Bispecific antibodies and methods of use thereof

Abstract

Certain embodiments of the invention provide bispecific antibodies against one or more populations of B lineage cells. Certain embodiments of the invention provide a method of depleting or inhibiting two or more populations of B lineage cells in a subject. Certain embodiments of the invention provide a method of treating a B lineage cell related disease or condition in a subject.

Description

Attorney Docket No.37188-20001.40BISPECIFIC ANTIBODIES AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application 63 / 701,101, filed on September 30, 2024, which is hereby incorporated by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (371882000140seqlist.xml; Size:71,642 bytes; and Date of Creation: September 16, 2025) is incorporated herein by reference in its entirety.FIELD

[0003] The present disclosure relates to bispecific antibodies including bispecific antibodies that bind to two different target cell types and methods of using the same.BACKGROUND

[0004] Depletion of pathogenic cells, such as B cells, plasma cells and cancer cells is a validated therapeutic approach for the treatment of many diseases, including autoimmune, allergic and chronic inflammatory diseases, as well as solid tumors, hematologic cancers, and transplant-related conditions. Despite the licensure and availability of several cytolytic or depleting antibodies and other protein therapeutics that deplete pathogenic cells, many patients fail to achieve clinical response or remission. There are still major limitations associated with available therapeutic approaches, including the inability to eliminate multiple pathogenic cell populations and incomplete depletion of cells in tissues. Thus, there remains a substantial unmet need for new cell-depleting treatments for improved efficacy in the treatment of immunologic and oncologic diseases.

[0005] Depleting monoclonal antibodies (mAbs) exert their therapeutic effect through multiple mechanisms, including mechanisms mediated by the antibody fragment crystallizable (Fc) region. These include complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). These functions depend on the interaction of the immunoglobulin G (IgG) antibody Fc domain with its cognate receptors, Fc gamma receptors (FcγRs). FcγRs are expressed at varying levels on effector cells such as natural killer (NK) cells, neutrophils,1MOFO-360553874Attorney Docket No.37188-20001.40monocytes, and macrophages. There are six main FcγRs in humans that, upon ligation of the IgG Fc domain, are thought to either activate (FcγRI / CD64, FcγRIIa / CD32a, and FcγRIIIa / CD16a) or inhibit (FcγRIIb / CD32b, FcRIIIb / CD16b) various downstream effector functions. Antigen affinity of the antibody Fab region has been shown to be important for effector function, whereby higher affinity mAbs elicit better ADCC activity due to better antigen binding (Tang, Y. et al. J Immunol.2007;179(5):2815-2823). Additionally, mAbs with modifications to the Fc domain, including post-translational glycoengineering and / or amino acid substitutions, have been shown to result in enhanced FcγR binding, ADCC / ADCP activity, and clinical responses (Desjarlais, J.R. et al. Drug Discov Today.2007;12(21-22):898-910; Ramsland, P.A. et al. J Immunol.2011;187(6):3208-3217; Richards, J.O. et al. Mol Cancer Ther.2008;7(8):2517-2527; Stavenhagen, J.B. et al. Cancer Res.2007;67(18):8882-8890; Bartsch, Y.C. et al. MAbs.2025;17(1):2465391; US20070275460; US20230057150). However, Fc-engineered therapeutic antibodies are still unable to sufficiently deplete the majority of target cells, particularly in tissues, and current approaches deplete only a single cell population, such as B cells or cancer cells (Looney, C.M. et al. Transplant Dir.2023;9(2):e1436; Vijayaraghavan, S. et al. Mol Cancer Ther.2020;19(10):2044-2056).

[0006] A novel antibody modality that is able to deplete multiple pathogenic cell populations while also enhancing effector function for better target cell depletion in tissues may deliver transformative clinical efficacy in the treatment of immunologic, oncologic and other diseases.SUMMARY OF THE INVENTION

[0007] Herein are provided dual-depleting bispecific antibodies that deplete multiple pathogenic cell populations in blood and tissues. The bispecific antibodies bind monovalently to two distinctly expressed cell surface antigens to deplete multiple pathogenic cell populations through FcγR engagement on effector cells. In human ex vivo tissue-resident mixed immune cell culture assays with native target and effector cells, bispecific antibodies demonstrated enhanced target cell depletion relative to monoclonal antibodies (mAbs) with identical complementarity determining regions (CDRs). Without wishing to be bound to theory, it is thought that enhanced effector depleting activity can be due to monovalent Fab-antigen binding that results in increased antibody opsonization on the target cell surface, increased effector cell activation, and / or alterations in FcγR expression on effector cells compared to mAbs. The depleting activity of bispecific antibodies can be further augmented 2MOFO-360553874Attorney Docket No.37188-20001.40by engineering the Fc domain, including but not limited to glycoengineering and / or amino acid mutations.

[0008] Certain embodiments of the invention provide bispecific antibodies that bind to two distinct cell surface antigens to deplete multiple pathogenic B cell lineage populations. The bispecific antibodies deplete cells across the B cell lineage, including immature B cells, naïve B cells, memory B cells, plasmablasts, and plasma cells, for example in certain embodiments through dual binding to one target that is expressed on B cells and to a second target that is expressed on plasma cells. In certain embodiments, the bispecific antibodies dually bind to either B cell activating factor receptor (BAFF-R) or CD19 or CD20 on B cells and to CD38 or CD19 or B-cell maturation antigen (BCMA) on plasma cells. The bispecific antibodies exhibit multiple effector mechanisms, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and could have better target cell depleting activity. In some embodiments, the first and second antigen targets are different.

[0009] Certain embodiments of the invention provide a bispecific antibody comprising: a first binding domain which binds BAFF-R, CD19, or CD20 and a second binding domain which binds CD38, CD19, or BCMA. In some embodiments, the first and second binding domains bind to different target antigens.

[0010] Certain embodiments of the invention provide a bispecific antibody comprising an anti-BAFF-R antigen binding domain, an anti-CD38 antigen binding domain, and a Fc region (e.g., engineered Fc).

[0011] Certain embodiments of the invention provide a bispecific antibody comprising an anti-CD19 antigen binding domain, an anti-BCMA antigen binding domain, and a Fc region (e.g., engineered Fc).

[0012] Certain embodiments of the invention provide a bispecific antibody comprising an anti-CD19 antigen binding domain, an anti-CD38 antigen binding domain, and a Fc region (e.g., engineered Fc).

[0013] Certain embodiments of the invention provide a bispecific antibody comprising an anti-CD20 antigen binding domain, an anti-CD38 antigen binding domain, and a Fc region (e.g., engineered Fc).

[0014] Certain embodiments of the invention provide a bispecific antibody comprising an anti-CD20 antigen binding domain, an anti-BCMA antigen binding domain, and a Fc region (e.g., engineered Fc).3MOFO-360553874Attorney Docket No.37188-20001.40

[0015] Certain embodiments of the invention provide a bispecific antibody comprising an anti- BAFF-R antigen binding domain, an anti-BCMA antigen binding domain, and a Fc region (e.g., engineered Fc).

[0016] Certain embodiments of the invention provide a pharmaceutical composition comprising a bispecific antibody described herein, and a pharmaceutically acceptable carrier.

[0017] Certain embodiments of the invention provide a method of depleting B cell lineage immune cells in a subject, comprising administering a bispecific antibody described herein to the subject.

[0018] Certain embodiments of the invention provide a method of treating a B lineage cell related disease or condition (e.g., an autoimmune disease, transplant-related condition, or hematologic cancer) of a subject in need of, comprising administering a bispecific antibody described herein to the subject.

[0019] Certain embodiments of the invention provide a bispecific antibody as described herein for the prophylactic or therapeutic treatment of an autoimmune disease, transplant-related condition, or hematologic cancer in a subject.

[0020] Certain embodiments of the invention provide a method of depleting various immune cell subsets of the B cell lineage upon binding to cells expressing BAFF-R and / or CD38 with the bispecific antibody as described herein.

[0021] Certain embodiments of the invention provide a method of depleting various immune cell subsets of the B cell lineage upon binding to cells expressing CD19 and / or BCMA with the bispecific antibody as described herein.

[0022] Certain embodiments of the invention provide a method of depleting various immune cell subsets of the B cell lineage upon binding to cells expressing CD19 and / or CD38 with the bispecific antibody as described herein.

[0023] Certain embodiments of the invention provide a method of depleting various immune cell subsets of the B cell lineage upon binding to cells expressing CD20 and / or CD38 with the bispecific antibody as described herein.

[0024] Certain embodiments of the invention provide a method of depleting various immune cell subsets of the B cell lineage upon binding to cells expressing CD20 and / or BCMA with the bispecific antibody as described herein.

[0025] Certain embodiments of the invention provide a method of depleting various immune cell subsets of the B cell lineage upon binding to cells expressing BAFF-R and / or BCMA with the bispecific antibody as described herein.4MOFO-360553874Attorney Docket No.37188-20001.40

[0026] Certain embodiments of the invention provide a method of enhancing cytolytic activity through monovalent binding for greater antibody opsonization of the target cell surface that results in better cell depletion activity than the single agent combination of monoclonal antibodies.

[0027] Certain embodiments of the invention provide a polypeptide as described herein (e.g., a polypeptide chain comprising an amino acid sequence described herein).

[0028] Certain embodiments of the invention provide a method as described herein.

[0029] Certain embodiments of the invention provide a bispecific antibody that binds to two different target antigens, wherein the bispecific antibody binds to a first target antigen expressed on a first target cell of the B lymphocyte lineage and a second target antigen expressed on a second target cell of the B lymphocyte lineage, wherein the first and second target antigens are different, wherein the first and second target cells are (or represent, or are expressed by) distinct cell populations of the B lymphocyte lineage, and wherein the bispecific antibody exhibits increased effector cell function, as compared to effector cell function of an antibody that bivalently binds to either the first or the second target antigen. In some embodiments, the first target cell is a B cell, and the second target cell is a plasma cell. In some embodiments, the first target antigen is selected from the group consisting of BAFF-R, CD19, and CD20. In some embodiments, the second target antigen is selected from the group consisting of BCMA, CD19, and CD38. In some embodiments, the first target antigen is BAFF-R, and the second target antigen is CD38. In some embodiments, the effector cell function is selected from the group consisting of antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the effector cell function is ADCC, ADCP, or ADCC and ADCP. In some embodiments, the enhanced effector cell function results in depletion of both the first target cell and the second target cell. In some embodiments, the bispecific antibody exhibits increased target cell depletion, as compared to target cell depletion by an antibody that bivalently binds to either the first or the second target antigen (e.g., a bivalent, monospecific antibody). In some embodiments, the bispecific antibody exhibits increased depletion of the first or second target cell, as compared to depletion of the first or second target cell by an antibody that bivalently binds to either the first or the second target antigen. In some embodiments, the bispecific antibody exhibits increased antibody opsonization, as compared to antibody opsonization by an antibody that bivalently binds to either the first or the second target antigen (e.g., a bivalent, monospecific antibody). In some embodiments, the antibody comprises an Fc region. In some embodiments, the Fc region comprises one or more modifications that result in enhanced effector cell function, as compared to an Fc region that lacks the one or more modifications. In some embodiments, the Fc 5MOFO-360553874Attorney Docket No.37188-20001.40region comprises one or more amino acid substitutions or deletions that increase effector cell function, as compared to effector cell function of an Fc region that lacks the amino acid substitution(s) or deletion(s). In some embodiments, the Fc region is afucosylated. In some embodiments, the bispecific antibody comprises two antibody heavy chain polypeptides, each comprising an Fc region.

[0030] Certain embodiments of the invention provide a bispecific antibody that binds to BAFF-R and CD38, wherein the antibody comprises a first heavy chain variable (VH) domain, a first light chain variable (VL) domain, a second heavy chain variable (VH) domain, and a second light chain variable (VL) domain, wherein: (a) the first VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; (b) the first VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; (c) the second VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:11, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:12, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:13; and (d) the second VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:14, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:15, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:16. In some embodiments, the antibody further comprises an Fc domain, wherein the Fc domain is afucosylated.

[0031] Certain embodiments of the invention provide a bispecific antibody that binds to BAFF-R and CD38, wherein the antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 9, a first light chain comprising the amino acid sequence of SEQ ID NO: 10, a second heavy chain comprising amino acid sequence SEQ ID NO: 19, and a second light chain comprising amino acid sequence SEQ ID NO: 20. In some embodiments, one or both of the first and second heavy chains is / are afucosylated.

[0032] Certain embodiments of the invention provide a method for treating or preventing progression of a B-cell-mediated disease or disorder, comprising administering an effective amount of the bispecific antibody of any one of the above embodiments to an individual in need thereof. Certain embodiments of the invention provide a method for depleting B cells in an individual in need thereof, comprising administering to the individual an effective amount of the bispecific antibody of any one of the above embodiments. Certain embodiments of the invention provide the bispecific antibody of any one of the above embodiments for use in a method for treating or preventing progression of a B-cell-mediated disease or disorder in an 6MOFO-360553874Attorney Docket No.37188-20001.40individual in need thereof, said method comprising administering an effective amount of the antibody to the individual. Certain embodiments of the invention provide the bispecific antibody of any one of the above embodiments for use in a method for depleting B cells in an individual in need thereof, said method comprising administering an effective amount of the antibody to the individual. Certain embodiments of the invention provide the use of the bispecific antibody of any one of the above embodiments in the manufacture of a medicament for treating or preventing progression of a B-cell-mediated disease or disorder in an individual in need thereof. Certain embodiments of the invention provide the use of the bispecific antibody of any one of the above embodiments in the manufacture of a medicament for depleting B cells in an individual in need thereof. In some embodiments, the individual is a human. Certain embodiments of the invention provide a pharmaceutical composition comprising the bispecific antibody of any one of the above embodiments and a pharmaceutically acceptable carrier.

[0033] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will become apparent to one of skill in the art. These and other embodiments of the present disclosure are further described by the detailed description that follows.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG.1 shows a diagram and table of bispecific antibodies. In the diagram, dark gray rectangles represent variable domains that recognize a first target antigen, as well as immunoglobulin G1 (IgG1) constant region heavy and light chains, and unfilled rectangles represent variable domains that recognize a second target cell antigen, as well as IgG1 constant region heavy and light chains. The table lists each construct’s identifier, each of the two variable domains, and the constant fragment (Fc) domain.

[0035] FIGS.2A & 2B show purification of a bispecific antibody MGE-2v1. FIG.2A is a trace from standard size exclusion chromatography by high-performance liquid chromatography (SEC-HPLC) of MGE-2v1. FIG.2B shows images of sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses of MGE-2v1 in non-reducing (NR) and reducing (R) conditions.

[0036] FIGS.3A-3C show target binding by bispecific antibody MGE-2v1. FIG.3A is a schematic representation of binding (optical density (O.D.) units) in enzyme-linked7MOFO-360553874Attorney Docket No.37188-20001.40immunosorbent assays (ELISAs) using recombinant BAFF-R protein at 1 µg / mL and MGE-2v1 at 0.06 to 1.0 µg / mL. FIG.3B is a schematic representation of binding (O.D. units) measured by ELISA using recombinant CD38 protein at 1 µg / mL and MGE-2v1 at 0.06 to 1.0 µg / mL. FIG.3C is a schematic representation of binding (O.D. units) in a bridging ELISA using recombinant CD38 at 1 µg / mL, MGE-2v1 at 0.06 to 1.0 µg / mL and BAFF-R at 1 µg / mL.

[0037] FIGS.4A & 4B show target cell depletion via ADCC or ADCP by bispecific antibody MGE-2v1. FIG.4A is a schematic representation of target cell depletion by ADCC (percentage of target cells remaining) in in vitro ADCC assays using peripheral blood mononuclear cells (PBMCs) and MGE-2v1 at 0.00006 to 5.0 µg / mL. FIG.4B is a schematic representation of target cell depletion by ADCP (percentage of phagocytosed cells) in in vitro ADCP assays using fluorescent-labeled B cells isolated from peripheral blood, peripheral blood monocyte-derived macrophages (MDMs), and MGE-2v1 at 0.001 to 20.0 µg / mL.

[0038] FIGS.5A-5D are schematic representations of depletion (percentage of cells remaining) of various target cell populations in an ex vivo bone marrow tissue-resident mixed immune cell culture assay with in vitro MDMs and MGE-2v1 (gray) or rituximab (anti-CD20 mAb; black) at 0.006 to 20.0 µg / mL. FIG.5A is a schematic representation of depletion of BAFF-R+ CD38+ immature B cells. FIG.5B is a schematic representation of depletion of BAFF-R+ CD38- naïve B cells. FIG.5C is a schematic representation of depletion of BAFF-R- CD38+ plasmablasts. FIG.5D is a schematic representation of depletion of BAFF-R-CD38+ plasma cells.

[0039] FIGS.6A & 6B show antibody opsonization by bispecific antibody MGE-2v1. FIG.6A is a schematic representation of antibody opsonization (percentage increase in antibody on surface) on one target cell population (naïve B cells) in PBMCs incubated with MGE-2v1 (gray) or ianalumab (anti-BAFF-R mAb; black) at 0.001 to 20.0 µg / mL. FIG.6B is a schematic representation of antibody opsonization (percentage increase in antibody on surface) on a second target cell population (plasmablasts) in tonsillar mononuclear cells incubated with MGE-2v1 (gray) or felzartamab / MOR-202 (anti-CD38 mAb; black) at 0.001 to 20.0 µg / mL

[0040] FIGS.7A & 7B show cell depletion by bispecific antibody MGE-2v1. FIG.7A is a schematic representation of depletion (percentage of cells remaining) of CD20+ CD38- naïve B cells in an ex vivo bone marrow tissue-resident mixed immune cell culture assay with in vitro MDMs and MGE-2v1, ianalumab (anti-BAFF-R mAb), or felzartamab (anti-CD38 mAb) at 25 µg / mL. FIG.7B is a schematic representation of depletion (percentage of cells 8MOFO-360553874Attorney Docket No.37188-20001.40remaining) of CD20- CD38+ plasma cells in an ex vivo bone marrow tissue-resident mixed immune cell culture assay with in vitro MDMs and MGE-2v1, ianalumab (anti-BAFF-R mAb), or felzartamab (anti-CD38 mAb) at 25 µg / mL.DETAILED DESCRIPTION

[0041] As used herein, the articles “a” and “an” refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means one or element or more than one element.

[0042] The term “therapeutically effective amount,” in reference to treating a disease state / condition, refers to an amount of an antibody either alone or as contained in a pharmaceutical composition that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state / condition when administered as a single dose or in multiple doses. Such effect need not be absolute to be beneficial.

[0043] The terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or decrease an undesired physiological change or disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean reducing risk of death, reducing the severity of the disorder, or prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0044] The term “Fc region” refers to a C-terminal region of an immunoglobulin heavy chain polypeptide. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. The term includes native sequence of Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at about position Cys226, or from about position Pro230, to the carboxyl-terminus of the Fc region (using herein the numbering system according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991). The C-9MOFO-360553874Attorney Docket No.37188-20001.40terminal lysine (Lys447) of the Fc region may or may not be present. One or more C-terminal residue(s) of Fc region may be absent or replaced by other amino acid substitution(s).

[0045] A “variant Fc” or “engineered Fc” refers to an Fc region that has been modified relative to a parent, native Fc region. A variant Fc may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising a modification or substitution at one or more amino acid positions in the Fc region. An engineered Fc also include modified glycosylation in the Fc region.

[0046] The terms “Fc receptor” or “FcR” refer to a receptor that binds to the Fc region of an antibody. There are three main classes of Fc receptors: (1) FcγR which binds to IgG, (2) FcαR which binds to IgA, and (3) FcεR which binds to IgE. The FcγR family includes several members, such as FcγI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16A), and FcγRIIIB (CD16B). The Fcγ receptors differ in their affinity for IgG and also have different affinities for the IgG subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0047] The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to, chimeric antibody, humanized antibody, human antibody, monoclonal antibody, single-domain antibody, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen- binding activity. An antibody may include a full-length immunoglobulin molecule or a portion of a full-length immunoglobulin molecule that contains an antigen binding site that specifically binds an antigen of a target of interest. The immunoglobulin can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species (e.g., human, or mouse). An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which theintact antibodybinds. Examples of antibody fragments include but are not limited to Fab, Fab', Fab'-SH, F(ab')2and scFv.

[0048] The term “antigen binding domain” or “binding domain” as used herein refers to one or more fragments of an antibody that retains the ability to specifically bind a target antigen. Examples of antigen binding domain includes, but not limited to, Fab (a monovalent fragment consisting of the VL, VH, CL and CH1 domains), single-chain variable fragment (scFv), single- domain antibody (nanobody or VHH), a VL (light chain variable region), and a VH (heavy chain variable region).10MOFO-360553874Attorney Docket No.37188-20001.40

[0049] A scFv is a fusion protein of the variable region of the heavy (VH) and light chains(VL)of an immunoglobulin that is connected by means of a linker peptide. The linker is usually short, about 10-25 amino acids in length. If flexibility is important, the linker will contain a significant number of glycines. If solubility is important, serines or threonines maybe utilizedin the linker. The linker may link the amino-terminus of the VHto the carboxy-terminus of the VL, or the linker may link the carboxy-terminus of the VH to the amino-terminus of the VL.

[0050] A Fv fragment is an antibody fragment, and contains a complete antigen recognition and binding site. The six CDRs of both the variable regions (VH and VL) interact with each other to form an antigen-binding site. However, a variable region (or a half Fv, which contains only three antigen-specific CDRs) alone is also known to be able to recognize and bind to an antigen, although its affinity is lower than the affinity of the entire binding site.

[0051] A Fab fragment (also referred to as F(ab)) also contains a light chain constant region and heavy chain constant region CH1. For example, papain digestion of an antibody produces two kinds of fragments: an antigen-binding fragment, called a Fab fragment, containing the variable regions of a heavy chain and light chain, which serve as an antigen-binding domain; and the remaining portion, which is called an “Fc” because it is readily crystallized. A Fab' fragment is different from a Fab fragment in that a Fab' fragment also has several residues derived from the carboxyl terminus of a heavy chain CH1 region, which contains one or more cysteine residues from the hinge region of an antibody. A Fab' fragment is, however, structurally equivalent to Fab in that both are antigen-binding fragments which comprise the variable regions of a heavy chain and light chain. Herein, an antigen-binding fragment comprising the variable regions of a heavy chain and light chain which may serve as an antigen-binding domain, and which is equivalent to that obtained by papain digestion, is referred to as a “Fab-like antibody,” even when it is not identical to an antibody fragment produced by protease digestion. Fab'-SH is Fab' with one or more cysteine residues having free thiol groups in its constant region. A F(ab') fragment is produced by cleaving the disulfide bond between the cysteine residues in the hinge region of F(ab')2. Other chemically crosslinked antibody fragments are also known to those skilled in the art. Pepsin digestion of an antibody yields two fragments; one is a F(ab')2fragment whichcomprises two antigen-binding domains, and the other is the remaining fragment (referred to as pFc'). Herein, an antibody fragment equivalent to that obtained bypepsin digestion is11MOFO-360553874Attorney Docket No.37188-20001.40referred to as a “F(ab')2-like antibody” when it comprises two antigen-binding domains.Such antibody fragments can also be produced, for example, by genetic engineering.

[0052] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In some embodiments, a chimeric antibody is a monoclonal antibody comprising a variable region from one source or species (e.g., mouse) and a constant region derived from a second source or species (e.g., human).

[0053] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human framework regions (FRs). In certain embodiments, a humanized antibody will comprise at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.

[0054] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, an antibody group wherein the antibodies constituting the group are homogeneous except for naturally occurring mutants that may exist in a small amount. Monoclonal antibodies are highly specific and interact with a single antigenic site. Furthermore, each monoclonal antibody targets a single antigenic determinant (epitope) on an antigen, as compared to common polyclonal antibody preparations that typically contain various antibodies against diverse antigenic determinants. In addition to their specificity, monoclonal antibodies are advantageous in that they are typically produced from hybridoma cultures not contaminated with other immunoglobulins.

[0055] As used herein, the term “bispecific” antibody as used herein, refers to an antibody having binding affinity for at least two distinct epitopes. The distinct epitopes may be located on one protein or may be located on different proteins. Bispecific antibodies of the present invention may or may not display symmetrical monovalent binding to two distinct epitopes.

[0056] As used herein, the term “monospecific” refers to an antibody that has binding affinity for only one epitope.

[0057] The terms “identical” or percent “identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 12MOFO-360553874Attorney Docket No.37188-20001.4097%, at least 98%, or at least 99% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal X, Clustal W, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used.

[0058] The terms “protein,” “peptide” and “polypeptide” are used interchangeably herein. The term “variant” polypeptide refers to a polypeptide derived from the native protein but has substitution of one or more amino acids at one or more sites in the native protein, or deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and / or C-terminal end of the native protein; or deletion or addition of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.

[0059] Thus, the polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the polypeptides can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art.

[0060] Individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are “conservatively modified variations,” where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I);Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q).13MOFO-360553874Attorney Docket No.37188-20001.40

[0061] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucl. Acids Res.,19:508 (1991); Ohtsuka et al., JBC, 260:2605 (1985);Rossolini et al., Mol. Cell. Probes, 8:91 (1994).

[0062] A "nucleic acid fragment" is a fraction of a given nucleic acid molecule.Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term "nucleotide sequence" refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms "nucleic acid," "nucleic acid molecule," "nucleic acid fragment," "nucleic acid sequence or segment," or "polynucleotide" may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene.

[0063] A "variant" of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis that encode the native protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the invention will have at least 40, 50, 60, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%,14MOFO-360553874Attorney Docket No.37188-20001.4092%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence.

[0064] “Conservatively modified variations” of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are "silent variations" which are one species of "conservatively modified variations." Every nucleic acid sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. One of skill will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each "silent variation" of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0065] The invention encompasses isolated or substantially purified nucleic acid or protein compositions. In the context of the present invention, an "isolated" or "purified" DNA molecule or an "isolated" or "purified" polypeptide is a DNA molecule or polypeptide that exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of 15MOFO-360553874Attorney Docket No.37188-20001.40contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, culture medium may represent less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals. Fragments and variants of the disclosed nucleotide sequences and proteins or partial-length proteins encoded thereby are also encompassed by the present invention.

[0066] A “vector" is defined to include, inter alia, any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).

[0067] "Expression cassette" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development. Such expression cassettes will comprise the transcriptional initiation region of the invention linked to a nucleotide sequence of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

[0068] "Promoter" refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription."Promoter" includes a minimal promoter that is a short DNA sequence comprised of a16MOFO-360553874Attorney Docket No.37188-20001.40TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. "Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.

[0069] "Expression" refers to the transcription and / or translation in a cell of an endogenous gene, transgene, as well as the transcription and stable accumulation of sense (mRNA) or functional RNA. In the case of antisense constructs, expression may refer to the transcription of the antisense DNA only. Expression may also refer to the production of protein.

[0070] The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window.

[0071] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that has the same function or biological activity as screened or selected for in the originally transformed cell are included herein.17MOFO-360553874Attorney Docket No.37188-20001.40Bispecific Antibodies

[0072] Described herein include bispecific antibodies that broadly deplete immune cell populations across the B cell lineage, nucleic acids encoding these bispecific antibodies, and methods of making and using these proteins. The bispecific antibodies can simultaneously bind to one antigen expressed on B cells, such as BAFF-R (TNFRSF13C, BR3), CD19, or CD20, and one antigen expressed on plasma cells, such as CD38, CD19, or BCMA, to deplete multiple B cell subsets, including immature, naïve, and memory B cells and plasmablasts, and plasma cells. In another aspect, the bispecific antibodies exhibit increased cytolytic activity due to enhanced antibody opsonization on the target cell surface resulting from monovalent binding that results in better cell depletion than the combination of mAbs thereof.

[0073] Accordingly, certain embodiments of the invention provide a bispecific antibody comprising a first binding domain which binds BAFF-R, CD19, or CD20 and a second binding domain which binds CD38, CD19, or BCMA. In some embodiments, the first and second binding domains bind to different target antigens. In some embodiments, when the first binding domain binds CD19, the second binding domain does not also bind CD19. In some embodiments, when the second binding domain binds CD19, the first binding domain does not also bind CD19.

[0074] The term “B cell activating factor receptor” or “BAFF-R” refers to human BAFF-R protein (see NCBI accession number Q96RJ3). The term “CD19” refers to human CD19 protein (see NCBI accession number P15391). The term “CD20” refers to human CD20 protein (see NCBI accession number P11836). The term “CD38” refers to human CD38 protein (see NCBI accession number P28907). The term “B Cell Maturation Antigen) or “BCMA” refers to human BCMA protein (see NCBI accession number Q02223).

[0075] In certain embodiments, the bispecific antibody of the present invention comprises a first binding domain (e.g., antigen binding domain) and a second binding domain (e.g., antigen binding domain). In some embodiments, the first binding domain binds BAFF-R. In some embodiments, the first binding domain binds CD19. In other embodiments, the first binding domain binds CD20. In some embodiments, the second binding domain binds CD38. In other embodiments, the second binding domain binds BCMA.

[0076] In certain embodiments, the first binding domain binds BAFF-R and the second binding domain binds CD38. In certain embodiments the first binding domain binds CD19 and the second binding domain binds BCMA. In certain embodiments, the first binding domain binds18MOFO-360553874Attorney Docket No.37188-20001.40CD19 and the second binding domain binds CD38. In certain embodiments, the first binding domain binds CD20 and the second binding domain binds CD38. In other embodiments, the first binding domain binds CD20 and the second binding domain binds BCMA. In some embodiments, the first binding domain binds BAFF-R and the second binding domain binds BCMA.

[0077] In certain embodiments, the bispecific antibody of the present invention binds to two different target antigens, wherein the bispecific antibody binds to a first target antigen expressed on a first target cell of the B lymphocyte lineage and a second target antigen expressed on a second target cell of the B lymphocyte lineage. In some embodiments, the first and second target cells are distinct cell populations of the B lymphocyte lineage. For example, in some embodiments, the first target cell is a B cell, and the second target cell is a plasma cell. In some embodiments, the first and second target antigens are not expressed by the same cell population of the B lymphocyte lineage. In some embodiments, the first and second target antigens are different. In some embodiments, the first and second target antigens are antigens from different polypeptides (i.e., the first and second target antigens do not represent different epitopes from the same polypeptide).

[0078] In certain embodiments, an antibody of the present disclosure binds to an antigen target monovalently if only one binding domain of the antibody specifically binds the antigen target. In certain embodiments, an antibody of the present disclosure binds to a cell type or target cell population monovalently if only one binding domain of the antibody specifically binds the cell type or target cell population. For the example, the antibody can be a bispecific antibody that binds monovalently to two distinct antigen targets or cell types / target cell populations, e.g., when each antigen binding domain of the antibody specifically binds to a different antigen target or cell type / target cell population. In some embodiments, the bispecific antibody comprises only one antigen binding domain that specifically binds to a particular antigen target or cell type / target cell population. In some embodiments, the antibody is a bispecific antibody that binds monovalently to two different antigen targets. In some embodiments, the antibody is a bispecific antibody that binds monovalently to two different cell types or target cell populations (e.g., by monovalently binding to two different antigen targets that are not expressed on the same cell type or target cell population).

[0079] In certain embodiments, an antibody of the present disclosure binds to an antigen target bivalently if two antigen binding domains of the antibody specifically bind the (same) antigen target. In some embodiments, the two antigen binding domains of the antibody can bind to the same or different, distinct epitopes of the same antigen target. In certain embodiments, an antibody of the present disclosure binds to a cell type or target cell19MOFO-360553874Attorney Docket No.37188-20001.40population bivalently if two antigen binding domains of the antibody specifically bind (same) the cell type or target cell population (e.g., by specifically binding to the same antigen target expressed by the cell type or target cell population, or by specifically binding to two different antigen targets that are both expressed by the same cell type or target cell population).

[0080] In certain embodiments, a bispecific antibody of the present disclosure depletes (e.g., is capable of depleting) a cell type or target cell population that expresses an antigen target to which the antibody binds monovalently. In certain embodiments, a bispecific antibody of the present disclosure depletes (e.g., is capable of depleting) a cell type or target cell population to which the antibody binds monovalently. In certain embodiments, a bispecific antibody of the present disclosure depletes (e.g., is capable of depleting) a cell type or target cell population that expresses an antigen target to which the antibody binds bivalently. In certain embodiments, a bispecific antibody of the present disclosure depletes (e.g., is capable of depleting) a cell type or target cell population to which the antibody binds bivalently (e.g., by bivalently binding to the same antigen target expressed by the cell type or target cell population, or by binding to two different antigen targets that are both expressed by the cell type or target cell population).

[0081] In certain embodiments, the first binding domain comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining regions (CDRs) as described in Table 1.

[0082] In certain embodiments, the first binding domain comprises an anti-BAFF-R VH, an anti- CD19 VH, or an anti-CD20 VH (e.g., see Table 1). In certain embodiments, the first binding domain comprises an anti-BAFF-R VL, an anti-CD19 VL, or an anti-CD20 VL as described in Table 1.

[0083] In certain embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL). In certain embodiments, the first binding domain comprises a Fab. In certain embodiments, the Fab comprises VL-CL and VH-CH1. In certain embodiments, the Fab has a crossmab construct that comprises VL-CH1 and VH-CL.

[0084] In certain embodiments, the second binding domain comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining regions (CDRs) as described herein (e.g., see Table 1).

[0085] In certain embodiments, the second binding domain comprises an anti-BCMA VH, or an anti-CD38 VH (e.g., see Table 1). In certain embodiments, the second binding domain comprises an anti-BCMA VL, or an anti-CD38 VL (e.g., see Table 1).

[0086] In certain embodiments, the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL). In certain embodiments, the second binding domain comprises a Fab. In certain embodiments, the Fab comprises VL-CL 20MOFO-360553874Attorney Docket No.37188-20001.40and VH-CH1. In certain embodiments, the Fab has a crossmab construct that comprises VL-CH1 and VH-CL.

[0087] In certain embodiments, a variable region (VH or VL) is directly linked to a constant region (e.g., CL or CH1). In certain embodiments, a variable region (VH or VL) is indirectly linked to a constant region (e.g., CL or CH1), e.g., via a linker sequence.

[0088] In certain embodiments, the antibody comprises a kappa light chain constant region.

[0089] In certain embodiments, the antibody comprises a lambda light chain constant region.

[0090] In certain embodiments, the antibody comprises a kappa light chain constant region and a lambda light chain constant region.

[0091] In certain embodiments, the first binding domain comprises a kappa light chain constant region and the second binding domain comprises a lambda light chain constant region.

[0092] In certain embodiments, the first binding domain comprises a lambda light chain constant region and the second binding domain comprises a kappa light chain constant region.

[0093] In certain embodiments, the bispecific antibody is an IgG1 antibody. In certain embodiments, the bispecific antibody is an IgG2 antibody. In certain embodiments, the bispecific antibody is an IgG3 antibody. In certain embodiments, the bispecific antibody is an IgG4 antibody. In certain embodiments, the bispecific antibody comprises engineered Fc region. In certain embodiments, the bispecific antibody comprises engineered Fc region (e.g., amino acid residue mutation and / or glycosylation modification) that has enhanced effector function(s), for example, in certain embodiments, the bispecific antibody comprises engineered Fc region that has enhanced binding with one or more FcγRs as compared to the parent wildtype Fc region. In certain embodiments, the bispecific antibody is an afucosylated antibody.

[0094] In certain embodiments, the bispecific antibody comprises an Fc region that comprises one or more modifications that result in enhanced effector cell function, as compared to an Fc region that lacks the one or more modifications (e.g., as compared to a wild-type Fc region). Exemplary modifications include, without limitation, altered carbohydrate modification (e.g., reduced fucosylation or afucosylation) and amino acid substitution(s) that enhance binding with one or more FcγRs as compared to the parent wildtype Fc region. In some embodiments, the effector cell function(s) comprise ADCC, ADCP, or ADCC and ADCP. In some embodiments, the enhanced effector cell function results in depletion of both the first target cell and the second target cell.21MOFO-360553874Attorney Docket No.37188-20001.40

[0095] In certain embodiments, the bispecific antibody comprises an engineered Fc region that has enhanced binding with neonatal Fc receptor (FcRn) as compared to the parent wildtype Fc region. In certain embodiments, the bispecific antibody is a human IgG antibody that has improved circulating half-life as compared to parent wildtype IgG antibody. In certain embodiments, the Fc region comprises M252Y / S254T / T256E (YTE) mutations.

[0096] Technologies for construction of bispecific antibodies are known in the art and also describe herein, for example, knob-into-hole format (Knob: T366W; Hole: T366S, L368A, and Y407V) can be used for the production of bispecific antibodies. In certain embodiments, the bispecific antibody comprises one heavy chain comprising T366W mutation and the other heavy chain comprising T366S, L368A, and Y407V mutations.

[0097] In certain embodiments, the bispecific antibody comprises one or more heavy chains (e.g., two heavy chains) that lack a C-terminal lysine. In some embodiments, the bispecific antibody comprises a heavy chain sequence listed in Table 1, but lacking the C-terminal lysine. As is known in the art, the C-terminal lysine of a heavy chain or Fc region may be removed, e.g., during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.

[0098] In certain embodiments, the bispecific antibody is a chimeric antibody. In certain embodiments, the bispecific antibody is a humanized antibody. In certain embodiments, the bispecific antibody is a human antibody.

[0099] By way of non-limiting examples, Table 1 summarizes sequences of the bispecific antibodies of the present invention. One of skill in the art will recognize that antibodies are “modular” in nature. Throughout the disclosure, various specific embodiments of the various “modules” composing the antibodies of the present invention are described. As specific non-limiting examples, various embodiments of variable heavy chain CDRs, variable heavy chains, variable light chain CDRs, and variable light chains are described. It is intended that all of the specific embodiments may be combined with each other as though each specific combination were explicitly described individually.22MOFO-360553874Attorney Docket No.37188-20001.4023MOFO-360553874Attorney Docket No.37188-20001.4024MOFO-360553874Attorney Docket No.37188-20001.4025MOFO-360553874Attorney Docket No.37188-20001.4026MOFO-360553874Attorney Docket No.37188-20001.4027MOFO-360553874Attorney Docket No.37188-20001.4028MOFO-360553874Attorney Docket No.37188-20001.40BAFF-R X CD38 bispecific antibodies (e.g., MGE-2v1)

[0100] In certain embodiments, the first binding domain binds BAFF-R and the second binding domain binds CD38.

[0101] In certain embodiments, the BAFF-R X CD38 bispecific antibody comprises an anti-BAFF-R binding domain as described herein, and an anti-CD38 binding domain as described herein.

[0102] In certain embodiments, the BAFF-R X CD38 bispecific antibody comprises an anti-BAFF-R heavy chain and an anti-BAFF-R light chain as described herein; and an anti-CD38 heavy chain and an anti-CD38 light chain as described herein.

[0103] In certain embodiments, the first binding domain (anti-BAFF-R binding domain) comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining regions (CDRs) selected from the group consisting of (a) CDR-H1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:1; (b) CDR-H2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:2); (c) CDR-H3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least95%, or 100% sequence identity to SEQ ID NO:3; (d) CDR-L1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:4; (e) CDR-L2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:5; and (f) CDR-L3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:6.

[0104] In certain embodiments, the anti-BAFF-R binding domain comprises CDR-H1 comprising amino acid sequence of SEQ ID NO:1, CDR-H2 comprising amino acid sequence of SEQ ID NO: 2, and CDR-H3 comprising amino acid sequence of SEQ ID NO: 3. In certain embodiments, the anti-BAFF-R binding domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, CDR-L2 comprising amino acid sequence of SEQ ID NO:5, and CDR-L3 comprising amino acid sequence of SEQ ID NO:6.

[0105] In certain embodiments, the anti-BAFF-R binding domain comprises (a) a heavy chain variable region (VH) and / or (b) a light chain variable region (VL) as described herein.

[0106] In certain embodiments, the anti-BAFF-R binding domain comprises:29MOFO-360553874Attorney Docket No.37188-20001.40

[0107] (a) a heavy chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:7; and / or

[0108] (b) a light chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:8).

[0109] In certain embodiments, the anti-BAFF-R binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:7 and a light chain variable region (VL) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:8. In some embodiments, the anti-BAFF-R binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:8.

[0110] In certain embodiments, the bispecific antibody (e.g., anti-BAFF-R arm) comprises: (a) a heavy chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:9, and / or b) a light chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:10.

[0111] In certain embodiments, the bispecific antibody comprises: (a) a heavy chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:9, and (b) a light chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:10.

[0112] In certain embodiments, the bispecific antibody (anti-BAFF-R arm of MGE-2v1) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a light chain comprising the amino acid sequence of SEQ ID NO:10.

[0113] In certain embodiments, the second binding domain (anti-CD38 binding domain) comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining regions (CDRs) selected from the group consisting of (a) CDR-H1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:11; (b) CDR-H2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:12; (c) CDR-H330MOFO-360553874Attorney Docket No.37188-20001.40comprising amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 13; (d) CDR-L1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:14; (e) CDR-L2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:15; and (f) CDR-L3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:16.

[0114] In certain embodiments, the anti-CD38 binding domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO:11, CDR-H2 comprising the amino acid sequence of SEQ ID NO:12, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:13. In certain embodiments, the anti-CD38 binding domain sequences comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO:14, CDR-L2 comprising the amino acid sequence of SEQ ID NO:15, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:16.

[0115] In certain embodiments, the anti-CD38 binding domain comprises CDR-H1 comprising amino acid sequence of SEQ ID NO:11, CDR-H2 comprising amino acid sequence of SEQ ID NO:12, CDR-H3 comprising amino acid sequence of SEQ ID NO:13, CDR-L1 comprising amino acid sequence of SEQ ID NO: 14, CDR-L2 comprising amino acid sequence of SEQ ID NO:15 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:16.

[0116] In certain embodiments, the anti-CD38 binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 17 and / or a light chain variable region (VL) comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:18.

[0117] In certain embodiments, the anti-CD38 binding domain comprises a VH comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 17 and a VL comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:18.

[0118] In certain embodiments, the anti-CD38 binding domain comprises a heavy chain variable region comprising amino acid sequence of SEQ ID NO:17 and a light chain variable region comprising amino acid sequence of SEQ ID NO:18.31MOFO-360553874Attorney Docket No.37188-20001.40

[0119] In certain embodiments, the bispecific antibody (anti-CD38) arm comprises a heavy chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:19, and / or light chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:20.

[0120] In certain embodiments, the bispecific antibody comprises a heavy chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:19 and a light chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:20.

[0121] In certain embodiments, the bispecific antibody MGE-2v1 (anti-CD38 arm) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:19 and a light chain comprising the amino acid sequence of SEQ ID NO:20.CD19 X BCMA bispecific antibodies (e.g., MGE-2v2)

[0122] In certain embodiments, the first binding domain binds CD19 and the second binding domain binds BCMA.

[0123] In certain embodiments, the CD19 X BCMA bispecific antibody comprises an anti-CD19 binding domain as described herein, and an anti-BCMA binding domain as described in Table 1. In certain embodiments, the CD19 X BCMA bispecific antibody comprises an anti-CD19 heavy chain and an anti-CD19 light chain as described herein; and an anti-BCMA heavy chain and an anti-BCMA light chain as described in Table 1.

[0124] In certain embodiments, the first binding domain (anti-CD19 binding domain) comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining regions (CDRs) selected from the group consisting of (a) CDR-H1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:41; (b) CDR-H2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:42); (c) CDR-H3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least95%, or 100% sequence identity to SEQ ID NO:43; (d) CDR-L1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:44; (e) CDR-L2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ 32MOFO-360553874Attorney Docket No.37188-20001.40ID NO:45; and (f) CDR-L3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:46.

[0125] In certain embodiments, the anti-CD19 binding domain comprises CDR-H1 comprising amino acid sequence of SEQ ID NO:41, CDR-H2 comprising amino acid sequence of SEQ ID NO: 42, and CDR-H3 comprising amino acid sequence of SEQ ID NO: 43. In certain embodiments, the anti-CD19 binding domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO:44, CDR-L2 comprising amino acid sequence of SEQ ID NO:45, and CDR-L3 comprising amino acid sequence of SEQ ID NO:46.

[0126] In certain embodiments, the anti-CD19 binding domain comprises (a) a heavy chain variable region (VH) and / or (b) a light chain variable region (VL) as described herein.

[0127] In certain embodiments, the anti-CD19 binding domain comprises a heavy chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or 100%) sequence identity to SEQ ID NO:47 and / or a light chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:48.

[0128] In certain embodiments, the anti-CD19 binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:47 and a light chain variable region (VL) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:48. In some embodiments, the anti-CD19 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:47 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:48.

[0129] In certain embodiments, the bispecific antibody (e.g., anti-CD19 arm) comprises a heavy chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:49, and / or a light chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:50.

[0130] In certain embodiments, the bispecific antibody comprises a heavy chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence 33MOFO-360553874Attorney Docket No.37188-20001.40identity to SEQ ID NO:49, and a light chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:50.

[0131] In certain embodiments, the bispecific antibody (anti-CD19 arm of MGE-2v1) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:49 and a light chain comprising the amino acid sequence of SEQ ID NO:50.

[0132] In certain embodiments, the second binding domain (anti-BCMA binding domain) comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining regions (CDRs) selected from the group consisting of (a) CDR-H1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:31; (b) CDR-H2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:32; (c) CDR-H3 comprising amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 33; (d) CDR-L1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:34; (e) CDR-L2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:35; and (f) CDR-L3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:36.

[0133] In certain embodiments, the anti-BCMA binding domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO:31, CDR-H2 comprising the amino acid sequence of SEQ ID NO:32, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:33. In certain embodiments, the anti-CD38 binding domain sequences comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO:34, CDR-L2 comprising the amino acid sequence of SEQ ID NO:35, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:36.

[0134] In certain embodiments, the anti-BCMA binding domain comprises CDR-H1 comprising amino acid sequence of SEQ ID NO:31, CDR-H2 comprising amino acid sequence of SEQ ID NO:32, CDR-H3 comprising amino acid sequence of SEQ ID NO:33, CDR-L1 comprising amino acid sequence of SEQ ID NO: 34, CDR-L2 comprising amino acid sequence of SEQ ID NO:35 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:36.

[0135] In certain embodiments, the anti-BCMA binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,34MOFO-360553874Attorney Docket No.37188-20001.4098%, 99%, or 100%) sequence identity to SEQ ID NO: 37 and / or a light chain variable region (VL) comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:38.

[0136] In certain embodiments, the anti-BCMA binding domain comprises a VH comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 37 and a VL comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:38.

[0137] In certain embodiments, the anti-BCMA binding domain comprises a heavy chain variable region comprising amino acid sequence of SEQ ID NO:37 and a light chain variable region comprising amino acid sequence of SEQ ID NO:38.

[0138] In certain embodiments, the bispecific antibody (anti-BCMA arm) comprises a heavy chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:39, and / or light chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:40.

[0139] In certain embodiments, the bispecific antibody (anti-BCMA arm) comprises a heavy chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:39 and a light chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:40.

[0140] In certain embodiments, the bispecific antibody MGE-2v2 (anti-BCMA arm) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:39 and a light chain comprising the amino acid sequence of SEQ ID NO:40.CD19 X CD38 bispecific antibodies (e.g., MGE-2v3, MGE-2v4)

[0141] In certain embodiments, the first binding domain binds CD19 and the second binding domain binds CD38.

[0142] In certain embodiments, the CD19 X CD38 bispecific antibody comprises an anti-CD19 binding domain as described herein, and an anti-CD38 binding domain as described herein. In certain embodiments, the CD19 X CD38 bispecific antibody comprises an anti-35MOFO-360553874Attorney Docket No.37188-20001.40CD19 heavy chain and an anti-CD19 light chain as described herein; and an anti-CD38 heavy chain and an anti-CD38 light chain.

[0143] In certain embodiments, the first binding domain (anti-CD19 binding domain) comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining regions (CDRs) selected from the group consisting of (a) CDR-H1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:41; (b) CDR-H2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:42); (c) CDR-H3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least95%, or 100% sequence identity to SEQ ID NO:43; (d) CDR-L1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:44; (e) CDR-L2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:45; and (f) CDR-L3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:46.

[0144] In certain embodiments, the anti-CD19 binding domain comprises CDR-H1 comprising amino acid sequence of SEQ ID NO:41, CDR-H2 comprising amino acid sequence of SEQ ID NO: 42, and CDR-H3 comprising amino acid sequence of SEQ ID NO: 43. In certain embodiments, the anti-CD19 binding domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO:44, CDR-L2 comprising amino acid sequence of SEQ ID NO:45, and CDR-L3 comprising amino acid sequence of SEQ ID NO:46.

[0145] In certain embodiments, the anti-CD19 binding domain comprises (a) a heavy chain variable region (VH) and / or (b) a light chain variable region (VL) as described herein. In certain embodiments, the anti-CD19 binding domain comprises a heavy chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or 100%) sequence identity to SEQ ID NO:47 and / or a light chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:48.

[0146] In certain embodiments, the anti-CD19 binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:47 and a light chain variable region (VL) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 36MOFO-360553874Attorney Docket No.37188-20001.40100% sequence identity to SEQ ID NO:48. In some embodiments, the anti-CD19 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:47 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:48.

[0147] In certain embodiments, the bispecific antibody (e.g., anti-CD19 arm) comprises a heavy chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:49, and / or a light chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:50.

[0148] In certain embodiments, the bispecific antibody comprises a heavy chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:49, and a light chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:50.

[0149] In certain embodiments, the bispecific antibody (anti-CD19 arm of MGE-2v3) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:49 and a light chain comprising the amino acid sequence of SEQ ID NO:50.

[0150] In certain embodiments, the second binding domain (anti-CD38 binding domain) comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining regions (CDRs) selected from the group consisting of (a) CDR-H1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:21; (b) CDR-H2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:22; (c) CDR-H3 comprising amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 23; (d) CDR-L1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:24; (e) CDR-L2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:25; and (f) CDR-L3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:26.

[0151] In certain embodiments, the anti-CD38 binding domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO:21, CDR-H2 comprising the amino acid sequence of SEQ ID NO:22, and CDR-H3 comprising the amino acid sequence of SEQ ID 37MOFO-360553874Attorney Docket No.37188-20001.40NO:23. In certain embodiments, the anti-CD38 binding domain sequences comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO:24, CDR-L2 comprising the amino acid sequence of SEQ ID NO:25, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:26.

[0152] In certain embodiments, the anti-CD38 binding domain comprises CDR-H1 comprising amino acid sequence of SEQ ID NO:21, CDR-H2 comprising amino acid sequence of SEQ ID NO:22, CDR-H3 comprising amino acid sequence of SEQ ID NO:23, CDR-L1 comprising amino acid sequence of SEQ ID NO: 24, CDR-L2 comprising amino acid sequence of SEQ ID NO:25 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:26.

[0153] In certain embodiments, the anti-CD38 binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 27 and / or a light chain variable region (VL) comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:28.

[0154] In certain embodiments, the anti-CD38 binding domain comprises a VH comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 27 and a VL comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:28.

[0155] In certain embodiments, the anti-CD38 binding domain comprises a heavy chain variable region comprising amino acid sequence of SEQ ID NO:27 and a light chain variable region comprising amino acid sequence of SEQ ID NO:28.

[0156] In certain embodiments, the bispecific antibody (anti-CD38) arm comprises a heavy chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:29, and / or light chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:30.

[0157] In certain embodiments, the bispecific antibody comprises a heavy chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence38MOFO-360553874Attorney Docket No.37188-20001.40identity to SEQ ID NO:29 and a light chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:30.

[0158] In certain embodiments, the bispecific antibody MGE-2v3 (anti-CD38 arm) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:29 and a light chain comprising the amino acid sequence of SEQ ID NO:30.

[0159] In certain embodiments, the first binding domain (anti-CD38 binding domain, such as in MGE-2v4) comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining regions (CDRs) selected from the group consisting of (a) CDR-H1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:11; (b) CDR-H2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:12); (c) CDR-H3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least95%, or 100% sequence identity to SEQ ID NO:13; (d) CDR-L1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:14; (e) CDR-L2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:15; and (f) CDR-L3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:16.

[0160] In certain embodiments, the anti-CD38 binding domain comprises CDR-H1 comprising amino acid sequence of SEQ ID NO:11, CDR-H2 comprising amino acid sequence of SEQ ID NO:12, and CDR-H3 comprising amino acid sequence of SEQ ID NO:13. In certain embodiments, the anti-CD38 binding domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO:14, CDR-L2 comprising amino acid sequence of SEQ ID NO:15, and CDR-L3 comprising amino acid sequence of SEQ ID NO:16.

[0161] In certain embodiments, the anti-CD38 binding domain comprises (a) a heavy chain variable region (VH) and / or (b) a light chain variable region (VL) as described herein.

[0162] In certain embodiments, the anti-CD38 binding domain comprises a heavy chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or 100%) sequence identity to SEQ ID NO:17 and / or a light chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:18.39MOFO-360553874Attorney Docket No.37188-20001.40

[0163] In certain embodiments, the anti-CD38 binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:17 and a light chain variable region (VL) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:18. In some embodiments, the anti-CD38 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:18.

[0164] In certain embodiments, the bispecific antibody (e.g., anti-CD38 arm) comprises a heavy chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:19, and / or a light chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:20.

[0165] In certain embodiments, the bispecific antibody comprises a heavy chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:19, and a light chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:20.

[0166] In certain embodiments, the bispecific antibody (anti-CD38 arm of MGE-2v4) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:19 and a light chain comprising the amino acid sequence of SEQ ID NO:20.CD20 X CD38 bispecific antibodies (e.g., MGE-2v5, MGE-2v6)

[0167] In certain embodiments, the first binding domain binds CD20 and the second binding domain binds CD38.

[0168] In certain embodiments, the CD20 X CD38 bispecific antibody comprises an anti-CD20 binding domain as described herein, and an anti-CD38 binding domain as described herein.

[0169] In certain embodiments, the CD20 X CD38 bispecific antibody comprises an anti-CD20 heavy chain and an anti-CD20 light chain as described herein; and an anti-CD38 heavy chain and an anti-CD38 light chain.

[0170] In certain embodiments, the first binding domain (anti-CD20 binding domain, such as in MGE-2v5) comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining 40MOFO-360553874Attorney Docket No.37188-20001.40regions (CDRs) selected from the group consisting of (a) CDR-H1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:51; (b) CDR-H2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:52); (c) CDR-H3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least95%, or 100% sequence identity to SEQ ID NO:53; (d) CDR-L1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:54; (e) CDR-L2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:55; and (f) CDR-L3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:56.

[0171] In certain embodiments, the anti-CD20 binding domain comprises CDR-H1 comprising amino acid sequence of SEQ ID NO:51, CDR-H2 comprising amino acid sequence of SEQ ID NO: 52, and CDR-H3 comprising amino acid sequence of SEQ ID NO: 53. In certain embodiments, the anti-CD20 binding domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO:54, CDR-L2 comprising amino acid sequence of SEQ ID NO:55, and CDR-L3 comprising amino acid sequence of SEQ ID NO:56.

[0172] In certain embodiments, the anti-CD20 binding domain comprises (a) a heavy chain variable region (VH) and / or (b) a light chain variable region (VL) as described herein.

[0173] In certain embodiments, the anti-CD20 binding domain comprises a heavy chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or 100%) sequence identity to SEQ ID NO:57 and / or a light chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:58.

[0174] In certain embodiments, the anti-CD20 binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:57 and a light chain variable region (VL) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:58. In some embodiments, the anti-CD20 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:57 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:58.41MOFO-360553874Attorney Docket No.37188-20001.40

[0175] In certain embodiments, the bispecific antibody (e.g., anti-CD20 arm) comprises a heavy chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:59, and / or a light chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:60.

[0176] In certain embodiments, the bispecific antibody comprises a heavy chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:59, and a light chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:60.

[0177] In certain embodiments, the bispecific antibody (anti-CD20 arm of MGE-2v5) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:59 and a light chain comprising the amino acid sequence of SEQ ID NO:60.

[0178] In certain embodiments, the first binding domain (anti-CD20 binding domain) comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining regions (CDRs) selected from the group consisting of (a) CDR-H1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:61; (b) CDR-H2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:62; (c) CDR-H3 comprising amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 63; (d) CDR-L1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:64; (e) CDR-L2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:65; and (f) CDR-L3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:66.

[0179] In certain embodiments, the anti-CD20 binding domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO:61, CDR-H2 comprising the amino acid sequence of SEQ ID NO:62, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:63. In certain embodiments, the anti-CD38 binding domain sequences comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO:64, CDR-L2 comprising the amino acid sequence of SEQ ID NO:65, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:66.42MOFO-360553874Attorney Docket No.37188-20001.40

[0180] In certain embodiments, the anti-CD20 binding domain comprises CDR-H1 comprising amino acid sequence of SEQ ID NO:61, CDR-H2 comprising amino acid sequence of SEQ ID NO:62, CDR-H3 comprising amino acid sequence of SEQ ID NO:63, CDR-L1 comprising amino acid sequence of SEQ ID NO: 64, CDR-L2 comprising amino acid sequence of SEQ ID NO:65 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:66.

[0181] In certain embodiments, the anti-CD20 binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 67 and / or a light chain variable region (VL) comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:68.

[0182] In certain embodiments, the anti-CD20 binding domain comprises a VH comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 67 and a VL comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:68.

[0183] In certain embodiments, the anti-CD20 binding domain comprises a heavy chain variable region comprising amino acid sequence of SEQ ID NO:67 and a light chain variable region comprising amino acid sequence of SEQ ID NO:68.

[0184] In certain embodiments, the bispecific antibody (anti-CD20) arm comprises a heavy chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:69, and / or light chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:70.

[0185] In certain embodiments, the bispecific antibody comprises a heavy chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:69 and a light chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:70.

[0186] In certain embodiments, the bispecific antibody MGE-2v6 (anti-CD20 arm) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:69 and a light chain comprising the amino acid sequence of SEQ ID NO:70.43MOFO-360553874Attorney Docket No.37188-20001.40

[0187] In certain embodiments, the second binding domain (anti-CD38 binding domain, such as in MGE-2v5, or MGE-2v6) comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining regions (CDRs) selected from the group consisting of (a) CDR-H1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:11; (b) CDR-H2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:12); (c) CDR-H3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least95%, or 100% sequence identity to SEQ ID NO:13; (d) CDR-L1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:14; (e) CDR-L2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:15; and (f) CDR-L3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:16.

[0188] In certain embodiments, certain embodiments, the anti-CD38 binding domain comprises CDR-H1 comprising amino acid sequence of SEQ ID NO:11, CDR-H2 comprising amino acid sequence of SEQ ID NO: 12, and CDR-H3 comprising amino acid sequence of SEQ ID NO:13. In certain embodiments, the anti-CD38 binding domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO:14, CDR-L2 comprising amino acid sequence of SEQ ID NO:15, and CDR-L3 comprising amino acid sequence of SEQ ID NO:16.

[0189] In certain embodiments, the anti-CD38 binding domain comprises (a) a heavy chain variable region (VH) and / or (b) a light chain variable region (VL) as described herein.

[0190] In certain embodiments, the anti-CD38 binding domain comprises a heavy chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or 100%) sequence identity to SEQ ID NO:17 and / or a light chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:18.

[0191] In certain embodiments, the anti-CD38 binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:17 and a light chain variable region (VL) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 44MOFO-360553874Attorney Docket No.37188-20001.40100% sequence identity to SEQ ID NO:18. In some embodiments, the anti-CD38 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:18.

[0192] In certain embodiments, the bispecific antibody (e.g., anti-CD38 arm) comprises a heavy chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:71, and / or a light chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:20.

[0193] In certain embodiments, the bispecific antibody comprises a heavy chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:71, and a light chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:20.

[0194] In certain embodiments, the bispecific antibody (anti-CD38 arm of MGE-2v5 or MGE-2v6) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:71 and a light chain comprising the amino acid sequence of SEQ ID NO:20.CD20 X BCMA bispecific antibodies (e.g., MGE-2v7)

[0195] In certain embodiments, the first binding domain binds CD20 and the second binding domain binds BCMA.

[0196] In certain embodiments, the CD20 X BCMA bispecific antibody comprises an anti-CD20 binding domain as described herein, and an anti-BCMA binding domain as described herein.

[0197] In certain embodiments, the CD20 X BCMA bispecific antibody comprises an anti-CD20 heavy chain and an anti-CD20 light chain as described herein; and an anti-BCMA heavy chain and an anti-BCMA light chain as described herein (e.g., Table 1).

[0198] In certain embodiments, the first binding domain (anti-CD20 binding domain) comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining regions (CDRs) selected from the group consisting of (a) CDR-H1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:51; (b) CDR-H2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:52); (c) CDR-H345MOFO-360553874Attorney Docket No.37188-20001.40comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:53; (d) CDR-L1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:54; (e) CDR-L2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:55; and (f) CDR-L3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:56.

[0199] In certain embodiments, the anti-CD20 binding domain comprises CDR-H1 comprising amino acid sequence of SEQ ID NO:51, CDR-H2 comprising amino acid sequence of SEQ ID NO: 52, and CDR-H3 comprising amino acid sequence of SEQ ID NO: 53. In certain embodiments, the anti-CD20 binding domain comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO:54, CDR-L2 comprising amino acid sequence of SEQ ID NO:55, and CDR-L3 comprising amino acid sequence of SEQ ID NO:56.

[0200] In certain embodiments, the anti-CD20 binding domain comprises (a) a heavy chain variable region (VH) and / or (b) a light chain variable region (VL) as described herein.

[0201] In certain embodiments, the anti-CD20 binding domain comprises a heavy chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99% or 100%) sequence identity to SEQ ID NO:57 and / or a light chain variable region comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:58.

[0202] In certain embodiments, the anti-CD20 binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:57 and a light chain variable region (VL) comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:58. In some embodiments, the anti-CD20 binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:57 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:58.

[0203] In certain embodiments, the bispecific antibody (e.g., anti-CD20 arm) comprises a heavy chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:59, and / or a light chain comprising an amino acid 46MOFO-360553874Attorney Docket No.37188-20001.40sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:60.

[0204] In certain embodiments, the bispecific antibody comprises a heavy chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:59, and a light chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:60.

[0205] In certain embodiments, the bispecific antibody (anti-CD20 arm of MGE-2v7) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:59 and a light chain comprising the amino acid sequence of SEQ ID NO:60.

[0206] In certain embodiments, the second binding domain (anti-BCMA binding domain) comprises one or more (e.g., 3, 4, 5, or 6) complementarity-determining regions (CDRs) selected from the group consisting of (a) CDR-H1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:31; (b) CDR-H2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:32; (c) CDR-H3 comprising amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 33; (d) CDR-L1 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:34; (e) CDR-L2 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:35; and (f) CDR-L3 comprising an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:36.

[0207] In certain embodiments, the anti-BCMA binding domain comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO:31, CDR-H2 comprising the amino acid sequence of SEQ ID NO:32, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:33. In certain embodiments, the anti-BCMA binding domain sequences comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO:34, CDR-L2 comprising the amino acid sequence of SEQ ID NO:35, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:36.

[0208] In certain embodiments, the anti-BCMA binding domain comprises CDR-H1 comprising amino acid sequence of SEQ ID NO:31, CDR-H2 comprising amino acid sequence of SEQ ID NO:32, CDR-H3 comprising amino acid sequence of SEQ ID NO:33, CDR-L1 comprising amino acid sequence of SEQ ID NO: 34, CDR-L2 comprising amino 47MOFO-360553874Attorney Docket No.37188-20001.40acid sequence of SEQ ID NO:35 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:36.

[0209] In certain embodiments, the anti-BCMA binding domain comprises a heavy chain variable region (VH) comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 37 and / or a light chain variable region (VL) comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:38.

[0210] In certain embodiments, the anti-BCMA binding domain comprises a VH comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO: 37 and a VL comprising an amino acid sequence that has at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO:38.

[0211] In certain embodiments, the anti-BCMA binding domain comprises a heavy chain variable region comprising amino acid sequence of SEQ ID NO:37 and a light chain variable region comprising amino acid sequence of SEQ ID NO:38.

[0212] In certain embodiments, the bispecific antibody (anti-BCMA arm) comprises a heavy chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:39, and / or light chain comprising an amino acid sequence that has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO:40.

[0213] In certain embodiments, the bispecific antibody comprises a heavy chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:39 and a light chain comprising an amino acid sequence that has at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO:40.

[0214] In certain embodiments, the bispecific antibody MGE-2v7 (anti-BCMA arm) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:39 and a light chain comprising the amino acid sequence of SEQ ID NO:40.

[0215] Certain embodiments of the invention provide a method of depleting immune cells, comprising contacting a cell(s) expressing BAFF-R and / or CD38 with a bispecific antibody.

[0216] Certain embodiments of the invention provide a method of depleting immune cells, comprising contacting a cell(s) expressing CD19 and / or BCMA with a bispecific antibody.48MOFO-360553874Attorney Docket No.37188-20001.40

[0217] Certain embodiments of the invention provide a method of depleting immune cells, comprising contacting a cell(s) expressing CD19 and / or CD38 with a bispecific antibody.

[0218] Certain embodiments of the invention provide a method of depleting immune cells, comprising contacting a cell(s) expressing CD20 and / or CD38 with a bispecific antibody.

[0219] Certain embodiments of the invention provide a method of depleting immune cells, comprising contacting a cell(s) expressing CD20 and / or BCMA with a bispecific antibody.

[0220] Certain embodiments of the invention provide a method of depleting immune cells, comprising contacting a cell(s) expressing BAFF-R and / or BCMA with a bispecific antibody.

[0221] In certain embodiments, the immune cell(s) is cell from the B cell lineage (e.g., a naïve B cell, memory B cell, plasmablast, or plasma cell).

[0222] In certain embodiments, the contacting is conducted in vitro.

[0223] In certain embodiments, the contacting is conducted in vivo.

[0224] In certain embodiments, the bispecific antibody exhibits increased effector cell function, e.g., as compared to effector cell function of an antibody that bivalently binds to either the first or the second target antigen.

[0225] In certain embodiments, the bispecific antibody of the present disclosure exhibits increased target cell depletion, e.g., as compared to target cell depletion by an antibody that bivalently binds to either the first or the second target antigen (e.g., a bivalent, monospecific antibody). For instance, in some embodiments, the bispecific antibody exhibits depletion of both first and second target cells of the present disclosure, and the bispecific antibody exhibits increased depletion of the first target cell as compared to depletion of the first target cell by an antibody that bivalently binds to the first target antigen (e.g., a bivalent, monospecific antibody). In other embodiments, the bispecific antibody exhibits depletion of both first and second target cells of the present disclosure, and the bispecific antibody exhibits increased depletion of the second target cell as compared to depletion of the second target cell by an antibody that bivalently binds to the second target antigen (e.g., a bivalent, monospecific antibody). In certain embodiments, the target cells are depleted by ADCC, ADCP, or ADCC and ADCP. Assays for measuring cell depletion (e.g., by ADCC or ADCP) are known in the art; non-limiting exemplary assays are described in Examples 4 and 5 infra.

[0226] In certain embodiments, the bispecific antibody of the present disclosure increased antibody opsonization, e.g., as compared to antibody opsonization by an antibody that bivalently binds to either the first or the second target antigen (e.g., a bivalent, monospecific antibody).Assays for measuring opsonization are known in the art; a non-limiting exemplary assay is described in Example 6 infra.49MOFO-360553874Attorney Docket No.37188-20001.40

[0227] In certain embodiments, the amount of a bispecific antibody bound on a target cell surface is at least 15%, 20%, 25%, 30%, 40%, 45%, 50%, or more than a bivalent monospecific control antibody. In certain embodiments, the amount of a bispecific antibody bound on a target cell (e.g., naïve B, or plasmablast) surface is at least 15%, 20%, 25%, 30%, 40%, 45%, 50%, or more than a bivalent monospecific control antibody that comprises an identical binding domain (e.g., anti-BAFF-R or anti-CD38 binding domain), for example, in an opsonization assay described herein (also see Example 1), such as when target cells are contacted with antibody at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 60ug / mL.

[0228] In certain embodiments, a bispecific antibody described herein results in enhanced ADCP mediated depletion of a target cell that is enhanced by at least 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80% or more than a monospecific control antibody that binds bivalently to a single target (e.g., one of the two antigen targets of the bispecific antibody). In certain embodiments, a bispecific antibody described herein results in enhanced ADCP mediated depletion of a target cell (e.g., naïve B, or plasmablast) that is enhanced by at least 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80% or more than a monospecific control antibody that comprises an identical binding domain (e.g., anti- BAFF-R or anti-CD38 binding domain) and bivalently to a single target via the identical binding domain, for example, in an ADCP assay described herein.

[0229] Certain embodiments of the invention provide a method of depleting B cell lineage immune cells (e.g., a naïve B cell, memory B cell, plasmablast, and / or plasma cell) in a subject, comprising administering a bispecific antibody described herein to the subject.

[0230] In certain embodiments, the cell numbers of one or more (e.g., two or more) subsets or populations of B cell lineage immune cells in the subject are reduced, for example, by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In certain embodiments, the cell numbers of two or more subsets or populations of B cell lineage immune cells selected from the group consisting of immature B cells, naïve B cells, memory B cells, plasmablasts, and plasma cells are each independently reduced in the subject, for example, by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In certain embodiments, the cell numbers of naïve B cells and plasmablasts are each independently reduced in the subject, for example, by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In certain embodiments, the cell numbers of naïve B cells and plasma cells are each independently reduced in the subject, for example, by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.50MOFO-360553874Attorney Docket No.37188-20001.40

[0231] Certain embodiments of the invention provide a method of treating a B lineage cell related disease or condition in a subject in need of, comprising administering a bispecific antibody described herein to the subject.

[0232] In certain embodiments, the B lineage cell related disease or condition is an autoimmune disease, a hematologic cancer, a transplant related condition such as rejection of transplant, or graft-vs-host disease.

[0233] Certain embodiments of the invention provide a method of treating an autoimmune disease or hematologic cancer, preventing rejection of an organ or stem cell transplant of a subject in need of, comprising administering a bispecific antibody described herein to the subject (e.g., a therapeutically effective amount of protein is administered).

[0234] In certain embodiments, treating refers to a clinical intervention designed to alter the course of clinical pathology of a disease or disorder of the present disclosure (e.g., in an individual in need thereof), including, for example, reducing, mitigating, or eliminating one or more symptoms associated with a disease or disorder. In some embodiments, treating does not encompass prophylactic measures. In some embodiments, treating includes increasing the quality of life of those suffering from a disease or disorder, decreasing the dose of other medications required for treating the disease or disorder, reducing the frequency of recurrence of the disease or disorder, lessening severity of the disease or disorder, delaying the development or progression of the disease or disorder, and / or prolonging survival of individual being treated.

[0235] In certain embodiments, the subject has an autoimmune disease.

[0236] In certain embodiments, the subject has a hematologic cancer.

[0237] In certain embodiments, the subject is a transplantation recipient. In certain embodiments, transplant-related immune response is inhibited in the subject. In certain embodiments, transplant engraftment success rate is increased. In certain embodiments, transplant rejection or rejection severity is prevented or mitigated.

[0238] In certain embodiments, the method further comprises administering one or more additional therapeutic agent(s) to the subject. In certain embodiments, the additional therapeutic agent is an immunosuppressant.

[0239] In certain embodiments, the method does not comprise administering an additional therapeutic agent (e.g., immunosuppressant) to the subject.

[0240] In certain embodiments, the subject has an autoimmune disease. In certain embodiments, the autoimmune disease is rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, type 1 diabetes, multiple sclerosis, Sjogren’s syndrome, systemic and / or 51MOFO-360553874Attorney Docket No.37188-20001.40cutaneous lupus erythematosus, lupus nephritis, idiopathic inflammatory myopathies, systemic sclerosis, renal autoimmune disease, glomerular autoimmune disease, autoimmune nephritis, immunoglobulin A (IgA) nephropathy, membranous nephropathy, immunoglobulin G4 (IgG4)-related disease, neuromyelitis optica, focal segmental glomerulosclerosis (FSGS), anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, granulomatosis with polyangiitis, microscopic polyangiitis, amyloid light chain (AL) amyloidosis, antibody-mediated rejection, bullous pemphigoid, membranous nephropathy, immune thrombocytopenia, hemolytic anemia, primary biliary and / or sclerosing cholangitis, thyroid eye disease, Grave’s hyperthyroidism, ulcerative colitis, Crohn’s disease, celiac disease, hidradenitis suppurativa, myasthenia gravis, psoriasis pernicious anemia, vitiligo, reactive arthritis, Behçet’s disease, vasculitis, Addison’s disease, ankylosing spondylitis, alopecia areata, or uveitis.

[0241] In certain embodiments, the subject has a hematologic cancer associated with malignant B cells and / or plasma cells. In certain embodiments, the hematologic cancer is multiple myeloma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, cutaneous B cell lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia, small lymphocytic leukemia, marginal zone lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), hairy cell leukemia, primary central nervous system lymphoma, primary intraocular lymphoma, plasmacytoma, multiple myeloma, monoclonal gammopathy of undetermined significance, or extramedullary plasmacytoma.

[0242] In certain embodiments, the subject is a transplant recipient. In certain embodiments, the subject is a stem cell transplant recipient. In certain embodiments, the subject has or is at risk of developing organ rejection or graft-vs-host disease. In certain embodiments, the subject is a solid organ (e.g., kidney, lung, liver, or heart) transplant recipient.

[0243] In certain embodiments, the subject is a human, or a non-human primate. In certain embodiments, the subject is a human.

[0244] In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a rodent (e.g., mouse or rat). In certain embodiments, the mammal is a cow, horse, sheep, goat, pig, dog, cat, or hamster.

[0245] In certain embodiments, the bispecific antibody described herein is administered intravenously, subcutaneously, intradermally, intramuscularly, or intrathecally.

[0246] Certain embodiments of the invention provide a bispecific antibody as described herein for use in medical therapy.52MOFO-360553874Attorney Docket No.37188-20001.40

[0247] Certain embodiments of the invention provide a bispecific antibody as described herein for the prophylactic or therapeutic treatment of a B lineage cell related disease or condition.

[0248] Certain embodiments of the invention provide a bispecific antibody as described herein for the inhibition of transplant related immune response in a subject.

[0249] Certain embodiments of the invention provide a bispecific antibody as described herein to prepare a medicament for the treatment of a B lineage cell related disease or condition in a subject.Nucleic Acids, Expression Cassettes, Vectors and Cells

[0250] Certain embodiments of the invention provide nucleic acid(s) (e.g., an isolated nucleic acid) encoding a polypeptide or a bispecific antibody (e.g., heavy chain and / or light chain) as described herein. In certain embodiments, the polypeptide further comprises a signal peptide (e.g., SEQ ID NO:72, 73, or 74) at the N terminal of the polypeptide. In certain embodiments, the nucleic acid further comprises a promoter. In certain embodiments, the isolated nucleic acid encoding a bispecific antibody as described herein is DNA. In certain embodiments, the isolated nucleic acid encoding a bispecific antibody described herein is mRNA.

[0251] Certain embodiments of the invention provide an expression cassette comprising a nucleic acid as described herein and a promoter.

[0252] Certain embodiments of the invention provide a vector (e.g., a plasmid or phagemid) comprising a nucleic acid or an expression cassette as described herein.

[0253] Certain embodiments of the invention provide a host cell (e.g., mammalian cell, such as CHO cell) comprising a nucleic acid, expression cassette or vector as described herein. In certain embodiments, the host cell (e.g., mammalian cell such as CHO) lacks or has reduced expression of certain fucosylation enzyme(s), such as the enzyme Fucosyltransferase 8 (FUT8).Compositions and Administration

[0254] Certain embodiments of the invention provide a pharmaceutical composition comprising a bispecific antibody described herein, and a pharmaceutically acceptable carrier. In certain embodiments, the protein is present in a liquid composition (e.g., saline, D5W, or buffered solution).53MOFO-360553874Attorney Docket No.37188-20001.40

[0255] In certain embodiments, the protein is present in a solid composition. In certain embodiments, the protein is present in a lyophilized composition, which may be reconstituted with proper solution prior to administration as a liquid. In certain embodiments, the lyophilized composition further comprises one or more excipients selected from the group consisting of a cryo-lyoprotectant (e.g., trehalose, sucrose) and a bulking agent (e.g., mannitol, glycine).

[0256] For in vivo use, a bispecific antibody of the invention is generally incorporated into a pharmaceutical composition prior to administration. Within such compositions, the antibody may be present as active ingredient(s) (i.e., are present at levels sufficient to provide a statistically significant effect on the symptoms of a relevant disease, as measured using a representative assay). A pharmaceutical composition may comprise an antibody in combination with any pharmaceutically acceptable carrier(s) known to those skilled in the art to be suitable for the particular mode of administration. In addition, other pharmaceutically active ingredients (including other therapeutic agents) may, but need not, be present within the composition.

[0257] Compositions for injection will commonly comprise a solution of the antibody dissolved in a pharmaceutically acceptable carrier. Among the acceptable vehicles and solvents that can be employed are water and an isotonic solution of one or more salts such as sodium chloride, e.g., Ringer’s solution. Pharmaceutical compositions desirably are sterile and generally free of undesirable matter. These pharmaceutical compositions can be sterilized by conventional, well known sterilization techniques. The pharmaceutical compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The pharmaceutical compositions may contain salt, sugar, preservative, surfactant, or any other suitable excipient.

[0258] The pharmaceutical composition may contain any suitable concentration of the antibody. The concentration of the antibody in the pharmaceutical composition can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient’s needs. In certain embodiments, the concentration of antibody in a solution formulation for injection will range from about 0.1% (w / w) to about 10% (w / w), or more such as 100 mg of antibody per milliliter of the formulation.54MOFO-360553874Attorney Docket No.37188-20001.40

[0259] In certain embodiments, the antibody of the present invention may be systemically administered, e.g., intravenously, in combination with a pharmaceutically acceptable carrier. In certain embodiments, the antibody may be administered intravenously, subcutaneously, intradermally, intramuscularly, intraperitoneally, or intrathecally by infusion or injection. In certain embodiments, the antibody of the present invention may be locally administered into a particular tissue, structure, or organ.

[0260] Frequency of administration can range from multiple doses to a single dose per week, or less frequently (e.g., single dose per month or every two to three months or every six to 18 months). In certain embodiments, the antibody of the present invention may be administered, e.g., intravenously or subcutaneously, to a mammal in need of, for example, about once every two weeks, once every three weeks, once every month, once every five weeks, or once every six weeks. In certain embodiments, the antibody may be administered about once every month, once every two or three months, or once every six or 12 or 18 months. In certain embodiments, the antibody may be administered about once every week. In some embodiments, the antibody is administered from about once per month to about five times per week

[0261] In one embodiment, the antibody is administered to the patient parenterally. Dosing of the antibody can be by any suitable route, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein. The antibody dose can range from about 5 mg / kg (body weight) to about 50 mg / kg, from about 10 μg / kg to about 5 mg / kg, or from about 100 μg / kg to about 1 mg / kg. The antibody dose can be about 100, 200, 300, 400, or 500 μg / kg. The antibody dose can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / kg, or more. The antibody dose can also be outside of these ranges, depending on the type and severity of the disorder being treated. The antibody dose for a patient can be adjusted by physician or pharmacist.

[0262] In certain embodiments, the antibody may be delivered using a device (e.g., a device comprising one or two chambers, for example, containing the antibody, and / or liquid). In certain embodiments, the device is an injector (e.g., a self-injector device comprising the antibody).

[0263] The following description is presented to enable a person of ordinary skill in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described 55MOFO-360553874Attorney Docket No.37188-20001.40herein will be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein and shown, but are to be accorded the scope consistent with the claims.EXAMPLES

[0264] BAFF-R, CD19, and CD20 are cell surface antigens highly expressed on the surface of B cells. Targeting of BAFF-R with a mAb has been shown to antagonize BAFF signaling and deplete BAFF-R+ B cells (McWilliams EM, et al. Blood Adv.2019 Feb 12;3(3):447-460). Targeting of CD19 with a mAb has been shown to deplete circulating B cells and has demonstrated clinical efficacy in autoimmune diseases and hematologic cancers (Nie et al; Pirosa et al). Several mAbs targeting CD20 have been shown to deplete B cells and have demonstrated clinical efficacy in autoimmune diseases and hematologic cancers (Casan et al).

[0265] CD38 and BCMA are cell surface antigens highly expressed on the surface of plasma cells. Targeting of CD38 with a mAb results in depletion of CD38+ plasmablasts and plasma cells (Moreno L et al. Clin Cancer Res.2019 May 15;25(10):3176-3187; Korver W et al. J Pharmacol Exp Ther.2019 Aug;370(2):182-196). Targeting of BCMA with an antibody drug conjugate, chimeric antigen receptor (CAR)-T cells, or a bispecific T cell engaging antibody results in depletion of plasmablasts and plasma cells and has demonstrated clinical efficacy in hematologic cancers and autoimmune diseases; however, targeting of BCMA with a mAb has not been well described in the literature (Trudel, S. et al. Blood Cancer J.2019;9(4):37; Qin, C. et al. Signal Transduc Target Ther.2023;8(1):5; Hagen, M. et al. N Engl J Med.2024;391(9):867-869).

[0266] Given their specific and non-overlapping expression on cells across the B cell lineage, the B cell antigens (BAFF-R, CD19, or CD20) and the plasma cell antigens (CD38 or BCMA) are candidate antigens for combinatorial targeting. However, a bispecific antibody that simultaneously engages both antigens and results in robust depletion of multiple cell populations requires a complex, non-conventional construct. Whether a certain single molecule construct could be expressed with decent yield while maintaining proper binding, blocking, and depleting activity warrants investigation.

[0267] While individual targeting of BAFF-R, CD19, CD20, or CD38 with a cytolytic mAb has demonstrated clinical activity, many autoimmune diseases and some hematologic cancers involve multiple pathogenic B cell populations, including both earlier-stage B cells and 56MOFO-360553874Attorney Docket No.37188-20001.40plasma cells. Thus, broad depletion of multiple B cell subtypes may be helpful to achieve improved clinical efficacy. Dual targeting of both a B cell antigen and a plasma cell antigen through using or administering two separate single agents may be possible; however, there are many inherent challenges with the development, regulatory approval, and commercialization of multi-drug combinations or co- formulations. One major challenge of combination treatment is development of an acceptable dosing regimen when contending with agents with two differing pharmacokinetic (PK) profiles.

[0268] Development of a single bispecific antibody that robustly targets multiple cell surface antigens for broad depletion of immune cell subsets is a means to obviate the concerns of mismatched PK and offer a convenient dosing regimen for patients. Dosing convenience can be further augmented by engineering the bispecific molecule for satisfactory protein expression and developing it as a high-concentration subcutaneous (s.c.) formulation.

[0269] Herein are bispecific antibodies that simultaneously target both a B cell antigen and a plasma cell antigen with a single molecule to deplete multiple B cell, plasmablast, and plasma cell populations. The bispecific antibodies exhibit multiple unique innovations that result in more potent and deeper target cell depletion. First, the bispecific antibodies were designed to engage with two unique cell surface antigens with differential, non-overlapping expression across target cell types. This innovation results in broader activity across pathogenic cell populations. Second, the bispecific antibodies exhibit enhanced opsonization on the target cell surface as a result of monovalent rather than bivalent binding. Enhanced opsonization through monovalent binding is a unique attribute of the bispecific antibodies that results in deeper depletion of multiple B cell and plasma cell populations. Third, the bispecific antibodies exhibit enhanced effector cell activation and FcγR engagement, even at very low antibody concentrations. Importantly, this increase in effector cell activation occurs in the absence of aberrant cytokine release. These unique innovations translate into robust activity in vivo and ex vivo human samples from patients with autoimmune disease. Without wishing to be bound to theory, it is thought that dual-targeting and monovalent binding are distinct advantages that will translate into better clinical efficacy that cannot be achieved with mAbs or a mAb single agent combination. Moreover, the bispecific antibodies of the present invention are positioned for development in a formulation with convenient dosing, given their robust functional activity, compelling biophysical properties and potential to be engineered for half-life extension.

[0270] In sum, these data show that the bispecific antibodies have excellent therapeutic potential across autoimmune diseases, such as rheumatoid arthritis, psoriatic arthritis,57MOFO-360553874Attorney Docket No.37188-20001.40juvenile idiopathic arthritis, type 1 diabetes, multiple sclerosis, antibody-mediated rejection, Sjogren’s syndrome, systemic and / or cutaneous lupus erythematosus, lupus nephritis, idiopathic inflammatory myopathies, systemic sclerosis, renal autoimmune disease, glomerular autoimmune disease, autoimmune nephritis, immunoglobulin A (IgA) nephropathy, membranous nephropathy, immunoglobulin G4 (IgG4)-related disease, neuromyelitis optica, focal segmental glomerulosclerosis (FSGS), anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, granulomatosis with polyangiitis, microscopic polyangiitis, amyloid light chain (AL) amyloidosis, bullous pemphigoid, membranous nephropathy, immune thrombocytopenia, hemolytic anemia, primary biliary and / or sclerosing cholangitis, thyroid eye disease, Grave’s hyperthyroidism, ulcerative colitis, Crohn’s disease, celiac disease, hidradenitis suppurativa, myasthenia gravis, psoriasis pernicious anemia, vitiligo, reactive arthritis, Behçet’s disease, vasculitis, Addison’s disease, ankylosing spondylitis, alopecia areata, or uveitis.

[0271] MRG-2v1 also has strong potential in the treatment of hematologic cancers such as multiple myeloma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, cutaneous B cell lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia, small lymphocytic leukemia, marginal zone lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), hairy cell leukemia, primary central nervous system lymphoma, primary intraocular lymphoma, plasmacytoma, multiple myeloma, monoclonal gammopathy of undetermined significance, or extramedullary plasmacytoma. MGE-2v1 also has potential in the treatment or prophylaxis of conditions related to organ or stem cell transplant, including prophylaxis of organ rejection after solid organ transplant, antibody-mediated rejection, or graft- versus-host disease.

[0272] The objective of this series of experiments was to create a bispecific antibody that (1) binds to two different target antigens, (2) is highly active in biological assays, and (3) has favorable biophysical properties. A schematic design of an exemplary bispecific antibody that dually targets two antigens is illustrated in FIG.1.Example 1: Generation of bispecific antibodies

[0273] Bispecific antibodies were designed for monovalent binding to each of its binding targets. The bispecific antibodies with different variable domains that bind to different monovalent binding targets that were generated are summarized in FIG.1 and Table 2 below.58MOFO-360553874Attorney Docket No.37188-20001.40

[0274] MGE-2v1 was designed for monovalent binding to both BAFF-R and CD38. It was constructed using an afucosylated IgG1 backbone to augment Fcγ receptor (FcγR) interactions and effector function. One antigen-binding fragment (Fab) of the IgG1 comprises the complementarity-determining region (CDR) sequences of the anti-BAFF-R mAb ianalumab (VAY-736). The other Fab of the IgG1 comprises the CDR sequences of the anti-CD38 mAb felzartamab (MOR202). A knob-into-hole format (Knob: T366W; Hole: T366S, L368A, and Y407V) was used to ensure correct heavy chain assembly. Each Fab of MGE-2v1 comprises a different light chain (kappa for anti-BAFF-R and lambda for anti-CD38), which enhances proper light chain pairing. All other constructs in FIG.1 / Table 2 (i.e., MGE2v2, MGE2v3, MGE2v4, MGE2v5, MGE2v6, MGE2v7 and MGE2v8) use knob-into-hole mutations to ensure correct heavy chain assembly as described for MGE2-v1 and variable heavy-variable light chain (VH-VL) domain swap (ie VH-VL CrossMab) to ensure correct light chain pairing. The numbering is according to the EU index in Kabat.Example 2: Biophysical properties of MGE-2v1

[0275] To determine whether the construct exhibited acceptable biophysical properties, the bispecific antibody MGE-2v1 was generated as described in Example 1 above and tested. To generate the protein, an expression vector encoding each target were transfected into FUT-8 knockout Chinese hamster ovary (CHO) cell line and protein was purified by protein A and size exclusion chromatography by high-performance liquid chromatography (SEC-HPLC). Analytical characterization of MGE-2v1 by SEC-HPLC showed several products with one main peak (FIG.2A). SEC-HPLC revealed MGE-2v1 to be 68.4% monomer.59MOFO-360553874Attorney Docket No.37188-20001.40

[0276] MGE-2v1 was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to have an estimated size of approximately 162 kDa (FIG.2B), which is consistent with the predicted molecular weight of the heterodimeric bispecific antibody

[0277] Based on the above data, MGE-2v1 exhibited acceptable protein expression, biophysical properties, and thermal stability and was amenable for further evaluation.Example 3: Target binding by bispecific antibodies

[0278] To confirm that the bispecific antibodies described in the above Examples were able to bind both intended targets, the use of enzyme-linked immunosorbent assays (ELISAs) with recombinant proteins was employed. Recombinant human BAFF-R or CD38 were coated on 96 well plates at 1.0 μg / mL followed by titration of MGE-2v1 at 0.06 to 1 μg / mL. Binding was detected using an anti-human IgG horseradish peroxidase (HRP) at 0.08 μg / mL. Optical density (O.D.) units were measured by ELISA and plotted in O.D. units as a schematic representation of binding to BAFF-R (FIG.3A) or to CD38 (FIG.3B). Bridging ELISA was used to assess simultaneous dual target binding by coating 96 well plates with 1.0 μg / mL recombinant human CD38. MGE-2v1 was titrated at 0.06 to 1.0 μg / ml followed by addition of recombinant human biotinylated BAFF-R at 1.0 μg / ml. Streptavidin HRP at 0.04 μg / mL was used for detection, and O.D. was measured by ELISA. Data were plotted in O.D. units as a schematic representation of dual binding to both BAFF-R and CD38 (FIG.3C). These data confirm that MGE-2v1 is capable of dual binding to both targets.Example 4: ADCC- and ADCP-mediated cell depletion by bispecific antibodies

[0279] The objective of this set of experiments was to determine whether the bispecific antibodies of the present invention can deplete target cells through various Fc-mediated effector mechanisms. It has previously been demonstrated that a bispecific antibody targeting two tumor-associated antigens on the same cancer cell can result in ADCC of tumor cells (Grugan, K.D. et al. MAbs.2017;9(1):114-126), but this has not been shown for a bispecific antibody targeting normal, non-malignant cells in which the targets are expressed on two distinct cell populations. The ADCC activity of MGE-2v1 on normal target cells was assessed using PBMC cultures isolated from peripheral blood from healthy human subjects. PBMCs were cultured overnight in 96 well plates at a density of approximately 0.20 million cells per well with MGE-2v1 at 0.00006 to 5.0 μg / mL. The following day, cells were stained to identify viable cells and fluorophore-conjugated anti-CD19 was used to identify B cells by flow cytometry. Data were plotted as percentage of B cells remaining as a schematic60MOFO-360553874Attorney Docket No.37188-20001.40representation of target cell depletion by ADCC (FIG.4A). MGE-2v1 effectively depleted up to approximately 90% of target cells via ADCC in a dose-dependent fashion

[0280] While a bispecific antibody has previously been shown to result in macrophage-mediated trogocytosis of tumor cells targeting two antigens on the same cancer cell (Vijayaraghavan, S. et al. Mol Cancer Ther.2020;19(10):2044-2056), there is a lack of robust data demonstrating ADCP activity of a bispecific antibody. ADCP activity of MGE-2v1 was assessed by co-culturing human monocyte-derived macrophages (MDMs) and isolated human B cells. Monocytes were isolated from human peripheral blood by magnetic separation. MDMs were generated by culturing monocytes with 50 ng / mL macrophage-stimulating colony factor (M-CSF) for 5-7 days. B cells were isolated from peripheral blood via magnetic separation, labeled with CellTrace Violet (CTV) and incubated with MGE-2v1 at 0.001 to 20.0 μg / mL for 15-30 minutes at 4ºC, followed by washing. MDMs were labeled with CellTrace Far Red (FR) and were cultured with isolated B cells at 1:4 effector to target ratio for 30 minutes at 37ºC. The number of CTV+ FR+ cells were quantified by flow cytometry and plotted as percentage of cells phagocytosed as a schematic representation of target cell depletion by ADCP (FIG.4B). Surprisingly, MGE-2v1 exhibited robust, dose-dependent ADCP, with over 80% of MDMs phagocytosing target cells at high antibody concentrations. These data confirm that MGE-2v1 exhibits robust cell depleting activity through two mechanisms.Example 5: Depletion of multiple distinct cell populations

[0281] The objective of this set of experiments was to establish whether the bispecific antibodies of the present invention can deplete multiple cell populations, including cells that express only one of the two target antigens (binding target) and to which the bispecific antibody binds monovalently. It has been previously shown that a bispecific antibody that binds two different antigens that are both expressed on tumor cells can deplete said cells through ADCC and trogocytosis (Grugan, K.D. et al. MAbs.2017;9(1):114-126;Vijayaraghavan, S. et al. Mol Cancer Ther.2020;19(10):2044-2056). Additionally, the results in Example 4 above demonstrates the ability of MGE-2v1 to significantly deplete target cells via ADCC. However, it has not previously been demonstrated whether a binding molecule with two different antigen binding domains can deplete multiple distinct cell populations, each with a different pattern of antigen expression.

[0282] ADCP activity of MGE-2v1 on multiple B cell populations was compared with that of rituximab, a depleting anti-CD20 mAb that is the current clinical standard-of-care in the 61MOFO-360553874Attorney Docket No.37188-20001.40treatment of numerous autoimmune diseases and hematologic cancers. Human bone marrow or tonsillar mononuclear cells were cultured with MGE-2v1 or rituximab at 0.006 to 20.0 μg / mL for 30 minutes at 4ºC to allow for opsonization. MDMs were generated from peripheral blood monocytes as described in Example 4 and added at 1:5 effector to target ratio with the bone marrow or tonsillar cells and cultured overnight at 37ºC. Cells were stained to identify viable cells and with fluorophore-conjugated anti-CD19 to identify total B cells. Specific B cell subsets were further delineated by flow cytometry: immature B cells (7AAD-CD3-CD19+,IgD+,CD38+), naïve B cells (7AAD-CD3-CD19+IgD-CD27-), plasmablasts (7AAD-CD3-CD24-CD38++HLADR+), and plasma cells (7AAD-CD3-CD24-IgD-CD27+CD38+HLADR-). Data were plotted as percentage of cells remaining as a schematic representation of target cell depletion by ADCP.

[0283] As expected, MGE-2v1 depleted immature B cells, which express both of the MGE-2v1 target antigens (BAFF-R and CD38), in a dose-dependent manner (FIG.5A). The depth and potency of depletion was similar to that seen with rituximab. Surprisingly, MGE-2v1 depleted naïve B cells, which express only one of the two target antigens (BAFF-R), more potently than rituximab (FIG.5B). Similarly, MGE-2v1 also depleted plasmablasts and plasma cells, which express only one of the two target antigens (CD38), in a dose-dependent manner (FIGS.5C & 5D). In contrast, rituximab showed little to no effect on plasmablasts and plasma cells, a finding that is consistent with previous reports (Rehnberg M. et al. Arthritis Res Ther.11, R123 (2009)).

[0284] These data confirm that the bispecific antibodies potently deplete multiple distinct immune cell populations with differential expression patterns of the respective target antigens. The bispecific antibodies can robustly deplete cells that express either one or both target antigens – that is, they can deplete cells to which they bind monovalently or bivalently.Example 6: Opsonization of bispecific antibodies on the target cell surface

[0285] Previous studies have demonstrated that high IgG opsonization on target cells and tight clustering of FcγRs on the macrophage surface are required for efficient ADCP (Zhang, Y. et al. Proc Natl Acad Sci USA.2010;107(45):19332-19337; Kern, N. et al. Elife.2021;10:e68311). The objective of this set of experiments was to evaluate the level of bispecific antibody opsonization on target cells compared with mAbs that bind the same antigen, but with bivalent rather than monovalent binding. Human PBMCs or tonsillar mononuclear cells were cultured for 30 minutes at 4°C with the indicated antibodies. Cells were washed and stained with an anti-human IgG fluorophore-conjugated antibody to 62MOFO-360553874Attorney Docket No.37188-20001.40detect bound antibody on the cell surface by flow cytometry. Naïve B cells were identified in PBMCs and plasmablasts were identified in tonsil by flow cytometry as described in Example 5. Data were plotted as percentage change in surface antibody relative to untreated samples.

[0286] MGE-2v1 showed dose-dependent opsonization on the surface of naïve B cells (BAFF- R+CD38-) (FIG.6A). Compared with ianalumab (anti-BAFF-R mAb), MGE-2v1 exhibited approximately 50% greater antibody opsonization on the B cell surface at certain equivalent antibody concentrations. Similarly, MGE-2v1 showed greater opsonization on the surface of plasmablasts (CD38+BAFF-R-) compared to felzartamab (anti-CD38 mAb) at certain equivalent concentrations (FIG.6B). These results were unexpected for two reasons. One, the observed increase in MGE-2v1 opsonization occurred on cells expressing only one of the two target antigens of MGE-2v1 (BAFF-R or CD38) and thus were not the result of enhanced avidity due to dual binding to BAFF-R and CD38 in cis. Two, MGE-2v1 comprises anti-BAFF-R and anti-CD38 CDR sequences that are identical to the CDR sequences of ianalumab and felzartamab, respectively. Without wishing to be bound to theory, it is thought that the increase in opsonization seen with MGE-2v1 can be attributed to monovalent binding to each of its target antigens.

[0287] Ianalumab, felzartamab, and other standard mAbs are bivalent and are comprised of a single Fc region and two antigen binding domains; thus, at saturating concentrations, a 1:2 ratio of mAb Fc to antigen on the target cell surface is expected. In other words, bivalent binding results in half as many Fc regions as target receptors, lower antibody opsonization, and therefore suboptimal effector cell recruitment. Conversely, monovalent binding is expected to result in a 1:1 ratio of target receptors to mAb Fc regions on the cell surface. That is, compared with bivalent binding, monovalent binding leads to greater antibody opsonization, which we hypothesize will result in increased engagement with Fc gamma receptors (FcγRs) on effector cells. While this is not well-described in the literature, there aresome data correlating antibody valency with effector cell function, however thesestudies focused on bivalent binding mAbs (Mazor, Y. et al. PLoS One 2016 Jun 20;11(6):e0157788). Increased antibody opsonization on target cells is an unexpected finding for a bispecific antibody and provides mechanistic rationale for improved target cell depletion by MGE-2v1.Example 7: Target cell depletion by bispecific antibodies compared to mAbs with identical CDRs63MOFO-360553874Attorney Docket No.37188-20001.40

[0288] The objective of this set of experiments was to compare the functional activity of MGE-2v1 versus precedent mAbs containing identical CDR sequences. For these experiments, ADCP assays followed by cell quantification by flow cytometry were utilized as described in Example 5. This assay system primarily assesses macrophage ADCP rather than other forms of target cell depletion, as there are few natural killer (NK) cells and no complement components in the assay system.

[0289] Depletion of naïve B cells by MRG-2v1 was approximately 50% greater than with ianalumab, the anti-BAFF-R mAb with which it shares identical CDR sequences (FIG.7A). Conversely, felzartamab (anti-CD38 mAb) showed no depletion of naïve B cells, which was likely due to the low expression of CD38 on these cells (Burns, M. et al. Int J Mol Sci. 2021 Feb 28;22(5):2424). These data confirm the hypothesis that heightened MGE-2v1 opsonization (as described above in Example 6 and FIGs.6A & 6B) from monovalent binding contributes to better target cell depletion than bivalent binding.

[0290] Analogously, depletion of plasmablasts was approximately 80% greater with MGE-2v1 compared with felzartamab (FIG.7B). Ianalulumab showed no activity against plasmablasts, providing additional evidence that MRG-2v1’s enhanced target cell depletion is the result of monovalent binding and hypothesized greater Fc engagement rather than dual-target engagement in cis. These results are highly unexpected, since several studies have indicated that monovalent Fab-antigen interaction is too weak for optimal activity and that bivalent antibodies are much more effective than monovalent antigen-binding domains in neutralizing pathogens (Edeling, M.A. et al. PLoS Pathog.2014;10(4):e1004072; Yan, R. et al. Cell Res.2021;31(5):517-525). Recent studies have described binding molecules with a single, monovalent antigen binding domain or biparatropic binding molecules that exhibit increased hexamerization and complement dependent cytotoxicity (CDC; WO2023069982; WO2023069986). These findings exemplify for the first time that a bispecific antibody that binds each of its targets in a monovalent fashion improves Fc-mediated target cell depletion.

[0291] All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.64MOFO-360553874

Claims

Attorney Docket No.37188-20001.40CLAIMSWhat is claimed is:

1. A bispecific antibody that binds to two different target antigens, wherein the bispecific antibody binds to a first target antigen expressed on a first target cell of the B lymphocyte lineage and a second target antigen expressed on a second target cell of the B lymphocyte lineage, wherein the first and second target antigens are different, wherein the first and second target cells are distinct cell populations of the B lymphocyte lineage, and wherein the bispecific antibody exhibits increased effector cell function, as compared to effector cell function of an antibody that bivalently binds to either the first or the second target antigen.

2. The bispecific antibody of claim 1, wherein the first target cell is a B cell, and the second target cell is a plasma cell.

3. The bispecific antibody of claim 2, wherein the first target antigen is selected from the group consisting of BAFF-R, CD19, and CD20.

4. The bispecific antibody of claim 2, wherein the second target antigen is selected from the group consisting of BCMA, CD19, and CD38.

5. The bispecific antibody of claim 2, wherein the first target antigen is BAFF-R, and the second target antigen is CD38.

6. The bispecific antibody of claim 1, wherein the effector cell function is selected from the group consisting of antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).

7. The bispecific antibody of claim 6, wherein the effector cell function is ADCC.

8. The bispecific antibody of claim 6, wherein the effector cell function is ADCP.

9. The bispecific antibody of claim 6, wherein the effector cell function is ADCC and ADCP.

10. The bispecific antibody of claim 9, wherein the enhanced effector cell function results in depletion of both the first target cell and the second target cell.

11. The bispecific antibody of claim 10, wherein the bispecific antibody exhibits increased depletion of the first or second target cell, as compared to depletion of the first or second target cell by an antibody that bivalently binds to either the first or the second target antigen.

12. The bispecific antibody of claim 10, wherein the bispecific antibody exhibits increased antibody opsonization, as compared to opsonization by an antibody that bivalently binds to either the first or the second target antigen.

13. The bispecific antibody of claim 1, wherein the antibody comprises an Fc region.65MOFO-360553874Attorney Docket No.37188-20001.4014. The bispecific antibody of claim 13, wherein the Fc region comprises one or more modifications that result in enhanced effector cell function, as compared to an Fc region that lacks the one or more modifications.

15. The bispecific antibody of claim 14, wherein the Fc region comprises one or more amino acid substitutions or deletions that increase effector cell function, as compared to effector cell function of an Fc region that lacks the amino acid substitution(s) or deletion(s).

16. The bispecific antibody of claim 14 or claim 15, wherein the Fc region is afucosylated.

17. A bispecific antibody that binds to BAFF-R and CD38, wherein the antibody comprises a first heavy chain variable (VH) domain, a first light chain variable (VL) domain, a second heavy chain variable (VH) domain, and a second light chain variable (VL) domain, wherein:(a) the first VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:3;(b) the first VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:6;(c) the second VH domain comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO:11, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:12, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:13; and(d) the second VL domain comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO:14, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:15, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:16.

18. The bispecific antibody of claim 17, further comprising an Fc domain, wherein the Fc domain is afucosylated.

19. A bispecific antibody that binds to BAFF-R and CD38, wherein the antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 9, a first light chain comprising the amino acid sequence of SEQ ID NO: 10, a second heavy chain comprising amino acid sequence SEQ ID NO: 19, and a second light chain comprising amino acid sequence SEQ ID NO: 20.

20. The bispecific antibody of claim 19, wherein one or both of the first and second heavy chains is / are afucosylated.

21. A method for treating or preventing progression of a B-cell-mediated disease or disorder, comprising administering an effective amount of the bispecific antibody of any one of claims 1-20 to an individual in need thereof.66MOFO-360553874Attorney Docket No.37188-20001.4022. The method of claim 21, wherein the B-cell-mediated disease or disorder is an autoimmune disease, a hematologic cancer, a transplant-related condition, or graft-versus-host disease.

23. A method for depleting B cells in an individual in need thereof, comprising administering to the individual an effective amount of the bispecific antibody of any one of claims 1-20.

24. The method of any one of claims 21-23, wherein the individual is a human.67MOFO-360553874

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