a T lymphocyte, in particular a cytotoxic T lymphocyte, selected from the group consisting of proliferation, differentiation, secretion of cytokines, release of cytotoxic effector molecules, cytotoxic activity and expression of activation markers. In one aspect, binding of a (multispecific) antibody to CD3 without concomitant binding to CD19 does not result in T cell activation.

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

Preferably, the T cell according to any aspects of the invention is a cytotoxic T cell. In some aspects, the T cell is a CD4 ⁺ or CD8 ⁺ T cell, in particular a CD8 ⁺ T cell.

a) First antigen-binding domain

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

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

In one aspect, the antigen-binding domain that binds to CD3 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 aspect, the antigen-binding domain that binds to CD3 is a Fab molecule.

In preferred aspects, 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 heavy and light chains of the Fab are/are replaced by each other. In such aspects, the antigen-binding domain(s) that bind to CD19 is/are preferably a standard Fab molecule. In aspects in which more than one antigen-binding domain, in particular a Fab molecule that binds to CD19, is present in a (multispecific) antibody, the antigen-binding domain that binds to CD3 is preferably a crossover Fab molecule and the antigen-binding domain that binds to CD19 are standard Fab molecules.

In alternative aspects, the antigen-binding domain that binds to CD3 is a standard Fab molecule. In such aspects, the antigen-binding domain(s) that binds to 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 heavy and light chains of the Fab are replaced/substituted with each other. In aspects in which more than one antigen-binding domain, in particular a Fab molecule that binds to CD3, is present in a (multispecific) antibody, the antigen-binding domain that binds to CD19 is preferably a crossover Fab molecule and the antigen-binding domains that bind to CD3 are standard Fab molecules.

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

In one aspect, the (multispecific) antibody has no more than one antigen-binding domain that binds to CD3 (i.e., the antibody provides monovalent binding to CD3).

b) The second (and third) antigen-binding domain

The (multispecific) antibody of the invention comprises at least one antigen-binding domain (a second and optionally a third antigen-binding domain), in particular a Fab molecule that binds to CD19. The second antigen-binding domain is capable of directing the (multispecific) antibody to a target site, for example to a particular cell type that expresses CD19.

In one aspect, 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 aspect, the antigen-binding domain that binds to CD19 is a Fab molecule.

In certain aspects, the (multispecific) antibody comprises two antigen-binding domains, in particular Fab molecules, that bind to CD19. In a preferred aspect, all of these antigen-binding domains are identical, i.e. they have the same molecular format (e.g., a standard or crossover Fab molecule) and comprise the same amino acid sequences, comprising the same amino acid substitutions in the CH1 and CL domains, as described herein (if any). In one aspect, the (multispecific) antibody comprises no more than two antigen-binding domains, in particular Fab molecules, that bind to CD19.

In preferred aspects, the antigen binding domain(s) that binds to CD19 is/are a standard Fab molecule. In such aspects, the antigen binding domain(s) 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 heavy and light chains of the Fab are/are replaced with each other.

In alternative aspects, the antigen-binding domain(s) that binds to CD19 is/are 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 heavy and light chains of the Fab are/are substituted for each other. In such aspects, the antigen-binding domain(s) that binds to CD3 is a standard Fab molecule.

In one aspect, the second (and, if present, the third) antigen-binding domain comprises a human constant region. In one aspect, the second (and, if present, the third) antigen-binding domain is a Fab molecule comprising a human constant region, in particular a human CH1 and/or CL domain. Exemplary sequences of human constant domains are set forth in SEQ ID NOs:52 and 53 (human kappa and lambda CL domains, respectively) and SEQ ID NO:54 (human IgG1 heavy chain CH1-CH2-CH3 _constant domains). In one aspect, the second (and, if present, the third) antigen-binding domain comprises a light chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: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 amino acid mutations described herein as "charge modifications" and/or may comprise a deletion or substitution of one or more (in particular two) N-terminal amino acids if it belongs to a Fab crossover molecule. In some aspects, the second (and, if present, the third) antigen-binding domain comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of the CH1 domain contained in the amino acid sequence of SEQ ID NO: 54. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise amino acid mutations, described herein as "charge modifications."

In one aspect, the second (and, if present, the third) antigen-binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:15, HCDR 2 of SEQ ID NO:16 and HCDR 3 of SEQ ID NO:17, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO:19, LCDR 2 of SEQ ID NO:20 and LCDR 3 of SEQ ID NO:21.

In one aspect, the second (and, if present, the third) antigen-binding domain is (or is derived from) a humanized antibody. In one aspect, the second (and, if present, the third) antigen-binding domain is a humanized antigen-binding domain (i.e., an antigen-binding domain of a humanized antibody). In one aspect, the VH and/or VL of the second (and, if present, the third) antigen-binding domain is a humanized variable region.

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

In one aspect, the VH of the second (and, if present, 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, third) antigen-binding domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:18. In one aspect, the VH of the second (and, if present, third) antigen-binding domain comprises a sequence that 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, third) antigen-binding domain comprises a sequence that is at least approximately 98% identical to the amino acid sequence of SEQ ID NO:18. In certain aspects, the VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions compared to a reference sequence, but an antibody comprising the sequence retains the ability to bind to CD19. 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:18. In some aspects, the substitutions, insertions, or deletions are in regions outside of the CDRs (i.e., in the FRs). In one aspect, the VH domain of the second (and, if present, third) antigen-binding domain comprises the amino acid sequence of SEQ ID NO:18. Optionally, the VH of the second (and, if present, third) antigen-binding domain comprises the amino acid sequence of SEQ ID NO:18, including post-translational modifications of this sequence.

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

In one aspect, the VH of the second (and, if present, third) antigen-binding domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18, and the VL of the second (and, if present, third) antigen-binding domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 22. In one aspect, the VH of the second (and, if present, third) antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 18, and the VL of the second (and, if present, third) antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 22.

In a further aspect, the second (and, if present, third) 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.

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

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

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

In one aspect, the VH of the second (and, if present, third) antigen-binding domain comprises the VH heavy chain CDR sequences of SEQ ID NO: 18 and a framework region with at least 95%, 96%, 97%, 98% or 99% sequence identity to the VH framework sequence of SEQ ID NO: 18. In one aspect, the VH of the second (and, if present, third) antigen-binding domain comprises the VH heavy chain CDR sequences of SEQ ID NO: 18 and a framework region with at least 95% sequence identity to the VH framework sequence of SEQ ID NO: 18. In another aspect, the VH of the second (and, if present, third) antigen-binding domain comprises the VH heavy chain CDR sequences of SEQ ID NO: 18 and a framework region with at least 98% sequence identity to the VH framework sequence of SEQ ID NO: 18.

In one aspect, the VL of the second (and, if present, third) antigen-binding domain comprises the VL light chain CDR sequences of SEQ ID NO:22 and a framework region with at least 95%, 96%, 97%, 98% or 99% sequence identity to the VL framework sequence of SEQ ID NO:22. In one aspect, the VL of the second (and, if present, third) antigen-binding domain comprises the VL light chain CDR sequences of SEQ ID NO:22 and a framework region with at least 95% sequence identity to the VL framework sequence of SEQ ID NO:22. In another aspect, the VL of the second (and, if present, third) antigen-binding domain comprises the VL light chain CDR sequences of SEQ ID NO:22 and a framework region with at least 98% sequence identity to the VL framework sequence of SEQ ID NO:22.

In another aspect, the second (and, if present, the third) antigen-binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:28, HCDR 2 of SEQ ID NO:29 and HCDR 3 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, LCDR 2 of SEQ ID NO:33 and LCDR 3 of SEQ ID NO:34.

In one aspect, the second (and, if present, the third) antigen-binding domain is (or is derived from) a humanized antibody. In one aspect, the second (and, if present, the third) antigen-binding domain is a humanized antigen-binding domain (i.e., an antigen-binding domain of a humanized antibody). In one aspect, the VH and/or VL of the second (and, if present, the third) antigen-binding domain is a humanized variable region.

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

In one aspect, the VH of the second (and, if present, 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, third) antigen-binding domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:31. In one aspect, the VH of the second (and, if present, third) antigen-binding domain comprises a sequence that 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, third) antigen-binding domain comprises a sequence that is at least approximately 98% identical to the amino acid sequence of SEQ ID NO:31. In certain aspects, the VH sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody comprising the sequence retains the ability to bind to CD19. In certain aspects, a total of 1-10 amino acids have been substituted, inserted, and/or deleted in the amino acid sequence of SEQ ID NO:31. In some aspects, the substitutions, insertions, or deletions are in regions outside of the CDRs (i.e., in the FRs). In one aspect, the VH domain of the second (and, if present, third) antigen-binding domain comprises the amino acid sequence of SEQ ID NO:31. Optionally, the VH of the second (and, if present, third) antigen-binding domain comprises the amino acid sequence of SEQ ID NO:31, including post-translational modifications of this sequence.

In one aspect, the VL of the second (and, if present, 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, third) antigen-binding domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:35. In one aspect, the VL of the second (and, if present, third) antigen-binding domain comprises a sequence that 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 a sequence that is at least approximately 98% identical to the amino acid sequence of SEQ ID NO:35. In certain aspects, the VL sequence having at least 95%, 96%, 97%, 98%, or 99% identity comprises substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody comprising the sequence retains the ability to bind to CD19. In certain aspects, a total of 1-10 amino acids have been substituted, inserted, and/or deleted in the amino acid sequence of SEQ ID NO:35. In some aspects, the substitutions, insertions, or deletions are in regions outside of the CDRs (i.e., in the FRs). In one aspect, the VL domain 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, third) antigen-binding domain comprises the amino acid sequence of SEQ ID NO:35, including post-translational modifications of this sequence.

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

In a further aspect, the second (and, if present, third) 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.

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

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

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

In one aspect, the VH of the second (and, if present, third) antigen-binding domain comprises the VH heavy chain CDR sequences of SEQ ID NO:31 and a framework region with at least 95%, 96%, 97%, 98% or 99% sequence identity to the VH framework sequence of SEQ ID NO:31. In one aspect, the VH of the second (and, if present, third) antigen-binding domain comprises the VH heavy chain CDR sequences of SEQ ID NO:31 and a framework region with at least 95% sequence identity to the VH framework sequence of SEQ ID NO:31. In another aspect, the VH of the second (and, if present, third) antigen-binding domain comprises the VH heavy chain CDR sequences of SEQ ID NO:31 and a framework region with at least 98% sequence identity to the VH framework sequence of SEQ ID NO:31.

In one aspect, the VL of the second (and, if present, third) antigen-binding domain comprises the VL light chain CDR sequences of SEQ ID NO:35 and a framework region with at least 95%, 96%, 97%, 98% or 99% sequence identity to the VL framework sequence of SEQ ID NO:35. In one aspect, the VL of the second (and, if present, third) antigen-binding domain comprises the VL light chain CDR sequences of SEQ ID NO:35 and a framework region with at least 95% sequence identity to the VL framework sequence of SEQ ID NO:35. In another aspect, the VL of the second (and, if present, third) antigen-binding domain comprises the VL light chain CDR sequences of SEQ ID NO:35 and a framework region with at least 98% sequence identity to the VL framework sequence of SEQ ID NO:35.

c) Charge modifications

The (multispecific) antibody of the invention may comprise amino acid substitutions in the Fab molecules contained therein that are particularly effective in reducing mispairing of light chains with mismatched heavy chains (Bence Jones-type side products) that may occur when producing Fab-based multispecific antibodies with VH/VL exchange in one (or more in the case of molecules comprising more than two antigen-binding Fab molecules) of their binding arms (see also PCT Publication No. WO 2015/150447, incorporated herein by reference in its entirety, in particular the examples therein). The ratio of desired (multispecific) antibody versus undesirable side products, in particular Bence-Jones type side products that arise in multispecific antibodies with a VH/VL domain exchange in one of their binding arms, can be improved by introducing charged amino acids with opposite charges at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modifications").

Accordingly, in some aspects, wherein 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 (in particular, in the first antigen-binding domain) the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other,

i) in the CL constant domain of the second (and, if present, third) antigen-binding domain, the amino acid at position 124 is substituted with a positively charged amino acid (Kabat numbering), and in the CH1 constant domain of the second (and, if present, third) antigen-binding domain, the amino acid at position 147 or the amino acid at position 213 is substituted with a negatively charged amino acid (numbering in accordance with the EU index according to Kabat); or

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

The (multispecific) antibody does not contain both modifications mentioned in i) and ii). The constant domains CL and CH1 of the antigen-binding domain containing the VH/VL exchange are not substituted for each other (i.e. remain unexchanged).

In a more specific aspect

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

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

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

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

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

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

In an even more preferred aspect, in the CL constant domain of the second (and, if present, third) antigen-binding domain, the amino acid at position 124 is substituted with lysine (K) (Kabat numbering) and the amino acid at position 123 is substituted with arginine (R) (Kabat numbering), and in the CH1 constant domain of the second (and, if present, third) antigen-binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (Kabat EU numbering) and the amino acid at position 213 is substituted with glutamic acid (E) (Kabat EU numbering).

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

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

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

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

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

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

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

In a preferred aspect, the (multispecific) antibody of the invention comprises:

(a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:2, HCDR 2 of SEQ ID NO:3 and HCDR 3 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, LCDR 2 of SEQ ID NO:9 and LCDR 3 of SEQ ID NO:10, and

(b) a second and optionally third antigen-binding domain that binds CD19;

wherein in the constant domain CL of the second (and, if present, third) antigen-binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (Kabat numbering) (in a preferred aspect, independently with lysine (K) or arginine (R)) and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (Kabat numbering) (in a preferred aspect, independently with lysine (K) or arginine (R)), 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 with glutamic acid (E) or aspartic acid (D) (numbering according to the EU index according to Kabat) and the amino acid at position 213 is independently substituted with glutamic acid acid (E) or aspartic acid (D) (numbering according to the EU index according to Kabat).

d) Formats of multispecific antibodies

The (multispecific) antibody according to the present invention can have various configurations. Typical configurations are illustrated in Fig. 1.

In preferred aspects, the antigen-binding domains contained in the (multispecific) antibody are Fab molecules. In such aspects, the first, second, third, etc. antigen-binding domain may be referred to herein as the first, second, third, etc. Fab molecule, respectively.

In one aspect, the first and second antigen-binding domains of the (multispecific) antibody are fused to each other, optionally via a peptide linker. In preferred aspects, each of the first and second antigen-binding domains is a Fab molecule. In one such aspect, the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain. In another such aspect, the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain. In aspects in which (i) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain or (ii) the second antigen-binding domain is 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 further be fused to each other, optionally via a peptide linker.

A (multispecific) antibody with one antigen-binding domain (such as a Fab molecule) capable of specifically binding to a second antigen, such as a target cell antigen such as CD19 (e.g. as shown in Fig. 1A, D, G, I, M, H), is particularly useful in cases where internalization of the second antigen can be expected following binding to 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 the internalization of the second antigen, thereby reducing its availability.

In other cases, however, it is advantageous to have a (multispecific) antibody comprising two or more antigen-binding domains (such as Fab molecules) specific for a second antigen, such as a target cell antigen such as CD19 (see the examples illustrated in Fig. 1B, 1C, 1E, 1E, 1K, 1K, 1P or 1R), for example to optimize targeting to the target site or to allow cross-linking of target cell antigens.

Accordingly, in preferred aspects, the (multispecific) antibody according to the present invention comprises a third antigen-binding domain.

In one aspect, the third antigen-binding domain binds to CD19. In one aspect, the third antigen-binding domain is a Fab molecule.

In one aspect, the third antigen-binding domain is identical to the second antigen-binding domain.

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

Like the second antigen-binding domain, the third antigen-binding domain is preferably a standard Fab molecule. However, aspects are also provided in which the second and third antigen-binding domains are crossover Fab molecules (and the first antigen-binding domain is a standard Fab molecule). Thus, in preferred aspects, each of the second and third antigen-binding domains is a standard 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 heavy and light chains of the Fab are replaced/substituted with each other. In other aspects, each of the second and third antigen-binding domains is a crossover Fab molecule and the first antigen-binding domain is a standard Fab molecule.

In the case of a third antigen-binding domain, in a preferred aspect the first antigen-binding domain binds to CD3 and the second and third antigen-binding domains bind to CD19.

In preferred aspects, the (multispecific) antibody of the invention comprises an Fc domain consisting of a first and a second subunit. The first and second subunits of the Fc domain are capable of stable association.

The (multispecific) antibody according to the invention may have different configurations, i.e. the first, second (and optionally the third) antigen-binding domain 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. If the Fab molecule is fused to the N-terminus of the Fc domain subunit, the fusion typically occurs via the hinge region of the immunoglobulin.

In some aspects, each of the first and second antigen-binding domains is 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 aspects, the second antigen-binding domain can be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain or to the N-terminus of another Fc domain subunit. In such preferred aspects, the second antigen-binding domain is a standard Fab molecule and the first antigen-binding domain is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the VH and VL variable domains or the CL and CH1 constant domains of the Fab heavy and light chains are replaced/substituted for each other. In other such aspects, the second antigen-binding domain is a crossover Fab molecule and the first antigen-binding domain is a standard Fab molecule.

In one aspect, each of the first and second antigen-binding domains is a Fab molecule, the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain. In a particular aspect, the (multispecific) antibody consists essentially of first and second Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Such a configuration is schematically depicted in Fig. 1G and 1M (in these examples, the first antigen-binding domain 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 be further fused to each other.

In another aspect, each of the first and second antigen-binding domains is a Fab molecule, and each of the first and second antigen-binding domains is fused at the C-terminus of a Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. In a particular aspect, the (multispecific) antibody consists essentially of first and second Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein each of the first and second Fab molecules is fused at the C-terminus of a Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. Such a configuration is schematically depicted in Fig. 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 standard Fab molecule). The first and second Fab molecules can be fused to the Fc domain directly or via a peptide linker. In a preferred aspect, each of the first and second Fab molecules is fused to the Fc domain via a hinge region of an immunoglobulin. In a particular aspect, the hinge region of an immunoglobulin is a hinge region of human IgG ₁ , in particular when the Fc domain is the Fc domain of human IgG ₁ .

In some aspects, each of the first and second antigen-binding domains is 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 aspects, 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 (as described above) to the N-terminus of another Fc domain subunit. In such preferred aspects, said second antigen-binding domain is a standard 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 replaced/substituted with each other. In other such aspects, said second antigen-binding domain is a crossover Fab molecule and said first antigen-binding domain is a standard Fab molecule.

In one aspect, each of the first and second antigen-binding domains is a Fab molecule, the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain. In a particular aspect, the (multispecific) antibody consists essentially of first and second Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Such a configuration is schematically depicted in Fig. 1H and 1H (in these examples, the first antigen-binding domain is a VH/VL crossover Fab molecule and the second antigen-binding domain is a standard Fab molecule). Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be further fused to each other.

In some aspects, the third antigen-binding domain, in particular the third Fab molecule, is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second Fc domain subunit. In such preferred aspects, each of said second and third antigen-binding domains is a standard 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 heavy and light chains of the Fab are replaced/substituted with each other. In other aspects, each of said second and third antigen-binding domains is a crossover Fab molecule, and the first antigen-binding domain is a standard Fab molecule.

In such a preferred aspect, each of the first and third antigen-binding domains is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits, 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 particular aspect, the (multispecific) antibody consists essentially of the first, second and third Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first Fc domain subunit, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit. Such a configuration is schematically depicted in Figs. 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 a standard Fab molecule) and Figs. 1K and 1P (in these examples, the first antigen-binding domain is a standard Fab molecule and the second and third antigen-binding domains are a VH/VL crossover Fab molecule). The first and third Fab molecules can be fused to the Fc domain directly or via a peptide linker. In a preferred aspect, each of the first and third Fab molecules is fused to the Fc domain via an immunoglobulin hinge region. In a particular aspect, the immunoglobulin hinge region is a human IgG ₁ hinge region, in particular when the Fc domain is a human IgG ₁ Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be further fused to each other.

In another such aspect, each of the second and third antigen-binding domains is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits, 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 particular aspect, the (multispecific) antibody consists essentially of first, second and third Fab molecules, an Fc domain consisting of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first Fc domain subunit, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit. Such a configuration is schematically depicted in Figs. 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 a standard Fab molecule) and Figs. 1K and 1I (in these examples, the first antigen-binding domain is a standard Fab molecule and the second and third antigen-binding domains are a VH/VL crossover Fab molecule). The second and third Fab molecules may be fused to the Fc domain directly or via a peptide linker. In a preferred aspect, each of the second and third Fab molecules is fused to the Fc domain via an immunoglobulin hinge region. In a particular aspect, the immunoglobulin hinge region is a human IgG ₁ hinge region, in particular when the Fc domain is a human IgG ₁ Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may be further fused to each other.

In (multispecific) antibody configurations in which a Fab molecule is fused at the C-terminus of a Fab heavy chain to the N-terminus of each of the Fc domain subunits via an immunoglobulin hinge region, the two Fab molecules, the hinge regions and the Fc domain advantageously form an immunoglobulin molecule. In a preferred aspect, the immunoglobulin molecule is an immunoglobulin of the IgG class. In an even more preferred aspect, the immunoglobulin is an immunoglobulin of the IgG ₁ subclass. In another aspect, the immunoglobulin is an immunoglobulin of the IgG ₄ subclass. In a further preferred aspect, the immunoglobulin is a human immunoglobulin. In other aspects, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one aspect, the immunoglobulin comprises a human constant region, in particular a human Fc region.

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 configuration of the first and second Fab molecules, the Fab light chain of the first molecule may be fused at the C-terminus to the N-terminus of the Fab light chain of the second Fab molecule or the Fab light chain of the second molecule may be fused at the C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. Fusion of the Fab light chains of the first and second Fab molecules further reduces mispairing of mismatched Fab heavy and light chains and also reduces the number of plasmids required for expression of some of the (multispecific) antibodies of the invention.

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 to 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 . "n" is generally an integer from 1 to 10, typically from 2 to 4. In one aspect, said peptide linker is at least 5 amino acids long, in one aspect from 5 to 100, in a further aspect from 10 to 50 amino acids long. In one aspect, said peptide linker is (GxS) _n or (GxS) _n G _m , where G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6 and m=0, 1, 2 or 3) or (x=4, n=1, 2, 3, 4 or 5 and m=0, 1, 2, 3, 4 or 5), in one aspect x=4 and n=2 or 3, in a further aspect x=4 and n=2, in yet another further aspect x=4, n=1 and m=5. In one aspect, said peptide linker is (G ₄ S) ₂ . In another aspect, said peptide linker is G ₄ SG ₅ . A particularly suitable peptide linker for fusing the Fab light chains of the first and second Fab molecules to each other is ( _G4S ) ₂ . An exemplary peptide linker suitable for joining the Fab heavy chains of the first and second Fab fragments comprises the sequence (D)-( _G4S ) ₂ (SEQ ID NOs 48 and 49). Another such particularly suitable linker comprises the sequence (D) _-G4SG5 (SEQ ID NOs 50 and 51). In addition, _the linkers may comprise an immunoglobulin hinge region (or a portion thereof). In particular, if the Fab molecule is fused to the N-terminus of the Fc domain subunit, it may be fused via an immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

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

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

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

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

In certain aspects, the (multispecific) antibody does not comprise an Fc domain. In such preferred aspects, each of said second and, if present, third antigen-binding domains is a standard 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 heavy and light chains of the Fab are replaced/substituted with each other. In other such aspects, each of said second and, if present, third antigen-binding domains is a crossover Fab molecule, and the first antigen-binding domain is a standard Fab molecule.

In one such aspect, the (multispecific) antibody is preferably comprised of first and second antigen-binding domains 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 heavy chain of the first antigen-binding domain. Such a configuration is schematically depicted in Figs. 1C and 1X (in these examples, the first antigen-binding domain is a VH/VL crossover Fab molecule and the second antigen-binding domain is a standard Fab molecule).

In another such aspect, the (multispecific) antibody is preferably comprised of first and second antigen-binding domains and optionally one or more peptide linkers, wherein 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 heavy chain of the second antigen-binding domain. Such a configuration is schematically depicted in Figs. 1T and 1C (in these examples, the first antigen-binding domain is a VH/VL crossover Fab molecule and the second antigen-binding domain is a standard Fab molecule).

In some aspects, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the (multispecific) antibody further comprises a third antigen-binding domain, in particular a third Fab molecule, wherein said third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In certain such aspects, the (multispecific) antibody consists essentially of the first, second and third Fab molecules and, optionally, one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. Such a configuration is schematically depicted in Fig. 1U and 1III (in these examples, the first antigen-binding domain is a VH/VL crossover Fab molecule, and each of the second and third antigen-binding domains is a standard Fab molecule) and Figs. 1U and 1AA (in these examples, the first antigen-binding domain is a standard Fab molecule, and each of the second and third antigen-binding domains is a VH/VL crossover Fab molecule).

In some aspects, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of a second Fab molecule, and the (multispecific) antibody further comprises a third antigen-binding domain, in particular a third Fab molecule, wherein said third Fab molecule is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the second Fab molecule. In certain such aspects, the (multispecific) antibody consists essentially of the first, second and third Fab molecules and, optionally, one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the third Fab molecule is fused at the N-terminus of the Fab heavy chain to the C-terminus of the Fab heavy chain of the second Fab molecule. Such a configuration is schematically depicted in Fig. 1F and 1G (in these examples, the first antigen-binding domain is a VH/VL crossover Fab molecule, and each of the second and third antigen-binding domains is a standard Fab molecule) and Figs. 1E and 1Y (in these examples, the first antigen-binding domain is a standard Fab molecule, and each of the second and third antigen-binding domains is a VH/VL crossover Fab molecule).

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

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

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

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

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

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

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

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

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

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

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

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

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 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 substituted for each other, and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO:2, HCDR 2 with SEQ ID NO:3 and HCDR 3 with SEQ ID NO:5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 with SEQ ID NO:8, LCDR 2 with SEQ ID NO:9 and LCDR 3 with SEQ ID NO:10;

b) a second antigen-binding domain that binds to CD 19, wherein the second antigen-binding domain is a (standard) Fab molecule;

c) Fc domain, consisting of the first and second subunits; and

(i) the first antigen-binding domain according to a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain according to b), and the second antigen-binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c), or

(ii) the second antigen-binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain according to a), and the first antigen-binding domain according to a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c).

In a preferred 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 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 substituted for each other, and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO:2, HCDR 2 with SEQ ID NO:3 and HCDR 3 with SEQ ID NO:5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 with SEQ ID NO:8, LCDR 2 with SEQ ID NO:9 and LCDR 3 with SEQ ID NO:10;

b) a second and third antigen-binding domain that binds to CD 19, wherein each of the second and third antigen-binding domains is a (standard) Fab molecule; and

c) Fc domain, consisting of the first and second subunits; wherein

(i) the first antigen-binding domain of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain of b), and the second antigen-binding domain of b) and the third antigen-binding domain of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c), or

(ii) the second antigen-binding domain of b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain of a), and the first antigen-binding domain of a) and the third antigen-binding domain of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

In another aspect, the invention provides a (multispecific) antibody comprising:

a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule 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 substituted for each other, and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 with SEQ ID NO:2, HCDR 2 with SEQ ID NO:3 and HCDR 3 with SEQ ID NO:5, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 with SEQ ID NO:8, LCDR 2 with SEQ ID NO:9 and LCDR 3 with SEQ ID NO:10;

b) a second antigen-binding domain that binds to CD 19, wherein the second antigen-binding domain is a (standard) Fab molecule;

c) Fc domain, consisting of the first and second subunits;

moreover

(i) the first antigen-binding domain of a) and the second antigen-binding domain of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c).

In all of the different configurations of the (multispecific) antibody according to the invention, the amino acid substitutions ("charge modifications") described herein, if present, may be in the CH1 and CL domains of the second and (if present) third antigen-binding domain/Fab molecule, or in the CH1 and CL domains of the first antigen-binding domain/Fab molecule. Preferably, they are in the CH1 and CL domains of the second and (if present) third antigen-binding domain/Fab molecule. According to the concept of the invention, if the amino acid substitutions described herein are made in the second (and, if present, third) antigen-binding domain/Fab molecule, such amino acid substitutions are not made in the first antigen-binding domain/Fab molecule. Conversely, if the amino acid substitutions described herein are made in the first antigen-binding domain/Fab molecule, no such amino acid substitutions are made in the second (and, if present, third) antigen-binding domain/Fab molecule. The amino acid substitutions are preferably made in (multispecific) antibodies comprising a Fab molecule in which the variable domains VL and VH1 of the Fab light chain and the Fab heavy chain are substituted for each other.

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

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 in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:2, HCDR 2 of SEQ ID NO:3 and HCDR 3 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, LCDR 2 of SEQ ID NO:9 and LCDR 3 of SEQ ID NO:10;

b) a second antigen-binding domain that binds to CD19, wherein the second antigen-binding domain is a (standard) Fab molecule;

c) Fc domain, consisting of the first and second subunits;

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

moreover

(i) the first antigen-binding domain according to a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain according to b), and the second antigen-binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c), or

(ii) the second antigen-binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain according to a), and the first antigen-binding domain according to a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c).

In a preferred 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 in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:2, HCDR 2 of SEQ ID NO:3 and HCDR 3 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, LCDR 2 of SEQ ID NO:9 and LCDR 3 of SEQ ID NO:10;

b) a second and third antigen-binding domain that binds to CD19, wherein each of the second and third antigen-binding domains is a (standard) Fab molecule; and

c) Fc domain, consisting of the first and second subunits;

wherein in the constant domain CL of the second antigen-binding domain according to b) and the third antigen-binding domain according to b), the amino acid at position 124 is substituted with lysine (K) (Kabat numbering) and the amino acid at position 123 is substituted with lysine (K) or arginine (R) (Kabat numbering) (most preferably arginine (R)), and wherein in the constant domain CH1 of the second antigen-binding domain according to b) and the third antigen-binding domain according to

b) the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat) and the amino acid at position 213 is substituted with glutamic acid (E) (numbering according to the EU index according to Kabat); and

moreover

(i) the first antigen-binding domain of a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain of b), and the second antigen-binding domain of b) and the third antigen-binding domain of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c), or

(ii) the second antigen-binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain according to a), and the first antigen-binding domain according to a) and the third antigen-binding domain according to b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c).

In another aspect, the invention provides a (multispecific) antibody comprising:

a) a first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:2, HCDR 2 of SEQ ID NO:3 and HCDR 3 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, LCDR 2 of SEQ ID NO:9 and LCDR 3 of SEQ ID NO:10;

b) a second antigen-binding domain that binds to CD19, wherein the second antigen-binding domain is a (standard) Fab molecule;

c) Fc domain, consisting of the first and second subunits;

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

wherein the first antigen-binding domain according to a) and the second antigen-binding domain according to b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c).

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

In a preferred aspect, the invention provides a (multispecific) antibody comprising:

a) a first antigen-binding domain that binds CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:2, HCDR 2 of SEQ ID NO:3 and HCDR 3 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, LCDR 2 of SEQ ID NO:9 and LCDR 3 of SEQ ID NO:10;

b) a second and third antigen-binding domains that bind to CD 19, wherein each of the second and third antigen-binding domains is a (standard) Fab molecule and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:15, HCDR 2 of SEQ ID NO:16 and HCDR 3 of SEQ ID NO:17, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO:19, LCDR 2 of SEQ ID NO:20 and LCDR 3 of SEQ ID NO:21;

c) Fc domain, consisting of the first and second subunits; wherein

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

and additionally

the second antigen-binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain according to a), and the first antigen-binding domain according to a) and the third antigen-binding domain according to b), are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c).

In a further preferred 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 in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and comprises 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) a second and third antigen-binding domains that bind to CD19, wherein each of the second and third antigen-binding domains is a (standard) 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;

c) Fc domain, consisting of the first and second subunits; wherein

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

and additionally

the second antigen-binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain according to a), and the first antigen-binding domain according to a) and the third antigen-binding domain according to b), are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c).

In a further preferred aspect, the invention provides a (multispecific) antibody comprising:

a) a first antigen-binding domain that binds CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:2, HCDR 2 of SEQ ID NO:3 and HCDR 3 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, LCDR 2 of SEQ ID NO:9 and LCDR 3 of SEQ ID NO:10;

b) a second and third antigen-binding domains that bind to CD19, wherein each of the second and third antigen-binding domains is a (standard) Fab molecule and comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:28, HCDR 2 of SEQ ID NO:29 and HCDR 3 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, LCDR 2 of SEQ ID NO:33 and LCDR 3 of SEQ ID NO:34;

c) Fc domain, consisting of the first and second subunits; wherein

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

and additionally

the second antigen-binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain according to a), and the first antigen-binding domain according to a) and the third antigen-binding domain according to b), are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c).

In a further preferred 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 in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are substituted for each other, and comprises 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) a second and third antigen-binding domains that bind to CD19, wherein each of the second and third antigen-binding domains is a (standard) 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) Fc domain, consisting of the first and second subunits; and

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

and additionally

the second antigen-binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain according to a), and the first antigen-binding domain according to a) and the third antigen-binding domain according to b), are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c).

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

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

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

In a further aspect according to these aspects of the invention, the Fc domain is the Fc domain of human IgG ₁ .

In a preferred particular aspect, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:23 or SEQ ID NO:39 (particularly 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 (particularly two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:25, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to sequence of SEQ ID NO:27. In a further preferred particular aspect, 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.

In one aspect, the invention provides a (multispecific) antibody that binds 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 (particularly 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 (particularly two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: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 invention provides a (multispecific) antibody that binds to CD3 and CD19, comprising 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.

In a preferred particular aspect, the (multispecific) antibody comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:36 or SEQ ID NO:40 (in particular 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 (in particular two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:38, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to sequence of SEQ ID NO:27. In a further preferred particular aspect, the (multispecific) antibody comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:40 (in particular SEQ ID NO:36), a polypeptide comprising the amino acid sequence of SEQ ID NO:37, a polypeptide (in particular 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.

In one aspect, the invention provides a (multispecific) antibody that binds 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 (in particular 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 (in particular two polypeptides) comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: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 invention provides a (multispecific) antibody that binds to CD3 and CD19, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:40 (in particular SEQ ID NO:36), a polypeptide comprising the amino acid sequence of SEQ ID NO:37, a polypeptide (in particular 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.

8. Variants of Fc domains

In preferred aspects, the (multispecific) antibody of the invention comprises an Fc domain consisting of a first and a second subunit.

The Fc domain of a (multispecific) antibody consists of a pair of polypeptide chains containing the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which contains the constant domains of the heavy chain CH2 and CH3 of IgG. Two subunits of the Fc domain are capable of stable association with each other. In one aspect, the (multispecific) antibody of the invention contains no more than one Fc domain.

In one aspect, the Fc domain of the (multispecific) antibody is an IgG Fc domain. In a preferred aspect, the Fc domain is an IgG ₁ Fc domain. In another aspect, the Fc domain is an IgG ₁ Fc domain. In a more specific aspect, the Fc domain is an IgG ₄ Fc domain comprising an amino acid substitution at position S228 (numbering according to the EU Kabat index), in particular an amino acid substitution S228P. This amino acid substitution reduces the in vivo turnover of the Fab arms of IgG ₄ antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further preferred aspect, the Fc domain is a human Fc domain. In an even more preferred aspect, the Fc domain is a human IgG ₁ Fc domain. A typical sequence of the Fc region of human IgG ₁ is given in SEQ ID NO:47.

a) Modifications of the Fc domain that promote heterodimerization

The (multispecific) antibodies according to the invention comprise different antigen-binding domains which can be fused to one or the other of the two Fc domain subunits, so that the two Fc domain subunits are typically contained in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization lead to several possible combinations of the two polypeptides. Therefore, in order to improve the yield and purity of (multispecific) antibodies in recombinant production, it will be expedient to introduce a modification into the Fc domain of the (multispecific) antibody that facilitates the association of the desired polypeptides.

Accordingly, in preferred aspects, the Fc domain of the (multispecific) antibody according to the invention comprises a modification that facilitates the association of the first and second subunits of the Fc domain. The site of the most intense protein-protein interaction between the two subunits of the Fc domain of human IgG is the CH3 domain of the Fc domain. Thus, in one aspect, said modification is in the CH3 domain of the Fc domain.

There are several approaches for modification of the CH3 domain of the Fc domain to promote heterodimerization, which are well described, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. As a rule, in all such approaches, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit The Fc domains are designed in a complementary manner such that each CH3 domain (or the heavy chain containing it) can no longer form a homodimer with itself, but is forced to form a heterodimer with another complementarily engineered CH3 domain (so that heterodimerization of the first and second CH3 domains occurs and no homodimerization occurs between the first two or the second two CH3 domains). These different approaches to improve heavy chain heterodimerization are envisaged as different alternatives in combination with heavy-light chain modifications (e.g. swapping/replacing VH and VL in one binding arm and introducing substitutions of oppositely charged amino acids in the CH1/CL interface) in the (multispecific) antibody, which reduces heavy/light chain mispairing and Bence-Jones side products.

In a particular aspect, said modifications that facilitate the association of the first and second Fc domain subunits are a so-called knob-into-hole modification, comprising a modification of the knob in one of the two Fc domain subunits and a modification of the hole in the other of the two Fc domain subunits.

Knob-into-hole technology is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, this method involves introducing a bump ("knob") at the interface of a first polypeptide and a corresponding cavity ("cavity") at the interface of a second polypeptide, such that the bump can be positioned in the cavity to promote heterodimer formation and hinder homodimer formation. The bumps are constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensating cavities of identical or similar size to the protrusions are created on the contact surface of the second polypeptide by replacing large amino acid side chains with smaller ones (for example, alanine or threonine).

Accordingly, in a preferred aspect, in the CH3 domain of the first subunit of the Fc domain of the (multispecific) antibody, the amino acid residue is replaced by an amino acid residue having a larger side chain volume, thereby creating a bulge in the CH3 domain of the first subunit that can be accommodated in a cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, the amino acid residue is replaced by an amino acid residue having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit that can accommodate the bulge in the CH3 domain of the first subunit.

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

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

The bulge and cavity can be created by altering the nucleic acid encoding the polypeptides, for example by site-directed mutagenesis, or by peptide synthesis.

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

In a further aspect, in the first subunit of the Fc domain, additionally the serine residue at position 354 is substituted with a cysteine residue (S354C) or the glutamic acid residue at position 356 is substituted with a cysteine residue (E356C) (in particular, the serine residue at position 354 is substituted with a cysteine residue), and in the second subunit of the Fc domain, additionally the tyrosine residue at position 349 is substituted with a cysteine residue (Y349C) (numbering according to the EU index according to Kabat). The introduction of two cysteine residues leads to the formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

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

In a preferred aspect, the antigen binding domain that binds to CD3 is fused (optionally via a second antigen binding domain that binds to CD 19 and/or a peptide linker) to a first subunit of the Fc domain (containing a "knob" modification). Without being limited by theory, fusing the antigen binding domain that binds to CD3 to a knob-containing subunit of the Fc domain will (further) minimize the creation of antibodies containing two antigen binding domains that bind to CD3 (due to steric hindrance of the two knob-containing polypeptides).

Other CH3 modification techniques for stimulating heterodimerization are provided as alternative embodiments according to the invention and are described, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

In one aspect, the heterodimerization approach described in EP 1 870 459 is used as an alternative. This approach is based on the introduction of charged amino acids with opposite charges at certain amino acid positions in the interface of the CH3/CH3 domains between the two subunits of the Fc domain. A particular aspect for the (multispecific) antibody of the invention are the amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and the amino acid mutations D399K; E357K in the other of the two CH3 domains of the Fc domain (numbering according to the EU index according to Kabat).

In another aspect, the (multispecific) antibody of the invention comprises the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations T366S, L368A, Y407V 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 in accordance with the EU index according to Kabat).

In another aspect, the (multispecific) antibody of the invention comprises the amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the 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 domains 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 numbering according to the EU index according to Kabat).

In one aspect, the heterodimerization approach described in WO 2013/157953 is used as an alternative. In one aspect, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (numbering according to the EU index according to Kabat). In a further aspect, the first CH3 domain comprises an additional amino acid mutation L351K. In a further aspect, the second CH3 domain comprises an additional amino acid mutation selected from Y349E, Y349D and L368E (in particular L368E) (numbering according to the EU index according to Kabat).

In one aspect, the heterodimerization approach described in WO 2012/058768 is used as an alternative. In one aspect, 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 aspect, the second CH3 domain comprises an additional amino acid mutation at position T411, D399, S400, F405, N390 or K392, such as 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, e) K392V, K392M, K392R, K392L, K392F or K392E (numbering according to the EU index according to Kabat). In a further aspect, the first CH3 domain comprises the amino acid mutations L351Y, Y407A, and the second CH3 domain comprises the amino acid mutations T366V, K409F. In a further aspect, the first CH3 domain comprises the amino acid mutation Y407A, and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further aspect, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to the EU index according to Kabat).

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

In one aspect, the heterodimerization approach described in WO 2011/090762, which also uses the knob-into-hole technology described above, is used as an alternative. In one aspect, the first CH3 domain comprises the amino acid mutation T366W and the second CH3 domain comprises the amino acid mutation Y407A. In one aspect, the first CH3 domain comprises the amino acid mutation T366Y and the second CH3 domain comprises the amino acid mutation Y407T (numbering according to the EU index according to Kabat).

In one aspect, the (multispecific) antibody or its Fc domain is of the IgG2 subclass, and alternatively the heterodimerization approach described in WO 2010/129304 is used.

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

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

In another aspect, the heterodimerization approach described in WO 2007/110205 can be used as an alternative.

In one aspect, 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 EU index according to Kabat).

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

The Fc domain confers favorable pharmacokinetic properties to the (multispecific) antibody, including a long serum half-life that facilitates good accumulation in the target tissue and a favorable tissue-to-blood distribution ratio. However, at the same time, it may result in undesirable targeting of the (multispecific) antibody to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. In addition, co-activation of Fc receptor signaling pathways may result in cytokine release, which, in combination with the T cell activating properties and the long half-life of the (multispecific) antibody, leads to excessive cytokine receptor activation and severe side effects after systemic administration. Activation of (Fc receptor-bearing) immune cells other than T cells may even reduce the effectiveness of the (multispecific) antibody due to potential destruction of T cells, for example by NK cells.

Accordingly, in preferred aspects, the Fc domain of the (multispecific) antibody according to the invention exhibits reduced Fc receptor binding affinity and/or reduced effector function compared to the native IgG ₁ Fc domain. In one such aspect, the Fc domain (or a (multispecific) antibody comprising said Fc domain) exhibits less than 50%, in particular less than 20%, more particularly less than 10% and most particularly less than 5% of the binding affinity for the Fc receptor compared to the native Fc domain of IgG ₁ (or a (multispecific) antibody comprising a native Fc domain of IgG ₁ ), and/or less than 50%, in particular less than 20%, more particularly less than 10% and most particularly less than 5% of the effector function compared to the native Fc domain of IgG ₁ (or a (multispecific) antibody comprising a native Fc domain of IgG ₁ ). In one aspect, the Fc domain (or a (multispecific) antibody comprising said Fc domain) substantially does not bind to an Fc receptor and/or does not induce an effector function. In a preferred aspect, the Fc receptor is an Fcy receptor. In one aspect, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcy receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one aspect, the effector function is one or more functions selected from the group consisting of CDC, ADCC, ADCP and cytokine secretion. In a preferred aspect, the effector function is ADCC. In one aspect, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the native Fc domain of IgG ₁ . Substantially similar binding to FcRn is achieved when the Fc domain (or (multispecific) antibody comprising said Fc domain) exhibits greater than about 70%, in particular greater than about 80%, more particularly greater than about 90% of the binding affinity of the native Fc domain of IgG ₁ (or (multispecific) antibody comprising the native Fc domain of IgG ₁ ) to FcRn.

In certain aspects, the Fc domain is engineered to have reduced binding affinity for an Fc receptor and/or reduced effector function compared to a non-engineered Fc domain. In preferred aspects, the Fc domain of a (multispecific) antibody comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain for an Fc receptor and/or 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 aspect, the amino acid mutation reduces the binding affinity of the Fc domain for an Fc receptor. In one aspect, the amino acid mutation reduces the binding affinity of the Fc domain for an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In aspects where more than one amino acid mutation is present that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to the Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one aspect, the (multispecific) antibody comprising an engineered Fc domain exhibits less than 20%, in particular less than 10%, more particularly less than 5%, of the binding affinity to the Fc receptor compared to the (multispecific) antibody comprising a non-engineered Fc domain. In a preferred aspect, the Fc receptor is an Fcy receptor. In some aspects, the Fc receptor is a human Fc receptor. In some aspects, the Fc receptor is an activating Fc receptor. In a particular aspect, the Fc receptor is a human activating Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, most particularly human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some aspects, the binding affinity for a complement component is also reduced, in particular the binding affinity for C1q. In one aspect, the binding affinity for the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. maintaining the binding affinity of the Fc domain for said receptor is achieved when the Fc domain (or (multispecific) antibody comprising said Fc domain) exhibits more than about 70% of the binding affinity of the non-engineered form of the Fc domain (or (multispecific) antibody comprising said non-engineered form of the Fc domain) for FcRn. The Fc domain or (multispecific) antibodies of the invention comprising said Fc domain may exhibit more than about 80% and even more than about 90% of such affinity. In certain aspects, the Fc domain of the (multispecific) antibody is engineered to have reduced effector function compared to the non-engineered Fc domain. Reduced effector function may include, but is not limited to, one or more of the following: 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 that induces apoptosis, reduced cross-linking of target-bound antibodies, reduced maturation of dendritic cells, or reduced priming of T cells. In one aspect, the reduced effector function is one or more functions selected from the group of reduced CDC, reduced ADCC, reduced ADCF, and reduced cytokine secretion. In a preferred aspect, the reduced effector function is reduced ADCC. In one aspect, 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 aspect, an amino acid mutation that reduces the binding affinity of an Fc domain to an Fc receptor and/or effector function is an amino acid substitution. In one aspect, 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 the EU index of Kabat). In a more particular aspect, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numbering according to the EU index of Kabat). In some aspects, the Fc domain comprises the amino acid substitutions L234A and L235A (numbering according to the EU index of Kabat). In one such aspect, the Fc domain is an IgG ₁ Fc domain, in particular a human IgG ₁ Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more particular aspect, the amino acid substitution is P329A or P329G, in particular P329G (numbering according to the EU index according to Kabat). In one aspect, the Fc domain comprises an amino acid substitution at position P329 and an additional amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbering according to the EU index according to Kabat). In a more particular aspect, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In preferred aspects, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numbering according to the EU index according to Kabat). In more preferred aspects, the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG"). In particular, in preferred aspects, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (numbering according to the EU index according to Kabat), i.e., in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is substituted with an alanine residue (L234A), the leucine residue at position 235 is substituted with an alanine residue (L235A) and the proline residue at position 329 is substituted with a glycine residue (P329G) (numbering according to the EU index according to Kabat).

In one such aspect, the Fc domain is an IgG ₁ Fc domain, in particular a human IgG ₁ Fc domain. The amino acid substitution combination "P329G LALA" substantially completely abolishes Fc receptor (as well as complement) binding of the human IgG ₁ Fc domain, as described in PCT Publication No. WO 2012/130831, which is incorporated herein by reference in its entirety. WO 2012/130831 also describes methods for producing such mutant Fc domains and methods for determining properties thereof, such as Fc receptor binding or effector functions.

IgG ₄ antibodies exhibit reduced binding affinity for Fc receptors and reduced effector functions compared to IgG ₁ antibodies. Therefore, in some aspects, the Fc domain of the (multispecific) antibodies of the invention is the Fc domain of IgG ₄ , in particular the Fc domain of human IgG _4. In one aspect, the Fc domain of IgG ₄ comprises an amino acid substitution at position S228, in particular the amino acid substitution S228P (numbering according to the EU index according to Kabat). To further reduce the binding affinity for the Fc receptor and/or the effector function, in one aspect, the Fc domain of IgG ₄ comprises an amino acid substitution at position L235, in particular the amino acid substitution L235E (numbering according to the EU index according to Kabat). In another aspect, the Fc domain of IgG ₄ comprises an amino acid substitution at position P329, in particular the amino acid substitution P329G (numbering according to the EU index according to Kabat). In a preferred aspect, the Fc domain of IgG ₄ comprises amino acid substitutions at positions S228, L235 and P329, in particular the amino acid substitutions S228P, L235E and P329G (numbering according to the EU index according to Kabat). Such mutant Fc domains of IgG ₄ and their Fcγ receptor binding properties are described in PCT Publication No. WO 2012/130831, which is incorporated herein by reference in its entirety.

In a preferred aspect, the Fc domain exhibiting reduced binding affinity for the Fc receptor and/or reduced effector function compared to the native IgG ₁ Fc domain is the human IgG ₁ Fc domain comprising the amino acid substitutions L234A, L235A and, optionally, P329G, or the human IgG ₄ Fc domain comprising the amino acid substitutions S228P, L235E and, optionally, P329G (numbering according to the EU index according to Kabat).

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

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

Mutant Fc domains can be produced by deleting, substituting, inserting or modifying amino acids using genetic or chemical methods well known in the art. Genetic methods can include site-specific mutagenesis of the coding DNA sequence, PCR, gene synthesis, etc. Correct nucleotide changes can be verified, for example, by sequencing.

Binding to Fc receptors can be readily determined, for example, by ELISA or surface plasmon resonance (SPR), using standard equipment such as the BIAcore instrument (GE Healthcare) and Fc receptors that can be produced by recombinant expression. Alternatively, the binding affinity of Fc domains or (multispecific) Fc domain-containing antibodies for Fc receptors can be assessed using cell lines known to express specific Fc receptors, such as NK cells expressing the FcγIIIa receptor.

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

In some aspects, the binding of the Fc domain to a complement component, in particular C1q, is reduced. Accordingly, in some aspects in which the Fc domain is engineered to have reduced effector function, said reduced effector function comprises reduced CDC. C1q binding assays can be performed to determine whether an Fc domain or a (multispecific) antibody comprising an Fc domain is capable of binding C1q and therefore has CDC activity. See, for example, the C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. Complement activation can be assessed using the CDC assay (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045–1052 (2003); and Cragg and Glennie, Blood 103, 2738–2743 (2004)).

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

B. Polynucleotides

The invention further provides an isolated polynucleotide encoding the antibody of the invention. Said isolated polynucleotide may be a single polynucleotide or a plurality of polynucleotides.

Polynucleotides encoding the (multispecific) antibodies of the invention can be expressed as a single polynucleotide that encodes the entire antibody, or as multiple (e.g., two or more) co-expressed polynucleotides. The polypeptides encoded by

co-expressed polynucleotides may be combined, for example, by disulfide bonds or other means, to form a functional antibody. For example, a portion of the light chain of an antibody may be encoded by a separate polynucleotide from a portion of the antibody comprising the heavy chain of the antibody. When co-expressed, the heavy chain polypeptides will 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 (or portions thereof) may be encoded by a separate polynucleotide from a portion of the antibody comprising the other of two Fc domain subunits and, optionally, a Fab molecule (or portion thereof). When co-expressed, the Fc domain subunits will associate to form the Fc domain.

In some aspects, the isolated polynucleotide encodes the entire antibody molecule of the invention as described herein. In other aspects, the isolated polynucleotide encodes a polypeptide contained in an antibody of the invention as described herein.

In certain aspects, the polynucleotide or nucleic acid is DNA. In other aspects, the polynucleotide of the present invention is RNA, such as in the form of messenger RNA (mRNA). The RNA of the present invention may be single-stranded or double-stranded.

B. Recombinant methods

The antibodies of the invention can be produced, for example, by solid-phase peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding an antibody, for example, as described above, are isolated and inserted into one or more vectors for subsequent cloning and/or expression in a host cell. Such a polynucleotide can be readily isolated and sequenced using conventional procedures. In one aspect, a vector is provided, in particular an expression vector, comprising a polynucleotide (including but not limited to one polynucleotide or a plurality of polynucleotides) of the invention. Methods well known to those skilled in the art can be used to construct expression vectors comprising an antibody coding sequence along with appropriate transcriptional/translational regulatory signals. These methods include in vitro recombinant DNA technologies, synthetic technologies, 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, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y (1989). The expression vector may be part of a plasmid, a virus, or may be a nucleic acid fragment. Expression vectors include an expression cassette into which a polynucleotide encoding an antibody (i.e., the coding region) is cloned in operative linkage to a promoter and/or other transcriptional or translational regulatory elements. As used herein, a "coding region" is the portion of a nucleic acid that consists of codons that are translated into amino acids. Although a "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it may be considered part of the coding region, if present, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc., are not part of the coding region. Two or more coding regions may be present in a single polynucleotide construct, such as a single vector, or in separate polynucleotide constructs, such as separate (different) vectors. Furthermore, any vector may contain a single coding region or may contain two or more coding regions, such as a vector of the present invention may encode one or more polypeptides that are separated after or during translation into final proteins by proteolytic cleavage. In addition, the vector, polynucleotide or nucleic acid of the invention may encode heterologous coding regions, fused or not fused to a polynucleotide encoding an antibody of the invention or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. A functional linkage occurs when a coding region of a gene product, such as a polypeptide, is linked to one or more regulatory sequences such that expression of the gene product is influenced or controlled by these regulatory sequences. Two DNA fragments (such as a polypeptide coding region and an associated promoter) are "operably linked" if induction of the promoter results in transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product or does not interfere with the ability to transcribe a DNA template. Thus, a promoter region is operably linked to a nucleic acid encoding a polypeptide if the promoter is capable of influencing the transcription of the nucleic acid. The promoter may be a cell-specific promoter that causes sufficient transcription of DNA only in certain cells. In addition to the promoter, other transcriptional regulatory elements, such as enhancers, operators, repressors, and transcription termination signals, may be operably linked to the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional regulatory regions are described herein. Various transcriptional regulatory regions are known to those skilled in the art. These include, but are not limited to, transcriptional regulatory regions that are functional in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g., the immediate early promoter in combination with intron-A), simian virus 40 (e.g., the early promoter), and retroviruses (such as, for example, Rous sarcoma virus). Other transcriptional regulatory regions include those derived from vertebrate genes, such as the actin, heat shock protein, bovine growth hormone, and rabbit p-globin genes, as well as other sequences capable of regulating gene expression in eukaryotic cells. Additional suitable transcriptional regulatory regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., tetracycline-inducible promoters). Likewise, various translational regulatory elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly the internal ribosome entry site or IRES, also referred to as the CITE sequence). The expression cassette may also contain other elements such as an origin of replication and/or chromosomal integration elements such as the retroviral long terminal repeats (LTRs) or the inverted terminal repeats (ITRs) of adeno-associated virus (AAV).

The coding regions of the polynucleotide and nucleic acid of the present invention may be linked to additional coding regions that encode secretory or signal peptides that direct the secretion of the polypeptide encoded by the 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 of the invention or a fragment thereof. 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 after initiation of export of the nascent protein chain through the rough endoplasmic reticulum. Those skilled in the art will recognize 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 form the secreted or "mature" form of the polypeptide. In certain aspects, a native signal peptide, such as an immunoglobulin heavy chain or light chain signal peptide, or a functional derivative of such a sequence that retains the ability to direct secretion of a polypeptide that is operably linked to it, is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the wild-type leader sequence can be replaced with a human tissue plasminogen activator (TPA) or mouse β-glucuronidase leader sequence.

DNA encoding a short protein sequence that can be used to facilitate subsequent purification (e.g., a histidine tag) or labeling of the antibody can be included within or at the ends of the antibody-encoding (fragment) polypeptide.

In a further aspect, a host cell is provided comprising a polynucleotide (i.e., one polynucleotide or a plurality of polynucleotides) of the invention. In certain aspects, a host cell is provided comprising a vector of the invention. The polynucleotides and vectors may comprise any of the elements, individually or in combination, described herein in connection with polynucleotides and vectors, respectively. In one such aspect, the host cell comprises (e.g., has been transformed or transfected with) one or more vectors comprising one or more polynucleotides that encode an antibody (or portion thereof) of the invention. As used herein, the term "host cell" refers to any type of cellular system that can be engineered to generate an antibody of the invention or fragments thereof. Host cells suitable for replicating and supporting expression of antibodies are well known in the art. Such cells can be transfected or transduced with a specific expression vector if desired, and large quantities of vector-containing cells can be grown to seed large-scale fermenters to produce sufficient quantities of antibodies for clinical applications. Suitable host cells include prokaryotic microorganisms such as E. coli or various eukaryotic cells such as Chinese hamster ovary (CHO) cells, insect cells, etc. For example, the polypeptides can be produced in bacteria, particularly if glycosylation is not required. After expression, the polypeptide can be isolated from the bacterial cell paste into a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for cloning or expression of polypeptide-encoding vectors, including strains of fungi and yeast whose glycosylation pathways have been "humanized", resulting in a polypeptide with a partially or fully human glycosylation profile. See Gerngross, Nat Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for expression of (glycosylated) polypeptides are also obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfection of Spodonmera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be used. Other examples of useful mammalian host cell lines include the SV40-transformed CV1 monkey kidney cell line (COS-7), the human embryonic kidney cell line (293 or 293T cells, described, for example, in Graham et al., J Gen Virol 36, 59 (1977)), newborn hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, described, for example, 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), brown rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (described, for example, in Mather et al., Annals NY Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr ^- CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp2/0. For a review of some mammalian host cell lines 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 cultured mammalian cells, yeast cells, insect cells, bacterial cells and plant cells, to name a few, but also cells in a transgenic animal, a transgenic plant or in a cultured plant or animal tissue. In one aspect, the host cell is a eukaryotic cell, in particular a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., a Y0, NSO, Sp20 cell). In one aspect, the host cell is not a cell in a human body.

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

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

The components of the (multispecific) antibody of the invention may be genetically fused to each other. The (multispecific) antibody may be designed so that its components are directly fused to each other or indirectly via a linker sequence. The composition and length of the linker may be determined according to methods well known in the art and may be tested for effectiveness. Examples of linker sequences between different components of the (multispecific) antibodies are provided herein. Additional sequences may also be included if necessary to include a cleavage site to separate the individual fusion components, such as an endopeptidase recognition sequence.

Antibodies prepared as described herein can be purified by methods known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. In the case of purification by affinity chromatography, the antibody, ligand, receptor, or antigen to which the antibody binds can be used. For example, a protein A or protein G matrix can be used to affinity chromatograph the antibodies of the invention. The sequential steps of protein A or G affinity chromatography and size exclusion chromatography can be used to isolate the antibody substantially as described in the examples. Antibody purity can be determined by any of a number of well-known analytical methods, including gel electrophoresis, high-pressure liquid chromatography, etc.

G. Analysis

Identification, screening or characterization of the antibodies provided herein with respect to their physical/chemical properties and/or biological activity can be carried out using various assays known in the art.

1. Binding assays

Binding (affinity) to an Fc receptor or target antigen can be determined, for example, by surface plasmon resonance (SPR) using standard equipment such as the BIAcore instrument (GE Healthcare) and receptors or target proteins that can be produced by recombinant expression. Alternatively, binding of antibodies to different receptors or target antigens can be assessed using cell lines expressing a particular receptor or target antigen, for example by flow cytometry (FACS).

A specific illustrative and exemplary aspect for determining CD3 binding activity is described below.

In one aspect, CD3 binding activity is determined using SPR as follows:

SPR was performed on a Biacore T200 instrument (GE Healthcare). The capture anti-Fab antibody (GE Healthcare, #28958325) was immobilized on a CM5 S-series sensor chip (GE Healthcare) using standard amine coupling chemistry at a surface density of 4000-6000 resonance units (RU). HBS-P+ (10 mM HEPES, 150 mM NaCl, pH 7.4, 0.05% P20 surfactant) was used as running and dilution buffer. Anti-CD3 antibodies at a concentration of 2 μg/mL (in 20 mM His, 140 mM NaCl, pH 6.0) were injected over approximately 60 s at a flow rate of 5 μL/min. The CD3 antigen used is a heterodimer of the ectodomains of CD3 delta and CD3 epsilon fused to a human Fc domain with knob-into-hole modifications and a C-terminal Avi tag (see SEQ ID NOs 41 and 42). The CD3 antigen is injected at a concentration of 10 μg/mL for 120 s and dissociation is monitored at a flow rate of 5 μL/min for approximately 120 s. The chip surface is restored by two successive injections of 10 mM glycine, pH 2.1, for approximately 60 s each. Bulk refractive index differences are corrected by subtracting blank injections and by subtracting the response obtained from the flow cell blank control. The binding response is assessed 5 s after the end of the injection. To normalize the binding signal, CD3 binding is divided by the anti-Fab response (the signal (RE) obtained after capture of the anti-CD3 antibody on the immobilized anti-Fab antibody). The binding activity of an antibody to CD3 after a particular treatment compared to the binding activity of an antibody to CD3 after another treatment (also called the relative active concentration (RAC)) is calculated by comparing the binding activity of an antibody sample after a particular treatment to the binding activity of the corresponding antibody sample after another treatment.

2. Activity analysis

The biological activity of the (multispecific) antibodies of the invention can be measured using various assays, as described in the examples. The biological activity can, for example, include induction of T cell proliferation, induction of signaling in T cells, induction of expression of activation markers in T cells, induction of cytokine secretion by T cells, induction of lysis of target cells such as B cells, and induction of tumor regression and/or improved survival. D. Compositions, Formulations and Routes of Administration

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

Additionally, a method is provided for producing an antibody of the invention in a form suitable for in vivo administration, comprising (a) producing an antibody in accordance with the invention and (b) formulating the antibody with at least one pharmaceutically acceptable carrier, wherein the antibody preparation is formulated for in vivo administration.

The pharmaceutical compositions of the present invention comprise an effective amount of an antibody dissolved or dispersed in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" refers to molecular entities and compositions that are generally nontoxic to recipients at the dosages and concentrations employed, i.e., do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as, for example, a human, as the case may be. The preparation of a pharmaceutical composition that contains an antibody and, optionally, a further active ingredient, will be apparent to one skilled in the art in light of the present invention and as illustrated in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Furthermore, when administered to an animal (e.g., a human), it will be understood that the preparations must meet the standards of sterility, pyrogen-freeness, general safety, and purity required by the FDA's Office of Biological Standards or the appropriate authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, a "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavors, colors, the like materials, and combinations thereof, as would be known to one 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 where any conventional carrier is incompatible with the active ingredient, its use in pharmaceutical compositions is envisaged.

The antibody of the invention (and any additional therapeutic agent) can be administered by any suitable route, including parenteral, intrapulmonary and intranasal, and, if necessary for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Dosing can be by any convenient route, for example by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is short-term or chronic.

Parenteral compositions include those designed for administration by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. For injection, the antibodies of the invention can be formulated in aqueous solutions, in particular physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer. The solution may contain auxiliary agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the antibodies can be in powder form for constitution with a suitable vehicle, for example, sterile, pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the antibodies of the invention in the required amount into a suitable solvent with various other ingredients listed below, as needed. Sterility can be readily ensured, for example, by filtration through sterile filter membranes. In general, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred methods of preparation are vacuum drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional required ingredient from a previously sterile-filtered liquid medium. If necessary, the liquid medium should be suitably buffered and the liquid diluent first rendered isotonic with a sufficient amount of saline or glucose before injection. The composition should be stable under the conditions of manufacture and storage and protected against the contaminating action of microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to the minimum safe level, for example, 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 (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than 10 residues) polypeptides; 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal-containing complexes (e.g. Zn-protein complexes) and/or nonionic surfactants such as polyethyleneglycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension such as sodium carboxymethylcellulose, sorbitol, dextran and the like. Optionally, the suspension may also contain suitable stabilizers or substances which increase the solubility of the compounds to enable the preparation of highly concentrated solutions. In addition, suspensions of the active compounds can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleates or triglycerides, or liposomes.

The active ingredients can also be incorporated into microcapsules prepared, for example, by coacervation processes or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described 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, the matrices being in the form of shaped articles, for example films or microcapsules. In certain aspects, prolonged absorption of the injectable composition can be achieved by using absorption delaying agents in the compositions, such as, for example, aluminum monostearate, gelatin, or combinations thereof.

In addition to the compositions described above, the antibodies can also be formulated as depot preparations. Such prolonged-release formulations can be administered by implantation (e.g., subcutaneous or intramuscular) 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 poorly soluble derivatives, e.g., as a poorly soluble salt.

Pharmaceutical compositions containing the antibodies of the invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, embedding or lyophilizing processes. The pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients that facilitate processing of proteins into preparations that can be used for pharmaceutical purposes. The appropriate formulation depends on the chosen route of administration.

Antibodies can be formulated as a composition in free acid or base form, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acids or bases. These include acid addition salts, for example, formed with free amino groups of the protein composition or formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with free carboxyl groups can also be prepared from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts are generally more soluble in aqueous and other protic solvents than the corresponding free base forms.

E. 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 types or autoimmune diseases.

For use in therapeutic methods, the antibodies of the invention should be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the location of delivery of the agent, the route of administration, the schedule of administration, and other factors known to practitioners.

In one aspect, antibodies of the invention are provided for use as a medicine. In further aspects, antibodies of the invention are provided for use in treating a disease. In certain aspects, antibodies of the invention are provided for use in a method of treatment. In one aspect, the invention provides an antibody of the invention for use in treating a disease in an individual in need thereof. In certain aspects, the 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 an antibody. In certain aspects, the disease is a proliferative disorder. In specific aspects, the disease is a cancer, in particular a CD19-expressing cancer. In a specific aspect, the disease is a B-cell cancer. The B-cell cancer in one aspect 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 other aspects, the disease is an autoimmune disease. In a specific aspect, the disease is lupus, in particular systemic lupus erythematosus (SLE) or lupus nephritis (LN).

In certain aspects, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as an anti-cancer agent if the disease to be treated is cancer, or an immunosuppressive agent if the disease to be treated is an autoimmune disease. In further aspects, the invention provides an antibody of the invention for use in inducing lysis of a target cell, in particular a B cell. In certain aspects, the invention provides an antibody of the invention for use in a method for inducing lysis of a target cell, in particular a B cell, in an individual, comprising administering to the individual an effective amount of the antibody to induce lysis of the target cell. The "individual" according to any of the above aspects is a mammal, preferably a human.

In a further aspect, the invention provides the use of an antibody of the invention in the manufacture or preparation of a medicament. In one aspect, the medicament is for 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 to an individual having the disease an effective amount of the medicament. In certain aspects, the disease is a proliferative disorder. In specific aspects, the disease is a cancer, in particular a CD19-expressing cancer. In a specific aspect, the disease is a B-cell cancer. The B-cell cancer in one aspect 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 other aspects, the disease is an autoimmune disease. In a specific aspect, the disease is lupus, in particular 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, such as an anti-cancer agent if the disease to be treated is cancer, or an immunosuppressant if the disease to be treated is an autoimmune disease. In a further aspect, the medicament is for inducing lysis of a target cell, in particular a B cell. In a further aspect, the medicament is for use in a method for inducing lysis of a target cell, in particular a B cell, in an individual, comprising administering to the individual an effective amount of a medicament for inducing lysis of the target cell. The "individual" according to any of the above aspects may be a mammal, preferably a human.

In a further aspect, the invention provides a method of treating a disease. In one aspect, the method comprises administering to an individual having such a disease an effective amount of an antibody of the invention. In one aspect, said individual is administered a composition comprising an antibody of the invention in a pharmaceutically acceptable form. In certain aspects, the disease is a proliferative disorder. In specific aspects, the disease is a cancer, in particular a CD19-expressing cancer. In a specific aspect, the disease is a B-cell cancer. The B-cell cancer in one aspect 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 other aspects, the disease is an autoimmune disease. In a specific aspect, the disease is lupus, in particular systemic lupus erythematosus (SLE) or lupus nephritis (LN). In certain aspects, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as an anticancer agent if the disease to be treated is cancer, or an immunosuppressive agent if the disease to be treated is an autoimmune disease. The "individual" according to any of the above aspects may be a mammal, preferably a human.

In a further aspect, the present invention provides a method for inducing lysis of a target cell, in particular a CD19-expressing cell, such as a B cell. In one aspect, the method comprises contacting the target cell with an antibody of the invention in the presence of a T cell, in particular a cytotoxic T cell. In a further aspect, there is provided a method for inducing lysis of a target cell, in particular a CD19-expressing cell, such as a B cell, 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 cell. In one aspect, the "individual" is a human.

It is understood by a person skilled in the art that in many cases an antibody may not provide a cure, but may only provide a partial beneficial effect. In some aspects, a physiological change that has some beneficial effect is also considered therapeutically beneficial. Thus, in some aspects, an amount of an antibody that provides a physiological change is considered an "effective amount". The subject, patient, individual in need of treatment is typically a mammal, in particular a human.

In some aspects, an effective amount of an antibody of the invention is administered to an individual to treat a disease.

For the prevention or treatment of a disease, the appropriate dose of an antibody of the invention (used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the route of administration, the body weight of the patient, the type of antibody, the severity and course of the disease, whether the antibody is administered prophylactically or therapeutically, previous or concurrent therapeutic interventions, the patient's medical history and response to the antibody, and the judgment of the attending physician. The practitioner responsible for administration will in any event determine the concentration of the active ingredient(s) in the composition and the appropriate dose(s) for each individual subject. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations at different times, bolus administration, and pulse infusion.

The antibody is preferably administered to a patient at one time or over a series of treatment courses. Depending on the type and severity of the disease, an initial candidate dosage for administration to a patient may be from about 1 mcg/kg to 15 mg/kg (e.g., 0.1 mg/kg 10 mg/kg) of the antibody, such as in one or more separate administrations or as a continuous infusion. One typical daily dose may range from about 1 mcg/kg to 100 mg/kg or more, depending on the factors mentioned above. In the case of repeated administrations over several days or more, depending on the condition, the treatment is generally carried out until the desired degree of suppression of the symptoms of the disease is achieved. One typical dosage of the antibody is in the range of from about 0.005 mg/kg to about 10 mg/kg. In other non-limiting examples, the dose can also be from about 1 microgram/kg body weight, about 5 micrograms/kg body weight, about 10 micrograms/kg body weight, about 50 micrograms/kg body weight, about 100 micrograms/kg body weight, about 200 micrograms/kg body weight, about 350 micrograms/kg body weight, about 500 micrograms/kg body weight, about 1 milligram/kg body weight, about 5 milligrams/kg body weight, about 10 milligrams/kg body weight, about 50 milligrams/kg body weight, about 100 milligrams/kg body weight, about 200 milligrams/kg body weight, about 350 milligrams/kg body weight, about 500 milligrams/kg body weight to about 1000 mg/kg body weight or more per administration, and any range resulting therefrom. In non-limiting examples of the range obtained from the values listed herein, a dose in the range of about 5 mg/kg body weight to about 100 mg/kg body weight, about 5 micrograms/kg body weight to about 500 milligrams/kg body weight, etc. can be administered, taking into account the above numerical values. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg, or 10 mg/kg (or any combination thereof) can be administered to a patient. Such doses can be administered at intervals, such as once a week or once every three weeks (e.g., so that the patient receives from about two to about twenty, or, for example, about six doses of the antibody). An initial higher loading dose can be administered, followed by one or more smaller doses. Other dosing schedules can be used. The effectiveness of such therapy can be easily monitored using conventional techniques and assays.

The antibodies of the invention are generally used in an amount effective to achieve the intended purpose. When used to treat or prevent a disease state, the antibodies of the invention or pharmaceutical compositions thereof are administered or used in an effective amount.

For systemic administration, the effective dose can first be estimated from an in vitro assay, such as a cell culture assay. The dose for use in animal models can then be formulated to achieve a circulating concentration range that includes the IC ₅₀ determined in cell culture. Such information can be used to more accurately determine doses useful in humans.

Initial dosages can also be estimated from in vivo data, such as animal models, using techniques that are well known in the art.

The amount and interval of dosage can be individually adjusted to provide plasma levels of antibodies sufficient to maintain a therapeutic effect. Usual dosages for patients when administered by injection are in the range of about 0.1 to 50 mg/kg/day, typically about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels can be achieved by administering multiple doses every day. Plasma levels can be measured, for example, by HPLC.

An effective dose of the antibodies of the invention will generally provide a therapeutic benefit without significant toxicity. The toxicity and therapeutic efficacy of an antibody can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD ₅₀ /ED ₅₀ . Antibodies that exhibit high therapeutic indices are preferred. In one aspect, an antibody of the invention exhibits a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a dosage range suitable for human use. The dosage is preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending on a number of factors, such as the dosage form employed, the route of administration employed, the condition of the subject, etc. The exact composition, route of administration, and dosage may be selected by the individual physician taking into account the patient's condition (see, e.g., Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).

The treating physician of patients treated with antibodies of the invention should know how and when to stop, interrupt or adjust the administration due to toxicity, organ dysfunction, etc. Conversely, the treating physician should also know how to adjust the treatment to higher levels if the clinical response has been insufficient (avoiding toxicity). The amount of the administered dose in the treatment 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 can, for example, be assessed in part by standard assessment methods. In addition, the dose and possibly the frequency of dosing will also vary according to the age, body weight and response of each individual patient.

Antibodies of the invention can be administered in combination with one or more other agents in therapy. For example, an antibody of the invention can be administered together with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent administered to treat a symptom of a disease in an individual in need of such treatment. Such an additional therapeutic agent can include any active ingredients suitable for the particular disease being treated, preferably those that have complementary activity and do not negatively affect each other. In certain aspects, the additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptosis inducers. In certain aspects, the additional therapeutic agent is an anticancer agent, such as a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, a tumor cell apoptosis activator, or an antiangiogenic agent. In other aspects, the additional therapeutic agent is an immunosuppressant. In a specific example, 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.

Such other agents are suitably present in the combination in amounts that are effective for the intended purpose. The effective amount of such other agents depends on the amount of antibody used, the type of disorder or treatment, and other factors discussed above. Antibodies are generally used in the same dosages and by the routes described herein, or in dosages that are from about 1% to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

Such combination therapies noted above include combined administration (when two or more therapeutic agents are included in one or separate compositions) as well as separate administration, in which case the administration of the antibody of the invention may occur before, simultaneously and/or after the administration of an additional therapeutic agent and/or adjuvant. The antibodies of the invention may also be used in combination with radiation therapy.

F. Finished products

In another aspect of the invention, there is provided a finished article containing materials used in the treatment, prevention and/or diagnosis of the above-described disorders. The finished article comprises a container and a label or package insert located thereon or associated therewith. Suitable containers include, for example, bottles, vials, syringes, bags for intravenous solution, etc. The containers can be made of a number of materials, such as glass or plastic. The container contains a composition that is effective alone or in combination with another composition for the treatment, prevention and/or diagnosis of a pathological condition, and can have a sterile entry port (for example, the container can be a bag for intravenous solution or a vial with a stopper pierced by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat the selected pathological condition. In addition, the finished article may comprise (a) a first container containing a composition, wherein said composition comprises an antibody of the invention; and (b) a second container containing a composition, wherein said composition comprises an additional cytotoxic or other therapeutic agent. The finished article in this aspect of the present invention may further comprise a package insert indicating that the compositions can be used to treat a particular pathological condition. Alternatively or additionally, the finished article 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 also comprise other materials required from a commercial and user point of view, including other buffers, diluents, filters, needles and syringes.

I. Methods and compositions for diagnostics and detection

In certain aspects, any of the antibodies provided herein are useful for detecting the presence of their target (e.g., CD3 or CD19) in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection. In certain aspects, the biological sample includes a cell or tissue, such as prostate tissue.

In one embodiment, an antibody of the invention is provided for use in a diagnostic or detection method. In a further aspect, a method is provided for detecting the presence of CD3 or CD19 in a biological sample. In certain aspects, the method comprises contacting the biological sample with an antibody of the present invention under conditions that allow the antibody to bind 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, an antibody of the invention is used to select subjects for therapy with an antibody that binds CD3 and/or CD19, for example, if CD3 and/or CD19 is a biomarker for patient selection.

Typical disorders that can be diagnosed using the antibody of the invention include cancer, in particular B-cell cancer.

In certain aspects, an antibody according to the present invention is provided, wherein the antibody is labeled. Labels include, but are not limited to, labels or moieties that are directly detectable (e.g., fluorescent, chromophore, electron-dense, chemiluminescent, and radioactive labels), as well as moieties such as enzymes or ligands that are indirectly detectable, such as by an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, radioactive isotopes ^32P , ^14C , ^125I , ^3H , and ^131I , fluorophores such as rare earth metal chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase coupled to an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, etc.

IV. EXAMPLES

Examples of methods and compositions according to the invention are given below. It should be understood that a number of other aspects can be realized in practice taking into account the general description given above.

Example 1 - Creation of an optimized CD3 binding fragment

Starting from a previously described (see, e.g., WO 2014/131712, incorporated herein by reference) CD3 binding fragment, herein referred to as “CD3 _orig ” and comprising the VH and VL sequences of SEQ ID NOs 6 and 11, respectively, we aimed to optimize the properties of this binding fragment by removing two asparagine deamidation sequence motifs at positions 97 and 100 of the Kabat CDR3 of the heavy chain.

To this end, we generated a phage display-ready heavy chain antibody library with both asparagines at Kabat positions 97 and 100 deleted and additionally randomized H1, H2, and H3 CDRs to compensate for the loss of affinity caused by the substitution of Asn97 and Asn100 through an affinity maturation process.

This library was applied to filamentous phage via fusion with the minimal coat protein p3 (Marks et al. (1991) J Mol Biol 222, 581-597) and selected for binding to recombinant CD3ε.

During the initial screening, 10 candidate clones were identified that demonstrated acceptable binding to the recombinant antigen as measured by SPR, in the form of Fab fragments (produced by E. coli).

However, only one of these clones demonstrated acceptable binding activity to CD3 expressing cells as measured by flow cytometry after conversion to IgG format.

The selected clone, herein referred to as “CD3 _opt ” and containing the VH and VL sequences of SEQ ID NO 7 and 11, respectively, was further evaluated and converted to a bispecific format as described below.

Example 2 - Binding of an optimized CD3 binding fragment to CD3

Binding to recombinant CD3

Binding to recombinant CD3 was determined by surface plasmon resonance (SPR) for the optimized CD3 binding fragment “CD3 _opt ” and the original CD3 binding fragment “CD3 _orig ”, both in the human _IgG1 format with the P329G L234A L235A mutations (“PGLALA”, EU numbering) in the Fc region (SEQ ID NOs 12 and 14 (CD3 _orig ) and SEQ ID NOs 13 and 14 (CD3 _opt )).

To evaluate the effect of deamidation site removal and its impact on antibody stability, binding of the original and optimized CD3 binding fragment to recombinant CD3 was studied after temperature stress for 14 days at 37°C or 40°C. Samples stored at -80°C were used as a reference. Reference and 40°C-stressed samples were in 20 mM His, 140 mM NaCl, pH 6.0, and 37°C-stressed samples were in PBS, pH 7.4, all at a concentration of 1.2-1.3 mg/mL. After the stress period (14 days), samples were back-dialyzed into PBS in 20 mM His, 140 mM NaCl, pH 6.0 for additional analysis.

The relative active concentration (RAC) of the samples was determined by the SPR method as follows.

SPR was performed on a Biacore T200 instrument (GE Healthcare). The capture anti-Fab antibody (GE Healthcare, #28958325) was immobilized on a CM5 S-series sensor chip (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% P20 surfactant) was used as running and dilution buffer. Anti-CD3 antibody at a concentration of 2 μg/mL was injected for 60 s at a flow rate of 5 μL/min. CD3 antigen (see below) was injected at a concentration of 10 μg/mL for 120 s and dissociation was monitored at a flow rate of 5 μL/min. for 120 s. The chip surface was restored by two successive injections of 10 mM glycine, pH 2.1, for 60 s each. The volumetric refractive index difference was corrected by subtracting the control injections and by subtracting the response obtained from the control flow cell. For the binding response assessment, the binding response was determined 5 s after the end of the injection. To normalize the binding signal, CD3 binding was divided by the anti-Fab response (the signal (PE) obtained after capture of the anti-CD3 antibody on the immobilized anti-Fab antibody). The relative concentration of the active substance was calculated by referencing each temperature-treated sample to the corresponding untreated sample.

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

The results of this experiment are illustrated in Fig. 2. As can be seen, the optimized CD3 binding fragment CD3 _opt exhibited markedly improved binding to CD3 after temperature stress (2 weeks at 37°C, pH 7.4) compared to the original CD3 binding fragment CD3 _orig. This result demonstrates that the removal of the deamidation site was successful and resulted in an antibody with superior stability properties related to its in vivo half-life as well as the antibody composition at neutral pH.

CD3 binding on Jurkat cells

CD3 binding on the human Jurkat NFAT reporter T cell line was determined by FACS for the optimized CD3 binding fragment "CD3 _opt " and the original CD3 binding fragment "CD3 _orig ", both in the human _IgG1 format with P329G L234A L235A mutations ("PGLALA", EU numbering) in the Fc region (SEQ ID NOs 12 and 14 (CD3 _orig ) and SEQ ID NOs 13 and 14 (CD3 _opt )).

Jurkat-NFAT reporter cells (GloResponse Jurkat NFAT-RE-luc2P; Promega #CS176501) are a human acute lymphocytic leukemia reporter cell line with the NFAT promoter expressing human CD3. Cells were cultured in RPMI1640, 2 g/L glucose, 2 g/L _NaHCO3 , 10% FBS, 25 mM HEPES, 2 mM L-glutamine, 1 x NEAA, 1 x sodium pyruvate at 0.1-0.5 million cells/ml. A final concentration of 200 μg/ml hygromycin B was added each time the cells were passaged.

For binding analysis, Jurkat NFAT cells were harvested, washed in PBS and resuspended in FACS buffer. Antibody staining was performed in a 96-well round-bottom plate. Therefore, 100,000 to 200,000 cells were seeded per well. The plates were centrifuged for 4 min at 400 × g and the supernatant was removed. The antibodies to be studied were diluted in FACS buffer and 20 μl of the antibody solution were added to the wells for 30 min at 4°C. To remove unbound antibody, cells were washed twice with FACS buffer before addition of diluted secondary antibody (PE-conjugated AffiniPure P(ab')2 fragment goat anti-human IgG, Fcg fragment-specific; Jackson ImmunoResearch #109-116-170). After 30 min of incubation at 4°C, unbound secondary antibody was washed away. Before measurement, cells were resuspended in 200 μl FACS buffer and analyzed by flow cytometry using a BD Canto II.

As shown in Fig. 3, the optimized CD3 binding fragment “CD3 _opt ” and the original CD3 binding fragment “CD3 _orig ” bound comparably well to CD3 on Jurkat cells.

Example 3 - Functional activity of the optimized CD3 binding fragment

The functional activity of the optimized CD3 binding fragment "CD3_wholesale" were examined in a Jurkat cell reporter assay and compared with the activity of the original CD3 binding fragment "CD3_orig" To investigate the functional activity of IgG, anti-PGLALA expressing CHO cells were co-incubated with Jurkat NFAT reporter cells in the presence of increasing concentrations of IgG₁Human PGLALA to CD3_wholesaleor IgG₁Human PGLALA to CD3_orig. Activation of CD3 on Jurkat NFAT reporter cells following T cell cross-linking induces luciferase production, and luminescence can be measured as a marker of activation. IgG₁human to CD3_origdt was included as a negative control that cannot bind to anti-PGLALA expressing CHO cells and therefore cannot cross-link to Jurkat NFAT cells. A schematic illustration of this assay is shown in Fig. 4.

Anti-PGLALA expressing CHO cells are CHO-K1 cells engineered to express on their surface an antibody that specifically binds to human IgG ₁ Fc(PGLALA) (see WO 2017/072210, incorporated herein by reference). These cells were cultured in DMEM/F12 containing 5% FBS+1% GluMax. Jurkat NFAT reporter cells are as described in Example 2.

Upon simultaneous binding of CD3 huIgG ₁ PGLALA to anti-PGLALA antibody expressed on CHO and CD3 expressed on Jurkat-NFAT reporter cells, the NFAT promoter is activated and results in expression of active firefly luciferase. The intensity of the luminescence signal (obtained after addition of luciferase substrate) is proportional to the intensity of CD3 activation and signaling. Jurkat-NFAT reporter cells were grown in suspension and cultured in RPMI1640, 2 g/L glucose, 2 g/L NaHCO3, 10% FBS, 25 mM HEPES, 2 mM L-glutamine, 1 x NEAA, 1 x sodium pyruvate at 0.1-0.5 million cells/ml, 200 μg/ml hygromycin. For the assay, CHO cells were harvested and viability was determined using ViCell. 30,000 target cells/well were seeded in a flat-bottomed 96-well white-walled plate (Greiner bio-one #655098) in 100 μl of medium and 50 μl/well of diluted antibodies or medium (for controls) were added to the CHO cells. Jurkat-NFAT reporter cells were then harvested and viability was determined using ViCell. Cells were resuspended at 1.2 million cells/ml in cell culture medium without hygromycin B and added to CHO cells at 60,000 cells/well (50 μl/well) to give a final effector to target (E:T) ratio of 2:1 and a final volume of 200 μl per well. Then 4 μl of GloSensor (Promega #E1291) (2% of the final volume) were added to each well. The cells were incubated for 24 h at 37°C in a humidified incubator. At the end of the incubation time, luminescence was recorded using a TECAN Spark 10M.

As shown in Fig. 5, the optimized CD3 binding fragment CD3 _opt had activity on Jurkat NFAT cells after cross-linking similar to CD3 _orig .

Example 4 - Generation of a T-cell bispecific antibody containing an optimized CD3 binding fragment

The optimized CD3 binding fragment identified in Example 1 (“CD3 _opt ”, SEQ ID NOs: 7 (VH) and 11 (VL)) was used to generate T cell bispecific antibodies (TCBs) targeting CD3 and CD19 (“CD19-TCB”) using the anti-CD19 antibodies 2B11 or 018 as the CD19 binding fragment (SEQ ID NOs: 15-22 or 28-35, respectively).

A schematic illustration of the TCB molecules is shown in Fig. 6A, and their complete sequences are given in SEQ ID NOs:39, 24, 25, and 27 (2B11) and SEQ ID NOs:36, 37, 38, and 27 (O18).

Corresponding molecules containing either the above-mentioned antibodies to CD19 as the target cell antigen-binding fragment and CD3 _orig as the CD3-binding fragment were also obtained (SEQ ID NOs:39, 24, 25, and 26 (2B11) and SEQ ID NOs:40, 37, 38, and 26 (O18)).

The heavy and light chain variable region DNA sequences were subcloned in frame with the heavy chain constant region or the light chain constant region previously inserted into the corresponding mammalian recipient expression vectors, as shown in Fig. 6B-D.

To improve the correct pairing of light chains with their corresponding heavy chains, mutations were introduced into human CL (E123R, Q124K) and human CH1 (K147E, K213E) of the CD19-binding molecule Fab.

To ensure correct pairing of heavy chains (formation of a heterodimeric molecule), “knob-into-hole” mutations were introduced into the constant region of the antibody heavy chains (T366W/S354C and T366S/L368A/Y407V/Y349C, respectively).

In addition, mutations P329G, L234A and L235A were introduced into the constant region of the heavy chains of the antibody to abolish binding to Fcγ receptors. The TCBs were produced by Evitria (Switzerland) using their own vector system by standard (non-PCR-based) cloning techniques and using suspension-adapted CHO K1 cells (originally obtained from ATCC and adapted for serum-free growth in suspension culture by Evitria). Evitria produced them using their own animal component-free and serum-free media (eviGrow and eviMake2) and their own transfection reagent (eviFect). Cells were transfected with the appropriate expression vectors at a ratio of 1:1:2:1 (knob heavy chain vector:hole heavy chain vector:CD3 light chain vector:CD19 light chain vector). The supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter).

As an alternative to production at Evitria, TSV molecules were produced in-house by transient transfection of HEK293 cells with EBNA. Cells were centrifuged and the medium was replaced with pre-warmed CD CHO medium (Thermo Fisher, #10743029). Expression vectors were mixed in CD CHO medium, polyethyleneimine (PEI, Polysciences Inc, #23966-1) was added, the solution was vortexed and incubated for 10 min at room temperature. Cells (2 million/ml) were then mixed with the vector/PEI solution, transferred to a flask and incubated for 3 h at 37°C in a shaking incubator with 5% CO ₂ . After incubation, Excell medium with supplements (80% of the total volume) was added (W. Zhou and A. Kantardjieff, Mammalian Cell Cultures for Biologies Manufacturing, DOI: 10.1007/978-3-642-54050-9; 2014). One day after transfection, supplements (Feed, 12% of the total volume) were added. Cell supernatants were collected after 7 days by centrifugation and subsequent filtration (0.2 μm filter).

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

The concentrations of purified proteins were determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated from the amino acid sequence according to Pace et al., Protein Science, 1995, 4, 2411-1423. The purity and molecular weight of proteins were analyzed by CE-SDS in the presence or absence of reducing agent using a LabChipGXII (Perkin Elmer). Determination of the aggregate content was performed by HPLC chromatography at 25°C using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated in running buffer (25 mM K ₂ HPO ₄ , 125 mM NaCl, 200 mM L-arginine monohydrochloride, pH 6.7, or 200 mM KH ₂ PO ₄ , 250 mM KCl, pH 6.2, respectively).

The results of biochemical and biophysical analysis of the obtained TSV molecules are presented in Table 1.

All four TSV molecules could be obtained in good quality.

Example 5 - Functional characteristics of optimized CD3 antibodies (multispecific) based on surface plasmon resonance (SPR) data

SPR experiments were performed using the CD19-TCB molecules prepared in Example 4 on a Biacore T200 instrument at 25°C with HBS-EP+ as a running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20 (GE Healthcare, #BR-1006-69)). Anti-Fc(P329G) IgG (an antibody that specifically binds to human IgG ₁ Fc(P329G); “anti-PG antibody” - see WO 2017/072210, incorporated herein by reference) was directly immobilized by amine coupling onto a C1 chip (GE Healthcare). The different TCB molecules were captured at 25 nM. Three-fold dilution series (0.14 to 100 nM in HBS-EP) of CD19 antigen (human CD19 extracellular domain (ECD) - Fc fusion; see SEQ ID NOS:43 and 44) or CD3 antigen (CD3ε/δ - Fc fusion; see Example 2, SEQ ID NOS:41 and 42) were passed through the ligand at 30 fpm for 240 s to record the association phase. The dissociation phase was monitored for 1500 s (CD19 antigen) or 800 s (CD3 antigen) and initiated by switching from sample solution to HBS-EP+. The chip surface was recovered after each cycle using two injections of 10 mM glycine, pH 2.1, for 60 s. Bulk refractive index differences were corrected by subtracting the response obtained from the reference flow cell (without captured TCB). Affinity constants were obtained from kinetic rate constants by fitting a 1:1 Langmuir binding model using Biaeval software (GE Healthcare). Measurements were performed in triplicate with independent serial dilutions.

Rate constants for 1:1 Langmuir binding were determined for the four tested TCBs to recombinant human CD19 (Table 2) and to recombinant human CD3 (Table 3).

The different binding moieties have similar affinities at different TCBs. The CD19 binding moiety 2B11 has a K _D of approximately 0.4 nM at the respective TCBs. The CD19 binding moiety 018 has a slightly lower affinity with a K _D of approximately 1.1 nM at the respective TCBs. TCBs with the CD3 binding moiety CD3 _orig or CD3 _opt have comparable affinities to CD3, with a K D of approximately 3.4 nM. While the K _D of interaction with CD19 and CD3 antigens are similar, the kinetics differ. CD19 dissociates more slowly than CD3, but CD3 associates more rapidly than CD19, resulting in similar K _D values.

Example 6 - Binding of CD19-TCB molecules with an optimized anti-CD3 antibody to human CD19 and human CD3 expressing cells

The binding of the CD19-TCB molecules prepared in Example 4 to human CD19 and human CD3 expressing target cells was tested. Two CD19 expressing cell lines with different CD19 expression levels were used. Nalm-6, an acute lymphoblastic leukemia (ALL) cell line with high CD19 expression, and Z-138 (mantle cell lymphoma) with intermediate expression levels. CD3 binding was assessed using an immortalized T lymphocyte line (Jurkat cell line). Briefly, cells were harvested, counted, tested for viability, and resuspended at 1× ¹⁰⁶ cells/mL in FACS buffer (PBS+2% FCS+5 mM EDTA+0.25% sodium azide). 100 μl of cell suspension (containing 0.1× ¹⁰⁶ cells) were incubated in a round-bottomed 96-well plate for 30 min at 4°C with increasing concentrations of CD19-TCB molecules (200 nM - 0.05 nM on Jurkat cells; 200 nM - 0.0002 nM on Z-128 and Nalm-6 cells), washed twice with cold FACS buffer, and reincubated for another 30 min. at 4°C with a secondary antibody specific for AffiniPure PE-conjugated goat anti-human IgG F(ab')2 Fcγ fragment (Jackson Immuno Research Lab PE #109-116-170), washed twice with cold FACS buffer and immediately analyzed by FACS using FACS CantoII (FlowJo 10.5.3 software). Binding curves and EC50 values for binding were calculated using GraphPad Prism 7.

The results are shown in Fig. 7 and Tables 4 and 5. CD19-TCB molecules containing CD3 _opt as the CD3 binding fragment show comparable (or slightly better) CD3 binding relative to molecules containing the CD3 binding fragment CD3 _orig (Fig. 7A and Table 4).

CD19-TCB molecules (containing either 2B11 or 018 as CD19 binding moieties) show comparable binding to CD19-expressing cells (Fig. 7B, C and Table 5). For Z-138 cells, EC50 values could not be calculated because the binding curve did not reach saturation.

Example 7 - Tumor cell lysis and T cell activation induced by CD19-TCB molecules with optimized anti-CD3 antibody

Lysis of CD19-expressing tumor cells and subsequent T cell activation mediated by the CD19-TCB molecules obtained in Example 4 were assessed in Nalm-6 (ALL) cells and Z-138 (mantle cell lymphoma) cells. Human PBMCs were used as effectors and tumor lysis was detected after 20 h of incubation with the different TCB molecules.

Peripheral blood mononuclear cells (PBMCs) were prepared by density gradient centrifugation of enriched lymphocyte preparations (buffy coats) obtained from a healthy human donor. Fresh blood was diluted in sterile PBS and stratified in a Histopaque gradient (Sigma, #H8889). After centrifugation (450 × g, 30 min, no braking, room temperature), the blood plasma above the PBMC-containing interphase was decanted and PBMCs were transferred to a new Falcon tube, which was then filled with 50 ml PBS. The mixture was centrifuged (350 × g, 10 min, room temperature), the supernatant was removed, and the PBMC pellet was incubated in red blood cell lysis solution for 5 min at 37°C before washing with sterile PBS (centrifugation 300 × g, 10 min). The resulting PBMC population was resuspended in PBS and counted automatically (ViCell). 50 million PBMC per cryovial were frozen in RPMI1640 medium (Gibco, #21870076) containing 10% FCS and 1% GlutaMAX (Gibco) containing 10% DMSO (Sigma, #D2650). PBMC were thawed on the day of analysis and counted again automatically (ViCell). The required number was washed once with sterile PBS. B cell depletion was performed using CD20 microbeads (Miltenyi, #130-091-104) according to the manufacturer's instructions. B cell-depleted PBMCs were counted (ViCell) and resuspended at 5 x ¹⁰⁶ cells/ml in RPMI1640 medium containing 10% FCS and 1% GlutaMAX.

For the killing assay, 0.25 million B-cell-depleted PBMCs were added to 96-well U-bottom plates. Briefly, target cells were collected, washed, and plated at a density of 50,000 cells/well, resulting in a final effector-to-target (E:T) ratio of 5:1. TCB molecules were added at the indicated concentrations (range 0.02 pM–1000 pM, in triplicate). CD107a (LAMP-1) was directly stained in the assay (anti-human CD107a PE antibody; Biolegend, #328608).

Tumor cell lysis was assessed after 20 h of incubation at 37°C, 5% _CO2 by quantifying LDH released into cell supernatants by apoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11644793001). Maximum target cell lysis (=100%) was achieved by incubating target cells with 1% Triton X-100. Minimum lysis (=0%) refers to target cells that were co-incubated with effector cells without the bispecific construct.

To assess T cell activation following tumor cell lysis, PBMCs were centrifuged at 400 × g for 4 min and washed twice with FACS buffer. Briefly, cells were washed twice with PBS followed by live/dead staining (Zombie Aqua Fixed Viability Kit; Biolegend, #423102, 20 min at RT). After repeated washing first with PBS followed by FACS buffer, surface staining for CD3 (PE-Cy5 anti-human CD3 antibody; BD Pharmigen, #555341), CD4 (BV605 anti-human CD4 antibody; Biolegend, #317438), CD8 (BV711 anti-human CD8 antibody; Biolegend, #301044), CD25 (PE-Cy7 anti-human CD25 antibody; Biolegend, #302612), and CD69 (BV421 anti-human CD69 antibody; Biolegend, #310930) was performed according to the supplier's directions. Cells were washed twice with 150 µl/well FACS buffer and fixed with 120 µl/well 1x lysis solution (BD Biosciences, #349202). Samples were analyzed on a BD FACS Fortessa (FlowJo 10.5.3 software).

Fig. 8 shows that CD19-TCB molecules induced on-target killing of CD19+ target cells. The four different CD19-TCB molecules were generally comparable in inducing lysis of CD19-expressing tumor cells. Figs. 9 and 10 show that CD19-TCBs containing the CD3 _opt binding fragment exhibited slightly better induction of T cell activation after tumor killing (CD25, CD69, and CD107 expression on CD8 and CD4 T cells upon killing of Nalm-6 or Z-138 target cells) compared to molecules containing the CD3 _orig binding fragment. No effect on T cell activation was observed with the CD19 binding fragments 2B11 and 018.

Example 8 - Determination of the thermal stability of the CD19-TCB molecule of the optimized anti-CD3 antibody

The thermal stability of the CD19-TCB molecule containing the optimized anti-CD3 antibody CD3 _onT (and the CD19 binding fragment 2B11, see Example 4) was monitored by static light scattering (SLS) by applying a temperature ramp using the Uncle system (Unchained Labs, USA).

9 μg of filtered protein sample with a protein concentration of 1 mg/mL was added to the Uncle device. The temperature was ramped from 30 to 90°C with 0.1°C/min to obtain the scattering intensity at 266 nm.

The results are shown in Table 9.

Example 9 - Characterization of the CD19-TCB Molecule Using Optimized Anti-CD3 Antibodies by Surface Plasmon Resonance (SPR) Data after Stress

To confirm the effect of deamidation site removal and its effect on antibody stability, CD19-TCB molecule containing the optimized anti-CD3 antibody CD3 _opt (and the CD19 binding fragment 2B11, see Example 4) were incubated for 14 days at 37°C, pH 7.4 and at 40°C, pH 6 and then analyzed by SPR for their ability to bind to human CD3ε/δ. Samples stored at -80°C, pH 6, were used as reference. Reference and 40°C-stressed samples were in 20 mM His, 140 mM NaCl, pH 6.0, and 37°C-stressed samples were in PBS, pH 7.4, all at a concentration of 1.0 mg/mL. After the stress period (14 days), reverse dialysis of samples was performed in PBS in 20 mM His, 140 mM NaCl, pH 6.0 for additional analysis.

All SPR experiments were performed on a Biacore T200 instrument (GE Healthcare) at 25°C with HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) as running and dilution buffers. Biotinylated human CD3ε/δ (see Example 2, SEQ ID NOs:41 and 42) and biotinylated anti-huIgG (Capture Select, Thermo Scientific, #7103262100) were immobilized on a S-series SA sensor chip (GE Healthcare, #29104992) to obtain a surface density of at least 1000 resonance units (RU). TCB at a concentration of 2 μg/mL was injected for 30 s at a flow rate of 5 μL/min. and monitored for dissociation for 120 s. The surface was restored by injecting 10 mM glycine, pH 1.5, for 60 s. Differences in bulk refractive index were corrected by subtracting blank injections and by subtracting the response obtained from a blank control flow cell. The binding response was evaluated 5 s after the end of injection. To normalize the binding signal, CD3 binding was divided by the anti-huIgG response (signal (PE) obtained after TCB capture on immobilized anti-huIgG). Relative binding activity was calculated by comparing each temperature-stressed sample with the corresponding unstressed sample.

As shown in Table 10, the binding of CD19-TCB containing the optimized CD3 antibody binding fragment CD3 _opt to CD3ε/δ is essentially unaffected following stress, consistent with the results for the CD3 binding fragment alone (Example 2).

Example 10 - In vivo B-cell depletion and cytokine release induced by CD19-TCB molecule with optimized anti-CD3 antibody

To understand the potency and safety profile of the CD19-TCB molecule, containing the optimized anti-CD3 antibody CD3 _opt and the CD19 binding fragment 2B11, an in vivo mode of action study was performed assessing peripheral B cell depletion and cytokine release in humanized NSG mice.

Female NSG mice (4–5 weeks old at the start of the experiment) (Jackson Laboratory) were maintained in pathogen-free conditions with 12 h light/12 h dark cycles according to established guidelines (GV-Solas; Felasa; TierschG). The experimental protocol was reviewed and approved by the local authorities (ZH223-17). After arrival, the animals were kept for one week for habituation to the new environment and for observation. Continuous monitoring of health status was performed on an ongoing basis.

Female NSG mice were injected i.p. with 15 mg/kg busulfan, followed one day later by i.v. injection of 1 x 10 ⁵ human hematopoietic cells isolated from umbilical cord blood. At 14–16 weeks after stem cell injection, mice were bled sublingually and analyzed by flow cytometry for successful humanization. Mice with effective engraftment were randomized according to human T cell frequency to different treatment groups. After randomization, mice from the three groups were pretreated once with obinutuzumab (Gazyva®) (30 mg/kg) as a measure to prevent excessive cytokine release.

Seven days after this pretreatment, on day 0, all groups received CD19-TCB at different doses, CD20-TCB (a TCB targeting CD20 and containing the CD3 binding fragment of the CD3 _orig ), or vehicle. Three different doses (0.5, 0.15, and 0.05 mg/kg) of CD19-TCB were injected. CD20-TCB (0.15 mg/kg) with and without Gazyva® pretreatment was used as a comparator. All mice were injected with 200 μl of the respective solution intravenously. Three mice per group were bled at 4 h, 24 h, and 72 h after therapy (day 0).

The study design is shown in Fig. 11, and the study groups are summarized in Table 11.

At the end (day 3), mice were sacrificed and spleens, lymph nodes (LNs) and bone marrow (BM) were collected, weighed and single cell suspensions were prepared by enzymatic digestion with Liberase and DNase for subsequent FACS analysis. Single spleen cells as well as whole blood samples were stained for human CD45, CD19, CD20 and analyzed on a BD Fortessa flow cytometer. In addition, sera from three blood collection time points were analyzed for cytokine content by Multiplex assay.

Fig. 12 shows the change in body weight (%) in the treatment groups. The CD19-TCB molecule caused a smaller drop in body weight compared to CD20-TCB treatment. This drop in body weight was independent of the dose used. Furthermore, analysis of cytokines in the serum of treated animals showed a peak of elevated cytokine levels 4 hours after treatment with the CD20-TCB molecule (which could be reduced by pre-treatment with Gazyva® (GPT)), whereas only low levels of cytokines were detected for the CD19-TCB molecule (Fig. 13).

ImmunoPD data (Fig. 14) on the kinetics of B cell depletion in blood showed a strong depletion of CD19+CD20+ B cells (mean CD19+ or CD20+ cell counts/µl blood+/- PCA) over time by CD19-TCB and confirmed a dose-dependency. This B cell depletion effect was also evident in all lymphatic organs analyzed 72 hours after CD19-TCB treatment as strong as CD20-TCB (data not shown).

Example 11 - Control of Tumor Growth in a Mouse Xenograft Experiment Using the CD19-TCB Molecule with Optimized Anti-CD3 Antibody

To evaluate the antitumor efficacy of the CD19-TCB molecule containing the optimized anti-CD3 antibody CD3 _opt and the CD19 binding fragment 2B11 in vivo, humanized NSG mice were inoculated with patient-derived CD19+ lymphoma xenograft (PDX) cells from a patient who relapsed after R-CHOP treatment. When the tumor volume reached 200 mm ³ , mice were randomized into groups of 8 based on their tumor size. They were then injected weekly with 0.5 mg/kg CD19-TCB or vehicle (i.v.), as shown in Fig. 15. To evaluate the effect of CD19-TCB on tumor growth, tumor volumes were calculated from capillary measurements twice or thrice weekly.

As a result, daily treatment with CD19-TCB exhibited significant tumor growth control compared with vehicle treatment (Fig. 16). These data demonstrated that weekly dosing of 0.5 mg/kg CD19-TCB is effective in PDX lymphoma-bearing huNSG mice and indicate that lymphoma patients who relapsed after R-CHOP treatment may benefit from treatment with CD19-TCB.

Although the foregoing invention has been described in detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be taken as limiting the scope of the invention. The contents of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety.

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SEQUENCE LIST

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<120> ANTIBODIES BINDING TO CD3 AND CD19

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Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

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Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

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Pro Gln Leu Leu Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro

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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Leu

85 90 95

Leu Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 23

<211> 674

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Pro Gln Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gln Ala Val Val Thr

225 230 235 240

Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr

245 250 255

Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp

260 265 270

Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr

275 280 285

Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu

290 295 300

Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu

305 310 315 320

Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly

325 330 335

Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro

340 345 350

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

355 360 365

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

370 375 380

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

385 390 395 400

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

405 410 415

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

420 425 430

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

435 440 445

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

450 455 460

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

465 470 475 480

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

485 490 495

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

500 505 510

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

515 520 525

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

530 535 540

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

545 550 555 560

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

565 570 575

Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val

580 585 590

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

595 600 605

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

610 615 620

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

625 630 635 640

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

645 650 655

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

660 665 670

Ser Pro

<210> 24

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Pro Gln Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 25

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 25

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Glu Thr Ser

20 25 30

Thr Gly Thr Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Leu

85 90 95

Leu Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg

115 120 125

Lys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 26

<211> 232

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 26

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 27

<211> 232

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 27

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gln Phe Ser Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Thr Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr

100 105 110

Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 28

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 28

Asp Tyr Ile Met His

1 5

<210> 29

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 29

Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe Gln

1 5 10 15

Gly

<210> 30

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 30

Gly Thr Tyr Tyr Tyr Gly Ser Ala Leu Phe Asp Tyr

1 5 10

<210> 31

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 31

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Ala Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 32

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 32

Lys Ser Ser Gln Ser Leu Glu Asn Pro Asn Gly Asn Thr Tyr Leu Asn

1 5 10 15

<210> 33

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 33

Arg Val Ser Lys Arg Phe Ser

1 5

<210> 34

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 34

Leu Gln Leu Thr His Val Pro Tyr Thr

1 5

<210> 35

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 35

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Glu Asn Pro

20 25 30

Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Leu

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 36

<211> 674

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 36

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Ala Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gln Ala Val Val Thr

225 230 235 240

Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr

245 250 255

Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp

260 265 270

Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr

275 280 285

Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu

290 295 300

Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu

305 310 315 320

Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly

325 330 335

Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro

340 345 350

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

355 360 365

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

370 375 380

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

385 390 395 400

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

405 410 415

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

420 425 430

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

435 440 445

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

450 455 460

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

465 470 475 480

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

485 490 495

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

500 505 510

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

515 520 525

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

530 535 540

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

545 550 555 560

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

565 570 575

Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val

580 585 590

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

595 600 605

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

610 615 620

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

625 630 635 640

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

645 650 655

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

660 665 670

Ser Pro

<210> 37

<211> 449

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 37

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Ala Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 38

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 38

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Glu Asn Pro

20 25 30

Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Leu

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg

115 120 125

Lys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 39

<211> 674

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 39

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Pro Gln Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr

225 230 235 240

Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr

245 250 255

Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp

260 265 270

Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr

275 280 285

Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu

290 295 300

Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu

305 310 315 320

Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly

325 330 335

Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro

340 345 350

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

355 360 365

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

370 375 380

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

385 390 395 400

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

405 410 415

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

420 425 430

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

435 440 445

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

450 455 460

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

465 470 475 480

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

485 490 495

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

500 505 510

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

515 520 525

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

530 535 540

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

545 550 555 560

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

565 570 575

Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val

580 585 590

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

595 600 605

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

610 615 620

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

625 630 635 640

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

645 650 655

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

660 665 670

Ser Pro

<210> 40

<211> 674

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 40

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Ala Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr

225 230 235 240

Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr

245 250 255

Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp

260 265 270

Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr

275 280 285

Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu

290 295 300

Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu

305 310 315 320

Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly

325 330 335

Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro

340 345 350

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

355 360 365

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

370 375 380

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

385 390 395 400

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

405 410 415

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

420 425 430

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

435 440 445

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

450 455 460

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

465 470 475 480

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

485 490 495

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

500 505 510

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

515 520 525

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

530 535 540

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro

545 550 555 560

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

565 570 575

Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val

580 585 590

Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

595 600 605

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

610 615 620

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

625 630 635 640

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

645 650 655

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

660 665 670

Ser Pro

<210> 41

<211> 360

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 41

Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys

1 5 10 15

Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro

20 25 30

Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp

35 40 45

Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys

50 55 60

Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg

65 70 75 80

Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg

85 90 95

Val Ser Glu Asn Cys Val Asp Glu Gln Leu Tyr Phe Gln Gly Gly Ser

100 105 110

Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

115 120 125

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

130 135 140

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

145 150 155 160

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

165 170 175

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

180 185 190

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

195 200 205

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

210 215 220

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

225 230 235 240

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

245 250 255

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

260 265 270

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

275 280 285

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

290 295 300

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

305 310 315 320

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

325 330 335

Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu

340 345 350

Ala Gln Lys Ile Glu Trp His Glu

355 360

<210> 42

<211> 325

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 42

Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn Cys

1 5 10 15

Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Ser

20 25 30

Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly

35 40 45

Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Thr

50 55 60

Val Gln Val His Tyr Arg Met Cys Arg Ser Glu Gln Leu Tyr Phe Gln

65 70 75 80

Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

85 90 95

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

100 105 110

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

115 120 125

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

130 135 140

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

145 150 155 160

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

165 170 175

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

180 185 190

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

195 200 205

Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

210 215 220

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

225 230 235 240

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

245 250 255

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr

260 265 270

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

275 280 285

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

290 295 300

Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys

305 310 315 320

Ile Glu Trp His Glu

325

<210> 43

<211> 534

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 43

Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp Asn Ala Val

1 5 10 15

Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln Gln Leu Thr

20 25 30

Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu Ser Leu Gly

35 40 45

Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile Trp Leu Phe

50 55 60

Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu Cys Gln Pro

65 70 75 80

Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr Val Asn Val

85 90 95

Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp Leu Gly Gly

100 105 110

Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro Ser Ser Pro

115 120 125

Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala Lys Asp Arg

130 135 140

Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro Arg Asp Ser

145 150 155 160

Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro Gly Ser Thr

165 170 175

Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser Arg Gly Pro

180 185 190

Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser Leu Leu Ser

195 200 205

Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp Val Met Glu

210 215 220

Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala Gly Lys Tyr

225 230 235 240

Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu Glu Ile Thr

245 250 255

Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly Gly Trp Lys

260 265 270

Val Asp Ala Ser Gly Gly Ser Pro Thr Pro Pro Thr Pro Gly Gly Gly

275 280 285

Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

290 295 300

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

305 310 315 320

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

325 330 335

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

340 345 350

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

355 360 365

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

370 375 380

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

385 390 395 400

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

405 410 415

Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln

420 425 430

Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

435 440 445

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

450 455 460

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

465 470 475 480

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

485 490 495

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

500 505 510

Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln

515 520 525

Lys Ile Glu Trp His Glu

530

<210> 44

<211> 225

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 44

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro

225

<210> 45

<211> 186

<212> Protein

<213> Homo sapiens

<400> 45

Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys

1 5 10 15

Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro

20 25 30

Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp

35 40 45

Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys

50 55 60

Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg

65 70 75 80

Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg

85 90 95

Val Cys Glu Asn Cys Met Glu Met Asp Val Met Ser Val Ala Thr Ile

100 105 110

Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu Leu Leu Val Tyr

115 120 125

Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly

130 135 140

Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro

145 150 155 160

Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp

165 170 175

Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile

180 185

<210> 46

<211> 177

<212> Protein

<213> Macaca fascicularis

<400> 46

Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr Gln Thr Pro Tyr Gln

1 5 10 15

Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Ser Gln His Leu

20 25 30

Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys Asn Lys Glu Asp Ser

35 40 45

Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu Met Glu Gln Ser Gly

50 55 60

Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro Glu Asp Ala Ser His

65 70 75 80

His Leu Tyr Leu Lys Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp

85 90 95

Val Met Ala Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Leu

100 105 110

Gly Leu Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys

115 120 125

Ala Lys Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly

130 135 140

Gln Asn Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro

145 150 155 160

Ile Arg Lys Gly Gln Gln Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg

165 170 175

Ile

<210> 47

<211> 225

<212> Protein

<213> Homo sapiens

<400> 47

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro

225

<210> 48

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 48

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 49

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 49

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 50

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 50

Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly

1 5 10

<210> 51

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 51

Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly

1 5 10

<210> 52

<211> 107

<212> Protein

<213> Homo sapiens

<400> 52

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 53

<211> 105

<212> Protein

<213> Homo sapiens

<400> 53

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 54

<211> 328

<212> Protein

<213> Homo sapiens

<400> 54

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<---

Claims (47)

1. An antibody that binds to CD3 and CD19, wherein the antibody comprises: (a) a first antigen-binding domain that binds to CD3, comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 3 and HCDR 3 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, LCDR 2 of SEQ ID NO: 9 and LCDR 3 of SEQ ID NO: 10; and (b) a second and optionally third antigen binding domain that binds to CD19, comprising a VH comprising HCDR 1 of SEQ ID NO: 15, HCDR 2 of SEQ ID NO: 16 and HCDR 3 of SEQ ID NO: 17, and a VL comprising LCDR 1 of SEQ ID NO: 19, LCDR 2 of SEQ ID NO: 20 and LCDR 3 of SEQ ID NO: 21. 2. The antibody of claim 1, wherein the VH of the first antigen-binding domain comprises 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: 7, and/or the VL of the first antigen-binding domain comprises 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: 11. 3. The antibody of claim 1 or 2, wherein the VH of the second and, if present, the third antigen-binding domain comprises 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 the VL of the second and, if present, the third antigen-binding domain comprises 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. 4. 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 third antigen-binding domain that binds to CD19 (i) comprising a VH comprising HCDR 1 of SEQ ID NO: 15, HCDR 2 of SEQ ID NO: 16 and HCDR 3 of SEQ ID NO: 17, and a VL comprising LCDR 1 of SEQ ID NO: 19, LCDR 2 of SEQ ID NO: 20 and LCDR 3 of SEQ ID NO: 21; and/or (ii) comprising a VH sequence of SEQ ID NO: 18 and a VL sequence of SEQ ID NO: 22. 5. The antibody of any one of claims 1 to 4, wherein the first, second and/or, if present, third antigen-binding domain is a Fab molecule. 6. An antibody according to any one of claims 1-5, comprising an Fc domain consisting of a first and a second subunit. 7. The antibody of any one of claims 1 to 6, wherein the first antigen-binding domain is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1, in particular the variable domains VL and VH, of the Fab light chain and the Fab heavy chain are substituted for each other. 8. The antibody of any one of claims 1 to 7, wherein the second and, if present, the third antigen-binding domain is a standard Fab molecule. 9. The antibody of any one of claims 1 to 8, wherein the second and, if present, the third antigen-binding domain is a Fab molecule, wherein in the CL constant domain the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H), numbering according to Kabat, and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H), numbering according to Kabat, and in the CH1 constant domain the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D), numbering according to the EU index according to Kabat and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D), numbering according to the EU index according to Kabat). 10. The antibody of any one of claims 1 to 9, wherein the first and second antigen-binding domains are fused to each other, optionally via a peptide linker. 11. The antibody of any one of claims 1-10, wherein each of the first and second antigen-binding domains is a Fab molecule 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. 12. The antibody of any one of claims 1 to 11, wherein each of the first, second and, if present, third antigen-binding domains is a Fab molecule, and the antibody comprises an Fc domain consisting of a first and a second subunit; and wherein 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 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. 13. The antibody of claim 12, which consists of a first, second and third antigen-binding domain, 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, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain and the third antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. 14. The antibody of any one of claims 6 to 13, wherein the Fc domain is the Fc domain of IgG, in particular IgG ₁ . 15. The antibody of any one of claims 6 to 14, wherein the Fc domain is a human Fc domain. 16. The antibody of any one of claims 6 to 15, wherein the Fc domain is the Fc domain of human IgG ₁ . 17. The antibody of any one of claims 6-16, wherein the Fc comprises a modification that facilitates association of the first and second subunits of the Fc domain. 18. The antibody of any one of claims 6 to 17, wherein the amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced by an amino acid residue having a larger side chain volume, thereby creating a bulge in the CH3 domain of the first subunit that can be accommodated in a cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain the amino acid residue is replaced by an amino acid residue having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit that can accommodate the bulge in the CH3 domain of the first subunit. 19. The antibody of any one of claims 6 to 18, wherein in the first subunit of the Fc domain the threonine residue at position 366 is substituted with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is substituted with a valine residue (Y407V), and optionally (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) in the first subunit of the Fc domain, additionally the serine residue at position 354 is substituted with a cysteine residue (S354C) or the glutamic acid residue at position 356 is substituted with a cysteine residue (E356C), and in the second subunit of the Fc domain, additionally the tyrosine residue at position 349 is substituted with a cysteine residue (Y349C), numbering according to the EU index according to Kabat. 20. The antibody of any one of claims 6-19, wherein the Fc domain comprises one or more amino acid substitutions that reduce Fc receptor binding and/or effector function. 21. The antibody of any one of claims 6 to 20, wherein the Fc domain comprises an amino acid substitution at a position selected from the group E233, L234, L235, N297, P331 and P329, in particular at a position selected from the group L234, L235 and P329, numbered according to the EU index according to Kabat. 22. The antibody of any one of claims 6 to 21, wherein each Fc domain subunit comprises the amino acid substitutions L234A, L235A and P329G, numbered according to the EU index according to Kabat. 23. The antibody of any one of claims 1-22, comprising a) a first antigen-binding domain that binds CD3, wherein the first antigen-binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other, and wherein the variable region of the heavy chain comprises the amino acid sequence of SEQ ID NO: 7 and the variable region of the light chain comprises the amino acid sequence of SEQ ID NO: 11; b) a second and third antigen-binding domains that bind CD19, wherein the second and third antigen-binding domains each represent a standard Fab molecule and comprise 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; c) The Fc domain is formed by the first and second subunits; Where in the constant domain CL of the second and third antigen-binding domain according to b) the amino acid residue at position 124 is substituted with lysine (K), numbering according to Kabat, and the amino acid residue at position 123 is substituted with lysine (K) or arginine (R), numbering according to Kabat, more preferably arginine (R) and wherein the constant domain CH1 of the second and third binding domain according to b) the amino acid residue at position 147 is substituted with glutamic acid (E), numbering according to the EU index according to Kabat, and the amino acid residue at position 213 is substituted with glutamic acid (E), numbering according to the EU index according to Kabat; and where else the second antigen-binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain according to a), and the first antigen-binding domain according to a) and the third antigen-binding domain according to b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c). 24. The antibody of claim 23, wherein the Fc domain is the Fc domain of human IgG ₁ and wherein in the first subunit of the Fc domain the threonine residue at position 366 is substituted with a tryptophan residue (T366W) and the serine residue at position 354 is substituted with a cysteine residue (S354C) and in the second subunit of the Fc domain the tyrosine residue at position 407 is substituted with a valine residue (Y407V), the threonine residue at position 366 is substituted with a serine residue (T366S), the leucine residue at position 368 is substituted with an alanine residue (L368A) and the tyrosine residue at position 349 is substituted with a cysteine residue (Y349C), the numbering according to the EU index according to Kabat, and wherein additionally in each of the first and second In the Fc domain subunit, the leucine residue at position 234 is substituted by an alanine residue (L234A), the leucine residue at position 235 is substituted by an alanine residue (L235A), and the proline residue at position 329 is substituted by a glycine residue (P329G), numbered according to the EU index according to Kabat. 25. The antibody according to any one of claims 1-24, wherein the antibody comprises 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: 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%, 99%, or 100% identical to the sequence of SEQ ID NO: 24, a polypeptide, in particular 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. 26. An isolated polynucleotide encoding an antibody according to any one of claims 1-25. 27. A host cell for producing an antibody that binds to CD3 and CD19, comprising the isolated polynucleotide of claim 26. 28. A method for producing an antibody that binds to CD3 and CD19, comprising the steps of (a) culturing the host cell of claim 27 under conditions suitable for expression of the antibody, and optionally (b) isolating the antibody. 29. A pharmaceutical composition for treating cancer or an autoimmune disease, comprising an antibody according to any one of claims 1-25 and a pharmaceutically acceptable carrier. 30. Use of an antibody according to any one of claims 1-25 or a pharmaceutical composition according to claim 29 in the manufacture of a medicinal product for the treatment of cancer or an autoimmune disease. 31. The use according to claim 30, wherein the cancer is (i) a CD19-expressing cancer and/or (ii) a B-cell cancer, in particular a B-cell lymphoma or a B-cell leukemia, most preferably a non-Hodgkin's lymphoma or an acute lymphoblastic leukemia or a chronic lymphocytic leukemia. 32. The use according to claim 30, wherein the autoimmune disease is lupus, in particular systemic lupus erythematosus (SLE) or lupus nephritis (LN). 33. A method of treating a disease in an individual, comprising administering to said individual an effective amount of an antibody according to any one of claims 1-25 or a pharmaceutical composition according to claim 29, wherein the disease is cancer or an autoimmune disease. 34. The method of claim 33, wherein the cancer is (i) a CD19-expressing cancer and/or (ii) a B-cell cancer, in particular a B-cell lymphoma or a B-cell leukemia, most preferably a non-Hodgkin's lymphoma or an acute lymphoblastic leukemia or a chronic lymphocytic leukemia. 35. The method according to claim 33, wherein the autoimmune disease is lupus, in particular systemic lupus erythematosus (SLE) or lupus nephritis (LN).