Administration for treatment with anti-CD20 / anti-CD3 bispecific antibodies

Abstract

The present invention provides methods of administration for the treatment of cancer, such as B-cell proliferative disorders, with anti-cluster of differentiation 20 (CD20) / anti-cluster of differentiation 3 (CD3) bispecific antibodies. [Selection diagram] None

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on November 13, 2017, is designated 50474-150WO3_Sequence_Listing_11.13.17_ST25 and is 21,101 bytes in size.

[0002] The present invention relates to the treatment of cancer, such as B-cell proliferative disorders. More particularly, the present invention relates to the specific treatment of human patients with B-cell proliferative disorders using anti-cluster of differentiation 20 (CD20) / anti-cluster of differentiation 3 (CD3) bispecific antibodies. [Background technology]

[0003] Cancer is characterized by the uncontrolled proliferation of a subpopulation of cells. It is the leading cause of death in the developed world and the second leading cause of death in developing countries, with over 14 million new cancer cases diagnosed and over 8 million cancer deaths occurring each year. The National Cancer Institute estimates that over 500,000 Americans will die from cancer in 2016, accounting for nearly one in four deaths in the United States. As the elderly population grows, so does the incidence of cancer. Cancer treatment therefore represents a significant and growing societal burden.

[0004] Hematological cancers, in particular, are the second leading cause of cancer-related deaths. Hematological cancers include B-cell proliferative disorders, such as non-Hodgkin's lymphoma (NHL) (e.g., diffuse large B-cell lymphoma (DLBCL)), which progress rapidly and, if untreated, are fatal. While treatment with the monoclonal anti-cluster of differentiation 20 (CD20) antibody rituximab reduces the number of relapsed DLBCL patients, it becomes increasingly difficult to treat these patients with relapsed or refractory DLBCL. For these patients, alternative or secondary treatment modalities, such as bispecific antibody-based immunotherapy, can be particularly effective. Bispecific antibodies can simultaneously bind cell surface antigens on cytotoxic cells (e.g., T cells via binding to cluster of differentiation 3 (CD3)) and cancer cells (e.g., B cells via binding to CD20) with the intent that the bound cytotoxic cells will destroy the bound cancer cells. However, antibody-based immunotherapies such as these can be limited by unwanted effects, including cytokine-driven toxicities (e.g., cytokine release syndrome (CRS)), infusion-related reactions (IRR), severe tumor lysis syndrome (TLS), and central nervous system (CNS) toxicity.

[0005] Thus, there is an unmet need in the field for the development of effective methods of administering therapeutic bispecific antibodies (e.g., anti-CD20 / anti-CD3 bispecific antibodies) for the treatment of cancer (e.g., B-cell proliferative disorders) that achieve a more favorable benefit-risk profile. Summary of the Invention

[0006] The present invention relates to methods of treating a subject with cancer (e.g., a B-cell proliferative disorder) using an anti-cluster of differentiation 20 (CD20) / anti-cluster of differentiation 3 (CD3) bispecific antibody.

[0007] In one aspect, the invention features a method of treating a subject with cancer (e.g., a B-cell proliferative disorder) comprising administering to the subject a bispecific antibody that binds CD20 and CD3 in a dosing regimen including at least a first and a second dosing cycle, wherein: (a) the first dosing cycle comprises a first dose (C1D1), a second dose (C1D2), and a third dose (C1D3) of the bispecific antibody, wherein C1D1 and C1D2 are each no greater than C1D3; and (b) the second dosing cycle comprises a single dose (C2D1) of the bispecific antibody, wherein C2D1 is equal to or greater than C1D3, and wherein C1D1, C1D2, and C1D3 comprise a cumulative dose that is about 50% greater than the highest clarified dose of the bispecific antibody in the first dosing cycle of the non-fractionated dose escalation regimen, wherein the highest clarified dose is between about 0.2 mg and about 30 mg. In some embodiments, C1D3 is equal to the highest clarified dose of the bispecific antibody in the first administration cycle of the non-fractionated dose escalation regimen. In some embodiments, C1D2 and C1D1 are equal. In some embodiments, C1D2 is about 50% to about 250% greater than C1D1. In some embodiments, C1D3 is about 150% to about 300% greater than C1D2.

[0008] In another aspect, the invention features a method of treating a subject having a B-cell proliferative disorder, comprising administering to the subject a bispecific antibody that binds CD20 and CD3 in a dosing regimen having at least a first and a second dosing cycle, where (a) the first dosing cycle has a first dose (C1D1), a second dose (C1D2), and a third dose (C1D3) of the bispecific antibody, where C1D1 and C1D2 are each no greater than C1D3, C1D1 being between about 0.0056 mg and about 12.50 mg, C1D2 being between about 0.0125 mg and about 20.00 mg, and C1D3 being between about 0.0500 mg and about 50.00 mg, and (b) the second dosing cycle has a single dose of the bispecific antibody, where C2D1 is equal to or greater than C1D3 and is between about 0.0500 mg and about 50.00 mg. In some embodiments, (a) C1D1 is between about 0.02 mg and about 4.0 mg, C1D2 is between about 0.05 mg and about 20.0 mg, and C1D3 is between about 0.2 mg and about 50.0 mg, and (b) C2D1 is between about 0.2 mg and about 50.0 mg. In some embodiments, (a) C1D1 is between about 0.4 mg and about 4.0 mg, C1D2 is between about 1.0 mg and about 20.0 mg, and C1D3 is between about 3.0 mg and about 50.0 mg, and (b) C2D1 is between about 3.0 mg and about 50.0 mg. In some embodiments, (a) C1D1 is between about 0.4 mg and about 4.0 mg, C1D2 is between about 1.0 mg and about 20.0 mg, and C1D3 is between about 3.0 mg and about 20.0 mg, and (b) C2D1 is between about 3.0 mg and about 20.0 mg. In some embodiments, (a) C1D1 is between about 0.8 mg and about 3.0 mg, C1D2 is between about 1.0 mg and about 6.0 mg, and C1D3 is between about 3.0 mg and about 50.0 mg, and (b) C2D1 is between about 3.0 mg and about 50.0 mg.In some embodiments, (a) C1D1 is between about 0.8 mg and about 3.0 mg, C1D2 is between about 1.0 mg and about 6.0 mg, and C1D3 is between about 3.0 mg and about 20.0 mg, (b) C2D1 is between about 3.0 mg and about 20.0 mg, In some embodiments, (a) C1D1 is about 1.0 mg, C1D2 is about 2.0 mg, and C1D3 is between about 3.0 mg and about 50.0 mg (e.g., C1D3 is about 6.0 mg), and (b) C2D1 is between about 3.0 mg and about 50.0 mg (e.g., C2D1 is about 6.0 mg). In some embodiments, (a) C1D1 is about 1.0 mg, C1D2 is about 2.0 mg, and C1D3 is between about 3.0 mg and about 50.0 mg, and (b) C2D1 is equal to C1D3. In some embodiments, (a) C1D1 is about 1.0 mg, C1D2 is about 2.0 mg, and C1D3 is about 6.0 mg, and (b) C2D1 is equal to C1D3. In some embodiments, (a) C1D1 is between about 0.02 mg and about 4.0 mg, C1D2 is between about 0.05 mg and about 20.0 mg, and C1D3 is between about 0.2 mg and about 20.0 mg, and (b) C2D1 is between about 3.0 mg and about 20.0 mg. In some embodiments, (a) C1D1 is between about 0.4 mg and about 4.0 mg, C1D2 is between about 1.0 mg and about 20.0 mg, and C1D3 is between about 3.0 mg and about 20.0 mg, and (b) C2D1 is between about 3.0 mg and about 20.0 mg. In some embodiments, (a) C1D1 is between about 0.8 mg and about 3.0 mg, C1D2 is between about 1.0 mg and about 6.0 mg, and C1D3 is between about 3.0 mg and about 20.0 mg, and (b) C2D1 is between about 3.0 mg and about 20.0 mg. In some embodiments, (a) C1D1 is between about 0.8 mg and about 3.0 mg, C1D2 is between about 1.0 mg and about 6.0 mg, and C1D3 is between about 3.0 mg and about 6.0 mg; and (b) C2D1 is between about 3.0 mg and about 6.0 mg.

[0009] In some embodiments, (a) C1D1 is about 0.8 mg, C1D2 is about 2.0 mg, and C1D3 is about 4.2 mg, and (b) C2D1 is about 4.2 mg. In some embodiments, (a) C1D1 is about 1.0 mg, C1D2 is about 1.0 mg, and C1D3 is about 3.0 mg, and (b) C2D1 is about 3.0 mg. In some embodiments, (a) C1D1 is about 1.0 mg, C1D2 is about 2.0 mg, and C1D3 is about 6.0 mg, and (b) C2D1 is about 6.0 mg. In some embodiments, (a) C1D1 is about 0.8 mg, C1D2 is about 2.0 mg, and C1D3 is about 6.0 mg, and (b) C2D1 is about 6.0 mg. In some embodiments of any of the above aspects, the length of the first administration cycle is 21 days. In some embodiments, the method comprises administering C1D1, C1D2, and C1D3 to the subject on or about days 1, 8, and 15, respectively, of the first administration cycle.

[0010] In some embodiments of any of the above aspects, the length of the second administration cycle is 21 days. In some embodiments, the method comprises administering C2D1 to the subject on day 1 of the second administration cycle.

[0011] In some embodiments of any of the above aspects, the dosing regimen includes one or more additional administration cycles. In some embodiments, the dosing regimen includes 1 to 14 additional administration cycles. In some embodiments, the dosing regimen includes 1 to 6 additional administration cycles. In some embodiments, the length of each of the one or more additional administration cycles is 7 days, 14 days, 21 days, or 28 days. In some embodiments, the length of each of the one or more additional administration cycles is 21 days. In some embodiments, each of the one or more additional administration cycles comprises a single dose of the bispecific antibody. In some embodiments, the method comprises administering to the subject a single dose of the one or more additional administration cycles on day 1 of the one or more additional administration cycles.

[0012] In some embodiments of any of the above aspects, the bispecific antibody comprises an anti-CD20 arm having a first binding region comprising the following six hypervariable regions (HVRs): (a) HVR-H1 comprising the amino acid sequence GYTFTSYNMH (SEQ ID NO: 1), (b) HVR-H2 comprising the amino acid sequence AIYPGNGDTSYNQKFKG (SEQ ID NO: 2), (c) HVR-H3 comprising the amino acid sequence VVYYSNSYWYFDV (SEQ ID NO: 3), (d) HVR-L1 comprising the amino acid sequence RASSSVSYMH (SEQ ID NO: 4), (e) HVR-L2 comprising the amino acid sequence APSNLAS (SEQ ID NO: 5), and (f) HVR-L3 comprising the amino acid sequence QQWSFNPPT (SEQ ID NO: 6). In some embodiments, the bispecific antibody comprises an anti-CD20 arm having a first binding domain comprising: (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO:7, (b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO:8, or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, the first binding domain comprises a VH domain comprising the amino acid sequence SEQ ID NO:7 and a VL domain comprising the amino acid sequence SEQ ID NO:8. In some embodiments, the bispecific antibody comprises an anti-CD3 arm having a second binding domain comprising the following six HVRs: (a) HVR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 9); (b) HVR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 10); (c) HVR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 11); (d) HVR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 12); (e) HVR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 13); and (f) HVR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 14).In some embodiments, the bispecific antibody comprises an anti-CD3 arm with a second binding domain comprising: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO: 15, (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO: 16, or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, the second binding domain comprises a VH domain comprising the amino acid sequence SEQ ID NO: 15, and a VL domain comprising the amino acid sequence SEQ ID NO: 16.

[0013] In some embodiments of any of the above aspects, the bispecific antibody comprises an aglycosylation site mutation. In some embodiments, the aglycosylation site mutation reduces effector function of the bispecific antibody. In some embodiments, the aglycosylation site mutation is a substitution mutation. In some embodiments, the bispecific antibody comprises a substitution mutation in the Fc region that reduces effector function. In some embodiments, the substitution mutation is at amino acid residues N297, L234, L235, and / or D265 (EU numbering). In some embodiments, the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, D265A, and P329G. In some embodiments, the substitution mutation is at amino acid residue N297. In some embodiments, the substitution mutation is N297A.

[0014] In some embodiments of any of the above aspects, the bispecific antibody is a monoclonal antibody. In some embodiments of any of the above aspects, the bispecific antibody is a humanized antibody. In some embodiments of any of the above aspects, the bispecific antibody is a chimeric antibody. In some embodiments of any of the above aspects, the bispecific antibody is an antibody fragment that binds CD20 and CD3. In some embodiments, the antibody fragment is selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments of any of the above aspects, the bispecific antibody is a full-length antibody. In some embodiments of any of the above aspects, the bispecific antibody is an IgG antibody. In some embodiments, the IgG antibody is an IgG1 antibody.

[0015] In some embodiments of any of the above aspects, the bispecific antibody comprises one or more heavy chain constant domains, wherein the one or more heavy chain constant domains are selected from a first CH1 (CH11) domain, a first CH2 (CH21) domain, a first CH3 (CH31) domain, a second CH1 (CH12) domain, a second CH2 (CH22) domain, and a second CH3 (CH32) domain. In some embodiments, at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain. In some embodiments, the CH31 and CH32 domains each comprise a protrusion or cavity, and the protrusion or cavity in the CH31 domain can be positioned within the cavity or protrusion, respectively, in the CH32 domain. In some embodiments, the CH31 and CH32 domains meet at the interface between the protrusion and the cavity. In some embodiments, the CH21 and CH22 domains each comprise a protrusion or cavity, and the protrusion or cavity in the CH21 domain can be positioned within the cavity or protrusion, respectively, in the CH22 domain. In some embodiments, the CH21 and CH22 domains associate at the interface between the protrusion and the cavity.

[0016] In some embodiments of any of the above aspects, the bispecific antibody is administered to the subject as a monotherapy.

[0017] In other embodiments of any of the above aspects, the bispecific antibody is administered to the subject as a combination therapy. In some embodiments, the bispecific antibody is administered to the subject simultaneously with an additional therapeutic agent (e.g., atezolizumab). In other embodiments, the bispecific antibody is administered to the subject prior to administration of the additional therapeutic agent (e.g., atezolizumab). In some embodiments, the additional therapeutic agent is atezolizumab. In some embodiments, the method further comprises administering to the subject a first dose of atezolizumab concurrently with the bispecific antibody C2D1 on day 1 of a second administration cycle. In some embodiments, the method further comprises administering atezolizumab to the subject concurrently with a single dose of the bispecific antibody for one or more additional administration cycles on day 1 of one or more additional administration cycles. In some embodiments, atezolizumab is administered to the subject only concurrently with the bispecific antibody. In some embodiments, each dose of atezolizumab is about 1200 mg.

[0018] In yet other embodiments, the bispecific antibody is administered to a subject after administration of an additional therapeutic agent (e.g., obinutuzumab (GAZYVA®) or tocilizumab (ACTEMRA® / RoACTEMRA®)).

[0019] In some embodiments of any of the above aspects, the B-cell proliferative disorder is non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL). In some embodiments, the NHL is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the DLBCL is relapsed or refractory DLBCL. In some embodiments, the NHL is follicular lymphoma (FL). In some embodiments, the NHL is primary mediastinal (thymic) large B-cell lymphoma (PMLBCL).

[0020] In some embodiments of any of the above aspects, the administration is by intravenous infusion.

[0021] In some embodiments of any of the above aspects, the administration is subcutaneous.

[0022] In some embodiments of any one of the above aspects, the subject experiences a cytokine release syndrome (CRS) event, and the method further comprises administering to the subject an effective amount of an interleukin-6 receptor (IL-6R) antagonist (e.g., an anti-IL-6R antibody, e.g., tocilizumab (ACTEMRA® / RoACTEMRA®)) to manage the CRS event.

[0023] In some embodiments, tocilizumab is administered intravenously to a subject as a single dose of about 8 mg / kg.

[0024] In other embodiments, because the CRS event does not improve or worsen within 24 hours of treating the symptoms of the CRS event, the method further comprises administering one or more additional doses of an IL-6R antagonist (e.g., an anti-IL-6R antibody, e.g., tocilizumab) to the subject to manage the CRS event. In some embodiments, because the CRS event does not improve or worsen within 24 hours of treating the symptoms of the CRS event, the method further comprises administering one or more additional doses of tocilizumab to the subject to manage the CRS event. In some embodiments, the one or more additional doses of tocilizumab are administered intravenously to the subject at a single dose of about 8 mg / kg. In some embodiments, the method further comprises administering an effective amount of a corticosteroid to the subject. In some embodiments, the corticosteroid is administered intravenously to the subject. In some embodiments, the corticosteroid is methylprednisolone. In some embodiments, methylprednisolone is administered at a single dose of about 2 mg / kg per day. In other embodiments, the corticosteroid is dexamethasone. In some embodiments, dexamethasone is administered at a single dose of about 10 mg. DETAILED DESCRIPTION OF THE INVENTION

[0025] I. Definition The term "about" as used herein refers to a normal error range for the respective value, readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0026] For purposes herein, an "acceptor human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof or may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0027] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0028] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not possess such alterations, which improve the affinity of the antibody for its antigen.

[0029] The terms "anti-CD3 antibody" and "antibody that binds to CD3" refer to an antibody that is capable of binding to CD3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting CD3. In one embodiment, the extent of binding of an anti-CD3 antibody to an unrelated, non-CD3 protein is less than about 10% of the binding of the antibody to CD3, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CD3 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 In certain embodiments, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among CD3 from different species.

[0030] The terms "anti-CD20 antibody" and "antibody that binds to CD20" refer to an antibody that is capable of binding to CD20 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting CD20. In one embodiment, the extent of binding of an anti-CD20 antibody to an unrelated, non-CD20 protein is less than about 10% of the binding of the antibody to CD20, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CD20 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 In certain embodiments, the anti-CD20 antibody binds to an epitope of CD20 that is conserved among CD20 from different species.

[0031] The terms "anti-CD20 antibody / anti-CD3 antibody" and "antibody that binds to CD20 and CD3," or variants thereof, refer to a multispecific antibody (e.g., a bispecific antibody) that is capable of binding to CD20 and CD3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting CD20 and / or CD3. In one embodiment, the extent of binding of an anti-CD20 / anti-CD3 antibody to an unrelated non-CD3 protein and / or a non-CD20 protein is less than about 10% of the binding of the antibody to CD3 and / or CD20, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CD20 and CD3 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 In certain embodiments, the anti-CD20 / anti-CD3 antibody binds to an epitope on CD3 that is conserved among CD3 from different species and / or an epitope on CD20 that is conserved among CD20 from different species.

[0032] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0033] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0034] "Binding domain" refers to a portion of a compound or molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Binding domains include, but are not limited to, antibodies (e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), antibody fragments or portions thereof (e.g., Fab fragments, Fab'2, scFv antibodies, SMIPs, domain antibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies, and antibody VH and / or VL domains), receptors, ligands, aptamers, and other molecules with identified binding partners.

[0035] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents (such as thiotepa and cyclosphosphamide (CYTOXAN®)); alkylsulfonates (such as busulfan, improsulfan, and piposulfan); aziridines (such as benzodopa, carboquone, meturedopa, and uredopa); ethylenimines and methylameramines (such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, and methylameramine); acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocaproic acid). including amputatecin; bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (chlorambucil, chlorna chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, etc.; nitrosoureas (carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimnustine, etc.);Antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin γ1I and calicheamicin ωII (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral α4 integrin inhibitor; dynemicins (including dynemicin A); esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin , carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), pegylated liposomal doxorubicin (including CAELYX® and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites (such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU)); combretastatins; folic acid analogs (such as denopterin, methotrexate, pteropterin, trimetrexate); purine analogs (such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine);Pyrimidine analogues (such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine); androgens (such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone); antiadrenal agents (such as aminoglutethimide, mitotane, and trilostane); folic acid replenishers (such as folinic acid); aceglatone; and aldophosphamide glycosides glycoside); aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids (such as maytansine and ansamitocin); mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2'-trichlorotriethylamine; trichothecines (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa;Taxoids (e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), albumin-engineered paclitaxel nanoparticle formulation (ABRAXANE™), and docetaxel (TAXOTERE®, Rhome-Poultry) Rorer, Antony, France); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents (such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin); vincas (which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®)); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids (such as retinoic acids, including bexarotene (TARGRETIN®)); bisphosphonates (such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®)); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides (particularly those that inhibit the expression of genes in signaling pathways associated with abnormal cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R) (e.g., erlotinib (Tarceva™)));and VEGF-A (which reduces cell proliferation); vaccines (such as the THERATOPE® vaccine and gene therapy vaccines, e.g., the ALLOVECTIN® vaccine, the LEUVECTIN® vaccine, and the VAXID® vaccine); topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib, Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors (such as oblimersen sodium (GENASENSE®)); pixantrone; EGFR inhibitors; tyrosine kinase inhibitors; serine-threonine kinase inhibitors (such as rapamycin (sirolimus, RAPAMUNE®)); farnesyltransferase inhibitors (such as lonafarnib (SCH6636, SARASAR™)); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for the treatment regimen with oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™), and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

[0036] Chemotherapeutic agents, as defined herein, include "antihormonal agents" or "endocrine therapeutic agents" that act to modulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth. These include antiestrogens and selective estrogen receptor modulators (SERMs), such as tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON.cndot. toremifene; aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-dihydroxybenzoates (DHT), 4(6 ... )-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, holmestein, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole, and the like; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (1,3-dioxolanum). antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell growth, such as PKC-alpha, Raf, and H-Ras; ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME® ribozyme) and HER2 expression inhibitors; vaccines, such as gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; vinorelbine and esperamicin (see U.S. Pat. No. 4,675,187), as well as pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing; and combinations of two or more of the foregoing.

[0037] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0038] The term "cluster of differentiation 3" or "CD3," as used herein, unless otherwise indicated, refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), including, for example, the CD3ε, CD3γ, CD3α, and CD3β chains. The term encompasses "full-length" unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ) as well as any form of CD3 resulting from intracellular processing. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, the human CD3ε protein, which is 207 amino acids long (NCBI Reference SEQ ID NO: NP_000724), and the human CD3γ protein, which is 182 amino acids long (NCBI Reference SEQ ID NO: NP_000064).

[0039] As used herein, the term "cluster of differentiation 20" or "CD20," unless otherwise indicated, refers to any native CD20 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD20 and any form of CD20 that results from processing within the cell. The term also encompasses naturally occurring variants of CD20, including, for example, splice variants or allelic variants. CD20 includes, for example, the human CD20 protein (see, e.g., NCBI Reference SEQ ID NOs: NP_068769.2 and NP_690605.1), which is, for example, 297 amino acids in length and can be produced from, for example, a mutant mRNA transcript lacking part of the 5'UTR (see, e.g., NCBI Reference SEQ ID NO: NM_021950.3), or a longer mutant mRNA transcript (see, e.g., NCBI Reference SEQ ID NO: NM_152866.2).

[0040] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0041] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0042] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic agents include radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes such as nucleases and fragments thereof; antibiotics; toxins such as small molecule toxins or enzymatically active toxins (including fragments and / or mutant forms thereof) of bacterial, fungal, plant or animal origin; and various anti-tumor or anti-cancer agents disclosed below.

[0043] A "disorder" is any condition that may benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including conditions that predispose a mammal to the disease in question.

[0044] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In another embodiment, the cell proliferative disorder is a tumor.

[0045] The terms "B-cell proliferative disorder" or "B-cell malignancy" refer to disorders associated with some degree of abnormal B-cell proliferation and include, for example, lymphoma, leukemia, myeloma, and myelodysplastic syndrome. In one embodiment, the B-cell proliferative disorder is a lymphoma, such as, for example, non-Hodgkin's lymphoma (NHL), including, for example, diffuse large B-cell lymphoma (DLBCL) (e.g., relapsed or refractory DLBCL). In another embodiment, the B-cell proliferative disorder is a leukemia, such as chronic lymphocytic leukemia (CLL).

[0046] The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, hematological cancers such as mature B-cell cancers, including non-Hodgkin's lymphoma (NHL), such as diffuse large B-cell lymphoma (DLBCL), which excludes Hodgkin's lymphoma, but can be relapsed or refractory DLBCL. Other specific examples of cancer include germinal center B-cell-like (GCB) Diffuse Large B-cell Lymphoma (DLBCL), Activated B-cell-like (ABC) DLBCL, Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL), Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Marginal Zone Lymphoma (MZL), Small Lymphocytic Leukemia (SLL), Lymphoplasmacytic Lymphoma (LL), Waldenstrom's Hypergammaglobulinemia (WM), Central Nervous System Lymphoma (CNSL), Burkitt's Lymphoma (BL), B-cell Prolymphocytic Leukemia, Splenic Marginal Zone Lymphoma, Hepatocellular Carcinoma Alley cell leukemia, splenic lymphoma / leukemia, unclassifiable, diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, Waldenstrom's hypergammaglobulinemia, heavy chain disease, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, isolated bone plasmacytoma, extraskeletal plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, primary CNS Examples of cancer include DLBCL, primary cutaneous DLBCL, leg type, EBV-positive DLBCL in elderly patients, chronic inflammation-associated DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma (PMLBCL), intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma due to HHV8-associated multicentric Castleman disease, primary effusion lymphoma, unclassifiable B-cell lymphoma with features intermediate between DLBCL and Burkitt lymphoma, and unclassifiable B-cell lymphoma with features intermediate between DLBCL and classical Hodgkin lymphoma. Further examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and lymphoid malignancies, including leukemia or B-cell lymphoma.More specific examples of such cancers include, but are not limited to, multiple myeloma (MM); low-grade / follicular NHL; small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphocytic NHL; high-grade small non-dividing cell NHL; bulky mass disease NHL; AIDS-related lymphoma; and acute lymphocytic leukemia (ALL); chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD).

[0047] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor," as referred to herein, are not mutually exclusive.

[0048] The term "tumor antigen" as used herein can be understood as an antigen presented on tumor cells. These antigens may be presented on the cell surface with an extracellular portion often associated with the transmembrane and cytoplasmic portions of the molecule. These antigens are presented only by tumor cells and never by normal cells. Tumor antigens may be expressed only on tumor cells or may exhibit tumor-specific mutations compared to normal cells. In this case, they are referred to as tumor-specific antigens. Tumor antigens presented by both tumor cells and normal cells are more common and are referred to as tumor-associated antigens. These tumor-associated antigens may be overexpressed compared to normal cells, or may be accessible to antibodies that bind in tumor cells due to the structure of tumor tissue being smaller than that of normal tissue. In one embodiment, the tumor antigen is CD20.

[0049] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0050] An "effective amount" of a compound, e.g., an anti-CD20 / anti-CD3 antibody, or composition thereof (e.g., pharmaceutical composition) is at least the minimum amount necessary to achieve a desired therapeutic or prophylactic result, such as a measurable improvement or prevention of a particular disease (e.g., cancer, e.g., a B-cell proliferative disorder, e.g., NHL, e.g., DLBCL). An effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. In the case of prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of disease, its complications, and intermediate pathological phenotypes manifest during disease development. For therapeutic use, beneficial or desired results include clinical results such as a reduction in one or more symptoms resulting from the disease, an improvement in the quality of life of the person suffering from the disease, a reduction in the dose of other drugs required to treat the disease, an enhancement of the effect of another drug (e.g., by targeting), a delay in disease progression, and / or an increase in survival time. In the case of cancer or tumors, an effective amount of a drug may have the effect of reducing the number of cancer cells, reducing tumor size, inhibiting (i.e., slowing or desirably stopping) the invasion of cancer cells into peripheral organs, inhibiting (i.e., slowing or desirably stopping) tumor metastasis, inhibiting tumor growth to some extent, and / or alleviating to some extent one or more symptoms associated with the disorder. An effective amount may be administered in one or more administrations. For purposes of this invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in the clinical field, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in relation to the administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in combination with one or more other agents, a desired result can be or is achieved.

[0051] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0052] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0053] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0054] A "human antibody" is one that possesses an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source utilizing a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Methods described in Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991); Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner et al., J. Immunol., 147(1):86-95 (1991) are also available for preparing human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenogeneic mice, that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). See, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), also for human antibodies produced by human B cell hybridoma technology.

[0055] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is derived from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III in Kabat et al., supra.

[0056] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of its HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of its FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0057] The term "hypervariable region" or "HVR," as used herein, refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity determining region" or "CDR") and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen-contacting residues ("antigen contacts"). Generally, antibodies contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein are: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and (d) A combination of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0058] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0059] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including, but not limited to, a cytotoxic agent.

[0060] A "subject" or "individual" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject or individual is a human.

[0061] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0062] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies that contain, for example, naturally occurring mutations or that arise during production of the monoclonal antibody preparation, in which such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0063] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

[0064] "Native antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0065] The term "package insert" is used to refer to instructions customarily included in commercial packaging of therapeutic products that contain information about the indications, uses, dosages, administration, concomitant therapies, contraindications, and / or warnings regarding the use of such therapeutic product.

[0066] As used herein, the term "protein," unless otherwise indicated, refers to any naturally occurring protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed proteins and any form of a protein that results from processing within a cell. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants.

[0067] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across the full length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been submitted, along with user documentation, to the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0068] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (alternatively, it may be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) may be expressed as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as perfect matches by the sequence alignment program ALIGN-2 in that program's alignment of A with B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0069] The term "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredient contained therein is effective and that does not contain additional ingredients that are unacceptably toxic to a subject to which the formulation may be administered.

[0070] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0071] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to a clinical intervention aimed at altering the natural history of the individual being treated and may be performed prophylactically or during the clinical pathological process. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or remission of disease symptoms, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the development of disease or slow the progression of disease.

[0072] As used herein, "delaying the progression" of a disorder or disease means postponing, preventing, slowing, retarding, stabilizing, and / or delaying the development of the disease or disorder (e.g., a B-cell proliferative disorder, e.g., NHL, e.g., DLBCL). This delay can be of varying duration depending on the history of the disease and / or the individual being treated. As will be apparent to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the onset of metastases, can be delayed.

[0073] "Reducing" or "inhibiting" refers to the ability to produce an overall decrease, e.g., of 20% or more, 50% or more, or 75%, 85%, 90%, 95% or more. In certain embodiments, reducing or inhibiting refers to a reduction or inhibition of undesirable events such as cytokine-driven toxicity (e.g., cytokine release syndrome (CRS)), infusion-related reactions (IRR), macrophage activation syndrome (MAS), neurotoxicity, severe tumor lysis syndrome (TLS), neutropenia, thrombocytopenia, liver enzyme elevation, and / or central nervous system (CNS) toxicity following treatment with an anti-CD20 / anti-CD3 bispecific antibody using a fractionated dose-escalation regimen of the invention compared to treatment with an anti-CD20 / anti-CD3 bispecific antibody using a non-fractionated regimen. In other embodiments, reducing or inhibiting can refer to antibody effector functions mediated by the antibody Fc region, specifically including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0074] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of a natural antibody generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al., Kuby Immunology, 6 th(See, e.g., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated from antibodies that bind to the antigen using a VH or VL domain to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0075] The term "PD-1 axis-binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with any one or more of its binding partners to eliminate T cell dysfunction resulting from signaling on the PD-1 signaling axis, thereby restoring or enhancing T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis-binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0076] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-1 with one or more of its binding partners, e.g., PD-L1, PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In specific aspects, PD-1 binding antagonists inhibit the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or prevent signaling resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative costimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes that mediated signaling through PD-1, rendering dysfunctional T cells less dysfunctional (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a specific embodiment, the PD-1 binding antagonist is MDX-1106 (nivolumab), as described herein. In another specific embodiment, the PD-1 binding antagonist is MK-3475 (lambrolizumab), as described herein. In another specific embodiment, the PD-1 binding antagonist is CT-011 (pidilizumab), as described herein. In another specific embodiment, the PD-1 binding antagonist is AMP-224, as described herein.

[0077] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-L1 with any one or more of its binding partners, e.g., PD-1 or B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or prevent signaling resulting from the interaction of PD-L1 with any one or more of its binding partners, e.g., PD-1 or B7-1. In one embodiment, the PD-L1-binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes that mediate PD-L1-mediated signaling, rendering the dysfunctional T cell less dysfunctional (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-L1-binding antagonist is an anti-PD-L1 antibody. In a specific embodiment, the anti-PD-L1 antibody is atezolizumab (CAS Registry Number: 1422185-06-5), also known as MPDL3280A and described herein. In another specific embodiment, the anti-PD-L1 antibody is YW243.55.S70, described herein. In another specific embodiment, the anti-PD-L1 antibody is MDX-1105, described herein. In yet another specific aspect, the anti-PD-L1 antibody is MEDI4736, described herein.

[0078] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or prevents signaling resulting from the interaction of PD-L2 with any one or more of its binding partners, e.g., PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In a specific aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or prevent signaling resulting from the interaction of PD-L2 with any one or more of its binding partners, e.g., PD-1. In one embodiment, the PD-L2 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed in T lymphocyte-mediated signaling via PD-L2, such that dysfunctional T cells are rendered less dysfunctional (e.g., enhance the effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

[0079] As used herein, a "week" is 7 days ± 2 days.

[0080] As used herein, "administering" refers to a method of providing a dosage of a compound (e.g., an anti-CD20 / anti-CD3 antibody of the invention) or composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising an anti-CD20 / anti-CD3 antibody) to a subject. The compounds and / or compositions used in the methods described herein can be administered, for example, intravenously (e.g., by intravenous infusion), subcutaneously, intramuscularly, intradermally, transcutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subconjunctivally, intravesicularly, transmucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, injection, infusion, continuous infusion, localized perfusion directly to target cells, by catheter, lavage, cream, or lipid composition. Methods of administration can vary depending on various factors (eg, the compound or composition being administered and the severity of the condition, disease, or disorder being treated).

[0081] II. Treatment method The present invention is based, in part, on methods for treating subjects with cancer (e.g., B-cell proliferative disorders) using a fractionated-dose escalation regimen with an anti-cluster of differentiation 20 (CD20) / anti-cluster of differentiation 3 (CD3) bispecific antibody. The methods are expected to reduce or prevent undesired therapeutic effects, including cytokine-driven toxicity (e.g., cytokine release syndrome (CRS)), infusion-related reactions (IRR), macrophage activation syndrome (MAS), neurotoxicity, severe tumor lysis syndrome (TLS), neutropenia, thrombocytopenia, elevated liver enzymes, and / or central nervous system (CNS) toxicity. Thus, these methods are useful for treating subjects while achieving a more favorable benefit-risk profile.

[0082] The present invention provides methods useful for treating a subject with cancer (e.g., a B-cell proliferative disorder, e.g., non-Hodgkin's lymphoma (NHL), e.g., diffuse large B-cell lymphoma (DLBCL) (e.g., relapsed or refractory DLBCL), follicular lymphoma (FL), or primary mediastinal (thymic) large B-cell lymphoma (PMLBCL)), comprising administering to the subject a bispecific antibody that binds to CD20 and CD3 (i.e., an anti-CD20 / anti-CD3 antibody) in a fractionated dose-escalation regimen.

[0083] The invention provides methods of treating a subject with cancer (e.g., a B-cell proliferative disorder) comprising administering to the subject a bispecific antibody that binds CD20 and CD3 in a dosing regimen having at least a first and a second dosing cycle, wherein: (a) the first dosing cycle comprises a first dose (C1D1), a second dose (C1D2), and a third dose (C1D3) of the bispecific antibody, wherein C1D1 and C1D2 are each no greater than C1D3; and (b) the second dosing cycle comprises a single dose (C2D1) of the bispecific antibody, wherein C2D1 is equal to or greater than C1D3, and wherein C1D1, C1D2, and C1D3 comprise a cumulative dose that is about 50% greater than the highest clarified dose (i.e., a dose that does not induce unacceptable toxicity in the patient) of the bispecific antibody in the first dosing cycle of the unfractionated dose escalation regimen, wherein the highest clarified dose is between about 0.05 mg and about 30 mg (e.g., For example, between about 0.05 mg and about 25 mg, for example, between about 0.05 mg and about 20 mg, for example, between about 0.05 mg and about 15 mg, for example, between about 0.05 mg and about 13 mg, for example, between about 0.1 mg and about 13 mg, for example, between about 0.2 mg and about 13 mg, for example, between about 0.2 mg and about 10 mg, for example, between about 0.2 mg and about 7.5 mg, for example, between about 0.4 mg and about 7.5 mg, for example, between about 0.4 mg and about 6 mg, for example, between about 0.4 mg and about 5 mg, for example, between about 0.8 mg and about 5 mg, for example, between about 1 mg and about 5 mg, for example, about 2.8 mg). In some embodiments, C1D3 is equal to the highest clarified dose of the bispecific antibody in the first administration cycle of the non-fractionated dose escalation regimen. In some embodiments, C1D2 and C1D1 are equal.In some embodiments, C1D2 is from about 50% to about 250% greater than C1D1 (e.g., C1D2 is from about 50% to about 225% greater than C1D1, e.g., C1D2 is from about 50% to about 200% greater than C1D1, e.g., C1D2 is from about 50% to about 175% greater than C1D1, e.g., C1D2 is from about 50% to about 150% greater than C1D1, e.g., C1D2 is from about 50% to about 125% greater than C1D1, e.g., C1D2 is from about 50% to about 100% greater than C1D1, e.g., C1D2 is from about 50% to about 75% greater than C1D1). In some embodiments, C1D3 is from about 150% to about 300% greater than C1D2 (e.g., C1D3 is from about 150% to about 275% greater than C1D2, e.g., C1D3 is from about 150% to about 250% greater than C1D2, e.g., C1D3 is from about 150% to about 233% greater than C1D2, e.g., C1D3 is from about 150% to about 225% greater than C1D2). For example, C1D3 is greater than C1D2 by about 150% to about 200%, for example, C1D3 is greater than C1D2 by about 150% to about 180%, for example, C1D3 is greater than C1D2 by about 150% to about 175%, for example, C1D3 is greater than C1D2 by about 150% to about 167%, for example, C1D3 is greater than C1D2 by about 150% to about 157%.

[0084] The invention also provides a method of treating a subject with cancer (e.g., a B-cell proliferative disorder) comprising administering to the subject a bispecific antibody that binds CD20 and CD3 in a dosing regimen comprising at least a first and a second dosing cycle, wherein: (a) the first dosing cycle comprises a first dose (C1D1), a second dose (C1D2), and a third dose (C1D3) of the bispecific antibody, wherein C1D1 and C1D2 are each no greater than C1D3, and C1D1 is between about 0.0056 mg and about 12.50 mg (e.g., between about 0.0075 mg and about 12.50 mg, e.g., between about 0.0075 mg and about 12 mg, e.g., between about 0.0075 mg and about 10 mg, e.g., between about 0.0075 mg and about 8 mg); For example, between about 0.010 mg and about 8 mg, for example, between about 0.010 mg and about 7 mg, for example, between about 0.010 mg and about 6 mg, for example, between about 0.010 mg and about 5 mg, for example, between about 0.010 mg and about 4 mg, for example, between about 0.010 mg and about 3.5 mg, for example, between about 0.015 mg and about 3.5 mg, for example, between about 0.020 mg and about 3.5 mg, for example, between about 0.020 mg and about 3.2 mg, for example, between about 0.8 mg and about 1 mg), and C1D2 is between about 0.0125 mg and about 20.00 mg (e.g., between about 0.0125 mg and about 17.5 mg, for example, between about 0.0125 mg and about 15 mg, for example, between about 0.020 mg and about 15 mg, for example, between about 0.025 mg and about 15 mg, for example, between about 0.030 mg and about 15 mg, for example, between about 0.035 mg and about 15 mg, for example, between about 0.040 mg and about 15 mg, for example, between about 0.045 mg and about 15 mg). mg, for example, between about 0.050 mg and about 15 mg, for example, between about 0.050 mg and about 12.5 mg, for example, between about 0.050 mg and about 10 mg, for example, between about 0.050 mg and about 7.5 mg, for example, between about 0.050 mg and about 5 mg, for example, about 1 mg or about 2 mg), and C1D3 is between about 0.0500 mg and about 50 mg (for example, between about 0.055 mg and about 50 mg, for example, between about 0.10 mg and about 50 mg, for example, about 1.(b) the second administration cycle has a single dose of bispecific antibody (C2D1), wherein C2D1 is equal to or greater than C1D3 and is between about 0.0500 mg and about 50 mg (e.g., between about 0.0500 mg and about 50 mg, e.g., between about 0.055 mg and about 50 mg, e.g., between about 0.10 mg and about 50 mg, e.g., between about 1.0 mg and about 50 mg); For example, between about 1.0 mg and about 35 mg, for example, between about 2 mg and about 10 mg, for example, between about 3.0 mg and about 6.0 mg, for example, about 3.0 mg, 4.2 mg, or 6 mg. In some examples, for example, (a) C1D1 is between about 0.02 mg and about 4.0 mg, C1D2 is between about 0.05 mg and about 20.0 mg, and C1D3 is between about 0.2 mg and about 50.0 mg; and (b) C2D1 is between about 3.0 mg and about 50.0 mg. In some examples, (a) C1D1 is between about 0.02 mg and about 4.0 mg, C1D2 is between about 0.05 mg and about 20.0 mg, and C1D3 is between about 0.2 mg and about 50.0 mg. D1 is between about 0.4 mg and about 4.0 mg, C1D2 is between about 1.0 mg and about 20.0 mg, C1D3 is between about 3.0 mg and about 50.0 mg, and (b) C2D1 is between about 3.0 mg and about 50.0 mg. In some examples, (a) C1D1 is between about 1.0 mg and about 3.0 mg, C1D2 is between about 2.0 mg and about 6.0 mg, and C1D3 is between about 6.0 mg and about 50.0 mg, and (b) C2D1 is between about 6.0 mg and about 50.0 mg. In some examples, In some examples, (a) C1D1 is about 1.0 mg, C1D2 is about 2.0 mg, and C1D3 is between about 6.0 mg and about 50.0 mg; and (b) C2D1 is between about 6.0 mg and about 50.0 mg. In some examples, (a) C1D1 is about 1.0 mg, C1D2 is about 2.0 mg, and C1D3 is between about 6.0 mg and about 50.0 mg; and (b) C2D1 is equal to C1D3. In some examples, (a) C1D1 is about 1.0 mg, C1D2 is about 2.0 mg, and C1D3 is about 20.0 mg, and (b) C2D1 is equal to C1D3. In some examples, for example, (a) C1D1 is between about 0.02 mg and 4.0 mg, C1D2 is between about 0.05 mg and about 20.0 mg, and C1D3 is between about 0.2 mg and about 20.0 mg, and (b) C2D1 is between about 0.2 mg and about 20.0 mg. In some examples, (a) C1D1 is between about 0.4 mg and about 4.0 mg, C1D2 is between about 1.0 mg and about 20.0 mg, and C1D3 is between about 3.0 mg and about 20.0 mg, and (b) C2D1 is between about 3.0 mg and about 20.0 mg. In another example, (a) C1D1 is between about 0.8 mg and about 3.0 mg, C1D2 is between about 1.0 mg and about 6.0 mg, and C1D3 is between about 3.0 mg and about 20.0 mg, and (b) C2D1 is between about 3.0 mg and about 20.0 mg. In another example, (a) C1D1 is between about 0.8 mg and about 3.0 mg, C1D2 is between about 1.0 mg and about 6.0 mg, and C1D3 is between about 3.0 mg and about 6.0 mg, and (b) C2D1 is between about 3.0 mg and about 6.0 mg.

[0085] In some embodiments, (a) C1D1 is about 0.8 mg, C1D2 is about 2.0 mg, and C1D3 is about 4.2 mg, and (b) C2D1 is about 4.2 mg. In some embodiments, (a) C1D1 is about 1.0 mg, C1D2 is about 1.0 mg, and C1D3 is about 3.0 mg, and (b) C2D1 is about 3.0 mg. In some embodiments, (a) C1D1 is about 1.0 mg, C1D2 is about 2.0 mg, and C1D3 is about 6.0 mg, and (b) C2D1 is about 6.0 mg. In some embodiments, (a) C1D1 is about 0.8 mg, C1D2 is about 2.0 mg, and C1D3 is about 6.0 mg, and (b) C2D1 is about 6.0 mg. The invention also provides a method of treating a subject with cancer (e.g., a B-cell proliferative disorder) comprising administering to the subject a bispecific antibody that binds CD20 and CD3 in a dosing regimen having a first dosing cycle and, optionally, a second dosing cycle, wherein (a) the first dosing cycle has a first dose (C1D1) and a second dose (C1D2) of the bispecific antibody, where C1D1 is not greater than the C1D2 dose, and optionally (b) the second dosing cycle has a single dose (C2D1) of the bispecific antibody, where C2D1 is greater than or equal to C1D2, and C1D1 and C1D2 have a cumulative dose that is about 50% greater than the highest clarified dose of the bispecific antibody in the first dosing cycle of the non-fractionated dose escalation regimen, where the highest clarified dose is between about 0.05 mg and about 30 mg (e.g., between about 0.05 mg and about 25 mg, e.g., , between about 0.05 mg and about 20 mg, for example, between about 0.05 mg and about 15 mg, for example, between about 0.05 mg and about 13 mg, for example, between about 0.1 mg and about 13 mg, for example, between about 0.2 mg and about 13 mg, for example, between about 0.2 mg and about 10 mg, for example, between about 0.2 mg and about 7.5 mg, for example, between about 0.4 mg and about 7.5 mg, for example, between about 0.4 mg and about 6 mg, for example, between about 0.4 mg and about 5 mg, for example, between about 0.8 mg and about 5 mg, for example, between about 1 mg and about 5 mg, for example, about 2.8 mg).In some embodiments, C1D2 is equal to the highest clarified dose of the bispecific antibody in the first dosing cycle of a non-fractionated dose escalation regimen. In some examples, C1D2 is from about 50% to about 250% greater than C1D1 (e.g., C1D2 is from about 50% to about 225% greater than C1D1, e.g., C1D2 is from about 50% to about 200% greater than C1D1, e.g., C1D2 is from about 50% to about 175% greater than C1D1, e.g., C1D2 is from about 50% to about 150% greater than C1D1, e.g., C1D2 is from about 50% to about 125% greater than C1D1, e.g., C1D2 is from about 50% to about 100% greater than C1D1, e.g., C1D2 is from about 50% to about 75% greater than C1D1).

[0086] In some examples, C1D1 is between about 0.0056 mg and about 12.50 mg (e.g., between about 0.0075 mg and about 12.50 mg, e.g., between about 0.0075 mg and about 12 mg, e.g., between about 0.0075 mg and about 10 mg, e.g., between about 0.0075 mg and about 8 mg, e.g., between about 0.010 mg and about 8 mg, e.g., between about 0.010 mg and about 7 mg, e.g., For example, between about 0.010 mg and about 6 mg, for example, between about 0.010 mg and about 5 mg, for example, between about 0.010 mg and about 4 mg, for example, between about 0.010 mg and about 3.5 mg, for example, between about 0.015 mg and about 3.5 mg, for example, between about 0.020 mg and about 3.5 mg, for example, between about 0.020 mg and about 3.2 mg, for example, about 0.8 mg or about 1 mg). In some examples, C1D2 is between about 0.0125 mg and about 19.44 mg (e.g., between about 0.0125 mg and about 17.5 mg, for example, between about 0.0125 mg and about 15 mg, for example, between about 0.020 mg and about 15 mg, for example, between about 0.025 mg and about 15 mg, for example, between about 0.030 mg and about 15 mg, for example, between about 0.035 mg and about 15 mg, for example, between about 0.040 mg and about 15 mg, for example, between about 0.045 mg and about 15 mg, for example, between about 0.050 mg and about 15 mg, for example, between about 0.050 mg and about 12.5 mg, for example, between about 0.050 mg and about 10 mg, for example, between about 0.050 mg and about 7.5 mg, for example, between about 0.050 mg and about 5 mg).In some examples, C1D2 is between about 0.0500 mg and about 50 mg (e.g., between about 0.055 mg and about 50 mg, e.g., between about 0.055 mg and about 45 mg, e.g., between about 0.055 mg and about 40 mg, e.g., between about 0.055 mg and about 35 mg, e.g., between about 0.055 mg and about 30 mg, e.g., between about 0.10 mg and about 30 mg, e.g., about between 0.15 mg and about 30 mg, for example, between about 0.15 mg and about 25 mg, for example, between about 0.15 mg and about 20 mg, for example, between about 0.15 mg and about 17.5 mg, for example, between about 0.15 mg and about 15 mg, for example, between about 0.20 mg and about 15 mg, for example, between about 0.20 mg and about 12.8 mg, for example, between about 0.20 mg and about 12.5 mg).

[0087] In some examples, C1D1 is between about 0.0056 mg and about 12.50 mg (e.g., between about 0.0075 mg and about 12.50 mg, for example, between about 0.0075 mg and about 12 mg, for example, between about 0.0075 mg and about 10 mg, for example, between about 0.0075 mg and about 8 mg, for example, between about 0.010 mg and about 8 mg, for example, between about 0.010 mg and about 7 mg, for example, between about 0.010 mg and about 6 mg, for example, between about 0.010 mg and about 5 mg, for example, between about 0.010 mg and about 4 mg, for example, between about 0.010 mg and about 3.5 mg, for example, between about 0.015 mg and about 3.5 mg, for example, between about 0.020 mg and about 3.5 mg, for example, between about 0.020 mg and about 3.2 mg, for example, about 1.6 mg), and C1D2 is between about 0.0125 mg and about 19.44 mg (e.g., between about 0.0125 mg and about 17.5 mg, for example, between about 0.0125 mg and about 15 mg, for example, between about 0.020 mg and about 15 mg, for example, between about 0.025 mg and about 15 mg, for example, between about 0.030 mg and about 15 mg, for example, between about 0.035 mg and about 15 mg, for example, between about 0.040 mg and about 15 mg, for example, between about 0.045 mg and about 15 mg, for example, between about 0.050 mg and about 15 mg, for example, between about 0.050 mg and about 12.5 mg, for example, between about 0.050 mg and about 10 mg, for example, between about 0.050 mg and about 7.5 mg, for example, between about 0.050 mg and about 5 mg).In some examples, C1D1 is between about 0.0056 mg and about 12.50 mg (e.g., between about 0.0075 mg and about 12.50 mg, e.g., between about 0.0075 mg and about 12 mg, e.g., between about 0.0075 mg and about 10 mg, e.g., between about 0.0075 mg and about 8 mg, e.g., between about 0.010 mg and about 8 mg, e.g., between about 0.010 mg and about 7 mg for example, between about 0.010 mg and about 6 mg, for example, between about 0.010 mg and about 5 mg, for example, between about 0.010 mg and about 4 mg, for example, between about 0.010 mg and about 3.5 mg, for example, between about 0.015 mg and about 3.5 mg, for example, between about 0.020 mg and about 3.5 mg, for example, between about 0.020 mg and about 3.2 mg, for example, about 1.6 mg ), and C1D2 is between about 0.0500 mg and about 50 mg (e.g., between about 0.055 mg and about 50 mg, for example, between about 0.055 mg and about 45 mg, for example, between about 0.055 mg and about 40 mg, for example, between about 0.055 mg and about 35 mg, for example, between about 0.055 mg and about 30 mg, for example, between about 0.10 mg and about 30 mg, for example, about 0.1 The dose may be between about 5 mg and about 30 mg, e.g., between about 0.15 mg and about 25 mg, e.g., between about 0.15 mg and about 20 mg, e.g., between about 0.15 mg and about 17.5 mg, e.g., between about 0.15 mg and about 15 mg, e.g., between about 0.20 mg and about 15 mg, e.g., between about 0.20 mg and about 12.8 mg, e.g., between about 0.20 mg and about 12.5 mg). In any of the above examples, the dosing regimen can comprise a first dosing cycle and, optionally, a second dosing cycle, where (a) the first dosing cycle consists of bispecific antibodies C1D1 and C1D2, and optionally (b) the second dosing cycle comprises bispecific antibody C2D1. In any of the above examples, the dosing regimen can have at least a first dosing cycle and a second dosing cycle. Thus, (a) the first administration cycle consists of bispecific antibodies C1D1 and C1D2, and (b) the second administration cycle consists of bispecific antibody C2D1.

[0088] In some examples, the methods described above can have a first administration cycle of 3 weeks or 21 days. In some examples, the methods can comprise administering C1D1, C1D2, and C1D3 to the subject on or about days 1, 8, and 15, respectively, of the first administration cycle.

[0089] In some instances, the methods described above can have a 3-weekly or 21-day second administration cycle. In some instances, the methods can comprise administering C2D1 to the subject on or about day 1 of the second administration cycle.

[0090] In some examples, the methods described above can include one or more additional administration cycles. In some examples, the administration regimen includes 1 to 14 additional administration cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 additional administration cycles (i.e., the administration regimen includes one or more additional administration cycle(s) C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, and C16)). In some examples, the administration regimen includes 1 to 6 additional administration cycles (i.e., the administration regimen includes one or more additional administration cycle(s) C3, C4, C5, C6, C7, and C8). In some embodiments, the length of each of the one or more additional administration cycles is 7 days, 14 days, 21 days, or 28 days. In some examples, the length of each of the one or more additional administration cycles is 3 weeks or 21 days. In some examples, each of the one or more additional administration cycles comprises a single dose of the bispecific antibody. In some examples, the method comprises administering to the subject a single dose of the one or more additional administration cycles on or about day 1 of the one or more additional administration cycles.

[0091] In some examples, the bispecific antibody is administered subcutaneously to a subject. In this embodiment, the bispecific antibody is administered at a dosage of between about 0.5 mg and about 40 mg. In some embodiments, the bispecific antibody is administered at a dosage of between about 1.0 and about 20 mg, between about 1.0 and about 10 mg, or between about 1.0 and about 5 mg. In one embodiment, the bispecific antibody is administered in a dosage of 1.6 mg. Subsequent dosages are administered in amounts equal to the initial subcutaneous dosage.

[0092] In some instances, the bispecific anti-CD20 / anti-CD3 antibody is administered to a subject as a monotherapy.

[0093] In other examples, the bispecific anti-CD20 / anti-CD3 antibody is administered to a subject in combination therapy. For example, the bispecific anti-CD20 / anti-CD3 antibody can be administered simultaneously with one or more additional therapeutic agents. In one example, the therapeutic agent is another antibody that targets CD20. In one example, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with one or more antibodies that target CD20 selected from the chimeric monoclonal CD20 antibody rituximab (RITUXAN®) or the monoclonal CD20 antibody obinutuzumab (GAZYVA®). In some examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with rituximab. In some examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with obinutuzumab. In some examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with obinutuzumab and rituximab. In other examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with tocilizumab (ACTEMRA® / RoACTEMRA®).

[0094] In some examples, the methods described above comprise administering a bispecific anti-CD20 / anti-CD3 antibody, with or without a CD20 monoclonal antibody, further comprising a chemotherapeutic agent and / or an antibody-drug conjugate (ADC). In some examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with one or more additional chemotherapeutic agents selected from cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP). In some examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with the ADC. In some examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with CHOP, with vincristine replacing the ADC. In some examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with an ADC selected from an anti-CD79b antibody drug conjugate (such as anti-CD79b-MC-vc-PAB-MMAE or an anti-CD79b antibody drug conjugate described in any one of U.S. Pat. No. 8,088,378 and / or U.S. Pat. No. 2014 / 0030280, or polatuzumab vedotin), an anti-CD19 antibody drug conjugate, an anti-CD22 antibody drug conjugate, an anti-CD45 antibody drug conjugate, and an anti-CD32 drug conjugate.

[0095] In some examples, the therapeutic agent is a biological modulator. In one example, the bispecific anti-CD20 / anti-CD3 antibody is used in combination with other therapeutic agents, such as BCL-2 inhibitors (such as GDC-0199 / ABT-199), lenalidomide (REVLIMID®), PI3K delta inhibitors (such as idelalisib (ZYDELIG®)), PD-1 axis binding antagonists, agonists, e.g., activating costimulatory molecules, e.g., CD40, CD226, CD28, OX40 (e.g., AgonOX), GITR, CD137 (TNFRSF9, 4-1 agonist antibodies, such as antibodies directed against CD27 (e.g., CDX-1127), HVEM, or CD127; antagonists, such as inhibitory costimulatory molecules, e.g., CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., 1-methyl-D-tryptophan (also known as 1-D-MT)), TIGIT , MICA / B, GITR (e.g., TRX518), or arginase, such as antagonist antibodies, ipilimumab (also known as MDX-010, MDX-101, or YERVOY®), tremelimumab (also known as ticilimumab or CP-675,206), urelumab (also known as BMS-663513), MGA271, antagonists directed against TGF-beta, such as metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR), e.g., adoptive transfer of T cells comprising a dominant-negative TGF-beta receptor, e.g., a dominant-negative TGF-beta type II receptor.

[0096] In some instances, for example, a bispecific anti-CD20 / anti-CD3 antibody is administered to a subject in a combination therapy that includes a PD-1 axis-binding antagonist. The PD-1 axis-binding antagonist can be administered before, after, and / or simultaneously with the bispecific anti-CD20 / anti-CD3 antibody. The PD-1 axis-binding antagonist can, in some instances, be a PD-1 binding antagonist, a PD-L1 binding antagonist, or a PD-L2 binding antagonist.

[0097] In some instances, the PD-1 binding antagonist is an anti-PD-1 antibody. For example, in some specific instances, the anti-PD-L1 antibody is atezolizumab (CAS Registry Number: 1422185-06-5). Atezolizumab (Genentech) is also known as MPDL3280A.

[0098] Atezolizumab contains (a) the sequences GFTFSDSWIH (SEQ ID NO: 33), AWISPYGGSTYYADSVKG (SEQ ID NO: 34), and RHWPGGFDY (SEQ ID NO: 35) for HVR-H1, HVR-H2, and HVR-H3, respectively, and (b) the sequences RASQDVSTAVA (SEQ ID NO: 36), SASFLYS (SEQ ID NO: 37), and QQYLYHPAT (SEQ ID NO: 38) for HVR-L1, HVR-L2, and HVR-L3, respectively.

[0099] Atezolizumab comprises heavy and light chain sequences, where (a) the heavy chain variable region sequence comprises the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 39), and (b) the light chain variable region sequence comprises the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 40).

[0100] Atezolizumab comprises heavy and light chain sequences, and (a) the heavy chain has the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP (b) the light chain comprises the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 42).

[0101] Thus, in some examples, these methods comprise administering a bispecific anti-CD20 / anti-CD3 antibody, including atezolizumab, with or without a CD20 monoclonal antibody. In some examples, the dose of atezolizumab is administered on day 1 of the second administration cycle concurrently with the bispecific antibody C2D1. In some examples, the first dose of atezolizumab is administered on day 1 of the second administration cycle concurrently with the bispecific antibody C2D1 (i.e., prior to its co-administration with the bispecific anti-CD20 / anti-CD3 antibody C2D1, the subject has not received atezolizumab according to the dosing regimen). In some examples, these methods further comprise administering atezolizumab to the subject on day 1 of one or more additional administration cycles concurrently with a single dose of the bispecific anti-CD20 / anti-CD3 antibody for one or more additional administration cycles. In some instances, atezolizumab is administered to the subject only simultaneously with the bispecific anti-CD20 / anti-CD3 antibody. In any of the above instances, each dose of atezolizumab can be about 1200 mg.

[0102] Accordingly, in some examples, the invention provides a method of treating a subject with cancer (e.g., a B-cell proliferative disorder) comprising administering to the subject a bispecific antibody that binds CD20 and CD3 in a dosing regimen having at least a first administration cycle and a second administration cycle, wherein: (a) the first administration cycle comprises a first dose (C1D1), a second dose (C1D2), and a third dose (C1D3) of the bispecific antibody, wherein C1D1 and C1D2 are each no greater than C1D3, and C1D1 is between about 0.02 mg and about 4.0 mg (e.g., about 0.8 mg or about 1.0 mg), C1D2 is between about 0.05 mg and about 20.0 mg (e.g., about 1 mg or 2 mg), and C1D3 is between about 0.2 mg and about 50.0 mg (e.g., between about 2 mg and about 10 mg, e.g., about 3.0 mg, 4.2 mg, or 6.0 mg); and (b) the second administration cycle comprises a first dose (C1D1), a second dose (C1D2), and a third dose (C1D3) of the bispecific antibody, wherein C1D1 and C1D2 are each no greater than C1D3, and C1D1 is between about 0.02 mg and about 4.0 mg (e.g., about 0.8 mg or about 1.0 mg), C1D2 is between about 0.05 mg and about 20.0 mg (e.g., about 1 mg or 2 mg), and C1D3 is between about 0.2 mg and about 50.0 mg (e.g., between about 2 mg and about 10 mg, e.g., about 3.0 mg, 4.2 mg, or 6.0 mg). The cycle has a single dose of bispecific antibody (C2D1), where C2D1 is about equal to or greater than C1D3 and is between about 0.2 mg and about 50.0 mg (e.g., between about 2 mg and about 10 mg, e.g., about 3.0 mg, 4.2 mg, or 6.0 mg), and each administration cycle of the administration regimen is 21 days in length, and C1D1, C1D2, and C1D3 are administered on days 1, 8, and 15, or about days 1, 8, and 15, respectively, of the first administration cycle. and on about day 15, wherein the bispecific antibody C2D1 is administered concurrently with the first dose of about 1200 mg of atezolizumab on day 1 of the second dosing cycle, and the dosing regimen optionally includes one to six additional dosing cycles, each 21 days in length, each having a single dose of the bispecific antibody approximately equal to C2D1 administered concurrently with the single dose of about 1200 mg of atezolizumab on day 1 of each of the one to six additional dosing cycles. Atezolizumab can only be administered to the subject concurrently with the bispecific anti-CD20 / anti-CD3 antibody in the dosing regimen. In examples such as these, the method can include administration of one or more additional therapeutic agents in accordance with the dosing regimen.For example, in certain instances, a bispecific anti-CD20 / anti-CD3 antibody can be administered simultaneously with obinutuzumab (GAZYVA®), or tocilizumab (ACTEMRA® / RoACTEMRA®), with the patient first receiving obinutuzumab (GAZYVA®) or tocilizumab (ACTEMRA® / RoACTEMRA®), followed by separate administration of the bispecific anti-CD20 / anti-CD3 antibody (e.g., the patient has been previously treated with obinutuzumab (GAZYVA®) or tocilizumab (ACTEMRA® / RoACTEMRA®)).

[0103] In some examples, the PD-1 binding antagonist is another anti-PD-1 antibody, such as an anti-PD-1 antibody selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody described in International Publication No. WO 2006 / 121168. MK-3475, also known as pembrolizumab or lambrolizumab, is an anti-PD-1 antibody described in International Publication No. WO 2009 / 114335. CT-011, also known as hBAT, hBAT-1, or pidilizumab, is an anti-PD-1 antibody described in International Publication No. WO 2009 / 101611. In another example, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1-binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In another example, the PD-1 binding antagonist is AMP-224. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in International Publication Nos. WO 2010 / 027827 and WO 2011 / 066342.

[0104] In other examples, the anti-PD-L1 antibody is selected from YW243.55.S70, MDX-1105, and MEDI4736 (durvalumab), and MSB0010718C (avelumab). Antibody YW243.55.S70 is an anti-PD-L1 antibody described in International Publication No. WO2010 / 077634. ​​MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in International Publication No. WO2007 / 005874. MEDI4736 (durvalumab) is an anti-PD-L1 monoclonal antibody described in International Publication No. WO2011 / 066389 and U.S. Publication No. 2013 / 034559. Examples of anti-PD-L1 antibodies useful in the methods of the invention, and methods for making them, are described in International Publication Nos. WO2010 / 077634, WO2007 / 005874, and WO2011 / 066389, as well as in U.S. Pat. No. 8,217,149 and U.S. Publication No. 2013 / 034559, which are incorporated herein by reference.

[0105] In other examples, the PD-L2 binding antagonist is an anti-PD-L2 antibody (e.g., a human, humanized, or chimeric anti-PD-L2 antibody). In some examples, the PD-L2 binding antagonist is an immunoadhesin.

[0106] In some examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with rituximab and one or more chemotherapeutic agents. In one example, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with rituximab and CHOP. In one example, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with rituximab and an ADC. In one example, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with rituximab and CHOP, with vincristine substituted for the ADC. In one example, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with an ADC selected from an anti-CD79b antibody drug conjugate (such as anti-CD79b-MC-vc-PAB-MMAE or an anti-CD79b antibody drug conjugate described in any one of U.S. Pat. No. 8,088,378 and / or U.S. Pat. No. 2014 / 0030280, or polatuzumab vedotin), an anti-CD19 antibody drug conjugate, an anti-CD22 antibody drug conjugate, an anti-CD45 antibody drug conjugate, and an anti-CD32 drug conjugate.

[0107] In some examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with rituximab and one or more biological modifiers, where the one or more biological modifiers are selected from the group consisting of a BCL-2 inhibitor (such as GDC-0199 / ABT-199), lenalidomide (REVLIMID®), a PI3K-delta inhibitor (such as idelalisib (ZYDELIG®)), a PD-1 axis binding antagonist, an agonist, e.g., an activating costimulatory molecule, e.g., CD40, CD226, antagonists, such as inhibitory costimulatory molecules, e.g., CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., 1-Met-3), IL-1, IL-2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-5 ... antagonist antibodies directed against, for example, til-D-tryptophan (also known as 1-D-MT), TIGIT, MICA / B, GITR (e.g., TRX518), or arginase; ipilimumab (also known as MDX-010, MDX-101, or YERVOY®), tremelimumab (also known as ticilimumab or CP-675, 206), urelumab (also known as BMS-663513), MGA271, TG Antagonists directed against F beta, such as metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR), such as adoptive transfer of T cells comprising a dominant negative TGF beta receptor, e.g., a dominant negative TGF beta type II receptor.

[0108] In some examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with rituximab, one or more chemotherapeutic agents, and one or more biological modifiers, including a BCL-2 inhibitor (such as GDC-0199 / ABT-199), lenalidomide (REVLIMID®), a PI3K-delta inhibitor (such as idelalisib (ZYDELIG®)), a PD-1 axis binding antagonist, an agonist, e.g., an activating costimulatory molecule, e.g., CD4 agonist antibodies directed against CD127, CD226, CD28, OX40 (e.g., AgonOX), GITR, CD137 (also known as TNFRSF9, 4-1BB, or ILA), CD27 (e.g., CDX-1127), HVEM, or CD127; antagonists, e.g., inhibitory costimulatory molecules, e.g., CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., For example, antagonist antibodies directed against 1-methyl-D-tryptophan (also known as 1-D-MT), TIGIT, MICA / B, GITR (e.g., TRX518), or arginase, ipilimumab (also known as MDX-010, MDX-101, or YERVOY®), tremelimumab (also known as ticilimumab or CP-675, 206), urelumab (also known as BMS-663513), MGA271 , antagonists directed against TGF beta, such as metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR), such as adoptive transfer of T cells comprising a dominant negative TGF beta receptor, e.g., a dominant negative TGF beta type II receptor.

[0109] In some examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with rituximab, an ADC, and one or more biological modifiers, where the one or more biological modifiers are selected from the group consisting of a BCL-2 inhibitor (such as GDC-0199 / ABT-199), lenalidomide (REVLIMID®), a PI3K-delta inhibitor (such as idelalisib (ZYDELIG®)), a PD-1 axis binding antagonist, an agonist, e.g., an activating costimulatory molecule, e.g., CD40, CD2 26, agonist antibodies directed against CD28, OX40 (e.g., AgonOX), GITR, CD137 (also known as TNFRSF9, 4-1BB, or ILA), CD27 (e.g., CDX-1127), HVEM, or CD127; antagonists, for example, inhibitory costimulatory molecules, such as CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., 1 -methyl-D-tryptophan (also known as 1-D-MT), TIGIT, MICA / B, GITR (e.g., TRX518), or arginase antagonist antibodies, such as ipilimumab (also known as MDX-010, MDX-101, or YERVOY®), tremelimumab (also known as ticilimumab or CP-675, 206), urelumab (also known as BMS-663513), MGA271, T Antagonists directed against TGF beta, such as metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR), for example, adoptive transfer of T cells comprising a dominant negative TGF beta receptor, for example, a dominant negative TGF beta type II receptor.

[0110] In some examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with obinutuzumab and one or more chemotherapeutic agents. In one example, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with obinutuzumab and CHOP. In one example, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with obinutuzumab and an ADC. In one example, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with obinutuzumab and CHOP, with vincristine substituted for the ADC. In one example, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with an ADC selected from an anti-CD79b antibody drug conjugate (such as anti-CD79b-MC-vc-PAB-MMAE or an anti-CD79b antibody drug conjugate described in any one of U.S. Pat. No. 8,088,378 and / or U.S. Pat. No. 2014 / 0030280, or polatuzumab vedotin), an anti-CD19 antibody drug conjugate, an anti-CD22 antibody drug conjugate, an anti-CD45 antibody drug conjugate, and an anti-CD32 drug conjugate.In one example, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with obinutuzumab and one or more biological modifiers, including a BCL-2 inhibitor (such as GDC-0199 / ABT-199), lenalidomide (REVLIMID®), a PI3K-delta inhibitor (such as idelalisib (ZYDELIG®)), a PD-1 axis binding antagonist, an agonist, e.g., an activating costimulatory molecule, e.g., CD40, CD226, CD 28, agonist antibodies such as antibodies directed against OX40 (e.g., AgonOX), GITR, CD137 (also known as TNFRSF9, 4-1BB, or ILA), CD27 (e.g., CDX-1127), HVEM, or CD127; antagonists, for example, inhibitory costimulatory molecules such as CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., 1-methyl- antagonist antibodies directed against, for example, l-D-tryptophan (also known as l-D-MT), TIGIT, MICA / B, GITR (e.g., TRX518), or arginase; ipilimumab (also known as MDX-010, MDX-101, or YERVOY®), tremelimumab (also known as ticilimumab or CP-675, 206), urelumab (also known as BMS-663513), MGA271, TGF- beta, such as metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR), e.g., adoptive transfer of T cells comprising a dominant negative TGF beta receptor, e.g., a dominant negative TGF beta type II receptor.

[0111] In some examples, the bispecific anti-CD20 / anti-CD3 antibody is administered simultaneously with obinutuzumab, an ADC, and one or more biological modifiers, where the one or more biological modifiers are selected from the group consisting of a BCL-2 inhibitor (such as GDC-0199 / ABT-199), lenalidomide (REVLIMID®), a PI3K-delta inhibitor (such as idelalisib (ZYDELIG®)), a PD-1 axis binding antagonist, an agonist, e.g., an activating costimulatory molecule, e.g., CD40, CD 226, CD28, OX40 (e.g., AgonOX), GITR, CD137 (also known as TNFRSF9, 4-1BB, or ILA), CD27 (e.g., CDX-1127), HVEM, or CD127; antagonists, e.g., inhibitory costimulatory molecules, e.g., CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO (e.g., antagonist antibodies directed against 1-methyl-D-tryptophan (also known as 1-D-MT), TIGIT, MICA / B, GITR (e.g., TRX518), or arginase, ipilimumab (also known as MDX-010, MDX-101, or YERVOY®), tremelimumab (also known as ticilimumab or CP-675, 206), urelumab (also known as BMS-663513), MGA271, T Antagonists directed against TGF beta, such as metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299k, and adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR), for example, adoptive transfer of T cells comprising a dominant negative TGF beta receptor, for example, a dominant negative TGF beta type II receptor.

[0112] In some examples, the additional therapy includes an alkylating agent. In one example, the alkylating agent is 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid and its salts. In one example, the alkylating agent is bendamustine.

[0113] In some examples, the additional therapy includes a BCL-2 inhibitor. In one embodiment, the BCL-2 inhibitor is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide and salts thereof. In one example, the BCL-2 inhibitor is venetoclax (CAS number: 1257044-40-8).

[0114] In some examples, the additional therapy includes a phosphoinositide 3-kinase (PI3K) inhibitor. In one example, the PI3K inhibitor inhibits the delta isoform of PI3K (i.e., P110δ). In some examples, the PI3K inhibitor is 5-fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone and its salts. In some examples, the PI3K inhibitor is idelalisib (CAS number: 870281-82-6). In one example, the PI3K inhibitor inhibits the alpha and delta isoforms of PI3K. In some examples, the PI3K inhibitor is 2-{3-[2-(1-isopropyl-3-methyl-1H-1,2-4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl]-1H-pyrazol-1-yl}-2-methylpropanamide and salts thereof.

[0115] In a further embodiment of the present invention, the additional therapy comprises a Bruton's tyrosine kinase (BTK) inhibitor. In one example, the BTK inhibitor is 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one and salts thereof. In one example, the BTK inhibitor is ibrutinib (CAS No.: 936563-96-1).

[0116] In some examples, the additional therapy includes thalidomide or a derivative thereof. In one example, the thalidomide or a derivative thereof is (RS)-3-(4-amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione and salts thereof. In one example, the thalidomide or a derivative thereof is lendalidomide (CAS number: 191732-72-6).

[0117] Where the methods described herein involve combination therapy, such as the specific combination therapies described above, the combination therapy includes co-administration of a bispecific anti-CD20 / anti-CD3 antibody with one or more additional therapeutic agents; such co-administration can be combined (the two or more therapeutic agents are in the same formulation or in separate formulations) or separate; in this case, administration of the anti-CD20 / anti-CD3 bispecific antibody can occur prior to, concurrently with, and / or after administration of the additional therapeutic agent or agents. In one embodiment, administration of the anti-CD20 / anti-CD3 bispecific antibody and administration of the additional therapeutic agent or agents, or exposure to radiation therapy, can occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other. In a particular example, the bispecific anti-CD20 / anti-CD3 antibody can be administered simultaneously with obinutuzumab (GAZYVA®), where the patient is administered obinutuzumab (GAZYVA®) first and then the bispecific anti-CD20 / anti-CD3 antibody is administered separately (e.g., the patient is pre-treated with obinutuzumab (GAZYVA®)). In another particular example, the bispecific anti-CD20 / anti-CD3 antibody can be administered simultaneously with tocilizumab (ACTEMRA® / RoACTEMRA®), where the patient is administered tocilizumab (ACTEMRA® / RoACTEMRA®) first and then the bispecific anti-CD20 / anti-CD3 antibody is administered separately (e.g., the patient is pre-treated with tocilizumab (ACTEMRA® / RoACTEMRA®)).

[0118] Any of these methods of the invention described herein can be useful for treating cancer, such as hematological cancers, including B-cell proliferative disorders / malignancies. In particular, B-cell proliferative disorders include, but are not limited to, non-Hodgkin's lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL) and other cancers that can be amenable to treatment with bispecific anti-CD20 / anti-CD3 antibodies according to these methods described herein and that are relapsed or refractory, including germinal center B-cell-like (GCB) diffuse large B-cell lymphoma (DLBCL), activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), and other cancers. Waldenstrom's hypergammaglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic lymphoma / leukemia, unclassifiable, diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, Waldenstrom's hypergammaglobulinemia, heavy chain disease, alpha heavy chain disease, gamma heavy chain disease, mu heavy chain disease, plasma cell myeloma, isolated bone plasmacytoma, extraskeletal plasmacytoma, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, pediatric follicular lymphoma, primary cutaneous follicle center lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, primary CNS DLBCL, primary cutaneous DLBCL, leg type, EBV-positive DLBCL in the elderly, chronic inflammation-associated DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma (PMLBCL), intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma due to HHV8-associated multicentric Castleman disease, primary effusion lymphoma; unclassifiable B-cell lymphoma with features intermediate between DLBCL and Burkitt lymphoma; and unclassifiable B-cell lymphoma with features intermediate between DLBCL and classical Hodgkin lymphoma.Further examples of B-cell proliferative disorders include, but are not limited to, multiple myeloma (MM); low-grade / follicular NHL; small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphocytic NHL; high-grade small non-dividing cell NHL; bulky mass disease NHL; AIDS-related lymphoma; and acute lymphoblastic leukemia (ALL); chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD). In certain instances, the B-cell proliferative disorder can be NHL (e.g., DLBCL (e.g., relapsed or refractory DLBCL), PMLBCL, or FL) or CLL.

[0119] The methods described herein can result in an improved benefit-risk profile for patients with cancer (e.g., a B-cell proliferative disorder, e.g., NHL (e.g., DLBCL (e.g., relapsed or refractory DLBCL), PMLBCL, or FL) or CLL) being treated with an anti-CD20 / anti-CD3 bispecific antibody. In some instances, treatment using the methods described herein that result in administering an anti-CD20 / anti-CD3 bispecific antibody in accordance with a fractionated dose-escalation regimen can result in a reduction in cytokine-driven toxicity (e.g., cytokine release syndrome (CRS)), infusion-related reactions (IRR), macrophage activation syndrome (MAS), and the like following treatment with an anti-CD20 / anti-CD3 bispecific antibody using a fractionated dose-escalation regimen of the invention compared to treatment with an anti-CD20 / anti-CD3 bispecific antibody using a non-fractionated regimen. ), neurotoxicity, severe tumor lysis syndrome (TLS), neutropenia, thrombocytopenia, elevated liver enzymes, and / or central nervous system (CNS) toxicity, resulting in a reduction (by 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more), or complete inhibition (100% reduction) of undesirable events.

[0120] These methods can comprise administering the anti-CD20 / anti-CD3 bispecific antibody (and / or any additional therapeutic agent) by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if desired for localized treatment, intralesional administration. Parenteral infusion includes intravenous, subcutaneous, intramuscular, intraarterial, and intraperitoneal routes of administration. In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody is administered by intravenous infusion. In other examples, the anti-CD20 / anti-CD3 bispecific antibody is administered subcutaneously. In some examples, an anti-CD20 / anti-CD3 bispecific antibody administered by intravenous infusion exhibits lower toxic reactions (i.e., fewer unwanted effects) in patients than the same anti-CD20 / anti-CD3 bispecific antibody administered by subcutaneous infusion, or vice versa.

[0121] For all of the methods described herein, the anti-CD20 / anti-CD3 bispecific antibody will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of drug delivery, the method of administration, the administration schedule, and other factors known to medical professionals. The anti-CD20 / anti-CD3 bispecific antibody need not be, but optionally is, formulated with one or more agents currently used to prevent or treat the disease in question. The effective amount of such other agents will depend on the amount of anti-CD20 / anti-CD3 bispecific antibody present in the formulation, the type of disease or treatment, and other factors discussed above. The anti-CD20 / anti-CD3 bispecific antibody can be appropriately administered to the patient over a course of treatment.

[0122] If the subject experiences an adverse event of cytokine release syndrome (CRS) after initiation of any of the methods described above, the methods can include additional treatment to treat the CRS event.

[0123] The National Cancer Institute's (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v4.0 includes a grading system for CRS, which was subsequently revised by Lee et al. (Blood. 124(2):188-95, 2014) to define CRS as mild, moderate, severe, or life-threatening despite stimulants. This revised CRS grading system is shown in Table 1 below. TIFF0007784795000001.tif102170

[0124] As shown in Table 1, CRS can lead to significant disability or death if not managed well. Current clinical management focuses on treating individual signs and symptoms, providing supportive care, and attempting to attenuate the inflammatory response using high doses of corticosteroids. However, this approach is not always successful, especially in cases of late intervention. CRS observed in patients during a two-phase fractionated-dose escalation regimen can be alternatively managed.

[0125] CRS is associated with elevations in a variety of cytokines, with marked increases in IFNγ, IL-6, and TNF-α levels. Emerging evidence specifically implicates IL-6 as a central mediator in CRS. IL-6 is a proinflammatory, multifunctional cytokine produced by a variety of cell types, and this cytokine has been shown to be involved in a wide variety of physiological processes, including T cell activation. Despite stimulatory agents, CRS is associated with high levels of IL-6 (Nagorsen et al. Cytokine. 25(1):31-5, 2004; Lee et al. Blood. 124(2):188-95, 2014; Doesegger et al. Clin. Transl. Immunology. 4(7):e39, 2015), and IL-6 correlates with CRS severity, with patients experiencing grade 4 or 5 CRS events containing even higher levels of IL-6 compared to patients who experience no CRS or milder CRS (grade 0-3) (Chen et al. J. Immunol. Methods. 434:1-8, 2016). Therefore, blocking the inflammatory effects of IL-6 with agents that inhibit IL-6-mediated signaling and manage the CRS observed in patients on a two-fractionated, dose-escalating regimen is an alternative to steroid treatment and is not expected to negatively affect T-cell function or reduce the efficacy or clinical usefulness of anti-CD20 / anti-CD3 bispecific antibody therapy in the treatment of B-cell proliferative disorders.

[0126] Tocilizumab (ACTEMRA® / RoACTEMRA®) is a recombinant, humanized, anti-human monoclonal antibody directed against the soluble, membrane-bound IL-6R that inhibits IL-6-mediated signaling (see, e.g., WO1992 / 019579, which is incorporated herein by reference in its entirety).

[0127] If the subject experiences a cytokine release syndrome (CRS) event after administration of the bispecific antibody, the method can further comprise administering to the subject an effective amount of an interleukin-6 receptor (IL-6R) antagonist (e.g., an anti-IL-6R antibody, e.g., tocilizumab (ACTEMRA® / RoACTEMRA®)) to manage the event. In some examples, tocilizumab is administered intravenously to the subject as a single dose of about 8 mg / kg. Other anti-IL-6R antibodies can be used in place of or in conjunction with tocilizumab, including sarilumab, bovalilizumab (ALX-0061), SA-237, and variants thereof.

[0128] If the subject has a CRS event that does not improve or worsen within 24 hours of administering the IL-6R antagonist to treat the symptoms of the CRS event, the method can further comprise administering one or more additional doses of an IL-6R antagonist (e.g., an anti-IL-6R antibody, e.g., tocilizumab) to the subject to manage the CRS event. If the CRS event is not managed through administration of the IL-6R antagonist, the subject can be administered a corticosteroid, such as methylprednisolone or dexamethasone.

[0129] Management of the CRS event can be tailored based on the stage of CRS and the presence of comorbidities. For example, if, after administration of the bispecific antibody, the subject has a Grade 2 cytokine release syndrome (CRS) event in the absence of comorbidities or in the presence of minimal comorbidities, the method can further comprise treating the symptoms of the Grade 2 CRS event while withholding treatment with the bispecific antibody. If the Grade 2 CRS event resolves to a Grade ≦1 CRS event for at least three consecutive days, the method can further comprise resuming treatment with the bispecific antibody at the same dosage. On the other hand, if the Grade 2 CRS event does not resolve or worsen to a Grade ≥ 3 CRS event within 24 hours of treating the symptoms of the Grade 2 CRS event, the method may further comprise administering to the subject an effective amount of an interleukin-6 receptor (IL-6R) antagonist (e.g., an anti-IL-6R antibody, e.g., tocilizumab (ACTEMRA® / RoACTEMRA®)) to manage the Grade 2 or Grade ≥ 3 CRS event. In some examples, tocilizumab is administered intravenously to the subject as a single dose of about 8 mg / kg. Other anti-IL-6R antibodies can be used in place of or in conjunction with tocilizumab, including sarilumab, bovalilizumab (ALX-0061), SA-237, and variants thereof.

[0130] If the subject experiences a grade 2 CRS event in the presence of extensive complications after administration of the bispecific antibody, the method may further comprise administering to the subject a first dose of an IL-6R antagonist (e.g., an anti-IL-6R antibody, e.g., tocilizumab (ACTEMRA® / RoACTEMRA®)) to manage the grade 2 CRS event while withholding treatment with the bispecific antibody. In some examples, the first dose of tocilizumab is administered intravenously to the subject in a single dose of about 8 mg / kg. Other anti-IL-6R antibodies may be used in place of or in conjunction with tocilizumab, including sarilumab, bovalilizumab (ALX-0061), SA-237, and variants thereof. In some examples, if the grade 2 CRS event resolves to a grade ≦1 CRS event within two weeks, the method may further comprise resuming treatment with the bispecific antibody at a reduced dose. In some examples, the reduced dose is the next highest clarified dose of the bispecific antibody in the first administration cycle of the non-fractionated dose escalation regimen. On the other hand, if the Grade 2 CRS event does not resolve or worsen to a Grade ≥ 3 CRS event within 24 hours of treating the symptoms of the Grade 2 CRS event, the method can further comprise administering one or more (e.g., 1, 2, 3, 4, or 5 or more) additional doses of an IL-6R antagonist (e.g., an anti-IL-6R antibody, e.g., tocilizumab) to the subject to manage the Grade 2 or Grade ≥ 3 CRS event. In some specific examples, since the Grade 2 CRS event does not resolve or worsen to a Grade ≥ 3 CRS event within 24 hours of treating the symptoms of the Grade 2 CRS event, the method can further comprise administering one or more additional doses of tocilizumab to the subject to manage the Grade 2 or Grade ≥ 3 CRS event. In some examples, one or more additional doses of tocilizumab are administered intravenously to the subject at a single dose of about 1 mg / kg to about 15 mg / kg, e.g., about 4 mg / kg to about 10 mg / kg, e.g., about 6 mg / kg to about 10 mg / kg, e.g., about 8 mg / kg.In some examples, the method further comprises administering an effective amount of a corticosteroid to the subject. The corticosteroid can be administered before, after, or simultaneously with one or more additional doses of tocilizumab or other anti-IL-6R antibody. In some examples, the corticosteroid is administered intravenously to the subject. In some examples, the corticosteroid is methylprednisolone. In some examples, the methylprednisolone is administered at a dose of about 1 mg / kg per day to about 5 mg / kg per day, for example, about 2 mg / kg per day. In some examples, the corticosteroid is dexamethasone. In some examples, the dexamethasone is administered at a dose of about 10 mg (e.g., a single dose of about 10 mg intravenously).

[0131] If the subject experiences a grade 3 CRS event after administration of the bispecific antibody, the method may further comprise administering to the subject a first dose of an IL-6R antagonist (e.g., an anti-IL-6R antibody, e.g., tocilizumab (ACTEMRA® / RoACTEMRA®)) to manage the grade 3 CRS event while withholding treatment with the bispecific antibody. In some examples, the first dose of tocilizumab is administered intravenously to the subject in a single dose of about 8 mg / kg. Other anti-IL-6R antibodies may be used in place of or in conjunction with tocilizumab, including sarilumab, bovalilizumab (ALX-0061), SA-237, and variants thereof. In some examples, since the grade 3 CRS event resolves to a grade ≦1 CRS event within two weeks, the method may further comprise resuming treatment with the bispecific antibody at a reduced dose. In some examples, the reduced dose is the next highest clarified dose of the bispecific antibody in the first administration cycle of the non-fractionated dose escalation regimen. In other examples, if the grade 3 CRS event does not resolve or worsen to a grade 4 CRS event within 24 hours of treating the symptoms of the grade 3 CRS event, the method can further comprise administering to the subject one or more (e.g., 1, 2, 3, 4, or 5 or more) additional doses of an IL-6R antagonist (e.g., an anti-IL-6R antibody, e.g., tocilizumab) to manage the grade 3 or grade 4 CRS event. In some specific examples, since the grade 3 CRS event does not resolve or worsen to a grade 4 CRS event within 24 hours of treating the symptoms of the grade 3 CRS event, the method can further comprise administering to the subject one or more additional doses of tocilizumab to manage the grade 3 or grade 4 CRS event. In some examples, one or more additional doses of tocilizumab are administered intravenously to the subject at a single dose of about 1 mg / kg to about 15 mg / kg, e.g., about 4 mg / kg to about 10 mg / kg, e.g., about 6 mg / kg to about 10 mg / kg, e.g., about 8 mg / kg.In some examples, the method further comprises administering an effective amount of a corticosteroid to the subject. The corticosteroid can be administered before, after, or simultaneously with one or more additional doses of tocilizumab or other anti-IL-6R antibody. In some examples, the corticosteroid is administered intravenously to the subject. In some examples, the corticosteroid is methylprednisolone. In some examples, the methylprednisolone is administered at a single dose of about 1 mg / kg per day to about 5 mg / kg per day, for example, about 2 mg / kg per day. In some examples, the corticosteroid is dexamethasone. In some examples, the dexamethasone is administered at a single dose of about 10 mg (e.g., a single dose of about 10 mg intravenously).

[0132] If the subject experiences a grade 4 CRS event after administration of the bispecific antibody, the method may further comprise administering to the subject a first dose of an IL-6R antagonist (e.g., an anti-IL-6R antibody, e.g., tocilizumab (ACTEMRA® / RoACTEMRA®)) to manage the grade 4 CRS event and persistently discontinuing treatment with the bispecific antibody. In some examples, the first dose of tocilizumab is administered intravenously to the subject in a single dose of about 8 mg / kg. Other anti-IL-6R antibodies may be used in place of or in conjunction with tocilizumab, including sarilumab, bovalilizumab (ALX-0061), SA-237, and variants thereof. In some examples, the grade 4 CRS event may resolve within 24 hours of treating the symptoms of the grade 4 CRS event. In other examples, if the grade 4 CRS event does not resolve within 24 hours of treating the symptoms of the grade 4 CRS event, the method can further comprise administering to the subject one or more additional doses of an IL-6R antagonist (e.g., an anti-IL-6R antibody, e.g., tocilizumab (ACTEMRA® / RoACTEMRA®))) to manage the grade 4 CRS event. In some particular examples, because the grade 4 CRS event does not resolve within 24 hours of treating the symptoms of the grade 4 CRS event, the method further comprises administering to the subject one or more (e.g., 1, 2, 3, 4, or 5 or more) additional doses of tocilizumab to manage the grade 4 CRS event. In some examples, the one or more additional doses of tocilizumab are administered intravenously to the subject at a single dose of about 1 mg / kg to about 15 mg / kg, for example, about 4 mg / kg to about 10 mg / kg, for example, about 6 mg / kg to about 10 mg / kg, for example, about 8 mg / kg. In some examples, the method further comprises administering an effective amount of a corticosteroid to the subject. The corticosteroid can be administered before, after, or simultaneously with the one or more additional doses of tocilizumab or other anti-IL-6R antibody. In some examples, the corticosteroid is administered intravenously to the subject.In some instances, the corticosteroid is methylprednisolone. In some instances, the methylprednisolone is administered at a single dose of about 1 mg / kg per day to about 5 mg / kg per day, for example, about 2 mg / kg per day. In some instances, the corticosteroid is dexamethasone. In some instances, the dexamethasone is administered at a single dose of about 10 mg (e.g., a single dose of about 10 mg intravenously).

[0133] A. Anti-CD20 / anti-CD3 bispecific antibody The methods described herein comprise administering a bispecific antibody that binds CD20 and CD3 (i.e., an anti-CD20 / anti-CD3 antibody) to a subject with cancer (e.g., a B-cell proliferative disorder, e.g., non-Hodgkin's lymphoma (NHL), e.g., diffuse large B-cell lymphoma (DLBCL), e.g., relapsed or refractory DLBCL).

[0134] In some examples, any of the methods described herein may comprise administering a bispecific antibody, wherein the bispecific antibody comprises an anti-CD20 arm having a first binding domain with at least one, two, three, four, five, or six HVRs selected from: (a) hypervariable region (HVR)-H1 comprising the amino acid sequence GYTFTSYNMH (SEQ ID NO: 1); (b) HVR-H2 comprising the amino acid sequence AIYPGNGDTSYNQKFKG (SEQ ID NO: 2); (c) HVR-H3 comprising the amino acid sequence VVYYSNSYWYFDV (SEQ ID NO: 3); (d) HVR-L1 comprising the amino acid sequence RASSSVSYMH (SEQ ID NO: 4); (e) HVR-L2 comprising the amino acid sequence APSNLAS (SEQ ID NO: 5); and (f) HVR-L3 comprising the amino acid sequence QQWSFNPPT (SEQ ID NO: 6). In some examples, the anti-CD20 / anti-CD3 bispecific antibody comprises at least one (e.g., one, two, three, or four) heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprising the sequences of SEQ ID NOs: 17 to 20, respectively, and / or at least one (e.g., one, two, three, or four) light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs: 21 to 24, respectively. In some examples, the bispecific antibody comprises an anti-CD20 arm comprising: (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 7, or to the amino acid sequence of SEQ ID NO: 7; (b) a light chain variable (VL) domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 8, or to the amino acid sequence of SEQ ID NO: 8; or (c) a first binding domain comprising a VH domain as in (a) and a VL domain as in (b). Thus, in some examples, the first binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 7, and a VL domain comprising the amino acid sequence of SEQ ID NO: 8.

[0135] In some examples, any of the methods described herein can comprise administering a bispecific antibody comprising an anti-CD3 arm having a second binding domain with at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 9); (b) HVR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 10); (c) HVR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 11); (d) HVR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 12); (e) HVR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 13); and (f) HVR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 14). In some examples, the anti-CD20 / anti-CD3 bispecific antibody comprises at least one (e.g., one, two, three, or four) heavy chain framework regions FR-H1, FR-H2, FR-H3, and FR-H4 comprising the sequences of SEQ ID NOs: 25 to 28, respectively, and / or at least one (e.g., one, two, three, or four) light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs: 29 to 32, respectively. In some examples, the bispecific antibody comprises an anti-CD3 arm comprising: (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 15, or to the amino acid sequence of SEQ ID NO: 15, (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16, or to the amino acid sequence of SEQ ID NO: 16, or (c) a second binding domain comprising a VH domain as in (a) and a VL domain as in (b). Thus, in some examples, the second binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 15, and a VL domain comprising the amino acid sequence of SEQ ID NO: 16.

[0136] In some examples, any of the methods described herein can comprise administering a bispecific antibody, wherein the bispecific antibody comprises at least one, two, three, four, or five amino acids selected from: (1) an HVR-H1 comprising the amino acid sequence GYTFTSYNMH (SEQ ID NO: 1); (b) an HVR-H2 comprising the amino acid sequence AIYPGNGDTSYNQKFKG (SEQ ID NO: 2); (c) an HVR-H3 comprising the amino acid sequence VVYYSNSYWYFDV (SEQ ID NO: 3); (d) an HVR-L1 comprising the amino acid sequence RASSSVSYMH (SEQ ID NO: 4); (e) an HVR-L2 comprising the amino acid sequence APSNLAS (SEQ ID NO: 5); and (f) an HVR-L3 comprising the amino acid sequence QQWSFNPPT (SEQ ID NO: 6). and (2) an anti-CD20 arm comprising a first binding domain comprising at least one, two, three, four, five, or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 9), (b) HVR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 10), (c) HVR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 11), (d) HVR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 12), (e) HVR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 13), and (f) HVR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 14).In some examples, an anti-CD20 / anti-CD3 bispecific antibody comprises (1) at least one (e.g., one, two, three, or four) positions of heavy chain framework region FR-H1, FR-H2, FR-H3, and FR-H4 comprising the sequences of SEQ ID NOs: 17-20, respectively, and / or at least one (e.g., one, two, three, or four) positions of light chain framework region FR-L1, FR-L2, FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs: 21-24, respectively, and (2) at least one (e.g., one, two, three, or four) positions of heavy chain framework region FR-H1, FR-H2, FR-H3, and FR-H4 comprising the sequences of SEQ ID NOs: 25-28, respectively, and / or at least one (e.g., one, two, three, or four) positions of light chain framework region FR-L1, FR-L2, FR-L3, and FR-L4 comprising the sequences of SEQ ID NOs: 29-32, respectively.In some examples, the anti-CD20 / anti-CD3 bispecific antibody comprises: (1) a first binding domain comprising: (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 7 or to the amino acid sequence of SEQ ID NO: 7; (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 8 or to the amino acid sequence of SEQ ID NO: 8; or (c) a VH domain as in (a) and a VL domain as in (b). and (2) an anti-CD3 arm comprising a second binding domain comprising: (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 15, or to the amino acid sequence of SEQ ID NO: 15; (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to SEQ ID NO: 16, or to the amino acid sequence of SEQ ID NO: 16; or (c) a VH domain as in (a) and a VL domain as in (b). In some examples, the anti-CD20 / anti-CD3 bispecific antibody comprises (1) a first binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO:7 and a VL domain comprising the amino acid sequence of SEQ ID NO:8, and (2) a second binding domain comprising a VH domain comprising the amino acid sequence of SEQ ID NO:15 and a VL domain comprising the amino acid sequence of SEQ ID NO:16.

[0137] Anti-CD20 / anti-CD3 bispecific antibodies can be produced using recombinant methods and compositions, such as those described in US Pat. No. 4,816,567.

[0138] In some instances, an anti-CD20 / anti-CD3 bispecific antibody according to any of the above embodiments may incorporate any of the features, alone or in combination, as described in sections 1 to 5 below.

[0139] 1. Antibody affinity In certain embodiments, the bispecific antibodies provided herein have a cytotoxicity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 It has a dissociation constant (Kd) of 1 M.

[0140] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of a Fab for an antigen is determined by binding the Fab to a minimal concentration ( 125 I) is measured by equilibrating with labeled antigen and then capturing the bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the conditions for the assay, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125[I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although this incubation can be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μL / well of scintillant (MICROSCINT-20™; Packard) is added, and the plate is counted for 10 minutes on a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that confers 20% or less of maximal binding is selected for use in competitive binding assays.

[0141] According to another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C with an immobilized antigen CM5 chip at approximately 10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of coupled protein. Following antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C at a flow rate of approximately 25 μL / min. on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). off / k on The on-rate is calculated as a ratio. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate is 10 6 M- 1 s- 1If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer such as a stopped-flow fitted spectrophotometer (Aviv Instruments) with a stirred cuvette or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic).

[0142] 2. Antibody fragments In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody is an antibody fragment, e.g., an antibody fragment that binds to both CD20 and CD3. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0143] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, European Patent No. 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0144] Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0145] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

[0146] 3. Chimeric and humanized antibodies In certain embodiments, the anti-CD20 / anti-CD3 antibody for use in the methods described herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al. Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0147] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the non-human parent antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. Optionally, a humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0148] Humanized antibodies and methods for their production are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633, (2008), and are also described, e.g., in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, and Kashmiri et al., Methods 36:25-34 (2005) (describing specificity-determining region (SDR) grafting), Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"), Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"), Osbourn et al., Methods 36:61-68 (2005), and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0149] Human framework regions that can be used for humanization include framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)), framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions obtained from screening of FR libraries (see, e.g., Baca et al. J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al. al., J. Biol. Chem. 271:22611-22618 (1996)).

[0150] 4. Knob-in-Hole Bispecific Antibody Engineering Anti-CD20 / anti-CD3 bispecific antibodies can be prepared as full-length antibodies or antibody fragments. Techniques for producing bispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, for example, U.S. Pat. No. 5,731,168). "Knob-in-hole" bispecific antibody engineering can be used to generate a first arm containing a knob and a second arm containing a hole to which the knob of the first arm can bind. In one embodiment, the knob of the bispecific antibody can be on the anti-CD3 arm. Alternatively, the knob of the bispecific antibody of the present invention can be on the anti-CD20 arm. In one embodiment, the hole of the bispecific antibody of the present invention can be on the anti-CD3 arm. Alternatively, the hole of the bispecific antibody of the present invention can be on the anti-CD20 arm. In some examples, the anti-CD20 / anti-CD3 bispecific antibody produced using the knobs-in-holes technology can comprise one or more heavy chain constant domains, where the one or more heavy chain constant domains are selected from a first CH1 (CH11) domain, a first CH2 (CH21) domain, a first CH3 (CH31) domain, a second CH1 (CH12) domain, a second CH2 (CH22) domain, and a second CH3 (CH32) domain. In some examples, at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain. In some examples, the CH31 and CH32 domains each comprise a protrusion or cavity, and the protrusion or cavity in the CH31 domain can be positioned within the cavity or protrusion in the CH32 domain, respectively. In some examples, the CH31 and CH32 domains meet at the interface between the protrusion and the cavity. In some instances, the CH21 and CH22 domains each comprise a protrusion or a cavity, and the protrusion or cavity in the CH21 domain can be positioned within the cavity or protrusion in the CH22 domain, respectively.In some instances, the CH21 and CH22 domains associate at the interface between the protrusion and the cavity.

[0151] Bispecific antibodies can also be engineered using immunoglobulin crossover (also known as Fab domain exchange or CrossMab format) technology (see, e.g., WO2009 / 080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)). Bispecific antibodies can also be produced by engineering electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004 A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)), using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 148(5):1547-1553 (1992)). Such antibodies can be produced by preparing trispecific antibodies as described in, for example, Tutt et al. J. Immunol. 147:60 (1991).

[0152] Anti-CD20 / anti-CD3 bispecific antibodies, or antibody fragments thereof, can also include "dual acting FAbs" or "DAFs" that contain antigen binding sites that bind to CD3 and CD20 (see, e.g., U.S. Publication No. 2008 / 0069820).

[0153] 5. Anti-CD20 / anti-CD3 bispecific antibody variants In some instances, amino acid sequence variants of the anti-CD20 / anti-CD3 bispecific antibodies described above are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the bispecific antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0154] Substitution, insertion, and deletion mutants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions." More substantial changes are shown in Table 2 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest, and the products can be screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. TIFF0007784795000002.tif223170 Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0155] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0156] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing will have modified (e.g., improved) certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0157] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be made at HVR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or at residues that contact the antigen, and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction of and reselection from secondary libraries is described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0158] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made within an HVR. Such modifications may, for example, be outside of the antigen-contact residues within the HVR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unaltered or has no more than one, two, or three amino acid substitutions.

[0159] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" and is described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, target residues or groups (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody's interaction with the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex can identify contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted as candidates for substitution or can be eliminated. The mutants can be screened to determine whether they have the desired properties.

[0160] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0161] B glycosylation mutants In some instances, the methods of the invention comprise administering to a subject in the context of a fractionated dose escalation regimen an anti-CD20 / anti-CD3 bispecific antibody variant that has been modified to increase or decrease the extent to which the bispecific antibody is glycosylated. Addition or deletion of glycosylation sites to an anti-CD20 / anti-CD3 antibody of the invention can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0162] If the bispecific antibody contains an Fc region, the carbohydrate attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are commonly attached to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modifications can be made to the antibodies of the present invention to generate antibody variants with certain improved properties.

[0163] In some examples, these methods comprise administering an anti-CD20 / anti-CD3 bispecific antibody variant having a glycan structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU numbering of Fc region residues) within the Fc region; however, Asn297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. 2003 / 0157108 (Presta, L.) and 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 013214 0, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1 (Presta, L); and WO2004 / 056312A1 (Adams et al.) (particularly in Example 11)), and knockout cell lines such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0164] In view of the above, in some examples, the methods of the invention comprise administering to a subject a mutant anti-CD20 / anti-CD3 bispecific antibody having an aglycosylation site mutation in accordance with a fractionated dose-escalation regimen. In some examples, the aglycosylation site mutation reduces the effector function of the bispecific antibody. In some examples, the aglycosylation site mutation is a substitution mutation. In some examples, the bispecific antibody comprises a substitution mutation in the Fc region that reduces effector function. In some examples, the substitution mutation is at amino acid residues N297, L234, L235, and / or D265 (EU numbering). In some examples, the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, D265A, and P329G. In some examples, the substitution mutation is at amino acid residue N297. In a preferred embodiment, the substitution mutation is N297A.

[0165] In another example, bispecific antibody variants containing bisected oligosaccharides are used in accordance with the methods of the present invention, e.g., where the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Pat. No. 6,602,684 (Umana et al.), and U.S. Patent Publication No. 2005 / 0123546 (Umana et al.). Antibody variants containing at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.), WO1998 / 58964 (Raju, S.), and WO1999 / 22764 (Raju, S.).

[0166] c.Fc region variants In some examples, anti-CD20 / anti-CD3 bispecific antibody variants have one or more amino acid modifications introduced into the Fc region of the bispecific antibody (i.e., Fc region variants (see, e.g., US2012 / 0251531)) and can be administered to a subject with cancer (e.g., a B-cell proliferative disorder) according to the methods of the invention. The Fc region variants can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0167] In some instances, bispecific Fc region antibody variants retain some, but not all, effector functions, making them desirable candidates for uses in which in vivo antibody half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al. J. Immunol. Methods 202:163 (1996); Cragg, MS et al. Blood. 101:1045-1052 (2003); and Cragg, MS and MJ Glennie Blood. 103:2738-2743 (2004)). FcRn binding and bioclearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al. Int'l. Immunol. 18(12):1759-1769 (2006)).

[0168] Antibodies with reduced effector function include antibodies with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent Nos. 6,737,056 and 8,219,149). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine (U.S. Patent Nos. 7,332,581 and 8,219,149).

[0169] In certain instances, the proline at position 329 of the wild-type human Fc region in the antibody is substituted with glycine or arginine, or an amino acid residue large enough to disrupt the proline sandwich within the Fc / Fcγ receptor interface formed between proline 329 of the Fc and tryptophan residues Trp87 and Trp110 of FcgRIII (Sondermann et al. Nature. 406, 267-273 (2000)). In certain embodiments, the bispecific antibody has at least one additional amino acid substitution. In one embodiment, the additional amino acid substitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S; in yet another embodiment, the at least one additional amino acid substitution is L234A and L235A in the human IgG1 Fc region, or S228P and L235E in the human IgG4 Fc region (see, e.g., US2012 / 0251531); and in yet another embodiment, the at least one additional amino acid substitution is L234A, L235A, and P329G in the human IgG1 Fc region.

[0170] Certain antibody variants have been described with improved or reduced binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0171] In certain instances, the anti-CD20 / anti-CD3 bispecific antibody comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0172] In some instances, changes are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0173] Antibodies with increased half-lives and improved binding to neonatal Fc receptors (FcRn), which are responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent Publication No. 2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0174] See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0175] d. Cysteine ​​engineered antibody variants In certain embodiments, it may be desirable to generate cysteine-engineered anti-CD20 / anti-CD3 bispecific antibodies, e.g., "thioMAbs," in which one or more residues of the bispecific antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites on the antibody. By replacing those residues with cysteine, reactive thiol groups are positioned at accessible sites on the bispecific antibody, which can be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to generate immunoconjugates. In certain embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be produced, for example, as described in U.S. Pat. No. 7,521,541.

[0176] Thus, immunoconjugates of anti-CD20 / anti-CD3 bispecific antibodies conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioisotope, are specifically contemplated.

[0177] In some examples, the immunoconjugate may comprise a bispecific antibody or a compound selected from the group consisting of maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. EP 0425235 B1); auristatins, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 5,820,589); 8, and 7,498,298); dolastatins; calicheamicins or their derivatives (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes (such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel); trichothecenes; and antibody-drug conjugates (ADCs) conjugated to one or more drugs, including, but not limited to, CC1065.

[0178] In some examples, the immunoconjugate comprises a bispecific antibody conjugated to an enzymatically active toxin, or a fragment thereof, including, but not limited to, diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diansin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0179] In another embodiment, the immunoconjugate comprises a bispecific antibody conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu. When a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, such as tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as, again, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0180] Conjugates of anti-CD20 / anti-CD3 bispecific antibodies and cytotoxic agents can be made using a variety of bifunctional protein-linking agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker may also be a "cleavable linker" that facilitates the release of the cytotoxic drug within cells. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0181] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with crosslinker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0182] e. Other antibody derivatives In some instances, anti-CD20 / anti-CD3 bispecific antibodies can be modified to contain additional nonproteinaceous moieties that are known in the art and readily available and administered to a subject according to the methods described herein. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can be advantageous during manufacturing due to its stability in water. The polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[0183] In some examples, conjugates of antibodies and nonproteinaceous moieties are provided that can be selectively heated by exposure to radiation. In one example, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength, including, but not limited to, wavelengths that do not harm normal cells but heat the nonproteinaceous moiety to a temperature that kills cells in close proximity to the antibody-nonproteinaceous moiety.

[0184] B. Pharmaceutical Compositions and Formulations Pharmaceutical compositions and formulations of anti-CD20 / anti-CD3 bispecific antibodies can be prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations used, and include buffers (such as phosphate, citric acid, and other organic acids), antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins (such as serum albumin), and the like. Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, intercalating drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.), hydrophilic polymers (such as polyvinylpyrrolidone), amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine), monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin), chelating agents (such as EDTA), sugars (such as sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (such as sodium), metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants (such as polyethylene glycol (PEG)). Exemplary pharmaceutically acceptable carriers herein further include intercalating drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0185] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations comprising a histidine-acetate buffer.

[0186] The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an additional therapeutic agent (e.g., a chemotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, and / or an antihormonal agent, such as those mentioned hereinabove). Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0187] The active ingredient may also be incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, for example, by microcapsules prepared by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0188] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0189] Formulations to be used for in vivo administration are generally sterile. Sterilization may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0190] III.Manufactured products Another aspect of the present invention provides an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned diseases. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective, by itself or in combination with another composition, for the treatment, prevention, and / or diagnosis of a condition and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-CD20 / anti-CD3 bispecific antibody described herein. The label or package insert indicates that the composition is used to treat a selected condition (e.g., a B-cell proliferative disorder, e.g., non-Hodgkin's lymphoma (NHL), e.g., diffuse large B-cell lymphoma (DLBCL), e.g., relapsed or refractory DLBCL), and further includes information regarding at least one of the dosing regimens described herein. Moreover, the article of manufacture may include (a) a first container having disposed therein a composition comprising an anti-CD20 / anti-CD3 bispecific antibody described herein, and (b) a second container having disposed therein a composition comprising an additional cytotoxic agent or other therapeutic agent. Alternatively, or in addition, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. [Example]

[0191] IV. Working Examples The following are examples of methods of the present invention: In view of the above summary, it will be appreciated that various other embodiments may be practiced.

[0192] Example 1. Fractionated Dose-Escalating Regimen for Treatment of B-Cell Proliferative Disorders with Anti-Cluster of Differentiation 20 (CD20) / Anti-Cluster of Differentiation 3 (CD3) Bispecific Antibodies Using a "knob-in-hole" engineering design, a full-length IgG1 bispecific antibody that binds both CD20 and CD3 was produced (see, e.g., U.S. Patent No. 5,731,168). Safety and pharmacodynamics (PD) were consistent with its proposed mechanism of action in cynomolgus monkey toxicology studies. Toxicity associated with treatment with the anti-CD20 / anti-CD3 bispecific antibody was primarily driven by T cell stimulation, as evidenced by PD changes in cytokine levels and activated T cell numbers. In a repeat-dose toxicity study of the anti-CD20 / anti-CD3 bispecific antibody in cynomolgus monkeys, improvements in cytokine levels, T cell activation, and acute post-administration observations were primarily limited to the first dose and were reduced or negligible with subsequent doses. Therefore, a clinical plan was designed to control the degree of T cell stimulation through alternative dosing regimens.

[0193] In a previous dosing study, anti-CD20 / anti-CD3 bispecific antibodies were administered in an unfractionated fashion during cycle 1, with the entire dose given on cycle 1 day 1 (C1D1). Preclinical data on anti-CD20 / anti-CD3 bispecific antibodies indicated that two-step fractionation has the potential to reduce the risk of cytokine-driven toxicity. Therefore, we modified the protocol to administer anti-CD20 / anti-CD3 bispecific antibodies in accordance with cycle 1 of a two-step fractionated dose-escalation schedule.

[0194] To support this fractionated dosing schedule with anti-CD20 / anti-CD3 bispecific antibodies, an exploratory quantitative systems pharmacology (QSP) model was used to simulate the time course of systemic cytokine (IL6) and activated T cell profiles in cycle 1 after administration of anti-CD20 / anti-CD3 bispecific antibodies as a single agent using various dosing regimens in patients with non-Hodgkin's lymphoma (NHL). Model-based predictions of serum cytokine concentrations and activated T cell time profiles after two cycles of treatment with anti-CD20 / anti-CD3 bispecific antibodies as a single agent were used to compare unfractionated and two-phase fractionated dosing schedules. Modeling and simulations support achieving a more favorable benefit-risk profile through a two-phase fractionated dose-escalation regimen for treating hematological cancers, such as B-cell proliferative disorders (e.g., NHL, e.g., DLBCL), with anti-CD20 / anti-CD3 bispecific antibodies. Based on the above, a two-stage fractionated dose-escalation regimen is expected to reduce or inhibit cytokine-driven toxicities (e.g., cytokine release syndrome (CRS)), infusion-related reactions (IRR), macrophage activation syndrome (MAS), neurotoxicity, severe tumor lysis syndrome (TLS), neutropenia, thrombocytopenia, liver enzyme elevations, and / or central nervous system (CNS) toxicity.

[0195] The two-stage fractionated dose-escalation regimen begins simultaneously with the first dose cycle (C1) of the dose escalation, in which patients receive the anti-CD20 / anti-CD3 bispecific antibody on days 1, 8, and 15 of cycle 1 (C1D1, C1D8, and C1D15, respectively). From cycle 2 onward, the bispecific antibody is given as a single dose only on day 1 of each 21-day cycle, with day 1 of cycle 2 (C2D1) being approximately 7 days after the C1D15 dose. From cycle 2 onward, in which the bispecific antibody is given as a single dose only on day 1 of each 21-day cycle, the bispecific antibody can be administered simultaneously with atezolizumab on day 1 of each 21-day cycle. For logistical / scheduling reasons, the anti-CD20 / anti-CD3 bispecific antibody and atezolizumab are given ±2 days from the scheduled date (i.e., with a minimum of 19 days between doses).

[0196] The cumulative dose in Cycle 1 is approximately 50% greater than the highest cleared dose in the first cycle of the unfractionated dose-escalation regimen, and the starting C1D15 dose corresponds to the highest cleared dose in the first cycle of the unfractionated dose-escalation regimen. The C1D15 dose is the dose level administered on Day 1 of subsequent cycles (Cycles 2 through the final administration cycle). This dose escalation uses a standard 3+3 design.

[0197] Example 2. Management of cytokine release syndrome (CRS) with anti-interleukin-6 receptor (IL-6R) antibodies In the event that CRS is observed in a patient receiving an anti-CD20 / anti-CD3 bispecific antibody in accordance with the two-fractionated dose-escalation regimen described in Example 1, the observed CRS is appropriately managed.

[0198] CRS can lead to significant disability or death if not managed well. Current clinical management focuses on treating individual signs and symptoms, providing supportive care, and attempting to attenuate the inflammatory response using high doses of corticosteroids. However, this approach is not always successful, especially in cases of late intervention. CRS observed in patients can alternatively be managed during a two-phase fractionated dose-escalation regimen.

[0199] CRS is associated with elevations in a variety of cytokines, with marked increases in IFNγ, IL-6, and TNF-α levels. Emerging evidence specifically implicates IL-6 as a central mediator in CRS. IL-6 is a proinflammatory, multifunctional cytokine produced by a variety of cell types, and this cytokine has been shown to be involved in a wide variety of physiological processes, including T cell activation. Despite stimulatory agents, CRS is associated with high levels of IL-6 (Nagorsen et al. Cytokine. 25(1):31-5, 2004; Lee et al. Blood. 124(2):188-95, 2014; Doesegger et al. Clin. Transl. Immunology. 4(7):e39, 2015), and IL-6 correlates with CRS severity, with patients experiencing grade 4 or 5 CRS events containing even higher levels of IL-6 compared to patients who experience no CRS or milder CRS (grade 0-3) (Chen et al. J. Immunol. Methods. 434:1-8, 2016). Therefore, blocking the inflammatory effects of IL-6 with agents that inhibit IL-6-mediated signaling and manage the CRS observed in patients on a two-fractionated, dose-escalating regimen is an alternative to steroid treatment and is not expected to negatively affect T-cell function or reduce the efficacy or clinical usefulness of anti-CD20 / anti-CD3 bispecific antibody therapy in the treatment of B-cell proliferative disorders.

[0200] Tocilizumab (ACTEMRA® / RoACTEMRA®) is a recombinant, humanized, anti-human monoclonal antibody directed against the soluble, membrane-bound IL-6R that inhibits IL-6-mediated signaling (see, e.g., WO1992 / 019579, which is incorporated herein by reference in its entirety).

[0201] In the event of a Grade 1 CRS episode, management can be achieved without tocilizumab therapy, and CRS symptoms are treated symptomatically, e.g., with antihistamines, antipyretics, and / or analgesics as needed. In addition, fever and neutropenia, if present, are treated, and patients are monitored for fluid balance and intravenous fluids are administered as clinically indicated.

[0202] Grade 2 CRS events are managed in patients with no (or minimally comorbid) complications by following the management scheme for grade 1 CRS events: immediately temporarily withholding (i.e., delaying administration of) anti-CD20 / anti-CD3 bispecific antibody treatment until CRS symptoms have resolved to grade ≤1 for 3 consecutive days, at which point the patient can receive the next dose of anti-CD20 / anti-CD3 bispecific antibody without a dose reduction approved by the medical monitor. Closely monitor cardiac and other organ function in patients with grade 2 CRS and administer oxygen as needed for hypoxia. If there is no clinical improvement within 24 hours, the medical monitor will be notified and allow the patient to receive tocilizumab as a single dose of 8 mg / kg intravenously. Grade 2 CRS events in patients with extensive comorbidities are managed by following the management scheme for grade 3 CRS events below.

[0203] In the event of a grade 3 CRS episode, the medical monitor will be notified immediately. Cardiopulmonary and organ function will be monitored, oxygen will be administered for hypoxia, and hemodynamic support and other supportive care (e.g., for fever and / or neutropenia) will be provided as needed. In addition, anti-CD20 / anti-CD3 bispecific antibody therapy will be immediately withheld, and the patient will receive tocilizumab intravenously at 8 mg / kg. If clinical improvement does not occur within 24 hours, the patient will receive a second dose of tocilizumab intravenously at 8 mg / kg, optionally with the initiation of intravenous corticosteroid therapy (e.g., methylprednisolone at 2 mg / kg / day or dexamethasone at 10 mg for neurological symptoms). Administration of the anti-CD20 / anti-CD3 bispecific antibody can be delayed for up to 2 weeks to allow the patient time to recover from CRS toxicity. If CRS symptoms resolve to grade ≤1 within 2 weeks after tocilizumab administration, then administration of the anti-CD20 / anti-CD3 bispecific antibody is continued at a reduced dose. The reduced dose of the anti-CD20 / anti-CD3 bispecific antibody is the second-highest cleared dose level evaluated during dose escalation (i.e., a non-fractionated dose-escalation regimen). If similar toxicity is observed at the reduced dose, then anti-CD20 / anti-CD3 bispecific antibody treatment is discontinued. Additionally, if the reduced dose is below the pharmacodynamic activity range of the anti-CD20 / anti-CD3 bispecific antibody, then anti-CD20 / anti-CD3 bispecific antibody treatment can also be discontinued (i.e., permanently stopped).

[0204] Grade 4 CRS events are managed as described above for grade 3 CRS events, but anti-CD20 / anti-CD3 bispecific antibody treatment is immediately discontinued (ie, permanently stopped).

[0205] Other embodiments The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, but the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. 1. A medicament for treating a subject having a B-cell proliferative disorder, comprising a full length bispecific antibody that binds to CD20 and CD3, wherein the full length bispecific antibody is administered to the subject in a dosing regimen having at least a first 21-day dosing cycle and a second 21-day dosing cycle; (a) the first administration cycle comprises a first dose (C1D1), a second dose (C1D2), and a third dose (C1D3) of the full length bispecific antibody, wherein the C1D1 is about 1.0 mg, the C1D2 is about 2.0 mg, and the C1D3 is between about 3.0 mg and about 50.0 mg; (b) the second administration cycle comprises a single dose of the full length bispecific antibody (C2D1), wherein the C2D1 is equal to or greater than the C1D3 and is between about 3.0 mg and about 50.0 mg; the dosing regimen comprises administering to the subject the C1D1, the C1D2, and the C1D3 on days 1, 8, and 15, respectively, of the first dosing cycle, wherein the first dosing cycle is a two-step fractionated ascending-dose dosing schedule; The full length bispecific antibody comprises an anti-CD20 arm comprising a first binding domain comprising the following six hypervariable regions (HVRs): The six hypervariable regions are: (a) HVR-H1 comprising the amino acid sequence GYTFTSYNMH (SEQ ID NO: 1); (b) HVR-H2 comprising the amino acid sequence AIYPGNGDTSYNQKFKG (SEQ ID NO: 2); (c) HVR-H3 comprising the amino acid sequence VVYYSNSYWYFDV (SEQ ID NO: 3); (d) HVR-L1 comprising the amino acid sequence RASSSVSYMH (SEQ ID NO: 4); (e) HVR-L2 comprising the amino acid sequence APSNLAS (SEQ ID NO: 5); and (f) an HVR-L3 comprising the amino acid sequence QQWSFNPPT (SEQ ID NO: 6); and The full length bispecific antibody comprises an anti-CD3 arm comprising a second binding domain comprising the following six HVRs: The six HVRs are: (a) HVR-H1 comprising the amino acid sequence NYYIH (SEQ ID NO: 9); (b) HVR-H2 comprising the amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 10); (c) HVR-H3 comprising the amino acid sequence DSYSNYYFDY (SEQ ID NO: 11); (d) HVR-L1 comprising the amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 12); (e) HVR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO: 13), and (f) A pharmaceutical comprising HVR-L3 comprising the amino acid sequence TQSFILRT (SEQ ID NO: 14).

2. The pharmaceutical composition of claim 1, wherein the administration regimen comprises administering the C2D1 to the subject on day 1 of the second administration cycle.

3. The pharmaceutical composition of claim 1 or 2, wherein the administration regimen includes one or more additional administration cycles.

4. The method of claim 3, wherein the administration regimen comprises one to six additional administration cycles.

5. 5. The method of claim 3, wherein each of the one or more additional administration cycles is 21 days or 28 days long.

6. The medicament of any one of claims 3 to 5, wherein each of the one or more additional administration cycles comprises a single dose of the full length bispecific antibody.

7. The pharmaceutical composition of claim 6, wherein the dosing regimen comprises administering to the subject the single dose of the one or more additional dosing cycles on day 1 of the one or more additional dosing cycles.

8. 8. The method of claim 6 or 7, wherein each single dose of the one or more additional administration cycles is an amount equivalent to C2D1.

9. The full-length bispecific antibody comprises: (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7; (b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain according to (a) and the VL domain according to (b); The medicament of any one of claims 1 to 8, comprising an anti-CD20 arm comprising a first binding domain comprising:

10. The pharmaceutical composition of claim 9 , wherein the first binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO:

8.

11. The full-length bispecific antibody comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16; or (c) the VH domain according to (a) and the VL domain according to (b); The medicament of any one of claims 1 to 10, comprising an anti-CD3 arm comprising a second binding domain comprising:

12. The pharmaceutical composition of claim 11 , wherein the second binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and a VL domain comprising the amino acid sequence of SEQ ID NO:

16.

13. (i) the first binding domain comprises: (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7; (b) a light chain variable (VL) domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 8; or (c) the VH domain described in (a) and the VL domain described in (b); (ii) The pharmaceutical according to any one of claims 9 to 12, wherein the second binding domain comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16; or (c) the VH domain described in (a) and the VL domain described in (b).

14. (i) the first binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8; (ii) The pharmaceutical according to claim 13, wherein the second binding domain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and a VL domain comprising the amino acid sequence of SEQ ID NO:

16.

15. The pharmaceutical of any one of claims 1 to 14, wherein the full-length bispecific antibody comprises an aglycosylation site mutation.

16. The pharmaceutical according to claim 15 , wherein the aglycosylation site mutation reduces the effector function of the full-length bispecific antibody.

17. The pharmaceutical according to claim 15 or 16, wherein the aglycosylation site mutation is a substitution mutation.

18. The pharmaceutical of claim 17 , wherein the full-length bispecific antibody comprises a substitution mutation in the Fc region that reduces effector function.

19. The pharmaceutical of claim 18, wherein the substitution mutation is at amino acid residues N297, L234, L235, D265, and / or P329 (EU numbering).

20. The pharmaceutical composition of claim 19, wherein the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, D265A, and P329G.

21. The pharmaceutical according to claim 19 or 20, wherein the substitution mutation is at amino acid residue N297.

22. The pharmaceutical of claim 21, wherein the substitution mutation is N297A.

23. The pharmaceutical of claim 14 , wherein the full-length bispecific antibody comprises an N297G substitution mutation in the Fc region.

24. The pharmaceutical according to any one of claims 1 to 23, wherein the full-length bispecific antibody is a humanized antibody.

25. The pharmaceutical according to any one of claims 1 to 24, wherein the full-length bispecific antibody is a chimeric antibody.

26. The pharmaceutical according to any one of claims 1 to 25, wherein the full-length bispecific antibody is an IgG antibody.

27. The IgG antibody is IgG １ The pharmaceutical according to claim 26, which is an antibody.

28. The full-length bispecific antibody comprises one or more heavy chain constant domains, the one or more heavy chain constant domains comprising a first CH1 (CH1 １ ) domain, first CH2 (CH2 １ ) domain, first CH3 (CH3 １ ) domain, second CH1 (CH1 ２ ) domain, second CH2 (CH2 ２ ) domain, and the second CH3 (CH3 ２ 28. The pharmaceutical of any one of claims 1 to 27, wherein the domain is selected from the group consisting of α- and β-actin.

29. 29. The pharmaceutical of claim 28, wherein at least one of the one or more heavy chain constant domains is paired with another heavy chain constant domain.

30. CH3 １ and CH3 ２ The domains each contain a protrusion or a cavity, and the CH3 １ The protrusion or cavity within the domain is ２ The pharmaceutical agent according to claim 28 or 29, which is positionable within the cavity or protrusion, respectively, within the domain.

31. CH3 １ and CH3 ２ The pharmaceutical of claim 30, wherein the domains associate at the interface between the protrusion and the cavity.

32. CH2 １ and CH2 ２ The domains each contain a protrusion or a cavity, and the CH2 １ The protrusion or cavity within the domain is ２ The pharmaceutical agent according to any one of claims 28 to 31, which is positionable within the cavity or protrusion, respectively, within the domain.

33. CH2 １ and CH2 ２ The method of claim 32, wherein the domains associate at the interface between the protrusion and the cavity.

34. The pharmaceutical of claim 14, wherein the full-length bispecific antibody comprises a knob-in-hole modification, wherein the anti-CD3 arm comprises a hole and the anti-CD20 arm comprises a knob.

35. The pharmaceutical according to any one of claims 1 to 34, wherein the full-length bispecific antibody is administered to the subject as a monotherapy.

36. The pharmaceutical according to any one of claims 1 to 34, wherein the full-length bispecific antibody is administered to the subject together with one or more additional therapeutic agents.

37. The pharmaceutical according to claim 36, wherein the full-length bispecific antibody is administered to the subject simultaneously with the one or more additional therapeutic agents.

38. The pharmaceutical according to claim 36, wherein the full-length bispecific antibody is administered to the subject prior to administration of the one or more additional therapeutic agents.

39. The pharmaceutical agent of claim 37 or 38, wherein the one or more additional therapeutic agents is atezolizumab.

40. 40. The pharmaceutical agent of any one of claims 1 to 39, wherein the dosing regimen further comprises administering to the subject a first dose of atezolizumab concurrently with the full length bispecific antibody C2D1 on day 1 of the second dosing cycle.

41. 41. The pharmaceutical agent of claim 40, wherein the dosing regimen further comprises administering atezolizumab to the subject concurrently with the single dose of the full length bispecific antibody for one or more additional dosing cycles on day 1 of the one or more additional dosing cycles.

42. 42. The medicament of claim 41, wherein atezolizumab is administered to the subject only simultaneously with the full length bispecific antibody.

43. 43. The pharmaceutical agent of any one of claims 39 to 42, wherein each dose of atezolizumab is about 1200 mg.

44. The pharmaceutical of claim 36, wherein the full-length bispecific antibody is administered to the subject after the administration of the one or more additional therapeutic agents.

45. 45. The pharmaceutical composition of claim 44, wherein the one or more additional therapeutic agents is obinutuzumab.

46. The pharmaceutical composition of claim 44, wherein the one or more additional therapeutic agents is tocilizumab.

47. The pharmaceutical of claim 36, wherein the one or more additional therapeutic agents are antibody drug conjugates (ADCs).

48. The pharmaceutical agent of claim 47, wherein the ADC is an anti-CD79b ADC.

49. The medicament of claim 48, wherein the anti-CD79b ADC is polatuzumab vedotin.

50. 50. The pharmaceutical of any one of claims 1 to 49, wherein the B-cell proliferative disorder is non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL).

51. The pharmaceutical agent of claim 50, wherein the NHL is diffuse large B-cell lymphoma (DLBCL), primary mediastinal (thymic) large B-cell lymphoma (PMLBCL), mantle cell lymphoma (MCL), or follicular lymphoma (FL).

52. The pharmaceutical agent of claim 51, wherein the DLBCL is relapsed or refractory DLBCL.

53. The pharmaceutical according to any one of claims 1 to 52, wherein the full-length bispecific antibody is administered by intravenous infusion.

54. The pharmaceutical according to any one of claims 1 to 52, wherein the full-length bispecific antibody is administered subcutaneously.

55. 55. The medicament of any one of claims 1 to 54, wherein the subject has a cytokine release syndrome (CRS) event, and the dosing regimen further comprises treating the symptoms of the CRS event while withholding treatment with the full length bispecific antibody.

56. 56. The pharmaceutical composition of claim 55, wherein the dosing regimen further comprises administering to the subject an effective amount of tocilizumab to treat the CRS event.

57. 57. The method of claim 56, wherein tocilizumab is administered intravenously to the subject as a single dose of about 8 mg / kg.

58. 58. The pharmaceutical agent of claim 57, wherein the CRS event does not improve or worsen within 24 hours of treating the symptoms of the CRS event, and the dosing regimen further comprises administering one or more additional doses of tocilizumab to the subject to manage the CRS event.

59. 59. The method of claim 58, wherein the one or more additional doses of tocilizumab are administered intravenously to the subject at a single dose of about 8 mg / kg.

60. 60. The pharmaceutical of claim 58 or 59, wherein the administration regimen further comprises administering to the subject an effective amount of a corticosteroid.

61. 61. The method of claim 60, wherein the corticosteroid is administered intravenously to the subject.

62. 62. The pharmaceutical of claim 60 or 61, wherein the corticosteroid is methylprednisolone.

63. 63. The pharmaceutical composition of claim 62, wherein methylprednisolone is administered at a dosage of about 2 mg / kg per day.

64. 62. The pharmaceutical of claim 60 or 61, wherein the corticosteroid is dexamethasone.

65. 65. The pharmaceutical composition of claim 64, wherein dexamethasone is administered at a dosage of about 10 mg.

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