Trispecific antibody targeting CD79b, CD20, and CD3

Abstract

Provided herein are multispecific antibodies, including trispecific antibodies that bind CD79b, CD20, and CD3, and bispecific antibodies that bind CD79b and CD3, and multispecific antigen-binding fragments thereof. Also described are related polynucleotides capable of encoding the provided multispecific antibodies or multispecific antigen-binding fragments, cells expressing the provided multispecific antibodies or multispecific antigen-binding fragments, and related vectors and detectably labeled multispecific antibodies or multispecific antigen-binding fragments. In addition, methods of producing and using the provided multispecific antibodies and multispecific antigen-binding fragments are described. Further provided herein are isolated antibodies and antigen-binding fragments thereof that bind CD79b. Also described are related polynucleotides capable of encoding the provided CD79b-specific antibodies or antigen-binding fragments, cells expressing the provided CD79b-specific antibodies or antigen-binding fragments, and related vectors and detectably labeled CD79b-specific antibodies or antigen-binding fragments. In addition, methods of producing and using the provided CD79b-specific antibodies and antigen-binding fragments are described.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Application No. 63 / 165,501, filed on March 24, 2021, and U.S. Provisional Application No. 63 / 286,309, filed on December 6, 2021. The entire contents of the foregoing applications are hereby incorporated by reference in their entirety into this specification.

[0002] (Sequence Listing) This application has been electronically filed in ASCII format and includes a sequence listing that is hereby incorporated by reference in its entirety into this specification. The ASCII copy was created on January 28, 2022, has the name PRD4135USNP1_SL.txt, and is 357,539 bytes in size.

[0003] (Field of the Invention) The disclosure provided herein relates to multispecific antibodies that bind to cluster of differentiation 79B protein (CD79b), cluster of differentiation 20 (CD20), and cluster of differentiation 3 (CD3), bispecific antibodies that bind to CD79b and CD3, monoclonal antibodies that bind to CD79b, and methods of producing and using the described antibodies.

[0004] (Background) Non-Hodgkin lymphoma (NHL) accounts for approximately 4% of all cancers. Despite improvements in available therapies, relapsed / refractory (R / R, sometimes referred to as r / r) NHL is characterized by a uniformly poor prognosis. Adoptive immunotherapy using T cells genetically engineered to express a chimeric antigen receptor (CAR) has shown promising results for the treatment of CD19-positive B-cell malignancies. However, even with an initial overall response rate of approximately 60–80%, only 40% of patients achieve long-term complete remission [1, 2]. Clinical data demonstrating disease relapse due to CD19 antigen loss have emerged in both acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL) patients [2, 4]. Therefore, there is a need for novel surface antigens to target.

[0005] T-cell redirecting is a powerful and novel approach that can address the unmet medical need of patients with B-cell malignancies where the disease no longer responds to standard chemotherapy or immunotherapy. CD20×CD3 and CD19×CD3 bispecific antibodies have shown promising clinical response rates (10–12), providing proof of concept (PoC) that this therapeutic approach can be highly effective in the clinical management of B-cell malignancies.

[0006] B cells, or B lymphocytes, are a central component of the adaptive immune system, producing antibodies, acting as antigen-presenting cells, secreting cytokines, and differentiating into memory B cells upon activation to respond to several different pathogens [5]. B cells circulate in the blood and lymphatic system. In lymphoid organs, upon encountering their cognate antigen and together with additional signals from T helper cells, B cells can differentiate into effector plasma cells. These cells secrete specific antibodies that circulate in the blood to target and eliminate antigens or pathogens.

[0007] To detect antigens or pathogens, B cells have B cell receptors (BCRs) on their cell surface. The B cell receptor is a multi-component receptor composed of a transmembrane immunoglobulin molecule (mIg), as well as a disulfide-bonded heterodimer of CD79a (Igα) and CD79b (Igβ). CD79b is highly expressed in a wide range of B cell lymphomas. Its expression plays a role in the cancer cell survival rate of most DLBCL tumor models. Therefore, the development of resistance to CD79b-targeted agents due to antigen loss is unlikely to occur and can be an attractive target for the development of new immunotherapy approaches. Clinically, polatuzumab (POLIVY™), an antibody-drug conjugate (ADC) molecule that targets CD79b, was recently approved as a treatment for r / rDLBCL [7]. Treatment with polatuzumab increases the complete response (CR) and duration of response (DOR) rates when combined with standard therapy (bendamustine and rituximab), validating CD79b as a valuable clinical target [8].

[0008] The CD20 molecule (also called human B lymphocyte-restricted differentiation antigen or Bp35) is a hydrophobic transmembrane protein that is overexpressed on most hematological malignancies in the B cell lineage. CD20 is found on the surface of more than 90% of B cells from peripheral blood or lymphoid organs, is expressed during early pre-B cell development, and persists until plasma cell differentiation. CD20 is present on both normal and malignant B cells. Specifically, CD20 is expressed in more than 90% of B cell non-Hodgkin lymphomas (NHLs) (Anderson et al. (1984) Blood 63(6):1424-1433).

[0009] Targeting one or more lymphoma tumor antigens and engaging T cells can result in efficient killing of malignant plasma cells and minimal residual disease (MRD) negativity. Dual CD79b and CD20 targeting prevents tumor antigen escape, targets clonal populations (e.g., capturing tumor cells that do not express sufficient CD79b or CD20 alone), and improves tumor efficacy via an avidity effect. The use of a low-affinity CD3-engaging arm can reduce the potential risk of cytokine release syndrome (CRS).

[0010] Therefore, there is a need for therapeutic antibodies that target both CD79b and CD20 for the treatment of B-cell malignancies such as B-cell lymphoma and non-Hodgkin lymphoma.

[0011] (Summary) In one aspect, provided herein are multispecific antibodies that bind, or specifically bind, to CD79b, CD20, and / or CD3, as well as multispecific antigen-binding fragments thereof. In some embodiments, provided herein are trispecific antibodies that bind, or specifically bind, to CD79b, CD20, and CD3 (“CD79b×CD20×CD3”), as well as trispecific antigen-binding fragments thereof. In some embodiments, provided herein are bispecific antibodies that bind to CD79b and CD3 (“CD79b×CD3”), as well as bispecific antigen-binding fragments thereof. Also described are related polynucleotides capable of encoding the provided multispecific antibodies or multispecific antigen-binding fragments, cells expressing the provided multispecific antibodies or multispecific antigen-binding fragments, as well as related vectors and detectably labeled multispecific antibodies or multispecific antigen-binding fragments. In addition, methods of using the provided multispecific antibodies are described. For example, the multispecific antibodies and multispecific antigen-binding fragments can be used to treat cancer (e.g., CD79b- and / or CD20-expressing cancer), and the multispecific antibodies can be used to diagnose or monitor the progression, regression, or stability of CD79b- and / or CD20-expressing cancer to determine whether a patient needs to be treated for cancer or to determine whether a subject is suffering from CD79b- and / or CD20-expressing cancer, and thus may be suitable for treatment with a CD79b- and / or CD20-specific anti-cancer therapeutic agent, e.g., a CD79b×CD20×CD3 trispecific antibody or a CD79b×CD3 bispecific antibody described herein.

[0012] Redirecting T lymphocytes to tumor cells expressing CD79b and / or CD20 by the TCR / CD3 complex presents an attractive alternative approach. The TCR / CD3 complex of T lymphocytes consists of either a TCR alpha (α) / beta (β) or TCR gamma (γ) / delta (δ) heterodimer co-expressed at the cell surface with invariant subunits of CD3 labeled with gamma (γ), delta (δ), epsilon (ε), zeta (ζ), and eta (η). In some embodiments, the multispecific antibodies or multispecific antigen-binding fragments described herein specifically bind to CD3ε.

[0013] CD79b×CD20×CD3-trispecific antibody In some embodiments, provided herein are isolated CD79b×CD20×CD3 trispecific antibodies or antigen-binding fragments. In some embodiments, the isolated CD79b×CD20×CD3 trispecific antibody or its trispecific binding fragment comprises (a) a first antigen-binding arm comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), (b) a second antigen-binding arm comprising a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2), and (c) a third antigen-binding arm comprising a third heavy chain variable domain (VH3) and a third light chain variable domain (VL3). In some embodiments, the first antigen arm binds to an epitope on CD79b, the second antigen-binding arm binds to an epitope on CD3, and the third antigen-binding arm binds to an epitope on CD20.

[0014] According to all aspects of the present invention, the CD79b×CD20×CD3 trispecific antibody or antigen-binding fragment can bind to the conformational epitope of CD79 composed of residues 30 to 42 (SEDRYRNPKGSAC, SEQ ID NO: 253), 50 to 52 (PRF), 81 to 86 (EMENP, SEQ ID NO: 254), and 144 to 148 (GFSTL, SEQ ID NO: 255). The residue numbers are the residue numbers of human CD79B (P40259).

[0015] According to all aspects of the present invention, the CD79b×CD20×CD3 trispecific antibody or antigen-binding fragment can bind to the conformational epitope of CD3 that includes residues 54 to 58 (GSEIL, SEQ ID NO: 257), 74 to 75 (NI), and 100 to 105 (PRGSKP, SEQ ID NO: 258). The residue numbers are the residue numbers of human CD3E (P07766).

[0016] In some embodiments, an isolated CD79b×CD20×CD3 trispecific antibody, or a bispecific antigen-binding fragment thereof, comprises: a) a first heavy chain portion (HC1); b) a light chain portion; and c) a second heavy chain portion (HC2), wherein HC1 and LC form a first antigen-binding site that specifically binds to a first antigen, HC2 comprises a second antigen-binding site that specifically binds to a second antigen, and HC1 or HC2 further comprises a third antigen-binding site that specifically binds to a third antigen, and each of HC1 and HC2 comprises a Fragment crystallizable (Fc) domain that includes a CH2-CH3 domain. In some embodiments, the first antigen-binding arm of the trispecific antibody or its trispecific binding fragment comprises a first heavy chain portion (HC1) that includes VH1 and a light chain portion (LC) that includes VL1. VH1 and VL1 form a first antigen-binding domain that binds to a first antigen. The second antigen-binding arm of the trispecific antibody or its trispecific binding fragment comprises a second heavy chain portion (HC2) that includes a VH2 domain. The VH2 of HC2 forms a second antigen-binding domain that binds to a second antigen. HC1 or HC2 is further linked to a third antigen-binding arm that includes VH3, which forms a third antigen-binding domain that binds to a third antigen. Each of HC1 and HC2 optionally comprises a Fragment crystallizable (Fc) domain, and the Fc domain includes a constant heavy chain region 2 (CH2) and CH3. In some embodiments, the first antigen is cluster of differentiation 79b (CD79b), the second antigen is cluster of differentiation 3 (CD3), and the third antigen is cluster of differentiation 20 (CD20). In some embodiments, the first antigen is cluster of differentiation 79b (CD79b), the second antigen is cluster of differentiation 20 (CD20), and the third antigen is cluster of differentiation 3 (CD3).

[0017] Some embodiments of the CD79b×CD20×CD3 trispecific antibody or its trispecific binding fragment are further described in the section of forms and examples for carrying out the following invention.

[0018] In some embodiments, the CD79b-binding arm (or "CD79b-specific arm") of the CD79b×CD20×CD3 trispecific antibody is derived from the CD79b antibodies described herein (e.g., antibodies having the CDR sequences listed in Table 1a). In some embodiments, the CD79b-binding arm of the CD79b×CD20×CD3 trispecific antibody comprises any one variable heavy (VH) domain and any one variable light (VL) domain selected from Table 1b. In some embodiments, the CD79b-binding arm of the CD79b×CD20×CD3 trispecific antibody is derived from the CD79b antibody CD9B374 as described herein.

[0019] In some embodiments, the CD20-binding arm (or "CD20-specific arm") of the CD79b×CD20×CD3 trispecific antibody is derived from the CD20 antibodies described herein (e.g., antibodies having the CDR sequences listed in Table 2a). In some embodiments, the CD20-binding arm of the CD79b×CD20×CD3 trispecific antibody comprises any one VH domain and any one VL domain selected from Table 2b. In some embodiments, the CD20-binding arm of the CD79b×CD20×CD3 trispecific antibody is derived from the CD20 antibody C20B648.

[0020] In some embodiments, the CD3-binding arm (or "CD3-specific arm") of a CD79b×CD20×CD3 trispecific antibody is derived from a CD3 antibody described herein (e.g., an antibody having the CDR sequences listed in Table 3). In some embodiments, the CD3-binding arm of a CD79b×CD20×CD3 trispecific antibody comprises any one VH domain and any one VL domain selected from Table 3. In some embodiments, the CD3-binding arm of a CD79b×CD20×CD3 trispecific antibody is derived from monoclonal antibody CD3W245. In some embodiments, the CD3-binding arm of a CD79b×CD20×CD3 trispecific antibody is derived from monoclonal antibody CD3B2030.

[0021] In some embodiments, the CD79b, CD20, and / or CD3-specific arms of a CD79b×CD20×CD3 multispecific antibody or antigen-binding fragment are IgG or derivatives thereof. The IgG class is divided in humans into four isotypes: IgG1, IgG2, IgG3, and IgG4. These share greater than 95% homology in the amino acid sequence of the Fc region but show major differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of the antibody binds to Fc receptors (FcγR) on the surface of immune effector cells such as natural killer and macrophages, leading to phagocytosis or lysis of the target cell. In CDC, the antibody kills the target cell by triggering the complement cascade on the cell surface.

[0022] For many therapeutic antibody applications, Fc-mediated effector functions do not contribute to the mechanism of action. These Fc-mediated effector functions can be detrimental by causing off-target toxicity and can pose a safety risk. Modification of effector functions can be achieved by genetically engineering the Fc region to reduce its binding to FcγR or complement factors. The binding of IgG to activating (FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb) and inhibitory (FcγRIIb) FcγRs or the first component of complement (C1q) is determined by residues located in the hinge region and CH2 domain. When mutations are introduced into IgG1, IgG2, and IgG4, Fc function is reduced or silenced.

[0023] In one embodiment, the antibody comprises an Fc region having one or more of the following properties: (a) reduced effector function compared to the parental Fc, (b) reduced affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIb, and / or FcγRIIIa, (c) reduced affinity for FcγRI, (d) reduced affinity for FcγRIIa, (e) reduced affinity for FcγRIIb, (f) reduced affinity for FcγRIIIb, or (g) reduced affinity for FcγRIIIa.

[0024] In some embodiments, the CD3 - specific antibody or antigen - binding fragment from which the CD3 - specificity arm of the trispecific antibody is derived is IgG or a derivative thereof. In some embodiments, the CD3 - specific antibody or antigen - binding fragment from which the CD3 - specificity arm of the trispecific antibody is derived is IgG1 or a derivative thereof. In some embodiments, for example, the Fc region of a CD3 - specific IgG1 antibody from which the CD3 - binding arm is obtained contains L234A, L235A, and D265S substitutions in its Fc region. In some embodiments, the CD3 - specific antibody or antigen - binding fragment from which the CD3 - specificity arm of the trispecific antibody is derived is IgG4 or a derivative thereof. In some embodiments, for example, the Fc region of a CD3 - specific IgG4 antibody from which the CD3 - binding arm is obtained contains S228P, L234A, L235A, F405L, and R409K substitutions in its Fc region. In some embodiments, the CD3 - specific antibody or antigen - binding fragment from which the CD3 - specificity arm of the trispecific antibody is derived binds to CD3ε on primary human T cells and / or primary cynomolgus monkey T cells. In some embodiments, the CD3 - specific antibody or antigen - binding fragment from which the CD3 - specificity arm of the trispecific antibody is derived activates primary human CD4+ T cells and / or primary cynomolgus monkey CD4+ T cells.

[0025] In addition to the CD79b×CD20×CD3 multispecific antibodies described, polynucleotide sequences capable of encoding the described CD79b×CD20×CD3 multispecific antibodies are also provided. In some embodiments, isolated synthetic polynucleotides encoding one or more CDRs of the heavy chain variable domain and / or one or more CDRs of the light chain variable domain of each antigen-binding arm of a CD79b×CD20×CD3 trispecific antibody or trispecific binding fragment are provided. In some embodiments, isolated synthetic polynucleotides encoding one or more heavy chain variable domains (HC1 and / or HC2) and / or one or more light chain variable domains of a CD79b×CD20×CD3 trispecific antibody or trispecific binding fragment are provided. In some embodiments, isolated synthetic polynucleotides encoding one or more polypeptide chains of the first, second, and / or third antigen-binding arms of a CD79b×CD20×CD3 trispecific antibody or trispecific binding fragment are provided. Vectors containing the described polynucleotides are also provided, and similarly, cells expressing the CD79b×CD20×CD3 multispecific antibody are provided herein. In another embodiment, isolated cells expressing a trispecific antibody or trispecific binding fragment are provided. Also described are cells capable of expressing the disclosed vectors. These cells can be mammalian cells (e.g., 293 cells, 293F cells, CHO cells), insect cells (e.g., Sf7 cells), yeast cells, plant cells, or bacterial cells (e.g., E. coli). The described antibodies can also be produced by hybridoma cells. In some embodiments, methods for producing a CD79b×CD20×CD3 trispecific antibody or trispecific binding fragment by culturing cells are provided.

[0026] Also provided herein is a pharmaceutical composition comprising a CD79b×CD20×CD3 trispecific antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

[0027] Methods of using a CD79B×CD20×CD3 trispecific antibody Also disclosed are methods of using the described CD79b×CD20×CD3 trispecific antibodies and their trispecific antigen-binding fragments. For example, CD79b×CD20×CD3 multispecific antibodies and their trispecific antigen-binding fragments may be useful in the treatment of subjects in need of treatment for CD79b and / or CD20-expressing cancers. In some embodiments, the CD79b and / or CD20-expressing cancers are lymphomas such as diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), or Waldenstrom macroglobulinemia (WM). In some embodiments, the CD79b and / or CD20-expressing cancers are lymphomas in a recurrent or refractory form, such as diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), or Waldenstrom macroglobulinemia (WM) in a recurrent or refractory form.

[0028] In a subject in need of treatment for CD79b and / or CD20-expressing cancer, the described method of doing so comprises administering to the subject a therapeutically effective amount of the described CD79b×CD20×CD3 trispecific antibody or its trispecific antigen-binding fragment. In some embodiments, the subject is a mammal, preferably a human. In a preferred embodiment, a method for treating a subject having cancer is provided by administering a therapeutically effective amount of a CD79b×CD20×CD3 trispecific antibody or trispecific antigen-binding fragment to a patient in need of treatment for cancer for a time sufficient to treat the cancer.

[0029] In this specification, there is further provided a method for inhibiting the growth or proliferation of cancer cells by administering a therapeutically effective amount of a CD79b×CD20×CD3 trispecific antibody or trispecific antigen-binding fragment to inhibit the growth or proliferation of cancer cells.

[0030] Also, in this specification, there is provided a method for redirecting T cells to cancer by administering a therapeutically effective amount of a CD79b×CD20×CD3 trispecific antibody or trispecific antigen-binding fragment to redirect T cells to CD79b- and / or CD20-expressing cancer cells.

[0031] Those skilled in the art will understand that the methods of using the described CD79b×CD20×CD3 multispecific antibodies and their multispecific antigen-binding fragments can be defined in the form of pharmaceutical use, for example, in the form of CD79b×CD20×CD3 multispecific antibodies and multispecific antigen-binding fragments for use in the treatment of diseases defined herein, particularly cancer. Those skilled in the art will also understand that the methods of using the described CD79b×CD20×CD3 multispecific antibodies and their multispecific antigen-binding fragments can be defined in the so-called Swiss form, for example, in the form of using CD79b×CD20×CD3 multispecific antibodies and multispecific antigen-binding fragments to manufacture a medicament for the treatment of diseases defined herein, particularly cancer. This applies throughout the present disclosure.

[0032] CD79b×CD20×CD3 Specific Antibody Kit Disclosed herein is a kit comprising a CD79b×CD20×CD3 multispecific antibody. Using the disclosed kit, a method of using the CD79b×CD20×CD3 multispecific antibody provided herein, or other methods known to those skilled in the art, can be carried out. In some embodiments, the disclosed kit may comprise the antibody described herein and a reagent for use in the treatment of CD20- and / or CD79b-expressing cancers. Accordingly, the disclosed kit may include one or more of a trispecific antibody or its trispecific antigen-binding fragment as described herein, a container for containing the antibody or fragment when not in use, and / or instructions for use of the antibody or fragment, an antibody or fragment immobilized on a solid support, and / or an antibody or fragment in a detectably labeled form.

[0033] CD79b×CD3 bispecific antibody In some embodiments, provided herein is an isolated CD79b×CD3 bispecific antibody or antigen-binding fragment.

[0034] In some embodiments, the isolated CD79b×CD3 bispecific antibody or its bispecific binding fragment comprises: (a) a first antigen-binding arm comprising a first heavy-chain variable domain (VH1) and a first light-chain variable domain (VL1); and (b) a second antigen-binding arm comprising a second heavy-chain variable domain (VH2) and a second light-chain variable domain (VL2), wherein the first antigen-binding arm binds to the epitope CD79b and the second antigen-binding arm binds to an epitope on CD3.

[0035] According to all aspects of the present invention, the CD79b×CD3 bispecific antibody, or its bispecific binding fragment, can bind to the conformational epitope of CD79 composed of residues 30-42 (SEDRYRNPKGSAC, SEQ ID NO: 253), 50-52 (PRF), 81-86 (EMENP, SEQ ID NO: 254), and 144-148 (GFSTL, SEQ ID NO: 255). The residue numbers are the residue numbers of human CD79B (P40259).

[0036] According to all aspects of the present invention, the CD79b×CD20×CD3 bispecific antibody, or its bispecific binding fragment, can bind to the conformational epitope of CD3 that includes residues 54-58 (GSEIL, SEQ ID NO: 257), 74-75 (NI), and 100-105 (PRGSKP, SEQ ID NO: 258). The residue numbers are the residue numbers of human CD3E (P07766).

[0037] In some embodiments, the isolated CD79b×CD3 bispecific antibody, or its bispecific antigen-binding fragment, comprises a) a first heavy chain portion (HC1), b) a light chain portion, and c) a second heavy chain portion (HC2), wherein HC1 and LC form a first antigen-binding site that specifically binds to a first antigen, HC2 comprises a second antigen-binding site that specifically binds to a second antigen, and HC1 and HC2 each comprise a fragment crystallizable (Fc) domain that includes a CH2-CH3 domain.

[0038] In some embodiments, the first antigen-binding arm of a CD79b×CD3 bispecific antibody, or a bispecific binding fragment thereof, comprises a first heavy chain portion (HC1) comprising VH1 and a light chain portion (LC) comprising VL1. VH1 of HC1 and VL1 of LC form a first antigen-binding domain that binds to a first antigen. The second antigen-binding arm of the bispecific antibody or a bispecific binding fragment thereof comprises a second heavy chain portion (HC2) comprising VH2. VH2 of HC2 forms a second antigen-binding domain that binds to a second antigen, and each of HC1 and HC2 optionally comprises a fragment crystallizable (Fc) domain comprising a CH2-CH3 domain. In some embodiments, the first antigen is cluster of differentiation 79b (CD79b) and the second antigen is cluster of differentiation 3 (CD3). In some embodiments, the first antigen is cluster of differentiation 79b (CD79b) and the second antigen is cluster of differentiation 3 (CD3). Some embodiments of the CD79b×CD3 bispecific antibody, or a bispecific binding fragment thereof, are further described in the Forms and Examples sections for carrying out the following invention.

[0039] In some embodiments, the CD79b-binding arm (or "CD79b-specific arm") of a CD79b×CD3 bispecific antibody is derived from a CD79b antibody described herein (e.g., an antibody having the CDR sequences listed in Table 1a). In some embodiments, the CD79b-binding arm of a CD79b×CD3 bispecific antibody comprises any one VH domain and any one VL domain selected from Table 1b.

[0040] In some embodiments, the CD3-binding arm (or "CD3-specific arm") of a CD79b×CD3 bispecific antibody is derived from a CD3 antibody described herein (e.g., an antibody having the CDR sequences listed in Table 3). In some embodiments, the CD3-binding arm of a CD79b×CD3 bispecific antibody comprises any one VH domain and any one VL domain selected from Table 3.

[0041] In some embodiments, the CD79b or CD3 specific arm of the CD79b×CD3 bispecific antibody or antigen-binding fragment is IgG or a derivative thereof.

[0042] In one embodiment, the antibody comprises an Fc region having one or more of the following properties: (a) reduced effector function when compared to the parental Fc, (b) reduced affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIb and / or FcγRIIIa, (c) reduced affinity for FcγRI, (d) reduced affinity for FcγRIIa, (e) reduced affinity for FcγRIIb, (f) reduced affinity for FcγRIIIb or (g) reduced affinity for FcγRIIIa.

[0043] In some embodiments, the CD3 specific antibody or antigen-binding fragment from which the CD3 specific arm of the bispecific antibody is derived is IgG or a derivative thereof. In some embodiments, the CD3 specific antibody or antigen-binding fragment from which the CD3 specific arm of the bispecific antibody is derived is IgG1 or a derivative thereof. In some embodiments, for example, the Fc region of a CD3 specific IgG1 antibody from which the CD3 binding arm is obtained comprises L234A, L235A, and D265S substitutions in its Fc region. In some embodiments, the CD3 specific antibody or antigen-binding fragment from which the CD3 specific arm of the bispecific antibody is derived is IgG4 or a derivative thereof. In some embodiments, for example, the Fc region of a CD3 specific IgG4 antibody from which the CD3 binding arm is obtained comprises S228P, L234A, L235A, F405L, and R409K substitutions in its Fc region. In some embodiments, the CD3 specific antibody or antigen-binding fragment from which the CD3 specific arm of the bispecific antibody is derived binds to CD3ε on primary human T cells and / or primary cynomolgus monkey T cells. In some embodiments, the CD3 specific antibody or antigen-binding fragment from which the CD3 specific arm of the bispecific antibody is derived activates primary human CD4+ T cells and / or primary cynomolgus monkey CD4+ T cells.

[0044] In addition to the described CD79b×CD3 bispecific antibodies, polynucleotide sequences capable of encoding the described CD79b×CD3 bispecific antibodies are also provided. In some embodiments, isolated synthetic polynucleotides encoding one or more CDRs of the heavy chain variable domain and / or one or more CDRs of the light chain variable domain of each antigen-binding arm of a CD79b×CD3 bispecific antibody or bispecific binding fragment are provided. In some embodiments, isolated synthetic polynucleotides encoding one or more heavy chain variable domains (such as HC1 and / or HC2) and / or one or more light chain variable domains of a CD79b×CD3 bispecific antibody or bispecific binding fragment are provided. In some embodiments, isolated synthetic polynucleotides encoding one or more polypeptide chains of the first and / or second antigen-binding arms of a CD79b×CD3 bispecific antibody or bispecific binding fragment are provided. Vectors containing the described polynucleotides are also provided, and similarly, cells expressing a CD79b×CD3 bispecific antibody are provided herein. In another embodiment, isolated cells expressing a bispecific antibody or bispecific binding fragment are provided. Also described are cells capable of expressing the disclosed vectors. These cells can be mammalian cells (e.g., 293 cells, 293F cells, CHO cells), insect cells (e.g., Sf7 cells), yeast cells, plant cells, or bacterial cells (e.g., E. coli). The described antibodies can also be produced by hybridoma cells. In some embodiments, a method for generating a CD79b×CD3 bispecific antibody or bispecific binding fragment by culturing cells is provided.

[0045] Further provided herein is a pharmaceutical composition comprising a CD79b×CD3 bispecific antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

[0046] Methods of using a CD79b×CD3 bispecific antibody Also disclosed are methods of using the described CD79b×CD3 bispecific antibodies and their bispecific antigen-binding fragments. For example, the CD79b×CD3 bispecific antibodies and their bispecific antigen-binding fragments may be useful in the treatment of CD79b-expressing cancers in a subject in need of treatment for CD79b-expressing cancer. In some embodiments, the CD79b-expressing cancer is a lymphoma such as diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), or Waldenström macroglobulinemia (WM). In some embodiments, the CD79b-expressing cancer is a recurrent or refractory form of lymphoma.

[0047] In a subject in need of treatment for CD79b-expressing cancer, the described method of doing so comprises administering to the subject a therapeutically effective amount of the described CD79b×CD3 bispecific antibody or its bispecific antigen-binding fragment. In some embodiments, the subject is a mammal, preferably a human. In a preferred embodiment, a method for treating a subject having cancer is provided by administering a therapeutically effective amount of a CD79b×CD3 bispecific antibody or bispecific antigen-binding fragment to a patient in need of treatment for a sufficient time to treat the cancer.

[0048] Also provided is a method for inhibiting the growth or proliferation of cancer cells by administering a therapeutically effective amount of a CD79b×CD3 bispecific antibody or bispecific antigen-binding fragment to inhibit the growth or proliferation of cancer cells.

[0049] Also provided herein is a method of redirecting T cells to CD79b-expressing cancer cells by administering a therapeutically effective amount of a CD79b×CD3 bispecific antibody or bispecific binding fragment to redirect T cells to cancer.

[0050] CD79b×CD3 Specific Antibody Kit Disclosed herein is a kit comprising the disclosed CD79b×CD3 bispecific antibody. Using the described kit, a method of using the CD79b×CD3 bispecific antibody provided herein or other methods known to those skilled in the art can be performed. In some embodiments, the described kit may include the antibody described herein and a reagent for use in the treatment of CD79b-expressing cancer. Thus, the described kit may include one or more of a trispecific antibody or its bispecific antigen-binding fragment as described herein, a container for containing the antibody or fragment when not using them, and / or instructions for use for using the antibody or fragment, an antibody or fragment immobilized on a solid support, and / or an antibody or fragment in a detectably labeled form.

[0051] CD79b-specific antibody Also provided herein are antibodies that bind to CD79b and antigen-binding fragments thereof. Also provided are related polynucleotides capable of encoding the provided CD79b-specific antibodies and antigen-binding fragments, cells expressing the provided antibodies and antigen-binding fragments, and related vectors, as well as detectably labeled antibodies and antigen-binding fragments. In addition, methods of using the provided antibodies and antigen-binding fragments are described. For example, the CD79b-specific antibodies and antigen-binding fragments can be used to treat cancer (e.g., CD79b-expressing cancer), and the CD79b-specific antibodies and antigen-binding fragments can be used to diagnose or monitor the progression, regression, or stability of CD79b-expressing cancer to determine whether a patient needs to be treated for cancer or to determine whether a subject has or will have CD79b-expressing cancer, and thus may be suitable for treatment with a CD79b-specific anti-cancer therapeutic agent, such as a multispecific antibody against CD79b and CD3 as described herein. Some aspects of the CD79b-specific antibody or antigen-binding fragment are further described in the following detailed description and examples sections.

[0052] According to all aspects of the present invention, the CD79b-specific antibody or antigen-binding fragment can bind to a conformational epitope of CD79 composed of residues 30-42 (SEDRYRNPKGSAC, SEQ ID NO: 253), 50-52 (PRF), 81-86 (EMENP, SEQ ID NO: 254), and 144-148 (GFSTL, SEQ ID NO: 255). The residue numbers are the residue numbers of human CD79B (P40259).

[0053] Methods of using CD79b-specific antibodies Methods of using the described CD79b-specific antibodies or antigen-binding fragments are also disclosed. Specific antibodies used in the methods considered in this section include those having the set of CDRs described for the antibodies in Table 1a. For example, these antibodies or antigen-binding fragments can be useful in the treatment of cancer by interfering with CD79b receptor interactions or, when the antibody is conjugated to a toxin, by targeting the toxin in this way to CD79b-expressing cancer. Further, these antibodies or antigen-binding fragments can be used to detect the presence of CD79b in a biological sample, such as blood or serum, to quantify the amount of CD79b in a biological sample, such as blood or serum, to diagnose CD79b-expressing cancer, to determine a method of treating a subject suffering from cancer, or to monitor the progression of CD79b-expressing cancer in a subject. In some embodiments, the CD79b-expressing cancer can be a lymphoma such as diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), or Waldenström macroglobulinemia (WM). In some embodiments, the CD79b-expressing cancer is a recurrent or refractory form of lymphoma.

[0054] The methods described may be performed before a subject undergoes treatment for CD79b-expressing cancer, e.g., treatment with a multispecific antibody against CD79b and CD3. Further, the methods described may be performed after a subject has undergone treatment for CD79b-expressing cancer, e.g., treatment with a multispecific antibody against CD79b and CD3 as described herein.

[0055] The methods described for detecting CD79b in a biological sample include exposing the biological sample to one or more of the CD79b-specific antibodies or antigen-binding fragments described herein.

[0056] Also, the methods described for diagnosing CD79b-expressing cancer in a subject involve exposing a biological sample to one or more of the CD79b-specific antibodies or antigen-binding fragments described herein. However, the method includes a step of quantifying the amount of CD79b present in the sample, a step of comparing the amount of CD79b present in the sample to a known standard or reference sample, and a step of determining whether the subject's CD79b level falls within the levels of CD79b associated with cancer.

[0057] Also described herein are methods for monitoring CD79b-expressing cancer in a subject. The methods described include exposing a biological sample to one or more of the CD79b-specific antibodies or antigen-binding fragments described herein, a step of quantifying the amount of CD79b present in the sample that is bound to the antibody or its antigen-binding fragment, a step of comparing the amount of CD79b present in the sample to either a known standard or reference sample or the amount of CD79b in a similar sample previously obtained from the subject, and a step of determining whether the subject's CD79b level indicates progression, regression, or no change in the cancer based on the differences in the amount of CD79b in the compared samples.

[0058] The sample obtained from or derived from a subject is a biological sample, such as urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells, tissue, surgically resected tumor tissue, biopsy, fine needle aspirate sample, or histological preparation.

[0059] The described CD79b-specific antibodies or antigen-binding fragments can be labeled for use in the described methods or other methods known to those of skill in the art. For example, the antibodies or antigen-binding fragments thereof described herein can be labeled with a radiolabel, a fluorescent label, an epitope tag, biotin, a chromophore label, an ECL label, an enzyme, ruthenium, 111 In-DOTA, 111 They may be labeled with In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase, and beta-galactosidase, or polyhistidine or similar such labels known in the art.

[0060] CD79b-specific antibody kit Kits containing the disclosed CD79b-specific antibodies or antigen-binding fragments thereof are described herein. The described kits can be used to perform methods using the CD79b-specific antibodies or antigen-binding fragments provided herein, or other methods known to those of skill in the art. In some embodiments, the described kits may include the antibodies or antigen-binding fragments described herein and reagents for use in detecting the presence of CD79b in a biological sample.

[0061] Thus, the described kits may include one or more of the antibodies or antigen-binding fragments thereof described herein, a container for housing the antibody or fragment when not in use, instructions for using the antibody or fragment, and the antibody or fragment immobilized on a solid support and / or in detectably labeled form as described herein.

Brief Description of the Drawings

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[0064] (Detailed description of the embodiments) B-cell malignancies encompass various cancers, including CLL, MCL, DLBCL, FL, and MZL, and represent a significant unmet medical need. After the success of small molecule inhibitors targeting the BCR or apoptosis pathways, the T-cell redirected approach has been selected as one of the major areas of research and development. Off-the-shelf preparations of T-cell engagers offer new treatment options for a wide range of patients, from frontline patients to heavily pre-treated patients, including those who have progressed after chimeric antigen receptor (CAR)-T cell therapy (28).

[0065] Non-Hodgkin lymphoma (NHL) is a heterogeneous group of malignancies of B- or T-cell origin, accounting for approximately 4% of all malignancies in the United States (29). Approximately 80% of NHLs are of B-cell lineage and express B-cell differentiation antigens, including CD19, CD20, CD22, and CD79b. These surface antigens represent extremely important targets for current standard therapies. However, despite improvements in available therapies, B-NHL uniformly has a poor prognosis, with 30% - 50% of DLBCL patients relapsing after rituximab-cyclophosphamide-hydroxydaunorubicin-oncovin-prednisone / prednisolone regimen (R-CHOP) therapy, and only 40% of patients with relapsed and refractory (R / R) disease achieving long-term complete remission with CD19 CAR-T therapy (30). New clinical trial data using CD20×CD3 bispecific antibodies appear to demonstrate that T-cell redirection can address the urgent unmet need of this growing population of patients whose disease no longer responds to standard chemotherapy or immunotherapy.

[0066] Both CD79b and CD20 are well-validated therapeutic targets expressed in most B-cell malignancies and thus serve as good surface antigens for use in treatment modalities. Dual antigen recognition on B cells by trispecific T-cell redirected antibodies as described herein has the potential to enhance tumor binding via the avidity effect, maximize tumor eradication in the presence of heterogeneous cell populations, and prevent the occurrence of tumor antigen escape observed with CD19 and CD20 targeted therapies (31 - 34).

[0067] DLBCL is the most common subset of aggressive B-NHL, accounting for approximately 30% to 58% of all new cases of NHL diagnosed worldwide each year. The patient share in the R / R setting is dominant in the United States and Europe by regimens of rituximab-ifosfamide-carboplatin-etoposide (R-ICE), rituximab-(dose-adjusted) etoposide-prednisolone-oncovin-cyclophosphamide-hydroxydaunorubicin (R-[DA]-EPOCH), and rituximab-dexamethasone-high-dose ara-C cytarabine-platinol (R-DHAP), followed by rituximab-etoposide-solu-medrol-high-dose ara-C cytarabine-platinol (R-ESHAP)) and bendamustine-rituximab (BR) regimens.

[0068] CAR-T therapy has been recently approved in this setting and is expected to show significant uptake over the next decade. The entry of three anti-CD19 CAR-T therapies, namely Yescarta, Kymriah, and Breyanzi, into the R / R DLBCL space indicates the entry of a new drug class for the treatment of NHL. All three therapies target chemotherapy-refractory patients, including those who have experienced disease progression after allogeneic stem-cell transplantation (ASCT). The high cost associated with CAR-T therapy, the logistics involved in the preparation of the constructs, and the acute toxicity associated with the treatment hinder their uptake and alternative treatment options are needed.

[0069] Monoclonal antibodies dominate the R / R FL setting in terms of patient share. The 2017 R / R patient share leader in the United States was BR, followed by R-CHOP. Lenalidomide + rituximab, the so-called R 2 regimen was approved in 2019 and is recommended by the National Comprehensive Cancer Network (NCCN) guidelines as a treatment option for this patient population. Zydelig (idelalisib), a phosphoinositide 3-kinase (PI3K) inhibitor, is also approved for patients who have received two prior lines. Ukoniq (umbralisib) and Tazverik (tasemetostat) were approved in 2020 for R / R patients after two or more lines of therapy. The unmet need is particularly high for early R / R patients with limited effective treatment options.

[0070] The multispecific antibodies and multispecific antigen-binding fragments described herein address these and other related needs.

[0071] Definitions Throughout this specification and the claims, various terms are used with respect to the aspects of this specification. Unless otherwise indicated, such terms are to be given their ordinary meaning in the relevant art. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0072] As used in this specification and the appended "claims", the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, a reference to "a cell" includes combinations of two or more cells.

[0073] As used herein, the term "about", when referring to a measured value, such as an amount, a duration, etc., means including a deviation of up to ±10% from the specified value. Similarly, such deviations are appropriate for performing the disclosed methods. Unless otherwise stated, all numbers expressing amounts of ingredients, and properties such as molecular weights and reaction conditions, used in this specification and the claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the reported number of significant digits and by applying ordinary rounding techniques.

[0074] The numerical ranges and parameters setting forth the broad scope of the invention are approximations, but the numerical values set forth in the specific examples are reported as precisely as possible. Nevertheless, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in the respective test measurements.

[0075] "Isolated" means that a biological component (e.g., a nucleic acid, peptide, or protein) is substantially separated from other biological components of the organism in which these components are naturally present, i.e., other chromosomes and extrachromosomal DNA and RNA, as well as proteins, and is produced separately from or purified away from these other components. Thus, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. "Isolated" nucleic acids, peptides, and proteins can be part of a composition and are considered isolated even if such composition is not part of the natural environment of the nucleic acid, peptide, or protein. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids. As used herein, an "isolated" antibody or antigen-binding fragment is intended to mean an antibody or antigen-binding fragment that is substantially free of other antibodies or antigen-binding fragments having various antigen specificities (e.g., an isolated antibody that specifically binds to CD79b is substantially free of antibodies that specifically bind to antigens other than CD79b). However, an isolated antibody that specifically binds to an epitope, isoform, or variant of CD79b may have cross-reactivity, for example, with other related antigens from other species (e.g., CD79b interspecies homologs).

[0076] The term "polynucleotide", which is also synonymously referred to as "nucleic acid molecule", "nucleotide", or "nucleic acid", refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA (which may be single-stranded, or more typically, double-stranded, or a mixture of single-stranded and double-stranded regions), but is not limited thereto. In addition, "polynucleotide" refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA having a backbone modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications may be made to DNA and RNA. Thus, "polynucleotide" typically encompasses chemically, enzymatically, or metabolically modified forms of naturally occurring polynucleotides, as well as chemical forms having the characteristics of viral and cellular DNA and RNA. "Polynucleotide" also includes relatively short nucleic acid chains (often referred to as oligonucleotides).

[0077] "Synthetic nucleic acid sequence", "synthetic polynucleotide", "synthetic oligonucleotide", "synthetic DNA", or "synthetic RNA", as used herein, refers to a nucleic acid sequence, polynucleotide, oligonucleotide, DNA, or RNA that is different from what is found in nature or has a chemical modification not found in nature by having a sequence different from what is found in nature. The definition of synthetic nucleic acids includes, but is not limited to, DNA sequences created using biotechnology tools. Such tools include, but are not limited to, recombinant DNA technology, chemical synthesis, or the directed use of nucleases (so-called "genome editing" or "gene optimization" techniques). The meaning of "substantially the same" can vary depending on the context in which the term is used. Since there can be natural sequence variations in the heavy and light chains and the genes encoding them, there are expected to be some variations within the amino acid sequences described herein or the genes encoding antibodies or antigen-binding fragments that have little or no effect on their intrinsic binding properties (e.g., specificity and affinity). Such predictions are due in part to the degeneracy of the genetic code and the evolutionary success of conserved amino acid sequence variations. Such variations do not change the properties of the encoded protein so much that they can be recognized. Thus, in the context of nucleic acid sequences, "substantially the same" means at least 65% identity between two or more sequences. Preferably, the term means at least 70% identity, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, and more preferably at least 99% or more identity between two or more sequences.The identity (%) between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap (i.e., % homology = number of identical positions / total number of positions × 100). This consideration is necessary to derive the optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988). This algorithm is incorporated into the ALIGN program (version 2.0) and uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the identity (%) between two amino acid sequences may be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0078] The degree of variation that can occur within the amino acid sequence of a protein without substantially affecting the function of the protein is much smaller than the degree of variation of a nucleic acid sequence. This is because the same degeneracy principle does not apply to amino acid sequences. Thus, in the context of an antibody or antigen-binding fragment, "substantially the same" means an antibody or antigen-binding fragment having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the recited antibody or antigen-binding fragment. Other embodiments do not share significant identity with the antibodies and antigen-binding fragments described herein, but have a framework, scaffold, or other non-binding region that incorporates one or more CDRs or other sequences required to confer binding, and have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such sequences described herein, and include antibodies or antigen-binding fragments having such identity.

[0079] A "clone" is a population of cells obtained from a single cell or a common ancestor by mitosis. A "cell line" is a clone of primary cells that can be stably grown in vitro for many generations. In some of the examples provided herein, cells are transformed by introducing DNA into the cells.

[0080] The terms "expressing" and "producing" are used interchangeably herein and mean the biosynthesis of a gene product. These terms include the transcription of a gene into RNA. These terms also include the translation of the RNA into one or more polypeptides and further include all natural post-transcriptional and post-translational modifications. The expression or production of an antibody or an antigen-binding fragment thereof may be in the cytoplasm of a cell or in the extracellular environment, such as in a growth medium for cell culture.

[0081] The term "treating" or "treatment" refers to any success or sign of success in attenuating or ameliorating an injury, pathology, or medical condition, including remission, alleviation, reduction of symptoms or making the medical condition more tolerable to the patient, slowing the rate of degeneration or decline, alleviating the ultimate debilitation due to degeneration, improving the physical or mental health of the patient, or extending the lifespan. Treatment may be evaluated by objective or subjective parameters. Such parameters include the results of a physical examination, neurological examination, or psychiatric evaluation.

[0082] An "effective amount" or "therapeutically effective amount" means an amount effective to achieve a desired therapeutic result at the required dosage and for the required period of time. The therapeutically effective amounts described herein may vary according to factors such as the condition of the individual, age, gender, and weight of the individual, and the ability of the antibody to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the antibody or antibody portion.

[0083] "Antibody" refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY), including various monomeric, polymeric, and chimeric forms, unless otherwise specified. Polyclonal antibodies, monoclonal antibodies (mAb), and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, are specifically included within the term "antibody".

[0084] The term "antigen-binding arm" refers to a portion of an antibody that includes an antigen-binding domain or site that binds to an antigen (e.g., CD79b, CD20, or CD3) and optionally includes one or more other antibody regions (e.g., the Fc domain). An antigen-binding arm is an example of an "antigen-binding region". An "antigen-binding fragment" is any proteinaceous structure that can exhibit binding affinity for a particular antigen. Similarly, a "bispecific binding fragment" or "trispecific binding fragment" is any proteinaceous structure that can exhibit binding affinity for two or three antigens, respectively. As used herein, the terms "antigen-binding fragment", "bispecific binding fragment", or "trispecific binding fragment" preferably refer to a fragment of an antigen-binding arm that contains an antigen-binding domain. Binding fragments include those provided by any known method, such as enzymatic cleavage, peptide synthesis, and recombinant methods. Some antigen-binding fragments are composed of intact antibody portions that retain the antigen-binding specificity of the parental antibody molecule. For example, a binding fragment may include at least one variable region (either the variable region of the heavy or light chain) or one or more CDRs of an antibody known to bind a particular antigen.Examples of suitable binding fragments include diabodies and single-chain molecules, as well as Fab, F(ab’)2, Fc, Fabc, and Fv molecules, single-chain (Sc) antibodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy chain and one light chain, monovalent fragments consisting of VL, VH, CL, and CH1 domains, or the monovalent antibodies described in WO 2007 / 059782, bivalent fragments containing two Fab fragments linked by a disulfide bond in the hinge region, Fd fragments consisting essentially of VH and CH1 domains, Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody, dAb fragments consisting essentially of a VH domain (Ward et al., Nature, 341:544-546, 1989; Trends Biotechnol. 2003 Nov.;21(11):484-90), camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan.;5(1):111-24), isolated complementarity determining regions (CDRs), etc., but are not limited thereto. All antibody isotypes may be used to generate binding fragments. Further, the binding fragments may include proteinaceous frameworks other than antibodies, such as protein scaffolds, which can successfully incorporate polypeptide segments in a direction that confers affinity for a given antigen of interest. Antigen-binding fragments, bispecific binding fragments, or trispecific binding fragments may be generated recombinantly or by enzymatic or chemical degradation of intact antibodies. The expressions “antibody or its antigen-binding fragment”, “bispecific antibody or its bispecific binding fragment”, or “trispecific antibody or its trispecific binding fragment” may be used to indicate that they incorporate one or more amino acid segments of the antibodies referred to within that expression.

[0085] The term "antigen-binding domain" refers to the proteinaceous structure of the antigen-binding arm that exhibits binding affinity for a specific antigen. This proteinaceous structure is mediated by the complementarity-determining regions (CDRs) of the antigen-binding domain.

[0086] The terms "CDR" and its plural form "CDRs" refer to the complementarity-determining regions (CDRs), three of which constitute the binding properties of the light-chain variable region (CDRL1, CDRL2, and CDRL3), and three of which constitute the binding properties of the heavy-chain variable region (CDRH1, CDRH2, and CDRH3). CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences that include the scaffold region or framework region. The exact definitional boundaries and lengths of CDRs vary depending on the various classification and numbering systems. Thus, CDRs can be referred to by the Kabat definition, the Chothia definition, the contact definition, or any other boundary definition. The CDRs described herein are referred to by the AbM definition. Despite the different boundaries, each of these systems has some overlap in the elements that constitute the so-called "hypervariable regions" within the variable sequences. Thus, the definitions of CDRs by these systems may differ in terms of the length and boundary regions for the adjacent framework regions. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. NIH Publication No. 91-3242 (1991), Chothia et al., "Canonical Structures For the Hypervariable Regions of Immunoglobulins," J. Mol. Biol. 196:901 (1987), and MacCallum et al., "Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography," J. Mol. Biol. 262:732 (1996), each of which is incorporated herein by reference in its entirety.

[0087] Typically, a CDR forms a loop structure that can be classified as a canonical structure. The term "canonical structure" refers to the backbone conformation adopted by the antigen-binding (CDR) loop. From studies of comparative structures, it has been found that 5 out of 6 antigen-binding loops have a very limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Thus, corresponding loops between antibodies can have very similar three-dimensional structures even though a high degree of amino acid sequence variability is seen in most of those loops (Chothia et al., "Canonical Structures For the Hypervariable Regions of Immunoglobulins," J. Mol. Biol. 196:901 (1987), Chothia et al., "Conformations of Immunoglobulin Hypervariable Regions," I 342:877 (1989); Martin and Thornton, "Structural Families in Loops of Homologous Proteins: Automatic Classification, Modelling and Application to Antibodies," J. Mol. Biol. 263:800 (1996), each of which is incorporated by reference in its entirety). Furthermore, there is a relationship between the loop structure adopted and the amino acid sequence around it. The conformation of a particular canonical class is determined by the length of the loop, the important positions within the loop, and the amino acid residues within the conserved framework (i.e., outside the loop). Thus, based on the presence of these important amino acid residues, an assignment to a particular canonical class can be made.

[0088] As used herein, the term "Fc" refers to the crystallizable domain fragment of an antibody that includes two constant heavy chain (CH) regions, CH2 and CH3. In this specification, the amino acid residues of the Fc region are typically numbered according to the EU numbering scheme (Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969). These residues can be readily designated according to alternative numbering schemes, such as those of IMGT and Kabat (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication n° 91-3242, pp 662, 680, 689 (1991)), as will be readily understood by those skilled in the art. For example, L234 according to EU numbering can also be represented as L247 according to Kabat numbering.

[0089] The term "polypeptide" is used interchangeably with the term "protein" and, in its broadest sense, refers to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds, such as ester, ether, etc. As used herein, the term "amino acid" refers to either natural and / or non-natural amino acids or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics. Peptides of three or more amino acids are generally referred to as oligopeptides when the peptide chain is short. When the peptide chain is long, the peptide is generally referred to as a polypeptide or a protein.

[0090] When used in the context of an antibody or antibody fragment, "specifically binds" or "binds specifically" or derivatives thereof means binding to one or more epitopes of a target protein without preferentially binding to other molecules in a sample containing a variety of mixed molecules via a domain encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. Typically, an antibody has a K -8 less than about 1×10 d M and binds to a homologous antigen. Phrases such as "[antigen] - specific" antibody (e.g., CD79b - specific antibody) mean to convey that the antibody listed specifically binds to the antigen listed. Whenever the term "binds" is used herein, it is intended to include "specifically binds", and these terms may be exchanged as desired.

[0091] As used herein, the term "chimeric" refers to an antibody or antigen - binding fragment thereof that has at least some portion of at least one variable domain derived from an antibody amino acid sequence of a non - human mammal, rodent, or reptile, while the remaining portions thereof are derived from a human.

[0092] A "vector" is a replicon, such as a plasmid, phage, cosmid, or virus, into which another nucleic acid segment can be operably inserted so as to cause the replication or expression of the segment.

[0093] As used herein, the term "host cell" can be any type of cell, such as, for example, primary cells, cells in culture, or cells derived from cell lines. In some embodiments, the host cell is ex vivo or in vitro. In some embodiments, the host cell is not a human totipotent or human pluripotent stem cell. In certain embodiments, the term "host cell" refers to a cell that has been transfected with a nucleic acid molecule, and progeny or potential progeny of such a cell. Progeny of such cells may not be identical to the parental cell transfected with the nucleic acid molecule due to, for example, mutations or environmental influences that may occur in subsequent generation or integration of the nucleic acid molecule into the host cell genome. The terms "expression" and "production" are used interchangeably herein and mean the biosynthesis of a gene product. These terms include the transcription of a gene into RNA. These terms also include the translation of the RNA into one or more polypeptides, and further include all natural post-transcriptional and post-translational modifications.

[0094] The term "subject" means human and non-human animals, and includes all vertebrates, such as, for example, mammals and non-mammals, such as, for example, non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In many embodiments of the methods described, the subject is human.

[0095] As used herein, the term "redirect" or "redirecting" refers to the ability of a multispecific antibody (e.g., a CD79b×CD20×CD3 antibody, a CD79b×CD3 antibody) described to effectively transport the activity of a T cell from its native cognate specificity to reactivity against CD79b and / or CD20 expressing cells. As used herein, the term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject (e.g., surgically removed tumor tissue, biopsies including fine needle aspirates), as well as fluids, cells, or tissues present within a subject. In some embodiments, the sample is a body fluid. A body fluid is typically a liquid at physiological temperature, present in and collected from, exuded from, or extracted from a subject or biological resource, and may include naturally occurring fluids. Certain body fluids obtained from specific tissues, organs, or local regions and certain other body fluids may be more suitable overall or systemically in a subject or biological resource. Examples of body fluids include blood, serum and serous fluids, plasma, lymph, urine, saliva, cyst fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascitic fluid, e.g., related to non-solid tumors, pleural, pericardial, peritoneal, abdominal and other body cavity fluids, fluids collected by bronchial lavage, and the like. Body fluids may also include solutions that have contacted a subject or biological resource, e.g., cell and organ culture media including cell or organ conditioning media, wash fluids, and the like. As used herein, the term "sample" encompasses materials removed from or present within a subject. Related aspects of the invention can be practiced ex vivo or in vitro based on a sample isolated as needed.

[0096] The "known standard" may be a solution having a known amount or concentration of CD79b and / or CD20. This solution may be a naturally occurring solution, for example, a sample from a patient known to have early, intermediate, late, progressive, or static cancer, or this solution may be a synthetic solution such as a buffered aqueous solution having a known amount of CD79b and / or CD20 diluted therein. The known standard described herein may include CD79b and / or CD20 isolated from a subject, recombinant or purified CD79b and / or CD20 protein, or values of CD79b and / or CD20 concentrations related to a disease state.

[0097] The "cluster of differentiation antigen CD79B protein" or "CD79b" refers to Igβ, a B cell antigen receptor (BCR) signaling component. Amino acid sequences of various isoforms are retrievable from GenBank accession numbers AAH32651.1, EAW94232.1, AAH02975.2, NP_000617.1, and NP_001035022.1. The amino acid sequence of the full-length CD79b sequence is shown below. The sequence includes an extracellular domain (residues 29-159) and a cytoplasmic domain (residues 181-229). MARLALSPVPSHWMVALLLLLSAEPVPAARSEDRYRNPKGSACSRIWQSPRFIARKRGFTVKMHCYMNSASGNVSWLWKQEMDENPQQLKLEKGRMEESQNESLATLTIQGIRFEDNGIYFCQQKCNNTSEVYQGCGTELRVMGFSTLAQLKQRNTLKDGIIMIQTLLIILFIIVPIFLLLDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE (SEQ ID NO: 252)

[0098] "Cluster of Differentiation 20" or "CD20" refers to an antigenic determinant known to be detectable on B cells. Human CD20 is also called membrane-spanning 4-domains, subfamily A, member 1 (MS4A1). For example, the amino acid sequences of human and murine can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD20 can be found under accession numbers NP_690605.1 and NP_068769.2, and the nucleic acid sequences encoding transcript variants 1 and 3 of human CD20 can be found under accession numbers NM_152866.2 and NM_021950.3, respectively.

[0099] The term "CD3" means the human CD3 protein multisubunit complex. The CD3 protein multisubunit complex is composed of six distinguishable polypeptide chains. These include the CD3γ chain (SwissProt P09693), the CD3δ chain (SwissProt P04234), two CD3ε chains (SwissProt P07766), and a CD3ζ chain homodimer (SwissProt 20963), which associates with the T cell receptor α and β chains. The term "CD3" includes, unless otherwise specified, any CD3 variant, isoform, and interspecies homolog that can be expressed by cells (including T cells) natively or on cells transfected with the gene or cDNA encoding those polypeptides.

[0100] The "CD79b×CD20×CD3 antibody" is a multispecific antibody, optionally a trispecific antibody, which contains three different antigen-binding arms, one of which binds to the antigen CD79b, one of which binds to the antigen CD20, and one of which binds to CD3. The "CD79b×CD3 antibody" is a multispecific antibody, optionally a bispecific antibody, and the bispecific antibody contains two different antigen-binding arms, one of which binds to the antigen CD79b and one of which binds to CD3. The "CD20×CD3 antibody" is a multispecific antibody, optionally a bispecific antibody, and the bispecific antibody contains two different antigen-binding arms, one of which binds to the antigen CD20 and one of which binds to CD3. The term "multispecific antibody" is used herein in the broadest sense and specifically includes antibodies having polyepitope specificity. Multispecific antibodies include antibodies containing heavy chain variable domains (VH) and light chain variable domains (VL), where the VHVL unit is an antibody having polyepitope specificity, an antibody having two or more V L domains and V H domains, where each VHVL unit binds to a different epitope, an antibody having two or more single variable domains, where each single variable domain binds to a different epitope, a full-length antibody, and an antibody containing one or more antibody fragments, and an antibody containing covalently or non-covalently linked antibody fragments, but are not limited thereto.

[0101] Multispecific antibodies can be bispecific antibodies, trispecific antibodies, diabodies, or similar molecules (for an explanation of diabodies, see, for example, PNAS USA 90(14), 6444-8 (1993)). The bispecific antibodies, trispecific antibodies, diabodies, etc. provided herein can bind to any suitable target in addition to a portion of CD79b or CD20. The term "bispecific antibody" is to be understood as an antibody having two different antigen-binding arms defined by different antibody sequences. The term "trispecific antibody" is to be understood as an antibody having three different antigen-binding arms defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes within one target.

[0102] A "reference sample" is a sample that can be used to evaluate the characteristics of a sample being compared by comparing it to another sample, e.g., a test sample. The reference sample has some characteristic that has been evaluated and functions as a standard when compared to the test sample. For example, a reference sample can be used as a benchmark for CD79b or CD20 levels indicative of a subject having cancer. The reference sample does not necessarily have to be analyzed in parallel with the test sample, and thus in some cases, the reference sample can be a predetermined numerical value or range for characterizing a given condition, such as CD79b or CD20 levels that are an indicator of cancer in a subject. This term also includes samples used for comparison purposes in a physiological or pathological state, e.g., a state where CD79b or CD20 expression is known to be associated with cancer, but the amount of CD79b or CD20 is unknown.

[0103] "Recurrent" refers to the recurrence of a disease or the signs and symptoms of a disease after a period of improvement following pretreatment with a therapeutic agent.

[0104] "Refractory" refers to a disease that does not respond to treatment. A refractory disease can be resistant to treatment before or at the start of treatment, or a refractory disease can become a resistant disease during treatment.

[0105] When used in the context of the progression of CD79b and / or CD20-expressing cancer, the term "progression" includes the change of cancer from a less severe state to a more severe state. This can include an increase such as the number or severity of tumors, the degree of metastasis, and the rate at which the cancer grows or spreads. For example, "progression of colon cancer" can include the progression of such cancer from a less severe state to a more severe state, such as progression from stage I to stage II, from stage II to stage III, etc.

[0106] When used in the context of the regression of CD79b and / or CD20-expressing cancer, the term "regression" includes the change of cancer from a more severe state to a less severe state. It is considered that this can include a reduction such as the number or severity of tumors, the degree of metastasis, and the rate at which the cancer grows or spreads. For example, "regression of colon cancer" can include the regression of such cancer from a more severe state to a less severe state, such as recovery (progression) from stage III to stage II, from stage II to stage I, etc.

[0107] When used in the context of stable CD79b and / or CD20-expressing cancer, the term "stable" is intended to describe a pathological condition that has not significantly changed or has changed sufficiently over a clinically relevant period so as to be considered a progressive or regressive cancer.

[0108] The embodiments described herein are not limited to a particular method, reagent, compound, composition, or biological system and can, of course, be variously modified.

[0109] Bispecific antibody Provided herein are bispecific antibodies that bind to CD79b, CD20, and CD3, as well as bispecific binding fragments thereof. Such antibodies or antibody fragments may enable more specific targeting to a particular subset of cells as compared to antibodies that target only one or two of these targets.

[0110] Trispecific antibody In some embodiments, provided herein are trispecific antibodies and trispecific antigen-binding fragments thereof that bind to CD79b, CD20, and CD3. This can be achieved, for example, by creating a molecule that includes a first antigen-binding arm that binds to CD79b, a second antigen-binding arm that binds to CD3, and a third antigen-binding arm that binds to CD20. The antigen-binding arms can take any form that allows for specific recognition of the target. For example, the binding arm can be a heavy-chain variable domain, Fv (a combination of a heavy-chain variable domain and a light-chain variable domain), single-chain Fv (scFv), Fab, a binding domain based on a fibronectin type III domain (such as the Centyrin molecule of Janssen Biotech, Inc., derived from fibronectin or based on a consensus of fibronectin-derived type III domains, or derived from tenascin or based on a consensus of tenascin-derived type III domains; see, for example, International Publication Nos. 2010 / 051274 and 2010 / 093627), or may include these. In certain embodiments, the trispecific antibody includes three antigen-binding arms. In some embodiments, the trispecific antibody is composed of an antibody (e.g., in an IgG format) in which an additional antigen-binding arm, for example in the form of a single-chain variable fragment, is fused to the N-terminus or C-terminus of one of the heavy chains or one of the light chains of the antibody.

[0111] Accordingly, provided is a trispecific molecule that includes three different antigen-binding arms that bind to CD79b, CD20, and CD3, respectively.

[0112] In some embodiments, an isolated CD79b×CD20×CD3 multispecific antibody comprises (a) a first antigen-binding arm comprising a first heavy-chain variable domain (VH1) and a first light-chain variable domain (VL1), and (b) A second antigen-binding arm comprising a second heavy-chain variable domain (VH2) and a second light-chain variable domain (VL2), (c) A third antigen-binding arm comprising a third heavy-chain variable domain (VH3) and a third light-chain variable domain (VL3), and the first antigen-binding arm binds to an epitope on CD79b, the second antigen-binding arm binds to an epitope on CD3, and the third antigen-binding arm binds to an epitope on CD20.

[0113] In some embodiments, VH1 comprises an amino acid sequence selected from SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, and SEQ ID NO: 71.

[0114] In some embodiments, VL1 comprises an amino acid sequence selected from SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, and SEQ ID NO: 73.

[0115] In some aspects, VH2 comprises an amino acid sequence selected from SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 196, and SEQ ID NO: 206.

[0116] In some embodiments, VL2 comprises an amino acid sequence selected from SEQ ID NO: 99, SEQ ID NO: 109, SEQ ID NO: 201, and SEQ ID NO: 211.

[0117] In some embodiments, VH3 comprises an amino acid sequence selected from SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, and SEQ ID NO: 138.

[0118] In some embodiments, VL3 comprises an amino acid sequence selected from SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 136, and SEQ ID NO: 140.

[0119] In some embodiments, VH1 and VL1 of the antigen-binding arms that bind to the CD79b epitope are present in a diabody, Fab, Fab’, F(ab’)2, Fv, scFv, Fd, disulfide-stabilized Fv fragment (dsFv), or disulfide-stabilized diabody (ds diabody).

[0120] In some embodiments, VH2 and VL2 of the antigen-binding arms that bind to the CD3 epitope are present in a diabody, Fab, Fab’, F(ab’)2, Fv, scFv, Fd, disulfide-stabilized Fv fragment (dsFv), or disulfide-stabilized diabody (ds diabody).

[0121] In some embodiments, VH3 and VL3 of the antigen-binding arms that bind to the CD20 epitope are present in a diabody, Fab, Fab’, F(ab’)2, Fv, scFv, Fd, disulfide-stabilized Fv fragment (dsFv), or disulfide-stabilized diabody (ds diabody).

[0122] In some embodiments, the first antigen-binding arm of the CD79b×CD20×CD3 multispecific antibody comprises a first heavy-chain portion (HC1) containing VH1 and a light-chain portion (LC) containing VL1, and VH1 and VL1 pair to form a first antigen-binding domain that binds to a first antigen. In some embodiments, HC1 comprises, from the N-terminus to the C-terminus, VH1, a first heavy-chain constant domain (CH1), and a first Fc domain. In some embodiments, VH1 and CH1 of HC1, together with LC, form a fragment antigen-binding (Fab) domain.

[0123] In some embodiments, VH1 of the first antigen-binding arm is linked to VH3 of the third antigen-binding arm via the first Fc domain. In some embodiments, the first Fc domain of the first antigen-binding arm is linked to the third antigen-binding arm via a first linker (L1), thereby forming the linked first and third antigen-binding arms. The linked first and third antigen-binding arms may include, from the N-terminus to the C-terminus, VH1, CH1 domain, and Fc domain of the first antigen-binding arm, the first linker, and the third antigen-binding arm. In some embodiments, the third antigen-binding arm is a single-chain variable fragment (scFv) formed from VH3 and VL3 of the third antigen-binding arm.

[0124] In some embodiments, the second antigen-binding arm of the CD79b×CD20×CD3 multispecific antibody includes a second heavy chain portion (HC2) that includes a second heavy chain variable domain (VH2) that forms a second antigen-binding domain that binds to a second antigen. In some embodiments, the second binding arm includes, from the N-terminus to the C-terminus, a single-chain variable fragment (scFv) formed from VH2 and VL2, and a second Fc domain.

[0125] In some embodiments, VH2 of the second antigen-binding arm is linked to VH3 of the third antigen-binding arm via the second Fc domain. In some embodiments, the second Fc domain of the second antigen-binding arm is linked to the third antigen-binding arm via a linker, thereby forming the linked second and third antigen-binding arms. The linked second and third antigen-binding arms may include, from the N-terminus to the C-terminus, the second antigen-binding domain, the second Fc domain, the first linker, and the third antigen-binding arm. In some embodiments, the third antigen-binding arm is a single-chain variable fragment (scFv) formed from VH3 and VL3 of the third antigen-binding arm.

[0126] In a preferred embodiment, the CD79b×CD20×CD3 multispecific antibody is a trispecific antibody comprising a CD79b-specific binding arm that includes a first heavy chain portion (HC1) having VH1 and a light chain portion (LC) having VL1. VH1 and VL1 pair up to form a first antigen-binding domain that binds to CD79b. The second antigen-binding arm of the trispecific antibody includes a second heavy chain portion (HC2) having VH2 that forms a second antigen-binding domain that binds to a second antigen. HC1 of the CD79b-specific binding arm or HC2 of the second antigen-binding arm is linked to a third antigen-binding arm that includes a VH3 domain that forms a third antigen-binding domain that binds to a third antigen. In some embodiments, the second antigen is CD20 and the third antigen is CD3. In some embodiments, the second antigen is CD3 and the third antigen is CD20.

[0127] In one embodiment, the CD79b×CD20×CD3 multispecific antibody is a trispecific antibody comprising a CD79b-specific binding arm that includes HC1 having VH1 and LC having VL1. VH1 and VL1 pair up to form a first CD79b-specific antigen-binding domain that binds to CD79b. The second antigen-binding arm includes VH2 and VL2 that form a second antigen-binding domain that binds to CD3. The third antigen-binding arm is linked to the second antigen-binding arm and includes VH3 and VL3 that form a third antigen-binding domain that binds to CD20.

[0128] In one embodiment, the CD79b×CD20×CD3 multispecific antibody is a trispecific antibody comprising a CD79b-specific binding arm that includes HC1 having VH1 and LC having VL1. VH1 and VL1 pair up to form a first CD79b-specific antigen-binding domain that binds to CD79b. The second antigen-binding arm includes VH2 and VL2 that form a second antigen-binding domain that binds to CD20. The third antigen-binding arm is linked to the second antigen-binding arm and includes VH3 and VL3 that form a third antigen-binding domain that binds to CD3.

[0129] In one embodiment, the CD79b×CD20×CD3 multispecific antibody is a trispecific antibody comprising a CD79b-specific binding arm comprising HC1 having VH1 and LC having VL1. VH1 and VL1 pair to form a first CD79b-specific antigen-binding domain that binds to CD79b. The second antigen-binding arm comprises VH2 and VL2 that form a second antigen-binding domain that binds to CD3. The third antigen-binding arm is linked to the first CD79b-specific antigen-binding arm and comprises VH3 and VL3 that form a third antigen-binding domain that binds to CD20.

[0130] In one embodiment, the CD79b×CD20×CD3 multispecific antibody is a trispecific antibody comprising a CD79b-specific binding arm comprising HC1 having VH1 and LC having VL1. VH1 and VL1 pair to form a first CD79b-specific antigen-binding domain that binds to CD79b. The second antigen-binding arm comprises VH2 and VL2 that form a second antigen-binding domain that binds to CD20. The third antigen-binding arm is linked to the first CD79b-specific antigen-binding arm and comprises VH3 and VL3 that form a third antigen-binding domain that binds to CD3.

[0131] In some embodiments, HC1 having VH1 and LC having VL1 of the first antigen-binding arm form an antigen-binding fragment (Fab) comprising the first antigen-binding domain. In some embodiments, VH2 and VL2 of the second antigen-binding arm form a single-chain variable fragment (scFv) comprising the second antigen-binding domain. In some embodiments, VH3 and VL3 of the third antigen-binding arm form a single-chain variable fragment (scFv) comprising the third antigen-binding domain.

[0132] In one embodiment, the CD79b-binding arm comprises an antigen-binding fragment (Fab), the CD3-binding arm comprises a single-chain variable fragment (scFv), and the CD20-binding arm comprises a single-chain variable fragment (scFv).

[0133] In some embodiments, the CD79b-binding arm of the trispecific antibody comprises HC1 and LC. HC1 can include a constant heavy chain region (CH1, CH2, and CH3) and VH1. LC can include VL1. VH1 and VL1 bind to form a CD79b antigen-binding domain.

[0134] In some embodiments, the CD3-binding arm of the trispecific antibody comprises HC2. HC2 can include a constant heavy chain region (CH2 and CH3) and a single-chain variable fragment (scFv) attached to the N-terminus of the CH2 region, and the scFv includes a CD3 antigen-binding domain.

[0135] In some embodiments, the trispecific antibody further comprises a CD20 antigen-binding arm that binds to the C-terminus of the CH3 region of the CD3-binding arm to form a CD3 / CD20-binding arm. In some embodiments, the CD20 antigen-binding arm comprises a second single-chain variable fragment (scFv). In some embodiments, the CD3 / CD20 arm can have a structure of an scFV containing a CD3-binding domain, CH2 and CH3 regions, and an scFv containing a CD20-binding domain.

[0136] Bispecific antibody In some embodiments, provided herein are bispecific antibodies that bind to CD79b and CD3 and bispecific antigen-binding fragments thereof. This can be achieved, for example, by creating a molecule that includes a first antigen-binding arm that binds to CD79b and a second antigen-binding arm that binds to CD3. The antigen-binding arms can take any form that allows for specific recognition of the target. For example, the binding region can be a heavy chain variable domain, Fv (a combination of a heavy chain variable domain and a light chain variable domain), single-chain Fv (scFv), Fab, a binding domain based on a fibronectin type III domain (from fibronectin or consensus of type III domains from fibronectin, or from tenascin or consensus of type III domains from tenascin, e.g., Centyrin molecules from Janssen Biotech, Inc., see, e.g., International Publication Nos. 2010 / 051274 and 2010 / 093627), or can include these. Thus, provided are bispecific molecules that include three different antigen-binding arms that bind to CD79b and CD3, respectively.

[0137] In some embodiments, the CD79b×CD3 bispecific antibody includes (a) a first antigen-binding arm that includes a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), and (b) a second antigen-binding arm that includes a first heavy chain variable domain (VH2) and a first light chain variable domain (VL2), wherein the first antigen-binding arm binds to an epitope on CD79b and the second antigen-binding arm binds to an epitope on CD3.

[0138] In some embodiments, VH1 and VL1 of the antigen-binding arm that binds to the CD79b epitope are present in a diabody, Fab, Fab’, F(ab’)2, Fv, scFv, Fd, disulfide-stabilized Fv fragment (dsFv), or disulfide-stabilized diabody (ds diabody).

[0139] In some embodiments, VH2 and VL2 of the antigen-binding arms that bind to the CD3 epitope are present in a diabody, Fab, Fab’, F(ab’)2, Fv, scFv, Fd, disulfide-stabilized Fv fragment (dsFv), or disulfide-stabilized diabody (ds diabody).

[0140] In some embodiments, the first antigen-binding arm comprises a first heavy-chain portion (HC1) comprising VH1 and a light chain (LC) comprising VL1. In some embodiments, HC1 comprises, from the N-terminus to the C-terminus, VH1, a first heavy-chain constant domain (CH1), and a first Fc domain. In some embodiments, VH1 and CH1 of HC1, together with LC, form a fragment antigen-binding (Fab) domain.

[0141] In some embodiments, the second binding arm comprises, from the N-terminus to the C-terminus, a single-chain variable fragment (scFv) formed from VH2 and VL2, and a second Fc domain.

[0142] In some embodiments, the first binding arm of the CD79b×CD3 bispecific antibody comprises HC1 comprising VH1 and LC comprising VL1. VH1 and VL1 pair to form a first antigen-binding domain that binds to the first antigen of the bispecific antibody. The second antigen-binding arm of the bispecific antibody comprises a second heavy-chain portion (HC2) having VH2 that forms a second antigen-binding domain that specifically binds to the second antigen. HC1 and HC2 each comprise a fragment crystallizable (Fc) domain comprising a CH2-CH3 domain.

[0143] In one embodiment, the CD79b×CD3 bispecific antibody comprises a CD79b-specific binding arm comprising HC1 having VH1 and LC having VL1. The VH1 and VL1 domains pair to form a first antigen-binding domain that binds to CD79b. The second antigen-binding arm of the bispecific antibody comprises HC2 having VH2 that forms a second antigen-binding domain that binds to CD3.

[0144] In one embodiment, the CD79b×CD3 bispecific antibody comprises a CD3-specific binding arm comprising HC1 having VH1 and LC having VL1. The VH1 and VL1 domains pair to form a first antigen-binding domain that binds to CD3. The second antigen-binding arm of the bispecific antibody comprises HC2 having VH2, which forms a second antigen-binding domain that binds to CD79b.

[0145] In some embodiments, HC1 having VH1 and LC having VL1 form an antigen-binding fragment (Fab) comprising the first antigen-binding domain. In some embodiments, HC2 having VH2 forms a single-chain variable fragment (scFv) comprising the second antigen-binding domain together with VL2.

[0146] In one embodiment, the CD79b-binding arm comprises an antigen-binding fragment (Fab), and the CD3-binding arm comprises a single-chain variable fragment (scFv).

[0147] In one embodiment, the CD3-binding arm comprises an antigen-binding fragment (Fab), and the CD79b-binding arm comprises a single-chain variable fragment (scFv).

[0148] In some embodiments, the multispecific antibodies (e.g., trispecific antibodies, bispecific antibodies) of the present invention include antibodies having a full-length antibody structure. As used herein, "full-length antibody" means an antibody having two full-length antibody heavy chains and two full-length antibody light chains. A full-length antibody heavy chain (HC) includes a heavy chain variable domain and constant domains, VH, CH1, CH2, and CH3. A full-length antibody light chain (LC) includes a light chain variable domain and constant domains, VL and CL. The full-length antibody may lack the C-terminal lysine (K) in either one or both of the heavy chains. The term "Fab arm" or "half molecule" means a pair of heavy chain-light chains that specifically binds to an antigen. In some embodiments, one of the antigen-binding domains is a non-antibody-based binding domain, e.g., a fibronectin type III domain, e.g., a binding domain based on Centyrin.

[0149] CD79b binding arm The multispecific antibodies (e.g., trispecific or bispecific antibodies) described herein include antigen-binding arms specific for CD79b. In some embodiments, the CD79b binding arm binds to CD79b. In some embodiments, the CD79b binding arm binds to human CD79b and cynomolgus monkey CD79b. In some embodiments, the CD79b binding arm binds to human CD79b but not to cynomolgus monkey CD79b. In some embodiments, the CD79b binding arm binds to an epitope comprising one or more residues from the extracellular domain (ECD) of CD79b. In some embodiments, the CD79b binding arm binds to one or more residues of a polypeptide having the amino acid sequence of SEQ ID NO: 252. In some embodiments, the CD79b binding arm binds to residues 30-42 (SEDRYRNPKGSAC, SEQ ID NO: 253), residues 50-52 (PRF), residues 81-86 (EMENP, SEQ ID NO: 254), and / or residues 144-148 (GFSTL, SEQ ID NO: 255) of human CD79b. Such CD79b binding arms can bind to CD79b with an affinity of 5×10 -7 M or less, e.g., 1×10 -7 M or less, 5×10 -8 M or less, 1×10 -8 M or less, 5×10 -9 M or less, 1×10 -9 M, or 5×10 -10 M or less. In one embodiment, the CD79b binding arm binds to CD79b with an affinity of about 1×10 -11 M to 1×10 -9 M. In one embodiment, the CD79b binding arm binds to CD79b with an affinity of about 1×10 -11 M, about 2×10 -11 M, about 3×10 -11 M, about 4×10 -11 M, about 5×10 -11 M, about 6×10 -11 M, about 7×10 -11 M, about 8×10 -11 M, about 9×10 -11M, approximately 1×10 -10 M, approximately 2×10 -10 M, approximately 3×10 -10 M, approximately 4×10 -10 M, approximately 5×10 -10 M, approximately 6×10 -10 M, approximately 7×10 -10 M, approximately 8×10 -10 M, approximately 9×10 -10 M, approximately 1×10 -9 binds to CD79b with an affinity for M.

[0150] Tables 1a and 1b provide an overview of the CDRs (defined by AbM) and VH and VL sequences of several exemplary CD79b-specific antibodies described herein.

[0151] [Table 1]

[0152] [Table 2]

[0153] In some embodiments, the CD79b binding arm comprises a heavy chain variable domain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 1a. In some embodiments, the CD79b binding arm comprises a light chain variable region comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 1a. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 1a and a light chain variable region comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 1a. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 1a and a light chain variable region comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 1a, and an antibody or antigen binding that competes to obtain binding to CD79b.

[0154] In some embodiments, the CD79b binding arm comprises the heavy chain variable domain of any one of the antibodies described in Table 1b. In some embodiments, the CD79b binding arm comprises the light chain variable region of any one of the antibodies described in 1b. In some embodiments, the CD79b binding arm comprises the heavy chain variable domain of any one of the antibodies described in Table 1b and the light chain variable region of any one of the antibodies described in Table 1b. In some embodiments, the CD79b binding arm competes with an antibody or antigen binding comprising the heavy chain variable domain of any one of the antibodies described in Table 1b and the light chain variable region of any one of the antibodies described in Table 1b for binding to CD79b.

[0155] In some embodiments, the CD79b binding arm comprises a heavy chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 1a. In some embodiments, the CD79b binding arm comprises a light chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 1a. In some embodiments, the CD79b binding arm comprises a heavy chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 1a and a light chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 1a.

[0156] In some embodiments, the CD79b binding arm comprises a heavy chain comprising the heavy chain variable domain of any one of the antibodies described in Table 1b. In some embodiments, the CD79b binding arm comprises a light chain comprising the light chain variable domain of any one of the antibodies described in Table 1b. In some embodiments, the CD79b binding arm comprises a heavy chain comprising the heavy chain variable domain of any one of the antibodies described in Table 1b and a light chain comprising the light chain variable domain of any one of the antibodies described in Table 1b.

[0157] In some embodiments, the CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 1, a heavy chain CDR2 comprising SEQ ID NO: 2, and a heavy chain CDR3 comprising SEQ ID NO: 3. In some embodiments, the CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 1, a heavy chain CDR2 comprising SEQ ID NO: 2, a heavy chain CDR3 comprising SEQ ID NO: 3, a light chain CDR1 comprising SEQ ID NO: 4, a light chain CDR2 comprising SEQ ID NO: 5, and a light chain CDR3 comprising SEQ ID NO: 6. The CD79b binding arm may comprise a human framework sequence. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 35. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 35 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 37. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 36. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 36 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 38 or 213.

[0158] In some embodiments, the CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 13, a heavy chain CDR2 comprising SEQ ID NO: 8, and a heavy chain CDR3 comprising SEQ ID NO: 9. In some embodiments, the CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 13, a heavy chain CDR2 comprising SEQ ID NO: 8, a heavy chain CDR3 comprising SEQ ID NO: 9, a light chain CDR1 comprising SEQ ID NO: 10, a light chain CDR2 comprising SEQ ID NO: 11, and a light chain CDR3 comprising SEQ ID NO: 12. The CD79b binding arm may comprise a human framework sequence. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 39. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 39 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 41. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 40. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 40 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 42.

[0159] In some embodiments, the CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 7, a heavy chain CDR2 comprising SEQ ID NO: 8, and a heavy chain CDR3 comprising SEQ ID NO: 9. In some embodiments, the CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 7, a heavy chain CDR2 comprising SEQ ID NO: 8, a heavy chain CDR3 comprising SEQ ID NO: 9, a light chain CDR1 comprising SEQ ID NO: 10, a light chain CDR2 comprising SEQ ID NO: 11, and a light chain CDR3 comprising SEQ ID NO: 12. The CD79b binding arm may comprise a human framework sequence. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 43. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 43 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 41. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 44. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 44 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 34.

[0160] In some embodiments, the CD79b-binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 14, a heavy chain CDR2 comprising SEQ ID NO: 15, and a heavy chain CDR3 comprising SEQ ID NO: 16. In some embodiments, the CD79b-binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 14, a heavy chain CDR2 comprising SEQ ID NO: 15, a heavy chain CDR3 comprising SEQ ID NO: 16, a light chain CDR1 comprising SEQ ID NO: 17, a light chain CDR2 comprising SEQ ID NO: 5, and a light chain CDR3 comprising SEQ ID NO: 6. The CD79b-binding arm may comprise a human framework sequence. In some embodiments, the CD79b-binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 45. In some embodiments, the CD79b-binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 45 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 47. In some embodiments, the CD79b-binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 46. In some embodiments, the CD79b-binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 46 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 48 or 214.

[0161] In some embodiments, the CD79b-binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 18, a heavy chain CDR2 comprising SEQ ID NO: 8, and a heavy chain CDR3 comprising SEQ ID NO: 19. In some embodiments, the CD79b-binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 18, a heavy chain CDR2 comprising SEQ ID NO: 8, a heavy chain CDR3 comprising SEQ ID NO: 19, a light chain CDR1 comprising SEQ ID NO: 20, a light chain CDR2 comprising SEQ ID NO: 21, and a light chain CDR3 comprising SEQ ID NO: 12. The CD79b-binding arm may comprise a human framework sequence. In some embodiments, the CD79b-binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 49. In some embodiments, the CD79b-binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 49 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 51. In some embodiments, the CD79b-binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 50. In some embodiments, the CD79b-binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 50 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 52.

[0162] In some embodiments, the CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 13, a heavy chain CDR2 comprising SEQ ID NO: 8, and a heavy chain CDR3 comprising SEQ ID NO: 9. In some embodiments, the CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 13, a heavy chain CDR2 comprising SEQ ID NO: 8, a heavy chain CDR3 comprising SEQ ID NO: 9, a light chain CDR1 comprising SEQ ID NO: 10, a light chain CDR2 comprising SEQ ID NO: 11, and a light chain CDR3 comprising SEQ ID NO: 12. The CD79b binding arm may comprise a human framework sequence. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 39. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 39 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 53. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 40. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 40 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 54.

[0163] In some embodiments, a CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 13, a heavy chain CDR2 comprising SEQ ID NO: 8, and a heavy chain CDR3 comprising SEQ ID NO: 9. In some embodiments, a CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 13, a heavy chain CDR2 comprising SEQ ID NO: 8, a heavy chain CDR3 comprising SEQ ID NO: 9, a light chain CDR1 comprising SEQ ID NO: 10, a light chain CDR2 comprising SEQ ID NO: 11, and a light chain CDR3 comprising SEQ ID NO: 12. A CD79b binding arm may comprise human framework sequences. In some embodiments, a CD79b binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 55. In some embodiments, a CD79b binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 55 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 57. In some embodiments, a CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence substantially the same as or identical to SEQ ID NO: 56. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence substantially the same as or identical to SEQ ID NO:56 and a light chain variable domain encoded by a nucleotide sequence substantially the same as or identical to SEQ ID NO:58.

[0164] In some embodiments, the CD79b-binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 22, a heavy chain CDR2 comprising SEQ ID NO: 23, and a heavy chain CDR3 comprising SEQ ID NO: 24. In some embodiments, the CD79b-binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 22, a heavy chain CDR2 comprising SEQ ID NO: 23, a heavy chain CDR3 comprising SEQ ID NO: 24, a light chain CDR1 comprising SEQ ID NO: 25, a light chain CDR2 comprising SEQ ID NO: 5, and a light chain CDR3 comprising SEQ ID NO: 6. The CD79b-binding arm may comprise a human framework sequence. In some embodiments, the CD79b-binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 59. In some embodiments, the CD79b-binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 59 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 61. In some embodiments, the CD79b-binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 60. In some embodiments, the CD79b-binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 60 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 62.

[0165] In some embodiments, the CD79b-binding arm comprises a heavy-chain CDR1 comprising SEQ ID NO: 22, a heavy-chain CDR2 comprising SEQ ID NO: 26, and a heavy-chain CDR3 comprising SEQ ID NO: 27. In some embodiments, the CD79b-binding arm comprises a heavy-chain CDR1 comprising SEQ ID NO: 22, a heavy-chain CDR2 comprising SEQ ID NO: 26, a heavy-chain CDR3 comprising SEQ ID NO: 27, a light-chain CDR1 comprising SEQ ID NO: 28, a light-chain CDR2 comprising SEQ ID NO: 5, and a light-chain CDR3 comprising SEQ ID NO: 29. The CD79b-binding arm may comprise a human framework sequence. In some embodiments, the CD79b-binding arm comprises a heavy-chain variable domain that is substantially the same as or identical to SEQ ID NO: 63. In some embodiments, the CD79b-binding arm comprises a heavy-chain variable domain that is substantially the same as or identical to SEQ ID NO: 63 and a light-chain variable domain that is substantially the same as or identical to SEQ ID NO: 65. In some embodiments, the CD79b-binding arm comprises a heavy-chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 64. In some embodiments, the CD79b-binding arm comprises a heavy-chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 64 and a light-chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 66.

[0166] In some embodiments, the CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 14, a heavy chain CDR2 comprising SEQ ID NO: 15, and a heavy chain CDR3 comprising SEQ ID NO: 16. In some embodiments, the CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 14, a heavy chain CDR2 comprising SEQ ID NO: 15, a heavy chain CDR3 comprising SEQ ID NO: 16, a light chain CDR1 comprising SEQ ID NO: 17, a light chain CDR2 comprising SEQ ID NO: 5, and a light chain CDR3 comprising SEQ ID NO: 6. The CD79b binding arm may comprise a human framework sequence. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 67. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 67 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 69. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 68. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 68 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 70.

[0167] In some embodiments, the CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 30, a heavy chain CDR2 comprising SEQ ID NO: 31, and a heavy chain CDR3 comprising SEQ ID NO: 32. In some embodiments, the CD79b binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 30, a heavy chain CDR2 comprising SEQ ID NO: 31, a heavy chain CDR3 comprising SEQ ID NO: 32, a light chain CDR1 comprising SEQ ID NO: 33, a light chain CDR2 comprising SEQ ID NO: 5, and a light chain CDR3 comprising SEQ ID NO: 6. The CD79b binding arm may comprise a human framework sequence. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 71. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 71 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 73. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 72. In some embodiments, the CD79b binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 72 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 74.

[0168] The CD79b binding arm can be derived from any species by recombinant techniques. For example, the CD79b binding arm can be derived from mouse, rat, goat, horse, pig, cow, chicken, rabbit, camel, donkey, human, or chimeric versions thereof. For use in administration to humans, antigen-binding fragments derived from non-human sources may be genetically or structurally altered to be less antigenic when administered to a human patient. In some embodiments, the CD79b binding arm comprises an antigen-binding fragment that is chimeric.

[0169] In some embodiments, the CD79b-binding arm comprises a humanized antigen-binding fragment. The humanized antigen-binding fragment may be a chimeric immunoglobulin, an immunoglobulin chain thereof, or a fragment (e.g., Fv, Fab, Fab’, F(ab’)2, or other antigen-binding subsequence of an antibody) that includes minimal sequences derived from a non-human immunoglobulin. In most cases, a humanized antibody or antigen-binding fragment is a human immunoglobulin (recipient antibody) or antigen-binding fragment in which residues from the recipient's complementarity-determining regions (CDRs) have been replaced by residues from the CDRs of a non-human species, such as a mouse, rat, or rabbit (donor antibody) that have the desired specificity, affinity, and capacity. Generally, a humanized antibody or antigen-binding fragment will comprise substantially all of at least one, typically two, variable domains. Within the variable domains, all or substantially all of the CDR regions correspond to the CDR regions of the non-human immunoglobulin, and all or substantially all of the framework regions are framework regions of human immunoglobulin sequences. The humanized antibody antigen-binding fragment may include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0170] CD20-binding arm The multispecific antibodies described herein (e.g., trispecific or bispecific antibodies) may include an antigen-binding arm specific for CD20. In some embodiments, the CD20-binding arm binds to CD20. In some embodiments, the CD20-binding arm binds to human CD20 and cynomolgus CD20, preferably to its extracellular domain. In some embodiments, the CD20-binding arm binds to human CD20 but not to cynomolgus CD20. In some embodiments, the CD20-binding arm binds to an epitope on CD20 as rituximab. In some embodiments, the CD20-binding arm is 5×10 -7 M or less, e.g., 1×10 -7 M or less, 5×10 -8 M or less, 1×10 -8 M or less, 5×10 -9 M or less, or 1×10 -9It can bind to CD20 with an affinity of less than 1 μM. In one embodiment, the CD20-binding arm has an affinity of about 1×10 -9 μM, about 2×10 -9 μM, about 3×10 -9 μM, about 4×10 -9 μM, about 5×10 -9 μM, about 6×10 -9 μM, about 7×10 -9 μM, about 8×10 -9 μM, about 9×10 -9 μM, about 1×10 -8 μM, about 2×10 -8 μM, about 3×10 -8 μM, about 4×10 -8 μM, about 5×10 -8 μM, about 6×10 -8 μM, about 7×10 -8 μM, about 8×10 -8 μM, about 9×10 -8 or about 1×10 -7 μM and binds to CD20.

[0171] In some embodiments, the CD20-binding arm comprises heavy chain CDR1, CDR2, and CDR3 derived from the antibody clones described in Table 2a. In some embodiments, the CD20-binding arm comprises light chain CDR1, CDR2, and CDR3 derived from the antibody clones described in Table 2a. In some embodiments, the CD20-binding arm comprises heavy chain CDR​1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 derived from the antibody clones described in Table 2a. In some exemplary embodiments, the CD20-binding arm comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of clone C20B648.

[0172] In some exemplary embodiments, the CD20-binding arm comprises the heavy chain variable domain derived from the antibody clones described in Table 2b. In some exemplary embodiments, the CD20-binding arm comprises the heavy chain variable domain obtained from the antibody clones as described in Table 2b and the light chain variable domain.

[0173] Tables 2a and 2b provide an overview of the CDRs and VH and VL sequences of some exemplary CD20 - specific antibodies described herein.

[0174] [Table 3]

[0175] [Table 4]

[0176] In some embodiments, the CD20 - binding arm comprises a heavy - chain variable domain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 2a. In some embodiments, the CD20 - binding arm comprises a light - chain variable domain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 2a. In some embodiments, the CD20 - binding arm comprises a heavy - chain variable domain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 2a and a light - chain variable domain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 2a. In some embodiments, the CD20 - binding arm comprises an antibody or antigen - binding that comprises a heavy chain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 2a and a light chain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 2a and competes to obtain binding to CD20.

[0177] In some embodiments, the CD20-binding arm comprises the heavy chain variable domain of any one of the antibodies described in Table 2b. In some embodiments, the CD20-binding arm comprises the light chain variable region of any one of the antibodies described in 2b. In some embodiments, the CD20-binding arm comprises the heavy chain variable domain of any one of the antibodies described in Table 2b and the light chain variable region of any one of the antibodies described in Table 2b. In some embodiments, the CD20-binding arm competes with an antibody or antigen binding comprising the heavy chain variable domain of any one of the antibodies described in Table 2b and the light chain variable region of any one of the antibodies described in Table 2b for binding to CD20.

[0178] In some embodiments, the CD20-binding arm comprises a heavy chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 2a. In some embodiments, the CD20-binding arm comprises a light chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 2a. In some embodiments, the CD20-binding arm comprises a heavy chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 2 and a light chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 2a.

[0179] In some embodiments, the CD20-binding arm comprises a heavy chain comprising the heavy chain variable domain of any one of the antibodies described in Table 2b. In some embodiments, the CD20-binding arm comprises a light chain comprising the light chain variable domain of any one of the antibodies described in Table 2b. In some embodiments, the CD20-binding arm comprises a heavy chain comprising the heavy chain variable domain of any one of the antibodies described in Table 2b and a light chain comprising the light chain variable domain of any one of the antibodies described in Table 2b.

[0180] In some embodiments, the CD20-binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 115, a heavy chain CDR2 comprising SEQ ID NO: 116, and a heavy chain CDR3 comprising SEQ ID NO: 117. In some embodiments, the CD20-binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 115, a heavy chain CDR2 comprising SEQ ID NO: 116, a heavy chain CDR3 comprising SEQ ID NO: 117, a light chain CDR1 comprising SEQ ID NO: 118, a light chain CDR2 comprising SEQ ID NO: 119, and a light chain CDR3 comprising SEQ ID NO: 120. The CD20-binding arm may comprise a human framework sequence. In some embodiments, the CD20-binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 126. In some embodiments, the CD20-binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 126 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 128. In some embodiments, the CD20-binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 127. In some embodiments, the CD20-binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 127 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 129.

[0181] In some embodiments, the CD20-binding arm comprises a heavy-chain CDR1 comprising SEQ ID NO: 121, a heavy-chain CDR2 comprising SEQ ID NO: 122, and a heavy-chain CDR3 comprising SEQ ID NO: 123. In some embodiments, the CD20-binding arm comprises a heavy-chain CDR1 comprising SEQ ID NO: 121, a heavy-chain CDR2 comprising SEQ ID NO: 122, a heavy-chain CDR3 comprising SEQ ID NO: 123, a light-chain CDR1 comprising SEQ ID NO: 124, a light-chain CDR2 comprising SEQ ID NO: 119, and a light-chain CDR3 comprising SEQ ID NO: 125. The CD20-binding arm may comprise a human framework sequence. In some embodiments, the CD20-binding arm comprises a heavy-chain variable domain that is substantially the same as or identical to SEQ ID NO: 130. In some embodiments, the CD20-binding arm comprises a heavy-chain variable domain that is substantially the same as or identical to SEQ ID NO: 130 and a light-chain variable domain that is substantially the same as or identical to SEQ ID NO: 132. In some embodiments, the CD20-binding arm comprises a heavy-chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 131. In some embodiments, the CD20-binding arm comprises a heavy-chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 131 and a light-chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 133.

[0182] In some embodiments, the CD20 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 115, a heavy chain CDR2 comprising SEQ ID NO: 116, and a heavy chain CDR3 comprising SEQ ID NO: 95. In some embodiments, the CD20 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 115, a heavy chain CDR2 comprising SEQ ID NO: 116, a heavy chain CDR3 comprising SEQ ID NO: 95, a light chain CDR1 comprising SEQ ID NO: 96, a light chain CDR2 comprising SEQ ID NO: 119, and a light chain CDR3 comprising SEQ ID NO: 125. The CD20 binding arm may comprise a human framework sequence. In some embodiments, the CD20 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 134. In some embodiments, the CD20 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 134 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 136. In some embodiments, the CD20 binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 135. In some embodiments, the CD20 binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 135 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 137.

[0183] In some embodiments, the CD20 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 121, a heavy chain CDR2 comprising SEQ ID NO: 116, and a heavy chain CDR3 comprising SEQ ID NO: 123. In some embodiments, the CD20 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 121, a heavy chain CDR2 comprising SEQ ID NO: 116, a heavy chain CDR3 comprising SEQ ID NO: 123, a light chain CDR1 comprising SEQ ID NO: 124, a light chain CDR2 comprising SEQ ID NO: 119, and a light chain CDR3 comprising SEQ ID NO: 125. The CD20 binding arm may comprise a human framework sequence. In some embodiments, the CD20 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 138. In some embodiments, the CD20 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 138 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 140. In some embodiments, the CD20 binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 139. In some embodiments, the CD20 binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 139 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 141.

[0184] The CD20 binding arm can be derived from any species by recombinant techniques. For example, the CD20 binding arm can be derived from mouse, rat, goat, horse, pig, cow, chicken, rabbit, camel, donkey, human, or chimeric versions thereof. For use in administration to humans, antigen-binding fragments derived from non-humans may be genetically or structurally altered to be less antigenic when administered to a human patient. In some embodiments, the CD20 binding arm comprises an antigen-binding fragment that is chimeric.

[0185] In some embodiments, the CD20 binding arm comprises a humanized antigen-binding fragment. The humanized antigen-binding fragment may be a chimeric immunoglobulin, an immunoglobulin chain thereof, or a fragment (e.g., Fv, Fab, Fab’, F(ab’)2, or other antigen-binding subsequence of an antibody) that includes minimal sequences derived from a non-human immunoglobulin. In most cases, the humanized antibody or antigen-binding fragment is a human immunoglobulin (recipient antibody) or antigen-binding fragment in which residues from the recipient's complementarity determining regions (CDRs) have been replaced by residues from the CDRs of a non-human species, such as a mouse, rat, or rabbit (donor antibody) that have the desired specificity, affinity, and potency. Generally, the humanized antibody or antigen-binding fragment will include substantially all of at least one, typically two variable domains. Within the variable domains, all or substantially all of the CDR regions correspond to the CDR regions of the non-human immunoglobulin, and all or substantially all of the framework regions are framework regions of the human immunoglobulin sequence. The humanized antibody antigen-binding fragment may include at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0186] CD3 binding arm The multispecific antibodies described herein (e.g., trispecific or bispecific antibodies) include an antigen-binding arm that binds to CD3. In some preferred embodiments, the CD3-specific arm of the multispecific antibodies described herein is derived from a CD3-specific antibody that binds to and activates human primary T cells and / or cynomolgus primary T cells. In some embodiments, the CD3 binding arm binds to an epitope at the N-terminus of CD3ε. In some embodiments, the CD3 binding arm binds to residues 54-58 (GSEIL, SEQ ID NO: 257), residues 74-75 (NI), and / or residues 100-105 (PRGSKP, SEQ ID NO: 258) of human CD3ε. In some embodiments, the CD3 binding arm binds to residues 22-35 (QDGNEEMGGITQTP (SEQ ID NO: 256)) of the CD3ε chain. Such CD3 binding arms are at 5×10 -7 M or less, e.g., 1×10-7 Less than M, 5×10 -8 Less than M, 1×10 -8 Less than M, 5×10 -9 Less than M, or 1×10 -9 It can bind to CD3 with an affinity of less than M. In some embodiments, the CD3-binding arm is about 1×10 -8 M, about 2×10 -8 M, about 3×10 -8 M, about 4×10 -8 M, about 5×10 -8 M, about 6×10 -8 M, about 7×10 -8 M, about 8×10 -8 M, about 9×10 -8 M, or about 1×10 -8 M and binds to CD3.

[0187] In some embodiments, such CD3-binding arms can have weak binding to CD3 (e.g., low micromolar or weaker). Weak CD3 binding can result in better T cell compatibility, less T cell exhaustion, a lower risk of cytokine release syndrome (CRS), better safety, and / or enable combinations of co-stimulation to enhance T cell persistence.

[0188] Human CD3ε is described in UniProt P07766 (CD3E_HUMAN). The anti-CD3ε antibody reported in the art is SP34 (Yang SJ, The Journal of Immunology (1986) 137; 1097-1100). SP34 reacts with CD3 of both primates and humans. SP34 is available from Pharmingen. A further anti-CD3 antibody described in the prior art is UCHT-1 (see WO 2000041474). A further anti-CD3 antibody described in the prior art is BC-3 (Fred Hutchinson Cancer Research Institute; used in Phase I / II trials of GvHD, Anasetti et al., Transplantation 54:844 (1992)). SP34 is different from UCHT-1 and BC-3 in that SP-34 recognizes an epitope that exists only on the ε chain of CD3 (Salmeron et al., (1991) J. Immunol. 147:3047), while UCHT-1 and BC-3 recognize an epitope contributed by both the ε chain and the γ chain. The sequences of antibodies having the same sequence as that of antibody SP34 are referred to in WO 2008119565, WO 2008119566, WO 2008119567, WO 2010037836, WO 2010037837, and WO 2010037838. A sequence having 96% identity to the VH of antibody SP34 is referred to in US Pat. No. 8,236,308 (WO 2007042261).

[0189] In some embodiments, the CD3 binding arm contacts an epitope comprising the six N-terminal amino acids of CD3ε. In some embodiments, the CD3-specific binding arm of the multispecific antibody is obtained from the mouse monoclonal antibody SP34, mouse IgG3 / lambda isotype. In some embodiments, the CD3 binding arm comprises the CDRs of antibody SP34. Such a CD3 binding arm is 5×10 -7 M or less, for example, 1×10 -7 M or less, 5×10 -8Less than M, 1×10 -8 Less than M, 5×10 -9 Less than M, or 1×10 -9 It can bind to CD3 with an affinity of less than M. The CD3-specific binding arm may be the arm of a humanized version of the mouse monoclonal antibody SP34. An anti-CD3 antibody from which the CD3-specific arm is obtained may be humanized using Human framework adaptation (HFA).

[0190] In some embodiments, the CD3-binding arm comprises the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 3. In some embodiments, the CD3-binding arm comprises the light chain CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 3. In some embodiments, the CD3-binding arm comprises a heavy chain comprising the CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 3 and a light chain comprising the CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 3. In some embodiments of the multispecific antibody, the CD3-binding arm comprises a pair of heavy and light chains selected from Table 3.

[0191] Table 3 provides an overview of examples of some CD3-specific antibodies described herein.

[0192]

Table 5-1

[0193]

Table 5-2

[0194] The characteristics of some CD3-specific antibodies or antigen-binding fragments can be found, for example, in U.S. Patent Nos. 10,562,968 and 10,072,088, and U.S. Patent Application Publication No. 2019 / 0382481, the contents of each of which are hereby incorporated by reference in their entirety.

[0195] In some embodiments, the CD3-binding arm comprises a heavy-chain variable domain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 3. In some embodiments, the CD3-binding arm comprises a light-chain variable domain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 3. In some embodiments, the CD3-binding arm comprises a heavy-chain variable domain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 3 and a light-chain variable domain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 3. In some embodiments, the CD3-binding arm comprises an antibody or antigen-binding that comprises a heavy chain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 3 and a light chain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 3, and competes to obtain binding to CD3.

[0196] In some embodiments, the CD3-binding arm comprises a heavy chain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 3. In some embodiments, the CD3-binding arm comprises a light chain comprising any one of the CDR1, CDR2, and CDR3 of the antibodies described in Table 3. In some embodiments, the CD3-binding arm comprises a heavy chain comprising any one of the CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 3 and a light chain comprising any one of the CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 3.

[0197] In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 76, a heavy chain CDR2 comprising SEQ ID NO: 77, and a heavy chain CDR3 comprising SEQ ID NO: 78. In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 76, a heavy chain CDR2 comprising SEQ ID NO: 77, a heavy chain CDR3 comprising SEQ ID NO: 78, a light chain CDR1 comprising SEQ ID NO: 79, a light chain CDR2 comprising SEQ ID NO: 80, and a light chain CDR3 comprising SEQ ID NO: 81. The CD3 binding arm may comprise a human framework sequence. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 97. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 97 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 99. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 98. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 98 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 100.

[0198] In some embodiments, the CD3-binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 76, a heavy chain CDR2 comprising SEQ ID NO: 77, and a heavy chain CDR3 comprising SEQ ID NO: 75. In some embodiments, the CD3-binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 76, a heavy chain CDR2 comprising SEQ ID NO: 77, a heavy chain CDR3 comprising SEQ ID NO: 75, a light chain CDR1 comprising SEQ ID NO: 79, a light chain CDR2 comprising SEQ ID NO: 80, and a light chain CDR3 comprising SEQ ID NO: 81. The CD3-binding arm may comprise a human framework sequence. In some embodiments, the CD3-binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 101. In some embodiments, the CD3-binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 101 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 99. In some embodiments, the CD3-binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 102. In some embodiments, the CD3-binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 102 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 100.

[0199] In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 76, a heavy chain CDR2 comprising SEQ ID NO: 77, and a heavy chain CDR3 comprising SEQ ID NO: 78. In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 76, a heavy chain CDR2 comprising SEQ ID NO: 77, a heavy chain CDR3 comprising SEQ ID NO: 78, a light chain CDR1 comprising SEQ ID NO: 79, a light chain CDR2 comprising SEQ ID NO: 80, and a light chain CDR3 comprising SEQ ID NO: 81. The CD3 binding arm may comprise a human framework sequence. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 103. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 103 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 99. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 104. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 104 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 100.

[0200] In some embodiments, the CD3-binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 76, a heavy chain CDR2 comprising SEQ ID NO: 77, and a heavy chain CDR3 comprising SEQ ID NO: 82. In some embodiments, the CD3-binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 76, a heavy chain CDR2 comprising SEQ ID NO: 77, a heavy chain CDR3 comprising SEQ ID NO: 82, a light chain CDR1 comprising SEQ ID NO: 79, a light chain CDR2 comprising SEQ ID NO: 80, and a light chain CDR3 comprising SEQ ID NO: 81. The CD3-binding arm may comprise a human framework sequence. In some embodiments, the CD3-binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 105. In some embodiments, the CD3-binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 105 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 99. In some embodiments, the CD3-binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 106. In some embodiments, the CD3-binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 106 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 100.

[0201] In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 83, a heavy chain CDR2 comprising SEQ ID NO: 84, and a heavy chain CDR3 comprising SEQ ID NO: 85. In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 83, a heavy chain CDR2 comprising SEQ ID NO: 84, a heavy chain CDR3 comprising SEQ ID NO: 85, a light chain CDR1 comprising SEQ ID NO: 86, a light chain CDR2 comprising SEQ ID NO: 87, and a light chain CDR3 comprising SEQ ID NO: 88. The CD3 binding arm may comprise a human framework sequence. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 107. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 107 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 109. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 108. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 108 and a light chain variable domain encoded by a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 110.

[0202] In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 193, a heavy chain CDR2 comprising SEQ ID NO: 194, and a heavy chain CDR3 comprising SEQ ID NO: 195. In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 193, a heavy chain CDR2 comprising SEQ ID NO: 194, a heavy chain CDR3 comprising SEQ ID NO: 195, a light chain CDR1 comprising SEQ ID NO: 198, a light chain CDR2 comprising SEQ ID NO: 199, and a light chain CDR3 comprising SEQ ID NO: 200. The CD3 binding arm may comprise a human framework sequence. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 196. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 196 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 201.

[0203] In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 203, a heavy chain CDR2 comprising SEQ ID NO: 204, and a heavy chain CDR3 comprising SEQ ID NO: 205. In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 203, a heavy chain CDR2 comprising SEQ ID NO: 204, a heavy chain CDR3 comprising SEQ ID NO: 205, a light chain CDR1 comprising SEQ ID NO: 208, a light chain CDR2 comprising SEQ ID NO: 209, and a light chain CDR3 comprising SEQ ID NO: 210. The CD3 binding arm may comprise a human framework sequence. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 206. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain that is substantially the same as or identical to SEQ ID NO: 206 and a light chain variable domain that is substantially the same as or identical to SEQ ID NO: 211.

[0204] In some embodiments, the CDRs of the heavy and / or light chains are derived from known anti-CD3 antibodies such as muromonab-CD3 (OKT3), otrexup (TRX4), teprotumumab (MGA031), besilesomab (Nuvion), TR-66 or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, Fl 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, and WT-31.

[0205] In some embodiments, the CD3 binding arm is IgG or a derivative thereof. In some embodiments, the CD3 binding arm is IgG1, IgG2, IgG3, or IgG4. In some embodiments where the CD3 binding arm has an IgG4 isotype, the binding arm contains S228P, L234A, L235A, F405L, and R409K substitutions in the Fc region. In some embodiments, the antibody or antigen-binding fragment binds to CD3ε on primary human T cells. In some embodiments, the antibody or antigen-binding fragment binds to CD3ε on primary cynomolgus T cells. In some embodiments, the antibody or antigen-binding fragment binds to CD3ε on primary human and cynomolgus T cells. In some embodiments, the antibody or antigen-binding fragment activates primary human CD3+ T cells. In some embodiments, the antibody or antigen-binding fragment activates primary cynomolgus CD4+ T cells.

[0206] In some embodiments, the multispecific antibodies described herein may employ any format described in the art for multispecific antibodies. In some embodiments, the multispecific antibodies described herein are constructed based on a bispecific antibody format. This can be achieved by adding a third antigen-binding arm to a bispecific antibody. Various formats of bispecific antibodies have been described to date and have been reviewed more recently by Chames and Baty (2009) Curr Opin Drug Disc Dev 12:276. In some embodiments, the multispecific antibody is a bispecific antibody, which is a bispecific antibody obtained by diabody, crossbody, or controlled Fab arm exchange such as those described in the present disclosure.

[0207] In some embodiments, multispecific antibodies include IgG-like molecules with complementary CH3 domains that force heterodimer formation, recombinant IgG-like dual targeting molecules (the two sides of the molecule each comprise an Fab fragment or portion of an Fab fragment of at least two different antibodies); IgG fusion molecules (in which a full-length IgG antibody is fused to an extra Fab fragment or portion of an Fab fragment); Fc fusion molecules (in which a single-chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, Fc region, or portion thereof); Fab fusion molecules (in which different Fab fragments are fused together); ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (in which different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule).

[0208] In some embodiments, IgG-like molecules with complementary CH3 domain molecules include Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs-into-Holes (Genentech), CrossMAbs (Roche) and electrostatically tuned (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonic (Merus), DuoBody (Genmab A / S), and other asymmetric variants (e.g., Zymeworks).

[0209] In some embodiments, recombinant IgG-like dual targeting molecules include Dual Targeting (DT)-Ig (GSK / Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), and CovX-body (CovX / Pfizer).

[0210] In some embodiments, examples of IgG fusion molecules include Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ), and BsAb (Zymogenetics), HERCULES (Biogen Idec), and TvAb (Roche).

[0211] In some embodiments, examples of Fc fusion molecules include ScFv / Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics), and Dual(ScFv).sub.2-Fab (National Research Center for Antibody Medicine--China).

[0212] In some embodiments, Fab fusion bispecific antibodies include F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol), and Fab-Fv (UCB-Celltech). ScFv-, diabody-based antibodies and domain antibodies include Bispecific T Cell Engager (BiTE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack), and COMBODY (Epigen Biotech), dual-targeting nanobodies (Ablynx), dual-targeting heavy chain only domain antibody, but are not limited thereto.

[0213] Full-length multispecific antibodies of the present disclosure may be generated, for example, using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies, either in vitro in a cell-free environment or by co-expression, by introducing substitutions in the heavy chain CH3 interface in each half molecule to favor heterodimerization of two antibody half molecules with distinct specificities. The Fab arm exchange reaction is the result of disulfide bond isomerization and dissociation-association of the CH3 domains. Heavy chain disulfide bonds in the hinge region of the monospecific parent antibody are reduced. A free cysteine occurring in one of the monospecific parent antibodies forms an intra-heavy chain disulfide bond with a cysteine residue in the second monospecific parent antibody molecule, while the CH3 domains of the parent antibody open and reform upon dissociation-association. The CH3 domains of the Fab arms may be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding a different epitope, e.g., an epitope on CD79b (or CD20) and an epitope on CD3. A third antigen-binding arm can then be introduced into the bispecific antibody, e.g., at the C-terminus of the first or second heavy chain, which can bind a third epitope, e.g., CD20 (or CD79b).

[0214] As used herein, "homodimerization" refers to the interaction of two heavy chains with identical CH3 amino acid sequences. As used herein, "homodimer" refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

[0215] As used herein, "heterodimerization" refers to the interaction of two heavy chains with non-identical CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

[0216] The "knob-in-hole" strategy (see, e.g., WO 2006 / 028936) may be used to generate full-length multispecific antibodies. Briefly stated, selected amino acids that form the interface of the CH3 domain in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with the "hole" and the heavy chain with the "knob". Exemplary pairs of CH3 substitutions that form knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V (EU numbering), represented as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain).

[0217] In some embodiments of the multispecific antibodies or multispecific binding fragments described herein, one of the Fc domains comprises the mutations T366S, L368A, and Y407V, and the other Fc domain comprises the mutation T366W. In some embodiments, the Fc domain of the first heavy chain portion (HC1) of the first antigen-binding arm (e.g., the CD79b-binding arm) comprises the mutations T366S, L368A, and Y407V, and the Fc domain of the second heavy chain portion (HC2) of the second antigen-binding arm and / or the third antigen-binding arm (e.g., the CD3 / CD20-binding arm of a trispecific antibody or the CD3-binding arm of a bispecific antibody) comprises the mutation T366W. In some embodiments, the Fc domain of HC2 of the second antigen-binding arm and / or the third antigen-binding arm (e.g., the CD3 / CD20-binding arm of a trispecific antibody or the CD3-binding arm of a bispecific antibody) comprises the mutations T366S, L368A, and Y407V, and the Fc domain of HC1 of the first antigen-binding arm (e.g., the CD79b-binding arm) comprises the mutation T366W.

[0218] Other strategies, such as promoting heavy chain heterodimer formation using electrostatic interactions by substituting positively charged residues on one CH3 surface and negatively charged residues on a second CH3 surface, may be used as described in U.S. Patent Application Publication No. 2010 / 0015133, U.S. Patent Application Publication No. 2009 / 0182127, U.S. Patent Application Publication No. 2010 / 028637, or U.S. Patent Application Publication No. 2011 / 0123532. In other strategies, heterodimer formation may be promoted by the following substitutions: L351Y_F405AY407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V K409F Y407A / T366A_K409F, or T350V_L351Y_F405A Y407V / T350V_T366L_K392L_T394W (expressed as modified positions in the first CH3 domain of the first heavy chain / modified positions in the second CH3 domain of the second heavy chain) as described in U.S. Patent Application Publication No. 2012 / 0149876 or U.S. Patent Application Publication No. 2013 / 0195849 (Zymeworks).

[0219] In addition to the above method, the multispecific antibodies of the present invention can be generated by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and forming a trispecific heterodimeric antibody from the two parental monospecific homodimeric antibodies in an in vitro cell-free environment according to the method described in WO 2011 / 131746 under reducing conditions that isomerize disulfide bonds. In this method, a first monospecific bivalent antibody (e.g., an anti-CD79b antibody) and a second monospecific bivalent antibody (e.g., an anti-CD3 antibody) are engineered to have specific substitutions in the CH3 domain that promote the stability of the heterodimer. These antibodies are incubated together under reducing conditions sufficient for the cysteine in the hinge region to isomerize the disulfide bond, thereby generating a multispecific antibody by Fab arm exchange. The incubation conditions may optimally be returned to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, an incubation of at least 90 minutes may be used at a temperature of at least 20 °C in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol at pH 5-8, e.g., pH 7.0 or pH 7.4.

[0220] In some embodiments, the multispecific antibody or antigen-binding fragment is an IgG or a derivative thereof. The IgG class is divided in humans into four isotypes: IgG1, IgG2, IgG3, and IgG4. These share greater than 95% homology in the amino acid sequence of the Fc region but show major differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of the antibody binds to Fc receptors (FcγR) on the surface of immune effector cells such as natural killer and macrophages, resulting in phagocytosis or lysis of the target cell. In CDC, the antibody kills the target cell by triggering the complement cascade on the cell surface. The antibodies described herein include antibodies having the described features of the variable domains in combination with any of the IgG isotypes, including modified versions in which the Fc sequence is modified to provide different effector functions.

[0221] For many therapeutic antibody applications, Fc-mediated effector functions do not contribute to the mechanism of action. These Fc-mediated effector functions can be detrimental by causing off-target toxicity and can pose a safety risk. Modification of effector functions can be achieved by genetically engineering the Fc region to reduce its binding to FcγR or complement factors. The binding of IgG to activating (FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb) and inhibitory (FcγRIIb) FcγRs or the first component of complement (C1q) is determined by residues located in the hinge region and CH2 domain. When mutations are introduced into IgG1, IgG2, and IgG4, the Fc function is reduced or silenced. The antibodies described herein may include these modifications.

[0222] In one embodiment, the antibody comprises an Fc region having one or more of the following characteristics: (a) reduced effector function when compared to the parental Fc, (b) reduced affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIb, and / or FcγRIIIa, (c) reduced affinity for FcγRI, (d) reduced affinity for FcγRIIa, (e) reduced affinity for FcγRIIb, (f) reduced affinity for FcγRIIIb, or (g) reduced affinity for FcγRIIIa.

[0223] In some embodiments, the antibody or antigen-binding fragment is an IgG or derivative thereof, such as an IgG1, IgG2, IgG3, and IgG4 isotype. In some embodiments where the antibody has an IgG1 isotype, the antibody comprises in its Fc region the L234A, L235A, D265S, and / or K409R substitutions. In some embodiments where the antibody has an IgG4 isotype, the antibody comprises in its Fc region the S228P, L234A, and L235A substitutions. The antibodies described herein may include these modifications.

[0224] In some embodiments, each of one or more Fc domains of the heavy chain portions (e.g., HC1 and / or HC2) described herein comprises one or more mutations selected from L234A, L235A, and D265S. In some embodiments, the Fc domains of the heavy chain portions (e.g., HC1 and HC2) each comprise the mutations L234A, L235A, and D265S.

[0225] In some embodiments, the Fc domain of one of the heavy chain portions of the multispecific antibodies described herein further comprises one or more mutations that reduce Fc binding to Protein A. In some embodiments, the Fc domain of one of the heavy chain portions comprises the mutation H435R and / or Y436F. In some embodiments, the Fc domain of HC2 of the second antigen-binding arm and / or the third antigen-binding arm (e.g., the CD3 / CD20-binding arm of a trispecific antibody, or the CD3-binding arm of a bispecific antibody) comprises the mutation H435R and / or Y436F.

[0226] In various embodiments of the trispecific antibodies described herein, the third antigen-binding arm is operably linked to the Fc domain of the first or second antigen-binding arm via a linker. In some embodiments, the linker is a peptide linker and can comprise any naturally occurring amino acid. Exemplary amino acids that can be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker should be of sufficient length to link the third antigen-binding arm and the first or second antigen-binding arm in a manner that allows them to form the correct three-dimensional conformation relative to each other so that they retain the desired activity, such as binding to a third antigen (e.g., CD3 or CD20).

[0227] In some embodiments of the trispecific antibodies described herein, HC1 comprises, from N-terminus to C-terminus, a VH1 associated with a first antigen-binding arm, a CH1 domain, an Fc domain, a linker, and a third antigen-binding arm.

[0228] In some embodiments of the trispecific antibodies described herein, HC2 comprises, from N-terminus to C-terminus, a second antigen-binding arm, an Fc domain, a linker, and a third antigen-binding arm.

[0229] In various embodiments, the scFvs used in the multispecific antibodies described herein comprise, from N- to C-terminus, a VH, a linker, and a VL (VH-L-VL) or a VL, a linker, and a VH (VL-L-VH). In some embodiments, the scFvs comprise, from N- to C-terminus, a VL, a linker, and a VH (VL-L-VH). In some embodiments, the scFvs comprise, from N- to C-terminus, a VH, a linker, and a VH (VL-L-VH).

[0230] Linkers used in the present disclosure can be about 5 to 50 amino acids in length. In some embodiments, the linker is about 10 to 40 amino acids in length. In some embodiments, the linker is about 10 to 35 amino acids in length. In some embodiments, the linker is about 10 to 30 amino acids in length. In some embodiments, the linker is about 10 to 25 amino acids in length. In some embodiments, the linker is about 10 to 20 amino acids in length. In some embodiments, the linker is about 15 to 20 amino acids in length. In some embodiments, the linker is 6 amino acids in length. In some embodiments, the linker is 7 amino acids in length. In some embodiments, the linker is 8 amino acids in length. In some embodiments, the linker is 9 amino acids in length. In some embodiments, the linker is 10 amino acids in length. In some embodiments, the linker is 11 amino acids in length. In some embodiments, the linker is 12 amino acids in length. In some embodiments, the linker is 13 amino acids in length. In some embodiments, the linker is 14 amino acids in length. In some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker is 16 amino acids in length. In some embodiments, the linker is 17 amino acids in length. In some embodiments, the linker is 18 amino acids in length. In some embodiments, the linker is 19 amino acids in length. In some embodiments, the linker is 20 amino acids in length. In some embodiments, the linker is 21 amino acids in length. In some embodiments, the linker is 22 amino acids in length. In some embodiments, the linker is 23 amino acids in length. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker is 25 amino acids in length. In some embodiments, the linker is 26 amino acids in length. In some embodiments, the linker is 27 amino acids in length. In some embodiments, the linker is 28 amino acids in length. In some embodiments, the linker is 29 amino acids in length. In some embodiments, the linker is 30 amino acids in length. In some embodiments, the linker is 31 amino acids in length.In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker is 33 amino acids in length. In some embodiments, the linker is 34 amino acids in length. In some embodiments, the linker is 35 amino acids in length. In some embodiments, the linker is 36 amino acids in length. In some embodiments, the linker is 37 amino acids in length. In some embodiments, the linker is 38 amino acids in length. In some embodiments, the linker is 39 amino acids in length. In some embodiments, the linker is 40 amino acids in length. Exemplary linkers that can be used are GIy-rich linkers, GIy- and Ser-containing linkers, GIy- and Ala-containing linkers, Ala- and Ser-containing linkers, and other flexible linkers.

[0231] Other linker sequences can include portions of an immunoglobulin hinge region, CL, or CH1, derived from an immunoglobulin heavy or light chain isotype. Exemplary linkers that can be used are shown in Table 4. Additional linkers are described, for example, in WO 2019 / 060695.

[0232] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:215.

[0233] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:216.

[0234] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:217.

[0235] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:218.

[0236] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:219.

[0237] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:220.

[0238] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 221.

[0239] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 222.

[0240] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 223.

[0241] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 224.

[0242] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 225.

[0243] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 226.

[0244] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 227.

[0245] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 228.

[0246] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 229.

[0247] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 230.

[0248] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 231.

[0249] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 232.

[0250] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 233.

[0251] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 234.

[0252] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 235.

[0253] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 236.

[0254] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 237.

[0255] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 238.

[0256] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 239.

[0257] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 240.

[0258] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 241.

[0259] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 242.

[0260] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 243.

[0261] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 244.

[0262] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 245.

[0263] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 246.

[0264] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 247.

[0265] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 248.

[0266] [Table 6]

[0267] In some embodiments, the trispecific antibody or trispecific antibody fragment of the present disclosure comprises a single polypeptide comprising a CD79b binding arm and a CD3 and CD20 binding arm (also referred to herein as the CD3 / CD20 arm). The CD79b binding arm comprises a heavy chain portion (HC1) and a light chain (LC). The CD3 / CD20 binding arm comprises, from the N-terminus to the C-terminus, an scFv that binds to CD3, heavy chain constant regions CH2 and CH3, and an scFv that binds to CD20. The trispecific molecules disclosed herein can be any one of the antibodies described in Table 5. In some embodiments, the trispecific antibody or trispecific antibody fragment of the present disclosure can be encoded by a nucleotide sequence encoding any one of the CD79b arms and / or CD3 / CD20 arms of the antibodies described in Table 5.

[0268] Table 5 provides an overview of examples of several CD79b×CD20×CD3 trispecific antibodies described herein.

[0269] [Table 7]

[0270] Table 5 shows the internal names of each antigen-binding arm present in various trispecific antibodies. The CDR sequences of the CD79b arm are provided in Table 1, and the CDR sequences of the CD3 / CD20 arm are provided in Tables 3 and 2a, respectively. Thus, for example, the trispecific antibody C923B169 comprises CDR1, 2, 3, 4, 5, 6; 77, 76, 75, 79, 80, 81; 121, 122, 123, 124, 119 and 125.

[0271] In some embodiments, HC1 of the CD79b×CD20×CD3 trispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 172, 176, 180, or 191. In some embodiments, the nucleotide sequence encoding HC1 of the CD79b×CD20×CD3 trispecific antibody comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 173, 177, 181, or 192.

[0272] In some embodiments, LC of the CD79b×CD20×CD3 trispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 174, 178, or 182. In some embodiments, the nucleotide sequence encoding LC of the CD79b×CD20×CD3 trispecific antibody comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 175, 179, 183, or 188.

[0273] In some embodiments, the CD3 / CD20 arm of the CD79b×CD20×CD3 trispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 142, 144, 148, 150, 152, 154, 156, 158, 160, 162, 166, 168, or 170. In some embodiments, the nucleotide sequence encoding the CD3 / CD20 arm of the CD79b×CD20×CD3 trispecific antibody comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 143, 145, 149, 151, 153, 155, 157, 159, 161, 163, 167, 169, or 171.

[0274] In some embodiments, HC1 of the CD79b×CD20×CD3 trispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 172, and LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 174. In some embodiments, the nucleotide sequence encoding HC1 of the CD79b×CD20×CD3 trispecific antibody comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 173, and the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 175.

[0275] In some embodiments, the HC1 of the CD79bxCD20xCD3 trispecific antibody comprises substantially the same or identical amino acid sequence as SEQ ID NO: 176, and the LC comprises substantially the same or identical amino acid sequence as SEQ ID NO: 178. In some embodiments, the nucleotide sequence encoding the HC1 of the CD79bxCD20xCD3 trispecific antibody comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 177, and the nucleotide sequence encoding the LC comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 179.

[0276] In some embodiments, the HC1 of the CD79bxCD20xCD3 trispecific antibody comprises substantially the same or identical amino acid sequence as SEQ ID NO: 180, and the LC comprises substantially the same or identical amino acid sequence as SEQ ID NO: 182. In some embodiments, the nucleotide sequence encoding the HC1 of the CD79bxCD20xCD3 trispecific antibody comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 181, and the nucleotide sequence encoding the LC comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 183 or 188.

[0277] In some embodiments, the HC1 of the CD79bxCD20xCD3 trispecific antibody comprises substantially the same or identical amino acid sequence as SEQ ID NO: 191, and the LC comprises substantially the same or identical amino acid sequence as SEQ ID NO: 182. In some embodiments, the nucleotide sequence encoding the HC1 of the CD79bxCD20xCD3 trispecific antibody comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 192, and the nucleotide sequence encoding the LC comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 183.

[0278] In some embodiments of the CD79bxCD20xCD3 trispecific antibody, the HC1 comprises substantially the same as or identical to an amino acid sequence of SEQ ID NO: 172, the LC comprises substantially the same as or identical to an amino acid sequence of SEQ ID NO: 174, and the CD3 / CD20 binding arm comprises substantially the same as or identical to an amino acid sequence of SEQ ID NO: 142. In some embodiments, the nucleotide sequence encoding the HC1 comprises substantially the same as or identical to a nucleotide sequence of SEQ ID NO: 173, the nucleotide sequence encoding the LC comprises substantially the same as or identical to a nucleotide sequence of SEQ ID NO: 175, and the nucleotide sequence encoding the CD3 / CD20 arm comprises substantially the same as or identical to a nucleotide sequence of SEQ ID NO: 143.

[0279] In some embodiments of the CD79bxCD20xCD3 trispecific antibody, the HC1 comprises substantially the same as or identical to an amino acid sequence of SEQ ID NO: 176, the LC comprises substantially the same as or identical to an amino acid sequence of SEQ ID NO: 178, and the CD3 / CD20 binding arm comprises substantially the same as or identical to an amino acid sequence of SEQ ID NO: 142. In some embodiments, the nucleotide sequence encoding the HC1 comprises substantially the same as or identical to a nucleotide sequence of SEQ ID NO: 177, the nucleotide sequence encoding the LC comprises substantially the same as or identical to a nucleotide sequence of SEQ ID NO: 179, and the nucleotide sequence encoding the CD3 / CD20 arm comprises substantially the same as or identical to a nucleotide sequence of SEQ ID NO: 143.

[0280] In some embodiments of the CD79b×CD20×CD3 trispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 180, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 182, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 142. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 181, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 183, and the nucleotide sequence encoding the CD3 / CD20 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 143.

[0281] In some embodiments of the CD79b×CD20×CD3 trispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 172, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 174, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 144. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 173, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 175, and the nucleotide sequence encoding the CD3 / CD20 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 145.

[0282] In some embodiments of the CD79b×CD20×CD3 trispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 176, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 178, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 144. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 177, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 179, and the nucleotide sequence encoding the CD3 / CD20 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 145.

[0283] In some embodiments of the CD79b×CD20×CD3 trispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 180, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 182, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 144. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 181, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 183, and the nucleotide sequence encoding the CD3 / CD20 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 145.

[0284] In some embodiments of the CD79bxCD20xCD3 trispecific antibody, the HC1 comprises substantially the same as or identical to an amino acid sequence of SEQ ID NO: 180, the LC comprises substantially the same as or identical to an amino acid sequence of SEQ ID NO: 182, and the CD3 / CD20 binding arm comprises substantially the same as or identical to an amino acid sequence of SEQ ID NO: 148. In some embodiments, the nucleotide sequence encoding the HC1 comprises substantially the same as or identical to a nucleotide sequence of SEQ ID NO: 181, the nucleotide sequence encoding the LC comprises substantially the same as or identical to a nucleotide sequence of SEQ ID NO: 183, and the nucleotide sequence encoding the CD3 / CD20 arm comprises substantially the same as or identical to a nucleotide sequence of SEQ ID NO: 149.

[0285] In some embodiments of the CD79bxCD20xCD3 trispecific antibody, the HC1 comprises substantially the same as or identical to an amino acid sequence of SEQ ID NO: 180, the LC comprises substantially the same as or identical to an amino acid sequence of SEQ ID NO: 182, and the CD3 / CD20 binding arm comprises substantially the same as or identical to an amino acid sequence of SEQ ID NO: 150. In some embodiments, the nucleotide sequence encoding the HC1 comprises substantially the same as or identical to a nucleotide sequence of SEQ ID NO: 181, the nucleotide sequence encoding the LC comprises substantially the same as or identical to a nucleotide sequence of SEQ ID NO: 188, and the nucleotide sequence encoding the CD3 / CD20 arm comprises substantially the same as or identical to a nucleotide sequence of SEQ ID NO: 151.

[0286] In some embodiments of the CD79b×CD20×CD3 trispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 180, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 182, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 152. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 181, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 188, and the nucleotide sequence encoding the CD3 / CD20 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 153.

[0287] In some embodiments of the CD79b×CD20×CD3 trispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 180, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 182, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 154. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 181, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 188, and the nucleotide sequence encoding the CD3 / CD20 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 155.

[0288] In some embodiments of the CD79b×CD20×CD3 trispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 180, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 182, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 156. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 181, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 188, and the nucleotide sequence encoding the CD3 / CD20 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 157.

[0289] In some embodiments of the CD79b×CD20×CD3 trispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 180, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 182, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 158. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 181, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 188, and the nucleotide sequence encoding the CD3 / CD20 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 159.

[0290] In some embodiments of the CD79b×CD20×CD3 trispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 180, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 182, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 160. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 181, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 188, and the nucleotide sequence encoding the CD3 / CD20 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 161.

[0291] In some embodiments of the CD79b×CD20×CD3 trispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 180, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 182, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 162. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 181, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 188, and the nucleotide sequence encoding the CD3 / CD20 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 163.

[0292] In some embodiments of the CD79b×CD20×CD3 trispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 191, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 182, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 166. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 192, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 183, and the nucleotide sequence encoding the CD3 / CD20 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 167.

[0293] In some embodiments of the CD79b×CD20×CD3 trispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 172, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 174, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 168. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 173, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 175, and the nucleotide sequence encoding the CD3 / CD20 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 169.

[0294] In some embodiments of the CD79b×CD20×CD3 trispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 172, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 174, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 170. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 173, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 175, and the nucleotide sequence encoding the CD3 / CD20 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 171.

[0295] In one embodiment, HC1 of the CD79b×CD20×CD3 trispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 172. In one embodiment, HC1 of the CD79b×CD20×CD3 trispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 173.

[0296] In one embodiment, LC of the CD79b×CD20×CD3 trispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 174. In one embodiment, LC of the CD79b×CD20×CD3 trispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 175.

[0297] In one embodiment, CD3 / CD20 of the CD79b×CD20×CD3 trispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 168. In one embodiment, CD3 / CD20 of the CD79b×CD20×CD3 trispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 169.

[0298] In one embodiment, CD3 / CD20 of the CD79b×CD20×CD3 trispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 170. In one embodiment, CD3 / CD20 of the CD79b×CD20×CD3 trispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 171.

[0299] In one embodiment, provided herein is an isolated trispecific antibody or a trispecific binding fragment thereof, which a) a CD79b binding arm comprising a heavy chain (HC1) and a light chain (LC), and b) a CD3 / CD20 binding arm, and HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 172, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 174, and the CD3 / CD20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 168.

[0300] In one embodiment, provided herein is an isolated trispecific antibody or a trispecific binding fragment thereof, which a) a CD79b binding arm comprising a heavy chain (HC1) and a light chain (LC), and b) a CD3 / CD20 binding arm, and HC1 comprises the amino acid sequence of SEQ ID NO: 172, LC comprises the amino acid sequence of SEQ ID NO: 174, and the CD3 / CD20 binding arm comprises the amino acid sequence of SEQ ID NO: 168.

[0301] In one embodiment, provided herein is an isolated trispecific antibody or a trispecific binding fragment thereof, which a) a CD79b binding arm comprising a heavy chain (HC1) and a light chain (LC), and b) a CD3 / CD20 binding arm, and HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 172, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 174, and the CD3 / Cd20 binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 170.

[0302] In one embodiment, provided herein is an isolated trispecific antibody or a trispecific binding fragment thereof, which a) a CD79b-binding arm comprising a heavy chain (HC1) and a light chain (LC), and b) a CD3 / CD20-binding arm, and HC1 comprises the amino acid sequence of SEQ ID NO: 172, LC comprises the amino acid sequence of SEQ ID NO: 174, and the CD3 / CD20-binding arm comprises the amino acid sequence of SEQ ID NO: 170.

[0303] In one embodiment, the CD79b×CD20×CD3 trispecific antibody is C923B168.

[0304] In one embodiment, the CD79b×CD20×CD3 trispecific antibody is C923B169.

[0305] In some embodiments, the bispecific antibody or bispecific antibody fragment of the present disclosure comprises a CD79b-binding arm comprising HC1 and LC of any one of the antibodies described in Table 6, and / or a CD3-binding arm. In some embodiments, the bispecific antibody or bispecific antibody fragment of the present disclosure may be encoded by a nucleotide sequence encoding HC1, LC, and / or a CD3-binding arm of any one of the antibodies described in Table 6.

[0306] Table 6 provides an overview of examples of several CD79b×CD3 bispecific antibodies described herein.

[0307]

Table 8

[0308] In some embodiments, HC1 of the CD79b×CD3 bispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 172, 176, or 180. In some embodiments, the nucleotide sequence encoding HC1 of the CD79b×CD3 bispecific antibody comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 173, 177, or 181.

[0309] In some embodiments, the LC of the CD79b×CD3 bispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 174, 178, or 182. In some embodiments, the nucleotide sequence encoding the LC of the CD79b×CD3 bispecific antibody comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 175, 179, or 183.

[0310] In some embodiments, the CD3 of the CD79b×CD3 bispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 164 or 189. In some embodiments, the nucleotide sequence encoding the CD3 arm of the CD79b×CD3 bispecific antibody comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 165 or 190.

[0311] In some embodiments, the HC1 of the CD79b×CD3 bispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 172, and the LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 174. In some embodiments, the nucleotide sequence encoding the HC1 of the CD79b×CD3 bispecific antibody comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 173, and the nucleotide sequence encoding the LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 175.

[0312] In some embodiments, the HC1 of the CD79b×CD3 bispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 176, and the LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 178. In some embodiments, the nucleotide sequence encoding the HC1 of the CD79b×CD3 bispecific antibody comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 177, and the nucleotide sequence encoding the LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 179.

[0313] In some embodiments, HC1 of the CD79b×CD3 bispecific antibody comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 180, and LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 182. In some embodiments, the nucleotide sequence encoding HC1 of the CD79b×CD3 bispecific antibody comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 181, and the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 183 or 188.

[0314] In some embodiments of the CD79b×CD3 bispecific antibody, in some embodiments, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 172, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 174, and the CD3 arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 164. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 173, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 175, and the nucleotide sequence encoding the CD3 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 165.

[0315] In some embodiments of the CD79b×CD3 bispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 176, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 178, and the CD3 arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 164. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 177, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 179, and the nucleotide sequence encoding the CD3 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 165.

[0316] In some embodiments of the CD79b x CD3 bispecific antibody, the HC1 comprises substantially the same or identical amino acid sequence as SEQ ID NO: 180, the LC comprises substantially the same or identical amino acid sequence as SEQ ID NO: 182, and the CD3 arm comprises substantially the same or identical amino acid sequence as SEQ ID NO: 164. In some embodiments, the nucleotide sequence encoding the HC1 comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 181, the nucleotide sequence encoding the LC comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 183, and the nucleotide sequence encoding the CD3 arm comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 165.

[0317] In some embodiments of the CD79bxCD3 bispecific antibody, the HC1 comprises substantially the same or identical amino acid sequence as SEQ ID NO: 172, the LC comprises substantially the same or identical amino acid sequence as SEQ ID NO: 174, and the CD3 arm comprises substantially the same or identical amino acid sequence as SEQ ID NO: 189. In some embodiments, the nucleotide sequence encoding the HC1 comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 173, the nucleotide sequence encoding the LC comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 175, and the nucleotide sequence encoding the CD3 arm comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 190.

[0318] In some embodiments of the CD79b x CD3 bispecific antibody, the HC1 comprises substantially the same or identical amino acid sequence as SEQ ID NO: 176, the LC comprises substantially the same or identical amino acid sequence as SEQ ID NO: 178, and the CD3 arm comprises substantially the same or identical amino acid sequence as SEQ ID NO: 189. In some embodiments, the nucleotide sequence encoding the HC1 comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 177, the nucleotide sequence encoding the LC comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 179, and the nucleotide sequence encoding the CD3 arm comprises substantially the same or identical nucleotide sequence as SEQ ID NO: 190.

[0319] In some embodiments of the CD79b×CD3 bispecific antibody, HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 180, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 182, and the CD3 arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO: 189. In some embodiments, the nucleotide sequence encoding HC1 comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 181, the nucleotide sequence encoding LC comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 183, and the nucleotide sequence encoding the CD3 arm comprises a nucleotide sequence that is substantially the same as or identical to SEQ ID NO: 190.

[0320] In addition to the multispecific antibodies or antigen-binding fragments described, polynucleotide sequences capable of encoding the multispecific antibodies or antigen-binding fragments described are also provided. Vectors comprising the polynucleotides described are also provided, and similarly, cells expressing the multispecific antibodies or antigen-binding fragments provided herein are provided. Also described are cells capable of expressing the disclosed vectors. These cells may be mammalian cells (e.g., 293F cells, CHO cells), insect cells (e.g., Sf7 cells), yeast cells, plant cells, or bacterial cells (e.g., E. coli). The antibodies described can also be produced by hybridoma cells. The antibodies described can also be produced recombinantly.

[0321] Polynucleotides encoding recombinant antigen-binding proteins are also within the scope of the present disclosure. In some embodiments, the polynucleotides described (and the peptides they encode) comprise a leader sequence. Any leader sequence known in the art can be utilized. The leader sequence can include, but is not limited to, a restriction site or a translation initiation site.

[0322] The multispecific antibodies or antigen-binding fragments described herein include variants having one or more amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described multispecific antibodies or antigen-binding fragments. In the context of the present invention, unless otherwise specified, the following notations are used to describe mutations. i) A substitution of an amino acid at a given position is described, for example, as K409R. K409R means the substitution of lysine at position 409 with arginine. ii) For a particular variant, specific three-letter or one-letter codes are used to denote amino acid residues, including the codes Xaa and X. Thus, the substitution of arginine for lysine at position 409 is designated K409R, or the substitution of any amino acid residue for lysine at position 409 is designated K409X. In the case of a deletion of lysine at position 409, this deletion is indicated by K409 * as shown. One of ordinary skill in the art can generate variants having one or more amino acid substitutions, deletions, or additions.

[0323] These variants include (a) variants in which one or more amino acid residues have been substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids have been added to or deleted from the polypeptide, (c) variants in which one or more amino acids include substituents, and (d) variants in which the polypeptide is fused to another peptide or polypeptide, such as a fusion partner, protein tag, or other chemical moiety, that can confer useful properties to the polypeptide, such as an epitope for an antibody, a polyhistidine sequence, a biotin moiety, etc. The antibodies or antigen-binding fragments described herein may include variants in which an amino acid residue from one species has been substituted with the corresponding residue in another species, either at a conserved or non-conserved position. In other embodiments, amino acid residues at non-conserved positions are substituted with conserved or non-conserved residues. Techniques for obtaining these variants are known to those of skill in the art and include genetic (deletion, mutation, etc.), chemical, and enzymatic techniques.

[0324] The multispecific antibodies or antigen-binding fragments described herein can embody several antibody isotypes, such as IgM, IgD, IgG, IgA, and IgE. In some embodiments, the antibody isotype is an isotype of IgG1, IgG2, IgG3, or IgG4, preferably an isotype of IgG1 or IgG4. The specificity of an antibody or its antigen-binding fragment is mainly determined by the amino acid sequence and arrangement of the CDRs. Thus, the CDRs of one isotype can be changed to another isotype without changing the antigen specificity. Alternatively, techniques (isotype switching) have been established to switch one antibody isotype to another in hybridomas without changing the antigen specificity. Thus, such antibody isotypes are within the scope of the antibodies or antigen-binding fragments described.

[0325] Vectors containing the polynucleotides described herein are also provided. The vector can be an expression vector. Thus, recombinant expression vectors containing a sequence encoding a polypeptide of interest are contemplated to be within the scope of the present disclosure. The expression vector may contain one or more additional sequences such as, but not limited to, control sequences (e.g., promoters, enhancers), selectable markers, and polyadenylation signals. Vectors for transforming a wide range of host cells are well known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bactermids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and other bacterial, yeast, and viral vectors.

[0326] Recombinant expression vectors within the scope of this description comprise a nucleic acid fragment of synthetic, genomic, or cDNA origin encoding at least one recombinant protein that can be operably linked to suitable regulatory elements. Such regulatory elements can include a transcriptional promoter, a sequence encoding a suitable mRNA ribosomal binding site, and sequences that control transcription and translation termination. Expression vectors, particularly mammalian expression vectors, can also contain one or more non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences (e.g., essential ribosomal binding sites), a polyadenylation site, splice donor and acceptor sites, or a transcription termination sequence. An origin of replication that confers the ability to replicate in a host can also be incorporated.

[0327] Transcriptional and translational control sequences in expression vectors used to transform vertebrate cells can be provided by viral sources. Exemplary vectors can be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

[0328] In some embodiments, the coding sequence of the multispecific antibody or antigen-binding fragment is placed under the control of a strong constitutive promoter, such as a promoter for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, etc. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the described embodiments. Such viral promoters include, but are not limited to, the cytomegalovirus (CMV) immediate-early promoter, the early and late promoters of SV40, the mouse mammary tumor virus (MMTV) promoter, Moloney leukemia virus, human immunodeficiency virus (HIV), Epstein Barr virus (EBV), Rous sarcoma virus (RSV), and the long terminal repeat (LTR) of other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. In one embodiment, the coding sequence of the multispecific antibody or its antigen-binding fragment is placed under the control of an inducible promoter, such as a metallothionein promoter, a tetracycline-inducible promoter, a doxycycline-inducible promoter, one or more interferon-stimulated response elements (ISREs), such as a promoter containing protein kinase R 2’,5’-oligoadenylate synthetase, the Mx gene, ADAR1, etc.

[0329] The vectors described herein may contain one or more internal ribosome entry sites (IRES). Inclusion of an IRES sequence in a fusion vector may be beneficial for enhancing expression of some proteins. In some embodiments, the vector system will contain one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. The components of the vector may be closely linked, positioned to provide optimal spacing for expression of the gene product (i.e., by introducing "spacer" nucleotides between ORFs), or otherwise positioned. Regulatory elements, such as IRES motifs, may also be positioned to provide optimal spacing for expression.

[0330] The vector may contain a selection marker known in the art. Selection markers include positive and negative selection markers, such as antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene, puromycin resistance gene, blasticidin resistance gene), glutamate synthase gene, HSV-TK or HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). The nucleic acid sequence or cloning site encoding the selection marker may be located upstream or downstream of the nucleic acid sequence or cloning site encoding the polypeptide of interest.

[0331] Using the vectors described herein, various cells can be transformed with the genes encoding the described antibodies or antigen-binding fragments. For example, vectors can be used to generate cells that produce multispecific antibodies or antigen-binding fragments. Accordingly, another aspect features a host cell transformed with a vector comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that binds to CD79b, CD20, and CD3, such as the antibodies or antigen-binding fragments described and exemplified herein.

[0332] Numerous techniques for introducing foreign genes into cells are known in the art and can be used to create recombinant cells for the purpose of performing the methods described according to the various embodiments described and exemplified herein. The technique used must stably introduce the heterologous gene sequence into the host cell such that the heterologous gene sequence is heritable and expressible by the progeny of the cell and does not impair the essential growth and physiological functions of the recipient cell. Techniques that can be used include chromosome introduction methods (e.g., cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carriers), viral vector introduction (e.g., recombinant DNA viruses, recombinant RNA viruses), etc. (Cline, 29 Pharmac. Ther. 69-92 (1985)). Cells can also be transformed using calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts by mammalian cells.

[0333] Cells suitable for use in the expression of the multispecific antibodies or antigen-binding fragments described herein are preferably eukaryotic cells, more preferably cells of plant, rodent, or human origin, such as, for example, but not limited to, NSO, CHO, CHOK1, perC.6, Tk-ts13, BHK, HEK293 cells, COS-7, T98G, CV-1 / EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2 / 0 myeloma cells, and BHK cell lines. Additionally, the expression of the antibody may be achieved using hybridoma cells. Methods for generating hybridomas are well established in the art.

[0334] Cells transformed with the expression vectors described herein may be selected or screened for the recombinant expression of the antibodies or antigen-binding fragments described herein. Recombinant positive cells are grown and screened for subclones that exhibit, for example, the ability to produce proteins with desired phenotypes, such as high levels of expression, enhanced growth characteristics, or desired biochemical properties, by protein modification or altered post-translational modification. These phenotypes may be due to the native properties or mutations of a given subclone. Mutations can occur by chemicals, UV wavelength light, irradiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or combinations of such methods.

[0335] Therapeutic Compositions and Methods of Treatment Using Multispecific Antibodies and Their Multispecific Antigen-Binding Fragments The multispecific antibodies discussed above, such as the CD79b×CD20×CD3 trispecific antibody or the CD79b×CD3 bispecific antibody discussed above, are useful in therapy. Specifically, the multispecific antibodies are useful for treating cancer. Also provided herein is a therapeutic composition for treating a proliferative disorder in a mammal, comprising a therapeutically effective amount of a multispecific antibody or multispecific antigen-binding fragment described herein and a pharmaceutically acceptable carrier. In some embodiments, the multispecific antibody is the CD79b×CD20×CD3 trispecific antibody described herein, or a CD79b×CD20×CD3 trispecific antigen-binding fragment thereof. In some embodiments, the bispecific antibody is the CD79b×CD3 bispecific antibody described herein, or a CD79b×CD3 bispecific antigen-binding fragment thereof. In one embodiment, the pharmaceutical composition is for the treatment of CD79b and / or CD20-expressing cancer, including (but not limited to) the following: CD79b and / or CD20-expressing B-cell cancers, such as B-cell lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM), multiple myeloma (MM), mucosa-associated lymphoid tissue (MALT) lymphoma, Hodgkin lymphoma, Burkitt lymphoma, hairy cell leukemia, or plasmacytoma cells, as well as other cancers in which CD79b and / or CD20 are expressed but have not yet been determined. Specific trispecific antibodies that may be used to treat blood cancers, such as the specific cancers discussed above, include antibodies C923B168 and C923B169.

[0336] In some embodiments, the CD79b×CD20×CD3 trispecific antibody or its binding fragment is utilized for the treatment of DLBCL, including DLBCL with relapsed / refractory (R / R) diffuse large B-cell lymphoma.

[0337] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or binding fragment thereof is utilized for the treatment of non-Hodgkin's lymphoma (including R / R non-Hodgkin's lymphoma), such as follicular lymphoma (FL) or marginal zone lymphoma (MZL).

[0338] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or binding fragment thereof is utilized for the treatment of mantle cell lymphoma (MCL), including R / R MCL.

[0339] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or binding fragment thereof is utilized for the treatment of chronic lymphocytic leukemia (CLL), including R / R CLL.

[0340] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or binding fragment thereof is utilized for the treatment of Waldenstrom's macroglobulinemia (WM), including R / R WM.

[0341] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or binding fragment thereof is utilized for the treatment of Burkitt's lymphoma (BL).

[0342] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or binding fragment thereof is used for the treatment of primary mediastinal large B cell lymphoma (PMBCL).

[0343] In some embodiments, the CD79bxCD20xCD3 trispecific antibody or binding fragment thereof is utilized for the treatment of multiple myeloma.

[0344] In some embodiments, the CD79b×CD20×CD3 trispecific antibody or a binding fragment thereof is utilized for the treatment of post-transplantation lymphoproliferative disorder (PTLD).

[0345] In some embodiments, the CD79b×CD20×CD3 trispecific antibody or a binding fragment thereof is utilized for the treatment of primary central nervous system lymphoma (PCNSL).

[0346] In some embodiments, the CD79b×CD20×CD3 trispecific antibody or a binding fragment thereof is utilized as a conditioning regimen and for post-SCT chemotherapy-free maintenance therapy or prevention of relapse intended to cure relapsed B-cell lymphoma, for autologous stem cell transplantation (autoSCT) and allogeneic stem cell transplantation (alloSCT).

[0347] In some embodiments, the CD79b×CD20×CD3 trispecific antibody or a binding fragment thereof is utilized for the local treatment of intraocular lymphoma (IOL), including cutaneous B-cell lymphoma (including DLBCL Leg type), bronchus-associated lymphoid tissue (BALT) lymphoma, primary intraocular lymphoma (PIOL) or secondary intraocular lymphoma (SIOL) of B-cell lineage. In these embodiments, the CD79b×CD20×CD3 trispecific antibody or a binding fragment thereof may utilize a new administration approach such as an inhaler or nebulizer for BALT lymphoma affecting the lungs.

[0348] Pharmaceutical compositions provided herein comprise a) an effective amount of a multispecific antibody or antibody fragment of the invention and b) a pharmaceutically acceptable carrier, which may be inert or physiologically active. In some embodiments, the multispecific antibody is a CD79bxCD20xCD3 trispecific antibody described herein, or a CD79bxCD20xCD3 trispecific antigen-binding fragment thereof. In some embodiments, the bispecific antibody is a CD79bxCD3 trispecific antibody described herein, or a CD79bxCD3 bispecific antigen-binding fragment thereof. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, antifungal agents, and the like that are physiologically compatible. Examples of suitable carriers, diluents, and / or excipients include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, as well as any combination thereof. In many cases, it is preferable to include an isotonic agent, such as a sugar, polyalcohol, or sodium chloride, in the composition. Specifically, relevant examples of suitable carriers include: (1) Dulbecco's phosphate buffered saline, pH about 7.4, with or without about 1 mg / mL to 25 mg / mL human serum albumin; (2) 0.9% saline (0.9% weight / volume sodium chloride (NaCl)); and (3) 5% (weight / volume) dextrose, which may also contain an antioxidant, e.g., tryptamine, and a stabilizer, e.g., Tween 20®.

[0349] The compositions of the present invention may be in various forms. Such forms include, for example, liquid, semi-solid, and solid dosage forms, with the preferred form depending on the intended mode of administration and therapeutic application. Typically, preferred compositions are in the form of an injectable or infusible solution. A preferred mode of administration is parenteral (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous). In a preferred embodiment, the compositions of the present invention are administered intravenously by bolus or continuous infusion over a period of time. In another preferred embodiment, the compositions are injected intramuscularly, subcutaneously, intra-articularly, intrasynovially, intratumorally, peritumorally, intralesionally, or perilesionally to exert local and systemic therapeutic effects.

[0350] Sterile compositions for parenteral administration can be prepared by incorporating the antibody, antibody fragment, or antibody conjugate of the present invention in the required amount in an appropriate solvent, followed by sterilization by microfiltration. As solvents or excipients, water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof can be used. In many cases, it is preferable to include an isotonic agent, such as a sugar, polyalcohol, or sodium chloride, in the composition. These compositions may also contain auxiliary agents, particularly wetting agents, isotonicity adjusting agents, emulsifying agents, dispersing agents, and stabilizing agents. Sterile compositions for parenteral administration can also be prepared in the form of sterile solid compositions that can be dissolved in sterile water or any other injectable sterile medium at the time of use.

[0351] Multispecific antibodies or antibody fragments may also be administered orally. Solid compositions for oral administration may be used in the form of tablets, pills, powders (gelatin capsules, sachets), or granules. In these compositions, the active ingredient according to the present invention is mixed with one or more inert diluents, such as starch, cellulose, sucrose, lactose, or silica, under an argon stream. These compositions may also contain substances other than diluents, such as one or more lubricants, such as magnesium stearate or talc, colorants, coatings (sugar-coated tablets), or glazes.

[0352] As liquid compositions for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups, and elixirs containing an inert diluent, for example, water, ethanol, glycerol, vegetable oil, or paraffin oil may be used. These compositions may contain substances other than the diluent, for example, wetting agents, sweeteners, thickening agents, flavoring agents, or stabilizing products.

[0353] The dosage is determined by the desired effect, the duration of treatment, and the route of administration used. The dosage is generally 5 mg to 1000 mg orally per day in adults, and the unit dosage ranges from 1 mg to 250 mg of the active substance. Generally, the physician determines the appropriate dosage according to the age, weight, and any other factors specific to the subject being treated.

[0354] Also provided herein is a method for killing CD79b and / or CD20+ cells by administering to a patient in need of killing of CD79b and / or CD20+ cells a multispecific antibody that binds to CD79b and / or CD20 and mobilizes T cells to kill the CD79b and / or CD20+ cells (i.e., T cell redirecting). Either the multispecific antibody or antibody fragment of the present invention may be used therapeutically. For example, in one embodiment, the CD79b×CD20×CD3 multispecific antibody may be used therapeutically to treat cancer in a subject.

[0355] In a preferred embodiment, the multispecific antibody or antibody fragment of the invention is used to treat a proliferative disorder in a mammal. In a more preferred embodiment, one of the pharmaceutical compositions disclosed above containing the multispecific antibody or antibody fragment of the invention is used to treat a proliferative disorder in a mammal. In one embodiment, the disorder is cancer. Specifically, the cancer is a CD79b and / or CD20-expressing cancer, including (but not limited to) the following: CD79b and / or CD20-expressing B cell cancers, such as B cell lymphoma, non-Hodgkin lymphoma, diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM), multiple myeloma (MM), mucosa-associated lymphoid tissue (MALT) lymphoma, Hodgkin lymphoma, Burkitt lymphoma, hairy cell leukemia, or plasmacytoma cells, as well as other cancers that have not yet been determined in which CD79b and / or CD20 are expressed. In some embodiments, the CD79b and / or CD20-expressing B cell cancers treated with the pharmaceutical compositions disclosed herein are recurrent or refractory forms of the cancer. In a preferred embodiment, the multispecific antibody is a CD79b×CD20×CD3 multispecific antibody as described herein, or a multispecific antigen-binding fragment thereof, more preferably a CD79b×CD20×CD3 trispecific antibody as described herein, or a CD79b×CD20×CD3 trispecific antigen-binding fragment thereof.

[0356] Accordingly, the pharmaceutical composition of the present invention is useful for the treatment or prevention of various cancers, including (but not limited to) the following: CD79b and / or CD20-expressing cancers, including (but not limited to) the following: CD79b and / or CD20-expressing B-cell cancers, such as B-cell lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM), multiple myeloma (MM), mucosa-associated lymphoid tissue (MALT) lymphoma, Hodgkin lymphoma, Burkitt lymphoma, hairy cell leukemia, or plasma cell tumors, as well as other cancers that have not yet been determined but in which CD79b and / or CD20 are expressed. In some embodiments, the CD79b and / or CD20-expressing B-cell cancers treated with the pharmaceutical composition disclosed herein are recurrent or refractory forms of the cancer.

[0357] Similarly, provided herein is a method for inhibiting the growth of a selected cell population, comprising contacting CD79b and / or CD20-expressing target cells, or a tissue containing such target cells, with an effective amount of the multispecific antibody or antibody fragment of the present invention, either alone or in combination with other cytotoxic or therapeutic agents, in the presence of peripheral blood mononuclear cells (PBMC). A CD79b×CD20×CD3 antibody that blocks the binding of ligands to CD79b and CD20 can block CD79b- and CD20-mediated signal transduction and result in the inhibition or cell death of target cells. In preferred embodiments, the multispecific antibody is a CD79b×CD20×CD3 multispecific antibody as described herein, or a multispecific antigen-binding fragment thereof, more preferably a CD79b×CD20×CD3 trispecific antibody as described herein, or a CD79b×CD20×CD3 trispecific antigen-binding fragment thereof.

[0358] In some embodiments, the methods described herein that include administration of a multispecific antibody or a pharmaceutical composition comprising the same further comprise administering another therapeutic agent. Suitable other therapeutic agents include, but are not limited to, chemotherapeutic agents, anti-CD20 agents, anti-CD19 agents, anti-CD22 agents, anti-CD37 agents, Bruton’s tyrosine kinase (BTK) inhibitors, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) inhibitors, immunomodulatory imide drugs (IMiDs), apoptosis-promoting B cell lymphoma 2 (Bcl-2) family inhibitors, phosphoinositide 3-kinase (PI3K) inhibitors, NFKB-inducing kinase (NIK) inhibitors, immune checkpoint inhibitors, CD28 co-stimulatory bispecific antibodies, or CD137 co-stimulatory bispecific antibodies, or combinations thereof.

[0359] In some embodiments, the other therapeutic agent is a chemotherapeutic drug regimen, such as rituximab-cyclophosphamide-hydroxy daunorubicin-oncovin-prednisone / prednisolone (R-CHOP), rituximab-ifosfamide-carboplatin-etoposide (R-ICE), rituximab-(dose-adjusted) etoposide-prednisolone-oncovin-cyclophosphamide-hydroxy daunorubicin (R[DA]EPOCH), rituximab-dexamethasone-high-dose ara-C cytarabine-platinol (R-DHAP), rituximab-etoposide-solumedrol-high-dose ara-C cytarabine-platinol (R-ESHAP), bendamustine-rituximab (BR), and lenalidomide + rituximab (R 2 ), Polivy (polatuzumab vedotin) + BR, or Monjuvi (tafasitamab-cxix) + lenalidomide regimen, and the like.

[0360] In some embodiments, the other therapeutic agent is an anti-CD79b agent (e.g., anti-CD79b CAR-T therapy), an anti-CD20 agent, e.g., a CD20×CD3 bispecific antibody (e.g., mosunetuzumab, glofitamab, odronextamab, IGM-2323, and epcoritamab). In some embodiments, the other therapeutic agent is an anti-CD19 agent (e.g., an anti-CD19 antibody (e.g., tafasitamab), an anti-CD19 CAR-T therapy (e.g., Yescarta, Kymriah, and Breyanzi), or a CD20×CD3 bispecific antibody). In some embodiments, the other therapeutic agent is an anti-CD22 antibody such as an anti-CD22 agent (e.g., moxetumomab pasudotox). In some embodiments, the other therapeutic agent is a Bruton's tyrosine kinase (BTK) inhibitor, e.g., ibrutinib, a covalent or non-covalent BTK inhibitor.

[0361] In some embodiments, the other agent is a mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) inhibitor. In some embodiments, the other therapeutic agent is an immunomodulatory imide drug (IMiD), e.g., lenalidomide. In some embodiments, the other therapeutic agent is an apoptosis-promoting B-cell lymphoma 2 (Bcl-2) family inhibitor such as venetoclax, and a Bcl-2-related protein A1 (BFL-1) inhibitor. In some embodiments, the other therapeutic agent is a phosphoinositide 3-kinase (PI3K) inhibitor, e.g., parsaclisib, idelalisib, and umbralisib. In some embodiments, the other therapeutic agent is an immune checkpoint inhibitor, e.g., an anti-PD-1 antibody, a T cell immunoreceptor with Ig and ITIM domains [TIGIT] having anti-T cell Ig, TIM-3, Ig and ITIM domains, and LAG-3. In some embodiments, the additional therapeutic agent is cytarabine, anthracycline, histamine dihydrochloride, selinexor, tasemetostat, or interleukin 2. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. The method for inhibiting the growth of a selected cell population can be carried out in vitro, in vivo, or ex vivo.

[0362] Examples of in vitro uses include the treatment of autologous bone marrow prior to transplant into the same patient to kill diseased or malignant cells, and graft-versus-host disease (GVHD), the treatment of cell cultures to kill all cells except for desired variants that do not express the target antigen, or to kill variants expressing unwanted antigens. Non-clinical in vitro use conditions are readily determined by one of skill in the art.

[0363] An example of a clinical ex vivo use is the removal of tumor cells from bone marrow prior to autologous transplantation in cancer treatment. Treatment can be performed as follows: Bone marrow is harvested from a patient or other individual, or a donated sample is used, and then incubated in serum-containing medium supplemented with a cytotoxic agent of the present invention. The concentration ranges from about 10 μM to 1 μM at 37°C for about 30 minutes to about 48 hours. The exact conditions of concentration and time of incubation, i.e., dosage, can be easily determined by one skilled in the art. After incubation, the bone marrow cells are washed with serum-containing medium and returned to the patient via intravenous injection according to known methods. In situations where the patient is undergoing other treatments, such as a course of myeloablative chemotherapy or total body irradiation, the treated bone marrow cells are cryopreserved in liquid nitrogen between the time of bone marrow collection and the time of reinfusion of the treated cells, using standard medical equipment.

[0364] For clinical in vivo use, a therapeutically effective amount of a multispecific antibody or antigen-binding fragment is administered to a subject in need thereof. For example, a CD79b×CD20×CD3 multispecific antibody and its multispecific antigen-binding fragment may be useful in the treatment of subjects in need of treatment of CD79b- and / or CD20-expressing cancers. In some embodiments, the CD79b- and / or CD20-expressing cancers are B cell cancers such as diffuse large B cell lymphoma (DLBCL). In preferred embodiments, the multispecific antibody is a CD79b×CD20×CD3 multispecific antibody as described herein, or a multispecific antigen-binding fragment thereof, more preferably a CD79b×CD20×CD3 trispecific antibody as described herein, or a CD79b×CD20×CD3 trispecific antigen-binding fragment thereof. In some embodiments, the subject is a mammal, preferably a human. In some embodiments, the multispecific antibody or antigen-binding fragment is administered as a sterile-tested solution.

[0365] The dosing regimen in the above methods for treatment and use is adjusted to provide an optimal desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally decreased or increased in cases where the urgency of the treatment situation dictates. Parenteral compositions may be formulated in unit dosage form for ease of administration and uniformity of dosage.

[0366] The efficient administration and dosing regimen of the multispecific antibodies and fragments depends on the disease or condition being treated and may be determined by one of ordinary skill in the art. Exemplary and non-limiting ranges of a therapeutically effective amount of the compounds of the invention are from about 0.001 to 10 mg / kg, such as from about 0.001 to 5 mg / kg, such as from about 0.001 to 2 mg / kg, such as from about 0.001 to 1 mg / kg, such as about 0.001, about 0.01, about 0.1, about 1, or about 10 mg / kg.

[0367] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe an effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start with a level of the dosage of the multispecific antibody or fragment utilized in the pharmaceutical composition that is less than that required to achieve the desired therapeutic effect and increase such dosage gradually until the desired effect is achieved. Generally, a preferred daily dose of the multispecific antibody of the present invention is the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Administration may be, for example, parenteral, such as intravenous, intramuscular, intratumoral (e.g., bone marrow), or subcutaneous. In one embodiment, the multispecific antibody or fragment may be administered by infusion in a weekly administration calculated as mg / m 2 It may be administered by infusion in a weekly administration calculated as mg / m. Such dosages can be based on the mg / kg dosages provided above, for example, according to the following: dosage (mg / kg) × body weight (e.g., 50 - 100 kg). Such administration may be repeated, for example, 1 - 8 times, such as 3 - 5 times. Administration may be by continuous infusion over a period of 2 - 24 hours, such as 2 - 12 hours. In one embodiment, the multispecific antibody or fragment may be administered by slow continuous infusion over a long period, such as more than 24 hours, to reduce toxic side effects.

[0368] In one embodiment, the multispecific antibody or fragment may be administered in a weekly administration calculated as a fixed dose, up to 8 times, such as 4 - 6 times, when administered once a week. Such a schedule may be repeated one or more times, if necessary, after 6 months or 12 months, for example. Such fixed dosages can be based on the mg / kg dosages provided above, assuming a body weight of 50 - 100 kg. The dosage can be determined or adjusted, for example, by measuring the amount in the blood at the time of administration of the multispecific antibody of the present invention by taking a biological sample and using an anti-idiotype antibody that targets the CD79b and / or CD20 antigen-binding arm of the multispecific antibody of the present invention.

[0369] In one embodiment, the multispecific antibody or fragment may be administered for maintenance therapy, for example, once weekly for a period of 6 months or more.

[0370] The multispecific antibody or fragment may also be administered prophylactically to reduce the risk of cancer progression, delay the onset of events in cancer progression, and / or reduce the risk of recurrence when the cancer has remitted.

[0371] The multispecific antibodies and fragments thereof described herein may also be administered in combination therapy, i.e., in combination with other therapeutic agents relevant to the disease or condition being treated. Thus, in one embodiment, the antibody-containing medicament is for combination with one or more additional therapeutic agents such as chemotherapeutic agents, anti-CD20 agents, anti-CD19 agents, anti-CD22 agents, anti-CD37 agents, Bruton's tyrosine kinase (BTK) inhibitors, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) inhibitors, immunomodulatory imide drugs (IMiDs), apoptosis-promoting B-cell lymphoma 2 (Bcl-2) family inhibitors, phosphoinositide 3-kinase (PI3K) inhibitors, NFKB-inducing kinase (NIK) inhibitors, immune checkpoint inhibitors, CD28 co-stimulatory bispecific antibodies, or CD137 co-stimulatory bispecific antibodies, or combinations thereof. In some embodiments, the other therapeutic agent is a chemotherapy drug regimen such as rituximab-cyclophosphamide-hydroxydaunorubicin-oncovin-prednisone / prednisolone (R-CHOP), rituximab-ifosfamide-carboplatin-etoposide (R-ICE), rituximab-(dose-adjusted) etoposide-prednisolone-oncovin-cyclophosphamide-hydroxydaunorubicin (R[DA]EPOCH), rituximab-dexamethasone-high-dose ara-C cytarabine-platinol (R-DHAP), rituximab-etoposide-solumedrol-high-dose ara C cytarabine-platinol (R-ESHAP), bendamustine-rituximab (BR), and lenalidomide + rituximab (R 2), Polivy (polatuzumab vedotin) + BR, or Monjuvi (tafasitamab-cxix) + lenalidomide regimens. In some embodiments, the other therapeutic agent is an anti-CD79b agent (e.g., anti-CD79b CAR-T therapy), an anti-CD20 agent, e.g., a CD20xCD3 bispecific antibody (e.g., mosunetuzumab, glofitamab, odronextamab, IGM-2323, and epcolitamab). In some embodiments, the other therapeutic agent is an anti-CD19 agent (e.g., an anti-CD19 antibody (e.g., tafasitamab), an anti-CD19 In some embodiments, the other therapeutic agent is a CAR-T therapy (e.g., Yescarta, Kymriah, and Breyanzi) or a CD20xCD3 bispecific antibody. In some embodiments, the other therapeutic agent is an anti-CD22 antibody, such as an anti-CD22 agent (e.g., moxetumomab pasudotox). In some embodiments, the other therapeutic agent is a Bruton's tyrosine kinase (BTK) inhibitor, e.g., ibrutinib, a covalent or non-covalent BTK inhibitor. In some embodiments, the other agent is a mucosal-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) inhibitor. In some embodiments, the other therapeutic agent is an immunomodulatory imide drug (IMiD), e.g., lenalidomide. In some embodiments, the other therapeutic agent is a proapoptotic B-cell lymphoma 2 (Bcl-2) family inhibitor, such as venetoclax, and Bcl-2-related protein A1 (Bcl-2-related protein A1). A1, BFL-1 inhibitors. In some embodiments, the other therapeutic agent is a phosphoinositide 3-kinase (PI3K) inhibitor, such as palsaclisib, idelalisib, and umbralisib. In some embodiments, the other therapeutic agent is an immune checkpoint inhibitor, such as an anti-PD-1 antibody, anti-T cell Ig, TIM-3, T cell immunoreceptor with Ig and ITIM domains [TIGIT], and LAG-3. In some embodiments, the other therapeutic agent is cytarabine, an anthracycline, histamine dihydrochloride, selinexor, tazemetostat, or interleukin-2. Such combined administration may be simultaneous, in any order, separately, or sequentially.In co - administration, the agents may be administered as one composition or as separate compositions, as needed.

[0372] In one embodiment, a method for treating a disorder in a subject in which cells expressing CD79b and / or CD20 are involved, the method comprising administering to a subject in need thereof a therapeutically effective amount of a multispecific antibody or fragment, such as a CD79b×CD20×CD3 multispecific antibody described herein, and radiation therapy is provided. In one embodiment, a method for treating or preventing cancer is provided, the method comprising administering to a subject in need thereof a therapeutically effective amount of a multispecific antibody or fragment, such as a CD79b×CD20×CD3 antibody described herein, and radiation therapy. Radiation therapy may include the administration of radiation or a radiopharmaceutical relevant to the patient being treated. The radiation source may be either outside or inside the patient being treated (radiation therapy may be in the form of, for example, external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that can be used to carry out such methods include, for example, radium, cesium - 137, iridium - 192, americium - 241, gold - 198, cobalt - 57, copper - 67, technetium - 99, iodine - 123, iodine - 131, actinium - 225, and indium - 111.

[0373] Kit Also provided herein are kits that include, for example, a described multispecific antibody or antigen-binding fragment thereof and instructions for using the antibody or fragment to kill a specific cell type. In a preferred embodiment, the multispecific antibody is a CD79bxCD20xCD3 multispecific antibody or multispecific antigen-binding fragment thereof as described herein, more preferably a CD79bxCD20xCD3 trispecific antibody or CD79bxCD20xCD3 trispecific antigen-binding fragment thereof as described herein. The instructions may include directions for using the multispecific antibody or antigen-binding fragment thereof in vitro, in vivo, or ex vivo.

[0374] Typically, the kit has a compartment containing the multispecific antibody or antigen-binding fragment thereof. The multispecific antibody or antigen-binding fragment thereof may be in lyophilized, liquid, or other form amenable to inclusion in the kit. The kit may also contain additional components required to perform the methods described in the kit's instructions, such as sterile solutions for reconstituting the lyophilized powder, additional agents for combining with the multispecific antibody or antigen-binding fragment thereof prior to administration to a patient, and tools to assist in administering the multispecific antibody or antigen-binding fragment thereof to a patient.

[0375] Diagnostic Use The multispecific antibodies and fragments described herein may also be used for diagnostic purposes. Accordingly, there are also provided diagnostic compositions comprising a multispecific antibody or fragment as defined herein and their use. In preferred embodiments, the multispecific antibody is a CD79b×CD20×CD3 multispecific antibody as described herein, or a multispecific antigen-binding fragment thereof, more preferably a CD79b×CD20×CD3 trispecific antibody as described herein, or a CD79b×CD20×CD3 trispecific antigen-binding fragment thereof. In one embodiment, the invention provides a kit for the diagnosis of cancer, comprising a container containing a CD79b×CD20×CD3 trispecific antibody and one or more reagents for detecting the binding of an antibody to CD79b and / or CD20. Reagents can include, for example, fluorescent tags, enzyme tags, or other detectable tags. The reagents may also include secondary or tertiary antibodies or reagents for an enzymatic reaction. In this case, the enzymatic reaction results in a product that can be visualized. For example, the multispecific antibodies or antigen-binding fragments thereof described herein may be labeled with a radiolabel, a fluorescent label, an epitope tag, biotin, a chromophore label, an ECL label, an enzyme, ruthenium, 111 In-DOTA, 111 In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase, and β-galactosidase, or polyhistidine, or may be labeled with similar such labels known in the art.

[0376] CD79b-specific antibody Isolated antibodies and antigen-binding fragments specific for CD79b are described herein. In some embodiments, the CD79b-specific antibodies and antigen-binding fragments bind to human CD79b. The overall structure of a CD79b-specific antibody molecule can include an antigen-binding domain comprising a heavy chain and a light chain, and an Fc domain that performs various functions including complement fixation and antibody-binding receptors.

[0377] In some embodiments, a CD79b-specific antibody or antigen-binding fragment thereof is provided, comprising a heavy chain comprising the CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1a. In some embodiments, a CD79b-specific antibody or antigen-binding fragment thereof is provided, comprising a heavy chain comprising the CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1a, and a light chain comprising the CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1a.

[0378] The heavy and light chain variable domains of the antibodies discussed in this section are suitable for inclusion in multispecific (e.g., bispecific or trispecific) constructs, in which one arm is an anti-CD79b arm. Exemplary trispecific constructs comprising the CD79b-specific antibodies, or antigen-binding fragments thereof, discussed in this section are provided herein.

[0379] In some embodiments, CD79b-specific antibodies and antigen-binding fragments bind to human CD79b and cynomolgus monkey CD79b. In some embodiments, CD79b-specific antibodies and antigen-binding fragments bind to human CD79b but not cynomolgus monkey CD79b. In some embodiments, CD79b-specific antibodies and antigen-binding fragments bind to an epitope comprising one or more residues from the CD79b extracellular domain (ECD). In some embodiments, a CD79b-binding arm binds to one or more residues of a polypeptide having the amino acid sequence of SEQ ID NO: 252. In some embodiments, a CD79b-binding arm binds to residues 30-42 (SEDRYRNPKGSAC, SEQ ID NO: 253), residues 50-52 (PRF), residues 81-86 (EMENP, SEQ ID NO: 254), and / or residues 144-148 (GFSTL, SEQ ID NO: 255) of human CD79b. Such CD79b-binding arms may be used in combination with a 5×10 -7 M or less, e.g., 1×10 -7 M or less, 5×10 -8 M or less, 1×10 -8 M or less, 5×10 -9 M or less, 1×10-9 M, or 5 x 10 -10 In one embodiment, the CD79b-binding arm can bind to CD79b with an affinity of about 1×10 M or less. -11 M~1×10 -9 In one embodiment, the CD79b-binding arm binds to CD79b with an affinity of about 1 x 10 -11 M, approx. 2 x 10 -11 M, about 3 x 10 -11 M, approx. 4 x 10 -11 M, about 5 x 10 -11 M, about 6 x 10 -11 M, about 7 x 10 -11 M, about 8 x 10 -11 M, about 9 x 10 -11 M, about 1 x 10 -10 M, about 2 x 10 -10 M, about 3 x 10 -10 M, approx. 4 x 10 -10 M, about 5 x 10 -10 M, about 6 x 10 -10 M, about 7 x 10 -10 M, about 8 x 10 -10 M, about 9 x 10 -10 M, or approximately 1 x 10 -9 Binds to CD79b with an affinity of M.

[0380] In humans, the IgG class is divided into four isotypes: IgG1, IgG2, IgG3, and IgG4. These share greater than 95% homology in the amino acid sequence of the Fc region but exhibit major differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (FcγR) on the surface of immune effector cells, such as natural killer cells and macrophages, resulting in the phagocytosis or lysis of the target cell. In CDC, the antibody kills the target cell by triggering the complement cascade on the cell surface. The antibodies described herein include antibodies having the described characteristics of the variable domain in combination with any of the IgG isotypes, including modified versions in which the Fc sequence has been modified to confer different effector functions.

[0381] For many therapeutic antibody applications, Fc-mediated effector functions are not responsible for the mechanism of action. These Fc-mediated effector functions can be harmful by causing extramechanistic toxicity and pose safety risks. Altered effector function can be achieved by genetically engineering the Fc region to reduce binding to FcγRs or complement factors. Binding of IgGs to activating (FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb) and inhibitory (FcγRIIb) FcγRs or the first component of complement (C1q) is determined by residues located in the hinge region and CH2 domain. Mutations introduced into IgG1, IgG2, and IgG4 reduce or silence Fc function. The antibodies described herein may contain these modifications.

[0382] In one embodiment, the antibody comprises an Fc region having one or more of the following properties: (a) reduced effector function when compared to the parent Fc; (b) reduced affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIb and / or FcγRIIIa; (c) reduced affinity for FcγRI; (d) reduced affinity for FcγRIIa; (e) reduced affinity for FcγRIIb; (f) reduced affinity for FcγRIIIb; or (g) reduced affinity for FcγRIIIa.

[0383] In some embodiments, the antibody or antigen-binding fragment is an IgG or derivative thereof, such as an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, where the antibody has an IgG1 isotype, the antibody comprises L234A, L235A, D265S, and / or K409R substitutions in its Fc region. In some embodiments, where the antibody has an IgG4 isotype, the antibody comprises K409R, S228P, L234A, and L235A substitutions in its Fc region. The antibodies described herein may comprise these modifications.

[0384] In some embodiments, the described antibodies may be capable of inhibiting APRIL binding at low nanomolar concentrations as measured by ELISA. 50

[0385] In some embodiments, the described antibodies bind to CD79b-positive multiple myeloma cell lines.

[0386] In addition to the described CD79b-specific antibodies and antigen-binding fragments, polynucleotide sequences capable of encoding the described antibodies and antigen-binding fragments are also provided. Vectors containing the described polynucleotides are also provided, and similarly, cells expressing the CD79b-specific antibodies and antigen-binding fragments provided herein are provided. Also described are cells capable of expressing the disclosed vectors. These cells may be mammalian cells (e.g., 293F cells, CHO cells), insect cells (e.g., Sf7 cells), yeast cells, plant cells, or bacterial cells (e.g., E. coli). The described antibodies can also be produced by hybridoma cells.

[0387] The described CD79b-specific antibodies or antigen-binding fragments include all isotypes, IgA, IgD, IgE, IgG, and IgM, as well as synthetic multimers of the four-chain immunoglobulin structure. The described antibodies or antigen-binding fragments also include the IgY isotype commonly found in the sera of female birds or hens and in the egg yolks of female birds or hens.

[0388] The CD79b-specific antibodies and antigen-binding fragments can be derived from any species by recombinant techniques. For example, the antibody or antigen-binding fragment may be murine, rat, goat, horse, pig, bovine, chicken, rabbit, camel, llama, human, or chimeric versions thereof. For use in administration to humans, non-human-derived antibodies or antigen-binding fragments may be genetically or structurally altered to be less antigenic when administered to a human patient.

[0389] ​In some embodiments, the antibody or antigen-binding fragment is chimeric. As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment thereof that has at least some portion of at least one variable domain derived from a non-human mammalian, rodent, or reptilian antibody amino acid sequence, while the remainder is of human origin.

[0390] In some embodiments, the antibody is a humanized antibody. A humanized antibody may be a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0391] The antibodies or antigen-binding fragments described herein may exist in various forms, but comprise one or more of the antibody CDRs shown in Table 1a.

[0392] Described herein are recombinant antibodies and antigen-binding fragments that bind to CD79b. In some embodiments, the CD79b-specific antibodies or antigen-binding fragments are human IgG or derivatives thereof. The CD79b-specific antibodies or antigen-binding fragments exemplified herein are human, although the exemplified antibodies or antigen-binding fragments may also be chimerized.

[0393] In some embodiments, the antibody or antigen-binding fragment is an IgG or derivative thereof, such as an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, where the antibody is of the IgG1 isotype, the antibody comprises the Fc region of IgG1 (SEQ ID NO: 249). SEQ ID NO: 249 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0394] In some embodiments, where the antibody is of the IgG1 isotype, the antibody comprises L234A, L235A, and D265S substitutions (underlined) in its Fc region (SEQ ID NO: 250). SEQ ID NO: 250

[0395] [Table 9]

[0396] In some embodiments where the antibody is of the IgG4 isotype, the antibody comprises S228P, L234A, and L235A substitutions in its Fc region (SEQ ID NO: 251). SEQ ID NO: 251

[0397] [Table 10]

[0398] The CD79b-specific antibodies defined by the CDR and / or variable domain sequences discussed in the above paragraphs may include these IgG Fc regions.

[0399] Also disclosed are isolated synthetic polynucleotides encoding an antibody or antigen-binding fragment that binds to CD79b. The isolated polynucleotides capable of encoding the variable domain segments provided herein may be included in the same or different vectors that generate the antibody or antigen-binding fragment.

[0400] Polynucleotides encoding recombinant antigen-binding proteins are also within the scope of the present disclosure. In some embodiments, the polynucleotides described (and the peptides they encode) include a leader sequence. Any leader sequence known in the art can be utilized. The leader sequence can include, but is not limited to, a restriction site or a translation initiation site.

[0401] The CD79b - specific antibodies or antigen - binding fragments described herein include variants having one or more amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described CD79b - specific antibodies or antigen - binding fragments. In the context of the present invention, unless otherwise specified, the following notations are used to describe mutations. i) An amino acid substitution at a given position is described, for example, as K409R. K409R means the substitution of lysine at position 409 with arginine. ii) For a particular variant, the specific three - letter or one - letter code is used to denote amino acid residues, including the codes Xaa and X. Thus, the substitution of lysine at position 409 with arginine is designated as K409R, or the substitution of lysine at position 409 with any amino acid residue is designated as K409X. In the case of a deletion of lysine at position 409, this deletion is indicated by K409 * as shown. One of ordinary skill in the art can generate variants having one or more amino acid substitutions, deletions, or additions.

[0402] These variants include: (a) variants in which one or more amino acid residues are substituted with conservative or non - conservative amino acids; (b) variants in which one or more amino acids are added to or deleted from the polypeptide; (c) variants in which one or more amino acids contain substituents; and (d) variants in which the polypeptide is fused with another peptide or polypeptide, such as a fusion partner, protein tag, or other chemical moiety, that can confer useful properties on the polypeptide, such as an epitope for an antibody, a polyhistidine sequence, a biotin moiety, etc. The antibodies or antigen - binding fragments described herein may include variants in which amino acid residues from one species are substituted with corresponding residues from another species, either at conservative or non - conservative positions. In other embodiments, amino acid residues at non - conservative positions are substituted with conservative or non - conservative residues. Methods for obtaining these variants are known to those of skill in the art and include genetic (deletion, mutation, etc.), chemical, and enzymatic methods.

[0403] The CD79b-specific antibodies or antigen-binding fragments described herein can embody several antibody isotypes, such as IgM, IgD, IgG, IgA, and IgE. In some embodiments, the antibody isotype is an isotype of IgG1, IgG2, IgG3, or IgG4, preferably an isotype of IgG1 or IgG4. The specificity of the antibody or its antigen-binding fragment is mainly determined by the amino acid sequence and arrangement of the CDRs. Thus, the CDRs of one isotype can be changed to another isotype without changing the antigen specificity. Alternatively, techniques (isotype switching) have been established to switch one antibody isotype to another in hybridomas without changing the antigen specificity. Thus, such antibody isotypes are within the scope of the antibodies or antigen-binding fragments described.

[0404] Vectors containing the polynucleotides described herein are also provided. The vector can be an expression vector. Thus, recombinant expression vectors containing a sequence encoding a polypeptide of interest are contemplated to be within the scope of the present disclosure. The expression vector may contain one or more additional sequences such as, but not limited to, control sequences (e.g., promoter, enhancer), selection markers, and polyadenylation signals. Vectors for transforming a wide range of host cells are well known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bactermids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and other bacterial, yeast, and viral vectors.

[0405] Recombinant expression vectors within the scope of this description comprise a nucleic acid fragment of synthetic, genomic, or cDNA origin encoding at least one recombinant protein that can be operably linked to suitable regulatory elements. Such regulatory elements can include a transcriptional promoter, a sequence encoding a suitable mRNA ribosomal binding site, and sequences that control transcription and translation termination. Expression vectors, particularly mammalian expression vectors, can also contain one or more non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences (e.g., essential ribosomal binding sites), a polyadenylation site, splice donor and acceptor sites, or a transcription termination sequence. An origin of replication that confers the ability to replicate in a host can also be incorporated.

[0406] Transcriptional and translational control sequences in expression vectors used to transform vertebrate cells can be provided by viral sources. Exemplary vectors can be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

[0407] In some embodiments, the coding sequence of the antibody or antigen-binding fragment is placed under the control of a strong constitutive promoter, such as a promoter for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, etc. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the described embodiments. Such viral promoters include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the early and late promoters of SV40, the mouse mammary tumor virus (MMTV) promoter, Moloney leukemia virus, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and the long terminal repeat sequences (LTRs) of other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. In one embodiment, the coding sequence of the CD79b-specific antibody or its antigen-binding fragment is placed under the control of an inducible promoter, such as a metallothionein promoter, a tetracycline-inducible promoter, a doxycycline-inducible promoter, one or more interferon-stimulated response elements (ISREs), such as a promoter containing protein kinase R 2’,5’-oligoadenylate synthetase, Mx gene, ADAR1, etc.

[0408] The vectors described herein may contain one or more internal ribosome entry sites (IRES). Inclusion of an IRES sequence in a fusion vector can be beneficial for enhancing the expression of some proteins. In some embodiments, the vector system will contain one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. The components of the vector may be linked in proximity or arranged to provide an optimal spacing for expressing the gene product (i.e., by introducing "spacer" nucleotides between ORFs) or otherwise positioned. Also, regulatory elements, such as IRES motifs, may be arranged to provide an optimal spacing for expression.

[0409] The vector may contain selectable markers known in the art. Selectable markers include positive and negative selectable markers, such as antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene, puromycin resistance gene, blasticidin resistance gene), glutamate synthase gene, HSV-TK for ganciclovir selection, HSV-TK derivatives, or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). The nucleic acid sequence or cloning site encoding the selectable marker may be upstream or downstream of the nucleic acid sequence or cloning site encoding the polypeptide of interest.

[0410] Using the vectors described herein, various cells can be transformed with a gene encoding the described antibody or antigen-binding fragment. For example, a vector can be used to generate CD79b-specific antibody or antigen-binding fragment-producing cells. Accordingly, another aspect features a host cell transformed with a vector comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that binds to CD79b, such as the antibodies or antigen-binding fragments described and exemplified herein.

[0411] Numerous methods for introducing foreign genes into cells are known in the art and can be used to create recombinant cells for the purpose of performing the methods described according to the various embodiments described and exemplified herein. The method used must stably introduce the heterologous gene sequence into the host cell such that the heterologous gene sequence is heritable and expressible by the progeny of the cell and does not impair the essential growth and physiological functions of the recipient cell. Methods that can be used include chromosome introduction methods (e.g., cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carriers), virus vector introduction (e.g., recombinant DNA virus, recombinant RNA virus), etc. (Cline, 29 Pharmac. Ther. 69-92 (1985)). Cells can also be transformed using calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts by mammalian cells.

[0412] Cells suitable for use in expressing the CD79b - specific antibodies or antigen - binding fragments described herein are preferably eukaryotic cells, more preferably cells of plant, rodent, or human origin, such as, inter alia, NSO, CHO, CHOK1, perC.6, Tk - ts13, BHK, HEK293 cells, COS - 7, T98G, CV - 1 / EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2 / 0 myeloma cells, and BHK cell lines, etc., but are not limited thereto. Additionally, the expression of the antibody may be achieved using hybridoma cells. Methods for generating hybridomas are well - established in the art.

[0413] Cells transformed with the expression vectors described herein may be selected or screened for the recombinant expression of the antibodies or antigen - binding fragments described herein. Recombinant positive cells are grown and screened for sub - clones that exhibit the ability to produce a protein with a desired phenotype, such as high - level expression, enhanced growth characteristics, or desired biochemical properties, for example, by protein modification or altered post - translational modification. These phenotypes can be due to the native properties or mutations of a given sub - clone. Mutations can occur by chemicals, UV - wavelength light, irradiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or combinations of such methods.

[0414] Methods of using CD79b - specific antibodies for treatment Provided herein are CD79b - specific antibodies or antigen - binding fragments thereof for use in therapy. Specifically, these antibodies or antigen - binding fragments may be useful for treating cancer, such as CD79b - expressing cancer. Accordingly, the present invention provides a method of treating cancer, comprising administering an antibody as described herein, such as a CD79b - specific antibody or antigen - binding fragment. For example, its use is by interfering with CD79b receptor interaction or, when the antibody is conjugated to a toxin, by targeting the toxin in such a way to CD79b - expressing cancer. In some embodiments, CD79b - expressing cancer includes lymphomas such as diffuse large B - cell lymphoma (DLBCL). Antibodies for use in these methods include those described hereinabove, such as CD79b - specific antibodies or antigen - binding fragments having the characteristics described in Tables 1a and 1b, such as CDR or variable domain sequences, and those in further investigations of these antibodies.

[0415] In some embodiments described herein, the immune effector properties of CD79b - specific antibodies are known to those skilled in the art and can be enhanced or silenced by Fc modification by the techniques described herein. For example, Fc effector functions, such as Clq binding,...

Claims

1. A trispecific antibody or a trispecific binding fragment thereof, comprising: a first antigen-binding arm that binds to an epitope on the cluster of differentiation 79B protein (CD79b), a second antigen-binding arm that binds to an epitope on the cluster of differentiation 3 (CD3), and a third antigen-binding arm that binds to an epitope on the cluster of differentiation 20 (CD20), wherein (i) the first antigen-binding arm comprises a) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively, b) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 13, 8, 9, 10, 11, and 12, respectively, c) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 7, 8, 9, 10, 11, and 12, respectively, d) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 14, 15, 16, 17, 5, and 6, respectively, e) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 18, 8, 19, 20, 21, and 12, respectively, f) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 22, 23, 24, 25, 5, and 6, respectively, g) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 22, 26, 27, 28, 5, and 29, respectively, or h) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 30, 31, 32, 33, 5, and 6, respectively; wherein (ii) the second antigen-binding arm comprises a) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 76, 77, 78, 79, 80, and 81, respectively, b) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 76, 77, 75, 79, 80, and 81, respectively, c) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 76, 77, 82, 79, 80, and 81, respectively, or d) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 83, 84, 85, 86, 87, and 88, respectively; and wherein (iii) the third antigen-binding arm comprises a) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 115, 116, 117, 118, 119, and 120, respectively, b) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 121, 122, 123, 124, 119, and 125, respectively, c) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 115, 116, 95, 96, 119, and 125, respectively, or d) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of SEQ ID NOs: 121, 116, 123, 124, 119, and 125, respectively; a trispecific antibody or a trispecific binding fragment thereof.

2. The first antigen-binding arm that binds to CD79b comprises a) VH1 of SEQ ID NO: 35 and VL1 of SEQ ID NO: 37, b) VH1 of SEQ ID NO: 39 and VL1 of SEQ ID NO: 41, c) VH1 of SEQ ID NO: 43 and VL1 of SEQ ID NO: 41, d) VH1 of SEQ ID NO: 45 and VL1 of SEQ ID NO: 47, e) VH1 of SEQ ID NO: 49 and VL1 of SEQ ID NO: 51, f) VH1 of SEQ ID NO: 39 and VL1 of SEQ ID NO: 53, g) VH1 of SEQ ID NO: 55 and VL1 of SEQ ID NO: 57, h) VH1 of SEQ ID NO: 59 and VL1 of SEQ ID NO: 61, i) VH1 of SEQ ID NO: 63 and VL1 of SEQ ID NO: 65, j) VH1 of SEQ ID NO: 67 and VL1 of SEQ ID NO: 69, or k) VH1 of SEQ ID NO: 71 and VL1 of SEQ ID NO: 73, and the second antigen-binding arm that binds to CD3 is a) VH2 of SEQ ID NO: 97 and VL2 of SEQ ID NO: 99, b) VH2 of SEQ ID NO: 101 and VL2 of SEQ ID NO: 99, c) VH2 of SEQ ID NO: 103 and VL2 of SEQ ID NO: 99, d) VH2 of SEQ ID NO: 105 and VL2 of SEQ ID NO: 99, or e) VH2 of SEQ ID NO: 107 and VL2 of SEQ ID NO: 109, and the third antigen-binding arm that binds to CD20 is a) VH3 of SEQ ID NO: 126 and VL3 of SEQ ID NO: 128, b) VH3 of SEQ ID NO: 130 and VL3 of SEQ ID NO: 132, c) VH3 of SEQ ID NO: 134 and VL3 of SEQ ID NO: 136, or d) VH3 of SEQ ID NO: 138 and VL3 of SEQ ID NO: 140, and the trispecific antibody or trispecific binding fragment according to claim 1.

3. The first antigen-binding arm that binds to CD79b comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively, the second antigen-binding arm that binds to CD3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 83, 84, 85, 86, 87, and 88, respectively, the third antigen-binding arm that binds to CD20 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 121, 122, 123, 124, 119, and 125, respectively, the trispecific antibody or trispecific binding fragment according to claim 1.

4. The first antigen-binding arm that binds to CD79b comprises VH1 of SEQ ID NO: 35 and VL1 of SEQ ID NO: 37, the second antigen-binding arm that binds to CD3 comprises VH2 of SEQ ID NO: 107 and VL2 of SEQ ID NO: 109, The triple - specific antibody or triple - specific binding fragment according to any one of claims 1 to 3, wherein the third antigen - binding arm that binds to CD20 comprises VH3 of SEQ ID NO: 130 and VL3 of SEQ ID NO:

132.

5. The first antigen - binding arm that binds to CD79b comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively, The second antigen - binding arm that binds to CD3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 76, 77, 75, 79, 80, and 81, respectively, The triple - specific antibody or triple - specific binding fragment according to claim 1, wherein the third antigen - binding arm that binds to CD20 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 121, 122, 123, 124, 119, and 125, respectively.

6. The first antigen - binding arm that binds to CD79b comprises VH1 of SEQ ID NO: 35 and VL1 of SEQ ID NO:

37. The second antigen - binding arm that binds to CD3 comprises VH2 of SEQ ID NO: 97 and VL2 of SEQ ID NO:

99. The triple - specific antibody or triple - specific binding fragment according to any one of claims 1, 2, and 5, wherein the third antigen - binding arm that binds to CD20 comprises VH3 of SEQ ID NO: 130 and VL3 of SEQ ID NO:

132.

7. a) The first antigen - binding arm comprises a heavy chain 1 (HC1) comprising the amino acid sequence of SEQ ID NO: 172 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 174, and the polypeptide comprising the second antigen - binding arm and the third antigen - binding arm comprises the amino acid sequence of SEQ ID NO:

142. b) The first antigen - binding arm comprises an HC1 comprising the amino acid sequence of SEQ ID NO: 176 and an LC comprising the amino acid sequence of SEQ ID NO: 178, and the polypeptide comprising the second antigen - binding arm and the third antigen - binding arm comprises the amino acid sequence of SEQ ID NO:

142. c) The first antigen - binding arm comprises an HC1 comprising the amino acid sequence of SEQ ID NO: 180 and an LC comprising the amino acid sequence of SEQ ID NO: 182, and the polypeptide comprising the second antigen - binding arm and the third antigen - binding arm comprises the amino acid sequence of SEQ ID NO:

142. d) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 172 and LC containing the amino acid sequence of SEQ ID NO: 174, and the polypeptide comprising the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 144, e) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 176 and LC containing the amino acid sequence of SEQ ID NO: 178, and the polypeptide comprising the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 144, f) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 180 and LC containing the amino acid sequence of SEQ ID NO: 182, and the polypeptide comprising the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 144, g) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 180 and LC containing the amino acid sequence of SEQ ID NO: 182, and the polypeptide comprising the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 148, h) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 180 and LC containing the amino acid sequence of SEQ ID NO: 182, and the polypeptide comprising the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 150, i) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 180 and LC containing the amino acid sequence of SEQ ID NO: 182, and the polypeptide comprising the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 152, j) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 180 and LC containing the amino acid sequence of SEQ ID NO: 182, and the polypeptide comprising the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 154, k) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 180 and LC containing the amino acid sequence of SEQ ID NO: 182, and the polypeptide comprising the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 156, l) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 180 and LC containing the amino acid sequence of SEQ ID NO: 182, and the polypeptide containing the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 158, m) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 180 and LC containing the amino acid sequence of SEQ ID NO: 182, and the polypeptide containing the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 160, n) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 180 and LC containing the amino acid sequence of SEQ ID NO: 182, and the polypeptide containing the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 162, o) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 191 and LC containing the amino acid sequence of SEQ ID NO: 182, and the polypeptide containing the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 166, p) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 172 and LC containing the amino acid sequence of SEQ ID NO: 174, and the polypeptide containing the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 168, or q) The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 172 and LC containing the amino acid sequence of SEQ ID NO: 174, and the polypeptide containing the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 170, the trispecific antibody or trispecific binding fragment according to any one of claims 1 to 6.

8. The first antigen-binding arm comprises HC1 containing the amino acid sequence of SEQ ID NO: 172 and LC containing the amino acid sequence of SEQ ID NO: 174, and the polypeptide containing the second antigen-binding arm and the third antigen-binding arm contains the amino acid sequence of SEQ ID NO: 170, the trispecific antibody or trispecific binding fragment according to any one of claims 1 to 7.

9. A synthetic polynucleotide encoding the trispecific antibody or trispecific binding fragment according to any one of claims 1 to 8.

10. A pharmaceutical composition for use in a method for performing it in a subject in need of cancer treatment, comprising the trispecific antibody or trispecific binding fragment according to any one of claims 1 to 8, wherein the method comprises administering to the subject a therapeutically effective amount of the trispecific antibody or trispecific binding fragment.

11. The pharmaceutical composition according to claim 10, wherein the cancer is diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), or Waldenström macroglobulinemia (WM).

12. An antibody or antigen-binding fragment thereof that binds to an epitope on the differentiation antigen group 79B protein (CD79b), a) SEQ ID NOs: 1, 2, 3, 4, 5, and 6, b) SEQ ID NOs: 13, 8, 9, 10, 11, and 12, c) SEQ ID NOs: 7, 8, 9, 10, 11, and 12, d) SEQ ID NOs: 14, 15, 16, 17, 5, and 6, e) SEQ ID NOs: 18, 8, 19, 20, 21, and 12, f) SEQ ID NOs: 22, 23, 24, 25, 5, and 6, g) SEQ ID NOs: 22, 26, 27, 28, 5, and 29, or h) an antibody or antigen-binding fragment comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 30, 31, 32, 33, 5, and 6, respectively.

13. a) VH of SEQ ID NO: 35 and VL of SEQ ID NO: 37, b) VH of SEQ ID NO: 39 and VL of SEQ ID NO: 41, c) VH of SEQ ID NO: 43 and VL of SEQ ID NO: 41, d) VH of SEQ ID NO: 45 and VL of SEQ ID NO: 47, e) VH of SEQ ID NO: 49 and VL of SEQ ID NO: 51, f) VH of SEQ ID NO: 39 and VL of SEQ ID NO: 53, g) VH of SEQ ID NO: 55 and VL of SEQ ID NO: 57, h) VH of SEQ ID NO: 59 and VL of SEQ ID NO: 61, i) VH of SEQ ID NO: 63 and VL of SEQ ID NO: 65, j) VH of SEQ ID NO: 67 and VL of SEQ ID NO: 69, or k) an antibody or antigen-binding fragment according to claim 12, comprising VH of SEQ ID NO: 71 and VL of SEQ ID NO:

73.

14. A synthetic polynucleotide encoding the antibody or antigen-binding fragment according to claim 12 or 13.

15. A bispecific antibody or a bispecific binding fragment thereof, comprising a first antigen-binding arm that binds to an epitope on the cluster of differentiation 79B protein (CD79b) and a second antigen-binding arm that binds to an epitope on the cluster of differentiation 3 (CD3), wherein the first antigen-binding arm a) has VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 1, 2, 3, 4, 5, and 6 respectively, b) has VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 13, 8, 9, 10, 11, and 12 respectively, c) has VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 7, 8, 9, 10, 11, and 12 respectively, d) has VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 14, 15, 16, 17, 5, and 6 respectively, e) has VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 18, 8, 19, 20, 21, and 12 respectively, f) has VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 22, 23, 24, 25, 5, and 6 respectively, g) has VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 22, 26, 27, 28, 5, and 29 respectively, or h) has VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 30, 31, 32, 33, 5, and 6 respectively, and wherein the second antigen-binding arm a) has VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 76, 77, 78, 79, 80, and 81 respectively, b) has VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 76, 77, 75, 79, 80, and 81 respectively, c) has VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 76, 77, 82, 79, 80, and 81 respectively, or d) has VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 selected from SEQ ID NOs: 83, 84, 85, 86, 87, and 88 respectively, A bispecific antibody or a bispecific binding fragment thereof.

16. The first antigen-binding arm that binds to CD79b a) comprises VH of SEQ ID NO: 35 and VL of SEQ ID NO: 37, b) comprises VH of SEQ ID NO: 39 and VL of SEQ ID NO: 41, c) comprises VH of SEQ ID NO: 43 and VL of SEQ ID NO: 41, d) comprises VH of SEQ ID NO: 45 and VL of SEQ ID NO: 47, e) comprises VH of SEQ ID NO: 49 and VL of SEQ ID NO: 51, f) comprises VH of SEQ ID NO: 39 and VL of SEQ ID NO: 53, g) comprises VH of SEQ ID NO: 55 and VL of SEQ ID NO: 57, h) comprises VH of SEQ ID NO: 59 and VL of SEQ ID NO: 61, i) comprises VH of SEQ ID NO: 63 and VL of SEQ ID NO: 65, j) comprises VH of SEQ ID NO: 67 and VL of SEQ ID NO: 69, or k) comprises VH of SEQ ID NO: 71 and VL of SEQ ID NO: 73, The second antigen-binding arm that binds to CD3 a) comprises VH of SEQ ID NO: 97 and VL of SEQ ID NO: 99, b) comprises VH of SEQ ID NO: 101 and VL of SEQ ID NO: 99, c) comprises VH of SEQ ID NO: 103 and VL of SEQ ID NO: 99, d) VH of SEQ ID NO: 105 and VL of SEQ ID NO: 99, or e) VH of SEQ ID NO: 107 and VL of SEQ ID NO: 109, comprising a bispecific antibody or bispecific binding fragment according to claim 15. The bispecific antibody or bispecific binding fragment according to claim 15. **Claim 17** The first antigen-binding arm comprises a heavy chain 1 (HC1) polypeptide and a light chain (LC) polypeptide, and the second antigen-binding arm comprises a second antigen-binding arm polypeptide, a) the HC1 comprises the amino acid sequence of SEQ ID NO: 172, the LC comprises the amino acid sequence of SEQ ID NO: 174, and the second antigen-binding arm polypeptide comprises the amino acid sequence of SEQ ID NO: 164, b) the HC1 comprises the amino acid sequence of SEQ ID NO: 176, the LC comprises the amino acid sequence of SEQ ID NO: 178, and the second antigen-binding arm polypeptide comprises the amino acid sequence of SEQ ID NO: 164, c) the HC1 comprises the amino acid sequence of SEQ ID NO: 180, the LC comprises the amino acid sequence of SEQ ID NO: 182, and the second antigen-binding arm polypeptide comprises the amino acid sequence of SEQ ID NO: 164, d) the HC1 comprises the amino acid sequence of SEQ ID NO: 172, the LC comprises the amino acid sequence of SEQ ID NO: 174, and the second antigen-binding arm polypeptide comprises the amino acid sequence of SEQ ID NO: 189, e) the HC1 comprises the amino acid sequence of SEQ ID NO: 176, the LC comprises the amino acid sequence of SEQ ID NO: 178, and the second antigen-binding arm polypeptide comprises the amino acid sequence of SEQ ID NO: 189, or f) the HC1 comprises the amino acid sequence of SEQ ID NO: 180, the LC comprises the amino acid sequence of SEQ ID NO: 182, and the second antigen-binding arm polypeptide comprises the amino acid sequence of SEQ ID NO: 189, the bispecific antibody or bispecific binding fragment according to claim 15 or 16. **Claim 18** A synthetic polynucleotide encoding the bispecific antibody or bispecific binding fragment according to any one of claims 15 to 17. **Claim 19** A pharmaceutical composition for use in a method for performing the same in a subject in need of treatment for cancer, comprising the bispecific antibody or bispecific binding fragment according to any one of claims 15 to 17, wherein the method comprises administering to the subject a therapeutically effective amount of the bispecific antibody or bispecific binding fragment. **Claim 20** The pharmaceutical composition according to claim 19, wherein the cancer is diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), or Waldenström macroglobulinemia (WM).

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