Antigen binding molecules

Abstract

The present invention relates to a pair of binding molecules comprising a complementary portion of an effector domain, such binding molecules being capable of forming a functional effector domain when bound to their target antigen on the surface of a cell. Specifically, the present invention relates to a pair of binding molecules wherein the complementary portion of the effector domain is complementary to an inert complementary domain without forming a functional effector domain, thereby providing advantageous properties to the binding molecules, such as producibility, stability and / or biological functionality.

Description

Technical Field

[0001] The present invention relates to binding molecule pairs that comprise complementary portions of effector domains, such binding molecules being capable of forming functional effector domains when bound to their target antigens on the surface of cells. Specifically, the present invention relates to binding molecule pairs in which the complementary portions of the effector domains are complementary to inert complementary domains without forming functional effector domains, thereby providing advantageous properties to the binding molecules, such as manufacturability, stability, and / or biological functionality. Background Art

[0002] T cell engagers or T cell bispecific antibodies (TCBs) are bispecific antibodies having one binding portion that recognizes a target cell antigen, such as a tumor antigen expressed on tumor cells, and another binding portion that recognizes the T cell receptor. TCBs have great promise as cancer immunotherapeutic agents. Crosslinking of CD3 with the target cell antigen triggers T cell activation, proliferation, and cytokine release, resulting in target cell killing (Bacac et al., Clin Cancer Res (2016) 22, 3286 - 97; Bacac et al., Oncoimmunology (2016) 5, e1203498).

[0003] However, due to the potency of T cell redirection methods, TCB therapy is sometimes associated with on - target off - tumor cytotoxic activity and safety liabilities caused by cytokine release. Especially in the treatment of solid tumors, in order to unleash the full potential of T cell bispecific antibodies, on - target off - tumor activity remains a challenge to be addressed.

[0004] To date, the clinical experience with T cell bispecific antibodies has shown that high durable response rates can be achieved in hematological malignancies, but remain very limited in solid tumors. Multiple T cell engagers have been stopped in phase I trials. Although the reasons for terminating these trials may vary, multiple trials have shown adverse events, with CRS frequently occurring despite prophylactic corticosteroid treatment and limited positive pharmacodynamic signals at relatively low doses.

[0005] The limited success of T cell bispecific antibodies, especially in the treatment of solid tumors, is due to the lack of tumor - specific or targets expressed only on physiologically tissues considered to be dispensable, such as CD19 or CD20 on B cells. So far, traditional solid tumor T cell bispecific antibodies have been hampered by limited efficacy driven by a narrow therapeutic window resulting from on - target off - tumor activation of T cells. For example, dose - limiting toxicity has been shown in clinical trials of catumaxomab for the treatment of solid tumors ( et al. (2015) Cancer Chemother Pharmacol 75, 1065 - 1073; Borlak et al. (2016) Oncotarget 7, 28059 - 28074). In fact, due to the potency of such therapeutic modalities and the lack of truly tumor - specific targets, the on - target off - tumor activity of T - cell conjugates has been a challenge for many years.

[0006] Therefore, there is a need to develop drugs with tumor - restricted activity, such as T - cell bispecific antibodies, to improve the therapeutic index and patient prognosis.

[0007] Novel methods are needed to achieve this goal, and although technically challenging, the generation of inactive prodrugs that are active primarily or only within the tumor microenvironment is an area of intense research.

[0008] To reduce the systemic toxicity common to T - cell bispecific antibodies, for example, Stuhler and colleagues described a “split” approach for on - target in - situ generation of a CD3 conjugate commonly used in this compound class (Banaszek et al. (2019) Nature Comm 10, 5387; PCT publication number WO 2013 / 104804). According to this concept, the functional antibody - binding fragment (Fv) of the CD3 conjugate is split into a VL domain and a VH domain, neither of which has CD3 - binding ability as separate domains, and the two V domains are contained in separate prodrug molecules, namely the CD3 - VH prodrug and the CD3 - VL prodrug, respectively.

[0009] However, the VL domain and VH domain as separate domains are generally less stable than the assembled Fv fragment and often have limited expression capacity (Ewert et al. (2003) J Molecular Biol 325, 531 - 553), because the VH domain tends to stabilize the VL domain and the VL domain can enhance the folding of the VH domain. In addition, the isolated V domains display hydrophobic surface patches in the interfacial region with the associated V domain and thus are prone to aggregation as separate domains, especially under stress conditions such as long - term storage or elevated temperature. Many CD3 conjugates commonly used industrially for T - cell bispecific antibodies are indeed subject to these limitations.

[0010] Therefore, there is still a need for improved “split” methods that can be applied, for example, to T - cell bispecific antibodies. SUMMARY OF THE INVENTION

[0011] The present invention relates to binding molecule pairs that comprise complementary portions of effector domains, such binding molecules being capable of forming functional effector domains when bound to their target antigens on the surface of cells.

[0012] The inventors have found that complementing the complementary portions of effector domains with inert complementary domains, without forming functional effector domains, provides advantageous properties to the binding molecules, including improved production capabilities (such as increased yields, easier purification), stability (including after exposure to stress conditions), and / or biological functionality (such as reduced assembly in the absence of cells expressing the target antigen of the binding molecule).

[0013] The complementary domains cover the (potentially hydrophobic) interface between portions of the effector domain and contribute to the folding and stability of the binding molecule. In addition, the complementary domains can be used as regulators of the association equilibrium of the binding molecule. In non-complementary binding molecules, the equilibrium of assembly depends on the local concentration of the binding molecule and the association rate constant of the effector domain portions. In the complementary methods described herein, assembly additionally depends on the dissociation rate constants of the portions of the effector domain and their respective complementary domains. Thus, for complementary binding molecules, their assembly equilibrium is shifted towards higher concentrations, such that target-independent assembly, such as assembly in the circulation, is disfavored, and target-dependent assembly on target cells is favored. By fine-tuning the interaction between the portions of the effector domain and the complementary domains, it may be possible to modulate the therapeutic window.

[0014] Accordingly, in a first aspect, the present invention provides binding molecule pairs that comprise

[0015] (a) a first binding molecule that comprises (i) a first antigen-binding domain capable of binding to a target antigen, (ii) a first portion of an effector domain, and (iii) a first complementary domain capable of associating with the first portion of the effector domain; and

[0016] (b) a second binding molecule that comprises (i) a second antigen-binding domain capable of binding to the target antigen, (ii) a second portion of the effector domain, and (iii) a second complementary domain capable of associating with the second portion of the effector domain;

[0017] wherein if the first antigen-binding domain and the second antigen-binding domain bind to their target antigen on the cell surface, the first and second portions of the effector domain are capable of associating with each other to form a functional effector domain,

[0018] wherein the first complementary domain and the second complementary domain associate with the first and second portions of the effector domain, respectively, and the first and second portions of the effector domain do not associate with each other.

[0019] In another aspect, the present invention provides a binding molecule that forms part of a binding molecule pair of the present invention.

[0020] In a further aspect of the present invention, there is provided an isolated polynucleotide encoding a binding molecule pair or a binding molecule of the present invention, and a host cell comprising the isolated polynucleotide of the present invention. In another aspect, there is provided a method for producing a binding molecule (pair), the method comprising the steps of: (a) culturing a host cell according to the present invention under conditions suitable for expressing the binding molecule (pair), and optionally (b) recovering the binding molecule (pair).

[0021] The present invention further provides a pharmaceutical composition comprising a binding molecule pair or a binding molecule of the present invention and a pharmaceutical carrier.

[0022] The present invention also includes methods of using the binding molecule pairs, binding molecules, and pharmaceutical compositions of the present invention. In one aspect, the present invention provides a binding molecule pair, a binding molecule, or a pharmaceutical composition according to the present invention for use as a medicament. In one aspect, there is provided a binding molecule pair, a binding molecule, or a pharmaceutical composition according to the present invention for the treatment of a disease. There is also provided the use of a binding molecule pair, a binding molecule, or a pharmaceutical composition according to the present invention in the manufacture of a medicament, and the use of a binding molecule pair, a binding molecule, or a pharmaceutical composition according to the present invention in the manufacture of a medicament for the treatment of a disease. The present invention also provides a method for treating a disease in an individual, comprising administering to the individual an effective amount of a binding molecule pair according to the present invention. Detailed Description

[0023] Definitions

[0024] Unless otherwise defined herein, the terms used herein are generally as used in the art.

[0025] As used herein, the terms "first", "second", or "third" with respect to antigen-binding domains, etc. are used for convenience of distinction when there is more than one of each type of moiety. Unless expressly stated, the use of these terms is not intended to impart a particular order or orientation to the moieties.

[0026] The term "binding molecule" as used herein refers to a polypeptide molecule (composed of one or more polypeptide chains) capable of binding to an antigen. The binding molecule can be derived from an antibody and typically comprises an antigen-binding domain.

[0027] The term "antibody" as used herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the desired antigen-binding activity.

[0028] "Antibody fragment" refers to a molecule that, other than a full antibody, contains a portion of a full antibody and binds to the antigen to which the full antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies; and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0029] The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody.

[0030] "Antigen-binding domain" is a molecular domain that is capable of binding to an antigen. The term particularly refers to the antigen-binding domain of an antibody, i.e., the portion of the antibody that contains regions that bind and are complementary to part or all of the antigen. Thus, in certain aspects, the antigen-binding domains herein are the antigen-binding domains of an antibody. Such antigen-binding domains can be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). Thus, in certain aspects, the antigen-binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH). Antigen-binding domains can also be provided by molecules from non-antibody sources, such as ankyrin repeat proteins or lipocalin-derived binding molecules

[0031] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have similar structures, each domain comprising four conserved framework regions (FRs) and complementarity-determining regions (CDRs). See, e.g., Kindt et al., Kuby Immunology, 6 thEd., W.H. Freeman & Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind a particular antigen can be isolated using, respectively, the VH or VL domains from an antibody that binds that antigen to screen a library of complementary VL or VH domains. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). As used herein, "Kabat numbering" with respect to variable region sequences refers to that described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0032] As used herein, the amino acid positions of all constant regions and constant domains of heavy and light chains are numbered according to the Kabat numbering system described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "according to Kabat numbering" or "Kabat numbering." Specifically, the Kabat numbering system (see pages 647 to 660 of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domain CL of κ and λ isotypes, and the Kabat EU index numbering system (see pages 661 to 723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3), which is further clarified herein by referring to this case as "according to Kabat EU index numbering" or "Kabat EU index numbering."

[0033] As used herein, the term "hypervariable region" or "HVR" refers to each region within the variable domain of an antibody that is highly variable in sequence and that defines antigen-binding specificity, such as "complementary determining regions" ("CDRs"). Generally, an antibody comprises six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include:

[0034] (a) Hypervariable loops occurring at the following amino acid residues: 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2), and 96 to 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0035] (b) CDRs occurring at the following amino acid residues: 24 to 34 (L1), 50 to 56 (L2), 89 to 97 (L3), 31 to 35b (H1), 50 to 65 (H2), and 95 to 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

[0036] (c) Antigen contact sites present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0037] Unless otherwise specified, CDRs are determined by the method described by Kabat et al. supra. Those skilled in the art will understand that CDR names can also be determined according to the method described by Chothia supra, the method described by MacCallum supra, or any other scientifically accepted naming system.

[0038] "Framework" or "FR" refers to the variable domain residues other than the complementarity determining regions (CDRs). The FRs of the variable domain typically consist of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically occur in the VH (or VL) in the following order: FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0039] Unless otherwise specified, CDR residues and other residues (e.g., FR residues) in the variable domain are numbered herein according to Kabat et al., supra.

[0040] The term "immunoglobulin molecule" as used herein refers to a protein having the structure of a naturally occurring antibody. For example, an IgG class immunoglobulin is a heterotetrameric glycoprotein of approximately 150,000 daltons, which is composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH) (also referred to as variable heavy chain domain or heavy chain variable region), followed by three constant domains (CH1, CH2, and CH3) (also referred to as heavy chain constant regions). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL) (also referred to as variable light chain domain or light chain variable region), followed by a constant light chain (CL) domain (also referred to as light chain constant region). The heavy chains of immunoglobulins can be assigned to one of the following five types: designated as α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of the following two types based on the amino acid sequence of their constant domains: designated as kappa (κ) and lambda (λ). An immunoglobulin substantially consists of two Fab molecules and an Fc domain linked by an immunoglobulin hinge region.

[0041] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region that its heavy chain has. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated as α, δ, ε, γ, and μ, respectively.

[0042] "Fab molecule" refers to a protein composed of the VH and CH1 domains of the heavy chain of an immunoglobulin ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain").

[0043] The term "multispecific" means that a binding molecule (e.g., an antibody) is capable of specifically binding to at least two different antigenic determinants. A multispecific binding molecule (e.g., an antibody) can be, for example, a bispecific binding molecule. Generally, a bispecific binding molecule contains two antigen-binding sites, each of which is specific for a different antigenic determinant. In certain aspects, a multispecific (e.g., bispecific) binding molecule is capable of binding two antigenic determinants simultaneously, particularly two antigenic determinants expressed on the same cell, adjacent cells, or cells in the same tissue.

[0044] As used herein, the term "valence" refers to the number of antigen-binding sites present in a binding molecule. Thus, the term "monovalent binding to an antigen" means that there is one (and no more than one) antigen-binding site specific for the antigen present in the binding molecule.

[0045] "Antigen-binding site" refers to the site of a binding molecule that provides an interaction with an antigen, i.e., one or more amino acid residues. For example, the antigen-binding site of an antibody contains amino acid residues from the complementarity-determining region (CDR). Native immunoglobulin molecules typically have two antigen-binding sites, and Fab molecules typically have a single antigen-binding site.

[0046] As used herein, the term "antigenic determinant" or "antigen" refers to a site on a polypeptide macromolecule (e.g., a stretch of contiguous amino acids or a conformational configuration composed of different regions of non-contiguous amino acids) to which an antigen-binding domain binds, thereby forming an antigen-binding domain-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, virus-infected cells, other diseased cells, immune cells, free substances in serum, and / or the extracellular matrix (ECM). In certain aspects, the antigen is a human protein.

[0047] "T cell antigen" refers to an antigenic determinant expressed on the surface of T lymphocytes.

[0048] As used herein, "activating T cell antigen" refers to an epitope expressed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes, that is capable of inducing T cell activation upon interaction with an antigen-binding molecule. Specifically, the interaction of the antigen-binding molecule with the activating T cell antigen can induce T cell activation by triggering the signal transduction cascade of the T cell receptor complex. In certain aspects, the activating T cell antigen is CD3, particularly the ε subunit of CD3.

[0049] As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and described herein.

[0050] Unless otherwise specified, "CD3" refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., human), non-human primates (e.g., cynomolgus monkey), and rodents (e.g., mouse and rat). The term encompasses "full-length" unprocessed CD3, as well as any form of CD3 produced by processing in cells. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one aspect, CD3 is human CD3, particularly the ε subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in SEQ ID NO:57 (without the signal peptide). See also UniProt (www.uniprot.org) entry number P07766 (version 209), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. In other aspects, CD3 is cynomolgus monkey (Macaca fascicularis) CD3, particularly cynomolgus monkey CD3ε. The amino acid sequence of cynomolgus monkey CD3ε is shown in SEQ ID NO:58 (without the signal peptide). See also NCBI GenBank accession number BAB71849.1. In certain aspects, the binding molecules of the invention bind to epitopes of CD3 that are conserved in CD3 antigens from different species, particularly human and cynomolgus monkey CD3. In specific aspects, the binding molecules bind to human CD3.

[0051] As used herein, "target antigen" refers to an epitope present on the surface of a target cell, such as a cell in a tumor (such as a cancer cell or a cell of the tumor stroma) (in which case, a "tumor antigen"). Preferably, the target antigen is not CD3, and / or is expressed on a cell different from CD3.

[0052] Unless otherwise indicated, "HER2" (also known as erbB-2 or CD340) refers to any native HER2 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses "full-length" unprocessed HER2, as well as any form of HER2 produced by processing in cells. The term also encompasses naturally occurring variants of HER2, such as splice variants or allelic variants. In one aspect, HER2 is human HER2. The amino acid sequence of human HER2 is shown in UniProt (www.uniprot.org) entry number Q9UK79 (version 95).

[0053] The terms "anti-[protein x] (e.g., CD3) antibody" and "antibody that binds to [protein x] (e.g., CD3)" refer to an antibody that is capable of binding to [protein x] (e.g., CD3) with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting [protein x] (e.g., CD3). In one aspect, the degree of binding of an anti-[protein x] (e.g., CD3) antibody to an unrelated, non-[protein x] (e.g., CD3) protein is less than about 10% of the binding of the antibody to [protein x] (e.g., CD3), as measured, for example, by surface plasmon resonance (SPR). In certain aspects, an antibody that binds to [protein x] (e.g., CD3) has a dissociation constant (K D ) of ≤1 μM, ≤500 nM, ≤200 nM, or ≤100 nM. As measured, for example, by SPR, when the K D of an antibody is 1 μM or less, the antibody is said to "specifically bind" to [protein x] (e.g., CD3). In certain aspects, an anti-[protein x] (e.g., CD3) antibody binds to an epitope of [protein x] (e.g., CD3) that is conserved in [protein x] (e.g., CD3) from different species.

[0054] The term "Fc domain" or "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by a host cell can undergo post-translational cleavage of one or more (especially 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 can include the full-length heavy chain, or the antibody can include a cleavage variant of the full-length heavy chain. This can be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index numbering). Thus, the C-terminal lysine (Lys447) of the Fc region or the C-terminal glycine (Gly446) and lysine (Lys447) can be present or can be absent. Unless otherwise specified, the amino acid sequence of a heavy chain containing an Fc region (or subunit of an Fc domain as defined herein) is represented herein as lacking the C-terminal glycine-lysine dipeptide. In one aspect, a heavy chain comprising an Fc region (subunit) as specified herein is included in a binding molecule according to the invention, the heavy chain comprising an additional C-terminal glycine-lysine dipeptide (G446 and K447, according to the Kabat EU index numbering). In one aspect, a heavy chain comprising an Fc region (subunit) as specified herein is included in a binding molecule according to the invention, the heavy chain comprising an additional C-terminal glycine residue (G446, according to the Kabat EU index numbering). Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain that is capable of stable self-association. For example, a subunit of an IgG Fc domain comprises IgG CH2 and IgG CH3 constant domains.

[0055] "Modifications that promote the association of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent a polypeptide containing Fc domain subunits from associating with the same polypeptide to form a homodimer. As used herein, "modifications that promote association" preferably include separate modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, wherein the modifications are complementary to each other to promote the association of the two Fc domain subunits. For example, the modifications that promote association can alter the structure or charge of one or both of the Fc domain subunits so as to render their association spatially or electrostatically favorable, respectively. Thus, (heterologous) dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, which may be different with respect to additional components (such as antigen-binding domains) fused to each subunit. In some aspects, the modifications that promote the association of the first and second subunits of the Fc domain include amino acid mutations in the Fc domain, specifically amino acid substitutions. In certain aspects, the modifications that promote the association of the first and second subunits of the Fc domain include separate amino acid mutations, specifically amino acid substitutions, in each of the two subunits of the Fc domain.

[0056] The term "effector function" refers to those biological activities that are attributable to the Fc region of an antibody and that vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), cytokine secretion, antigen uptake by immune complex-mediated antigen-presenting cells, downregulation of cell surface receptors (e.g., B-cell receptors), and B-cell activation.

[0057] "Activating Fc receptor" is an Fc receptor that, upon engagement by the Fc domain of an antibody, initiates a signaling event that stimulates a cell bearing the receptor to perform an effector function. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0058] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. The target cells are cells that specifically bind to an antibody or a derivative thereof that contains an Fc region, and this specific binding is typically through the protein moiety at the N-terminus of the Fc region. As used herein, the term "reduced ADCC" is defined as a decrease in the number of target cells lysed by the ADCC mechanism as defined above over a given time at a given antibody concentration in the culture medium surrounding the target cells, and / or an increase in the antibody concentration required to effect lysis of a given number of target cells over a given time by the ADCC mechanism in the culture medium surrounding the target cells. The reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cell but not engineered, using the same standard production, purification, formulation, and storage methods (such methods being known to those of skill in the art). For example, the reduction in ADCC mediated by an antibody containing an amino acid substitution that reduces ADCC in the Fc domain is relative to the ADCC mediated by the same antibody without that 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).

[0059] "Reduced binding" (e.g., reduced binding to an Fc receptor) refers to a decrease in the affinity for the corresponding interaction, as measured, for example, by SPR. For clarity, the term also includes reducing the affinity to zero (or below the detection limit of the assay method), i.e., completely eliminating the interaction. Conversely, "increased binding" refers to an increase in the binding affinity for the corresponding interaction.

[0060] "Non-antigen binding" associated with VH and / or VL means that VH and VL alone or in combination are unable to specifically bind to an antigen, particularly not to a human antigen. It can be determined, for example, by ELISA or surface plasmon resonance that such VH and / or VL do not specifically bind to an antigen (i.e., there is no binding distinguishable from non-specific interactions).

[0061] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of 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 those described herein. The preferred method for measuring affinity is surface plasmon resonance (SPR).

[0062] As used herein, the terms "engineered," "engineered," and "engineered" are considered to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modification of the amino acid sequence, glycosylation pattern, or side chain groups of individual amino acids, as well as combinations of these methods.

[0063] The term "amino acid mutation" as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be made to obtain the final construct, provided that the final construct has the desired characteristics, such as reduced binding to Fc receptors or increased association with another peptide. Amino acid sequence deletions and insertions include amino-terminal and / or carboxyl-terminal deletions and insertions of amino acids. Preferred amino acid mutations are amino acid substitutions. For the purpose of altering, for example, the binding characteristics of the Fc region, non-conservative amino acid substitutions, i.e., substituting one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include replacement with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Genetic or chemical methods well known in the art can be used to generate amino acid mutations. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. Methods for altering amino acid side chain groups by methods other than genetic engineering, such as chemical modification, are also contemplated as useful. Various names can be used herein to denote the same amino acid mutation. For example, substitution of proline at position 329 of the Fc domain with glycine can be denoted as 329G, G329, G 329 , P329G or Pro329Gly.

[0064] "Percent amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps (if necessary) to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. The alignment used to determine the percent amino acid sequence identity can be achieved in various ways within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. One of ordinary skill in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve the maximum alignment over the full length of the sequences being compared. Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate percent identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been filed with the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001 / 007611.

[0065] Unless otherwise indicated, for the purposes of this disclosure, percent amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with the BLOSUM50 comparison matrix. The FASTA program package is described by W.R. Pearson and D.J. Lipman (“Improved Tools for Biological Sequence Analysis”, PNAS 85 (1988) 2444-2448), W.R. Pearson (“Effective protein sequence comparison” Meth. Enzymol. 266 (1996) 227-258), and Pearson et al. (Genomics 46 (1997) 24-36), and is publicly available from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi may be used to compare sequences, using the ggsearch (global protein:protein) program with default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure that a global rather than a local alignment is performed. The percent amino acid identity is given in the output alignment header.

[0066] As used herein, “fusion” means that components (such as Fab molecules and Fc domain subunits) are linked directly or via one or more peptide linkers by peptide bonds.

[0067] The term "polynucleotide" or "nucleic acid molecule" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, in particular 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. Generally, nucleic acid molecules are described by a base sequence, wherein the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually represented from 5' to 3'. In the present context, 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 or RNA, and hybrid polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleobases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression, in vitro and / or in vivo (e.g., in a host or patient), of the binding molecule pairs or binding molecules of the present invention. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject to generate antibodies in vivo (see, for example, Stadler et al. (2017) Nature Medicine 23:815-817, or EP 2101 823B1).

[0068] An "isolated" nucleic acid molecule refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acid molecules include nucleic acid molecules that are contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0069] An "isolated polynucleotide (or nucleic acid) encoding [a binding molecule / binding molecule pair]" refers to one or more polynucleotide molecules encoding a polypeptide chain of a binding molecule, such as an antibody heavy and light chain (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.

[0070] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked thereto. The term includes vectors as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked thereto. Such vectors are referred to herein as "expression vectors".

[0071] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including progeny of such cell. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived from the primary transformed cell, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. This includes mutant progeny having the same function or biological activity as selected or screened in the original transformed cell. A host cell is any type of cell system that can be used to produce the binding molecules of the present invention. Host cells include cultured cells, such as cultured mammalian cells, such as, by way of example only, 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, and also include cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues. In one aspect, the host cells of the present invention are eukaryotic cells, particularly mammalian cells. In one aspect, the host cells are not cells within the human body.

[0072] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective and that contains no additional components that are unacceptably toxic to the subject to which the composition is to be administered.

[0073] A "pharmaceutical carrier" refers to the components of a pharmaceutical composition or formulation other than the active ingredient, which are non-toxic to the subject. Pharmaceutical carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0074] The term "cancer" refers to a physiological condition in mammals that is typically characterized by unregulated cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More non-limiting examples of cancer include hematological cancers (such as leukemia), bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, bile duct cancer, thyroid cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, skin cancer, squamous cell carcinoma, sarcoma, bone cancer, and kidney cancer. Other cell proliferation disorders include, but are not limited to, tumors located in the following sites: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral nervous systems), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases.

[0075] The so-called "solid tumor cancer" means a malignant tumor that forms discrete tumor masses (including tumor metastases) at specific locations within a patient's body, such as sarcoma or carcinoma (as opposed to, for example, blood cancers such as leukemia, which generally do not form solid tumors). Non-limiting examples of solid tumor cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, skin cancer, squamous cell carcinoma, bone cancer, liver cancer, and kidney cancer. Other solid tumor cancers contemplated in the context of the present invention include, but are not limited to, neoplasms located in the following sites: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central nervous system and peripheral nervous systems), lymphatic system, pelvis, skin, soft tissue, muscle, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases.

[0076] "[Protein x] (e.g., HER2)-positive cancer" or "cancer expressing [protein x] (e.g., HER2)" refers to a cancer characterized by the expression or overexpression of [protein x] (e.g., HER2) in cancer cells. The expression of [protein x] (e.g., HER2) can be determined, for example, by quantitative real-time PCR (measuring the [protein x] (e.g., HER2) mRNA level), immunohistochemistry (IHC), or western blot assay. In one aspect, the cancer expresses [protein x] (e.g., HER2). In one aspect, as determined by immunohistochemistry (IHC) using an antibody specific for [protein x] (e.g., HER2), the cancer expresses [protein x] (e.g., HER2) in at least 20%, preferably at least 50%, or at least 80% of the tumor cells.

[0077] As used herein, "treatment" (and its grammatical variants such as "treat" or "treating") refers to an attempt to alter the natural course of a disease in an individual being treated and can be performed prophylactically or as a clinical intervention that can be performed during the clinical pathologic process. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathologic consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and relieving or improving the prognosis. In some aspects, the molecules of the invention are useful for delaying the development of a disease or for slowing the progression of a disease.

[0078] "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 aspects, the individual or subject is a human.

[0079] An "effective amount" of an agent (e.g., a pharmaceutical composition) is an amount capable of effectively achieving a desired therapeutic or prophylactic result at a required dosage for a required period of time.

[0080] The term "package insert" is used to refer to the instructions typically included in the commercial packaging of a therapeutic product that contain information regarding indications, usage, dosage, administration, combination therapies, contraindications, and / or warnings relating to the use of such therapeutic product.

[0081] Binding molecular pair

[0082] In a first aspect, the invention provides a binding molecular pair comprising

[0083] (a) a first binding molecule comprising (i) a first antigen-binding domain capable of binding to a target antigen, (ii) a first portion of an effector domain, and (iii) a first complementary domain capable of associating with the first portion of the effector domain; and

[0084] (b) a second binding molecule comprising (i) a second antigen-binding domain capable of binding to the target antigen, (ii) a second portion of the effector domain, and (iii) a second complementary domain capable of associating with the second portion of the effector domain;

[0085] wherein if the first antigen-binding domain and the second antigen-binding domain bind to their target antigen on the cell surface, the first and second portions of the effector domain are capable of associating with each other to form a functional effector domain,

[0086] Wherein the first complementary domain and the second complementary domain associate with the first part and the second part of the effector domain, respectively, and the first part and the second part of the effector domain do not associate with each other.

[0087] The binding molecule pairs described above and herein can bind, alone or in combination, any of the features described below (unless the context dictates otherwise).

[0088] Effector domain

[0089] According to the invention, each binding molecule in a binding molecule pair comprises a part of an effector domain, which part of the effector domain is capable of forming a functional effector domain if the antigen-binding domain of the binding molecule binds to its target antigen on the cell surface. The separate parts of the effector domain are not functional effector domains (i.e., the separate parts of the effector domain do not have the functions of a complete effector domain).

[0090] In some aspects, the effector domain is a dimer.

[0091] A functional effector domain can exert biological functions such as binding to an antigen, activating a cell signaling pathway, blocking a receptor, etc.

[0092] In some aspects, the effector domain is an antigen-binding domain. In certain aspects, the effector domain is an anti-CD3 antigen-binding domain (i.e., an antigen-binding domain capable of binding to CD3).

[0093] In some aspects, the functional effector domain is capable of binding to an antigen. In some aspects, the separate parts of the effector domain are not capable of binding to an antigen. In certain aspects, the antigen is a T cell antigen, particularly an activating T cell antigen. In even more certain aspects, the antigen is CD3, particularly CD3ε. In some aspects, the antigen is human CD3.

[0094] In some aspects, the first part of the effector domain comprises a heavy chain variable region (VH) and the second part of the effector domain comprises a light chain variable region (VL). In some aspects, the first part of the effector domain is a heavy chain variable region (VH) and the second part of the effector domain is a light chain variable region (VL). In some aspects, the first part of the effector domain consists of a heavy chain variable region (VH) and the second part of the effector domain consists of a light chain variable region (VL). In some aspects, the effector domain is an Fv molecule.

[0095] In some aspects, the effector domain is a humanized antigen-binding domain (i.e., the antigen-binding domain of a humanized antibody). In some aspects, the VH and / or VL of the effector domain are humanized variable regions. In some aspects, the VH and / or VL of the effector domain contain a receptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0096] In certain aspects, the VH of the effector domain contains 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 the VL of the effector domain contains 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 further aspects, the VH of the effector domain contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:18, and / or the VL of the effector domain contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:22.

[0097] In some aspects, the VH of the effector domain contains one or more heavy chain framework sequences (i.e., FR1, FR2, FR3 and / or FR4 sequences) of the heavy chain variable region sequence of SEQ ID NO:18. In some aspects, the VH of the effector domain contains 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 some aspects, the VH of the effector domain contains an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:18. In some aspects, the VH of the effector domain contains an amino acid sequence that is at least about 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 contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the antigen-binding domain containing the sequence retains the ability to bind to CD3. In certain aspects, in the amino acid sequence of SEQ ID NO:69, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted. In certain aspects, the substitutions, insertions or deletions occur in regions outside the CDRs (i.e., in the FRs). In some aspects, the VH of the effector domain contains the amino acid sequence of SEQ ID NO:18. Optionally, the VH of the effector domain contains the VL sequence of SEQ ID NO:18, including post-translational modifications of the sequence.

[0098] In some aspects, the VL of the effector domain comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of the light chain variable region sequence of SEQ ID NO:22. In some aspects, the VL of the effector 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 some aspects, the VL of the effector domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:22. In some aspects, the VL of the effector domain comprises an amino acid sequence that is at least about 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 relative to the reference sequence, but the antigen-binding domain comprising the sequence retains the ability to bind to CD3. In certain aspects, in the amino acid sequence of SEQ ID NO:22, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In some aspects, the VL of the effector domain comprises the amino acid sequence of SEQ ID NO:22. Optionally, the VL of the effector domain comprises the amino acid sequence of SEQ ID NO:22, including post-translational modifications of the sequence.

[0099] In some aspects, the VH of the effector 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 effector 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 some aspects, the VH of the effector domain comprises the amino acid sequence of SEQ ID NO:18 and the VL of the effector domain comprises the amino acid sequence of SEQ ID NO:22.

[0100] In some aspects, the effector domain is an antigen-binding domain that binds to CD3, and the antigen-binding domain comprises: a VH comprising the amino acid sequence of SEQ ID NO:18 and a VL comprising the amino acid sequence of SEQ ID NO:22. In some aspects, the effector domain is an antigen-binding domain that binds to CD3, and the antigen-binding domain comprises: the VH sequence of SEQ ID NO:18 and the VL sequence of SEQ ID NO:22.

[0101] In some aspects, the effector domain is an antigen-binding domain that binds to CD3, and the antigen-binding domain comprises: a VH comprising the heavy-chain CDR sequences of the VH of SEQ ID NO:18, and a VL comprising the light-chain CDR sequences of the VL of SEQ ID NO:22. In some aspects, the antigen-binding domain comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH of SEQ ID NO:18 and the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL of SEQ ID NO:22.

[0102] In some aspects, the VH of the effector domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO:18 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the framework sequence of the VH of SEQ ID NO:18. In some aspects, the VH of the effector domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO:18 and a framework having at least 95% sequence identity to the framework sequence of the VH of SEQ ID NO:18. In some aspects, the VH of the effector domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO:18 and a framework having at least 98% sequence identity to the framework sequence of the VH of SEQ ID NO:18.

[0103] In some aspects, the VL of the effector domain comprises the light-chain CDR sequences of the VL of SEQ ID NO:22 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the framework sequence of the VL of SEQ ID NO:22. In some aspects, the VL of the effector domain comprises the light-chain CDR sequences of the VL of SEQ ID NO:22 and a framework having at least 95% sequence identity to the framework sequence of the VL of SEQ ID NO:22. In some aspects, the VL of the effector domain comprises the light-chain CDR sequences of the VL of SEQ ID NO:22 and a framework having at least 98% sequence identity to the framework sequence of the VL of SEQ ID NO:22.

[0104] In some aspects, the effector domain is an antigen-binding domain that binds to CD3, and the antigen-binding domain comprises the VH sequence in any of the aspects provided above and the VL sequence in any of the aspects provided above.

[0105] In a further specific aspect, the VH of the effector domain comprises heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:23, HCDR 2 of SEQ ID NO:24 and HCDR 3 of SEQ ID NO:25 and the VL of the effector binding domain comprises light chain complementarity determining region (LCDR) 1 of SEQ ID NO:27, LCDR 2 of SEQ ID NO:28 and LCDR 3 of SEQ ID NO:29. In some aspects, the VH of the effector 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:26, and / or the VL of the effector 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:30.

[0106] In some aspects, the VH of the effector domain comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3 and / or FR4 sequences) of the heavy chain variable region sequence of SEQ ID NO:26. In some aspects, the VH of the effector 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:26. In some aspects, the VH of the effector domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:26.. In some aspects, the VH of the effector domain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:26. In certain aspects, VH sequences having at least 95%, 96%, 97%, 98% or 99% identity comprise substitutions (e.g., conservative substitutions), insertions or deletions relative to a reference sequence, but the antigen-binding domain comprising the sequence retains the ability to bind to CD3. In certain aspects, in the amino acid sequence of SEQ ID NO:26, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted. In certain aspects, the substitutions, insertions or deletions occur in regions outside the CDRs (i.e., in the FRs). In some aspects, the VH of the effector domain comprises the amino acid sequence of SEQ ID NO:26. Optionally, the VH of the effector domain comprises the VL sequence of SEQ ID NO:26, including post-translational modifications of the sequence.

[0107] In some aspects, the VL of the effector domain comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of the light chain variable region sequence of SEQ ID NO:30. In some aspects, the VL of the effector 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:30. In some aspects, the VL of the effector domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:30. In some aspects, the VL of the effector domain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:30. 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 relative to the reference sequence, but the antigen-binding domain comprising the sequence retains the ability to bind to CD3. In certain aspects, in the amino acid sequence of SEQ ID NO:30, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In some aspects, the VL of the effector domain comprises the amino acid sequence of SEQ ID NO:30. Optionally, the VL of the effector domain comprises the amino acid sequence of SEQ ID NO:30, including post-translational modifications of the sequence.

[0108] In some aspects, the VH of the effector 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:26 and the VL of the effector 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:30. In some aspects, the VH of the effector domain comprises the amino acid sequence of SEQ ID NO:26 and the VL of the effector domain comprises the amino acid sequence of SEQ ID NO:30.

[0109] In some aspects, the effector domain is an antigen-binding domain that binds to CD3, the antigen-binding domain comprising: a VH comprising the amino acid sequence of SEQ ID NO:26 and a VL comprising the amino acid sequence of SEQ ID NO:30. In some aspects, the effector domain is an antigen-binding domain that binds to CD3, the antigen-binding domain comprising: the VH sequence of SEQ IDNO:26 and the VL sequence of SEQ ID NO:30.

[0110] In some aspects, the effector domain is an antigen-binding domain that binds to CD3, and the antigen-binding domain comprises: a VH comprising the heavy-chain CDR sequences of the VH of SEQ ID NO:26, and a VL comprising the light-chain CDR sequences of the VL of SEQ ID NO:30. In some aspects, the antigen-binding domain comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH of SEQ ID NO:26 and the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL of SEQ ID NO:30.

[0111] In some aspects, the VH of the effector domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO:26 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the framework sequence of the VH of SEQ ID NO:26. In some aspects, the VH of the effector domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO:26 and a framework having at least 95% sequence identity to the framework sequence of the VH of SEQ ID NO:26. In some aspects, the VH of the effector domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO:26 and a framework having at least 98% sequence identity to the framework sequence of the VH of SEQ ID NO:26.

[0112] In some aspects, the VL of the effector domain comprises the light-chain CDR sequences of the VL of SEQ ID NO:30 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the framework sequence of the VL of SEQ ID NO:30. In some aspects, the VL of the effector domain comprises the light-chain CDR sequences of the VL of SEQ ID NO:30 and a framework having at least 95% sequence identity to the framework sequence of the VL of SEQ ID NO:30. In some aspects, the VL of the effector domain comprises the light-chain CDR sequences of the VL of SEQ ID NO:30 and a framework having at least 98% sequence identity to the framework sequence of the VL of SEQ ID NO:30.

[0113] In some aspects, the effector domain is an antigen-binding domain that binds to CD3, and the antigen-binding domain comprises the VH sequence in any of the aspects provided above and the VL sequence in any of the aspects provided above.

[0114] In yet other aspects, the VH of the effector domain comprises heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:31, HCDR 2 of SEQ ID NO:32, and HCDR 3 of SEQ ID NO:33 and the VL of the antigen binding domain comprises light chain complementarity determining region (LCDR) 1 of SEQ ID NO:35, LCDR 2 of SEQ ID NO:36, and LCDR 3 of SEQ ID NO:37. In some aspects, the VH of the effector 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:34, and / or the VL of the effector 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:38.

[0115] In some aspects, the VH of the effector domain comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of the heavy chain variable region sequence of SEQ ID NO:34. In some aspects, the VH of the effector 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:34. In some aspects, the VH of the effector domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:34. In some aspects, the VH of the effector domain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:34. In certain aspects, the VH sequences having at least 95%, 96%, 97%, 98%, or 99% identity comprise substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the antigen binding domain comprising the sequence retains the ability to bind to CD3. In certain aspects, in the amino acid sequence of SEQ ID NO:34, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In certain aspects, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., in the FRs). In some aspects, the VH of the effector domain comprises the amino acid sequence of SEQ ID NO:34. Optionally, the VH of the effector domain comprises the amino acid sequence of SEQ ID NO:34, including post-translational modifications of the sequence.

[0116] In some aspects, the VL of the effector domain comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of the light chain variable region sequence of SEQ ID NO:38. In some aspects, the VL of the effector 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:38. In some aspects, the VL of the effector domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:38. In some aspects, the VL of the effector domain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:38. 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 relative to the reference sequence, but the antigen-binding domain comprising the sequence retains the ability to bind to CD3. In certain aspects, in the amino acid sequence of SEQ ID NO:38, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In some aspects, the VL of the effector domain comprises the amino acid sequence of SEQ ID NO:38. Optionally, the VL of the effector domain comprises the amino acid sequence of SEQ ID NO:38, including post-translational modifications of the sequence.

[0117] In some aspects, the VH of the effector 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:34 and the VL of the effector 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:38. In some aspects, the VH of the effector domain comprises the amino acid sequence of SEQ ID NO:34 and the VL of the effector domain comprises the amino acid sequence of SEQ ID NO:38.

[0118] In some aspects, the effector domain is an antigen-binding domain that binds to CD3, and the antigen-binding domain comprises: a VH comprising the amino acid sequence of SEQ ID NO:34 and a VL comprising the amino acid sequence of SEQ ID NO:38. In some aspects, the effector domain is an antigen-binding domain that binds to CD3, and the antigen-binding domain comprises: the VH sequence of SEQ IDNO:34 and the VL sequence of SEQ ID NO:38.

[0119] In some aspects, the effector domain is an antigen-binding domain that binds to CD3, and the antigen-binding domain comprises: a VH comprising the heavy-chain CDR sequences of the VH of SEQ ID NO: 34, and a VL comprising the light-chain CDR sequences of the VL of SEQ ID NO: 38. In some aspects, the antigen-binding domain comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH of SEQ ID NO: 34 and the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL of SEQ ID NO: 38.

[0120] In some aspects, the VH of the effector domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO: 34 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the framework sequence of the VH of SEQ ID NO: 34. In some aspects, the VH of the effector domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO: 34 and a framework having at least 95% sequence identity to the framework sequence of the VH of SEQ ID NO: 34. In some aspects, the VH of the effector domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO: 34 and a framework having at least 98% sequence identity to the framework sequence of the VH of SEQ ID NO: 34.

[0121] In some aspects, the VL of the effector domain comprises the light-chain CDR sequences of the VL of SEQ ID NO: 38 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the framework sequence of the VL of SEQ ID NO: 38. In some aspects, the VL of the effector domain comprises the light-chain CDR sequences of the VL of SEQ ID NO: 38 and a framework having at least 95% sequence identity to the framework sequence of the VL of SEQ ID NO: 38. In some aspects, the VL of the effector domain comprises the light-chain CDR sequences of the VL of SEQ ID NO: 38 and a framework having at least 98% sequence identity to the framework sequence of the VL of SEQ ID NO: 38.

[0122] In some aspects, the effector domain is an antigen-binding domain that binds to CD3, particularly an Fv molecule, and the antigen-binding domain comprises the VH sequence in any of the aspects provided above and the VL sequence in any of the aspects provided above.

[0123] In other aspects, the VH of the effector domain comprises heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:23, HCDR 2 of SEQ ID NO:24, and HCDR 3 of SEQ ID NO:60 and the VL of the effector domain comprises light chain complementarity determining region (LCDR) 1 of SEQ ID NO:27, LCDR 2 of SEQ ID NO:28, and LCDR 3 of SEQ ID NO:29. In some aspects, the VH of the effector 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:61, and / or the VL of the effector 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:30.

[0124] In some aspects, the VH of the effector domain comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of the heavy chain variable region sequence of SEQ ID NO:61. In some aspects, the VH of the effector 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:61. In some aspects, the VH of the effector domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:61. In some aspects, the VH of the effector domain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:61. 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 relative to the reference sequence, but the antigen-binding domain comprising the sequence retains the ability to bind to CD3. In certain aspects, in the amino acid sequence of SEQ ID NO:61, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In some aspects, the VH of the effector domain comprises the amino acid sequence of SEQ ID NO:61. Optionally, the VH of the effector domain comprises the amino acid sequence of SEQ ID NO:61, including post-translational modifications of the sequence.

[0125] In some aspects, the VL of the effector domain comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of the light chain variable region sequence of SEQ ID NO:30. In some aspects, the VL of the effector 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:30. In some aspects, the VL of the effector domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:30. In some aspects, the VL of the effector domain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:30. 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 relative to the reference sequence, but the antigen-binding domain comprising the sequence retains the ability to bind to CD3. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in the amino acid sequence of SEQ ID NO:30. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In some aspects, the VL of the effector domain comprises the amino acid sequence of SEQ ID NO:30. Optionally, the VL of the effector domain comprises the amino acid sequence of SEQ ID NO:30, including post-translational modifications of the sequence.

[0126] In some aspects, the VH of the effector 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:61 and the VL of the effector 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:30. In some aspects, the VH of the effector domain comprises the amino acid sequence of SEQ ID NO:61 and the VL of the effector domain comprises the amino acid sequence of SEQ ID NO:30.

[0127] In some aspects, the effector domain is an antigen-binding domain that binds to CD3, the antigen-binding domain comprising: a VH comprising the amino acid sequence of SEQ ID NO:61 and a VL comprising the amino acid sequence of SEQ ID NO:30. In some aspects, the effector domain is an antigen-binding domain that binds to CD3, the antigen-binding domain comprising: the VH sequence of SEQ IDNO:61 and the VL sequence of SEQ ID NO:30.

[0128] In some aspects, the effector domain is an antigen-binding domain that binds to CD3, and the antigen-binding domain comprises: a VH comprising the heavy-chain CDR sequences of the VH of SEQ ID NO:61, and a VL comprising the light-chain CDR sequences of the VL of SEQ ID NO:30. In some aspects, the antigen-binding domain comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH of SEQ ID NO:61 and the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL of SEQ ID NO:30.

[0129] In some aspects, the VH of the effector domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO:61 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the framework sequence of the VH of SEQ ID NO:61. In some aspects, the VH of the effector domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO:61 and a framework having at least 95% sequence identity with the framework sequence of the VH of SEQ ID NO:61. In some aspects, the VH of the effector domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO:61 and a framework having at least 98% sequence identity with the framework sequence of the VH of SEQ ID NO:61.

[0130] In some aspects, the VL of the effector domain comprises the light-chain CDR sequences of the VL of SEQ ID NO:30 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the framework sequence of the VL of SEQ ID NO:30. In some aspects, the VL of the effector domain comprises the light-chain CDR sequences of the VL of SEQ ID NO:30 and a framework having at least 95% sequence identity with the framework sequence of the VL of SEQ ID NO:30. In some aspects, the VL of the effector domain comprises the light-chain CDR sequences of the VL of SEQ ID NO:30 and a framework having at least 98% sequence identity with the framework sequence of the VL of SEQ ID NO:30.

[0131] In some aspects, the effector domain is an antigen-binding domain that binds to CD3, and the antigen-binding domain comprises the VH sequence in any of the aspects provided above and the VL sequence in any of the aspects provided above.

[0132] Complementary domain

[0133] According to the present invention, each binding molecule of the binding molecule pair comprises a complementary domain that associates with a portion of the effector domain in such a binding molecule, while the portions of the effector domain do not associate with each other.

[0134] The complementary domain is complementary to the corresponding part of the effector domain, but does not form a functional effector domain with the corresponding part of the effector domain.

[0135] In some aspects, the first and second complementary domains cannot form a functional effector domain with parts of the effector domain. In some aspects, when associated with parts of the effector domain, the first and second complementary domains do not form a functional effector domain. In some aspects, when associated with each other, the first part of the effector domain and the first complementary domain and / or the second part of the effector domain and the second complementary domain do not form a functional effector domain.

[0136] The complementary domains can also be complementary to each other.

[0137] In some aspects, the first and second complementary domains are capable of associating with each other. In some aspects, if the first and second parts of the effector domain associate with each other, the first and second complementary domains associate with each other.

[0138] In some aspects, the first complementary domain contains VL and the second complementary domain contains VH. In some aspects, the first complementary domain is VL and the second complementary domain is VH. In some aspects, the first complementary domain consists of VL and the second complementary domain consists of VH.

[0139] In some aspects, the VH and VL of the complementary domains are non-antigen-binding (either individually (i.e., not associated with each other) or combinatorially (i.e., associated with each other, as in an antigen-binding domain)).

[0140] In some aspects, the VH of the second complementary domain contains heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:47, HCDR 2 of SEQ ID NO:48, and HCDR 3 of SEQ ID NO:49, and the VL of the first complementary domain contains light chain complementarity determining region (LCDR) 1 of SEQ ID NO:51, LCDR 2 of SEQ ID NO:52, and LCDR 3 of SEQ ID NO:53. In some aspects, the VH of the second complementary domain contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:50, and / or the VL of the first complementary domain contains an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:54.

[0141] In some aspects, the VH of the second complementary domain comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of the heavy chain variable region sequence of SEQ ID NO:50. In some aspects, the VH of the second complementary 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:50. In some aspects, the VH of the second complementary domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:50. In some aspects, the VH of the second complementary domain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:50. In some aspects, the VH of the second complementary domain comprises the amino acid sequence of SEQ ID NO:50. Optionally, the VH of the second complementary domain comprises the amino acid sequence of SEQ ID NO:50, including post-translational modifications of the sequence.

[0142] In some aspects, the VL of the first complementary domain comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of the light chain variable region sequence of SEQ ID NO:54. In some aspects, the VL of the first complementary 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:54. In some aspects, the VL of the first complementary domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:54. In some aspects, the VL of the first complementary domain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:54. In some aspects, the VL of the first complementary domain comprises the amino acid sequence of SEQ ID NO:54. Optionally, the VL of the first complementary domain comprises the amino acid sequence of SEQ ID NO:54, including post-translational modifications of the sequence.

[0143] In some aspects, the VH of the second complementary 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:50 and the VL of the first complementary 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:54. In some aspects, the VH of the second complementary domain comprises the amino acid sequence of SEQ ID NO:50 and the VL of the first complementary domain comprises the amino acid sequence of SEQ ID NO:54.

[0144] In some aspects, the first and second complementary domains are portions of a non-antigen-binding domain comprising VH and VL, where the VH comprises the amino acid sequence of SEQ ID NO:50, and the VL comprises the amino acid sequence of SEQ ID NO:54. In some aspects, the first and second complementary domains are portions of a non-antigen-binding domain that comprises the VH sequence of SEQ ID NO:50 and the VL sequence of SEQ ID NO:54.

[0145] In some aspects, the first and second complementary domains are portions of a non-antigen-binding domain comprising VH and VL, where the VH comprises the heavy-chain CDR sequences of the VH of SEQ ID NO:50, and the VL comprises the light-chain CDR sequences of the VL of SEQ ID NO:54. In some aspects, the non-antigen-binding domain comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of the VH of SEQ ID NO:50 and the LCDR1, LCDR2, and LCDR3 amino acid sequences of the VL of SEQ ID NO:54.

[0146] In some aspects, the VH of the second complementary domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO:50 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the framework sequence of the VH of SEQ ID NO:50. In some aspects, the VH of the second complementary domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO:50 and a framework having at least 95% sequence identity to the framework sequence of the VH of SEQ ID NO:50. In some aspects, the VH of the second complementary domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO:50 and a framework having at least 98% sequence identity to the framework sequence of the VH of SEQ ID NO:50.

[0147] In some aspects, the VL of the first complementary domain comprises the light-chain CDR sequences of the VL of SEQ ID NO:54 and a framework having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the framework sequence of the VL of SEQ ID NO:54. In some aspects, the VL of the first complementary domain comprises the light-chain CDR sequences of the VL of SEQ ID NO:54 and a framework having at least 95% sequence identity to the framework sequence of the VL of SEQ ID NO:54. In some aspects, the VL of the first complementary domain comprises the light-chain CDR sequences of the VL of SEQ ID NO:54 and a framework having at least 98% sequence identity to the framework sequence of the VL of SEQ ID NO:54.

[0148] In some aspects, the first and second complementary domains are part of a non-antigen-binding domain that includes a VH sequence as provided in any of the aspects above and a VL sequence as provided in any of the aspects above.

[0149] In some aspects, the first part of the effector domain includes a first VH and the first complementary domain includes a first VL, and the second part of the effector domain includes a second VL and the second complementary domain includes a second VH. In some aspects, the first part of the effector domain is the first VH and the first complementary domain is the first VL, and the second part of the effector domain is the second VL and the second complementary domain is the second VH. In some aspects, the first part of the effector domain consists of the first VH and the first complementary domain consists of the first VL, and the second part of the effector domain consists of the second VL and the second complementary domain consists of the second VH.

[0150] In some aspects, the first part of the effector domain and the first complementary domain and / or the second part of the effector domain and the second complementary domain are fused to each other. In some aspects, the first part of the effector domain is fused to the N-terminus of the first complementary domain at its C-terminus, and / or the second part of the effector domain is fused to the N-terminus of the second complementary domain at its C-terminus.

[0151] Charged residue

[0152] Portions of the complementary domain and / or effector domain may include amino acid substitutions, particularly substitutions by charged amino acid residues, which modulate the affinity between portions of the complementary domain and / or effector domain. For example, introducing amino acid residues of opposite charge in two complementary domains will increase the affinity of the two complementary domains for each other (and thus increase the strength and propensity of the two complementary domains to associate with each other). Conversely, introducing amino acid residues of the same charge in two complementary domains will decrease the affinity of the two complementary domains for each other (and thus decrease the strength and propensity of the two complementary domains to associate with each other). The same can be analogously applied to the two parts of the effector domain.

[0153] In some aspects, each of the second VH and the first VL and / or each of the first VH and the second VL, particularly each of the second VH and the first VL, according to the aspects above, includes an amino acid substitution, wherein the amino acid residue is substituted with a charged replacement amino acid residue, where (i) the replacement amino acid residues in the VH and VL have opposite charges, or (ii) the replacement amino acid residues in the VH and VL have the same charge.

[0154] In another aspect, each of the first VH and first VL and / or each of the second VH and second VL contains an amino acid substitution, wherein the amino acid residue is substituted with a charged replacement amino acid residue, wherein (i) the replacement amino acid residues in VH and VL have opposite charges, or (ii) the replacement amino acid residues in VH and VL have the same charge.

[0155] In some aspects, (i) the replacement amino acid residue in VH is a positively charged amino acid residue and the replacement amino acid residue in VL is a negatively charged amino acid residue, or the replacement amino acid residue in VH is a negatively charged amino acid residue and the replacement amino acid residue in VL is a positively charged amino acid residue, or (ii) the replacement amino acid residues in VH and VL are each a positively charged amino acid residue, or the replacement amino acid residues in VH and VL are each a negatively charged amino acid residue. In some aspects, the positively charged amino acid residue is lysine (K), arginine (R), or histidine (H), particularly lysine (K) or arginine (R), and most particularly lysine (K). In some aspects, the negatively charged amino acid residue is glutamic acid (E) or aspartic acid (D), particularly glutamic acid (E).

[0156] In some aspects, the amino acid substitution is in the framework regions of VH and VL.

[0157] In some aspects, the amino acid substitution is at a position at the interface between VH and VL (when associated with each other).

[0158] In some aspects, the amino acid substitution is at position 39 of VH and position 38 of VL (according to Kabat EU index numbering).

[0159] In some aspects, the amino acid substitution in VH is Q39K or Q39E, and / or the amino acid substitution in VL is Q38K or Q38E (according to Kabat EU index numbering).

[0160] In some aspects, the amino acid substitution in VH is Q39K and the amino acid substitution in VL is Q38E, the amino acid substitution in VH is Q39E and the amino acid substitution in VL is Q38K, the amino acid substitution in VH is Q39K and the amino acid substitution in VL is Q38K, or the amino acid substitution in VH is Q39E and the amino acid substitution in VL is Q38E.

[0161] Suitable amino acid substitutions are also described, for example, in Igawa et al. (Prot Eng Des Sel (2010) 23, 667 - 677) or European Patent Application EP1870459 (A1) (incorporated herein by reference in its entirety).

[0162] Antigen-binding domain

[0163] According to the present invention, each binding molecule in the binding molecule pair comprises an antigen-binding domain that is capable of binding to one or more target antigens. The binding of the binding molecule to its target antigen on the cell surface via its antigen-binding domain allows the two parts of the effector domain to associate with each other and form a functional effector domain.

[0164] Without wishing to be bound by theory, if the two parts of the effector domain are close enough to each other (by the binding of the binding molecule to its target antigen on the cell surface), then they will dissociate from their respective complementary domains and associate with each other to form a functional effector domain.

[0165] The binding of each binding molecule can be monovalent (i.e., a binding molecule that contains only a single antigen-binding domain) or multivalent, e.g., divalent (i.e., a binding molecule that contains more than one, e.g., two, antigen-binding domains).

[0166] Each binding molecule can be monospecific (i.e., all binding domains of the binding molecule bind to the same target antigen) or multispecific, e.g., bispecific (i.e., at least one antigen-binding domain of the binding molecule binds to one target antigen and at least one antigen-binding domain of the binding molecule binds to a different target antigen).

[0167] Furthermore, the binding molecules included in the binding molecule pair according to the present invention can have the same or different binding specificities (i.e., the two binding molecules bind to the same target antigen or the two binding molecules bind to different target antigens).

[0168] In some aspects, the first binding molecule comprises a third antigen-binding domain that is capable of binding to a target antigen, and / or the second binding molecule comprises a fourth antigen-binding domain that is capable of binding to a target antigen.

[0169] In some aspects, each binding molecule comprises a single antigen-binding domain that is capable of binding to a target antigen. In other aspects, each binding molecule comprises two antigen-binding domains that are capable of binding to a target antigen.

[0170] In some aspects, the first, second, third (when present) and / or fourth (when present) antigen-binding domains are antigen-binding domains selected from the group consisting of: Fv molecules, scFv molecules, Fab molecules, and single-domain antibodies.

[0171] In some aspects, the first, second, third (when present) and / or fourth (when present) antigen-binding domains are Fab molecules. In some aspects, the first, second, third (when present) and fourth (when present) antigen-binding domains are each Fab molecules.

[0172] In some aspects, each binding molecule comprises a single antigen-binding domain capable of binding to a target antigen, wherein the antigen-binding domain is a Fab molecule. In other aspects, each binding molecule comprises two antigen-binding domains capable of binding to a target antigen, wherein the antigen-binding domains are Fab molecules.

[0173] In some aspects, the first and second antigen-binding domains bind to the same target antigen. In some aspects, the first, second, third (when present) and fourth (when present) antigen-binding domains bind to the same target antigen.

[0174] In some aspects, the first and second antigen-binding domains bind to different target antigens. In some aspects, the first, second, third (when present) and fourth (when present) antigen-binding domains bind to different target antigens.

[0175] In some aspects, the first and second antigen-binding domains bind to the same target antigen, and the third (when present) and fourth (when present) antigen-binding domains bind to the same target antigen, wherein the target antigen bound by the first and second antigen-binding domains is different from the target antigen bound by the third (when present) and fourth (when present) antigen-binding domains.

[0176] In certain aspects, the first and third (when present) antigen-binding domains bind to the same target antigen, and the second and fourth (when present) antigen-binding domains bind to the same target antigen, wherein the target antigen bound by the first and third (when present) antigen-binding domains is different from the target antigen bound by the second and fourth (when present) antigen-binding domains.

[0177] In some aspects, the effector domain is an antigen-binding domain, and the target antigens of the first, second, third (when present) and fourth (when present) antigen-binding domains are different from the antigen bound by the effector domain (i.e., the antigen that a functional effector domain can bind to, such as CD3). In some aspects, the effector domain is an antigen-binding domain, and the antigen-binding domains of the first and second binding molecules cannot bind to the antigen that the effector domain binds to (i.e., the antigen that a functional effector domain can bind to, such as CD3).

[0178] In some aspects, the target antigens of the first, second, third (when present) and fourth (when present) antigen-binding domains are not CD3, particularly not human CD3. In some aspects, the antigen-binding domains of the first and second binding molecules cannot bind to CD3, particularly not to human CD3.

[0179] In some aspects, the target antigens of the first, second, third (when present) and fourth (when present) antigen-binding domains are expressed on the same cell, on adjacent cells, or on cells in the same tissue (i.e., cells that are very close to each other, if not the same cell). In some aspects, the target antigens of the first, second, third (when present) and fourth (when present) antigen-binding domains are expressed on the same cell.

[0180] In some aspects, the cell is a tumor cell. (e.g., a cancer cell or a cell of the tumor stroma, particularly a cancer cell). In some aspects, the target antigens of the first, second, third (when present) and fourth (when present) antigen-binding domains are tumor antigens.

[0181] An exemplary target antigen is HER2.

[0182] In some aspects, the target antigen of the first, second, third (when present) and / or fourth (when present) antigen-binding domain is HER2. In some aspects, the target antigens of the first and second antigen-binding domains are HER2. In some aspects, the target antigens of the first, second, third (when present) and fourth (when present) antigen-binding domains are HER2.

[0183] In some aspects, the first, second, third (when present) and / or fourth (when present) antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0184] In some aspects, the antigen-binding domain is a humanized antigen-binding domain (i.e., the antigen-binding domain of a humanized antibody). In some aspects, the VH and / or VL of the antigen-binding domain are humanized variable regions. In some aspects, the VH and / or VL of the antigen-binding domain comprise a recipient human framework, such as a human immunoglobulin framework or a human consensus framework.

[0185] The following aspects relate to the VH and VL of the first, second, third (when present) and / or fourth (when present) antigen-binding domain, wherein the antigen-binding domain is capable of binding to HER2 (i.e., the target antigen of the antigen-binding domain is HER2).

[0186] In some aspects, the VH of the antigen-binding domain comprises heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:39, HCDR 2 of SEQ ID NO:40 and HCDR 3 of SEQ ID NO:41 and the VL of the antigen-binding domain comprises light chain complementarity determining region (LCDR) 1 of SEQ ID NO:43, LCDR 2 of SEQ ID NO:44 and LCDR 3 of SEQ ID NO:45. In further aspects, the VH of the 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:42, and / or the VL of the 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:46.

[0187] In some aspects, the VH of the antigen-binding domain comprises one or more heavy chain framework sequences (i.e., FR1, FR2, FR3 and / or FR4 sequences) of the heavy chain variable region sequence of SEQ ID NO:42. In some aspects, the VH of the 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:42. In some aspects, the VH of the antigen-binding domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:42. In some aspects, the VH of the antigen-binding domain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:42. 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 relative to the reference sequence, but the antigen-binding domain comprising the sequence retains the ability to bind to HER2. In certain aspects, in the amino acid sequence of SEQ ID NO:42, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted. In certain aspects, the substitutions, insertions or deletions occur in regions outside the CDRs (i.e., in the FRs). In some aspects, the VH of the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:42. Optionally, the VH of the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:42, including post-translational modifications of the sequence.

[0188] In some aspects, the VL of the antigen-binding domain comprises one or more light chain framework sequences (i.e., FR1, FR2, FR3, and / or FR4 sequences) of the light chain variable region sequence of SEQ ID NO:46. In some aspects, the VL of the 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:46. In some aspects, the VL of the antigen-binding domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO:46. In some aspects, the VL of the antigen-binding domain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO:46. 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 relative to the reference sequence, but the antigen-binding domain comprising the sequence retains the ability to bind to HER2. In certain aspects, in the amino acid sequence of SEQ ID NO:46, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In some aspects, the VL of the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:46. Optionally, the VL of the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:42, including post-translational modifications of the sequence.

[0189] In some aspects, the VH of the 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:42 and / or the VL of the 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:46. In some aspects, the VH of the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:42 and the VL of the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:46.

[0190] In some aspects, the first, second, third (when present) and / or fourth (when present) antigen-binding domains are antigen-binding domains that bind to HER2, the antigen-binding domain comprising a VH and a VL, the VH comprising the amino acid sequence of SEQ ID NO:42 and the VL comprising the amino acid sequence of SEQ ID NO:46. In some aspects, the first, second, third (when present) and / or fourth (when present) antigen-binding domains are antigen-binding domains that bind to HER2, the antigen-binding domain comprising the VH sequence of SEQ ID NO:42 and the VL sequence of SEQ ID NO:46.

[0191] In some aspects, the first, second, third (when present) and / or fourth (when present) antigen-binding domains are antigen-binding domains that bind to HER2, the antigen-binding domain comprising a VH and a VL, the VH comprising the heavy-chain CDR sequences of SEQ ID NO: 42 and a VL comprising the light-chain CDR sequences of a VL of SEQ ID NO: 46. In some aspects, the antigen-binding domain comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NO: 42 and the LCDR1, LCDR2 and LCDR3 amino acid sequences of the VL of SEQ ID NO: 46.

[0192] In some aspects, the VH of the antigen-binding domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO: 42 and a framework having at least 95%, 96%, 97%, 98% or 99% sequence identity to the framework sequences of the VH of SEQ ID NO: 42. In some aspects, the VH of the antigen-binding domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO: 42 and a framework having at least 95% sequence identity to the framework sequences of the VH of SEQ ID NO: 42. In some aspects, the VH of the antigen-binding domain comprises the heavy-chain CDR sequences of the VH of SEQ ID NO: 42 and a framework having at least 98% sequence identity to the framework sequences of the VH of SEQ ID NO: 42.

[0193] In some aspects, the VL of the antigen-binding domain comprises the light-chain CDR sequences of the VL of SEQ ID NO: 46 and a framework having at least 95%, 96%, 97%, 98% or 99% sequence identity to the framework sequences of the VL of SEQ ID NO: 46. In some aspects, the VL of the antigen-binding domain comprises the light-chain CDR sequences of the VL of SEQ ID NO: 46 and a framework having at least 95% sequence identity to the framework sequences of the VL of SEQ ID NO: 46. In some aspects, the VL of the antigen-binding domain comprises the light-chain CDR sequences of the VL of SEQ ID NO: 46 and a framework having at least 98% sequence identity to the framework sequences of the VL of SEQ ID NO: 46.

[0194] In some aspects, the first, second, third (when present) and / or fourth (when present) antigen-binding domains are antigen-binding domains that bind to HER2, particularly Fab molecules, the antigen-binding domain comprising the VH sequences in any of the aspects provided above and the VL sequences in any of the aspects provided above.

[0195] In some aspects, the first, second, third (when present) and fourth (when present) antigen-binding domains are each an antigen-binding domain that binds to HER2, particularly a Fab molecule, and the antigen-binding domain comprises a VH sequence in any of the aspects provided above and a VL sequence in any of the aspects provided above.

[0196] Fc domain

[0197] According to the invention, the first and / or second binding molecule may comprise an Fc domain.

[0198] The Fc domain of the binding molecule consists of a pair of polypeptide chains comprising the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, and each subunit of the dimer comprises CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stably associating with each other.

[0199] In some aspects, the first binding molecule comprises a first Fc domain consisting of a first and a second subunit, and / or the second binding molecule comprises a second Fc domain consisting of a first and a second subunit. In some aspects, the first binding molecule comprises a first Fc domain consisting of a first and a second subunit, and the second binding molecule comprises a second Fc domain consisting of a first and a second subunit. In some aspects, each binding molecule comprises no more than one Fc domain.

[0200] In some aspects, the Fc domain of the first and / or second is an IgG Fc domain. In certain aspects, the Fc domain is an IgG1 Fc domain. In other aspects, the Fc domain is an IgG4 Fc domain. In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution (particularly the amino acid substitution S228P) at position S228 (Kabat EU index number). This amino acid substitution reduces Fab arm exchange in vivo of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further aspect, the Fc domain is a human Fc domain. In certain aspects, the Fc domain is a human IgG1 Fc domain. An exemplary sequence of the human IgG1 Fc region is given in SEQ ID NO:59.

[0201] In some aspects, the Fc domain comprises a modification that promotes the association of the first and second subunits of the Fc domain. Fc domain modifications that promote heterodimerization are further described below.

[0202] In some aspects, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector functions. Modifications of the Fc domain that reduce Fc receptor binding and / or effector functions are further described below.

[0203] In some aspects, the first and third (when present) antigen-binding domains are each fused to a subunit of the first Fc domain, and / or the second and fourth (when present) antigen-binding domains are each fused to a subunit of the second Fc domain. In some aspects, the first and third (when present) antigen-binding domains are each fused at their C-terminus to the N-terminus of a subunit of the first Fc domain, and / or the second and fourth (when present) antigen-binding domains are each fused at their C-terminus to the N-terminus of a subunit of the second Fc domain.

[0204] a) Fc domain modifications that promote heterodimerization

[0205] The binding molecule according to the invention comprises a part of an effector domain, a complementary domain, and one or more antigen-binding domains that can be fused to one or the other of the two subunits of the Fc domain, so that the two subunits of the Fc domain are generally contained in two different polypeptide chains. The recombinant co-expression of these polypeptides and subsequent dimerization result in several possible combinations of the two polypeptides. In order to increase the yield and purity of the binding molecule in recombinant production, it would therefore be advantageous to introduce modifications in the Fc domain of the binding molecule that promote the association of the desired polypeptides.

[0206] Thus, in certain aspects, the Fc domain of the binding molecule according to the invention comprises modifications that promote the association of the first and second subunits of the Fc domain. The most extensive protein-protein interaction sites between the two subunits of the human IgG Fc domain are in the CH3 domain of the Fc domain. Thus, in some aspects, the modifications are in the CH3 domain of the Fc domain.

[0207] There are several methods for modifying the CH3 domain of the Fc domain to effect heterodimerization, which are described in detail, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO2013157954, WO 2013096291. Generally, in all such methods, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are engineered in a complementary manner such that each CH3 domain (or the heavy chain containing it) can no longer homodimerize with itself, but is forced to heterodimerize with the other CH3 domain that is complementarily engineered (such that the first and second CH3 domains heterodimerize and no homodimers are formed between two first or two second CH3 domains).

[0208] In a specific aspect, the modification that promotes the association of the first and second subunits of the Fc domain is the so-called "knob-into-hole" modification, which includes a "knob" modification in one subunit of the two subunits of the Fc domain and a "hole" modification in the other subunit of the two subunits of the Fc domain.

[0209] The 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). Generally, this method involves introducing a protrusion ("knob") at the interface of the first polypeptide and introducing a corresponding cavity ("hole") in the interface of the second polypeptide such that the protrusion can be positioned in the cavity to facilitate the formation of heterodimers and impede the formation of homodimers. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (such as tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (such as alanine or threonine).

[0210] Thus, in a preferred aspect, in the CH3 domain of the first subunit of the Fc domain of the binding molecule, amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit, which protrusion can be positioned within a cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit, and the protrusion within the CH3 domain of the first subunit can be positioned within this cavity.

[0211] Preferably, the amino acid residues having a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0212] Preferably, the amino acid residues having a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0213] The protrusion and the cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis.

[0214] In a specific aspect, in the CH3 domain of the first subunit ("pestle" subunit) of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit ("mortar" subunit) of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In some aspects, in the second subunit of the Fc domain, additionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (according to the Kabat EU index number).

[0215] In a further aspect, in the first subunit of the Fc domain, additionally, the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamate residue at position 356 is replaced with a cysteine residue (E356C) (particularly, the serine residue at position 354 is replaced with a cysteine residue), and in the second subunit of the Fc domain, additionally, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (according to the EU index number of Kabat). Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0216] In certain aspects, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (according to Kabat EU index numbering).

[0217] Other CH3 modification techniques for implementing heterodimerization are envisioned as alternatives to the present invention and are described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO2013 / 157954, WO 2013 / 096291.

[0218] In some aspects, the heterodimerization method described in EP 1870459 is alternatively used. This method is based on introducing charged amino acids with opposite charges at specific amino acid positions at the CH3 / CH3 domain interface between the two subunits of the Fc domain. A particular aspect of the binding molecule of the present invention is the amino acid mutation R409D; K370E and the amino acid mutation D399K in one of the two CH3 domains (of the Fc domain); E357K in the other CH3 domain of the Fc domain (according to Kabat EU index numbering).

[0219] In some aspects, the binding molecule of the present 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 mutation R409D; K370E and the amino acid mutation D399K in the CH3 domain of the first subunit of the Fc domain; E357K in the CH3 domain of the second subunit of the Fc domain (according to Kabat EU index numbering).

[0220] In some aspects, the binding molecule of the present 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 binding molecule comprises the amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain and additionally the amino acid mutation R409D; K370E and the amino acid mutation D399K in the CH3 domain of the first subunit of the Fc domain; E357K in the CH3 domain of the second subunit of the Fc domain (all according to the Kabat EU index number).

[0221] In some aspects, alternatively, the heterodimerization method described in WO 2013 / 157953 can be used. In some aspects, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (according to the Kabat EU index number). In a further aspect, the first CH3 domain comprises a further amino acid mutation L351K. In a further aspect, the second CH3 domain comprises a further amino acid mutation selected from Y349E, Y349D and L368E (especially L368E) (according to the Kabat EU index number).

[0222] In some aspects, the heterodimerization method described in WO 2012 / 058768 can alternatively be used. In some aspects, the first CH3 domain contains the amino acid mutations L351Y, Y407A and the second CH3 domain contains the amino acid mutations T366A, K409F. In a further aspect, the second CH3 domain contains further amino acid mutations at positions 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, f) K392V, K392M, K392R, K392L, K392F or K392E (according to Kabat EU index number). In a further aspect, the first CH3 domain contains the amino acid mutations L351Y, Y407A and the second CH3 domain contains the amino acid mutations T366V, K409F. In a further aspect, the first CH3 domain contains the amino acid mutation Y407A and the second CH3 domain contains the amino acid mutations T366A, K409F. In a further aspect, the second CH3 domain further contains the amino acid mutations K392E, T411E, D399R and S400R (according to Kabat EU index number).

[0223] In some aspects, the heterodimerization method described in WO 2011 / 143545 can alternatively be used, for example, for amino acid modifications at positions selected from the group consisting of 368 and 409 (according to Kabat EU index number).

[0224] In some aspects, the heterodimerization method described in WO 2011 / 090762 can alternatively be used, which also uses the above-mentioned stud-and-mortar structure technology. In some aspects, the first CH3 domain contains the amino acid mutation T366W and the second CH3 domain contains the amino acid mutation Y407A. In some aspects, the first CH3 domain contains the amino acid mutation T366Y and the second CH3 domain contains the amino acid mutation Y407T (according to Kabat EU index number).

[0225] In some aspects, the binding molecule or its Fc domain is of the IgG2 subclass and the heterodimerization method described in WO 2010 / 129304 can alternatively be used.

[0226] In an alternative aspect, the modifications that promote the association of the first and second subunits of the Fc domain include modifications that mediate an electrostatic steering effect, such as those described in PCT Publication WO 2009 / 089004. Generally, the method involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues such that homodimer formation becomes electrostatically unfavorable, but heterodimerization is electrostatically favorable. In some such aspects, the first CH3 domain contains an amino acid substitution of K392 or N392 with a negatively charged amino acid (such as glutamic acid (E) or aspartic acid (D), particularly K392D or N392D), and the second CH3 domain contains an amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (such as lysine (K) or arginine (R), particularly D399K, E356K, D356K, or E357K, and more particularly D399K and E356K). In a further aspect, the first CH3 domain further contains an amino acid substitution of K409 or R409 with a negatively charged amino acid (such as glutamic acid (E) or aspartic acid (D), particularly K409D or R409D). In a further aspect, the first CH3 domain further or alternatively contains an amino acid substitution of K439 and / or K370 with a negatively charged amino acid, (such as glutamic acid (E) or aspartic acid (D)) (all according to the Kabat EU index number).

[0227] In some aspects, the heterodimerization method described in WO 2007 / 147901 can alternatively be used. In some aspects, the first CH3 domain contains the amino acid mutations K253E, D282K, and K322D and the second CH3 domain contains the amino acid mutations D239K, E240K, and K292D (according to the Kabat EU index number).

[0228] In some aspects, the heterodimerization method described in WO 2007 / 110205 can alternatively be used.

[0229] In some aspects, the first subunit of the Fc domain contains the amino acid substitutions K392D and K409D and the second subunit of the Fc domain contains the amino acid substitutions D356K and D399K (according to the Kabat EU index number).

[0230] b) Fc domain modifications that reduce Fc receptor binding and / or effector function

[0231] The Fc domain confers favorable pharmacokinetic properties to the binding molecule, including a long serum half-life that contributes to good accumulation in target tissues and a favorable tissue-blood distribution ratio. However, at the same time, it may lead to the unwanted targeting of the binding molecule to cells expressing Fc receptors rather than the preferred antigen-carrying cells. In addition, co-activation of the Fc receptor signaling pathway may lead to cytokine release, which may result in over-activation of cytokine receptors and severe side effects upon systemic administration. Activation of immune cells other than T cells (carrying Fc receptors), due to potential destruction of T cells (e.g., by NK cells), may even reduce the efficacy of the binding molecule.

[0232] Thus, in certain aspects, compared to the native IgG1 Fc domain, the Fc domain of the binding molecule according to the present invention exhibits a reduced binding affinity for Fc receptors and / or reduced effector functions. In some such aspects, the Fc domain (or the binding molecule comprising the Fc domain) exhibits a binding affinity for Fc receptors that is less than 50%, particularly less than 20%, more particularly less than 10% and most particularly less than 5% compared to the native IgG1 Fc domain (or the binding molecule comprising the native IgG1 Fc domain), and / or effector functions that are less than 50%, particularly less than 20%, more particularly less than 10% and most particularly less than 5% compared to the native IgG1 Fc domain domain (or the binding molecule comprising the native IgG1 Fc domain). In some aspects, the Fc domain domain (or the binding molecule comprising the Fc domain) essentially does not bind to Fc receptors and / or induce effector functions. In certain aspects, the Fc receptor is an Fcγ receptor. In some aspects, the Fc receptor is a human Fc receptor. In some aspects, the Fc receptor is an activating Fc receptor. In specific aspects, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, and most specifically human FcγRIIIa. In some aspects, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP and cytokine secretion. In certain aspects, the effector function is ADCC. In some aspects, the Fc domain domain exhibits a substantially similar binding affinity for the neonatal Fc receptor (FcRn) compared to the native IgG1 Fc domain domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the binding molecule comprising the Fc domain) exhibits a binding affinity for FcRn that is greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of that of the native IgG1 Fc domain (or the binding molecule comprising the native IgG1 Fc domain).

[0233] In some aspects, the Fc domain is engineered to have a reduced binding affinity for Fc receptors and / or reduced effector function compared to a non-engineered Fc domain. In certain aspects, the Fc domain of the binding molecule comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain for Fc receptors 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 some aspects, the amino acid mutation reduces the binding affinity of the Fc domain for Fc receptors. In some aspects, the amino acid mutation reduces the binding affinity of the Fc domain for Fc receptors by at least 2-fold, at least 5-fold, or at least 10-fold. In aspects where there are more than one amino acid mutation that reduces the binding affinity of the Fc domain for Fc receptors, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain for Fc receptors by at least 10-fold, at least 20-fold, or even at least 50-fold. In some aspects, compared to a binding molecule comprising a non-engineered Fc domain, a binding molecule comprising an engineered Fc domain exhibits a binding affinity for Fc receptors of less than 20%, particularly less than 10%, more particularly less than 5%. In certain aspects, the Fc receptor is an Fcγ receptor. In some aspects, the Fc receptor is a human Fc receptor. In some aspects, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, and most specifically human FcγRIIIa. Preferably, the binding to each of these receptors is reduced. In some aspects, the binding affinity for a complementary component, particularly the specific binding affinity for C1q, is also reduced. In some aspects, the binding affinity for the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn is achieved when the Fc domain (or the binding molecule comprising the Fc domain) exhibits a binding affinity for FcRn that is greater than about 70% of that of the Fc domain in non-engineered form (or the binding molecule comprising the non-engineered form of the Fc domain), i.e., the binding affinity of the Fc domain for the receptor is maintained. The Fc domain or the binding molecule of the invention comprising the Fc domain can exhibit a binding affinity greater than about 80% or even greater than about 90% of this affinity. In certain aspects, the Fc domain of the binding molecule is engineered to have reduced effector function compared to a non-engineered Fc domain.Reduced effector function can 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 cell phagocytosis (ADCP), reduced cytokine secretion, reduced uptake of antigen by immune complex-mediated antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signal transduction-induced apoptosis, reduced cross-linking of target-binding antibodies, reduced dendritic cell maturation, or reduced T cell sensitization. In some aspects, the reduced effector function is one or more of the group consisting of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In certain aspects, the reduced effector function is reduced ADCC. In some aspects, the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or binding molecule comprising a non-engineered Fc domain).

[0234] In some aspects, amino acid mutations that reduce the binding affinity of the Fc domain for Fc receptors and / or effector functions are amino acid substitutions. In some aspects, the Fc domain comprises an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331, and P329 (according to Kabat EU index numbering). In a more specific aspect, the Fc domain comprises an amino acid substitution at a position selected from the group consisting of L234, L235, and P329 (according to Kabat EU index numbering). In some aspects, the Fc domain comprises the amino acid substitutions L234A and L235A (according to Kabat EU index numbering). In some such aspects, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In some aspects, the Fc domain comprises an amino acid substitution at position P329. In a more specific aspect, the amino acid substitution is P329A or P329G, particularly P329G (according to Kabat EU index numbering). In some aspects, the Fc domain comprises an amino acid substitution at position P329 and additional amino acid substitutions at positions selected from E233, L234, L235, N297, and P331 (according to Kabat EU index numbering). In a more specific aspect, the additional amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In certain aspects, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235 (according to Kabat EU index numbering). In a more specific aspect, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329GLALA", "PGLALA", or "LALAPG"). Specifically, in certain aspects, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (according to Kabat EU index numbering).

[0235] In some such aspects, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The amino acid substitution combination of "P329G LALA" almost completely eliminates the binding of the human IgG1 Fc domain to Fcγ receptors (as well as complement), as described in PCT Publication No. WO 2012 / 130831, the entire content of which is incorporated herein by reference. WO2012 / 130831 also describes methods for preparing such mutant Fc domains and methods for determining their properties such as Fc receptor binding or effector function.

[0236] Compared with IgG1 antibodies, IgG4 antibodies exhibit reduced binding affinity for Fc receptors and reduced effector function. Thus, in some aspects, the Fc domain of the binding molecule of the present invention is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In some aspects, the IgG4 Fc domain comprises an amino acid substitution at position S228, particularly the amino acid substitution S228P (according to the Kabat EU index number). To further reduce its binding affinity for Fc receptors and / or its effector function, in some aspects, the IgG4 Fc domain comprises an amino acid substitution at position L235, particularly the amino acid substitution L235E (according to the Kabat EU index number). In some aspects, the IgG4 Fc domain comprises an amino acid substitution at position P329, particularly the amino acid substitution P329G (according to the Kabat EU index number). In a preferred aspect, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235, and P329, particularly the amino acid substitutions S228P, L235E, and P329G (according to the Kabat EU index number). Such IgG4 Fc domain mutants and their Fcγ receptor binding properties are described in PCT Publication No. WO 2012 / 130831, the entire content of which is incorporated herein by reference.

[0237] In certain aspects, an Fc domain that exhibits reduced binding affinity for Fc receptors and / or reduced effector function compared to the native IgG1 Fc domain is a human IgG1 Fc domain comprising the amino acid substitutions L234A, L235A, and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E, and optionally P329G (according to the Kabat EU index number).

[0238] In some aspects, the N-glycosylation of the Fc domain has been eliminated. In some such aspects, the Fc domain contains an amino acid mutation at position N297, in particular an amino acid substitution replacing asparagine with alanine (N297A) or aspartic acid (N297D) (according to the Kabat EU index numbering).

[0239] In addition to the Fc domains described above and in PCT Publication No. WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or reduced effector function also include those Fc domains having substitutions in one or more of residues 238, 265, 269, 270, 297, 327, and 329 of the Fc domain (U.S. Patent No. 6,737,056) (according to the EU index numbering of Kabat). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0240] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis of the coding DNA sequence, PCR, gene synthesis, etc. Correct nucleotide changes can be verified, for example, by sequencing.

[0241] Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors can be obtained, for example, by recombinant expression. Alternatively, cell lines known to express specific Fc receptors, such as human NK cells expressing the FcγIIIa receptor, can be used to evaluate the binding affinity of an Fc domain or a binding molecule comprising an Fc domain for Fc receptors.

[0242] The effector functions of an Fc domain or a binding molecule comprising an Fc domain can be measured by methods known in the art. Examples of in vitro assays for assessing the ADCC activity of a target molecule 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, e.g., ACTI TM for flow cytometry; Non-radioactive cytotoxicity assay (Cell Technology, Inc., Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95,652-656 (1998).

[0243] In some aspects, the binding of the Fc domain to complement components, particularly C1q, is reduced. Thus, in some aspects, where the Fc domain is engineered to have reduced effector function, the reduced effector function includes reduced CDC. A C1q binding assay can be performed to determine whether an Fc domain or a binding molecule comprising an Fc domain is capable of binding C1q and thus has CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J Immunol Methods 202,163 (1996); Cragg et al., Blood 101,1045-1052 (2003); and Cragg and Glennie, Blood 103,2738-2743 (2004)).

[0244] FcRn binding and in vivo clearance / half-life determination 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).

[0245] Configuration of the binding molecule

[0246] The binding molecules according to the invention can have various molecular configurations, i.e., the domains of the binding molecule can be connected to each other in different ways.

[0247] In certain aspects, the binding molecule comprises an Fc domain consisting of a first and a second subunit, and (i) the antigen-binding domain is fused at its C-terminus to the N-terminus of one of the subunits of the Fc domain, (ii) a portion of the effector domain is fused at its N-terminus to the C-terminus of one of the subunits of the Fc domain, and (iii) the complementary domain is fused at its N-terminus to the C-terminus of the portion of the effector domain. In some such aspects, the effector domain is the antigen-binding domain, particularly an anti-CD3 antigen-binding domain.

[0248] In some aspects, the antigen-binding domain is a Fab molecule and is fused at the C-terminus of its heavy chain to the N-terminus of one of the subunits of the Fc domain. In some aspects, the portion of the effector domain comprises a VH or a VL (or consists of a VH or a VL) and is fused at its N-terminus to the C-terminus of one of the subunits of the Fc domain. In some aspects, the complementary domain comprises a VH or a VL (or consists of a VH or a VL), and is fused at its N-terminus to the C-terminus of the portion of the effector domain. In some such aspects, the effector domain is the antigen-binding domain, particularly an anti-CD3 antigen-binding domain.

[0249] In some aspects,

[0250] The first binding molecule comprises

[0251] (i) a first and optionally a third antigen-binding domain, (ii) a first Fc domain consisting of a first and a second subunit, (iii) a first portion of the effector domain, and (iv) a first complementary domain,

[0252] wherein

[0253] (a) the first and the third (when present) antigen-binding domains are fused at their C-termini to the N-terminus of one of the subunits of the first Fc domain,

[0254] (b) the first portion of the effector domain is fused at its N-terminus to the C-terminus of one of the subunits of the first Fc domain,

[0255] (c) The first complementary domain is fused at its N-terminus to the C-terminus of the first part of the effector domain; and

[0256] The second binding molecule comprises

[0257] (i) Second and optionally fourth antigen-binding domains, (ii) a second Fc domain consisting of a first and a second subunit, (iii) a second part of the effector domain, and (iv) a second complementary domain,

[0258] wherein

[0259] (a) The second and fourth (when present) antigen-binding domains are fused at their C-termini to the N-terminus of one of the subunits of the second Fc domain,

[0260] (b) The second part of the effector domain is fused at its N-terminus to the C-terminus of one of the subunits of the second Fc domain,

[0261] (c) The second complementary domain is fused at its N-terminus to the C-terminus of the second part of the effector domain.

[0262] In some such aspects, the effector domain is an antigen-binding domain, particularly an anti-CD3 antigen-binding domain.

[0263] In some aspects,

[0264] The first binding molecule comprises

[0265] (i) First and optionally third antigen-binding domains, (ii) a first Fc domain consisting of a first and a second subunit, (iii) a first part of the effector domain, and (iv) a first complementary domain,

[0266] wherein

[0267] (a) The first and third (when present) antigen-binding domains are fused at their C-termini to the N-terminus of one of the subunits of the first Fc domain,

[0268] (b) The first part of the effector domain comprises VH (or consists of VH) and is fused at its N-terminus to the C-terminus of one of the subunits of the first Fc domain,

[0269] (c) The first complementary domain comprises VL (or consists of VL) and is fused at its N-terminus to the C-terminus of the first part of the effector domain; and

[0270] The second binding molecule comprises

[0271] (i) A second and optionally a fourth antigen-binding domain, (ii) a second Fc domain consisting of a first and a second subunit, (iii) a second part of an effector domain, and (iv) a second complementary domain,

[0272] wherein

[0273] (a) The second and fourth (when present) antigen-binding domains are fused at their C-termini to the N-terminus of one of the subunits of the second Fc domain,

[0274] (b) The second part of the effector domain comprises VL (or consists of VL) and is fused at its N-terminus to the C-terminus of one of the subunits of the second Fc domain,

[0275] (c) The second complementary domain comprises VH (or consists of VH) and is fused at its N-terminus to the C-terminus of the second part of the effector domain.

[0276] In some such aspects, the effector domain is an anti-CD3 antigen-binding domain, particularly an anti-CD3 Fv molecule.

[0277] In some aspects,

[0278] The first binding molecule comprises

[0279] (i) A first and optionally a third antigen-binding domain, (ii) a first Fc domain consisting of a first and a second subunit, (iii) a first part of an effector domain, and (iv) a first complementary domain,

[0280] wherein

[0281] (a) The first antigen-binding domain is a Fab molecule and is fused at its C-terminus to the N-terminus of the first subunit of the first Fc domain,

[0282] (b) The third antigen-binding domain (when present) is a Fab molecule and is fused at its C-terminus to the N-terminus of the first subunit of the first Fc domain,

[0283] (c) The first part of the effector domain comprises VH (or consists of VH) and is fused at its N-terminus to the C-terminus of the first or second subunit of the first Fc domain,

[0284] (d) The first complementary domain comprises VL (or consists of VL) and is fused at its N-terminus to the C-terminus of the first part of the effector domain; and

[0285] The second binding molecule comprises

[0286] (i) The second and optionally the fourth antigen-binding domains, (ii) a second Fc domain consisting of a first and a second subunit, (iii) a second part of the effector domain, and (iv) a second complementary domain,

[0287] wherein

[0288] (a) The second antigen-binding domain is a Fab molecule and is fused at its C-terminus to the N-terminus of the first subunit of the second Fc domain,

[0289] (b) The fourth antigen-binding domain (when present) is a Fab molecule and is fused at its C-terminus to the N-terminus of the first subunit of the second Fc domain,

[0290] (c) The second part of the effector domain comprises VL (or consists of VL) and is fused at its N-terminus to the C-terminus of the first or second subunit of the second Fc domain,

[0291] (d) The second complementary domain comprises VH (or consists of VH) and is fused at its N-terminus to the C-terminus of the second part of the effector domain.

[0292] In some such aspects, the effector domain is an anti-CD3 antigen-binding domain, particularly an anti-CD3 Fv molecule.

[0293] In some aspects, the first binding molecule consists of a first antigen-binding domain, a first Fc domain, a first part of the effector domain, a first complementary domain, and optionally one or more peptide linkers, and / or the second binding molecule consists of a second antigen-binding domain, a second Fc domain, a second part of the effector domain, a second complementary domain, and optionally one or more peptide linkers. In some aspects, the first binding molecule consists of a first antigen-binding domain, a third antigen-binding domain, a first Fc domain, a first part of the effector domain, a first complementary domain, and optionally one or more peptide linkers, and / or the second binding molecule consists of a second antigen-binding domain, a fourth antigen-binding domain, a second Fc domain, a second part of the effector domain, a second complementary domain, and optionally one or more peptide linkers.

[0294] Peptide linker

[0295] The domains (antigen-binding domain, effector domain, complementary domain, Fc domain, etc.) of the binding molecule according to the invention can be fused directly to each other or via one or more peptide linkers.

[0296] The peptide linker contains one or more amino acids, typically about 2 - 20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n, (SG4) n , G4(SG4) n or (G4S) n G5 peptide linker. "n" is typically an integer from 1 to 10, usually 2 to 4. In some aspects, the peptide linker has a length of at least 5 amino acids, in some aspects a length of 5 to 100 amino acids, and in other aspects a length of 10 to 50 amino acids. In some aspects, the 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 some aspects, x = 4 and n = 2 or 3. In a further aspect, x = 4 and n = 2. In yet a further aspect, x = 4, n = 1 and m = 5. In some aspects, the peptide linker is (G4S)2. (SEQ ID NO:55). In other aspects, the peptide linker is (G4S)G5. Additionally, the linker may comprise (a portion of) an immunoglobulin hinge region. In particular, in the case of the fusion of the Fab molecule to the N-terminus of the Fc domain subunit, the fusion can be carried out via the immunoglobulin hinge region or a portion thereof with or without an additional peptide linker.

[0297] In some aspects, the first and third (when present) antigen-binding domains are each fused via an immunoglobulin hinge region to one of the subunits of the first Fc domain, and / or the second and fourth (when present) antigen-binding domains are each fused via an immunoglobulin hinge region to one of the subunits of the second Fc domain.

[0298] In some aspects, the first part of the effector domain and the first Fc domain and / or the second part of the effector domain and the second Fc domain are fused via a peptide linker. In some aspects, the first part of the effector domain is fused at its N-terminus via a peptide linker to the C-terminus of a subunit of the first Fc domain, and / or the second part of the effector domain is fused at its N-terminus via a peptide linker to the C-terminus of a subunit of the second Fc domain. An exemplary peptide linker suitable for fusing a part of the effector domain to the C-terminus of a subunit of the Fc domain is (G4S)2. (SEQ ID NO:55). Thus, in some aspects, the first part of the effector domain is fused at its N-terminus via a peptide linker to the C-terminus of a subunit of the first Fc domain, the peptide linker comprising the sequence (G4S)2. (SEQ ID NO:55), and / or the second part of the effector domain is fused at its N-terminus via a peptide linker to the C-terminus of a subunit of the second Fc domain, the peptide linker comprising the sequence (G4S)2. (SEQ ID NO:55). In certain aspects, the peptide linker consists of the sequence (G4S)2. (SEQ ID NO:55).

[0299] In some aspects, the first part of the effector domain and the first complementary domain and / or the second part of the effector domain and the second complementary domain are fused via a peptide linker. In some aspects, the first part of the effector domain is fused at its C-terminus via a peptide linker to the N-terminus of the first complementary domain, and / or the second part of the effector domain is fused at its C-terminus via a peptide linker to the N-terminus of the second complementary domain. An exemplary peptide linker suitable for fusing a complementary domain to the C-terminus of a part of the effector domain is (G4S)3GGSGG (SEQ ID NO:56). Thus, in some aspects, the first part of the effector domain is fused at its C-terminus via a peptide linker comprising the sequence of SEQ ID NO:56 to the N-terminus of the first complementary domain, and / or the second part of the effector domain is fused at its C-terminus via a peptide linker comprising the sequence of SEQ ID NO:56 to the N-terminus of the second complementary domain. In certain aspects, the peptide linker consists of the sequence of SEQ ID NO:56.

[0300] Binding molecule

[0301] The invention also provides binding molecules that form part of the binding molecule pairs of the invention. The binding molecules can be incorporated, alone or in combination, into any of the features of the binding molecule pairs described above and herein (unless the context dictates otherwise).

[0302] Polynucleotide

[0303] The present invention further provides isolated polynucleotides encoding the binding molecule pairs of the present invention. The present invention also provides isolated polynucleotides encoding binding molecules that form part of the binding molecule pairs of the present invention. The isolated polynucleotides can be a single polynucleotide or multiple polynucleotides.

[0304] The polynucleotides encoding the binding molecules of the present invention can be expressed as a single polynucleotide encoding a complete binding molecule pair or binding molecule, or as multiple (e.g., two or more) polynucleotides that are co-expressed. The polypeptides encoded by the co-expressed polynucleotides can associate, for example, via disulfide bonds or other means to form a functional binding molecule. For example, the light chain portion of an antigen-binding domain can be encoded separately by a polynucleotide from the portion of the binding molecule that includes the heavy chain of the antigen-binding domain. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an antigen-binding domain. In another example, a portion of a binding molecule that includes one subunit of two Fc domain subunits and optionally one or more Fab molecules (or a portion thereof) can be encoded separately by a polynucleotide from a portion of the binding molecule that includes the other subunit of the two Fc domain subunits and optionally a Fab molecule (or a portion thereof). When co-expressed, the Fc domain subunits will associate to form an Fc domain.

[0305] In some aspects, the isolated polynucleotides encode complete binding molecule pairs or complete binding molecules according to the present invention as described herein. In other aspects, the isolated polynucleotides encode polypeptides that are included in the binding molecule pairs or binding molecules according to the present invention as described herein.

[0306] In certain aspects, the polynucleotide or nucleic acid is DNA. In other aspects, the polynucleotides of the present invention are RNA, for example in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

[0307] Recombinant methods

[0308] The binding molecules of the present invention can be obtained, for example, by solid-phase peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the binding molecules, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such polynucleotides can be readily isolated and sequenced using conventional methods. In one aspect, vectors, particularly expression vectors, containing the polynucleotides of the present invention (i.e., a single polynucleotide or multiple polynucleotides) are provided. Expression vectors containing the coding sequence of the binding molecule and appropriate transcriptional / translational control signals can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in the following references: 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 can be a plasmid, part of a virus, or can be a nucleic acid fragment. The expression vector includes an expression cassette into which the polynucleotide encoding the binding molecule (i.e., the coding region) is cloned operably associated with a promoter and / or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid that consists of codons that are translated into amino acids. Although "stop codons" (TAG, TGA, or TAA) are not translated into amino acids, they can be considered part of the coding region if present, while any flanking sequences, such as promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' untranslated regions, etc., are not part of the coding region. Two or more coding regions can be present in a single polynucleotide construct (e.g., on a single vector), or in separate polynucleotide constructs (e.g., on separate (different) vectors). In addition, any vector can contain a single coding region, or can contain two or more coding regions. For example, the vectors of the present invention can encode one or more polypeptides that are separated into the final proteins by proteolytic cleavage after or during translation. Furthermore, the vectors, polynucleotides, or nucleic acids of the present invention can encode a heterologous coding region that is fused or not fused to the polynucleotide encoding the binding molecule of the present invention or its variant or derivative. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as a secretion signal peptide or a heterologous functional domain.Operable association is when the coding region of a gene product (such as a polypeptide) is associated in some manner with one or more regulatory sequences such that the expression of the gene product is under the influence or control of the regulatory sequences. If induction of promoter function results in transcription of mRNA encoding the desired gene product and if the nature of the bonding between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed, then the two DNA fragments (such as a polypeptide coding region and its associated promoter) are "operably associated". Thus, a promoter region will be operably associated with a nucleic acid encoding a polypeptide if the promoter is capable of influencing transcription of that nucleic acid. The promoter can be a cell-specific promoter that directs substantial transcription of DNA only in a predetermined cell. In addition to promoters, other transcriptional control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with a polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. A variety of transcriptional control regions are known to those of skill in the art. These transcriptional control regions include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (such as the immediate early promoter with intron-A), simian virus 40 (such as the early promoter), and retroviruses (such as, for example, Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes (such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers, and inducible promoters (such as tetracycline-inducible promoters). Similarly, various translational control elements are known to those of ordinary skill in the art. These translational control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (especially internal ribosome entry sites, or IRES, also known as CITE sequences). The expression cassette can also include other features, such as an origin of replication, and / or chromosomal integration elements, such as retroviral long terminal repeats (LTRs), or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0309] The polynucleotide and nucleic acid coding region can be associated with an additional coding region encoding a secretory or signal peptide that directs the secretion of the polypeptide encoded by the polynucleotide of the invention. For example, if secretion of the binding molecule is desired, the DNA encoding the signal sequence can be placed upstream of the nucleic acid encoding the binding molecule 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 once the growing protein chain has been initiated for export across the rough endoplasmic reticulum. Those of ordinary skill in the art know that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the translated polypeptide to produce the secreted or "mature" form of the polypeptide. In some aspects, a native signal peptide (e.g., immunoglobulin heavy or light chain signal peptide) or a functional derivative of the sequence that retains the ability to direct secretion of the polypeptide with which it is operably associated 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 the leader sequence of human tissue plasminogen activator (TPA) or murine β-glucuronidase.

[0310] DNA encoding a short protein sequence that can be used to facilitate subsequent purification (e.g., histidine tag) or to aid in labeling the binding molecule can be included within or at the terminus of the binding molecule (fragment) encoding polynucleotide.

[0311] In some aspects, host cells are provided that contain the polynucleotides of the invention (i.e., a single polynucleotide or multiple polynucleotides). In certain aspects, host cells are provided that contain the vectors of the invention. The polynucleotides and vectors can incorporate, either singly or in combination, any of the features described herein with respect to polynucleotides and vectors, respectively. In some such aspects, the host cell contains one or more vectors (e.g., has been transformed or transfected with one or more vectors), the one or more vectors containing one or more polynucleotides encoding a binding molecule pair or a (portion of a) binding molecule of the invention. As used herein, the term "host cell" refers to any kind of cell system that can be engineered to produce a binding molecule or a fragment thereof of the invention. Host cells suitable for replicating and supporting the expression of binding molecules (e.g., antibodies) are well known in the art. Such cells can be appropriately transfected or transduced with a particular expression vector, and large amounts of cells containing the vector can be grown for inoculating a large-scale fermenter to obtain sufficient amounts of the binding molecule for clinical use. Suitable host cells include prokaryotic microorganisms such as Escherichia coli, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, and the like. For example, polypeptides can be produced in bacteria, especially when glycosylation is not required. The polypeptide can be isolated from the bacterial cell paste in the soluble fraction after expression and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptides, including fungal and yeast strains in which the glycosylation pathway has been "humanized", resulting in the production of polypeptides with a partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for expressing (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains that can be used with insect cells have been identified, especially for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants) TMtechnique). Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293T cells, as described, for example, in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, as 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), dog kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT060562), TRI cells (as described, for example, in Mather et al., Annals N.Y.Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr - CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by B.K.C. Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as, by way of example only, mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, and also include cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues. In some aspects, the host cell is a eukaryotic cell, particularly a mammalian cell, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or lymphocytes (e.g., Y0, NS0, Sp20 cells). In some aspects, the host cell is not a cell in the human body.

[0312] Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing a polypeptide comprising an antigen-binding domain, such as a heavy or light chain of an antibody, can be engineered to also express the other antibody chain so that the expressed product is an antibody having a heavy and a light chain.

[0313] In one aspect, provided is a method of making a binding molecular pair according to the present invention, wherein the method comprises culturing a host cell provided herein that contains a polynucleotide encoding the binding molecular pair under conditions suitable for expressing the binding molecular pair docking, and optionally recovering the binding molecular pair from the host cell (or the host cell culture medium). Similarly, in one aspect, provided is a method of producing a binding molecule according to the present invention, wherein the method comprises culturing a host cell containing a polynucleotide encoding the binding molecule as provided herein under conditions suitable for expressing the binding molecule, and optionally recovering the binding molecule from the host cell (or the host cell culture medium).

[0314] The binding molecules prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen that binds to the binding molecule can be used. For example, for affinity chromatography purification of the binding molecules of the present invention, a matrix having protein A or protein G can be used. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to isolate the binding molecules substantially as described in the examples. The purity of the binding molecules can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, etc.

[0315] Compositions, Formulations, and Routes of Administration

[0316] In a further aspect, the present invention provides pharmaceutical compositions comprising the binding molecular pairs or binding molecules provided herein, for use in any of the following therapeutic methods, for example. In some aspects, the pharmaceutical composition comprises a binding molecular pair or binding molecule according to the present invention and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition comprises a binding molecular pair or binding molecule according to the present invention and at least one additional therapeutic agent as described below, for example. The binding molecules that form part of the binding molecular pair can be included in one and the same pharmaceutical composition, or in separate pharmaceutical compositions (i.e., each binding molecule that forms part of the binding molecular pair is in a separate pharmaceutical composition). Generally, they will be provided in separate pharmaceutical formulations to minimize the risk of the first and second parts of the effector domain associating with each other within the pharmaceutical composition. Thus, in a particular aspect, the present invention provides a first pharmaceutical composition comprising a first binding molecule and a pharmaceutically acceptable carrier, and a second pharmaceutical composition comprising a second binding molecule and a pharmaceutically acceptable carrier. In other aspects, the present invention provides a pharmaceutical composition comprising the first and second binding molecules of the present invention and a pharmaceutically acceptable carrier.

[0317] Also provided is a method of producing the binding molecule pair or binding molecule of the present invention in a form suitable for in vivo administration, the method comprising (a) obtaining the binding molecule pair or binding molecule according to the present invention, and (b) formulating the binding molecule pair or binding molecule with at least one pharmaceutical carrier, whereby a formulation of the binding molecule pair or binding molecule is prepared for in vivo administration.

[0318] The pharmaceutical composition of the present invention comprises an effective amount of the binding molecule dissolved or dispersed in a pharmaceutical carrier. The term "pharmaceutically acceptable" means that the molecular entity and composition are generally non-toxic to the recipient at the dosages and concentrations employed, i.e., do not produce adverse, allergic, or other untoward reactions when administered to an animal (e.g., a human), as the case may be. The preparation of pharmaceutical compositions containing the binding molecule and optionally additional active ingredients will be known to those skilled in the art, as exemplified by Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, which reference is incorporated herein by reference. In addition, for administration to animals (e.g., humans), it is understood that the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards or other corresponding authorities in other countries / regions. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutical carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, pp. 1289-1329, which reference is incorporated herein by reference). The use of such carriers in the pharmaceutical compositions is contemplated except in instances where any conventional carrier is incompatible with the active ingredient.

[0319] The binding molecule pair or binding molecule (and any additional therapeutic agent) of the present invention can be administered by any suitable means, including parenterally, intraluminally, and intranasally, and for local treatment, intralesional administration if desired. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, e.g., by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. In some aspects, the binding molecule of the present invention is administered (or will be administered) intravenously.

[0320] Parenteral compositions include those designed for administration by injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, the binding molecules of the present invention can be formulated in an aqueous solution, particularly in a physiologically compatible buffer (such as Hanks solution, Ringer solution, or saline buffer). The solution can contain formulatory agents, such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the binding molecules can be in powder form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. As needed, a sterile injectable solution is prepared by incorporating the binding molecules of the present invention in the desired amounts, along with the various other ingredients listed below, into a suitable solvent. For example, sterility can be readily achieved by filtration through a sterile filter membrane. Generally, a dispersion is prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing a base dispersion medium and / or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions, or emulsions, the preferred method of preparation is vacuum drying or lyophilization techniques, which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered liquid medium. If desired, the liquid medium should be appropriately buffered and should first be made isotonic with sufficient saline or glucose to dilute the liquid diluent before injection. The composition must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to a minimum, for example, at a safe level of less than 0.5 ng / mg protein. Suitable pharmaceutical carriers include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Aqueous injection suspensions can contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, etc. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound to allow the preparation of highly concentrated solutions.In addition, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil; or synthetic fatty acid esters such as ethyl oleate or triglycerides; or liposomes.

[0321] The active ingredient can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively); entrapped in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules); or entrapped in a coarse emulsion. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th edition, Mack Printing Company, 1990). Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing polypeptides, which matrices are in the form of shaped articles such as membranes or microcapsules. In certain aspects, the extended absorption of an injectable composition can be achieved by using a reagent that delays absorption (for example, aluminum monostearate, gelatin or a combination thereof) in the composition.

[0322] In addition to the compositions described previously, the binding molecules can also be formulated into long-acting preparations. Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the binding molecules can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or with ion-exchange resins, or formulated as a sparingly soluble derivative, for example, as a sparingly soluble salt.

[0323] The pharmaceutical compositions containing the binding molecules of the present invention can be produced by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilization processes. The pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants, which carriers, diluents, excipients or adjuvants facilitate the processing of the protein into a pharmaceutically useful preparation. The appropriate formulation depends on the chosen route of administration.

[0324] The binding molecules can be formulated into compositions in the free acid or base, neutral or salt form. Medicinal salts are salts that substantially retain the biological activity of the free acid or base. These medicinal salts include acid addition salts, for example, acid addition salts formed with the free amino groups of the protein composition, or acid addition salts formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid or mandelic acid). Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or organic bases, such as isopropylamine, trimethylamine, histidine or procaine. Medicinal salts tend to be more soluble in aqueous and other protonic solvents compared to the corresponding free base forms.

[0325] Therapeutic methods and compositions

[0326] Any of the binding molecules provided herein can be used in therapeutic methods. The binding molecules of the present invention can be used as immunotherapeutic agents, for example, for the treatment of cancer.

[0327] For use in therapeutic methods, the binding molecules of the present invention will be formulated, administered, and dosed in a manner that is 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 site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the practicing physician.

[0328] In therapeutic methods, the first and second binding molecules are used in combination. As used herein, "combination" (and its grammatical variants such as "combine / combining") includes a combination of the first and second binding molecules, wherein the first and second binding molecules are in the same or different containers, in the same or different pharmaceutical formulations, administered together or separately, simultaneously or in any order sequentially, and by the same or different routes of administration, provided that the first and second binding molecules can bind simultaneously to their target antigens on the cell surface via their antigen-binding domains. For example, "combining" the first and second binding molecules can mean administering the first binding molecule first in a particular pharmaceutical formulation and then the second binding molecule in another pharmaceutical formulation, or vice versa.

[0329] The first and second binding molecules can be administered in any suitable manner known in the art. In some aspects, the first and second binding molecules are administered sequentially (at different times). In other aspects, the first and second binding molecules are administered concurrently (at the same time). In some aspects, the first and second binding molecules are in separate compositions. In some aspects, the first and second binding molecules are in the same composition.

[0330] In one aspect, the binding molecule pair or binding molecule of the present invention is provided for use as a medicament. In a further aspect, the binding molecule pair or binding molecule of the present invention is provided for treating a disease. In certain aspects, the binding molecule pair or binding molecule of the present invention is provided for a method of treatment. In one aspect, the present invention provides the binding molecule pair or binding molecule of the present invention for treating a disease in an individual in need thereof. In one aspect, the present invention provides a method for treating an individual suffering from a disease with a binding molecule pair, the method comprising administering to the individual an effective amount of the binding molecule pair. In certain aspects, the disease is a proliferative disorder. In certain aspects, the disease is cancer. In some aspects, the cancer is a solid tumor cancer. In some aspects, the cancer is a cancer expressing the target antigen of the binding molecule pair or binding molecule. In some aspects (specifically, if the antigen-binding domain of the first and / or second binding molecule is capable of binding HER2), the cancer is a cancer expressing HER2. In a specific aspect, the cancer is breast cancer. In certain aspects, if the disease to be treated is cancer, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as an anti-cancer agent. In a further aspect, the present invention provides the binding molecule pair of the present invention for inducing lysis of target cells, particularly cancer cells. In one aspect, the present invention provides the binding molecule pair of the present invention for a method of inducing lysis of target cells, particularly cancer cells, in an individual, the method comprising administering to the individual an effective amount of the binding molecule pair to induce lysis of the target cells. An "individual" according to any of the above aspects is a mammal, preferably a human.

[0331] In a further aspect, the present invention provides the use of the binding molecule pair or binding molecule of the present 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 a method of treating a disease, the method comprising administering to an individual suffering from the disease an effective amount of the medicament. In certain aspects, the disease is a proliferative disorder. In certain aspects, the disease is cancer. In some aspects, the cancer is a solid tumor cancer. In some aspects, the cancer is a cancer expressing the target antigen of the binding molecule pair or binding molecule. In some aspects (specifically, if the antigen-binding domain of the first and / or second binding molecule is capable of binding HER2), the cancer is a cancer expressing HER2. In a specific aspect, the cancer is breast cancer. In some aspects, if the disease to be treated is cancer, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as an anti-cancer agent. In a further aspect, the medicament is for inducing lysis of target cells, particularly cancer cells. In a still further aspect, the medicament is for a method of inducing lysis of target cells, particularly cancer cells, in an individual, the method comprising administering to the individual an effective amount of the medicament to induce lysis of the target cells. An "individual" according to any of the above aspects may be a mammal, preferably a human.

[0332] In a further aspect, the present invention provides a method for treating a disease. In one aspect, the method comprises administering to an individual suffering from such a disease an effective amount of the binding molecule pair of the present invention. In some aspects, one or more compositions are administered to the individual, the compositions comprising the binding molecule pair of the present invention in a pharmaceutical form. In certain aspects, the disease is a proliferative disorder. In certain aspects, the disease is cancer. In some aspects, the cancer is a solid tumor cancer. In some aspects, the cancer is a cancer that expresses the target antigen of the binding molecule pair or the binding molecule. In some aspects (specifically, if the antigen-binding domain of the first and / or second binding molecule is capable of binding HER2), the cancer is a cancer that expresses HER2. In a specific aspect, the cancer is breast cancer. In certain aspects, if the disease to be treated is cancer, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as an anti-cancer agent. The "individual" according to any of the above aspects can be a mammal, preferably a human.

[0333] In a further aspect, the present invention provides a method for inducing lysis of target cells. In some aspects, the target cells are cells that express the target antigen of the binding molecule pair or the binding molecule. In some aspects (specifically, if the antigen-binding domain of the first and / or second binding molecule is capable of binding HER2), the target cells are cells that express HER2. In some aspects, the method comprises contacting the target cells with the binding molecule pair of the present invention in the presence of T cells, particularly cytotoxic T cells. In a further aspect, a method for inducing lysis of target cells in an individual is provided. In some aspects, the target cells are cells that express the target antigen of the binding molecule pair or the binding molecule. In some aspects (specifically, if the antigen-binding domain of the first and / or second binding molecule is capable of binding HER2), the target cells are cells that express HER2. In some such aspects, the method comprises administering to the individual an effective amount of the binding molecule pair of the present invention to induce lysis of the target cells. In one aspect, the "individual" is a human.

[0334] Those skilled in the art will readily recognize that in many cases, the binding molecule pair may not provide a cure, but may only provide partial benefits. In some aspects, physiological changes with certain benefits are also considered to have therapeutic benefits. Thus, in some aspects, the amount of the binding molecule pair that provides a physiological change is considered to be an "effective amount". The subject, patient or individual in need of treatment is typically a mammal, more particularly a human.

[0335] In some aspects, an effective amount of the binding molecule pair of the present invention is administered to an individual to treat a disease.

[0336] For the prevention or treatment of diseases, the appropriate dosage of the binding molecule pair of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the patient's body weight, the type of binding molecule, the severity and course of the disease, whether the binding molecule pair is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the fusion protein, and the discretion of the attending physician. In any case, the practitioner responsible for administration will determine the concentration of the active ingredient and the appropriate dosage for the individual subject. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulsed infusion.

[0337] The binding molecule pair is appropriately administered to a patient once or in a series of treatments. Depending on the type and severity of the disease, an initial candidate dose of each binding molecule of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg - 10 mg / kg) can be administered to the patient, for example, by one or more separate administrations or by continuous infusion. Depending on the above factors, a typical daily dose can range from about 1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, depending on the condition, the treatment generally continues until the desired suppression of the disease symptoms occurs. Such doses can be administered intermittently, for example, weekly or every three weeks (e.g., such that the patient receives about two to about twenty, or for example about six doses of the binding molecule pair). An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosing regimens may be useful. The progress of the therapy can be easily monitored by conventional techniques and assays.

[0338] The binding molecule pair of the present invention will generally be used in an amount effective to achieve the intended purpose. For use in the treatment or prevention of a disorder, the binding molecule pair of the present invention or its pharmaceutical composition is administered or applied in a therapeutically effective amount.

[0339] For systemic administration, the effective dose can initially be estimated based on in vitro assays such as cell culture assays. The dose can then be formulated in an animal model to achieve a circulating concentration range including, for example, the IC as assayed in cell culture. 50 Such information can be used to more accurately determine the useful dose for humans.

[0340] The initial dose can also be estimated using techniques well known in the art based on in vivo data (e.g., animal models).

[0341] The amount and interval of the dosage can be adjusted individually to provide a plasma level of the binding molecule sufficient to maintain the therapeutic effect. The range of the common patient dosage administered by injection is from about 0.1 mg / kg / day to 50 mg / kg / day, usually about 0.5 mg / kg / day to 1 mg / kg / day. A therapeutically effective plasma level can be achieved by administering multiple doses per day. The level in plasma can be measured, for example, by HPLC.

[0342] The binding molecule pair of the present invention at an effective dose will generally provide a therapeutic benefit without causing significant toxicity. The toxicity and therapeutic efficacy of the binding molecule pair can be determined by standard pharmacological procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50 (the dose that kills 50% of the population) and the ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxicity and efficacy is the therapeutic index, and the therapeutic index can be expressed as the ratio LD 50 / ED 50 . Binding molecule pairs showing a large therapeutic index are preferred. In some aspects, the binding molecule pair according to the present invention shows a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of doses suitable for humans. The dose is preferably in the range including the circulating concentration of the ED 50 with little or no toxicity. The dose can vary within this range depending on a variety of factors, such as the dosage form employed, the administration route utilized, the condition of the subject, etc. The exact formulation, administration route, and dose can be selected by the individual physician according to the condition of the patient (see, for example, Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Chapter 1, page 1, the entire content of which is incorporated herein by reference).

[0343] The attending physician of a patient being treated with the binding molecule pair of the present invention will know how and when to terminate, interrupt, or adjust the administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is inadequate (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. The size of the dose administered in the management of the target disorder will vary with the severity of the disorder to be treated, the administration route, etc. For example, the severity of the disorder can be evaluated, in part, by standard prognostic assessment methods. In addition, the dose and possible dose frequency will also vary according to the age, weight, and response of the individual patient.

[0344] Binding molecules (i.e., the first and second binding molecules) that form a binding molecule pair can be co-administered (where the binding molecules are included in the same composition or separate compositions) or administered separately. In the case of separate administration, the administration of the first binding molecule can occur before, simultaneously with, and / or after the administration of the second binding molecule.

[0345] The binding molecule pairs of the present invention can be co-administered in therapy with one or more other agents. For example, the binding molecule pairs of the present invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent that is administered to treat the symptoms or diseases of an individual in need of such treatment. Such additional therapeutic agents can comprise any active ingredient suitable for the specific disease being treated, preferably active ingredients having complementary activities that do not adversely affect each other. In certain aspects, the additional therapeutic agent is an immunomodulator, a cell growth inhibitor, a cell adhesion inhibitor, a cytotoxic agent, an apoptosis activator, or an agent that increases the sensitivity of cells to apoptosis-inducing agents. In certain aspects, the additional therapeutic agent is an anti-cancer agent, such as a microtubule-disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, hormone therapy, a kinase inhibitor, a receptor antagonist, a tumor cell apoptosis activator, or an anti-angiogenic agent.

[0346] Such other agents are present in combination in an amount effective for the intended purpose. The effective amount of such other agents depends on the amount of the binding molecule pair used, the type of disorder or treatment, and the other factors discussed above. The binding molecule pair is generally used at the same dose and route of administration as described herein, or at about 1% to 99% of the dose described herein, or at any dose and by a route determined to be suitable empirically / clinically.

[0347] Such combination therapies as described above encompass co-administration (where two or more therapeutic agents are included in the same composition or separate compositions), as well as separate administration. In the case of separate administration, the administration of the binding molecule pairs of the present invention can occur before, simultaneously with, and / or after the administration of the additional therapeutic agent and / or adjuvant. The binding molecule pairs of the present invention can also be used in combination with radiotherapy.

[0348] Article

[0349] In another aspect of the present invention, there is provided an article containing a substance that can be used for treating, preventing, and / or diagnosing the above-mentioned diseases (such as cancer). The article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition that, either alone or in combination with another composition, can be effectively used for treating, preventing, and / or diagnosing a condition, and the container can have a sterile access port (for example, the container can be an intravenous injection solution bag or vial having a stopper that can be pierced by a hypodermic needle). In some aspects, the composition contains the binding molecule pairs of the present invention. The article in these aspects can further include a label or package insert indicating that the composition can be used for treating a specific condition. In other aspects, the article includes (a) a first container containing a composition that contains a first binding molecule; and (b) a second container containing a composition that contains a second binding molecule. The article in these aspects can further include a package insert indicating that the compositions can be used in combination for treating a specific condition (such as cancer).

[0350] In some aspects, the present invention provides an article (kit) for treating a disease (such as cancer), which includes (i) a container containing a pharmaceutical composition, where the pharmaceutical composition contains the binding molecule pairs of the present invention and an optional pharmaceutical carrier, and optionally (ii) a label or package insert containing instructions for using the pharmaceutical composition in the treatment of the disease (such as cancer).

[0351] In other aspects, the present invention provides an article (kit) for treating a disease (such as cancer), which includes (i) a first container containing a first pharmaceutical composition, where the first pharmaceutical composition contains a first binding molecule of the binding molecule pairs of the present invention and an optional pharmaceutical carrier; (ii) a second container containing a second pharmaceutical composition, where the second pharmaceutical composition contains a second binding molecule of the binding molecule pairs of the present invention and an optional pharmaceutical carrier; and optionally (iii) a label or package insert containing instructions for using the first pharmaceutical composition and the second pharmaceutical composition in combination in the treatment of the disease (such as cancer).

[0352] Alternatively or additionally, the article can further include a second (or third) container containing a pharmaceutical buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and glucose solution. It can further include other substances desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0353] Method for forming a functional effector domain

[0354] In a further aspect, the present invention provides a method for forming a functional effector domain, which comprises contacting a pair of binding molecules of the present invention with a cell expressing a target antigen of the first and second antigen-binding domains under conditions that permit the first and second antigen-binding domains to bind to their target antigens on the cell surface.

[0355] Amino acid sequence

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362] Description of the drawings

[0363] Figure 1 . Schematic diagrams of exemplary split molecules according to the present invention. (A) A split molecule having bivalent binding to a target antigen and a non-complementary effector VL domain. (B) A split molecule of (A) having bivalent binding to a target antigen and a non-complementary effector VH domain. (C) A split molecule having monovalent binding to a target antigen and a non-complementary effector VL domain. (D) A split molecule having monovalent binding to a target antigen and a non-complementary effector VH domain. (E) A split molecule having monovalent binding to a target antigen and a V-complementary effector VL domain. (F) A split molecule having monovalent binding to a target antigen and a V-complementary effector VH domain. The circles in the Fc region represent modifications that promote Fc heterodimerization, such as "staple" modifications.

[0364] Figure 2 . Molecules with different split anti-CD3 conjugates were tested in a binding assay using Jurkat cells as target cells. Binding on Jurkat cells was determined by measuring the median fluorescence intensity (MFI) by flow cytometry as described in the method. The MFI results for the V9 conjugate (A), the P035.093 conjugate (B), the 40G5c conjugate (C), and the C22 conjugate (D) are shown, where complementary molecules are represented by grey circles and non-complementary molecules are represented by black circles.

[0365] P1AF1375 + P1AF1376: Split molecules with non-complementary V9 conjugates.

[0366] P1AF3870 + P1AF3871: Split molecules with V-complementary V9 conjugates.

[0367] P1AF2484 + P1AF2678: Split molecules with non-complementary P035.093 conjugates.

[0368] P1AF3868 + P1AF3869: Split molecules with V-complementary P035.093 conjugates.

[0369] P1AA9518 + P1AA9516: Split molecules with non-complementary 40G5c conjugates.

[0370] P1AF3865 + P1AF3867: Split molecules with V-complementary 40G5c conjugates.

[0371] P1AF2678 + P1AE6818: Split molecules with non-complementary C22 conjugates.

[0372] The format of all molecules is similar to the molecules shown in Table 2.

[0373] Figure 3 . Tumor cell killing of CD3+ T cells from healthy donors against the ovarian adenocarcinoma cell line SKOV-3 (HER-2 positive) was evaluated when treated with split molecules with different anti-CD3 conjugates. Tumor cell killing was measured by quantifying cell death using the CytotoxGlo kit (Promega) after 48 hours. The cell death results for the V9 conjugate (A), P035.093 conjugate (B), and 40G5c conjugate (C) are shown. Solid symbols represent treatment with the conjugate and open symbols represent treatment with the single prodrug.

[0374] P1AD4471: Control molecule (non-split HER2 x CD3 bispecific antibody (with HER2 bivalent and CD3 monovalent binding)).

[0375] P1AF3870 + P1AF3871: HER2-targeted split molecules with complementary V9 conjugates.

[0376] P1AF3868 + P1AF3869: HER2-targeted split molecules with complementary P035.093 conjugates.

[0377] P1AE6814 + P1AF2484: HER2-targeted split molecules with non-complementary P035.093 conjugates.

[0378] P1AF3865 + P1AF3867: HER2-targeted split molecules with complementary 40G5c conjugates.

[0379] P1AD9224 + P1AD9225: HER2-targeted split molecules with non-complementary 40G5c conjugates (having charged residues in the VH(Q39E) and VL(Q38K) domains as described above).

[0380] The format of all split molecules is similar to the molecules shown in Table 2.

[0381] Figure 4 . Tumor cell killing of ovarian adenocarcinoma cell line SKOV-3 (HER-2 positive) by CD3+ T cells from healthy donors was evaluated when treated with split molecules containing the anti-CD3 conjugate P035.093. Tumor cell killing was measured by quantifying cell death using the CytotoxGlo kit (Promega) after 48 hours. The cell death results of non-complementary (A) and complementary (B) molecules are shown. The positive control molecule (non-split HER2 x CD3 bispecific antibody) is shown as solid squares, the HER2+HER2 production pair is represented by solid circles, and HER2+ irrelevant conjugates are shown with open symbols.

[0382] P1AD4471: Control molecule (non-split HER2 x CD3 bispecific antibody).

[0383] P1AE6814 + P1AF2484: HER2-targeted split molecules with non-complementary P035.093 conjugates.

[0384] P1AF2484 + P1AG7469: Split molecules with non-complementary P035.093 conjugates having one HER2-targeted prodrug and one non-targeted (DP47) prodrug.

[0385] P1AF3868 + P1AF3869: HER2-targeted split molecules with complementary P035.093 conjugates.

[0386] P1AF3869 + P1AG5786: Split molecules with complementary P035.093 conjugates having one HER2-targeted prodrug and one non-targeted (DP47) prodrug.

[0387] The format of all split molecules is similar to the molecules shown in Table 2.

[0388] Figure 5. Tumor cell killing of the ovarian adenocarcinoma cell line SKOV-3 (HER-2 positive) was evaluated using CD3+ T cells from healthy donors. Tumor cell killing was measured by quantifying cell death after 48 hours using the CytotoxGlo kit (Promega). The cell death results of non-complementary molecules containing the 40G5c conjugate (A) and the C22 conjugate (B) are shown. The positive control molecule (non-split HER2 x CD3 bispecific antibody) is shown as a solid square, the HER2+HER2 production pair is represented by solid circles, and the HER2+ irrelevant conjugate is shown with open symbols.

[0389] P1AD4471: Control molecule (non-split HER2 x CD3 bispecific antibody).

[0390] P1AA9516 + P1AA9518: HER2-targeted split molecules with non-complementary 40G5c conjugates.

[0391] P1AA9518 + P1AG7808: Split molecules with non-complementary 40G5c conjugates and one HER2-targeted prodrug and one non-targeted (DP47) prodrug.

[0392] P1AE6814 + P1AE6818: HER2-targeted split molecules with non-complementary C22 conjugates.

[0393] P1AE6818 + P1AG7469: Split molecules with non-complementary C22 conjugates and one HER2-targeted prodrug and one non-targeted (DP47) prodrug.

[0394] All split molecules are in a format similar to the molecules shown in Table 2.

[0395] Figure 6 . Tumor cell killing of the ovarian adenocarcinoma cell line SKOV-3 (HER-2 overexpressor) and the prostate cancer cell line LNCaP (HER-2 low expressor) was evaluated using CD3+ T cells from healthy donors. Tumor cell killing was measured by quantifying cell death after 48 hours using the CytotoxGlo kit (Promega). The cell death results of the SKOV-3 cell line (A) and the LNCaP cell line (B), as well as the quantification of their respective antigen-binding sites (C), are shown. The HER2+HER2 production pairs are represented by solid circles, and the HER2+ irrelevant conjugate pairs are shown with open symbols.

[0396] P1AF3868 + P1AF3869: HER2-targeted split molecules with complementary P035.093 conjugates.

[0397] P1AF3869 + P1AG5786: A split molecule with one HER2-targeted prodrug and one non-targeted (DP47) prodrug having non-complementary P35.093 conjugates.

[0398] The format of all molecules is similar to the molecules shown in Table 2.

[0399] Figure 7 . Tumor cell killing of ovarian adenocarcinoma cell line SKOV-3 (HER-2 high expressor) and prostate cancer cell line LNCaP (HER-2 low expressor) was evaluated using CD3+ T cells from healthy donors. Tumor cell killing was measured by quantifying cell death using the CytotoxGlo kit (Promega) after 48 hours. The cell death results of the SKOV-3 cell line (A) and the LNCaP cell line (B) are shown. The non-complementary production pairs of various CD3 conjugates are shown as 40G5c (squares), C22 (circles), P035.093 (diamonds).

[0400] P1AA9518 + P1AA9516: A HER2-targeted split molecule with non-complementary 40G5c conjugates.

[0401] P1AE6818 + P1AE6814: A HER2-targeted split molecule with non-complementary C22 conjugates.

[0402] P1AF2484 + P1AE6814: A HER2-targeted split molecule with non-complementary P035.093 conjugates.

[0403] The format of all molecules is similar to the molecules shown in Table 2.

[0404] Figure 8 . Ex vivo analysis of human T cells from humanized mice. Equimolar doses of split molecules were injected into the mice, and blood was collected and analyzed 24 hours after treatment to evaluate target-independent CD3 conjugate assembly in circulation. Molecules binding to T cells were measured by flow cytometry using a PE-labeled anti-human IgG, Fc-specific secondary antibody. The median fluorescence intensity of CD4+ (A) and CD8+ T cells (B) in the blood from animals treated with uncomplementary and complementary P035.093 compounds is shown.

[0405] P1AE6814 + P1AF2484: A HER2-targeted split molecule with non-complementary P035.093 conjugates.

[0406] P1AF3868 + P1AF3869: A HER2-targeted split molecule with complementary P035.093 conjugates.

[0407] The format of all molecules is similar to the molecules shown in Table 2.

[0408] Figure 9 . Ex vivo analysis of human T cells from humanized mice. Mice were injected with equimolar doses of split molecules, and blood was collected and analyzed 24 hours after treatment to evaluate target-independent CD3 conjugate assembly in circulation. Molecules binding to T cells were measured by flow cytometry using a PE-labeled anti-human IgG, Fc-specific secondary antibody. The median fluorescence intensity of CD4+ (A) and CD8+ T cells (B) in blood from animals treated with uncomplemented and complemented V9 compounds is shown.

[0409] P1AF1375 + P1AF1376: Tumor antigen-targeted split molecule with uncomplemented V9 conjugate.

[0410] P1AF3870 + P1AF3871: HER2-targeted split molecule with complemented V9 conjugate.

[0411] The format of all molecules is similar to the molecules shown in Table 2.

[0412] Figure 10 . Ex vivo analysis of human T cells from humanized mice. Mice were injected with equimolar doses of split molecules, and blood was collected and analyzed 24 hours after treatment to evaluate target-independent CD3 conjugate assembly in circulation. Molecules binding to T cells were measured by flow cytometry using a PE-labeled anti-human IgG, Fc-specific secondary antibody. The median fluorescence intensity of CD4+ (A) and CD8+ T cells (B) in blood from animals treated with uncomplemented compounds based on C22 and 40G5c as CD3 conjugates is shown.

[0413] P1AF6814 + P1AF6818: HER2-targeted split molecule with uncomplemented C22 conjugate.

[0414] P1AF9516 + P1AF9518: HER2-targeted split molecule with uncomplemented 40G5c conjugate.

[0415] The format of all molecules is similar to the molecules shown in Table 2.

[0416] Examples

[0417] The following are examples of the methods and compositions of the present invention. It should be understood that various other aspects can be practiced given the general description provided above.

[0418] Example 1. Generation of split molecules with or without V-complementation.

[0419] 1.1 Construction of expression plasmids for split molecules

[0420] For the expression of split constructs as reported herein, a transcription unit containing the following functional elements is used:

[0421] - Immediate early enhancer and promoter from human cytomegalovirus (P-CMV), including intron A,

[0422] - Human heavy chain immunoglobulin 5'-untranslated region (5'UTR),

[0423] - Mouse immunoglobulin heavy chain signal sequence,

[0424] - Nucleic acid encoding the corresponding fusion polypeptide, and

[0425] - Bovine growth hormone polyadenylation sequence (BGH pA).

[0426] In addition to the expression unit / cassette containing the desired gene, the basic / standard mammalian expression plasmid contains

[0427] - Origin of replication from vector pUC18, which allows the plasmid to replicate in Escherichia coli, and

[0428] - β-lactamase gene, which confers ampicillin resistance in Escherichia coli.

[0429] 1.2 Expression of split molecules

[0430] Transient expression of split molecules is carried out using transfection reagent-free 293 (Novagen) or ExpiFectamine TM 293 transfection kit (ExpiFectamine TM 293 reagent, ExpiFectamine TM 293 transfection enhancer 1 and 2) in suspension-adapted HEK293F (FreeStyle 293-F cells; Invitrogen) cells or Expi293 (Expi293F TM cells; Thermofisher Scientific).

[0431] After thawing in a 125 ml shake flask, the cells are passaged at least four times by dilution (volume 30 ml) (incubated / shaken at 37 °C, 7% CO2, 85% humidity, 135 rpm). The cells are expanded to 3x10 5 cells / ml in a 250 ml volume. Three days later, the cells are harvested and re-inoculated at a density of 1.5x10 6 to 7x10 5 cells / ml in a 1 L shake flask in a 250 ml volume. After 24 hours, at approximately 1.4 - 3.0x10 6Transfection was carried out at a cell density of cells / ml.

[0432] Before transfection, 250 μg of plasmid-DNA was diluted to a final volume of 10 ml with pre-warmed (water bath; 37 °C) Opti-MEM (Gibco). The solution was gently mixed and incubated at room temperature for up to 5 minutes. For transfection with ExpiFectamine TM For transfection with ExpiFectamine TM 293 reagent was added to 12.5 ml of OptiMEM solution and incubated for an additional 5 minutes in a separate tube. Subsequently, the two solutions were combined, gently mixed, and incubated at room temperature for 15 - 20 minutes, and the complete mixture was added to a 1 L shake flask with 250 ml of Expi293 cell culture. For transfection without 293, 333.3 μl of transfection reagent without 293 was added directly to the DNA-OptiMEM solution. The resulting solution was gently mixed and incubated at room temperature for 15 - 20 minutes. The entire volume of the mixture was added to a 1 L shake flask with 250 ml of HEK293F culture.

[0433] Incubation was carried out by shaking the flask at 37 °C, 7% CO2, 85% humidity, 135 rpm for 6 to 7 days.

[0434] The supernatant was harvested by a first centrifugation step at 2,000 rpm, 4 °C, for 10 minutes. Subsequently, the supernatant was transferred to a new centrifuge bottle for a second centrifugation at 4,000 rpm, 4 °C, for 20 minutes. Thereafter, the cell-free supernatant was filtered through a 0.22 μm bottle-top filter and stored in the refrigerator (-20 °C).

[0435] 1.3 Purification of split molecules

[0436] The culture supernatant containing the split molecule is filtered and purified by two chromatographic steps. The antibody is captured by affinity chromatography using HiTrap MabSelect SuRe (GE Healthcare) equilibrated with PBS (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl), pH 7.4. Unbound proteins are removed by washing with the equilibration buffer, and the split molecule is recovered with 50 mM citrate buffer (pH 2.8) and immediately neutralized to pH 6.0 with 1 M Tris base (pH 9.0) after elution. Alternatively, the split molecule is eluted from the MabSelect SuRe column with 100 mM acetic acid (pH 3.0) and adjusted to pH 5.5. The protein sample recovered from affinity chromatography is analyzed by analytical SEC, and additional purification steps are performed according to the impurity profile. As an additional purification step, an ion exchange column POROS XS or POROS HS50, or a hydroxyapatite column Macro Prep CHT I type (BioRad), or a hydrophobic interaction chromatography (HIC) column TSkgel Ether-5PW (Tosoh Bioscience, Griesheim) is used. For hydroxyapatite chromatography, the MabSelect SuRe eluate is diluted with 10 mM sodium citrate pH 5.0 (1:5 vol / vol) and the pH is adjusted to 7.5 with 2 M Tris / HCl (H 9.0), or it is dialyzed against 50 mM acetate buffer (pH 7.5). After adding a small amount of calcium chloride, the protein solution is loaded onto a Macro Prep column equilibrated in 25 mM HEPES, 5 mM Na2HPO4, 50 mM NaCl, 0.1 mM CaCl2, 100 mM MES (pH 6.8) and eluted with a gradient of the same buffer with a final concentration of 1500 mM NaCl. On POROS XS ion exchange chromatography, the split protein is purified with a salt gradient of 0 to 550 mM NaCl in 20 mM His / His-HCl buffer at pH 5.5. Alternatively, the VL fusion protein is preferably prepared using a pH / NaCl gradient from pH 4.5 to 5 and 125 mM to 600 mM NaCl in 40 mM sodium acetate buffer for binding. The VH fusion protein is preferably prepared on a POROS XS column with a salt gradient of 5 to 500 mM NaCl in 20 mM Na-phosphate buffer (pH 5.5). For hydrophobic interaction chromatography, 40 mM sodium acetate buffer (pH 5.5) containing 1.5 M (NH4)2SO4 is used and elution is achieved with a shallow gradient towards salt-free content over 50 column volumes. In all cases, Superdex 200 is used. TMSize exclusion chromatography on a column (GE Healthcare) was used as a polishing step. Size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. Finally, the solution containing the split molecules was concentrated using an Ultrafree-CL centrifugal filter unit equipped with a Biomax-SK membrane (Millipore, Billerica, MA) and stored at -80 °C.

[0437] 1.4 Mass spectrometry analysis of split molecules

[0438] PNGase F was obtained from Roche Diagnostics GmbH (14.3 U / μl; solution in sodium phosphate, EDTA, and glycerol). Before digestion, it was freshly reconstituted from the lyophilized product to specifically cleave proteases in the hinge region of IgG antibodies.

[0439] Enzymatic deglycosylation with PNGase F

[0440] 50 μg of split molecules were diluted to a final concentration of 0.5 mg / ml with 10 mM sodium phosphate buffer (pH 7.1) and deglycosylated with 1 μl of PNGase F at 37 °C for 16 hours.

[0441] ESI-QTOF mass spectrometry

[0442] Subsequently, the digested samples were analyzed by LC-MS. Liquid chromatography was performed on a Waters Acquity UPLC (Waters) using a reversed-phase C18 column (Agilent PLRP-S column, 2.1 x 150 mm, 8 μm, (Agilent, Cat.-Nr.: PL1912-3802)). The aqueous mobile phase (mobile phase A) contained 0.1% (v / v) formic acid (FA) in HPLC-grade water. The organic mobile phase (mobile phase B) contained 0.1% FA in acetonitrile. The gradient used in this experiment is plotted in Table 1.

[0443] Table 1. Gradient for LC-MS.

[0444] Step (n) Time (min) % A1 (FA / water) % B1 (FA / acetonitrile) 1 0 90 10 2 1 75 25 3 16 55 45 4 17 5 95 5 19 5 95 6 19.5 90 10 7 21 90 10

[0445] Further chromatographic settings were as follows:

[0446] - Flow rate: 0.6 mL / min

[0447] - Temperature of the column oven: 75 °C

[0448] - Injection volume: 8 μl

[0449] The UPLC was coupled with an ESI-QTOF MS instrument (maXis 4G UHR-QTOF MS system (Bruker Daltonik)). Calibration was performed using sodium iodide. For the digested split molecules, data acquisition was carried out at 800 - 4000 m / z (isCID: 85 eV). The raw mass spectra were evaluated and converted into individual relative molar masses. For the visualization of the results, dedicated software was used to generate deconvoluted mass spectra.

[0450] The resulting split molecules are shown in Table 2. The molecules contain or do not contain complementary domains.

[0451] Table 2. Split molecules with (shaded) or without (non-shaded) V-complementarity.

[0452]

[0453] The purification yields of the various split molecules are shown in Table 3.

[0454] Table 3. Purification yields of split molecules with V-complementarity (shaded) or without V-complementarity (non-shaded).

[0455]

[0456]

[0457] For the CD3 binder V9, the complementarity of the individual VH and VL domains with the homologous DP47 V domain significantly increased the yield of the split construct. In particular, the purification yield of the split VH construct of V9 increased from 0.8 mg / L of the non-complementary split construct P1AE6819 to 20.8 mg / L of the DP47-complementary construct P1AF3870.

[0458] The V-complementarity of the CD3 binder P035.093 with the homologous V domain of DP47 decreased the purification yield to some extent, but significantly increased the stability and developability of the split P035.093 construct (see Example 2).

[0459] For 40G5c, the V-complementarity with DP47 greatly improved the purification process, since the non-complementary split constructs P1AA9518 and P1AA9516 could only be purified in sufficient yield by hydroxyapatite chromatography, while the V-complementary constructs P1AF3865 and P1AF3867 did not require such a delicate purification method to obtain sufficient amounts.

[0460] Example 2. Developability of split molecules.

[0461] 2.1 Physicochemical characterization

[0462] Apparent hydrophobicity

[0463] Apparent hydrophobicity was evaluated by determining the retention time on a hydrophobic HPLC column compared to known high- and low-hydrophobicity standard molecules.

[0464] PK prediction

[0465] The interaction with immobilized FcRn and heparin was evaluated on a specific HPLC column. The corresponding retention times were compared to established thresholds based on molecules known to have good PK and poor PK.

[0466] Thermal stability and aggregation propensity

[0467] Molecules were exposed to elevated temperature under a controlled gradient and the aggregation propensity (T agg , by SLS) was determined.

[0468] Results for apparent hydrophobicity, PK prediction, thermal stability, and aggregation propensity are shown in Table 4 below.

[0469] 2.2 Early stability assessment

[0470] Storage

[0471] Molecules were stored under conditions simulating physiology and shelf life, indicating conditions over a long period at relevant temperatures (physiological and stress conditions). Subsequent analysis focused on changes observed in the stored samples compared to untreated controls.

[0472] Analysis

[0473] The aggregation of molecules was evaluated using a SE-HPLC apparatus under conditions suitable for the molecule being evaluated. Fragmentation of molecules was evaluated using capillary gel electrophoresis. The functional integrity of molecules was evaluated using a specific binding assay against the target of the molecule.

[0474] Results of the early stability assessment are shown in Table 5 below.

[0475] Table 4. Apparent hydrophobicity, PK prediction, thermal stability, and aggregation propensity of split molecules with (shaded) or without (non-shaded) V-complementation. A = P1AA9516. B = P1AA9518, C = P1AE6814, D = P1AF2484, E = P1AF3868, F = P1AF3869, G = P1AF3870, H = P1AF3871.

[0476]

[0477] Table 5. Early stability assessment of split molecules with or without V-complementation (shaded or unshaded). A = P1AA9516. B = P1AA9518, C = P1AE6814, D = P1AF2484, E = P1AF3868, F = P1AF3869, G = P1AF3870, H = P1AF3871.

[0478]

[0479]

[0480] The thermal stability and stress data of constructs with a 40G5c split CD3 domain meet the acceptable criteria for the developability analysis of non-complementary split constructs P1AA9518 and P1AA9516 that no longer require V-complementation.

[0481] Compared with non-complementary split constructs P1AE6814 and P1AF2484, the V-complementation of P035.093 split constructs (P1AF3868 and P1AF3869) with DP47 significantly improved the developability and properties, especially in terms of apparent hydrophobicity and stress stability determined by SEC.

[0482] Example 3. Functional characterization of split molecules.

[0483] 3.1 Method

[0484] Flow cytometry binding on human T cells

[0485] Assess the binding of split molecules to human T cells. For complementary VH / VL pairs of split molecules, the molecules in RPMI + 10% FBS were titrated and added to the cells at an equimolar ratio. The plates were incubated at 4 °C for 60 minutes in the dark. To remove unbound molecules, the plates were washed twice with cold PBS and the cell pellet was resuspended in cold FACS buffer containing a PE-conjugated anti-human Fcγ-specific goat IgG F(ab)2 fragment (Jackson ImmunoResearch) as a secondary detection antibody and Zombie Aqua (Biolegend) for identifying live cells. The plates were incubated at 4 °C for 30 minutes in the dark, washed twice with cold PBS and resuspended in FACS buffer.

[0486] Cells were acquired using a FORTESSA flow cytometer (Becton Dickinson) and an automated HTS plate handling system.

[0487] Data was analyzed using FlowJo v10.8.1 (FlowJo LLC) applicable to PCs, Microsoft Excel (Microsoft Office Standard 2016), and TIBCO SpotFire v10.10.4 (TIBCO Software Inc).

[0488] T cell-mediated tumor cell killing

[0489] The molecule was tested in a tumor cell killing assay using freshly isolated human CD3+ T cells co-incubated with target cells. Tumor cell lysis was determined by quantifying extracellular protease activity released into the supernatant by apoptotic or necrotic cells as described below.

[0490] Target cells were detached using trypsin (Gibco), washed once with PBS and resuspended at a density of 0.2 mio cells / ml in growth medium (RPMI 1640 (Gibco) containing 10% FBS, 1% GlutaMax (Gibco)). 100 μl of the cell suspension (containing 20,000 cells) was seeded into 96-well flat-bottom plates and incubated overnight at 37 °C in an incubator. After Ficoll separation of blood from healthy donors, CD3+ T cells were isolated from PBMCs and viability was checked. Antibodies were diluted in assay medium at the indicated concentrations and the molecule was added to the target cells.

[0491] Assay medium was added to the appropriate wells to achieve equal volumes in each well. The isolated CD3+ T cells were resuspended at a density of 4 mio cells / ml and 50 μl was added to each well, resulting in 200,000 cells / well (E:T 10:1). To determine spontaneous death cell protease release, CD3+ effector cells and target cells were co-incubated as a negative control.

[0492] The assay was incubated for a total of 72 h at 37 °C, 5% CO2. As indicated in the figure, dead cell protease activity measurements were performed 24 h, 48 h, and 72 h after the start of the assay. For this, CytoTox-Glo TM Cytotoxicity Assay (Promega, #G9291) was adjusted to room temperature. 50 μl of the supernatant from each well was transferred to a 96-well white flat-bottom plate for analysis. Subsequently, 25 μl of substrate was added to each well and after incubation at room temperature for 15 minutes, luminescence was measured using a Perkin Elmer 2104 instrument.

[0493] Quantification of antigen density on target cells

[0494] With BD QuantibriteTM Flow cytometry was used to estimate the antibody binding per cell with beads (Becton Dickinson #340495) and a monoclonal PE-conjugated antibody targeting human CD340 (HER2) (Biolegend #324406). 200,000 cells from each cell line were harvested and washed twice with 200 μl of FACS buffer. Diluted antibodies (1:100) were prepared in FACS buffer. The cells were centrifuged and resuspended in 100 μl of the antibody solution and then incubated at 4 °C for 30 minutes. 100 μl of FACS buffer was added before centrifugation and the cells were washed twice with 200 μl of FACS buffer. Samples were then collected on a BD LSRFortessa TM cell analyzer together with BD Quantibrite TM beads. Calculations and analysis were performed according to the manufacturer's protocol. Data were analyzed using FlowJo v10.8.1 for PC (FlowJo LLC), Microsoft Excel (Microsoft Office Standard 2016), and TIBCO SpotFire v10.10.4 (TIBCO Software Inc).

[0495] Ex vivo analysis of human T cells from treated humanized mice

[0496] In one study, NSG-huNSG mice were shipped from Charles River and transferred internally with human stem cells according to an in-house protocol. When the tumors reached approximately 360 mm 3 in size, the complementary VH / VL pairs of the split molecule were injected intravenously into the humanized mice. The VH and VL molecules were administered at a dose of 1.53 to 1.71 mg / kg of the split molecule in 100 μl of vehicle buffer (20 mM histidine, 140 mM NaCl, pH 6.0) with an interval of approximately 20 minutes between the VH and VL molecule administrations, for a total injection volume of 200 μl. As a negative control, 200 μl of vehicle buffer was injected. In another experiment, CD34-transplanted NSG mice were shipped from Jackson Laboratories and injected with the complementary VH / VL pairs of the split molecule (2.5 to 2.8 mg / kg, 100 μl per molecule) as described above.

[0497] In both studies, blood was harvested from n = 3 to 4 mice 24 hours after treatment administration and collected in heparin tubes. Red blood cell lysis was performed on the blood samples and single cells were stained for human CD45, CD8a, CD25, CD69, CD4, and PE-labeled anti-human IgG (for detection of the therapeutic molecule bound to CD4+ and CD8+ T cells). Samples were collected on a BD LSRFortessa cell analyzer.

[0498] 3.2 Results

[0499] As Figure 2 shown, non-complementary molecular pairs consisting of assembled split V9(A) and split P035.093(B) bind to CD3+ Jurkat T cells in the absence of target cells, while CD3 conjugates 40G5c(C) and C22(D) do not bind to CD3+ Jurkat T cells. Importantly, complementation of split P035.093(A) and split V9(B) prevents assembly on T cells in the absence of target tumor cells.

[0500] As Figure 3 shown, treatment with split pairs (HER2+HER2) and positive control (non-split HER2 x CD3 T cell bispecific antibody (with bivalent HER2 binding and monovalent CD3 binding)) resulted in potent T cell-mediated killing of SKOV-3 tumor cells after 48 hours of co-incubation, while individual prodrugs showed no activity. Although split complementation reduced potency compared to non-complementary pairs, the results indicate that the split pairs tested (including complementary split pairs) are capable of inducing T cell-mediated tumor cell killing when both molecules bind to target cells and assemble an active CD3 conjugate. It also shows that the prodrug remains inactive in the absence of its cognate partner.

[0501] Split pairs with non-complementary CD3 conjugate P035.093 are shown in Figure 4 (A), where T cell-mediated tumor cell killing is caused by the production pair, but also by a pair of HER2 and an unrelated conjugate. The results show that assembly of the active complex can be caused by non-complementary P035.093, although only one of the two molecules can bind to tumor cells. As Figure 4 (B) shown, this unwanted assembly can be prevented by complementation. In fact, the HER2+HER2 complementary pair induces T cell-mediated tumor cell killing, while the complementary HER2+unrelated conjugate pair does not induce T cell-mediated tumor cell killing. These results show the importance of P035.093 complementation in restricting activity on target cells bound by two molecules, although this comes at the cost of a slightly reduced potency.

[0502] Split pairs with non-complementary CD3 conjugate 40G5c are shown in Figure 5As shown in (A), T cell-mediated tumor cell killing is caused by the production of pairs (HER2+HER2), but also by a pair of HER2 and an unrelated conjugate, although this occurs only to a limited extent at high concentrations. This result indicates that when both molecules can bind to the tumor cell, assembly of the active complex can occur with non-complementary 40G5c pairs, whereas if only one molecule can bind to the target cell, assembly of the functional complex occurs to a much lower extent. In Figure 5 Similar results for the CD3 conjugate C22 can be observed in (B). In summary, Figure 4 and Figure 5 the data in show that assembly of the functional complex may vary depending on the VH and VL modules and whether complementarity is used. This provides an opportunity for engineering to modulate the forces driving complex assembly.

[0503] As shown in Figure 6 (A), when both molecules can bind to the target cell, the split pair with the complementary CD3 conjugate P035.093 confirmed T cell-mediated target cell killing activity, whereas when only one molecule could bind to the target cell, no killing activity was observed. Assembly of the active complex does not occur on target cells with low antigen density, as shown in Figure 6 (B,C). In fact, this places additional requirements on the production assembly of the active complex. Not only do both molecules need to bind to the target cell, but sufficient antigen density is required to trigger T cell activation and target cell killing. The density threshold requirement can provide an additional safety benefit by restricting activity on cells expressing low levels of both targets, since the relevant targets for T cell redirection in oncology are often observed on healthy tissues, albeit at lower levels than on tumor cells.

[0504] As Figure 7 (B) shows, non-complementary split molecules may or may not be active against cells with low target expression, depending on their CD3 conjugates, although they are active against cells with high target expression ( Figure 7 (A)).

[0505] Ex vivo analysis of human T cells sampled from humanized mice 24 hours after treatment with the split pair showed in Figure 8 that non-complementary pairs using P035.093 as the CD3 conjugate can assemble without binding to the target pair and bind to CD3 on circulating CD4+ T cells (A) and CD8+ T cells (B) in the blood. Complementarity of the P035.093 CD3 module prevents assembly on T cells in circulation.

[0506] Ex vivo analysis of human T cells sampled from humanized mice 24 hours after treatment with the split pair showed in Figure 9It is shown that non-complementary pairs using V9 as a CD3 binder can assemble without binding to the target pair and bind to CD3 on circulating CD4+ T cells (A) and CD8+ T cells (B) in the blood. Complementation of the V9 CD3 module greatly reduces assembly on circulating T cells. In summary, Figure 8 and Figure 9 it is shown that when the molecule is not bound to its target pair, complementation of the split CD3 improves the behavior of the split compound in circulation to limit assembly on T cells.

[0507] Ex vivo analysis of human T cells sampled from humanized mice after 24 hours of treatment with the split pair is shown in Figure 10 that other CD3 binders (such as 40G5c and C22) do not require complementation to prevent binding to CD3 on circulating CD4+ T cells (A) and CD8+ T cells (B) in the blood.

[0508] ***

[0509] Although the present invention has been described in considerable detail by way of illustration and example for purposes of clarity of understanding, such description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are hereby expressly incorporated by reference in their entireties.

Claims

1. A binding molecule pair, comprising (a) a first binding molecule comprising (i) a first antigen-binding domain capable of binding to a target antigen, (ii) a first portion of an effector domain, and (iii) a first complementary domain capable of associating with the first portion of the effector domain; and (b) a second binding molecule comprising (i) a second antigen-binding domain capable of binding to a target antigen, (ii) a second portion of an effector domain, and (iii) a second complementary domain capable of associating with the second portion of the effector domain; wherein if the first antigen-binding domain and the second antigen-binding domain bind to their target antigen on the surface of a cell, the first portion and the second portion of the effector domain are capable of associating with each other to form a functional effector domain, wherein the first complementary domain and the second complementary domain associate with the first portion and the second portion of the effector domain, respectively, and the first portion and the second portion of the effector domain do not associate with each other.

2. The binding molecule pair according to claim 1, wherein the effector domain is an antigen-binding domain.

3. The binding molecule pair according to claim 1 or 2, wherein the effector domain is an anti-CD3 antigen-binding domain.

4. The binding molecule pair according to any one of the preceding claims, wherein the functional effector domain is capable of binding to an antigen.

5. The binding molecule pair according to claim 4, wherein the antigen is a T cell antigen, particularly an activating T cell antigen.

6. The binding molecule pair according to claim 4 or 5, wherein the antigen is CD3, particularly CD3ε.

7. The binding molecule pair according to any one of the preceding claims, wherein the first portion of the effector domain comprises a heavy chain variable region (VH) and the second portion of the effector domain comprises a light chain variable region (VL).

8. The binding molecule pair according to claim 7, wherein the VH comprises heavy chain complementarity determining regions (HCDRs) 1 of SEQ ID NO:15, HCDR 2 of SEQ ID NO:16, and HCDR 3 of SEQ ID NO:17, and the VL comprises light chain complementarity determining regions (LCDRs) 1 of SEQ ID NO:19, LCDR 2 of SEQ ID NO:20, and LCDR 3 of SEQ ID NO:

21.

9. The binding molecule pair according to claim 8, wherein the VH 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: 18, and / or the VL 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:

22.

10. The binding molecule pair according to claim 7, wherein the VH comprises heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 23, HCDR 2 of SEQ ID NO: 24, and HCDR 3 of SEQ ID NO: 25, and the VL comprises light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 27, LCDR 2 of SEQ ID NO: 28, and LCDR 3 of SEQ ID NO:

29.

11. The binding molecule pair according to claim 10, wherein the VH 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: 26, and / or the VL 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:

30.

12. The binding molecule pair according to claim 7, wherein the VH comprises heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 31, HCDR 2 of SEQ ID NO: 32, and HCDR 3 of SEQ ID NO: 33, and the VL comprises light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 35, LCDR 2 of SEQ ID NO: 36, and LCDR 3 of SEQ ID NO:

37.

13. The binding molecule pair according to claim 12, wherein the VH 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: 34, and / or the VL 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:

38.

14. The binding molecule pair according to claim 7, wherein the VH comprises heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 23, HCDR 2 of SEQ ID NO: 24, and HCDR 3 of SEQ ID NO: 60, and the VL comprises light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 27, LCDR 2 of SEQ ID NO: 28, and LCDR 3 of SEQ ID NO:

29.

15. The binding molecule pair according to claim 14, wherein the VH 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: 61, and / or the VL 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:

30.

16. The binding molecule pair according to any one of the preceding claims, wherein the first complementary domain and the second complementary domain cannot form a functional effector domain with a part of the effector domain.

17. The binding molecule pair according to any one of the preceding claims, wherein the first complementary domain comprises VL and the second complementary domain comprises VH.

18. The binding molecule pair according to claim 17, wherein the VH and the VL are non-antigen-binding.

19. The binding molecule pair according to claim 17 or 18, wherein the VH comprises heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 47, HCDR 2 of SEQ ID NO: 48, and HCDR 3 of SEQ ID NO: 49, and the VL comprises light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 51, LCDR 2 of SEQ ID NO: 52, and LCDR 3 of SEQ ID NO:

53.

20. The binding molecule pair according to claim 19, wherein the VH 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: 50, and / or the VL 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:

54.

21. The binding molecule pair according to any one of the preceding claims, wherein the first part of the effector domain comprises a first VH and the first complementary domain comprises a first VL, and the second part of the effector domain comprises a second VL and the second complementary domain comprises a second VH.

22. The binding molecule pair according to claim 21, wherein each and / or any of the second VH and the first VL and / or the first VH and the second VL, in particular each of the second VH and the first VL, comprises an amino acid substitution, wherein the amino acid residue is substituted with a charged replacement amino acid residue, wherein (i) the replacement amino acid residues in the VH and the VL have opposite charges, or (ii) the replacement amino acid residues in the VH and the VL have the same charge.

23. The binding molecule pair according to any one of the preceding claims, wherein the first binding molecule comprises a third antigen-binding domain capable of binding to a target antigen, and / or the second binding molecule comprises a fourth antigen-binding domain capable of binding to a target antigen.

24. The binding molecule pair according to any one of the preceding claims, wherein the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain (when present) and / or the fourth antigen-binding domain (when present) are antigen-binding domains selected from the group consisting of: Fv molecules, scFv molecules, Fab molecules and single-domain antibodies.

25. The binding molecule pair according to any one of the preceding claims, wherein the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain (when present) and / or the fourth antigen-binding domain (when present) are Fab molecules.

26. The binding molecule pair according to any one of the preceding claims, wherein the first antigen-binding domain and the second antigen-binding domain bind to the same target antigen.

27. The binding molecule pair according to any one of the preceding claims, wherein the first antigen-binding domain and the second antigen-binding domain bind to the same target antigen, and the third antigen-binding domain (when present) and the fourth antigen-binding domain (when present) bind to the same target antigen, wherein the target antigen bound by the first antigen-binding domain and the second antigen-binding domain is different from the target antigen bound by the third antigen-binding domain (when present) and the fourth antigen-binding domain (when present).

28. The binding molecule pair according to any one of claims 1 to 25, wherein the first antigen-binding domain and the second antigen-binding domain bind to different target antigens.

29. A binding molecule pair according to any one of claims 1 to 25 or 28, wherein the first antigen-binding domain and the third antigen-binding domain (when present) bind to the same target antigen, and the second antigen-binding domain and the fourth antigen-binding domain (when present) bind to the same target antigen, wherein the target antigen bound by the first antigen-binding domain and the third antigen-binding domain (when present) is different from the target antigen bound by the second antigen-binding domain and the fourth antigen-binding domain (when present).

30. A binding molecule pair according to any one of the preceding claims, wherein the target antigens of the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain (when present) and the fourth antigen-binding domain (when present) are tumor antigens.

31. A binding molecule pair according to any one of the preceding claims, wherein the target antigen of the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain (when present) and / or the fourth antigen-binding domain (when present) is HER2.

32. A binding molecule pair according to any one of the preceding claims, wherein the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain (when present) and / or the fourth antigen-binding domain (when present) comprise a heavy chain variable region (VH) and a light chain variable region (VL).

33. A binding molecule pair according to claim 32, wherein the VH comprises heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:39, HCDR 2 of SEQ ID NO:40 and HCDR 3 of SEQ ID NO:41, and the VL comprises light chain complementarity determining region (LCDR) 1 of SEQ ID NO:43, LCDR 2 of SEQ ID NO:44 and LCDR 3 of SEQ ID NO:

45.

34. A binding molecule pair according to claim 33, wherein the VH 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:42, and / or the VL 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:

46.

35. A binding molecule pair according to any one of the preceding claims, wherein the first binding molecule comprises a first Fc domain composed of a first subunit and a second subunit, and / or the second binding molecule comprises a second Fc domain composed of a first subunit and a second subunit.

36. The binding molecule pair according to claim 35, wherein the Fc domain is an IgG Fc domain, particularly an IgG1 Fc domain.

37. The binding molecule pair according to claim 35 or 36, wherein the Fc domain is a human Fc domain.

38. The binding molecule pair according to any one of claims 35 to 37, wherein the Fc domain is a human IgG1 Fc domain.

39. The binding molecule pair according to any one of claims 35 to 38, wherein the Fc domain comprises a modification that promotes the association of the first subunit and the second subunit of the Fc domain.

40. The binding molecule pair according to any one of claims 35 to 39, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector functions.

41. The binding molecule pair according to any one of the foregoing claims, wherein the first binding molecule comprises (i) a first antigen-binding domain and an optional third antigen-binding domain, (ii) an Fc domain composed of a first subunit and a second subunit, (iii) a first part of an effector domain, and (iv) a first complementary domain, wherein (a) the first antigen-binding domain and the third antigen-binding domain (when present) are fused at their C-terminus to the N-terminus of one of the subunits of the Fc domain, (b) the first part of the effector domain is fused at its N-terminus to the C-terminus of one of the subunits of the Fc domain, (c) the first complementary domain is fused at its N-terminus to the C-terminus of the first part of the effector domain; and the second binding molecule comprises (i) a second antigen-binding domain and an optional fourth antigen-binding domain, (ii) an Fc domain composed of a first subunit and a second subunit, (iii) a second part of an effector domain, and (iv) a second complementary domain, wherein (a) the second antigen-binding domain and the fourth antigen-binding domain (when present) are fused at their C-terminus to the N-terminus of one of the subunits of the Fc domain, (b) the second part of the effector domain is fused at its N-terminus to the C-terminus of one of the subunits of the Fc domain, (c) The second complementary domain is fused at its N-terminus to the C-terminus of the second part of the effector domain.

42. A binding molecule pair according to any one of the preceding claims, wherein the first part of the effector domain and the first complementary domain and / or the second part of the effector domain and the second complementary domain are fused via a peptide linker.

43. A binding molecule pair according to claim 42, wherein the peptide linker comprises the sequence of SEQ ID NO:

56.

44. A binding molecule that forms part of a binding molecule pair according to any one of the preceding claims.

45. An isolated polynucleotide encoding a binding molecule pair according to any one of claims 1 to 43 or a binding molecule according to claim 44.

46. A host cell comprising the isolated polynucleotide according to claim 45.

47. A method for producing a binding molecule (pair), the method comprising the steps of: (a) Culturing the host cell according to claim 46 under conditions suitable for expressing the binding molecule(s), and optionally (b) recovering the binding molecule(s).

48. A pharmaceutical composition comprising a binding molecule pair according to any one of claims 1 to 43 or a binding molecule according to claim 44 and a pharmaceutically acceptable carrier.

49. A binding molecule pair according to any one of claims 1 to 43, a binding molecule according to claim 44 or a pharmaceutical composition according to claim 48 for use as a medicament.

50. A binding molecule pair according to any one of claims 1 to 43, a binding molecule according to claim 44 or a pharmaceutical composition according to claim 48 for use in the treatment of a disease.

51. A binding molecule pair, binding molecule or pharmaceutical composition for use according to claim 50, wherein the disease is cancer.

52. Use of a binding molecule pair according to any one of claims 1 to 43, a binding molecule according to claim 44 or a pharmaceutical composition according to claim 48 in the manufacture of a medicament.

53. Use of a binding molecule pair according to any one of claims 1 to 43, a binding molecule according to claim 44 or a pharmaceutical composition according to claim 48 in the manufacture of a medicament for the treatment of a disease.

54. Use according to claim 53, wherein the disease is cancer.

55. A method for treating a disease in an individual, the method comprising administering to the individual an effective amount of a binding molecule pair according to any one of claims 1 to 43.

56. The method according to claim 55, wherein the disease is cancer.

57. An article (kit) intended for the treatment of a disease, the article (kit) comprising: (i) A container comprising a pharmaceutical composition, wherein the pharmaceutical composition comprises a binding molecule pair according to any one of claims 1 to 43 and an optional pharmaceutical carrier, and optionally (ii) a label or package insert comprising instructions for using the pharmaceutical composition in the treatment of the disease.

58. An article (kit) intended for the treatment of a disease, the article (kit) comprising: (i) A first container comprising a first pharmaceutical composition, wherein the first pharmaceutical composition comprises the first binding molecule of the binding molecule pair according to any one of claims 1 to 43 and an optional pharmaceutical carrier; (ii) A second container comprising a second pharmaceutical composition, wherein the second pharmaceutical composition comprises the second binding molecule of the binding molecule pair according to any one of claims 1 to 43 and an optional pharmaceutical carrier; and optionally (iii) a label or package insert comprising instructions for using the first pharmaceutical composition and the second pharmaceutical composition in combination in the treatment of the disease.

59. The article according to claim 57 or 58, wherein the disease is cancer.

60. A method for forming a functional effector domain, the method comprising contacting a pair of binding molecules according to any one of claims 1 to 43 with a cell expressing the target antigen of the first antigen-binding domain and the second antigen-binding domain, the contacting being carried out under conditions that permit the first antigen-binding domain and the second antigen-binding domain to bind to their target antigen on the surface of the cell.

61. The present invention as described above.

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