BR122021001361B1 - MULTI-SPECIFIC ANTIGEN BINDING MOLECULE - Google Patents

Abstract

The present invention provides multispecific antigen binding molecules and uses thereof. Multispecific antigen-binding molecules comprise a first antigen-binding domain that specifically binds a target molecule, and a second antigen-binding domain that specifically binds an internalizing effector protein. The multispecific antigen-binding molecules of the present invention may, in some embodiments, be bispecific antibodies that are capable of binding both a target molecule and an internalizing effector protein. In certain embodiments of the invention, simultaneous binding of the target molecule and the internalizing effector protein by the multispecific antigen binding molecule of the present invention results in attenuation of the activity of the target molecule to a greater degree than binding of the target molecule alone. In other embodiments of the invention, the target molecule is a tumor-associated antigen, and the simultaneous binding of the tumor-associated antigen and the internalizing effector protein by the multispecific antigen-binding molecule of the present invention causes or facilitates targeted killing of tumor cells. tumor.

Description

Split order of BR112014022692-0 of 03/13/2013 FIELD OF INVENTION

[001] The present invention relates to the field of therapeutic proteins and, in particular, to the field of therapeutic proteins that are capable of inactivating, blocking, attenuating, eliminating and/or reducing the concentration of one or more target molecules in vitro or in vivo.

BASICS OF THE INVENTION

[002] Therapeutic treatments often require the inactivation or blocking of one or more target molecules that act on a cell or in its vicinity. For example, antibody-based therapeutics often work by binding to a particular antigen expressed on a cell surface or to a soluble ligand, thereby interfering with the normal biological activity of the antigen. Antibodies and other binding constructs directed against various cytokines (e.g., IL-1, IL-4, IL-6, IL-13, IL-22, IL-25, IL-33, etc.), or their respective receptors , for example, have proven useful in treating a wide range of human ailments and diseases. Therapeutic agents of this type typically work by blocking the interaction between the cytokine and its receptor in order to attenuate or inhibit cellular signaling. In certain contexts, however, it would be therapeutically beneficial to inactivate or inhibit the activity of a target molecule in a manner that does not necessarily involve blocking its physical interaction with another component. One way in which such non-blocking attenuation of a target molecule could be achieved would be to reduce the extracellular or cellular surface concentration of the target molecule. Although genetic and nucleic acid-based strategies for reducing the amount or concentration of a given target molecule are known in the art, such strategies are often fraught with substantial technical complications and unintended side effects in therapeutic settings. Therefore, alternative non-blocking strategies are needed to facilitate the inactivation or attenuation of various target molecules for therapeutic purposes.

BRIEF SUMMARY OF THE INVENTION

[003] The present invention is based, at least in part, on the concept of attenuating or inactivating a target molecule by facilitating or making a physical connection between the target molecule and an internalizing effector protein. Through this type of intermolecular physical binding, the target molecule can be forced to be internalized into the cell along with the internalizing effector protein, and processed by the intracellular degrading machinery, or otherwise attenuated, sequestered, or inactivated. This mechanism represents a novel and inventive strategy to inactivate or attenuate the activity of a target molecule without necessarily blocking the interaction between the target molecule and its binding partners.

[004] In this way, the present invention provides a multispecific antigen-binding molecule that is capable of simultaneously binding a target molecule (T) and an internalizing effector protein (E). More specifically, the present invention provides a multispecific antigen-binding molecule comprising a first antigen-binding domain (D1), and a second antigen-binding domain (D2), wherein D1 specifically binds T, and D2 specifically binds E. , and in which the simultaneous binding of T and E by the multispecific antigen-binding molecule attenuates the activity of T to a greater degree than the binding of T by D1 alone. The greater attenuation of T activity may be due to forced internalization/degradation of T through its physical binding to E; however, other mechanisms of action are possible and are not excluded from the scope of the present invention.

[005] Furthermore, the present invention provides methods of using the multispecific antigen binding molecule to inactivate or attenuate the activity of a target molecule (T). In particular, the present invention provides a method for inactivating or attenuating the activity of T by placing T and an internalizing effector protein in contact (E) with a multispecific antigen-binding molecule, wherein the multispecific antigen-binding molecule comprises a first antigen-binding domain (D1) and a second antigen-binding domain (D2), wherein D1 specifically binds T, and wherein D2 specifically binds E; and in which the simultaneous binding of T and E by the multispecific antigen-binding molecule attenuates the activity of T to a greater degree than the binding of T by D1 alone.

[006] In certain embodiments of the present invention, D1 and/or D2 comprise at least one antibody variable region. For example, the multispecific antigen-binding molecule may, in some embodiments, be a bispecific antibody, wherein D1 comprises an antibody heavy and light chain variable region pair (HCVR/LCVR) that specifically binds T, and wherein D2 comprises a HCVR/LCVR pair that specifically binds E. Alternatively, D1 and/or D2 may comprise a peptide or polypeptide that specifically interacts with the target molecule (T) and/or the internalizing effector protein (E). For example, if the target molecule is a cell surface receptor, then D1 may comprise a portion of a ligand that specifically binds the cell surface receptor target molecule. Similarly, if the internalizing effector protein is a cell surface internalizing receptor, then D2 may comprise a portion of a ligand that specifically binds the cell surface internalizing receptor. In certain embodiments, D1 comprises an antibody variable region that specifically binds T, and D2 comprises a peptide or polypeptide that specifically binds E. In still other embodiments, D1 comprises a peptide or polypeptide that specifically binds T, and D2 comprises a variable region of antibody that specifically binds E. In either configuration, however, the end result is that T and E are capable of being physically linked, directly or indirectly, through the simultaneous binding of T and E by a multispecific antigen-binding molecule.

[007] Other modalities will become apparent from a review of the detailed description below.

BRIEF DESCRIPTION OF FIGURES

[008] Figure 1 (panels A-D) provides schematic representations of four general exemplary mechanisms of action for the multispecific antigen-binding molecules of the present invention. In each illustrated configuration, D1 is a first antigen-binding domain; D2 is a second antigen-binding domain; T is a target molecule; And it is an internalizing effector protein; and R is a receptor that internalizes upon E binding. Panel A represents the situation in which both T and E are associated with the membrane. Panel B represents the situation in which T is soluble and E is membrane associated. Panel C represents the situation in which T is membrane associated and E is a soluble protein that interacts with the cell, and is internalized into it, through the interaction of E and R. Panel D represents the situation in which T is soluble and E is a soluble protein that interacts with the cell, and is internalized into it, through the interaction of E and R.

[009] Figure 2 shows the results of an immunoprecipitation experiment performed on two different cells (cell-1 expressing FCYRI alone, and cell-2 expressing Krm2 and FCYRI) after incubation for different time values (0, 15, 30 and 60 minutes) with a multispecific antigen-binding molecule DKK1-mFc.

[0010] Figure 3 shows the relative IL-4-induced luminescence produced by Stat6-luc reporting HEK293 cells in the presence and absence of an anti-IL-4R/anti-CD63 multispecific antigen binding protein ("ab conjugate" ) or control constructs ("control 1" and "control 2") at various concentrations of IL-4.

[0011] Figure 4 shows the results of an experiment carried out in the same way as the experiment shown in figure 3, except that CD63 expression was significantly reduced in the reporting cell line by an siRNA directed against CD63.

[0012] Figure 5 shows the results of an experiment performed in a similar manner to the experiments shown in Figures 3 and 4, except that the reporting cells were incubated with the multispecific antigen binding protein ("Ab conjugate") or control constructs. ("control 1" and "control 2") for 2 hours or overnight before adding IL-4 ligand. The top row of bar graphs represents the results of experiments conducted on cells expressing normal levels of CD63 ("untransfected"), while the bottom row of bar graphs represents the results of experiments conducted on cells in which CD63 expression was significantly reduced. reduced in the reporting cell line by an siRNA directed against CD63.

[0013] Figure 6 shows the results of an experiment performed in a similar manner to the experiments shown in Figures 3 and 4, except that the reporting cells were incubated with the anti-IL-4R/anti-CD63 multispecific antigen binding protein ("Ab conjugate") or control constructs ("control 1" and "control 2") for 15 minutes, 30 minutes, 1 hour, or 2 hours before the addition of IL-4 ligand.

[0014] Figure 7 shows the results of an experiment in which Stat6-luc reporter cells were treated with 10 pM IL-4 in the presence of various dilutions of an anti-IL-4R x anti-CD63 bispecific antibody ("bispecific") , or control constructs (monospecific anti-IL-4R, or false bispecific that binds only IL-4R).

[0015] Figure 8 shows the results of experiments in which HEK293 cells were treated with a SOST construct labeled with a myc tag and a Ph-sensitive tag (which produces a fluorescent signal at low pH), along with various monospecific antibodies and bispecifics as shown. Results are expressed in terms of number of fluorescent spots (i.e., labeled vesicles) per cell. Panel A shows the results after incubation on ice for 3 hours, panel B shows the results after 1 hour of incubation at 37°C, and panel C shows the results after 3 hours of incubation at 37°C.

[0016] Figure 9 shows the results of experiments in which HEK293 cells were treated with fluorescently labeled lipopolysaccharide (LPS) from E. coli (Panel A) or S. minnesota (Panel B), along with an anti-CD63 x bispecific antibody. anti-LPS, control antibodies, or LPS alone, several times, followed by non-internalized (i.e., surface-bound) fluorophore termination. Fluorescent signal therefore reflects internalized LPS under the various conditions shown. Results are expressed in terms of number of fluorescent spots (i.e., labeled vesicles) per cell.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Before the present invention is described, it should be understood that this invention is not limited to the particular experimental methods and conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is intended to describe only particular embodiments, and should not be considered limiting, as the scope of the present invention will be limited only by the appended claims.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning commonly understood by those skilled in the technology to which the invention belongs. As used herein, the expression "about", when used in reference to a particular quoted numerical value, means that the value may vary from the quoted value by no more than 1%. For example, as used herein, the expression "about" about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0019] Although any method and material similar or equivalent to those described here may be used in the practice or testing of the present invention, the preferred methods and materials will now be described.

MULTI-SPECIFIC ANTIGEN BINDING MOLECULES

[0020] The present inventors have surprisingly discovered that an activity of the target molecule can be attenuated by linking the target molecule to an internalizing effector protein through a multispecific antigen binding molecule.

[0021] In this way, the present invention provides multispecific antigen-binding molecules comprising a first antigen-binding domain (also referred to herein as "D1"), and a second antigen-binding domain (also referred to herein as "D2" ). D1 and D2 each bind different molecules. D1 specifically binds a "target molecule". The target molecule is also referred to herein as "T". D2 specifically binds an "internalizing effector protein". The internalizing effector protein is also referred to herein as "E". According to the present invention, the simultaneous binding of T and E by the multispecific antigen binding molecule attenuates the activity of T to a greater degree than the binding of T by D1 alone. As used herein, the term “simultaneous binding” in the context of a multispecific antigen-binding molecule means that the multispecific antigen-binding molecule is capable of contacting both a target molecule (T) and an effector protein. internalizing (E) over at least some period of time under physiologically relevant conditions to facilitate physical binding between T and E. Binding of the multispecific antigen-binding molecule on the T and E components can be sequential, e.g. The multispecific antigen may first bind T and then bind E, or it may first bind E and then bind T. Either way, as long as T and E are both bound by the multispecific antigen-binding molecule for some period of time (independent of the sequential order of binding), the multispecific antigen binding molecule will be considered "simultaneously bound" T and E for purposes of the present disclosure. Without binding by theory, it is believed that the major inactivation of T is caused by the internalization and degrading redirection of T in a cell due to its physical binding to E. The multispecific antigen binding molecules of the present invention are thus used to inactivate and/or reducing the activity and/or extracellular concentration of a target molecule without directly blocking or antagonizing the function of the target molecule.

[0022] According to the present invention, a multispecific antigen-binding molecule may be a single multifunctional polypeptide, or it may be a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with each other. As will be evident from the present disclosure, any antigen-binding construct that has the ability to simultaneously bind a T and an E molecule is considered a multispecific antigen-binding molecule. Any of the multispecific antigen-binding molecules of the invention, or variants thereof, can be constructed using standard molecular biological techniques (e.g., recombinant DNA technology and protein expression), as those skilled in the art will appreciate.

ANTIGEN BINDING DOMAINS

[0023] The multispecific antigen-binding molecules of the present invention comprise at least two separate antigen-binding domains (D1 and D2). As used herein, the term "antigen-binding domain" means any peptide, polypeptide, nucleic acid molecule, scaffold molecule, peptide display molecule, or polypeptide-containing construct that is capable of specifically binding a particular antigen of interest. . The term "specifically binds" or similar, as used herein, means that the antigen-binding domain forms a complex with a particular antigen characterized by a dissociation constant (KD) of 500 pM or less, and does not bind other non-binding antigens. related under ordinary test conditions. "Unrelated antigens" are proteins, peptides or polypeptides that have less than 95% amino acid identity with each other.

[0024] Exemplary categories of antigen-binding domains that can be used in the context of the present invention include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that interact specifically with a particular antigen, proteins comprising a ligand-binding portion of a receptor that specifically binds a particular antigen, antigen-binding scaffolds (e.g., DARPins repeat proteins, HEAT, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins, etc., [see, for example, Boersma and Pluckthun, 2011, Opin 22:849-857, and references cited therein]), and aptamers or moieties. of these.

[0025] In certain embodiments, in which the target molecule or internalizing effector protein is a receptor molecule, an "antigen-binding domain", for the purposes of the present invention, may comprise or consist of a ligand or portion of a ligand which is specific for the receptor. For example, if the target molecule (T) is IL-4R, the D1 component of the multispecific antigen binding molecule may comprise the IL-4 ligand or a portion of the IL-4 ligand. , which is capable of interacting specifically with IL-4R; or, if the internalizing effector protein (E) is a transferrin receptor, the D2 component of the multispecific antigen-binding molecule may comprise transferrin or a portion of transferrin, which is capable to interact specifically with the transferrin receptor.

[0026] In certain embodiments, in which the target molecule or internalizing effector protein is a ligand that is specifically recognized by a particular receptor (e.g., a soluble target molecule), an "antigen-binding domain", for the purposes of of the present invention, may comprise or consist of the receptor or a ligand-binding portion of the receptor. For example, if the target molecule (T) is IL-6, the D1 component of the multispecific antigen-binding molecule may comprise the domain. of IL-6 receptor ligand binding; or, if the internalizing effector protein (E) is an indirectly internalized protein (as that term is defined elsewhere herein), the D2 component of the antigen-binding molecule; multispecific may comprise a ligand-binding domain of a specific receptor for E.

[0027] Methods for determining whether two molecules specifically bind to each other are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antigen-binding domain, as used in the context of the present invention, includes polypeptides that bind a particular antigen (e.g., a target molecule [T] or an internalizing effector protein [E]) or a portion thereof with a KD of less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM , less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.2 pM, less than about of 0.1 pM, or less than about 0.05 pM, as measured in a surface plasmon resonance assay.

[0028] The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows the analysis of interactions in real time by detecting changes in protein concentrations in a biosensor array, e.g. using the BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[0029] The term "KD", as used herein, means the equilibrium dissociation constant of a particular protein-protein interaction (e.g., antibody-antigen interaction). Unless otherwise indicated, the KD values disclosed herein refer to KD values determined by surface plasmon resonance assay at 25 °C.

ANTIBODIES AND ANTIBODY ANTIGEN BINDING FRAGMENTS

[0030] As previously indicated, an "antigen-binding domain" (D1 and/or D2) may comprise or consist of an antibody or antigen-binding fragment of an antibody. The term "antibody" as used herein means any antigen-binding molecule or molecular complex comprising at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., T or E ).The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). It comprises a heavy chain variable region (abbreviated here as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. abbreviated here as LCVR or VL) and a light chain constant region. The light chain constant region comprises a domain (CL1). , dispersed with regions that are more conserved, called structure regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the antibodies of the invention (or their antigen-binding portion) may be identical to human germline sequences, or may be naturally or artificially modified. A consensus amino acid sequence can be defined based on a side-by-side analysis of two or more CDRs.

[0031] The D1 and/or D2 components of the multispecific antigen-binding molecules of the present invention may comprise or consist of antigen-binding fragments of total antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically produced polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody can be derived, for example, from whole antibody molecules using any suitable standard technique such as proteolytic digestion or recombinant gene production techniques involving the manipulation and expression of variable and optionally constant domains of the antibody that encode the DNA. Such DNA is known and/or is readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and/or constant domains in a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids. , etc.

[0032] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a FR3-CDR3-FR4 peptide restricted. Other molecules produced, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc. ), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed by the term “antigen-binding fragment” as used herein.

[0033] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR that is adjacent to or in correct reading position with one or more framework sequences. In antigen-binding fragments with a VH domain associated with a VL domain, the VH and VL domains can be situated relative to each other in any suitable arrangement. For example, the variable region may be dimeric and contain dimers of VH-VH, VH-VL or VL-VL. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0034] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Exemplary non-limiting configurations of variable and constant domains that may be observed in an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be either directly linked to each other or can be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semiflexible link between adjacent variable and/or constant domains in a single molecule. polypeptide. Furthermore, an antigen-binding fragment may comprise a homodimer or heterodimer (or other multimer) of any of the previously listed variable and constant domain configurations in noncovalent association with each other and/or with one or more VH domains or monomeric VL (e.g. by disulfide bond(s)).

[0035] The multispecific antigen binding molecules of the present invention may comprise or consist of human antibodies and/or recombinant human antibodies, or fragments thereof. The term "human antibody" as used herein includes antibodies with variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may, however, include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in CDRs. and in particular CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline species of another mammal, such as a mouse, have been grafted onto the human framework sequences.

[0036] The multispecific antigen-binding molecules of the present invention may comprise or consist of recombinant human antibodies or antigen-binding fragments thereof. The term "recombinant human antibody" as used herein is intended to include all human antibodies that are prepared, expressed, raised or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell. (further described below), antibodies isolated from a recombinant combinatorial human antibody library (further described below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see, e.g., example, Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involve dividing human immunoglobulin gene sequences into other DNA sequences. Such human recombinant antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such human recombinant antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the Recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, cannot naturally exist in the human germline antibody repertoire in vivo.

BIESPECIFIC ANTIBODIES

[0037] According to certain embodiments, the multispecific antigen-binding molecules of the invention are bispecific antibodies; for example, bispecific antibodies comprising an antigen-binding arm that specifically binds a target molecule (T) and an antigen-binding arm that specifically binds an internalizing effector protein (E). Methods for making bispecific antibodies are known in the art and can be used to construct multispecific antigen-binding molecules of the present invention. Exemplary bispecific formats that may be used in the context of the present invention include, without limitation, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, double variable domain (DVD)-Ig, Quadroma, overhangs in holes, chain common light (e.g., common light chain with protrusions in holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, Dual Action Fab (DAF)-IgG, and bispecific formats of Mab2 (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of previous formats).

MULTIMERIZING COMPONENTS

[0038] The multispecific antigen binding molecules of the present invention, in certain embodiments, may also comprise one or more multimerizing components. Multimerizing components may function to maintain the association between antigen-binding domains (D1 and D2). As used herein, a "multimerizing component" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing component of the same or similar structure or constitution. For example, a multimerizing component may be a polypeptide comprising an immunoglobulin CH3 domain. A non-limiting example of a multimerizing component is an Fc portion of an immunoglobulin, for example, an Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group. In certain embodiments, the multimerizing component is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerizing component is a cysteine residue, or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coil-coil motif.

[0039] In certain embodiments, the multispecific antigen-binding molecules of the present invention comprise two multimerizing domains, M1 and M2, in which D1 is attached to M1 and D2 is attached to M2, and in which the association of M1 with M2 facilitates the physical binding of D1 and D2 to each other in a single multispecific antigen-binding molecule. In certain embodiments, M1 and M2 are identical with each other. For example, M1 may be an Fc domain with a particular amino acid sequence, and M2 is an Fc domain with the same amino acid sequence as M1. Alternatively, M1 and M2 may differ from each other at one or more amino acid positions. For example, M1 may comprise a first immunoglobulin (Ig) CH3 domain and M2 may comprise a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other in at least one amino acid, and wherein at least One less amino acid difference reduces binding of the targeting construct to protein A compared to a reference construct with identical M1 and M2 sequences. In one embodiment, the Ig CH3 domain of M1 binds protein A and the Ig CH3 domain of M2 contains a mutation that reduces or abolishes protein A binding such as an H95R modification (by IMGT exon numbering; by IMGT exon numbering). EU H435R). The CH3 of M2 may further comprise a modification of Y96F (by IMGT; by EU Y436F). Additional modifications that can be observed in CH3 of M2 include: D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU) in the case of an IgG1 Fc domain; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I per EU) in the case of an IgG2 Fc domain; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) in the case of an IgG4 Fc domain.

INTERNALIZING EFFECTOR PROTEINS (E)

[0040] In the context of the present invention, the D2 component of the multispecific antigen binding molecule specifically binds an internalizing effector protein ("E"). An internalizing effector protein is a protein that is capable of being internalized into a cell or that otherwise participates in or contributes to retrograde membrane trafficking. In some examples, the internalizing effector protein is a protein that undergoes transcytosis; that is, the protein is internalized from one side of a cell and transported to the other side of the cell (e.g., apical to basal). In many embodiments, the internalizing effector protein is a protein expressed on the cell surface or a soluble extracellular protein. However, the present invention also contemplates embodiments in which the internalizing effector protein is expressed in an intracellular compartment such as the endosome, endoplasmic reticulum, Golgi, lysosome, etc. For example, proteins involved in membrane trafficking retrogradation (e.g., pathways from early/recycling endosomes to the trans-Golgi network) can serve as internalizing effector proteins in various embodiments of the present invention. Either way, binding of D2 to an internalizing effector protein causes the entire multispecific antigen-binding molecule, and any molecule associated with it (e.g., a target molecule bound by D1), to also become internalized into the cell. As explained below, internalizing effector proteins include proteins that are directly internalized into a cell, as well as proteins that are indirectly internalized into a cell.

[0041] Internalizing effector proteins that are directly internalized into a cell include membrane-associated molecules with at least one extracellular domain (e.g., transmembrane proteins, GPI-anchored proteins, etc.), which undergo cellular internalization, and are preferably processed through an intracellular degrading and/or recycling pathway. Specific non-limiting examples of internalizing effector proteins that are directly internalized into a cell include, for example, CD63, MHC-I (e.g., HLA-B27), Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin, LDL receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein-2 (APLP2), apelin receptor (APLNR), MAL (myelin lymphocyte protein E, a.k.a. VIP17), IGF2R, vacuolar-type H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptors, glutathione receptor, leptin receptors, scavenger receptors (e.g., SCARA1-5, SCARB1-3, CD36), etc.

[0042] In embodiments in which E is a directly internalized effector protein, the D2 component of the multispecific antigen-binding molecule may be, for example, an antibody or antigen-binding fragment of an antibody that specifically binds E, or an ligand or portion of a ligand that specifically interacts with the effector protein. For example, if E is Kremen-1 or Kremen-2, the D2 component may comprise or consist of a Kremen ligand (e.g., DKK1) or Kremen-binding portion thereof. As another example, if E is a receptor molecule such as ASGR1, the D2 component may comprise or consist of a receptor-specific ligand (e.g., asialo-orosomucoid [ASOR] or Beta-GalNAc) or a binding moiety of its receiver.

[0043] Internalizing effector proteins that are indirectly internalized into a cell include proteins and polypeptides that do not internalize on their own, but become internalized in a cell after binding to, or otherwise associated with, a second protein or polypeptide that is directly internalized into the cell. Proteins that are indirectly internalized into a cell include, for example, soluble ligands that are capable of binding to a receptor molecule expressed on the internalizing cell surface. A non-limiting example of a soluble ligand that is (indirectly) internalized into a cell through its interaction with an internalizing cell surface-expressed receptor molecule is transferrin. In embodiments in which E is transferrin (or another indirectly internalized protein), the binding of D2 to E, and the interaction of E with the transferrin receptor (or another receptor molecule expressed on the internalizing cell surface), causes the entire multispecific antigen-binding molecule, and any molecule associated with it (e.g., a target molecule bound by D1), becomes internalized into the cell simultaneously with the internalization of E and its binding partner.

[0044] In embodiments in which E is an indirectly internalized effector protein such as a soluble ligand, the D2 component of the multispecific antigen-binding molecule may be, for example, an antibody or antigen-binding fragment of an antibody that binds specifically E, or a receptor or portion of a receptor that specifically interacts with the soluble effector protein. For example, if E is a cytokine, the component of D2 may comprise or consist of the corresponding cytokine receptor or ligand-binding portion thereof.

TARGET MOLECULES (T)

[0045] In the context of the present invention, the D1 component of the multispecific antigen binding molecule specifically binds a target molecule ("T"). A target molecule is any protein, polypeptide, or other macromolecule whose activity or extracellular concentration is desired to attenuate, reduce, or eliminate. In many examples, the target molecule to which D1 binds is a protein or polypeptide [i.e., a "target protein"]; however, the present invention also includes embodiments in which the target molecule ("T") is a carbohydrate, glycoprotein, lipid, lipoprotein, lipopolysaccharide, or other non-protein polymer or molecule to which D1 binds. According to the present invention, T can be a target protein expressed on the cell surface or a soluble target protein. Target binding by the multispecific antigen-binding molecule can occur in an extracellular or cell surface context. In certain embodiments, however, the multispecific antigen-binding molecule binds a target molecule within the cell, for example, in an intracellular component such as the endoplasmic reticulum, Golgi, endosome, lysosome, etc.

[0046] Examples of target molecules expressed on the cell surface include receptors expressed on the cell surface, membrane-bound ligands, ion channels, and any other multimeric polypeptide or monomeric component with an extracellular portion that is attached to a cell membrane or associated with she. Exemplary non-limiting cell surface expressed target molecules that can be targeted by the multispecific antigen binding molecule of the present invention include, for example, cytokine receptors (e.g., receptors for IL-1, IL-4, IL-6, IL -13, IL-22, IL-25, IL-33, etc.), as well as cell surface targets including other type 1 transmembrane receptors such as PRLR, G protein coupled receptors such as GCGR, ion channels such as Nav1.7, ASIC1 or ASIC2, non-receptor surface proteins such as MHC-I (e.g. HLA-B*27), etc.

[0047] In embodiments in which T is a target protein expressed on the cell surface, the D1 component of the multispecific antigen-binding molecule may be, for example, an antibody or antigen-binding fragment of an antibody that specifically binds T, or a ligand or portion of a ligand that specifically interacts with the target protein expressed on the cell surface. For example, if T is IL-4R, the D1 component may comprise or consist of IL-4 or a receptor binding portion thereof.

[0048] Examples of soluble target molecules include cytokines, growth factors, and other signaling ligands and proteins. Exemplary non-limiting soluble target protein that can be targeted by the multispecific antigen binding molecule of the present invention includes, for example, IL-1, IL-4, IL-6, IL-13, IL-22, IL-25, IL -33, SOST, DKK1, etc. Soluble target molecules also include, for example, non-human target molecules such as allergens (e.g., Fel D1, Betv1, CryJ1), pathogens (e.g., Candida albicans, S. aureus, etc.), and pathogenic molecules (e.g., example, lipopolysaccharide [LPS], lipothechoic acid [LTA], protein A., toxins, etc.). In embodiments in which T is a soluble target molecule, the D1 component of the multispecific antigen-binding molecule may be, for example, an antibody or antigen-binding fragment of an antibody that specifically binds T, or a receptor or portion of a receptor that specifically interacts with the soluble target molecule. For example, if T is IL-4, the D1 component may comprise or consist of IL-4R or a ligand-binding portion thereof.

[0049] Target molecules also include tumor-associated antigens, as described elsewhere herein.

pH-DEPENDENT BINDING

[0050] The present invention provides multispecific antigen-binding molecules comprising a first antigen-binding domain (D1) and a second antigen-binding domain (D2), wherein one or both of the antigen-binding domains (D1 and /or D2) binds its antigen (T or E) in a pH-dependent manner. For example, an antigen-binding domain (D1 and/or D2) may exhibit less binding to its antigen at acidic pH compared to neutral pH. Alternatively, an antigen-binding domain (D1 and/or D2) may exhibit greater binding to its antigen at acidic pH compared to neutral pH. Antigen-binding domains with pH-dependent binding characteristics can be obtained, for example, by screening a population of antibodies for lower (or higher) binding to a particular antigen at acidic pH compared to neutral pH. Additionally, modifications of the binding domain of antigen at the amino acid level can produce antigen-binding domains with pH-dependent characteristics. For example, by replacing one or more amino acids of an antigen-binding domain (e.g., in a CDR) with a histidine residue, an antigen-binding domain with lower antigen binding at acidic pH relative to neutral pH can be obtained. .

[0051] In certain embodiments, the present invention includes multispecific antigen-binding molecules comprising a D1 and/or D2 component that binds its respective antigen (T or E) at acidic pH with a KD that is at least about 3, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more times greater than the KD of the D1 and/or D2 component for binding to their respective antigen at neutral pH. pH-dependent binding can also be expressed in terms of the t/ of the antigen binding domain to its antigen at acidic pH compared to neutral pH. For example, the present invention includes multispecific antigen-binding molecules comprising a D1 and/or D2 component that binds their respective antigen (T or E) at acidic pH with a t/ that is at least about 5, 6, 7 , 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more times lower than the t/ of the D1 and/or D2 component for binding to their respective antigen at pH neutral.

[0052] Multispecific antigen binding molecules of the present invention that comprise a D1 and/or D2 component with lower antigen binding at acidic pH compared to neutral pH, when administered to animal subjects, may in certain embodiments exhibit slower clearance at from circulation compared to comparable molecules that do not exhibit pH-dependent binding characteristics. According to this aspect of the invention, there are provided multispecific antigen binding molecules with lower antigen binding in both T and/or E at acidic pH compared to neutral pH, which exhibit at least 2-fold slower clearance from the circulation. relative to comparable antigen-binding molecules that do not have lower antigen binding at acidic pH compared to neutral pH. Clearance rate can be expressed in terms of the half-life of the antibody, where slower clearance correlates with a longer half-life.

[0053] As used herein, the expression "acidic pH" means a pH of 6.0 or less. The term "acidic pH" includes pH values of about 6.0, 5.95, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5 .45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As used herein, the term "neutral pH" means a pH of about 7.0 to about 7.4. The term "neutral pH" includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

ATTENUATION OF TARGET MOLECULE ACTIVITY

[0054] As noted elsewhere herein, and as demonstrated by the functional examples hereinafter, the present inventors have discovered that simultaneous binding of a target molecule (T) and an internalizing effector protein (E) by an antigen-binding molecule Multispecific attenuates T activity to a greater degree than T binding by the first antigen-binding domain component (D1) of the multispecific antigen-binding molecule alone. As used herein, the term "attenuates T activity to a greater degree than binding of T by D1 alone" means that, in an assay in which T activity can be measured using cells expressing E, the level of T activity measured in the presence of a multispecific antigen-binding molecule is at least 10% lower than the level of T activity measured in the presence of a control construct containing D1 itself (i.e., not physically bound in the second domain antigen binding (D2)). For example, the level of T activity measured in the presence of the multispecific antigen binding molecule may be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% lower than the level of T activity measured in the presence of a control construct containing D1 by itself own.

[0055] An illustrative non-limiting assay format for determining whether a multispecific antigen-binding molecule attenuates the activity of a target molecule, to a greater degree than binding of the target molecule by the D1 domain alone, is shown in functional examples 1 and 2, here below. In example 1, for example, "T" is the interleukin-4 receptor (IL-4R), and "E" is CD63. The multispecific antigen binding molecule of example 1 is a 2-antibody conjugate comprising an anti-IL-4R mAb linked to an anti-CD63 mAb via a streptavidin/biotin linker. Thus, "D1" in this exemplary construct is the antigen-binding domain (HCVR/LCVR pair) of the anti-IL-4R antibody, and "D2" is the antigen-binding domain (HCVR/LCVR pair) of the anti-IL-4R antibody. CD63. For the experiments of Examples 1 and 2, a cell-based assay format was used that produced a reporter signal when IL-4R activity was stimulated by the addition of exogenous IL-4 ligand. The value of IL-4-induced reporter activity detected in the presence of the multispecific antigen-binding molecule was compared with the value of IL-4-induced reporter activity detected in the presence of control constructs containing the anti-IL-4R antibody, both connected to an irrelevant control immunoglobulin (control 1) as combined with the anti-CD63 antibody, but not physically attached to it (control 2). The control constructs thus produce the condition in which T is bound by D1 alone (i.e., in which D1 is not a part of the multispecific antigen-binding molecule per se). If the degree of activity of the target molecule (represented by the reporter signal) observed in the presence of the multispecific antigen-binding molecule is at least 10% less than the value of the activity of the target molecule in the presence of a control construct comprising the non-D1 component physically bound to the D2 component (e.g., control 1 or control 2), then, for the purposes of the present disclosure, it is concluded that "simultaneous binding of T and E by the multispecific antigen-binding molecule attenuates the activity of T to a greater degree than the binding of T by D1 alone.”

[0056] Binding of T by D1 alone may, in some embodiments, result in partial attenuation of T activity (as in the case of example 1 where treating reporting cells with an anti-IL-4R antibody alone [i.e. controls 1 and 2] caused a small level of attenuation of IL-4 signaling compared to untreated cells). In other embodiments, binding of T by D1 alone will not result in any detectable attenuation of T activity; that is, the biological activity of T may not be affected by the binding of T by D1 alone. In any case, however, simultaneous binding of T and E by a multispecific antigen-binding molecule of the invention will attenuate T activity to a greater degree than binding of T by D1 alone.

[0057] Alternative assay formats and variations on the assay format(s) exemplified here will be apparent to those skilled in the art, taking into account the nature of the specific target molecule and effector proteins for which any given molecule of multispecific antigen binding can be targeted. Any such format can be used in the context of the present invention to determine whether simultaneous binding of T and E by a multispecific antigen-binding molecule attenuates T activity to a greater degree than binding of T by D1 alone.

TUMOR TARGETING

[0058] In another aspect of the invention, multispecific antigen binding molecules are used to target tumor cells. According to this aspect of the invention, the target molecule "T" to which D1 binds is a tumor-associated antigen. In certain examples, the tumor-associated antigen is an antigen that is not ordinarily internalized. The internalizing effector protein "E" to which D2 binds may be tumor specific, or it may be expressed on both tumor and non-tumor cells of an individual. Any of the internalizing effector proteins mentioned anywhere herein can be targeted for anti-tumor applications of the invention.

[0059] As used herein, the term "tumor-associated antigen" includes proteins or polypeptides that are preferentially expressed on the surface of a tumor cell. The term "preferentially expressed" as used in this context means that the antigen is expressed on a tumor cell at a level that is at least 10% higher (e.g., 10%, 20%, 30%, 40%, 50% %, 60%, 70%, 80%, 90%, 100%, 110%, 150%, 200%, 400%, or more) than the level of antigen expression in non-tumor cells. In certain embodiments, the molecule. target is an antigen that is preferentially expressed on the surface of a tumor cell selected from the group consisting of a kidney tumor cell, a colon tumor cell, a breast tumor cell, an ovarian tumor cell, a skin tumor, a lung tumor cell, a prostate tumor cell, a pancreatic tumor cell, a glioblastoma cell, a head and neck tumor cell, and a melanoma cell. tumor proteins include, for example, AFP, ALK, BAGE, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EphA2, Fra-1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, - 2, -3, -4, -6, and - 12), MART-1, mesothelin, ML-IAP, Muc1, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase, and uroplakin-3.

[0060] The multispecific antigen binding molecule, according to this aspect of the invention, can be conjugated with a drug, toxin, radioisotope, or other substance that is detrimental to the viability of a cell. Alternatively, the drug or toxin may be a substance that does not directly kill a cell, but makes a cell more susceptible to killing by other external agents. In still other embodiments involving tumor targeting, the multispecific antigen-binding molecule of the invention is not itself conjugated to a drug, toxin, or radioisotope, but rather is administered in combination with a second specific antigen-binding molecule. to the target (T) (referred to herein as a "complicit molecule"), wherein the companion molecule is conjugated to a drug, toxin, or radioisotope. In such embodiments, the multispecific antigen-binding molecule will preferably bind to an epitope on the target molecule (T) that is distinct and/or non-overlapping with the epitope recognized by the confederate molecule (i.e., to allow simultaneous binding of the antigen-binding molecule). multispecific antigen and the target accomplice molecule).

[0061] In a related embodiment, the present invention also includes anti-tumor combinations, and therapeutic methods, comprising: (a) a toxin- or drug-conjugated antigen-binding molecule that specifically binds a tumor-associated antigen; and (b) a multispecific antigen-binding molecule comprising (i) a first binding domain that specifically binds an internalizing effector protein (e.g., with low affinity) and (ii) a second binding domain that specifically binds the antigen molecule. antigen binding conjugated to toxin or drug. In this embodiment, the multispecific antigen-binding molecule functions to bind the toxin- or drug-conjugated antigen-binding molecule to the internalizing effector protein, which thereby functions to physically bind the tumor-associated antigen to the internalizing effector protein. Internalization of the toxin-labeled anti-tumor associated antigen antibody through its connection with the internalizing effector protein would consequently result in killing of the targeted tumor cell.

[0062] In accordance with certain embodiments of the tumor-targeting aspects of the invention, the multispecific antigen-binding molecule (or companion antibody) may be conjugated to one or more cytotoxic drugs selected from the group consisting of: calicamycin, esperamycin, methotrexate , doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin, 5-fluorouracil, estramustine, vincristine, etoposide, doxorubicin, paclitaxel, larotaxel,thesistaxel, orataxel, docetaxel, dolastatin a10, auristatin E, auristatin PHE and maytansine-based compounds (e.g. DM1, DM4, etc.). The multispecific antigen-binding molecule (or companion antibody) may also, or alternatively, be conjugated to a toxin such as diphtheria toxin, Pseudomonas aeruginosa exotoxin A, ricin A chain, Abrin A chain, modeccin A chain, alpha-sarcina, Aleurites fordii proteins, diantin proteins, Phytolaca americana proteins, etc. The multispecific antigen binding molecule (or companion antibody) may also, or alternatively, be conjugated to one or more radioisotopes selected from the group consisting of c, t, , , , , u , , ,67 125 123 77 153 166 64 121 224 223 Cu, I, I, Br, Sm, Ho, Cu, Pb, Ra and Ra. Thus, this aspect of the invention includes multispecific antigen binding molecules that are antibody-drug conjugates (ADCs) or antibody-radioisotope conjugates (ARCs).

[0063] In the context of tumor killing applications, the D2 component may, under certain circumstances, bind with low affinity to the internalizing effector protein "E". Thus, the multispecific antigen-binding molecule will preferentially target tumor cells that express the tumor-associated antigen. As used herein, "low affinity" binding means that the affinity binding of the D2 component to the internalizing effector protein (E) is at least 10% weaker (e.g., 15% weaker, 25% weaker, 50% weaker, % weaker, 75% weaker, 90% weaker, etc.) than the binding affinity of the D1 component with the target molecule (T). In certain embodiments, "low affinity" binding means that the D2 component interacts with the internalizing effector protein (E) with a KD greater than about 10 nM to about 1 μM, as measured in a D2 plasmon resonance assay. surface at around 25°C.

[0064] Simultaneous binding of a multispecific antigen-binding molecule to an internalizing effector protein and a tumor-associated antigen will result in preferential internalization of the multispecific antigen-binding molecule into tumor cells. If, for example, the multispecific antigen-binding molecule is conjugated to a drug, toxin, or radioisotope (or if the multispecific antigen-binding molecule is administered in combination with a companion antibody that is conjugated to a drug, toxin, or radioisotope) , targeted internalization of tumor-associated antigen into the tumor cell through its binding on the multispecific antigen-binding molecule will result in extremely specific tumor cell killing.

PHARMACEUTICAL COMPOSITIONS AND METHODS OF ADMINISTRATION

[0065] The present invention includes pharmaceutical compositions comprising a multispecific antigen-binding molecule. The pharmaceutical compositions of the invention can be formulated with suitable carriers, excipients, and other agents that provide improved transfer, distribution, tolerance, and the like.

[0066] The present invention also includes methods for inactivating or attenuating the activity of a target molecule (T). The methods of the present invention comprise contacting a target molecule with a multispecific antigen binding molecule in the manner described herein. In certain embodiments, methods according to this aspect of the invention comprise administering a pharmaceutical composition comprising a multispecific antigen binding molecule to a patient for whom it is desirable and/or beneficial to inactivate, attenuate, or otherwise decrease the concentration extracellular target molecule.

[0067] Various delivery systems are known in the art and can be used to administer the pharmaceutical compositions of the present invention to a patient. Methods of administration that can be used in the context of the present invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The pharmaceutical compositions of the invention may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration can be systemic or local. For example, a pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. Furthermore, with respect to subcutaneous delivery, a pen delivery device can be used to administer a pharmaceutical composition of the present invention to a patient.

EXAMPLES

[0068] The following examples are presented in order to provide those skilled in the technology with a complete disclosure and description of how to manufacture and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors consider their invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. quantities, temperature, etc.), but some experimental errors and deviations must be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Example 1. Use of a Multispecific Antigen Binding Molecule to Induce Degradation of a Cell Surface Receptor through Binding with an Internalizing Effector Protein

[0069] As an initial proof-of-concept experiment, a multispecific antigen-binding molecule was created that is capable of binding (a) an internalizing effector molecule and (b) a cell surface receptor target molecule. In this example, the internalizing effector protein is Kremen-2 (Krm2), and the cell surface receptor target molecule is an Fc receptor (FCYRI [Fc-gamma-R1]).

[0070] Kremen molecules (Krm1 and Krm2) are cell surface proteins known to mediate WNT signaling by directing the internalization and degradation of the LRP5 and LRP6 signaling molecules of the WNT pathway. Internalization of LRP5/6 is accomplished through the soluble interacting protein DKK1. In particular, DKK1 binds Kremen to LRP5/6 on the cell surface, and by virtue of this binding, internalization of Kremen triggers the internalization and degradation of LRP5 and LRP6. (See Li et al., PLoS One 5(6):e11014).

[0071] The present inventors sought to exploit the Kremen binding properties of DKK1 and the Kremen internalization properties to induce the internalization of FCYRI. To facilitate Kremen-mediated internalization/degradation of FCYRI, a multispecific antigen-binding molecule was constructed consisting of DKK1 fused to a mouse Fc (DKK1-mFc, having the amino acid sequence of SEQ ID NO:1). As explained elsewhere herein, a multispecific antigen-binding molecule is defined as a molecule comprising a first antigen-binding domain (D1) that specifically binds a target molecule, and a second antigen-binding domain (D2) that binds specifically an internalizing effector protein. In this proof-of-concept example, the "first antigen-binding domain" is the component of mFc that specifically binds the FCYRI target molecule, and the "second antigen-binding domain" is the component of DKK1 that specifically binds the effector protein. internalizing Kremen.

[0072] An experiment was first conducted to determine whether DKK1-mFc can be endocytosed into cells in a Kremen-dependent manner. For this experiment, two cell lines were used: Cell-1, a HEK293 cell line bred to express FCYRI but not Kremen-2, and cell-2, a HEK293 cell line bred to express both FcyR1 and Kremen-2. A 1:10 dilution of DKK1-mFc-conditioned medium was added to the respective cell lines and allowed to incubate at 37°C for 90 minutes. After 90 minutes of incubation, cells were stained with Alexa-488-labeled anti-mouse IgG antibody to detect the DKK1-mFc molecule. Using fluorescence microscopy, we observed that virtually no DKK1-mFc was localized within cell-1 (which lacks Kremen); however, substantial amounts of DKK1-mFc were detected in Cell-2 expressing Kremen-2. Thus, these results show that the multispecific antigen-binding molecule DKK1-mFc can be internalized into cells in a Kremen-dependent manner.

[0073] Next, a time course experiment was conducted to determine whether DKK1-mFc can induce degradation of FcyR1 in a Kremen-dependent manner. A brief description of the experimental protocol is as follows: Cell-1 (which expresses only FcyR1) and cell-2 (which expresses Kremen-2 and FcyR1) were treated with 2 mg/mL of NHS-Sulfo-Biotin for 15 minutes on ice to label all proteins expressed on the cell surface. Cells were then washed and resuspended in 400 μL of medium and divided into four 100 μL aliquots that were treated with DKK1-mFc for varying amounts of time (0 minute, 15 minutes, 30 minutes, and 60 minutes) at 37 °C. After DKK1-mFc incubation, cells were pelleted and treated with protease inhibitors. Cell lysates from the different incubation time points were subjected to FCYRI immunoprecipitation. For immunoprecipitation of FCYRI, mouse anti-FcYR1 antibody was added to cell lysates and incubated for 1 hour at 4°C. Then protein G microspheres were added and the mixture was incubated for 1 hour at 4 °C. The microspheres were then washed and the proteins eluted and subjected to SDS-PAGE. Proteins were transferred to the membrane and probed with HRP-labeled streptavidin to reveal relative amounts of surface-exposed FcyR1 protein remaining in each sample. Results are shown in figure 2.

[0074] As illustrated in Figure 2, the amount of FcyR1 protein exposed on the surface in samples from Cell-1 (which expresses FcyR1, but not Kremen-2) remained relatively constant regardless of the amount of time the cells were exposed to DKK1- mFc. In contrast, the amount of surface-exposed FcyR1 protein in samples from Cell-2 (which expresses both Kremen-2 and FcyR1) decreased substantially with increasing incubation time with DKK1-mFc. Thus, this experiment demonstrates that DKK1-mFc induces degradation of cell surface-expressed FcyR1 in a Kremen-2-dependent manner.

[0075] Considered together, the above results show that a multispecific antigen-binding molecule that simultaneously binds a cell surface target molecule (FcyR1) and an internalizing effector protein (Kremen-2) can induce degradation of the target molecule in a cell-dependent manner. of the effector protein.

Example 2. IL-4R Activity is Attenuated Using a Multispecific Antigen Binding Molecule with Specificity for IL-4R and CD63

[0076] In an additional set of proof-of-concept experiments, a multispecific antigen-binding molecule was constructed that is capable of simultaneously binding a cell surface-expressed target molecule (i.e., IL-4R) and an expressed internalizing effector protein. on the cell surface (i.e. CD63). The purpose of these experiments was to determine whether IL-4R activity in a cell can be attenuated to a greater degree by physically attaching IL-4R to an effector molecule that is internalized and targeted for degradation in the lysosome (in this case, CD63). In other words, this Example was designed to test whether normal CD63 internalization and degradation can be used to force redirection of IL-4R internalization and degradation in a cell.

[0077] First, a multispecific antigen-binding molecule was constructed that is capable of binding both IL-4R and CD63. Specifically, a streptavidin-conjugated anti-IL-4R antibody and a biotinylated anti-CD63 antibody were combined in a 1:1 ratio to produce an anti-IL-4R:anti-CD63 conjugate (i.e., an antigen-binding molecule multispecific that specifically binds both IL-4R and CD63). The anti-IL-4R antibody used in this example is a fully human mAb raised against the extracellular domain of IL-4R. (The anti-IL-4R antibody comprised a heavy chain variable region with SEQ ID NO:3 and a light chain variable region with SEQ ID NO:4). The anti-CD63 antibody used in this example is the mouse anti-human CD63 mAb clone MEM-259, obtained from Biolegend (San Diego, CA), catalog No. 312002.

[0078] Two control constructs were also created: Control-1 = streptavidin-conjugated anti-IL-4R antibody combined in a 1:1 ratio with biotinylated control mouse IgG1kappa antibody; and Control-2 = streptavidin-conjugated anti-IL-4R antibody combined in a 1:1 ratio with non-biotinylated anti-CD63 antibody. The anti-IL-4R antibody used in the experimental constructs and controls for this Example is an antibody that is known to specifically bind IL-4R and only partially block IL-4-mediated signaling.

[0079] The experimental cell line used in this example is a HEK293 cell line containing a STAT6-luciferase reporter construct and additional STAT6 ("HEK293/STAT6-luc cells"). The cells used in this experiment express both IL-4R and CD63 on their surface. When treated with IL-4 in the absence of any inhibitor, this cell line produces a dose-dependent detectable chemiluminescence signal that reflects the extent of IL-4-mediated signaling.

[0080] In an initial experiment, the experimental anti-IL-4R/anti-CD63 multispecific molecule, or the control constructs, were added to HEK293/STAT6-luc cells so that the final concentration of anti-IL-4R antibody in the media it was 12.5 nM. Reporter signal was measured at higher concentrations of IL-4 in the presence and absence of the experimental constructs and controls (Figure 3). As seen in Figure 3, the multispecific anti-IL-4R/anti-CD63 molecule ("ab conjugate") inhibited IL-4-mediated signaling to a significantly greater degree than either control construct.

[0081] To confirm that the effect observed in figure 3 depended on CD63, the same experiment described previously was performed, except that CD63 expression was significantly reduced in the reporting cell line, using an siRNA directed against CD63. With significantly reduced CD63 expression, the greater inhibitory activity of the multispecific anti-IL-4R/anti-CD63 molecule was no longer observed (Figure 4). This result suggests that the ability of the multispecific anti-IL-4R/anti-CD63 molecule to attenuate IL-4-mediated signaling is due to the simultaneous binding of the multispecific molecule to IL-4R and CD63 and the consequent internalization and degradation of all the antibody-IL-4R-CD63 complex.

[0082] Similar experiments were then performed in which the multispecific anti-IL-4R/anti-CD63 molecule, or control constructs, were incubated naturally with the HEK293/STAT6-luc reporter cell line for various amounts of time before of the addition of IL-4. In a first set of such experiments, molecules were incubated naturally with the reporter cell line for 0 hour (i.e., added simultaneously with IL-4), 2 hours, or overnight before addition of 50 pM IL-4. . Luciferase activity was measured six hours after the addition of IL-4. Results are shown in Figure 5, top panel ("untransfected"). In an additional set of experiments, a similar protocol was performed, except that the experimental molecules or controls were incubated naturally with the reporting cell line for 15 minutes, 30 minutes, 1 hour, or 2 hours before the addition of 50 pM IL-4. Results are shown in figure 6.

[0083] The results summarized in figures 5 and 6 show that the multispecific anti-IL-4R/anti-CD63 molecule is capable of inhibiting IL-4-mediated signaling, and that this inhibitory effect is increased with longer incubation times. As with the initial set of experiments, it was confirmed using CD63 RNAsi that the inhibitory effect of the multispecific anti-IL-4R/anti-CD63 molecule depended on CD63 expression (Figure 5 lower panel ["CD63 RNAsi"]).

[0084] Briefly, this example additionally provides proof of concept for inhibiting a target molecule's activity through the use of a multispecific antigen-binding molecule, which is capable of simultaneously binding both the target molecule (in this case IL-4R) as an internalizing effector protein (in this case CD63) to thereby cause the redirection of internalization and degradation of the target molecule in a cell. Differently established, simultaneous binding of IL-4R and CD63 by the exemplary multispecific antigen-binding molecule attenuated IL-4R activity to a substantially greater degree (i.e., >10%) than binding of IL-4R by the constructs. controls alone.

Example 3. An Anti-IL-4R x Anti-CD63 Bispecific Antibody Attenuates IL-4R Activity in a CD63-Dependent Manner

[0085] The experiments in example 2 show here that a multispecific anti-IL-4R/anti-CD63 molecule inhibits IL-4-mediated signaling in a CD63-dependent manner. In those experiments, the multispecific antigen-binding molecule consisted of two separate monoclonal antibodies (anti-IL-4R and anti-CD63) that were connected via a biotin-streptavidin bond. To confirm that the results observed with this proof-of-concept multispecific antigen-binding molecule are generalizable to other multispecific antigen-binding molecule formats, a true bispecific antibody was constructed.

[0086] Standard bispecific antibody technology was used to construct a bispecific antibody consisting of a first arm specific for IL-4R and a second arm specific for CD63. The IL-4R-specific arm contained an anti-IL-4R heavy chain paired with a CD63-specific light chain. The CD63-specific light chain was paired with the IL-4R-specific heavy chain solely for the purposes of construction convenience; however, pairing the anti-IL-4R heavy chain with the anti-CD63 light chain retained full specificity for IL-4R and did not exhibit CD63 binding. The CD63-specific arm contained an anti-CD63 heavy chain paired with an anti-CD63 light chain (the same light chain as used in the IL-4R arm). The anti-IL-4R heavy chain (comprising SEQ ID NO:3) was derived from the total anti-IL-4R antibody in the manner used in example 2; however, the anti-CD63 light and heavy chains were derived from the anti-CD63 antibody designated H5C6, obtained from the Developmental Studies Hybridoma Bank (University of Iowa Department of Biology, Iowa City, IA). As with the total anti-IL-4R antibody used in Example 2, the anti-IL-4R bispecific antibody component used in this example exhibited only moderate IL-4R blocking activity by itself.

[0087] An IL-4 luciferase assay was performed to evaluate the blocking activity of the anti-IL-4R x anti-CD63 bispecific antibody. Briefly, serial dilutions of anti-IL-4R x anti-CD63 bispecific antibody or control molecules were added to HEK293/STAT6-luc reporter cells (see example 2). Under normal conditions, these cells produce a detectable luciferase signal when treated with IL-4. For this experiment, 10 pM IL-4 was then added to the cells, and luciferase activity was quantified for each antibody dilution used. The controls used in this assay were: (a) mock bispecific antibody that binds IL-4R with one arm and has a functional anti-CD63 non-arm (i.e., containing an anti-IL-4R heavy chain and an anti-IL-4R heavy chain). anti-CD63, both paired with the anti-IL-4R light chain); (b) monospecific anti-IL-4R antibody; and (c) buffer (PBS) only (no antibody). Results are shown in Figure 7. As shown in Figure 7, for the control samples used, luciferase activity remained relatively high even at the highest concentrations of the antibody, whereas for the bispecific antibody, luciferase activity decreased significantly as the concentration increased. of antibody increased. These results confirm that simultaneous binding of IL-4R and CD63 by a bispecific antibody causes substantial inhibition of IL-4R activity.

Example 4. Internalization of SOST Using a Multispecific Antigen Binding Molecule That Simultaneously Binds SOST and CD63

[0088] In this example, the ability of multispecific antigen binding molecules to promote the internalization of the soluble target molecule SOST (sclerostin) was evaluated. For these experiments, the target molecule was a fusion protein consisting of a human SOST protein labeled with a pHrodo™ moiety (Life Technologies, Carlsbad, CA) and a myc tag. The pHrodo™ fraction is a pH-sensitive dye that is virtually non-fluorescent at neutral pH and brightly fluorescent in an acidic environment such as the endosome. The fluorescent signal, therefore, can be used as an indicator of cellular internalization of the SOST fusion protein. The multispecific antigen-binding molecules for these experiments were bispecific antibodies with binding specificity for both CD63 (an internalizing effector protein) and the SOST fusion protein (a soluble target molecule), as described in more detail below.

[0089] The experiments were conducted as follows: Briefly, HEK293 cells were plated at 10,000 cells/well in 96-well plates coated with poly-D-lysine (Greiner Bio-One, Monroe, NC). After the cells had naturally settled overnight, the media were replaced with media containing antibody (5 μg/mL, as described below), pHrodo™-myc-labeled SOST (5 μg/mL), heparin (10 μg/mL), and Hoechst 33342. Cells were then incubated for either 3 hours on ice or 3 hours at 37°C. All cells were washed twice before imaging in PBS, and the number of fluorescent spots per cell, as well as the corresponding fluorescence intensity, were counted to establish the extent of cellular internalization of pHrodo-myc-labeled SOST in the presence of the various antibody constructs.

[0090] The antibodies used in this example were as follows: (1) monospecific anti-CD63 antibody (clone H5C6, Developmental Studies Hybridoma Bank, University of Iowa Department of Biology, Iowa City, IA); (2) anti-myc antibody (clone 9E10, Schiweck et al., 1997, FEBS Lett. 414(1):33-38); (3) anti-SOST antibody (an antibody having the light and heavy chain variable regions of the antibody designated "Ab-B" in US Patent No. 7,592,429); (4) anti-CD63 x anti-myc bispecific antibody (i.e., a multispecific antigen-binding molecule comprising an anti-CD63 arm derived from the H5C6 antibody and an anti-myc arm derived from 9E10); (5) anti-CD63 x anti-SOST #1 bispecific antibody (i.e., a multispecific antigen-binding molecule comprising an anti-CD63 arm derived from the H5C6 antibody and an anti-SOST arm derived from "Ab-B" ); and (6) bispecific anti-CD63 x anti-SOST #2 antibody (i.e., a multispecific antigen-binding molecule comprising an anti-CD63 arm derived from the H5C6 antibody and an anti-SOST arm derived from the antibody designated "Ab -20" in US Patent No. 7,592,429). The bispecific antibodies used in these experiments were assembled using the so-called “bumps in holes” methodology (see, for example, Ridgway et al., 1996, Protein Eng. 9(7):617-621).

[0091] Results of the internalization experiments are shown in figure 8. Figure 8 shows the number of spots (labeled vesicles) per cell, in the various treatment conditions tested. Considered together, the results of these experiments demonstrate that the bispecific constructs, which simultaneously bind CD63 and SOST (both directly and via the myc tag), caused the maximum amount of SOST internalization as reflected by fluorescence intensity and number of fluorescent puncta per cell. over time at 37 °C. Thus, the multispecific antigen-binding molecules used in this example are capable of effectively directing the internalization of a soluble target molecule.

Example 5. Changes in Bone Mineral Density in Mice Treated with a Multispecific Antigen-Binding Molecule that Binds CD63 and SOST

[0092] A multispecific anti-CD63 x anti-SOST antigen binding molecule, described in example 4, is then tested for its ability to increase bone mineral density in mice. Five groups of mice (about 6 mice per group) are used in these experiments. Treatment groups are as follows: (I) untreated negative control mice; (II) mice treated with a monospecific anti-SOST blocking antibody that is known to increase bone mineral density by itself (positive control); (III) mice treated with a bispecific antibody that specifically binds CD63 and SOST but does not inhibit SOST activity by itself or only slightly inhibits SOST activity by itself; (IV) mice treated with a parental anti-CD63 antibody (i.e., a monospecific antibody containing the same anti-CD63 antigen-binding domain as the bispecific antibody); and (V) mice treated with a parental anti-SOST antibody (i.e., a monospecific antibody containing the same anti-SOST antigen-binding domain as the bispecific antibody). The amount of antibody administered to mice in each group is about 10 to 25 mg/kg.

[0093] Mice in group III (treated with an anti-SOST x anti-CD63 bispecific antibody) are expected to show an increase in bone mineral density that is at least comparable to that observed in mice in group II (treated with a known blocking anti-SOST antibody), even though the anti-SOST bispecific antibody component does not show SOST activity by itself (confirmed by the mice in Group V expected not to show an increase in bone mineral density). The increase in bone mineral density that is expected in group III mice is believed to be driven by CD63-mediated internalization of SOST, as previously observed in the cellular experiments of example 4.

Example 6. Cellular Internalization of Lipopolysaccharide (LPS) Mediated by a Multispecific Antigen-Binding Molecule That Simultaneously Binds LPS and CD63

[0094] This Example illustrates the use of a multispecific antigen-binding molecule of the invention to direct the internalization of a non-protein target molecule, namely lipopolysaccharide (LPS). LPS is a component of the outer membrane of Gram-negative bacteria and is known to contribute to septic shock. Anti-LPS antibodies have been investigated as possible treatment agents for sepsis. The experiments of the present Example were designed to evaluate the ability of a multispecific antigen-binding molecule to promote the internalization of LPS.

[0095] The multispecific antigen binding molecule used in this example was a bispecific antibody with one arm directed to LPS (target) and the other arm directed to CD63 (internalizing effector protein). The anti-LPS arm was derived from the antibody known as WN1 222-5. (DiPadova et al., 1993, Infection and Immunity 61(9):3863-3872; Muller-Loennies et al., 2003, J. Biol. Chem. 278(28):25618-25627; Gomery et al., 2012 , Natl Acad. Sci USA 109(51):20877-20882; The anti-CD63 arm was derived from the H5C6 antibody (see example 4). The anti-LPS x anti-CD63 bispecific antibody (i.e., multispecific antigen-binding molecule) was assembled using the so-called “bumps in holes” methodology (see, e.g., Ridgway et al., 1996, Protein Eng. 9 (7):617-621).

[0096] Two species of LPS were used in these experiments: LPS from E. coli and LPS from Salmonella minnesota. Both versions were obtained as fluorescently labeled molecules (ALEXA-FLUOR®-488-labeled LPS, Life Technologies, Carlsbad, CA).

[0097] Experiments were conducted as follows: HEK293 cells were plated on PDL-coated 96-well imaging plates. After overnight, media was replaced with fresh media. Fluorescently labeled LPS (both E. coli- and S. minnesota-derived) was added into regular medium. Then, the anti-LPS x anti-CD63 bispecific antibody, or half of the control antibodies paired with mock Fc, was added to the samples. After various times of incubation at 37 °C (1 hour and 3 hours) or on ice (3 hours), cells from LPS-treated samples were processed as follows: washed - finished with anti-ALEXA-FLUOR®-488 antibody - washed & fixed. The anti-ALEXA-FLUOR®-488 antibody terminates fluorescence of the non-internalized (i.e., surface-bound) fluorophore. Thus, any fluorescence observed in samples treated with termination antibody is due to internalized LPS. The fluorescence level of each sample at the various time points was measured.

[0098] Figure 9 expresses the results of these experiments in terms of the number of labeled vesicles per cell. As shown in Figure 9, only cells treated with the anti-CD63 x anti-LPS bispecific antibody demonstrated significant numbers of labeled vesicles that increased over time. Cells treated with labeled LPS and control antibodies did not show appreciable numbers of fluorescent vesicles, indicating that LPS was not internalized under those treatment conditions.

[0099] This example therefore demonstrates that an anti-LPS x anti-CD63 bispecific antibody causes internalization of LPS into cells in a manner that requires simultaneous binding of LPS and CD63. Thus, these results support the use of multispecific antigen-binding molecules of the invention to promote cellular internalization of target molecules such as LPS, for the treatment of diseases and disorders such as sepsis.

[00100] The present invention should not be limited in scope by the specific embodiments described here. Of course, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications shall fall within the scope of the appended claims.

Claims (7)

1. Multispecific antigen-binding molecule, characterized by the fact that it comprises a first antigen-binding domain (D1) and a second antigen-binding domain (D2), wherein: D1 specifically binds to a target molecule ( T) wherein T is a tumor-associated protein or polypeptide expressed preferentially on the surface of a tumor cell; D2 binds to an internalization effector protein (E) that is expressed on the surface of tumor and non-tumor cells, and E undergoes cellular internalization and is processed through an intracellular degradative pathway; D2 binds to E with low affinity, such that the multispecific antigen-binding molecule preferentially targets T-expressing tumor cells and simultaneous binding of the multispecific antigen-binding molecule to T and E results in forced internalization and degradation of T in tumor cells through its physical connection with E; and E is transferrin receptor, ASGR1, or amyloid precursor protein-like protein 2 (APLP2). 2. Multispecific antigen-binding molecule according to claim 1, characterized by the fact that it is conjugated to a drug, toxin or radioisotope. 3. Multispecific antigen binding molecule according to claim 1, characterized in that T is selected from the group consisting of: IL-4R, IL-6R, PRLR, HLA-B27, AFP, ALK, BAGE proteins , β-catenin, brc abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6- AML, EphA2, Fra- 1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican- 3, GM3, gp100, Her2, HLA/B-raf, HLA/k- ras, HLA/MAGE-A3, hTERT,LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRAME, PSMA (LEAF1 ), proteins RAGE, Ras, RGS5, Rho, SART-1, SART 3, Steap-1, Steap-2, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase and uroplakin-3. 4. Multispecific antigen-binding molecule according to claim 1, characterized by the fact that: (a) D1 and/or D2 exhibit pH-dependent binding to their antigen; (b) D1 and/or D2 comprise at least one antibody variable region; (c) D1 is derived from an antigen-binding molecule that binds to but does not substantially inactivate T on its own; or (d) D1 comprises a linker, or portion of a linker, that specifically binds to T. 5. Multispecific antigen-binding molecule according to claim 4(a), characterized in that D1 binds to T with lower affinity at acidic pH compared to neutral pH; and/or where D2 binds to E with lower affinity at acidic pH compared to neutral pH. 6. Multispecific antigen binding molecule according to claim 4(b), characterized by the fact that D1 and/or D2 comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR), preferably wherein the multispecific antigen-binding molecule is a bispecific antibody. 7. Multispecific antigen binding molecule according to any one of claims 1 to 6, characterized by the fact that D2 interacts with E with a KD of 10 nM to 1 μM as measured in a surface plasmon resonance assay. 25°C.