Dosing for treatment with Anti-CD20 / Anti-CD3 bispecific antibody

Abstract

To provide methods of treating a B-cell proliferative disorder.SOLUTION: The present invention relates to methods of treating a B-cell proliferative disorder by administering an anti-CD20 / anti-CD3 bispecific antibody, and methods for reduction of adverse effects in response to the administration of the anti-CD20 / anti-CD3 bispecific antibody. The present invention further relates to combination treatment methods of treating a B-cell proliferative disorder.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference. Said ASCII copy created on July 22, 2021 It is named 51177-036002_Sequence_Listing_7.22.21_ST25 and is 21,026 bytes in size.

[0002] FIELD OF THE INVENTION The present invention relates to methods for treating diseases, particularly B-cell proliferative disorders, by administering anti-CD20 / anti-CD3 bispecific antibodies, and methods for reducing adverse effects in response to administration of anti-CD20 / anti-CD3 bispecific antibodies. [Background technology]

[0003] B-cell proliferative disorders represent a heterogeneous group of malignancies, including both leukemias and lymphomas. Lymphomas arise from lymphocytes and include two major categories: Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). In the United States, lymphomas of B-cell origin comprise approximately 80-85% of all non-Hodgkin's lymphoma cases, and there is significant heterogeneity within the B-cell subset based on the genotypic and phenotypic expression patterns of the B-cell of origin. For example, B-cell lymphoma subsets include slow-growing, indolent, and refractory diseases such as follicular lymphoma (FL) or chronic lymphocytic leukemia (CLL), as well as more aggressive subtypes such as mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL). Diffuse large B-cell lymphoma (DLBCL) is the most common type of NHL, accounting for approximately 30%-40% of all NHL diagnoses, followed by follicular lymphoma (FL; 20%-25% of all NHL diagnoses) and mantle cell lymphoma (MCL; 6%-10% of all NHL diagnoses). B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in adults, with approximately 15,000 new cases each year in the United States (American Cancer Society 2015).

[0004] Bispecific antibodies can simultaneously bind cell surface antigens on multiple 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 of destroying the cancer cells to which the bound cytotoxic cells are attached. Glofitamab is a T cell bispecific (TCB) antibody that targets CD20 expressed on B cells and the CD3 epsilon chain (CD3ε) present on T cells.

[0005] However, immunotherapy using anti-CD20 / anti-CD3 bispecific antibodies such as glofitamab can be limited by unwanted effects, including cytokine-induced toxicity (e.g., cytokine release syndrome (CRS)), infusion-reactive reactions (IRR), severe tumor lysis syndrome (TLS), and central nervous system (CNS) toxicity.

[0006] Thus, there is an unmet need in the field of developing effective methods of administering anti-CD20 / anti-CD3 bispecific antibodies (e.g., glofitamab) for the treatment of CD20-positive B-cell proliferative disorders (e.g., non-Hodgkin's lymphoma, NHL) that achieve a more favorable benefit-risk profile. Summary of the Invention

[0007] The present invention is based on the discovery that a specific dosing regimen can significantly reduce cytokine release-related side effects associated with administration of an anti-CD20 / anti-CD3 bispecific antibody (e.g., glofitamab) to a subject while achieving clinical efficacy.

[0008] In one aspect, the invention features a method of treating a subject having a CD20-positive B-cell proliferative disorder, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: (a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of the anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg and C1D2 is 10 mg; and (b) the second dosing cycle comprises a single dose (C2D1) of 16 or 30 mg of the anti-CD20 / anti-CD3 bispecific antibody.

[0009] In one embodiment, a single dose of the second dosing cycle comprises 30 mg of the anti-CD20 / anti-CD3 bispecific antibody.

[0010] In one embodiment, the first dose (C1D1) is administered on day 1 and the second dose (C1D2) is administered on day 8 of the first dosing cycle.

[0011] In one embodiment, the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle.

[0012] In one embodiment, the method of treating a subject with a CD20-positive B-cell proliferative disorder comprises 1 to 10 additional dosing cycles (C3D1 through C12D1). In one such embodiment, the 1 to 10 additional dosing cycles (C3D1 through C12D1) comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) is administered on day 1 of each additional dosing cycle.

[0013] In one embodiment, the method of treating a subject with a CD20-positive B-cell proliferative disorder comprises a total of 12 dosing cycles.

[0014] In one embodiment, one treatment cycle comprises 14 or 21 days. In one embodiment, one treatment cycle comprises 21 days.

[0015] In one embodiment, the CD20-positive B-cell proliferative disorder is non-Hodgkin's lymphoma (NHL). In one embodiment, the B-cell proliferative disorder is relapsed or refractory NHL. In one embodiment, the NHL is indolent NHL (iNHL) or aggressive NHL (aNHL). In one embodiment, the NHL is diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal large B-cell lymphoma (PMBCL), or marginal zone lymphoma (MZL). In one embodiment, the DLBCL is Richter's transformed. In one embodiment, the NHL is mantle cell lymphoma (MCL). In one embodiment, the MCL is relapsed or refractory (R / R) MCL. In one embodiment, the subject with R / R MCL has received at least one prior systemic treatment regimen including a Bruton's tyrosine kinase inhibitor (BTKi). In one embodiment, the BTKi comprises ibrutinib, acalabrutinib, or zanubrutinib.

[0016] In one embodiment, the NHL is follicular lymphoma (FL). In one embodiment, the FL is grade 1, 2, or 3a FL. In one embodiment, the FL is transformed FL. In one embodiment, the FL is relapsed or refractory (R / R) FL. In one embodiment, a subject suffering from FL: (a) has relapsed after at least two prior therapies or is refractory to at least two prior therapies; (b) relapsed after treatment with a phosphoinositide 3-kinase (PI3K) inhibitor or was resistant to treatment with a phosphoinositide 3-kinase (PI3K) inhibitor; (c) experiencing disease progression within 24 months of frontline treatment; and / or (d) have lesions with a sum of the products of the lesion diameters of ≥ 3,000 mm2; Being a high-risk subject.

[0017] In one embodiment, a population of subjects with a CD20-positive B-cell proliferative disorder exhibit cytokine release syndrome after administration of the bispecific antibody, and the rate of cytokine release syndrome of Grade 3 or higher (as defined by the American Society for Transplantation and Cellular Therapy, 2019; ASTCT) is about 5% or less.

[0018] In one embodiment, administration of the anti-CD20 / anti-CD3 bispecific antibody to multiple subjects results in a complete remission rate of at least about 70%.

[0019] In one embodiment, administration of an anti-CD20 / anti-CD3 bispecific antibody to multiple subjects results in a complete remission rate of at least about 70% in subjects with iNHL. In one embodiment, administration of an anti-CD20 / anti-CD3 bispecific antibody to multiple subjects results in a complete remission rate of at least about 70% in subjects with aNHL.

[0020] In one embodiment, administration of an anti-CD20 / anti-CD3 bispecific antibody to multiple subjects results in an overall response rate of at least about 80% in subjects with MCL. In one embodiment, administration of an anti-CD20 / anti-CD3 bispecific antibody to multiple subjects results in a complete remission rate of at least about 65% in subjects with MCL. In one embodiment, the MCL is relapsed or refractory (R / R) MCL. In one embodiment, subjects with R / R MCL have received at least one prior systemic treatment regimen comprising a Bruton's tyrosine kinase inhibitor (BTKi). In one embodiment, the BTKi comprises ibrutinib, acalabrutinib, or zanubrutinib.

[0021] In one embodiment, administration of an anti-CD20 / anti-CD3 bispecific antibody to multiple subjects results in an overall response rate of at least about 80% in subjects with FL. In one embodiment, the FL is grade 1, 2, or 3a FL. In one embodiment, the FL is transformed FL. In one embodiment, the FL is relapsed or refractory (R / R) FL.

[0022] In one embodiment, administration of an anti-CD20 / anti-CD3 bispecific antibody to multiple subjects comprises: (a) has relapsed after at least two prior therapies or is refractory to at least two prior therapies; (b) relapsed after treatment with a phosphoinositide 3-kinase (PI3K) inhibitor or was resistant to treatment with a phosphoinositide 3-kinase (PI3K) inhibitor; (c) experiencing disease progression within 24 months of frontline treatment; and / or (d) have lesions with a sum of the products of the lesion diameters of ≥ 3,000 mm2; It provides a complete metabolic response rate of at least about 40% in subjects with high-risk FL.

[0023] In a second aspect, there is provided a method of treating a subject with follicular lymphoma (FL), comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle:

[0024] (i) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody; (ii) the second dosing cycle comprises a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1); (iii) The third dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1).

[0025] In one embodiment, a single dose of the third dosing cycle (C3D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody.

[0026] In one embodiment, the first dose (C1D1) is administered on day 1 of the first dosing cycle and the second dose (C1D2) is administered on day 8 of the first dosing cycle.

[0027] In one embodiment, the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle.

[0028] In one embodiment, the single dose of the third dosing cycle (C3D1) is administered on day 1 of the third dosing cycle.

[0029] In one embodiment, the method of treating a subject with follicular lymphoma (FL) comprises 1 to 9 additional dosing cycles (C4D1 through C12D1). In one embodiment, the 1 to 9 additional dosing cycles (C4D1 through C12D1) comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C4D1 through C12D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C4D1 through C12D1) is administered on day 1 of each additional dosing cycle.

[0030] In one embodiment, the method of treating a subject with follicular lymphoma (FL) comprises a total of 12 dosing cycles.

[0031] In one embodiment, one treatment cycle comprises 14 or 21 days. In one embodiment, one treatment cycle comprises 21 days.

[0032] In one embodiment, the FL is grade 1, 2, or 3a FL. In one embodiment, the FL is transformed FL. In one embodiment, the FL is relapsed or refractory (R / R) FL. In one embodiment, a subject suffering from FL: (a) has relapsed after at least two prior therapies or is refractory to at least two prior therapies; (b) relapsed after treatment with a phosphoinositide 3-kinase (PI3K) inhibitor or was resistant to treatment with a phosphoinositide 3-kinase (PI3K) inhibitor; (c) experiencing disease progression within 24 months of frontline treatment; and / or (d) have lesions with a sum of the products of the lesion diameters of ≥ 3,000 mm2; Being a high-risk subject.

[0033] In one embodiment, administration of an anti-CD20 / anti-CD3 bispecific antibody to multiple subjects results in an overall response rate of at least about 80% in subjects with FL. In one embodiment, the subjects are high-risk subjects with R / R FL, and administration of an anti-CD20 / anti-CD3 bispecific antibody to multiple subjects results in a complete remission rate of at least about 40%. In one embodiment, a population of subjects with FL exhibit cytokine release syndrome after administration of the bispecific antibody, and the rate of cytokine release syndrome of grade 3 or higher (as defined by the American Society for Transplantation and Cellular Therapy, 2019; ASTCT) is about 3%.

[0034] In one embodiment, the method of treating a subject with a CD20-positive B-cell proliferative disorder is combined with administration of obnutuzumab or rituximab. In one embodiment, the method of treating a subject with follicular lymphoma (FL) is combined with administration of obnutuzumab or rituximab. In one embodiment, the method of treating a subject with MCL is combined with administration of obnutuzumab or rituximab. In one embodiment, the subject has MCL and has received at least two prior systemic therapies.

[0035] In one embodiment, obinutuzumab or rituximab is administered 7 days before the first dose of anti-CD20 / anti-CD3 bispecific antibody (C1D1). In one embodiment, obinutuzumab is administered in one single dose of 1000 mg. In one embodiment, obinutuzumab is administered in first and second doses of obinutuzumab of 1000 mg each. In one embodiment, the first and second doses of obinutuzumab are administered on the same day.

[0036] In one embodiment, 2000 mg of obinutuzumab is administered 7 days before the first dose (C1D1) of the anti-CD20 / anti-CD3 bispecific antibody.

[0037] In one embodiment, the first and second doses of obinutuzumab are administered on different days.

[0038] In one embodiment, the first dose of obinutuzumab is administered 7 days before the first dose of anti-CD20 / anti-CD3 bispecific antibody (C1D1), and the second dose of obinutuzumab is administered 1 day before the first dose of anti-CD20 / anti-CD3 bispecific antibody (C1D1).

[0039] In one embodiment, the subject with mantle cell lymphoma (MCL) has received at least two prior systemic therapies.

[0040] In one embodiment, obinutuzumab or rituximab is administered on day 1 of the second cycle (C2D1) and on day 1 of the subsequent cycle.

[0041] In one embodiment, obinutuzumab or rituximab is administered on day 1 of the second cycle (C2D1) and day 1 of the third cycle (C3D1) through day 1 of the twelfth cycle (C12D1).

[0042] In one embodiment, obinutuzumab is administered at a dose of 1000 mg.

[0043] In one embodiment, the patient is premedicated with a corticosteroid prior to the anti-CD20 / anti-CD3 bispecific antibody.

[0044] In one embodiment, the corticosteroid premedication includes prednisolone and methylprednisolone, and / or dexamethasone.

[0045] In one embodiment, corticosteroid premedication is given prior to the first dose (C1D1) of the anti-CD20 / anti-CD3 bispecific antibody.

[0046] In one embodiment, treatment is stopped after a total of 12 treatment cycles.

[0047] In one embodiment, the patient is treated with the methods described herein when a relapse occurs and / or when the disease progresses.

[0048] In a third aspect, a method is provided for treating a subject having a CD20-positive cell proliferative disorder, comprising administering to the subject an anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3.

[0049] In one embodiment, a method of treating a subject having a CD20-positive cell proliferative disorder is provided, comprising administering to the subject an anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3, wherein administering the anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3 to a plurality of humans results in a complete remission in at least about 60%, at least about 70%, or at least about 80% of the plurality of humans after treatment with the anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3.

[0050] In one embodiment, a method is provided for treating a subject having a CD20-positive cell proliferative disorder, comprising administering to the subject an anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3, wherein administering the anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3 to a plurality of humans results in an overall response in at least about 80%, at least about 85%, or at least about 90% of the plurality of humans following treatment with the anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3.

[0051] In one embodiment, a method of treating a subject having a CD20-positive cell proliferative disorder is provided, comprising administering to the subject an anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3, wherein administration of the anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3 to a human does not result in Grade 2 or higher CRS.

[0052] In one embodiment, the method includes a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: (a) a first dosing cycle comprising an anti-CD20 antibody, cyclophosphamide, doxorubicin, and a first dose of a corticosteroid (C1D1), but no dose of a bispecific antibody; (b) a second dosing cycle comprises a second dose (C2D1) of an anti-CD20 antibody, cyclophosphamide, doxorubicin, and a corticosteroid, and a first dose (C2D8) and a second dose (C2D15) of a bispecific antibody, wherein C2D8 of the bispecific antibody is about 2.5 mg and C2D15 is about 10 mg; (c) A third dosing cycle comprises a third dose of an anti-CD20 antibody, cyclophosphamide, doxorubicin, and a corticosteroid (C3D1), and a third dose of a bispecific antibody (C3D8), wherein the bispecific antibody C3D8 is about 30 mg.

[0053] In one embodiment, the anti-CD20 antibody, cyclophosphamide, doxorubicin, and corticosteroid are administered on day 1 of each dosing cycle. In one embodiment, the first dose (C2D8) of the bispecific antibody is administered on day 8 of the second dosing cycle, and the second dose (C2D15) is administered on day 15 of the second dosing cycle.

[0054] In one embodiment, the third dose of the bispecific antibody (C3D8) is administered on day 8 of the third dosing cycle.

[0055] In one embodiment, the method comprises one to five (C4 through C8) additional dosing cycles. In one embodiment, the one to five additional dosing cycles (C4 through C8) comprise a single dose of an anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a single dose of 30 mg of an anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the anti-CD20 antibody, cyclophosphamide, doxorubicin, and a corticosteroid is administered on day 1, and the single dose of the anti-CD20 / anti-CD3 bispecific antibody is administered on day 8 of each additional dosing cycle (C4 through C8).

[0056] In one embodiment, the corticosteroid is prednisone and the anti-CD20 antibody is rituximab.

[0057] In one embodiment, a method of treating a subject having a CD20-positive B-cell proliferative disorder is provided, comprising administering to the subject rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: (a) the first dosing cycle includes a first dose (C1D1) of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) without a dose of bispecific antibody; (b) a second dosing cycle comprises a second dose (C2D1) of R-CHOP and a first dose (C2D8) and a second dose (C2D15) of a bispecific antibody, wherein C2D8 of the bispecific antibody is about 2.5 mg and C2D15 is about 10 mg; (c) A third dosing cycle comprises a third dose of R-CHOP (C3D1) and a third dose of bispecific antibody (C3D8), wherein the bispecific antibody C3D8 is about 30 mg.

[0058] In one embodiment, R-CHOP is administered on day 1 of each dosing cycle. In one embodiment, the first dose of the bispecific antibody (C2D8) is administered on day 8 of the second dosing cycle, and the second dose (C2D15) is administered on day 15 of the second dosing cycle. In one embodiment, the third dose of the bispecific antibody (C3D8) is administered on day 8 of the third dosing cycle. In one embodiment, the method comprises one to five (C4 to C8) additional dosing cycles. In one embodiment, the one to five additional dosing cycles (C4 to C8) comprise a single dose of R-CHOP and a single dose of 30 mg of the anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, a single dose of R-CHOP is administered on day 1, and a single dose of the anti-CD20 / anti-CD3 bispecific antibody is administered on day 8 of each additional dosing cycle (C4 to C8). In one embodiment, in the first dosing cycle, rituximab is replaced by obinutuzumab.

[0059] In one embodiment, the method comprises a total of 6 dosing cycles. In one embodiment, one treatment cycle comprises 14 days or 21 days. In one embodiment, one treatment cycle comprises 21 days. In one embodiment, the CD20-positive B-cell proliferative disorder is previously untreated DLBCL. In one embodiment, the subject being treated has an international prognostic index [IPI] of 2 to 5.

[0060] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is administered intravenously.

[0061] In one embodiment, the subject is a human. In one embodiment, the human is a high-risk subject.

[0062] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds to CD20, wherein the antigen-binding domain is (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable region comprising Includes.

[0063] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds to CD20, wherein the antigen-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.

[0064] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds to CD3, wherein the antigen-binding domain is (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a light chain variable region comprising Includes.

[0065] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds to CD3, wherein the antigen-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.

[0066] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a cross-Fab molecule that contains an antigen-binding domain that specifically binds to CD3 and in which the variable or constant domains of the Fab heavy and light chains have been exchanged.

[0067] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises an IgG1 Fc domain comprising one or more amino acid substitutions that reduce binding to Fc receptors and / or decrease effector function.

[0068] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and P329G (numbering according to the Kabat EU index).

[0069] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises at least one Fab molecule comprising an antigen-binding domain that specifically binds CD20, wherein in the constant domain CL of the Fab molecule the amino acid at position 124 is substituted by lysine (K) (Kabat numbering) and the amino acid at position 123 is substituted by arginine (R) or lysine (K) (Kabat numbering); and in the constant domain CHI of the Fab molecule the amino acid at position 147 is substituted by glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is substituted by glutamic acid (E) (Kabat EU index numbering).

[0070] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises two antigen-binding domains that specifically bind to CD20 and one antigen-binding domain that specifically binds to CD3.

[0071] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is bivalent with respect to CD20 and monovalent with respect to CD3.

[0072] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (i) an antigen-binding domain that specifically binds to CD3, fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; (ii) a first antigen-binding domain that specifically binds to CD20 fused to the N-terminus of the Fab heavy chain of an antigen-binding domain that specifically binds to CD3 at the C-terminus of the Fab heavy chain; (iii) a second antigen-binding domain that specifically binds to CD20 fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fc domain second subunit; Includes.

[0073] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is glofitamab.

[0074] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody is provided for use in a method of treating a subject having a CD20-positive B-cell proliferative disorder, the method comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: (a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of the anti-CD20 / anti-CD3 bispecific antibody, wherein C1D1 is 2.5 mg and C1D2 is 10 mg; and (b) the second dosing cycle comprises a single dose (C2D1) of 16 or 30 mg of the anti-CD20 / anti-CD3 bispecific antibody.

[0075] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody is provided for use in a method of treating a subject with follicular lymphoma (FL), the method comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: (i) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody; (ii) the second dosing cycle comprises a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1); (iii) The third dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1).

[0076] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody is provided for use in a method of treating a subject having a CD20-positive cell proliferative disorder, the method comprising administering to the subject an anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3.

[0077] In one embodiment, there is provided a use of an anti-CD20 / anti-CD3 bispecific antibody in the manufacture of a medicament for the treatment of a CD20-positive cell proliferative disorder, said treatment comprising administering to a subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: (a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of the anti-CD20 / anti-CD3 bispecific antibody, wherein C1D1 is 2.5 mg and C1D2 is 10 mg; and (b) the second dosing cycle comprises a single dose (C2D1) of 16 or 30 mg of the anti-CD20 / anti-CD3 bispecific antibody.

[0078] In one embodiment, there is provided the use of an anti-CD20 / anti-CD3 bispecific antibody in the manufacture of a medicament for the treatment of a CD20-positive cell proliferative disorder, said treatment comprising administering to a subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: (i) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody; (ii) the second dosing cycle comprises a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1); (iii) The third dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1).

[0079] In one embodiment, there is provided the use of an anti-CD20 / anti-CD3 bispecific antibody in the manufacture of a medicament for the treatment of a CD20-positive cell proliferative disorder, said treatment comprising administering to a subject an anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid and a bispecific antibody that binds CD20 and CD3. [Brief explanation of the drawings]

[0080] The contents of the application file contain at least one drawing executed in color. Copies of this patent or this patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0081] [Figures 1A-1F] 1 shows the structure of an anti-CD20 / anti-CD3 bispecific antibody. [Figure 1G-1N] 1 shows the structure of an anti-CD20 / anti-CD3 bispecific antibody. [Figure 2] 1 shows the structure of glofitamab. [Figure 3]Study Design Overview: Glofitamab Monotherapy and Combination Therapy in R / R NHL Dose Escalation and Dose Expansion Cohorts. aQ2W Monotherapy Schedule; bQ3W Monotherapy Schedule; cQ2W Combination Schedule; †Patients in the Part III Dose Expansion Monotherapy Cohorts may receive glofitamab on a Q2W or Q3W dosing schedule with fixed dosing or Q3W with stepped dosing (Cycle 1 Phase or Expansion Phase) if supported by emerging data and / or recommended by the IMC. *Based on the determined MTD / OBD, both or one expansion cohort may be selected for monotherapy B3 and / or D3, B4 and / or D4, while C3 or E3 and C4 or E4 may be selected. §Required paired new baseline (C1D-7) and on-treatment tumor biopsies (C1D9) will be collected in a subset of patients. Abbreviations: Q2W = every 2 weeks; Q3W = every 3 weeks; SoA = schedule of assessment. [Figure 4] Overview of glofitamab step-up dosing schedule. 1000 mg obinutuzumab (pre-treatment with Gazyva, Gpt) was administered 7 days prior to glofitamab administration. Glofitamab IV step-up doses were administered on days 1 (C1D1) and 8 (C1D8) of cycle 1, starting on day 1 (C2D1) of cycle 2 at target doses: 2.5, 10, 16 mg or 2.5, 10, 30 mg. [Figure 5] Adverse events with an incidence of ≥10% or NCI-CTCAE grade 5. Abbreviations: AE, adverse event; NCI-CTCAE, National Cancer Institute Common Terminology Criteria for Adverse Events. [Figure 6] Incidence of cytokine release syndrome (Lee grade) by cycle and dose. Cytokine release syndrome events were primarily limited to cycles 1 and 2. Stepwise dosing of glofitamab allowed for administration of a high target dose (30 mg). Abbreviations: C, cycle. [Figure 7]Patient demographics and baseline disease characteristics in patients who received glofitamab at any dose and at RP2D (safety-evaluable patients). Abbreviations: CAR-T, chimeric antigen receptor T-cell; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; ECOG, Eastern Cooperative Oncology Group; PMBCL, primary mediastinal B-cell lymphoma; RP2D, recommended phase II dose. ‡Data were not available for all patients by the cutoff date. §Includes FL grade 3B (n=1), mantle cell lymphoma (n=1), DLBCL transformed from MZL (n=1), DLBCL transformed from isolated cervical immunoblastic lymphoma (n=1), and DLBCL transformed from Wanderström / immunocytoma (n=1). [Figure 8] Summary of adverse events in patients (safety-evaluable patients) who received glofitamab at any dose and at the RP2D. Abbreviations: ICANS, immune effector cell-associated neurotoxicity syndrome; RP2D, recommended Phase II dose. ‡Includes the terms "neutropenia" and "neutrophil count decreased." [Figure 9] Summary of efficacy data in patients receiving glofitamab by dose level and histology abbreviation: aNHL, aggressive non-Hodgkin's lymphoma; CI, confidence interval; CT, computed tomography; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; Gr, grade; MCL, mantle cell lymphoma; PET, positron emission tomography; PMBCL, primary mediastinal B-cell lymphoma; RP2D, recommended phase II dose; trFL, transformed follicular lymphoma; trMZL, transformed marginal zone lymphoma. *aNHL includes FL (Gr3B), DLBCL, trFL, PMBCL, MCL, trMZL, Richter's transformed, DLBCL, MCL, and DLBCL transformed from other histologies. [Figure 10]The high response to glofitamab was maintained with step-dose therapy. Complete remission was usually achieved early at the first or second response assessment (Cycle 3: ~44 days after prior obinutuzumab treatment; Cycle 6: ~107 days after prior obinutuzumab treatment). The efficacy population included all patients who participated in the study long enough to have the first required response assessment (Lugano criteria). Patients with missing or no response assessments were included as non-responders. Two patients with aNHL and six patients with iNHL did not have a response assessment reported at the clinical cut-off date (CCOD). [Figure 11] Frequency / severity of CRS: A: No change in glofitamab, set dosing. B: Step-dose glofitamab. Step-dose glofitamab allows for administration of a higher target dose. While the overall CRS rate was similar between the fixed-dose and step-dose cohorts, step-dose reduced the frequency of high-grade CRS (grade 2; 36.3% with the 10 mg fixed dosing vs. 30.7% with the step-dose cohort). *Multiple occurrences of CRS are counted toward the highest grade. †Based on observed events, the first C1 dose of 25 mg on the fixed dosing schedule was determined to exceed the maximum tolerated dose. ‡Two patients did not reach the first dose of glofitamab at CCOD. ￥A patient who experienced grade 4 CRS received 30 mg glofitamab as part of step-dose after a long treatment delay. [Figure 12]Overview of glofitamab phase dosing schedule for the FL1-3A patient cohort. In the expanded phase (eSUD) dosing for FL1-3A patients, an initial low dose of glofitamab (0.5 mg) was administered on C1D1, followed by 2.5 mg glofitamab on C1D8, followed by an intermediate dose of 10 mg on cycle 2 (C2D1). The first dose of the target therapeutic dose (30 mg) was administered on cycle 3 (C3D1). Data were compared between FL1-3A patient cohorts receiving glofitamab monotherapy in stepwise dosing (SUD) with 2.5 mg on C1D1, 10 mg on C1D8, and 16 or 30 mg on C2D1, and FL1-3A patient cohorts receiving glofitamab stepwise dosing (SUD) with 2.5 mg on C1D1, 10 mg on C1D8, and 30 mg on C2D1 in combination with 1000 mg Gazyva on C2D1 ("G-combo"). All cohorts received prior Gazyva treatment with 1000 mg Gazyva 7 days prior to the start of cycle 1 (C1D-7). [Figure 13] Kaplan-Meier plots are shown to determine the duration of complete remission for the efficacy-evaluable population of aggressive non-Hodgkin's lymphoma (aNHL) and indolent non-Hodgkin's lymphoma (iNHL) patients receiving glofitamab step-up dosing (SUD). The efficacy population includes all patients for whom a response assessment was performed or who were still on treatment at the time of the first scheduled response assessment. aNHL, aggressive non-Hodgkin's lymphoma; CI, confidence interval; CR, complete remission; iNHL, indolent non-Hodgkin's lymphoma; RP2D, recommended phase II dose. [Figure 14] Schematic overview of the NP40126 study design, Part 1 participants with relapsed / refractory non-Hodgkin's lymphoma, and the use of obinutuzumab in Cycle 1. Abbreviations: C = cycle; CHOP = cyclophosphamide (C), doxorubicin (H), vincristine (O), and prednisone (P); CR = complete response; d / c = discontinued; D = day; DLT = dose-limiting toxicity; EOInd = end of induction; EOT = end of treatment; G = obinutuzumab; IMC = internal monitoring committee; IV = intravenously; M = month; PR = partial response; Q2M = every 2 months; Q3M = every 3 months; R = rituximab; SD = stable disease. [Figure 15] Schematic overview of the NP40126 study design, Part 1 participants with relapsed / refractory non-Hodgkin's lymphoma, and the use of rituximab in Cycle 1. Abbreviations: C = cycle; CHOP = cyclophosphamide (C), doxorubicin (H), vincristine (O), and prednisone (P); CR = complete response; d / c = discontinued; D = day; DLT = dose-limiting toxicity; EOInd = end of induction; EOT = end of treatment; G = obinutuzumab; IMC = internal monitoring committee; IV = intravenously; M = month; PR = partial response; Q2M = every 2 months; Q3M = every 3 months; R = rituximab; SD = stable disease. [Figure 16] Schematic overview of the NP40126 study design, Part II participants with previously untreated diffuse large B-cell lymphoma, and the use of rituximab or obinutuzumab in cycle 1. Participants with previously untreated DLBCL may be offered the option of consolidation therapy with glofitamab (administered for up to six cycles). Abbreviations: C = cycle; CHOP = cyclophosphamide (C), doxorubicin (H), vincristine (O), and prednisone (P); CR = complete response; d / c = discontinued; D = day; DLT = dose-limiting toxicity; EOInd = end of induction; EOT = end of treatment; G = obinutuzumab; IMC = internal monitoring committee; IV = intravenous; M = month; PR = partial response; Q2M = every 2 months; Q3M = every 3 months; R = rituximab; SD = stable disease. DETAILED DESCRIPTION OF THE INVENTION

[0082] I. General Techniques The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in detail in the literature, for example, in "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F.M. Usubel et al., eds., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction" (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

[0083] II. Definition Terms used herein are used as commonly used in the art unless otherwise defined.

[0084] CD20 (also known as B lymphocyte antigen CD20, B lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5; the human protein is characterized by UniProt database entry number P11836) is a hydrophobic transmembrane protein expressed on pre-B and mature B lymphocytes with a molecular weight of approximately 35 kD (Valentine, MA et al., J. Biol. Chem. 264 (1989) 11282-11287; Tedder, TF, et al., Proc. Natl. Acad. Sci. USA 85 (1988) 208-212; Stamenkovic, I., et al., J. Exp. Med. 167 (1988) 1975-1980; Einfeld, DA, et al., EMBO J. 7 (1988) 711-717; Tedder, TF, et al. al., J. Immunol. 142 (1989) 2560-2568). The corresponding human gene is transmembrane 4 domain, subfamily A, member 1, also known as MS4A1. This gene encodes a member of the transmembrane 4A gene family. Members of this emerging protein family are characterized by common structural features and similar intron / exon splice boundaries and exhibit unique expression patterns in hematopoietic cells and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule that plays a role in the development and differentiation of B cells into plasma cells. This family member is localized to 11q12 within a cluster of family members. Alternative splicing of this gene results in two transcript variants encoding the same protein.

[0085] The term "CD20," as used herein, 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, as well as any form of CD20 that results from processing within the cell. The term also encompasses naturally occurring variants of CD20, such as splice variants or allelic variants. In one embodiment, the CD20 is human CD20.

[0086] The terms "anti-CD20 antibody" and "antibody that binds to CD20" refer to an antibody that can bind to CD20 with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in 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 some 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, e.g. 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 In some embodiments, the anti-CD20 antibody binds to an epitope of CD20 that is conserved among CD20 from different species.

[0087] "Type II anti-CD20 antibody" means an anti-CD20 antibody having the binding characteristics and biological activity of the type II anti-CD20 antibodies described in Cragg et al., Blood 103(2004)27382743; Cragg et al., Blood 101(2003)10451052, Klein et al., mAbs 5(2013),2233 and summarized in Table 1 below.

[0088] Table 1. Characteristics of Type I and Type II anti-CD20 antibodies TIFF2025160202000001.tif77170 * For IgG1 isotype

[0089] Examples of Type II anti-CD20 antibodies include, for example, obinutuzumab (GA101), tositumomab (B1), humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody disclosed in WO 2005 / 044859), 11B8 IgG1 (disclosed in WO 2004 / 035607), and AT80 IgG1. Examples of Type I anti-CD20 antibodies include, for example, rituximab, ofatumumab, veltuzumab, ocaratuzumab, ocrelizumab, PRO131921, ublituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (disclosed in WO 2005 / 103081), 2F2 IgG1 (disclosed in WO 2004 / 035607 and WO 2005 / 103081), and 2H7 IgG1 (disclosed in WO 2004 / 056312).

[0090] Unless otherwise indicated, "CD3" refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD3, as well as any form of CD3 resulting from intracellular processing. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, the CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is set forth in UniProt (www.uniprot.org) accession number P07766 (version 144) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. The amino acid sequence of CD3ε from cynomolgus monkeys [Macaca fascicularis] is shown in NCBI GenBank number BAB71849.1.

[0091] The terms "anti-CD20 / anti-CD3 bispecific antibody" and "bispecific antibody that binds CD20 and CD3" can be used interchangeably and refer to a bispecific antibody that can bind to both CD20 and CD3 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD20 and / or CD3. In one embodiment, the extent of binding of a bispecific antibody that binds CD20 and CD3 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 some embodiments, a bispecific antibody that binds 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, e.g. 10 -8 M to 10 -13 M, e.g. 10 -9 M to 10 -13 In some embodiments, the bispecific antibody that binds to CD20 and CD3 binds to an epitope of CD3 that is conserved among CD3 from different species and / or an epitope of CD20 that is conserved among CD20 from different species. An example of an anti-CD20 / anti-CD3 bispecific antibody is glofitamab.

[0092] As used herein, the term "cytokine release" or "cytokine release" is synonymous with "cytokine storm" or "cytokine release syndrome" (abbreviated "CRS") and refers to elevated levels of cytokines, particularly tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-2 (IL-2), and / or interleukin-8 (IL-8), in a subject's blood during or shortly thereafter (e.g., within one day) after administration of a therapeutic agent, resulting in adverse symptoms. Cytokine release is a type of infusion reaction (IRR), a common adverse drug reaction to a therapeutic agent that is temporally related to the administration of the therapeutic agent. IRR typically occurs during or shortly thereafter after administration of a therapeutic agent, i.e., typically within 24 hours after infusion, primarily during the initial infusion. In some cases, for example, after administration of CAR-T cells, CRS may occur only in a delayed manner, e.g., several days after administration, during CAR-T cell expansion. The incidence and severity typically decrease with subsequent infusions. Symptoms range from symptomatic discomfort to fatal events and may include fever, chills, dizziness, hypertension, hypotension, dyspnea, restlessness, sweating, flushing, skin rash, tachycardia, tachypnea, headache, tumor pain, nausea, vomiting, and / or organ failure.

[0093] As used herein, the term "amino acid mutation" is intended to encompass amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to arrive at a final construct, as long as the final construct has the desired characteristics, such as reduced binding to the Fc receptor. Deletions and insertions in the amino acid sequence include amino- and / or carboxy-terminal deletions and amino acid insertions. Particularly, amino acid mutations are amino acid substitutions. For example, to alter the binding characteristics of the Fc region, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include substitutions with non-naturally occurring amino acids or substitutions with naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is believed that methods other than genetic engineering, such as chemical modification to change the side chain group of an amino acid, may also be useful. Various notations may be used herein to indicate the same amino acid mutation. For example, a proline to glycine substitution at position 329 in the Fc region is represented by 329G, G329, G 329 , P329G or Pro329Gly.

[0094] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ), which can be expressed as the dissociation and association rate constants (k off and k on) is the ratio of the rate constants. Thus, equivalent affinities may involve different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by established methods known in the art. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0095] As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding moiety can target a binding entity (e.g., a cytokine or a second antigen-binding moiety) to a target site, such as a specific type of tumor cell or tumor stroma that bears the antigenic determinant. Antigen-binding moieties include antibodies and fragments thereof, as further defined herein. Preferred antigen-binding moieties comprise the antigen-binding domain of an antibody, including an antibody heavy chain variable region and an antibody light chain variable region. In some embodiments, an antigen-binding moiety may comprise an antibody constant region, as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of the two isotypes: κ and λ.

[0096] "Specifically binds" means that the binding is antigen-selective and can be distinguished from unwanted or non-specific interactions. The binding ability of an antigen-binding moiety to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and classical binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of the antigen-binding moiety to an unrelated protein is less than about 10% of the binding of the antigen-binding moiety to the antigen, as measured, for example, by SPR. In some embodiments, the antigen-binding portion that binds to an antigen, or an antigen-binding molecule comprising the antigen-binding portion, has a denaturing activity 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, e.g. 10 -8 M to 10 -13 M, e.g. 10 -9 M to 10 -13 Dissociation constant (K D ) "Decreased binding," e.g., decreased binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, e.g., as measured by SPR. For clarity, the term also includes a decrease in affinity to zero (or below the detection limit of the analytical method), i.e., a complete loss of interaction. Conversely, "increased binding" refers to an increase in the binding affinity of the respective interaction.

[0097] As used herein, the term "antigen-binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and derivatives thereof, such as fragments.

[0098] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to the site on a polypeptide macromolecule to which an antigen-binding moiety binds (e.g., a contiguous stretch of amino acids or a three-dimensional structure composed of distinct regions of non-contiguous amino acids), forming an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, free in serum, and / or within the extracellular matrix (ECM). Unless otherwise indicated, a protein referred to herein as an antigen (e.g., CD3) refers to any native form of the protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In certain embodiments, the antigen is a human protein. When a particular protein is referred to herein, the term encompasses not only the "full-length," unprocessed protein, but also any form of the protein resulting from processing within the cell. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants. An exemplary human protein useful as an antigen is CD3, particularly the epsilon subunit of CD3 (for the human sequence, UniProt number P07766 (version 130), NCBI RefSeq no. NP_000724.1; or for the cynomolgus monkey [Macaca fascicularis] sequence, UniProt number Q95LI5 (version 49), NCBI GenBank no. BAB71849.1). In some embodiments, the T cell-activating bispecific antigen-binding molecules of the invention bind to an epitope of CD3 or a target cell antigen that is conserved among CD3 or target cell antigens from different species.

[0099] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linked in a linear chain by amino bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids, and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be obtained from natural biological sources or produced by recombinant technology, but are not necessarily translated from a designated nucleic acid sequence. Polypeptides can be produced by any means, including chemical synthesis. Polypeptides of the invention can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2000 or more amino acids in size. Polypeptides may, but do not necessarily, have a defined three-dimensional structure. Polypeptides that have a defined three-dimensional structure are referred to as folded polypeptides, while polypeptides that do not have a defined three-dimensional structure but can adopt a number of different conformations are referred to as unfolded polypeptides.

[0100] An "isolated" polypeptide or variant or derivative thereof refers to a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of the present invention, as are native or recombinant polypeptides that have been separated, fragmented, or partially or substantially purified by any suitable technique.

[0101] "Percent (%) amino acid sequence identity" 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, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of 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 over 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, along with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, and is hereby 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. In situations where ALIGN-2 is used for amino acid sequence comparisons, the percent amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which can alternatively be written as a given amino acid sequence A having or containing a particular percent amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated 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 and 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 differs from the length of amino acid sequence B, the % amino acid sequence identity of A to B will differ from the % amino acid sequence identity of B to A. Unless otherwise indicated, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0102] The term "antibody" is used herein 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.

[0103] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a heavy chain that has a structure substantially similar to a native antibody structure or that contains an Fc region as defined herein.

[0104] An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an 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. As used herein, the term "antibody fragment" also encompasses single-domain antibodies.

[0105] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain, also called a light chain constant region. Immunoglobulin heavy chains may be assigned to one of five classes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), several of which can be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and an Fc domain connected via an immunoglobulin hinge region.

[0106] The term "antigen-binding domain" refers to a portion of an antibody that comprises an area that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). Preferably, the antigen-binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0107] 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, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6 th ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0108] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or an antibody of non-human origin that utilizes the human antibody repertoire or other human antibody-encoding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0109] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to HVRs of a non-human antibody and all or substantially all of the FRs correspond to FRs 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.

[0110] 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 structurally forms defined loops ("hypervariable loops") and / or contains residues that contact the antigen ("antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include: (a) Hypervariable loops occurring at amino acid residues 2632 (L1), 5052 (L2), 9196 (L3), 2632 (H1), 5355 (H2), and 96101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901917 (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) antigenic 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) comprising HVR amino acid residues 4656 (L2), 4756 (L2), 4856 (L2), 4956 (L2), 2635 (H1), 26-35b (H1), 4965 (H2), 93102 (H3), and 94102 (H3). Includes:

[0111] 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.

[0112] "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.

[0113] The "class" of an antibody refers to the type of constant domain or constant region carried 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.

[0114] The term "Fc domain" or "Fc region" is used herein 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. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined to stretch from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, antibodies produced by host cells upon expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise a full-length heavy chain or a cleaved variant of the full-length heavy chain (also referred to herein as a "cleaved variant heavy chain"). This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (K447), 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 (see also above). As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide that comprises the C-terminal constant region of an immunoglobulin heavy chain and is capable of stable self-association. For example, a subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

[0115] A "modification that promotes association of the first and second subunits of the Fc domain" refers to manipulation of the peptide backbone or post-translational modification of the Fc domain subunit that reduces or prevents association of the peptide containing the Fc domain subunit with an identical polypeptide to form homodimers. As used herein, a modification that promotes association specifically includes separate modifications made to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, where these modifications are complementary to each other to promote association of the two Fc domain subunits. For example, a modification that promotes association can alter the structure or charge of one or both of the Fc domain subunits to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, which may be non-identical in the sense that additional components fused to each subunit (e.g., antigen-binding moieties) are not the same. In some embodiments, a modification that promotes association includes an amino acid mutation, specifically an amino acid substitution, within the Fc domain. In certain embodiments, the association-promoting modifications comprise distinct amino acid mutations, specifically amino acid substitutions, in each of the two subunits of the Fc domain.

[0116] An "activating Fc receptor" is an Fc receptor that, upon engagement by the Fc region of an antibody, triggers signaling events that stimulate the receptor-bearing cell to exert effector function. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0117] The term "effector function," when used in reference to an antibody, refers to a biological activity attributable to the Fc region of an antibody and varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0118] As used herein, the term "effector cells" refers to a population of lymphocytes that express effector moiety receptors, e.g., cytokine receptors, and / or Fc receptors, on their surface, thereby binding effector moieties, e.g., cytokines, and / or the Fc region of antibodies, and contributing to the destruction of target cells, e.g., tumor cells. Effector cells mediate, for example, cytotoxic or phagocytic effects. Effector cells include, but are not limited to, effector T cells, e.g., CD8 + Cytotoxic T cells, CD4 + These include helper T cells, γδ T cells, NK cells, lymphokine-activated killer (LAK) cells, and macrophages / monocytes.

[0119] As used herein, the terms "engineer, engineered, engineering" are intended to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, glycosylation pattern, or side groups of individual amino acids, as well as combinations of such techniques. Specifically, "engineer" with the prefix "glyco" and the term "glycosylation engineering" include metabolic engineering of the glycosylation machinery of a cell, including genetic manipulation of the oligosaccharide synthesis pathway to achieve altered glycosylation of glycoproteins expressed in the cell. Furthermore, glycosylation engineering includes the influence of mutations and the cellular environment on glycosylation. In one embodiment, glycosylation engineering is alteration of glycosyltransferase activity. In certain embodiments, engineering results in altered glucosaminyltransferase activity and / or fucosyltransferase activity. Glycosylation engineering can be used to obtain "host cells with increased GnTIII activity" (e.g., host cells engineered to express increased levels of one or more polypeptides having β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity), "host cells with increased ManII activity" (e.g., host cells engineered to express increased levels of one or more polypeptides having α-mannosidase II (ManII) activity), or "host cells with reduced α(1,6)fucosyltransferase activity" (e.g., host cells engineered to express reduced levels of α(1,6)fucosyltransferase).

[0120] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived from the primary transformed cell, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. Host cells are any type of cell line that can be used to produce proteins used in the present invention. In one embodiment, the host cells are engineered to enable the production of antibodies with modified oligosaccharides. In some embodiments, the host cells are engineered to express elevated levels of one or more polypeptides with β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. In some embodiments, the host cells are further engineered to express elevated levels of one or more polypeptides with α-mannosidase II (ManII) activity. Host cells include cultured cells, such as cultured mammalian cells, CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, to name just a few, yeast cells, insect cells, and plant cells, but also cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissue.

[0121] As used herein, the term "polypeptide having GnTIII activity" refers to a polypeptide capable of catalyzing the addition of an N-acetylglucosamine (GlcNAc) residue in the β-1,4 linkage of the trimannosyl core of an N-linked oligosaccharide to a β-linked mannoside. This includes fusion polypeptides that exhibit enzymatic activity similar, but not necessarily identical, to that of β(1,4)-N-acetylglucosaminyltransferase III, also known as β-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase (EC 2.4.1.144) according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), with or without dose-dependent activity, as measured in a specific biological assay. If a dose-dependence exists, it need not be identical to that of GnTIII, but rather should be substantially similar to the dose-dependence for a given activity when compared to GnTIII (i.e., the candidate polypeptide will exhibit greater activity or at least about 25-fold, preferably at least about 10-fold, and most preferably at least about 3-fold less activity than GnTIII). In some embodiments, the polypeptide having GnTIII activity is a fusion polypeptide comprising the catalytic domain of GnTIII and the Golgi localization domain of a heterologous Golgi-resident polypeptide. In particular, the Golgi localization domain is the localization domain of mannosidase II or GnTI, most particularly the localization domain of mannosidase II. Alternatively, the Golgi localization domain is selected from the group consisting of the localization domain of mannosidase I, the localization domain of GnTII, and the localization domain of α1,6 core fucosyltransferase. Methods for producing such fusion polypeptides and using them to generate antibodies with enhanced effector function are disclosed in WO 2004 / 065540, U.S. Provisional Patent Application No. 60 / 495,142 and U.S. Patent Application Publication No. 2004 / 0241817, the entire contents of which are expressly incorporated herein by reference.

[0122] As used herein, the term "Golgi localization domain" refers to an amino acid sequence of a Golgi-resident polypeptide that is responsible for anchoring the polypeptide to a location within the Golgi complex. Typically, the localization domain comprises the amino-terminal "tail" of the enzyme.

[0123] As used herein, the term "polypeptide having ManII activity" refers to a polypeptide capable of catalyzing the hydrolysis of terminal 1,3- and 1,6-linked α-D-mannose residues in the branched GlcNAcMan5GlcNAc2 mannoses found in the middle of N-linked oligosaccharides. This includes polypeptides that exhibit enzymatic activity similar, but not necessarily identical, to that of Golgi α-mannosidase II, also known as mannosyloligosaccharide 1,3-1,6-α-mannosidase II (EC 3.2.1.114) according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB).

[0124] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. Target cells are cells to which an antibody or its fragment, including an Fc region, specifically binds, usually via a protein portion N-terminal to the Fc region. As used herein, the term "increased / decreased ADCC" is defined as either an increase / decrease in the number of target cells lysed in a given time period by the ADCC mechanism as defined above, at a given antibody concentration in the medium surrounding the target cells, and / or a decrease / increase in the antibody concentration in the medium surrounding the target cells required to lyse a given number of target cells in a given time period by the ADCC mechanism. The increase / decrease in ADCC is relative to unmanipulated ADCC mediated by the same antibody produced by the same type of host cell using the same standard production, purification, formulation, and storage methods (known to those skilled in the art). For example, the increase in antibody-mediated ADCC produced by host cells engineered to have an altered glycosylation pattern (e.g., to express glycosyltransferase, GnTIII, or other glycosyltransferase) by the methods described herein is relative to the ADCC mediated by the same antibody produced by unengineered host cells of the same type.

[0125] "Antibody with increased / decreased antibody-dependent cell-mediated cytotoxicity (ADCC)" means an antibody with increased / decreased ADCC as determined by any suitable method known to those of skill in the art. One acceptable in vitro ADCC assay is as follows: 1) the assay uses target cells known to express the target antigen recognized by the antigen-binding region of the antibody; 2) the assay uses human peripheral blood mononuclear cells (PBMCs) isolated from the blood of randomly selected healthy donors as effector cells; 3) The assay is performed according to the following protocol: i) Isolate PBMCs using standard density centrifugation procedures and collect 5x10 6 Suspend in RPMI cell culture medium at cells / ml; ii) Target cells were grown by standard tissue culture methods, harvested in exponential growth phase with greater than 90% viability, washed in RPMI cell culture medium, and incubated with 100 microcuries of 51 Cr, washed twice with cell culture medium, and diluted to 10 5 Resuspend at a density of cells / ml; iii) transferring 100 microliters of the final target cell suspension to each well of a 96-well microtiter plate; iv) serially diluting the antibody from 4000 ng / ml to 0.04 ng / ml in cell culture medium, and adding 50 microliters of the resulting antibody solution to target cells in a 96-well microtiter plate, testing various antibody concentrations in triplicate to cover the entire concentration range; v) For maximum release (MR) controls, three additional wells in the plate containing labeled target cells receive 50 microliters of a 2% ((vVN)) aqueous solution of non-ionic detergent (Nonidet, Sigma, St. Louis) instead of the antibody solution (iv above); vi) For spontaneous release (SR) controls, three additional wells in the plate containing labeled target cells receive 50 microliters of RPMI cell media instead of the antibody solution (iv above); vii) The 96-well microtiter plate is then centrifuged at 50×g for 1 minute and incubated for 1 hour at 4° C.; viii) adding 50 microliters of PBMC suspension (i above) to each well to give an effector to target ratio of 25:1, and placing the plate in an incubator at 37°C under a 5% CO2 atmosphere for 4 hours; ix) Recover cell-free supernatant from each well and quantify experimentally released radioactivity (ER) using a gamma counter; x) The percentage of specific lysis is calculated for each antibody concentration according to the formula (ER-MR) / (MR-SR) x 100 (ER is the mean radioactivity quantified for that antibody concentration (see ix above), MR is the mean radioactivity quantified for the MR control (see v above) (see ix above), and SR is the mean radioactivity quantified for the SR control (see vi above) (see ix above)); 4) "Increased / decreased ADCC" is defined as the increase / decrease in the maximum percentage of specific lysis observed within the antibody concentration range tested above, and / or the decrease / increase in antibody concentration required to achieve half the maximum percentage of specific lysis observed within the antibody concentration range tested above. The increase / decrease in ADCC is relative to the ADCC measured using the above assay, produced by the same type of host cell and mediated by the same antibody, but unmodified, using the same standard production, purification, formulation, and storage methods known to those skilled in the art.

[0126] 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, e.g., variants usually present in minor amounts, that contain, for example, naturally occurring mutations or that arise during the generation of the monoclonal antibody preparation. In contrast to polyclonal antibody preparations, which generally 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, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0127] As used herein, the terms "first," "second," "third," etc., when used in reference to antigen-binding portions or domains, are used for convenience to distinguish between various portions or domains when multiple portions or domains are present. The use of these terms is not intended to confer a particular order or orientation unless explicitly indicated as such.

[0128] The terms "multispecific" and "bispecific" mean that an antigen-binding molecule can specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each of which is specific for a different antigenic determinant. In some embodiments, a bispecific antigen-binding molecule can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.

[0129] As used herein, the term "valency" indicates that a specific number of antigen-binding sites are present in an antigen-binding molecule. Thus, the term "monovalent binding to an antigen" indicates that one (and at most one) antigen-binding site specific for the antigen is present in the antigen-binding molecule.

[0130] "Antigen-binding site" refers to the site of an antigen-binding molecule, i.e., one or more amino acid residues, that interacts with an antigen. For example, the antigen-binding site of an antibody comprises amino acid residues of the complementarity-determining regions (CDRs). A naturally occurring immunoglobulin molecule typically has two antigen-binding sites, while a Fab molecule typically has a single antigen-binding site.

[0131] As used herein, a "T cell activation antigen" refers to an antigenic determinant expressed by T lymphocytes, particularly cytotoxic T lymphocytes, which can induce or enhance T cell activation upon interaction with an antigen-binding molecule. Specifically, interaction of an antigen-binding molecule with a T cell activation antigen can induce T cell activation by triggering a signal transduction cascade in the T cell receptor complex. An exemplary T cell activation antigen is CD3. In one specific embodiment, the T cell activation antigen is CD3, particularly the epsilon subunit of CD3 (for the human sequence, UniProt number P07766 (version 130), NCBI RefSeq no. NP_000724; or for the cynomolgus monkey [Macaca fascicularis] sequence, UniProt number Q95LI5 (version 49), NCBI GenBank no. BAB71849.1).

[0132] As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. T cell activating therapeutic agents used in the present invention are capable of inducing T cell activation. Suitable assays for measuring T cell activation are known in the art and are described herein.

[0133] As used herein, "target cell antigen" refers to an antigenic determinant present on the surface of a target cell, e.g., a cell in a tumor, such as a cancer cell or a cell of the tumor stroma. In a specific embodiment, the target cell antigen is CD20, particularly human CD20 (see UniProt No. P11836).

[0134] As used herein, "B cell antigen" refers to an antigenic determinant present on the surface of a B lymphocyte, particularly a malignant B lymphocyte (in which case the antigen is also referred to as a "malignant B cell antigen").

[0135] As used herein, "T cell antigen" refers to an antigenic determinant displayed on the surface of T lymphocytes, particularly cytotoxic T lymphocytes.

[0136] A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain (a "Fab heavy chain") and the VL and CL domains of a light chain (a "Fab light chain").

[0137] "Chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor protein that includes an antigen-binding portion, such as a short-chain variable fragment (scFv) of a targeting antibody, a transmembrane domain, an intracellular T cell activation signaling domain (e.g., the CD3 zeta chain of a T cell receptor), and optionally one or more intracellular costimulatory domains (e.g., of CD28, CD27, CD137 (4-1BB), Ox40). CARs mediate antigen recognition, T cell activation, and, in the case of second-generation CARs, costimulation, which increases T cell functionality and persistence. For a review, see, e.g., Jackson et al., Nat Rev Clin Oncol (2016) 13, 370-383.

[0138] By "fused" is meant that the components (eg, a Fab molecule and an Fc domain subunit) are joined by a peptide bond, either directly or via one or more peptide linkers.

[0139] An "effective amount" of a drug is the amount necessary to produce a physiological change in the cells or tissue to which it is administered.

[0140] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at the necessary dosage and for the necessary period of time, to achieve a desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0141] "Therapeutic agent" means an active ingredient, e.g., of a pharmaceutical composition, that is administered to a subject in an attempt to alter the natural course of a disease in the treated subject, and can be performed prophylactically or in the course of clinical pathology. "Immunotherapeutic agent" refers to a therapeutic agent that is administered to a subject in an attempt to restore or enhance the subject's immune response, e.g., against a tumor.

[0142] The term "pharmaceutical composition" refers to a preparation in a form that allows for the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0143] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, 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.

[0144] The term "package insert" or "instructions for use" is used to refer to instructions customarily included in the commercial packaging of a therapeutic product, which contain information about the use, directions for use, dosage, administration, concomitant therapy, contraindications and / or precautions for use of such therapeutic product.

[0145] The term "combination therapy" as used herein encompasses combined administration (when two or more therapeutic agents are contained in the same or separate formulations) and separate administration, where administration of an antibody as reported herein can occur before, simultaneously with, and / or after administration of one or more additional therapeutic agents, preferably one or more antibodies.

[0146] A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which the variable or constant domains of the Fab heavy and light chains have been exchanged (i.e., replaced by one another); i.e., the crossover Fab molecule comprises a peptide chain composed of a light chain variable domain VL and a heavy chain constant domain 1 CH1 (VL-CH1, from N- to C-terminal), and a peptide chain composed of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL, from N- to C-terminal). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain constant domain 1 CH1 is referred to herein as the "heavy chain" of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule in which the constant domains of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the "heavy chain" of the (crossover) Fab molecule.

[0147] In contrast, a "typical" Fab molecule is one that comprises a heavy chain in its native form, i.e., composed of a heavy chain variable domain and a constant domain (VH-CH1, from N- to C-terminus), and a light chain comprising a light chain variable domain and a constant domain (VL-CL, from N- to C-terminus).

[0148] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), viral-derived RNA, or plasmid DNA (pDNA). Polynucleotides can contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0149] An "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from its native chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive- and negative-stranded forms and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, polynucleotides or nucleic acids may contain or contain regulatory elements, such as a promoter, ribosome binding site, or transcription terminator.

[0150] A nucleic acid or polynucleotide having a nucleotide sequence that is, for example, at least 95% "identical" to a reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the nucleotide sequence of the polynucleotide may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides, or up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. Such alterations of the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or at any position between these terminal positions, and may be scattered individually among the residues in the reference sequence or scattered in one or more contiguous groups within the reference sequence. In practical terms, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be conventionally determined using known computer programs, such as those described above for polypeptides (e.g., ALIGN-2).

[0151] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide with a set of specific nucleic acid elements that allows transcription of a specific nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In some embodiments, the expression cassettes of the invention comprise a polynucleotide sequence encoding a bispecific antigen-binding molecule of the invention, or a fragment thereof.

[0152] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce and direct the expression of a specific gene to which it is operably linked in a target cell. This term includes a vector as a self-replicating nucleic acid structure and a vector integrated into the genome of a host cell into which it is introduced. The expression vector of the present invention comprises an expression cassette. The expression vector enables the transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antigen-binding molecule of the present invention or a fragment thereof.

[0153] The term "about" as used herein refers to a normal error range for the respective value, which would be readily understood by one of ordinary skill in the art. Reference herein to a value or parameter preceded by "about" includes (and describes) embodiments that are directed to that value or parameter itself.

[0154] "B cell proliferative disorder" refers to a disorder in which the number of B cells in a patient is increased compared to the number of B cells in a healthy individual, particularly a disorder in which an increased number of B cells is the cause or evidence of the disorder. "CD20-positive B cell proliferative disorder" refers to a B cell proliferative disorder in which B cells, particularly malignant B cells (in addition to normal B cells), express CD20.

[0155] Exemplary B-cell proliferative disorders include non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL; (NOS)r / r DLBCL unless otherwise specified), high-grade B-cell lymphoma (HGBCL), primary mediastinal large B-cell lymphoma (PMBCL), DLBCL arising from FL [transformed FL; trFL]; Richter's transformation; follicular lymphoma (FL), including grade 1-3b FL; mantle cell lymphoma (MCL), and marginal zone lymphoma (MZL), including splenic, nodal, or extranodal MZL. In one embodiment, the CD20-positive B-cell proliferative disorder is relapsed or refractory NHL (e.g., relapsed or refractory DLBCL, relapsed or refractory FL, or relapsed or refractory MCL). "Previously untreated NHL" or "treatment-naive NHL" (e.g., previously untreated DLBCL or treatment-naive DLBCL) refers to disease that has not been previously treated. In one embodiment, the methods of treatment described herein are first-line treatments. In one embodiment, the methods of treatment are for subjects with histologically confirmed, previously untreated DLBCL (IPI 2-5) that is predicted to express CD20.

[0156] "Refractory disease" is defined as lack of complete remission to first-line therapy. In one embodiment, refractory disease is defined as lack of response to prior therapy or relapse within 6 months of prior therapy. In one embodiment, refractory disease is characterized by one or more of the following: best response to first-line therapy is progressive disease (PD), best response is stable disease (SD) after at least four cycles of first-line therapy (e.g., after four cycles of rituximab, cyclophosphamide, doxorubicin hydrochloride (hydroxydaunorubicin), vincristine sulfate (Oncovin), and prednisone, also abbreviated as R-CHOP), or best response is partial remission (PR) after at least six cycles with biopsy-proven residual disease or disease progression after partial remission. "Relapsed disease" is defined as complete remission to first-line therapy. In one embodiment, disease recurrence is biopsy-proven. In one embodiment, the patient has relapsed after or failed to respond to at least one prior systemic treatment regimen (including at least one prior regimen containing an anti-CD20 directed therapy, e.g., rituximab or obinutuzumab). In one embodiment, the patient has relapsed after or failed to respond to at least two prior systemic treatment regimens (including at least one prior regimen containing an anthracycline and at least one prior regimen containing an anti-CD20 directed therapy).

[0157] An "individual" or "subject" 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). Preferably, the individual or subject is human.

[0158] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of disease in the individual being treated, and can be performed prophylactically or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the condition, and improving recovery or prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

[0159] As used herein, "delaying progression" of a disorder or disease means delaying, preventing, slowing, retarding, stabilizing, and / or postponing the onset of the disease or disorder (e.g., a CD20-positive B-cell proliferative disorder, e.g., NHL, e.g., DLBCL). The 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 of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, in late-stage cancer, the development of central nervous system (CNS) metastases can be delayed.

[0160] "Reduce" or "inhibit" refers to the ability to cause an overall decrease, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more. For clarity, the term also includes a reduction to zero (or a value below the limit of detection of an analytical method), i.e., complete disappearance or elimination. In some embodiments, reduce or inhibit refers to a decrease or inhibition of undesirable events, such as cytokine-driven toxicity (e.g., cytokine release syndrome (CRS)), infusion reaction (IRR), macrophage activation syndrome (MAS), neurotoxicity, severe tumor lysis syndrome (TLS), neutropenia, thrombocytopenia, elevated liver enzymes, and / or central nervous system (CNS) toxicity, following treatment with an anti-CD20 / anti-CD3 bispecific antibody using a stepped dosing regimen of the invention for constant, preset dosing with a target dose of the bispecific antibody. 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). In other embodiments, reducing or inhibiting can refer to symptoms of the CD20-positive B-cell proliferative disorder being treated (e.g., NHL (e.g., DLBCL), FL (e.g., relapsed and / or refractory FL or transformed FL), MCL, high-grade B-cell lymphoma, or PMLBCL), the presence or size of metastases, or the size of the primary tumor.

[0161] As used herein, "administering" refers to a method of providing a subject with a dosage of a compound (e.g., an anti-CD20 / anti-CD3 bispecific antibody) or composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising an anti-CD20 / anti-CD3 bispecific antibody). The compounds and / or compositions utilized in the methods described herein can be administered intravenously (e.g., by intravenous infusion).

[0162] A "fixed" or "flat" dose of a therapeutic agent (e.g., a bispecific antibody) herein refers to a dose administered to a patient without regard to the patient's weight or body surface area (BSA). Thus, a fixed or flat dose can be expressed as a mg / kg dose or a mg / m 2 It is not provided as a dose, but as an absolute amount (eg, mg) of therapeutic agent.

[0163] As used herein, "target dose" refers to the dose of an anti-CD20 / anti-CD3 bispecific antibody that achieves a therapeutic effect, i.e., achieves the desired clinical efficacy. In the case of glofitamab, possible target doses are 16 mg or 30 mg. In a preferred embodiment, the target dose for glofitamab is 30 mg.

[0164] "Constant or preset dosing with a target dose" and "treatment regimen without a stepped dosing regimen" refer to a dosing schedule that uses the same dosage for the first and second cycles, and optionally for subsequent treatment cycles, as opposed to stepped dosing, which uses a lower dosage for the first one or two treatment cycles and reaches the target dose only in the second or subsequent treatment cycles.

[0165] As used herein, the term "therapeutic cycle" or "cycle" (abbreviation: "C") refers to a course of one or more doses of an anti-CD20 / anti-CD3 bispecific antibody repeated on a regular schedule, optionally with a rest period (no treatment) in between. In one aspect of the invention, a first therapeutic cycle comprises a first dose and a second dose of an anti-CD20 / anti-CD3 bispecific antibody, followed by a rest period. In one such embodiment, a first therapeutic cycle comprises a first dose of an anti-CD20 / anti-CD3 bispecific antibody on day 1 of the first cycle and a second dose of an anti-CD20 / anti-CD3 bispecific antibody on day 8 of the first cycle, followed by a 12-day rest period. In one embodiment, a second and subsequent cycle comprises a dose of an anti-CD20 / anti-CD3 bispecific antibody given on day 1 of the cycle, followed by a 20-day rest period. In one embodiment, a treatment cycle comprising one or more doses of an anti-CD20 / anti-CD3 bispecific antibody comprises 21 days and may further comprise one or more dosages of one or more other therapeutic agents, for example an anti-CD20 antibody, in particular obinutuzumab. A treatment schedule according to the invention may comprise two or more treatment cycles, or 3, 4, 5, 6, 7, 8, 9, 10, 11, in particular 12 treatment cycles.

[0166] An "individual response" or "response" can be assessed using any endpoint that indicates a benefit to the subject, including, but not limited to, (1) some inhibition, including slowing and complete halt, of disease progression (e.g., progression of a CD20-positive cell proliferative disorder, e.g., non-Hodgkin's lymphoma (NHL)); (2) reduction in tumor size; (3) inhibition (i.e., reduction, slowing, or complete halt) of cancer cell invasion into adjacent peripheral organs and / or tissues; (4) inhibition (i.e., reduction, slowing, or complete halt) of metastasis; (5) some alleviation of one or more symptoms associated with a CD20-positive B-cell proliferative disorder, e.g., a B-cell proliferative disorder; (6) increased or prolonged survival, including overall survival and progression-free survival; and / or (9) a decrease in mortality at some point after treatment.

[0167] As used herein, "complete response" or "CR" refers to the disappearance of all target lesions. In one embodiment, standard NHL response criteria are evaluated to determine CR. (Lugano Criteria, Cheson et al. J Clin Oncol. 2014 Sep 20;32(27):3059-3067.) CR can be determined by PET-CT ("complete metabolic response" or "CMR") or CT ("complete radiological response"). In some embodiments, complete response (CR) can be used interchangeably with "complete metabolic response" or "CMR." The Lugano criteria for assessing complete and partial responses based on PET-CT (complete metabolic response) and CT (complete radiological response) are detailed in Table 2 below.

[0168] Table 2: Lugano response criteria for malignant lymphoma (Cheson et al. 2014) TIFF2025160202000002.tif243170TIFF2025160202000003.tif50170

[0169] "Duration of complete response" (DOCR) is defined as the time from the first occurrence of a documented CR to documented disease progression or death from any cause, whichever occurs first. In one embodiment, DOCR is assessed based on the Lugano classification (Cheson et al. J Clin Oncol. 2014 Sep 20;32(27):3059-3067.).

[0170] "Duration of objective response" (DOR) is defined as the time from the first occurrence of a documented objective response to the time of disease progression, recurrence, or death from any cause. In one embodiment, DOR is assessed based on the Lugano classification (Cheson et al. J Clin Oncol. 2014 Sep 20;32(27):3059-3067.).

[0171] "Progression-free survival" (PFS) is defined as the time from initial treatment with an anti-CD20 / anti-CD3 bispecific antibody to the first occurrence of disease progression or death from any cause, whichever occurs first. In one embodiment, PFS is assessed according to the Lugano classification (Cheson et al. J Clin Oncol. 2014 Sep 20;32(27):3059-3067.).

[0172] "Overall survival" (OS) is defined as the time from first treatment with anti-CD20 / anti-CD3 bispecific antibody to the date of death from any cause.

[0173] "Time to first overall response" (TFOR) is defined as the time from treatment initiation to first documented response. In one embodiment, TFOR is assessed based on the Lugano classification (Cheson et al. J Clin Oncol. 2014 Sep 20;32(27):3059-3067.).

[0174] "Time to first complete remission" (TFCR) is defined as the time from treatment initiation to first documented complete remission. In one embodiment, TFCR is assessed based on the Lugano classification (Cheson et al. J Clin Oncol. 2014 Sep 20;32(27):3059-3067.).

[0175] As used herein, "objective response rate" refers to the sum of patients who achieved complete response (CR), partial response (PR), and stable disease (SD) in a patient population. In one embodiment, the objective response rate is assessed based on the Lugano classification (Cheson et al. J Clin Oncol. 2014 Sep 20;32(27):3059-3067.).

[0176] The "overall response rate" (ORR) is defined as the sum of the partial response (PR) rate and the complete response (CR) rate. In one embodiment, the overall response is evaluated based on the Lugano classification (Cheson et al. J Clin Oncol. 2014 Sep 20;32(27):3059-3067.).

[0177] A "high-risk subject" is a subject who has disease progression within 24 months of frontline treatment or is resistant to multiple drug classes. In one embodiment, a high-risk subject: (a) has relapsed after at least two prior therapies or is resistant to at least two prior therapies; (b) has relapsed after treatment with a phosphoinositide 3-kinase (PI3K) inhibitor or is resistant to treatment with a phosphoinositide 3-kinase (PI3K) inhibitor; (c) experiences disease progression within 24 months of frontline treatment; and / or (d) has a sum of the products of lesion diameters of ≥ 3,000 mm 2 The present invention includes subjects having a lesion in which:

[0178] III. Anti-CD20 / anti-CD3 bispecific antibody The present invention provides new dosages and combination therapies for anti-CD20 / anti-CD3 bispecific antibodies. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a polyclonal antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a human antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a humanized antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a chimeric antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a full-length antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is an IgG class antibody, particularly an IgG1 subclass antibody. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is a recombinant antibody.

[0179] In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody comprises an antibody fragment. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, and scFv fragments, as well as other fragments described below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Plückthun, 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 5,587,4585. See U.S. Patent No. 5,869,046 for a description of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have extended in vivo half-lives. In one embodiment, the antibody fragment is a Fab fragment or an scFv fragment.

[0180] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:64446448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129134 (2003).

[0181] 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).

[0182] 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 (e.g., E. coli or phage), as described herein.

[0183] In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody is a chimeric antibody. Some 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, a 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, a 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.

[0184] In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody is a humanized antibody. Typically, a non-human antibody is humanized to retain the specificity and affinity of the parent non-human antibody while reducing immunogenicity to humans. 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. A humanized antibody will also optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0185] Humanized antibodies and methods for their production are reviewed, e.g., by Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, e.g., by 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; 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. al., Methods 36:43-60 (2005) (describing "FR shuffling"); and 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).

[0186] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences 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 derived from FR library screening (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)).

[0187] In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0188] Human antibodies may be prepared by administering an immunogen to intact antibodies with human variable regions or to transgenic animals that have been modified to produce intact human antibodies in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci that replace endogenous immunoglobulin loci or that are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See, e.g., XENOMOUSE. TM See also U.S. Patent Nos. 6,075,181 and 6,150,584, which describe HUMAB® technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VelociMouse® technology. The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining with a different human constant region.

[0189] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0190] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0191] Binding domains contained in anti-CD20 / anti-CD3 bispecific antibodies can be isolated by screening combinatorial libraries for binding moieties with one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are reviewed, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and further described, for example, by McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0192] In a specific phage display method, repertoires of VH and VL genes can be individually cloned by polymerase chain reaction (PCR), randomly recombined into phage libraries, and then screened for antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). The phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high-affinity antibodies against the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J., 12:725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells, encoding highly variable CDR3 regions using PCR primers containing random sequences, and achieving in vitro rearrangement as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0193] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein. Techniques for making bispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-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, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by manipulating 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 generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 148(5):1547-1553 (1992)); al., J. Immunol., 152:5368 (1994)); and, for example, by preparing trispecific antibodies as described in Tutt et al. J. Immunol. 147:60 (1991).

[0194] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576).

[0195] The anti-CD20 / anti-CD3 bispecific antibodies herein also include "dual acting FAbs" or "DABs" that contain antigen-binding sites that bind to two different antigens (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0196] "Crossmab" antibodies are also included herein (see, e.g., WO2009080251, WO2009080252, WO2009080253, WO2009080254).

[0197] Another technique for generating bispecific antibody fragments is the "bispecific T cell induction" or BiTE® approach (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261, and WO 2008 / 119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain comprises two single-chain Fv (scFv) fragments, each having a variable heavy (VH) and a variable light (VL) domain, separated by a polypeptide linker of sufficient length to allow intramolecular association between them. The single polypeptide further comprises a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, which may be specific for a different cell type, such that cells of two different cell types are brought into proximity or anchored when each scFv engages its cognate epitope. One particular embodiment of this approach involves an scFv that recognizes a cell surface antigen expressed by an immune cell, e.g., the CD3 polypeptide on a T cell, joined to another scFv that recognizes a cell surface antigen expressed by a target cell, such as a malignant or tumor cell.

[0198] Being a single polypeptide, the bispecific T cell engager can be expressed using any prokaryotic or eukaryotic cell expression system known in the art, such as a CHO cell line. However, specific purification techniques (see, e.g., EP 1691833) may be necessary to separate the monomeric bispecific T cell engager from other multimeric species that may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted using a gradient of imidazole concentration. This eluate is further purified using anion exchange chromatography, and the polypeptide is eluted using a gradient of sodium chloride concentration. Finally, the eluate is subjected to size exclusion chromatography to separate the monomer from the multimeric species.

[0199] In some embodiments, anti-CD20 / anti-CD3 bispecific antibodies may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Moieties suitable for derivatization of anti-CD20 / anti-CD3 bispecific antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, 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 (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may 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 multiple polymers are attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization can 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 therapeutically under defined conditions, etc.

[0200] The anti-CD20 / anti-CD3 bispecific antibody may be 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 a fragment thereof), or a radioisotope.

[0201] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 106). 235); auristatins, e.g., monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or its derivatives (see U.S. Pat. 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. 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. 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 CC1065.

[0202] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody is conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Jatropha curcas protein, diansin protein, pokeweed protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0203] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody is 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 Radioactive conjugates, when used for detection, include radioactive atoms for scintigraphic examinations, e.g., TC 99m Or I 123 , or spin labels 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.

[0204] 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 International Publication No. WO 94 / 11026. The linker may also be a "cleavable linker" that facilitates the release of the cytotoxic drug within the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127131 (1992); U.S. Patent No. 5,208,020) can be used.

[0205] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is indicated for the treatment of cancer. In one embodiment, the cancer is a B-cell proliferative disorder. In one embodiment, the cancer is a CD20-positive B-cell proliferative disorder. In one embodiment, the cancer is non-Hodgkin's lymphoma (NHL). In one embodiment, the NHL is diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), DLBCL arising from FL [transformed FL; trFL], primary mediastinal large B-cell lymphoma (PMBCL), or marginal zone lymphoma (MZL). MZL can be classified into splenic MZL, nodal MZL, and extranodal MZL. In one embodiment, the DLBCL is Richter's transformed. In one embodiment, the NHL is mantle cell lymphoma (MCL). In one embodiment, the NHL is grade 1-3a follicular lymphoma (FL). In one embodiment, the CD20-positive B-cell proliferative disorder is a relapsed or refractory B-cell proliferative disorder. In one embodiment, the relapsed or refractory B-cell proliferative disorder is a relapsed or refractory NHL (e.g., relapsed or refractory DLBCL, relapsed or refractory FL, or relapsed or refractory MCL). In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody, e.g., glofitamab, is indicated for the treatment of relapsed or refractory diffuse large B-cell lymphoma (DLBCL), DLBCL arising from follicular lymphoma, and high-grade B-cell lymphoma (HGBCL) after two or more lines of systemic therapy.

[0206] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody, e.g., glofitamab, is indicated for the treatment of adult patients with relapsed or refractory large B-cell lymphoma, including diffuse large B-cell lymphoma (DLBCL), DLBCL arising from follicular lymphoma, high-grade B-cell lymphoma (HGBCL), and primary mediastinal B-cell lymphoma (PMBCL), unless otherwise specified, after two or more lines of systemic therapy.

[0207] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody, such as glofitamab, is indicated for the treatment of relapsed or refractory follicular lymphoma (FL) after two or more lines of systemic therapy.

[0208] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody, such as glofitamab, is indicated for the treatment of adult patients with relapsed or refractory follicular lymphoma (FL) after two or more lines of systemic therapy.

[0209] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody, such as glofitamab, is indicated for the treatment of relapsed or refractory mantle cell lymphoma (MCL) after two or more lines of systemic therapy.

[0210] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody, such as glofitamab, is indicated for the treatment of adult patients with relapsed or refractory mantle cell lymphoma (MCL) after two or more lines of systemic therapy.

[0211] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody, such as glofitamab, is indicated for the treatment of relapsed or refractory mantle cell lymphoma (MCL) after at least one line of systemic therapy comprising a Bruton's tyrosine kinase (BTK) inhibitor.

[0212] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody, e.g., glofitamab, is indicated for the treatment of adult patients with relapsed or refractory mantle cell lymphoma (MCL) after at least one line of systemic therapy comprising a Bruton's tyrosine kinase (BTK) inhibitor.

[0213] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody, e.g., glofitamab, is indicated for the treatment of previously untreated DLBCL, e.g., in combination with an anti-CD20 antibody, cyclophosphamide, doxorubicin, and a corticosteroid. In one embodiment, the corticosteroid is prednisone and the anti-CD20 antibody is rituximab.

[0214] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody specifically binds to CD3ε.

[0215] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibodies are antibody H2C (WO 2008 / 119567), antibody V9 (Rodrigues et al., Int J Cancer Suppl 7,45-50 (1992) and US Pat. No. 6,054,297), antibody FN18 (Nooij et al., Eur J Immunol 19,981-984(1986)), antibody SP34 (Pessano et al., EMBO J 4,337-340(1985)), antibody OKT3 (Kung et al., Science 206,347-349(1979)), antibody WT31 (Spits et al., J Immunol 135, 1922 (1985)), antibody UCHT1 (Burns et al., J Immunol 129, 1451-1457 (1982)), antibody 7D6 (Coulie et al., Eur J Immunol 21, 1703-1709 (1991)), or antibody Leu-4. In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody is a antibody described in WO 2005 / 040220, WO 2005 / 118635, WO 2007 / 042261, WO 2008 / 119567, WO 2008 / 119565, WO 2012 / 162067, WO 2013 / 158856, WO 2013 / 188693, WO 2013 / 186613, WO 2014 / 110601, WO 2015 / 110602, WO 2016 / 110604, WO 2017 / 110606, WO 2018 / 119567, WO 2018 / 119565, WO 2012 / 162067, WO 2013 / 158856, WO 2013 / 188693, WO 2013 / 186613, WO 2014 / 110601, WO 2015 / 110602, WO 2015 / 110604, WO 2015 / 110606, WO 2015 / 110608, WO 2015 / 110609 ... The antibody may comprise an antigen-binding portion that specifically binds to CD3 as described in Publication No. WO 2014 / 145806, WO 2014 / 191113, WO 2014 / 047231, WO 2015 / 095392, WO 2015 / 181098, WO 2015 / 001085, WO 2015 / 104346, WO 2015 / 172800, WO 2016 / 020444, or WO 2016 / 014974.

[0216] In some embodiments, the anti-CD20 / anti-CD3 bispecific antibody may comprise an antibody or antigen-binding portion derived from tuximab, obinutuzumab ocrelizumab, ofatumumab, ocaratuzumab, veltuzumab, and ublituximab.

[0217] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is XmAb® 13676. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is REGN1979. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is FBTA05 (Lymphomun). In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is glofitamab.

[0218] In some embodiments, the anti-CD20 / anti-CD3 bispecific antibodies may include generic, biosimilar, or non-comparable versions of the antibodies named herein.

[0219] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds to CD20, wherein the antigen-binding domain is (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable region comprising Includes.

[0220] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds CD20, which antigen-binding domain comprises a heavy chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 8. In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds CD20 comprising the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8.

[0221] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds to CD3, wherein the antigen-binding domain is: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a light chain variable region comprising Includes.

[0222] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds CD3, which antigen-binding domain comprises a heavy chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 and a light chain variable region sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 16. In a further embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds CD3 comprising the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16.

[0223] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody a) at least one antigen-binding domain that specifically binds to CD20, comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable region comprising at least one antigen-binding domain that specifically binds to CD20, comprising: b) at least one antigen-binding domain that specifically binds to CD3, comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a light chain variable region comprising at least one antigen-binding domain that specifically binds to CD3, comprising Includes:

[0224] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (i) at least one antigen-binding domain that specifically binds to CD20 comprising a heavy chain variable region sequence of SEQ ID NO: 7 and a light chain variable region sequence of SEQ ID NO: 8; and (ii) at least one antigen-binding domain that specifically binds to CD3, comprising the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16 Includes:

[0225] In one embodiment, the antigen-binding domain that specifically binds to CD3 of an anti-CD20 / anti-CD3 bispecific antibody is an antibody fragment, in particular a Fab molecule or an scFv molecule, more particularly a Fab molecule. In a specific embodiment, the antigen-binding domain that specifically binds to CD3 of an anti-CD20 / anti-CD3 bispecific antibody is a crossover Fab molecule in which the variable or constant domains of the Fab heavy and light chains are swapped (i.e., replaced by each other).

[0226] In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds to CD20 and one antigen-binding domain that specifically binds to CD3. In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody comprises a first antigen-binding domain that specifically binds to CD3 and a second and third antigen-binding domain that specifically bind to CD20. In one embodiment, the first antigen-binding domain is a crossover Fab molecule, and the second and third antigen-binding domains are each conventional Fab molecules. In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody further comprises an Fc domain. An anti-CD20 / anti-CD3 bispecific antibody may comprise modifications in the Fc region and / or antigen-binding domain described herein. In one embodiment, an anti-CD20 / anti-CD3 bispecific antibody comprises an IgG1 Fc domain comprising one or more amino acid substitutions that reduce binding to Fc receptors and / or reduce effector function. In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises an IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and P329G (numbering according to the Kabat EU index).

[0227] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (i) an antigen-binding domain that specifically binds to CD3, fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; (ii) a first antigen-binding domain that specifically binds to CD20 fused to the N-terminus of the Fab heavy chain of an antigen-binding domain that specifically binds to CD3 at the C-terminus of the Fab heavy chain; (iii) a second antigen-binding domain that specifically binds to CD20 fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain; Includes:

[0228] In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody is a) a first Fab molecule that specifically binds to CD3, in particular CD3 epsilon, in which the variable domains VL and VH of the Fab light and heavy chains are replaced by each other; b) a second Fab and a third Fab molecule that specifically binds to CD20, wherein in the constant domain CL of the second Fab and third Fab molecules the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), in particular by arginine (R) (numbering according to Kabat); and in the constant domain CHI of the second Fab and third Fab molecules the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index); and (c) an Fc domain composed of a first subunit and a second subunit that can stably associate; Includes:

[0229] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises two antigen-binding domains that specifically bind to CD20 and one antigen-binding domain that specifically binds to CD3.

[0230] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is bivalent with respect to CD20 and monovalent with respect to CD3.

[0231] In one embodiment, a first Fab molecule of a) is fused at the C-terminus of its Fab heavy chain to the N-terminus of one of the subunits of the Fc domain of c), a second Fab molecule of b) is fused at the C-terminus of the Fab heavy chain of the first Fab molecule of a) to the N-terminus of the heavy chain, and a third Fab molecule of b) is fused at the C-terminus of its Fab heavy chain to the N-terminus of the other subunit of the Fc domain of c).

[0232] In one embodiment, the first Fab molecule of a) comprises a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 15 and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 16.

[0233] In still further embodiments, the first Fab molecule of a) comprises the heavy chain variable region sequence of SEQ ID NO:15 and the light chain variable region sequence of SEQ ID NO:16.

[0234] In one embodiment, the second and third Fab molecules of b) each comprise a heavy chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 7 and a light chain variable region that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 8.

[0235] In one embodiment, the second and third Fab molecules of b) each comprise a heavy chain variable region sequence of SEQ ID NO:7 and a light chain variable region sequence of SEQ ID NO:8.

[0236] In certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody comprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 17, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 18, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 19, and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 20. In further specific embodiments, the bispecific antibody comprises the polypeptide sequence of SEQ ID NO: 17, the polypeptide sequence of SEQ ID NO: 18, the polypeptide sequence of SEQ ID NO: 19, and the polypeptide sequence of SEQ ID NO: 20. In further specific embodiments, the bispecific antibody comprises one polypeptide chain comprising SEQ ID NO: 17, one polypeptide chain comprising SEQ ID NO: 18, two polypeptide chains comprising SEQ ID NO: 19, and one polypeptide chain comprising SEQ ID NO: 20.

[0237] Certain anti-CD20 / anti-CD3 bispecific antibodies are described in WO 2016 / 020309 and European Patent Application Nos. EP 15188093 and EP 16169160, each of which is incorporated by reference in its entirety.

[0238] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody is glofitamab, described below.

[0239] Antibody Format The components of the anti-CD20 / anti-CD3 bispecific antibody can be fused to each other in a variety of configurations, exemplary configurations are shown in Figure 1.

[0240] In certain embodiments, the antigen-binding portion of an anti-CD20 / anti-CD3 bispecific antibody is a Fab molecule. In such embodiments, the first, second, third, etc. antigen-binding portion may be referred to herein as the first, second, third, etc. Fab molecule, respectively. Furthermore, in certain embodiments, the anti-CD20 / anti-CD3 bispecific antibody comprises an Fc domain composed of a first subunit and a second subunit that are capable of stably associating.

[0241] In some embodiments, the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

[0242] In one such embodiment, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. In a specific such embodiment, the anti-CD20 / anti-CD3 bispecific antibody consists essentially of first and second Fab molecules, an Fc domain composed of first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule. Such a structure is shown schematically in Figures 1G and 1K. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule may further be fused to each other.

[0243] In another embodiment, the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In this specific embodiment, the antibody consists essentially of first and second Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the first and second Fab molecules are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. Such a structure is shown schematically in Figures 1A and 1D. The first and second Fab molecules can be fused to the Fc domain directly or via a peptide linker. In a specific embodiment, the first and second Fab molecules are each fused to the Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1 Fc domain.

[0244] In other embodiments, the second Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. In one such embodiment, the first Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In this specific embodiment, the antibody consists essentially of first and second Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain. Such structures are shown schematically in Figures 1H and 1L. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can be further fused to each other.

[0245] Fab molecules can be fused to the Fc domain or to each other either directly or via a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and are described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S) n or G4 (SG4) n Peptide linkers are included. "n" is usually an integer from 1 to 10, typically from 2 to 4. In one embodiment, the peptide linker has a length of at least 5 amino acids, in one embodiment from 5 to 100 amino acids, and in a further embodiment from 10 to 50 amino acids. In one embodiment, the peptide linker has a length of (GxS) n or (GxS) n G mwherein G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, m=0, 1, 2 or 3), or (x=4, n=2, 3, 4 or 5, and m=0, 1, 2 or 3), in one embodiment x=4, n=2 or 3, and in a further embodiment x=4, n=2. In one embodiment, the peptide linker is (G4S)2. A peptide linker particularly suitable for fusing the Fab light chains of the first and second Fab molecules to each other is (G4S)2. An exemplary peptide linker suitable for connecting the Fab heavy chains of the first and second Fab fragments comprises the sequence (D)-(G4S)2. Another suitable such linker comprises the sequence (G4S)4. In addition, the linker can comprise (a portion of) an immunoglobulin hinge region. In particular, when a Fab molecule is fused to the N-terminus of an Fc domain subunit, the fusion may be via the immunoglobulin hinge region or a portion thereof, with or without an additional peptide linker.

[0246] Antibodies comprising a single antigen-binding moiety (e.g., a Fab molecule) capable of specifically binding to a target cell antigen (e.g., as shown in Figure 1A, D, G, H, K, L) are useful, particularly when internalization of the target cell antigen is expected following binding of the high-affinity antigen-binding moiety. In such cases, the presence of more than one antigen-binding moiety specific for the target cell antigen may enhance internalization of the target cell antigen, thereby reducing its availability.

[0247] However, in many other cases, it will be advantageous to have an antibody that comprises two or more antigen-binding portions (e.g., Fab molecules) specific for target cell antigens (see examples shown in Figures 1B, 1C, 1E, 1F, 1I, 1J, 1M, or 1N), for example, to optimize targeting to the target site or to allow cross-linking of target cell antigens.

[0248] Thus, in certain embodiments, an anti-CD20 / anti-CD3 bispecific antibody comprises two anti-CD20 binding moieties, e.g., two Fab molecules that target CD20. In one embodiment, the two Fab molecules that target CD20 are conventional Fab molecules. In one embodiment, the two Fab molecules that target CD20 comprise the same heavy and light chain amino acid sequences and have the same domain organization (i.e., conventional or crossover).

[0249] In alternative embodiments, the anti-CD20 / anti-CD3 bispecific antibody comprises two anti-CD3 binding moieties, e.g., two Fab molecules that target CD3. In one such embodiment, the two Fab molecules that target CD3 are both crossover Fab molecules (Fab molecules in which the variable domains VH and VL or the constant domains CL and CH1 of the Fab heavy and light chains are swapped / replaced by each other). In one such embodiment, the two Fab molecules that target CD3 comprise the same heavy and light chain amino acid sequences and have the same organization of domains (i.e., conventional or crossover).

[0250] In one embodiment, the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first or second subunit of the Fc domain.

[0251] In certain embodiments, the second and third Fab molecules are each fused at the C-terminus of their Fab heavy chains to the N-terminus of one of the subunits of the Fc domain, and the first Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In these specific embodiments, the antibody consists essentially of first, second, and third Fab molecules, an Fc domain composed of the first and second subunits, and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, the second Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such structures are shown schematically in Figures 1B and 1E (embodiments in which the third Fab molecule is a conventional Fab molecule and is identical to the second Fab molecule) and Figures 1I and 1M (embodiments in which the third Fab molecule is a crossover Fab molecule and is preferably identical to the first Fab molecule). The second and third Fab molecules can be fused to an Fc domain directly or via a peptide linker. In certain embodiments, the second and third Fab molecules are each fused to an Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can further be fused to each other.

[0252] In another embodiment, the second and third Fab molecules are each fused at the C-terminus of their Fab heavy chains to the N-terminus of one of the subunits of the Fc domain, and the first Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule. In a specific such embodiment, the antibody consists essentially of first, second, and third Fab molecules, the Fc domains of the first and second subunits, and optionally one or more peptide linkers, and the first Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, the second Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the first subunit of the Fc domain, and the third Fab molecule is fused at the C-terminus of its Fab heavy chain to the N-terminus of the second subunit of the Fc domain. Such structures are shown schematically in Figures 1C and 1F (embodiments in which the third Fab molecule is a conventional Fab molecule and is identical to the second Fab molecule) and Figures 1J and 1N (embodiments in which the third Fab molecule is a crossover Fab molecule and is identical to the first Fab molecule). The first and third Fab molecules can be fused to an Fc domain directly or via a peptide linker. In certain embodiments, the second and third Fab molecules are each fused to an Fc domain via an immunoglobulin hinge region. In a specific embodiment, the immunoglobulin hinge region is a human IgG1 hinge region, and in particular, the Fc domain is an IgG1 Fc domain. Optionally, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule can be further fused to each other.

[0253] In an antibody structure in which a Fab molecule is fused at the C-terminus of the Fab heavy chain via an immunoglobulin hinge region to the N-terminus of each of the Fc domain subunits, the two Fab molecules, hinge region, and Fc domain essentially form an immunoglobulin molecule. In a specific embodiment, the immunoglobulin molecule is an immunoglobulin of the IgG class. In a more specific embodiment, the immunoglobulin is an immunoglobulin of the IgG1 subclass. In another embodiment, the immunoglobulin is an immunoglobulin of the IgG4 subclass. In a still more specific embodiment, the immunoglobulin is a human immunoglobulin. In other embodiments, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin.

[0254] In some antibodies, the Fab light chain of a first Fab molecule and the Fab light chain of a second Fab molecule are fused to each other, optionally via a peptide linker. Depending on the structure of the first and second Fab molecules, the Fab light chain of the first Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule may be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. Fusing the Fab light chains of the first and second Fab molecules further reduces mispairing of mismatched Fab heavy and light chains and also reduces the number of plasmids required to express some of the antibodies.

[0255] In some embodiments, the antibody comprises a polypeptide (VL) in which the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), and the Fab heavy chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VL). (1) -CH1 (1) -CH2-CH3(-CH4)) and a polypeptide in which the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (2) -CH1 (2)In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule. (1) -CL (1) ) and a Fab light chain polypeptide (VL) of a second Fab molecule. (2) -CL (2) In some embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0256] In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain constant region is replaced by a light chain constant region), and the Fab light chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH). (1) -CL (1) -CH2-CH3(-CH4)) and a polypeptide in which the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (2) -CH1 (2) In some embodiments, the antibody comprises a polypeptide in which the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (VL (1) -CH1 (1) ) and a Fab light chain polypeptide (VL) of a second Fab molecule. (2) -CL (2) In some embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0257] In some embodiments, the antibody comprises a polypeptide in which the Fab light chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), the Fab heavy chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a second Fab molecule, and the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VL (1) -CH1 (1) -VH (2) -CH1 (2) In other embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the first Fab molecule, the Fab light chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), and the Fab heavy chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2) -VL (1) -CH1 (1) -CH2-CH3(-CH4)).

[0258] In some of these embodiments, the antibody comprises a crossover Fab light chain polypeptide (VH) of a first Fab molecule in which the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule. (1) -CL (1) ) and a Fab light chain polypeptide (VL) of a second Fab molecule. (2) -CL (2)In other of these embodiments, optionally, the antibody further comprises a polypeptide (VH) in which the Fab heavy chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule, and the Fab light chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of a second Fab molecule. (1) -CL (1) -VL (2) -CL (2) ), or a polypeptide in which the Fab light chain polypeptide of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, and the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VL (2) -CL (2) -VH (1) -CL (1) ) further includes.

[0259] The antibody according to these embodiments may comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and a Fab light chain polypeptide (VL (3) -CL (3) In some embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0260] In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain constant region is replaced by a light chain constant region), the Fab light chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the second Fab molecule, and the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit. (1) -CL (1) -VH (2) -CH1 (2) In other embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (i.e., the first Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain constant region is replaced by a light chain constant region), and the Fab light chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with an Fc domain subunit (VH (2) -CH1 (2) -VH (1) -CL (1) -CH2-CH3(-CH4)).

[0261] In some of these embodiments, the antibody comprises a crossover Fab light chain polypeptide (VL) of a first Fab molecule, in which the Fab light chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule. (1) -CH1 (1) ) and a Fab light chain polypeptide (VL) of a second Fab molecule. (2) -CL (2)In other of these embodiments, optionally, the antibody further comprises a polypeptide (VL) in which the Fab light chain variable region of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the first Fab molecule, and the Fab heavy chain constant region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain polypeptide of a second Fab molecule. (1) -CH1 (1) -VL (2) -CL (2) ), or a polypeptide in which the Fab light chain polypeptide of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the first Fab molecule, and the Fab heavy chain variable region of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the first Fab molecule (VL (2) -CL (2) -VH (1) -CL (1) ) further includes.

[0262] The antibody according to these embodiments may comprise (i) an Fc domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide in which the Fab heavy chain of a third Fab molecule shares a carboxy-terminal peptide bond with the Fc domain subunit (VH (3) -CH1 (3) -CH2-CH3(-CH4)) and a Fab light chain polypeptide (VL (3) -CL (3) In some embodiments, the polypeptides are covalently linked, for example, by a disulfide bond.

[0263] In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, and the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region). (1) -CH1 (1) -VL (2)-CH1 (2) In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) further includes.

[0264] In some embodiments, the antibody comprises a polypeptide (VL) in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), and the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule. (2) -CH1 (2) -VH (1) -CH1 (1) In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) further includes.

[0265] In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain constant region is replaced by a light chain constant region), and the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule. (2) -CL (2) -VH (1) -CH1 (1)In some embodiments, the antibody comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) ) and further.

[0266] In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of the second Fab molecule, and the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region). (3) -CH1 (3) -VH (1) -CH1 (1) -VL (2) -CH1 (2) In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) In some embodiments, the antibody further comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ) further includes.

[0267] In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of the second Fab molecule, and the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain constant region is replaced by a light chain constant region). (3) -CH1 (3) -VH (1) -CH1 (1) -VH (2) -CL (2) In some embodiments, the antibody comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) In some embodiments, the antibody further comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ) further includes.

[0268] In some embodiments, the antibody comprises a polypeptide (VL) in which the Fab light chain variable region of a second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a first Fab molecule, and the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule. (2) -CH1 (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3)In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) In some embodiments, the antibody further comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ) further includes.

[0269] In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, in which the heavy chain variable region is replaced by a light chain variable region), the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule, and the Fab heavy chain of the first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of a third Fab molecule. (2) -CL (2) -VH (1) -CH1 (1) -VH (3) -CH1 (3) In some embodiments, the antibody comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) In some embodiments, the antibody further comprises a Fab light chain polypeptide (VL) of a third Fab molecule. (3) -CL (3) ) further includes.

[0270] In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises crossover Fab heavy chains, where the heavy chain variable region is replaced by a light chain variable region), the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a third Fab molecule, and the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of a third Fab molecule (i.e., the third Fab molecule comprises crossover Fab heavy chains, where the heavy chain variable region is replaced by a light chain variable region). (1) -CH1 (1) -VL (2) -CH1 (2) -VL (3) -CH1 (3) In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) In some embodiments, the antibody further comprises a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule. (3) -CL (3) ) further includes.

[0271] In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain of a first Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a second Fab molecule (i.e., the second Fab molecule comprises crossover Fab heavy chains, where the heavy chain constant region is replaced by a light constant region), the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a third Fab molecule, and the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of a third Fab molecule (i.e., the third Fab molecule comprises crossover Fab heavy chains, where the heavy chain constant region is replaced by a light chain constant region). (1) -CH1 (1) -VH (2) -CL (2) -VH (3) -CL (3) In some embodiments, the antibody comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) In some embodiments, the antibody further comprises a polypeptide (VL) in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule. (3) -CH1 (3) ) further includes.

[0272] In some embodiments, the antibody comprises a polypeptide (VL) in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule (i.e., the third Fab molecule comprises crossover Fab heavy chains, where the heavy chain variable region is replaced by a light chain variable region), the Fab heavy chain constant region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain variable region of a second Fab molecule, the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises crossover Fab heavy chains, where the heavy chain variable region is replaced by a light chain variable region), and the Fab heavy chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule. (3) -CH1 (3) -VL (2) -CH1 (2) -VH (1) -CH1 (1) In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule. (2) -CL (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) In some embodiments, the antibody further comprises a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule. (3) -CL (3) ) further includes.

[0273] In some embodiments, the antibody comprises a polypeptide (VH) in which the Fab heavy chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the third Fab molecule (i.e., the third Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), the Fab light chain constant region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain variable region of a second Fab molecule, the Fab heavy chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab light chain constant region of the second Fab molecule (i.e., the second Fab molecule comprises a crossover Fab heavy chain, wherein the heavy chain constant region is replaced by a light chain constant region), and the Fab light chain constant region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain of the first Fab molecule. (3) -CL (3) -VH (2) -CL (2) -VH (1) -CH1 (1) In some embodiments, the antibody comprises a polypeptide in which the Fab light chain variable region of the second Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the second Fab molecule (VL (2) -CH1 (2) ) and the Fab light chain polypeptide (VL) of the first Fab molecule (1) -CL (1) In some embodiments, the antibody further comprises a polypeptide (VL) in which the Fab light chain variable region of the third Fab molecule shares a carboxy-terminal peptide bond with the Fab heavy chain constant region of the third Fab molecule. (3) -CH1 (3) ) further includes.

[0274] According to the above embodiments, antibody components (e.g., Fab molecules, Fc domains) can be fused directly or via various linkers, particularly peptide linkers comprising one or more amino acids, typically about 2-20 amino acids, as described herein or known in the art. Suitable non-immunogenic peptide linkers include, for example, (G4S) n , (SG4) n , (G4S)n or G4 (SG4) n A peptide linker is included, where n is an integer generally from 1 to 10, typically from 2 to 4.

[0275] Fc domain Anti-CD20 / anti-CD3 bispecific antibodies may contain an Fc domain consisting of a pair of polypeptide chains comprising the heavy chain domain of an antibody molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which contains the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain can stably associate with each other.

[0276] In one embodiment, the Fc domain is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a specific embodiment, the Fc domain is an IgG4 Fc domain containing an amino acid substitution at position S228 (Kabat numbering), in particular the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further specific embodiment, the Fc domain is human.

[0277] (i) Fc domain modifications that promote heterodimerization Anti-CD20 / anti-CD3 bispecific antibodies can contain different components (e.g., antigen-binding domains) fused to one or the other of the two subunits of the Fc domain, which are therefore typically contained in two non-identical polypeptide chains. Recombinant coexpression of these polypeptides and subsequent dimerization generates multiple possible combinations of the two polypeptides. Therefore, to improve the yield and purity of such antibodies in recombinant production, it would be advantageous to include modifications in the Fc domain of the antibody that promote association of the desired polypeptides.

[0278] Thus, in certain embodiments, the Fc domain comprises a modification that promotes the association of the first and second subunits of the Fc domain. The most extensive site of protein-protein interaction between the two subunits of a human IgG Fc domain is within the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is within the CH3 domain of the Fc domain.

[0279] Several approaches for modifying the CH3 domain of an Fc domain to force heterodimerization have been well described, for example in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches, both the CH3 domain of the first Fc subunit and the CH3 domain of the second Fc subunit are engineered to be complementary so that each CH3 domain (or the heavy chain containing it) can no longer homodimerize with itself but can heterodimerize with another complementary engineered CH3 domain (so that the first CH3 domain heterodimerizes with the second CH3 domain and no homodimers form between the two first CH3 domains or the two second CH3 domains). These different approaches to improved heavy chain heterodimerization are intended as different alternatives to reduce light chain mispairing and Bence-Jones-type by-products, combined with heavy chain-light chain modifications (e.g., variable or constant region swaps / replacements in Fab arms or introduction of oppositely charged amino acid substitutions at the CH1 / CL interface).

[0280] In a specific embodiment, the modification that promotes association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which comprises a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain.

[0281] Knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168, U.S. Patent No. 7,695,936, Ridgway et al., Prot Eng 9, 617-621 (1996), and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protuberance can locate within the cavity, promoting heterodimer formation and preventing homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of identical or similar size to the protuberance is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (eg, alanine or threonine).

[0282] Thus, in one specific embodiment, in the CH3 domain of a first subunit of an Fc domain, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance within the CH3 domain of the first subunit that can be positioned within a cavity within the CH3 domain of a second subunit, and in the CH3 domain of a second subunit of an Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit that can be positioned within the protuberance within the CH3 domain of the first subunit.

[0283] Preferably, said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0284] Preferably, said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0285] The protuberances and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0286] In one particular embodiment, in the CH3 domain of the first subunit of the Fc domain (the "knob" subunit), the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain (the "hole" subunit), the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, further, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0287] In yet a further embodiment, the first subunit of the Fc domain further comprises a replacement of the serine residue at position 354 with a cysteine ​​residue (S354C) or a replacement of the glutamic acid residue at position 356 with a cysteine ​​residue (E356C), and the second subunit of the Fc domain further comprises a replacement of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). Introduction of these two cysteine ​​residues allows for the formation of disulfide bridges between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0288] In one particular embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to the Kabat EU index).

[0289] In certain embodiments, the CD3 antigen-binding moieties described herein are fused to the first subunit of an Fc domain (comprising a "knob" modification). Without wishing to be bound by theory, fusion of the CD3 antigen-binding moiety to the knob-containing subunit of an Fc domain (also) minimizes the generation of bispecific antibodies comprising two CD3 antigen-binding moieties (steric clashes between the two knob-containing polypeptides).

[0290] Other techniques of CH3 modification to effect heterodimerization are contemplated as alternatives according to the present invention and are described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291.

[0291] In one embodiment, the heterodimerization approach described in EP 1 870 459 A1 is used instead. This approach is based on the introduction of oppositely charged amino acids at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. A preferred embodiment is the amino acid variants R409D;K370E in one of the two CH3 domains (of the Fc domain) and D399K;E357K in the other CH3 domain of the Fc domain (numbering according to Kabat).

[0292] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally the further amino acid mutations R409D;K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K;E357K in the CH3 domain of the second subunit of the Fc domain (numbering according to Kabat EU index).

[0293] In another embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises the amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the antibody comprises the amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally comprises the amino acid mutations R409D;K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K;E357K in the CH3 domain of the second subunit of the Fc domain (all numbering according to Kabat EU index).

[0294] In one embodiment, the heterodimerization approach described in WO 2013 / 157953 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (numbering according to the Kabat EU index). In a further embodiment, the first CH3 domain comprises the additional amino acid mutation L351K. In a further embodiment, the second CH3 domain comprises an additional amino acid mutation selected from Y349E, Y349D and L368E, preferably L368E (numbering according to the Kabat EU index).

[0295] In one embodiment, the heterodimerization approach described in WO 2012 / 058768 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A, and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain comprises an additional amino acid mutation at positions T411, D399, S400, F405, N390, or K392, such as (a) T411N, T411R, T411Q, T411K, T411D, T411E, or T411W; (b) D399R, D399W, D399Y, or D399K; (c) D399R, D399W, D399Y, or D399K; (d) S400E, S400D, S400R or S400K, (d) F405I, F405M, F405T, F405S, F405V or F405W, (e) N390R, N390K or N390D, (f) K392V, K392M, K392R, K392L, K392F or K392E (numbering according to Kabat EU index). In a further embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A and the second CH3 domain comprises the amino acid mutations T366V, K409F. In a further embodiment, the first CH3 domain comprises the amino acid mutation Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to the Kabat EU index).

[0296] In one embodiment, the heterodimerization approach described in WO 2011 / 143545 is used instead, e.g., with an amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to the Kabat EU index).

[0297] In one embodiment, the heterodimerization approach described in WO 2011 / 090762, which also employs the knob-into-hole technique described above, is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutation T366W and the second CH3 domain comprises the amino acid mutation Y407A. In one embodiment, the first CH3 domain comprises the amino acid mutation T366Y and the second CH3 domain comprises the amino acid mutation Y407T (numbering according to the Kabat EU index).

[0298] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody or its Fc domain is of the IgG2 subclass and the heterodimerization approach described in WO 2010 / 129304 is used.

[0299] In an alternative embodiment, the modification that promotes association of the first and second subunits of the Fc domain comprises a modification that mediates an electrostatic steering effect, e.g., as described in WO 2009 / 089004. Typically, this method involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues, such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. In one such embodiment, the first CH3 domain comprises an amino acid substitution at K392 or N392 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain comprises an amino acid substitution at D399, E356, D356, or E357 with a positively charged amino acid (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K, or E357K, more preferably D399K and E356K). In a further embodiment, the first CH3 domain further comprises an amino acid substitution at K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In a further embodiment, the first CH3 domain additionally or alternatively comprises an amino acid substitution at K439 and / or K370 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)) (all numbering according to the Kabat EU index).

[0300] In still further embodiments, the heterodimerization approach described in WO 2007 / 147901 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutations K253E, D282K, and K322D, and the second CH3 domain comprises the amino acid mutations D239K, E240K, and K292D (numbering according to the Kabat EU index).

[0301] In yet another embodiment, the heterodimerization approach described in WO 2007 / 110205 can be used instead.

[0302] In one embodiment, the first subunit of the Fc domain comprises the amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises the amino acid substitutions D356K and D399K (numbering according to the Kabat EU index).

[0303] (ii) Fc domain modifications that reduce Fc receptor binding and / or reduce effector function The Fc domain confers desirable pharmacokinetic properties to antibodies, such as anti-CD20 / anti-CD3 bispecific antibodies, including a long serum half-life that contributes to favorable accumulation in target tissues and a favorable tissue-to-blood distribution ratio. However, at the same time, the Fc domain can cause undesirable targeting of antibodies to cells that express Fc receptors rather than to preferred antigen-bearing cells. Furthermore, coactivation of Fc receptor signaling pathways can lead to cytokine release, which, combined with other immunostimulatory properties that antibodies may possess and their long half-life, can lead to excessive activation of cytokine receptors and severe side effects when administered systemically.

[0304] Thus, in certain embodiments, the Fc domain of an anti-CEA / anti-CD3 bispecific antibody exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a corresponding IgG1 Fc domain. In one such embodiment, the Fc domain (or a molecule comprising said Fc domain, e.g., an antibody) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the binding affinity to Fc receptors compared to a native IgG1 Fc domain (or a corresponding molecule comprising a native IgG1 Fc domain), and / or exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the effector function compared to a native IgG1 Fc domain (or a corresponding molecule comprising a native IgG1 Fc domain). In one embodiment, the Fc domain (or a molecule comprising said Fc domain, e.g., an antibody) does not substantially bind to and / or induce effector function of an Fc receptor. In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In a specific embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding to the neonatal Fc receptor (FcRn) compared to a native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or a molecule, e.g., an antibody, comprising the Fc domain) exhibits about 70%, particularly greater than about 80%, and more particularly greater than about 90% of the binding affinity for FcRn of a native IgG1 Fc domain (or a corresponding molecule comprising a native IgG1 Fc domain).

[0305] In some embodiments, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and / or reduced effector function compared to a non-engineered Fc domain. In certain embodiments, the Fc domain comprises one or more amino acid mutations that reduce the binding affinity and / or effector function of the Fc domain to an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where more than one amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor is present, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, a molecule, e.g., an antibody, comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, and more particularly less than 5% of the binding affinity to an Fc receptor compared to a corresponding molecule comprising a non-engineered Fc domain. In a specific embodiment, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, most particularly human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complement components, particularly C1q, is also reduced. In one embodiment, binding affinity to neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e., preservation of the binding affinity of the Fc domain to the receptor, is achieved when the Fc domain (or a molecule comprising the Fc domain, e.g., an antibody) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or a corresponding molecule comprising the non-engineered form of the Fc domain) to FcRn. The Fc domain, or a molecule comprising the Fc domain (e.g., an antibody), may exhibit greater than about 80%, and in some cases greater than about 90%, of such affinity. In some embodiments, the Fc domain is engineered to have reduced effector function compared to a non-engineered Fc domain. Reduced effector function can include, but is not limited to, one or more of: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent T cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling to induce apoptosis, reduced cross-linking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function is one or more selected from the group of reduced effector function, reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a specific embodiment, the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or a corresponding molecule comprising a non-engineered Fc domain).

[0306] In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or the effector function is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, and P329 (numbering according to the Kabat EU index). In a specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235, and P329 (numbering according to the Kabat EU index). In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to the Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbering according to Kabat EU index). In a specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numbering according to Kabat EU index). In a more particular embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor (as well as complement) binding of the human IgG1 Fc domain, as described in WO 2012 / 130831, which is incorporated herein by reference in its entirety.WO 2012 / 130831 also describes methods for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector function.

[0307] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector function compared to IgG1 antibodies. Accordingly, in some embodiments, the Fc domain is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P (numbering according to the Kabat EU index). To further reduce binding affinity to Fc receptors and / or its effector function, in one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numbering according to the Kabat EU index). In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numbering according to the Kabat EU index). In a specific embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically the amino acid substitutions S228P, L235E and P329G (numbering according to the Kabat EU index). Such IgG4 Fc domain variants and their Fcγ receptor binding properties are described in WO 2012 / 130831, which is incorporated herein by reference in its entirety.

[0308] In certain embodiments, the Fc domain that exhibits reduced binding affinity to an Fc receptor and / or effector function compared to a native IgG1 Fc domain is a human IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numbering according to the Kabat EU index).

[0309] In some embodiments, N-glycosylation of the Fc domain is eliminated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, specifically an amino acid substitution replacing asparagine with alanine (N297A), or aspartic acid (N297D), or glycine (N297G) (numbering according to the Kabat EU index).

[0310] In addition to the Fc domains described above and in WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or decreased effector function also include those containing substitutions of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (Kabat EU index numbering). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including so-called "DANA" Fc variants with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0311] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The correct nucleotide changes can be verified, for example, by sequencing.

[0312] Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (GE Healthcare), and such Fc receptors can be obtained by recombinant expression. Alternatively, the binding affinity of an Fc domain, or a molecule containing an Fc domain, to an Fc receptor can be assessed using a cell line known to express a particular Fc receptor, for example, human NK cells expressing the FcγIIIa receptor.

[0313] The effector function of an Fc domain, or a molecule containing an Fc domain (e.g., an antibody), can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al., Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays may be used (e.g., ACTI for flow cytometry). TM See non-radioactive cytotoxicity assays (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® 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 can be assessed in vivo in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci (USA) 95, 652656 (1998).

[0314] In some embodiments, binding of the Fc domain to complement components, specifically C1q, is reduced. Thus, in some embodiments in which the Fc domain is engineered to have reduced effector function, the reduced effector function includes reduced CDC. To determine whether an Fc domain or a molecule (e.g., an antibody) comprising an Fc domain can bind C1q and therefore has CDC activity, a C1q binding assay may be performed. See, e.g., the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0315] glofitamab In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody useful in the methods provided herein is glofitamab. Glofitamab (Proposed INN: List 121 WHO Drug Information, Vol. 33, No. 2, 2019, page 276, also known as CD20-TCB, RO7082859, or RG6026) is a novel T cell-inducing bispecific full-length antibody with a 2:1 molecular configuration for bivalent binding to CD20 on B cells and monovalent binding to CD3, particularly the CD3 epsilon chain (CD3ε), on T cells. Its CD3-binding region is fused head-to-tail to one of the CD20-binding regions via a flexible linker. This structure confers superior in vitro potency to glofitamab compared to other CD20-CD3 bispecific antibodies with a 1:1 configuration, and results in high anti-tumor efficacy in preclinical DLBCL models. The bivalency of CD20 preserves its efficacy in the presence of competing anti-CD20 antibodies, providing the opportunity for pre- or co-treatment with these agents. Glofitamab contains an engineered heterodimeric Fc region that completely abolishes binding to FcgRs and C1q. It simultaneously binds to human CD20-expressing tumor cells and CD3ε in the T cell receptor (TCR) complex on T cells, inducing tumor cell lysis in addition to T cell activation, proliferation, and cytokine release. Glofitamab-mediated B cell lysis is CD20-specific and does not occur in the absence of CD20 expression or simultaneous binding (cross-linking) of T cells to CD20-expressing cells. In addition to killing, T cells undergo activation upon CD3 cross-linking, as detected by increased T cell activation markers (CD25 and CD69), cytokine release (IFNγ, TNFα, IL-2, IL-6, IL-10), release of cytotoxic granules (granzyme B), and T cell proliferation. A schematic molecular structure of glofitamab is shown in Figure 2.

[0316] IV. Novel Dosing Schedules for Anti-CD20 / Anti-CD3 Bispecific Antibodies The present invention relates to new dosing schedules for anti-CD20 / anti-CD3 bispecific antibodies, in particular glofitamab, that result in an acceptable safety and efficacy profile, particularly with regard to side effects associated with cytokine release syndrome.

[0317] Bispecific antibody therapies that utilize T cell activation have been associated with cytokine release syndrome (CRS), a potentially life-threatening condition caused by the excessive release of cytokines by immune effector cells or target cells during an excessive and persistent immune response. CRS can be caused by a variety of factors, including infection with a pathogenic agent, or by drugs that activate or enhance the immune response, resulting in a pronounced and persistent immune response.

[0318] Regardless of the presence or absence of a precipitating agent, severe or life-threatening CRS is a medical emergency. If not managed properly, it can lead to significant disability or even fatal consequences. Current clinical responses focus on treating individual signs and symptoms, providing symptomatic treatment, and attempting to attenuate inflammation using high-dose corticosteroids. However, this approach is not always successful, especially in cases of late intervention. Furthermore, steroids can adversely affect T-cell function, potentially reducing the clinical usefulness of immunomodulatory therapies in cancer treatment.

[0319] Symptoms and grading of CRS CRS is graded according to the Modified Cytokine Release Syndrome Grading System established by Lee et al., Blood, 124:188-195, 2014 or Lee et al., Biol Blood Marrow Transplant, 25(4):625-638, 2019, as shown in Table 3. In addition to the diagnostic criteria, recommendations for the management of CRS based on its severity, including early intervention with corticosteroids and / or anti-cytokine therapy, are provided and are referenced in Tables 3 and 4.

[0320] Table 3: Cytokine Release Syndrome Grading System TIFF2025160202000004.tif240170TIFF2025160202000005.tif26170Lee 2014 Standard: Lee et al.,Blood,124:188-195,2014. ASTCT consensus grading: Lee et al., Biol Blood Marrow Transplant, 25(4):625-638, 2019. a Low-dose vasopressors: A single vasopressor at a dose less than that shown in Table 3. b High-dose vasopressors: As defined in Table 4. * Fever is defined as a temperature of ≥ 38°C not attributable to other causes. Patients with CRS then receive antipyretic or anticytokine therapy, such as tocilizumab or steroids, and fever is no longer required to grade CRS severity thereafter. In this case, CRS grading is driven by hypotension and / or hypoxia. †CRS grade is determined by the more severe event: hypotension or hypoxia not attributable to other causes. For example, a patient with a temperature of 39.5°C, hypotension requiring one vasopressor, and hypoxia requiring a low-flow nasal cannula is classified as having grade 3 CRS. ‡Low-flow nasal cannula is defined as oxygen delivered at ≤6 L / min. Low-flow also includes blow-by oxygen delivery, sometimes used in pediatrics. High-flow nasal cannula is defined as oxygen delivered at >6 L / min.

[0321] Table 4: High-dose vasopressors TIFF2025160202000006.tif85170min = minutes; VASST = Vasopressin and Septic Shock Test. a VASST vasopressor equivalence equation: Norepinephrine equivalent dose = [Norepinephrine (μg / min)] + [dopamine (μg / kg / min) ÷ 2] + [epinephrine (μg / min)] + [phenylephrine (μg / min) ÷ 10].

[0322] Mild to moderate symptoms of CRS and / or infusion reaction (IRR), including symptoms such as fever, headache, and myalgia, may be treated symptomatically with analgesics, antipyretics, and antihistamines as indicated. Severe or life-threatening symptoms of CRS and / or IRR, such as hypotension, tachycardia, dyspnea, or chest discomfort, should be treated aggressively with symptomatic and resuscitative measures as indicated, including the use of high-dose corticosteroids, intravenous fluids, admission to an intensive care unit, and other symptomatic treatments. Severe CRS may be associated with other clinical sequelae, such as disseminated intravascular coagulation, capillary leak syndrome, or macrophage activation syndrome (MAS). No standard of care has been established for severe or life-threatening CRS resulting from immune-based therapies; case reports and recommendations using anti-cytokine therapies such as tocilizumab have been published (Teachey et al., Blood, 121:5154-5157, 2013; Lee et al., Blood, 124:188-195, 2014; Maude et al., New Engl J Med, 371:1507-1517, 2014).

[0323] In a Phase I / II multicenter, open-label, dose-escalation study designed to evaluate the efficacy, safety, tolerability, and PK pharmacokinetics of glofitamab, a novel T cell-engineering bispecific full-length antibody (TCB), the maximum tolerated dose was determined using fixed, preset dosing. Due to its unique structure as described above, glofitamab is a highly potent molecule that can result in unwanted side effects, particularly those related to cytokine release syndrome (CRS).

[0324] In this study, obinutuzumab (Gazyva) pretreatment was used as a CRS mitigation strategy. Despite these strategies, a target glofitamab dose of 25 mg was not feasible due to unacceptable levels of severe side effects, particularly cytokine release syndrome (CRS) of grade 2 or higher. Therefore, there is a need to further discover mechanisms for reducing the risk of CRS in patients treated with glofitamab. The present inventors developed a statistical model to determine a phased dosing schedule with the goal of reducing the incidence of any severe (i.e., grade III) CRS. The present inventors discovered that a specific phased dosing regimen of glofitamab is a useful CRS mitigation strategy, allowing for the administration of a target dose of glofitamab as high as 30 mg, higher than the maximum tolerated dose, when using a fixed or preset dosing regimen, in cases where the risk of CRS grade 2 or higher is lower. New phased dosing is specifically tailored for anti-CD20 / anti-CD3 bispecific antibodies, particularly glofitamab. The clinical data for glofitamab provided in the Examples herein confirm an improved CRS profile. The novel stepwise regimen during the first cycle (C1) further improves the clinical benefit / risk profile of glofitamab by reducing the incidence and severity of CRS in the first cycle. Thus, according to the present invention, the dose of glofitamab is selected to achieve the desired clinical efficacy while effectively reducing the risk of severe CRS in subjects.

[0325] In a first aspect, the present invention provides a method of treating a subject having a CD20-positive B-cell proliferative disorder comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: (a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of the anti-CD20 / anti-CD3 bispecific antibody, wherein C1D1 is 2.5 mg and C1D2 is 10 mg; and (b) the second dosing cycle comprises a single dose (C2D1) of 16 or 30 mg of the anti-CD20 / anti-CD3 bispecific antibody.

[0326] In one embodiment, the single dose of the second dosing cycle comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody. The inventors of the present invention have found that a two-step dose escalation in the first cycle allows for safe administration of the target dose in the second cycle. In one embodiment, the first dose (C1D1) is administered on day 1 of the first dosing cycle, and the second dose (C1D2) is administered on day 8 of the first dosing cycle. In one embodiment, the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle.

[0327] In one aspect, the invention provides a method of treating a subject having a CD20-positive B-cell proliferative disorder, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: (a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg and is administered on day 1 of the first cycle and C1D2 is 10 mg and is administered on day 8 of the first cycle; and (b) the second dosing cycle comprises a single dose of 30 mg of the anti-CD20 / anti-CD3 bispecific antibody (C2D1), administered on day 1 of the second cycle.

[0328] In one embodiment, the method comprises 1 to 10 additional dosing cycles (C3D1 through C12D1). In one such embodiment, the 1 to 10 additional dosing cycles (C3D1 through C12D1) comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, a single dose of the additional dosing cycles (C3D1 through C12D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody.

[0329] In one embodiment, a single dose of the additional dosing cycles (C3D1 through C12D1) is administered on day 1 of each additional dosing cycle.

[0330] Accordingly, in one aspect, the present invention provides a method of treating a subject having a CD20-positive B-cell proliferative disorder comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising 2 to 12 cycles: (a) a first dosing cycle comprising a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg and C1D2 is 10 mg; (b) Subsequent dosing cycles comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1).

[0331] In one such embodiment, the subsequent dosing cycle comprises a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1).

[0332] Accordingly, in one aspect, the present invention provides a method of treating a subject having a CD20-positive B-cell proliferative disorder comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising 2 to 12 cycles: (a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg and is administered on day 1 of the first cycle and C1D2 is 10 mg and is administered on day 8 of the first cycle; (b) Subsequent dosing cycles comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1) administered on day 1 of each subsequent cycle.

[0333] In one such embodiment, subsequent dosing cycles comprise a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1) administered on day 1 of each subsequent cycle.

[0334] In one aspect, the invention provides a method of treating a subject having a CD20-positive B-cell proliferative disorder, comprising administering glofitamab to the subject in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: (a) a first dosing cycle comprising a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 2.5 mg and C1D2 is 10 mg; (b) The second dosing cycle comprises a single dose of 16 or 30 mg glofitamab (C2D1).

[0335] In one embodiment, the single dose of the second dosing cycle contains 30 mg of glofitamab. The inventors of the present invention have found that a two-step increase in the dose of glofitamab in the first cycle allows for safe administration of the target dose in the second cycle. In one embodiment, the first dose (C1D1) is administered on day 1 of the first dosing cycle, and the second dose (C1D2) is administered on day 8 of the first dosing cycle. In one embodiment, the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle.

[0336] In one aspect, the invention provides a method of treating a subject having a CD20-positive B-cell proliferative disorder, comprising administering glofitamab to the subject in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: (a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 2.5 mg and is administered on day 1 of the first cycle, and C1D2 is 10 mg and is administered on day 8 of the first cycle; (b) The second dosing cycle comprises a single dose of 30 mg glofitamab administered on day 1 of the second cycle (C2D1).

[0337] In some embodiments, the dosing regimen includes 6 to 15 additional dosing cycles (e.g., 6 to 10 additional dosing cycles (e.g., 6 additional dosing cycles, 7 additional dosing cycles, 8 additional dosing cycles, 9 additional dosing cycles, or 10 additional dosing cycles), or 11 to 15 additional dosing cycles (e.g., 11 additional dosing cycles, 12 additional dosing cycles, 13 additional dosing cycles, 14 additional dosing cycles, or 15 additional dosing cycles) after the second dosing cycle. In some embodiments, the additional dosing cycles are 21-day dosing cycles.

[0338] In one embodiment, the method comprises 1 to 10 additional dosing cycles (C3D1 through C12D1). In one such embodiment, the 1 to 10 additional dosing cycles (C3D1 through C12D1) comprise a single dose of 16 or 30 mg of glofitamab. In one embodiment, a single dose of the additional dosing cycles (C3D1 through C12D1) comprises 30 mg of glofitamab.

[0339] In one embodiment, a single dose of the additional dosing cycles (C3D1 through C12D1) is administered on day 1 of each additional dosing cycle.

[0340] Accordingly, in one aspect, the present invention provides a method of treating a subject having a CD20-positive B-cell proliferative disorder comprising administering glofitamab to the subject in a dosing regimen comprising 2 to 12 cycles: (a) a first dosing cycle comprising a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 2.5 mg and C1D2 is 10 mg; (b) The second dosing cycle comprises a single dose of 16 or 30 mg glofitamab (C2D1 to C12D1). In one such embodiment, subsequent dosing cycles comprise a single dose of 30 mg glofitamab (C2D1 to C12D1).

[0341] Accordingly, in one aspect, the present invention provides a method of treating a subject having a CD20-positive B-cell proliferative disorder comprising administering glofitamab to the subject in a dosing regimen comprising 2 to 12 cycles: (a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 2.5 mg and is administered on day 1 of the first cycle, and C1D2 is 10 mg and is administered on day 8 of the first cycle; (b) Subsequent dosing cycles comprise a single dose of 16 or 30 mg glofitamab administered on day 1 of each subsequent cycle (C2D1 through C12D1).

[0342] In one such embodiment, subsequent dosing cycles comprise a single dose of 30 mg glofitamab administered on day 1 of each subsequent cycle (C2D1 through C12D1).

[0343] In one embodiment, the method includes a total of 12 dosing cycles. In one embodiment, one treatment cycle includes 14 days or 21 days. In one embodiment, one treatment cycle includes 21 days.

[0344] In one embodiment, the CD20-positive B-cell proliferative disorder is non-Hodgkin's lymphoma (NHL). In one embodiment, the NHL is diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), DLBCL arising from FL [transformed FL; trFL], primary mediastinal large B-cell lymphoma (PMBCL), or marginal zone lymphoma (MZL). MZL can be classified into splenic MZL, nodal MZL, and extranodal MZL. In one embodiment, the DLBCL is Richter's transformed. In one embodiment, the NHL is mantle cell lymphoma (MCL). In one embodiment, the NHL is grade 1-3a follicular lymphoma (FL). In one embodiment, the CD20-positive B-cell proliferative disorder is a relapsed or refractory B-cell proliferative disorder. In one embodiment, the relapsed or refractory B-cell proliferative disorder is relapsed or refractory NHL (e.g., relapsed or refractory DLBCL, relapsed or refractory FL, or relapsed or refractory MCL). In one embodiment, the NHL is indolent NHL (iNHL) or aggressive NHL (aNHL).

[0345] In one embodiment, the patient has relapsed after or failed to respond to at least two prior systemic treatment regimens, including at least one prior regimen containing an anthracycline and at least one prior regimen containing an anti-CD20 directed therapy.

[0346] In one embodiment, the patient with DLBCL has relapsed after or failed to respond to at least two prior systemic treatment regimens.

[0347] In one embodiment, patients with PMBCL and trFL have relapsed after or failed to respond to at least two prior systemic treatment regimens, including at least one prior regimen containing an anthracycline and at least one prior regimen containing an anti-CD20 directed therapy.

[0348] In one embodiment, patients with grade 1-3a FL have relapsed after or failed to respond to at least two prior lines of systemic therapy and have received prior treatment with rituximab and an alkylating agent.

[0349] In one embodiment, subjects with CLL, Burkitt's lymphoma, and lymphoplasmacytic lymphoma are excluded from the methods of treatment described above.

[0350] In one embodiment, a stepwise dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein effectively reduces cytokine release in a subject associated with administration of the anti-CD20 / anti-CD3 bispecific antibody compared to a corresponding treatment regimen without the stepwise dosing schedule. In one embodiment, cytokine release is reduced by at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to a corresponding treatment regimen without the stepwise dosing schedule. Cytokines can be detected by methods known in the art, such as, for example, ELISA, FACS, or Luminex® assay.

[0351] The cytokine can be detected, for example, in a blood sample taken from the subject. In one embodiment, the concentration of the cytokine is in the subject's blood. In some embodiments, the cytokine is one or more cytokines selected from the group consisting of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-2 (IL-2), and interleukin-8 (IL-8), particularly the group consisting of TNF-α, IFN-γ, and IL-6. In some embodiments, the cytokine is TNF-α. In some embodiments, the cytokine is IFN-γ. In some embodiments, the cytokine is IL-6. In some embodiments, the cytokine is IL-10. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is IL-8.

[0352] In some embodiments, the step-dosing schedules of anti-CD20 / anti-CD3 bispecific antibodies (e.g., glofitamab) provided herein increase the safety of the anti-CD20 / anti-CD3 bispecific antibodies (e.g., glofitamab) compared to a corresponding treatment regimen without the step-dosing regimen of anti-CD20 / anti-CD3 bispecific antibodies (e.g., glofitamab) (i.e., with a fixed dosing regimen of the invention). In some embodiments, the step-dosing schedules of anti-CD20 / anti-CD3 bispecific antibodies provided herein reduce adverse events in a subject compared to a corresponding treatment regimen without the step-dosing regimen of anti-CD20 / anti-CD3 bispecific antibodies. In some embodiments, the treatment regimen reduces the toxicity of the anti-CD20 / anti-CD3 bispecific antibodies compared to a corresponding treatment regimen without the step-dosing regimen of anti-CD20 / anti-CD3 bispecific antibodies.

[0353] In one embodiment, a population of subjects exhibit cytokine release syndrome after administration of an anti-CD20 / anti-CD3 bispecific antibody, and the rate of Grade 2 or higher cytokine release syndrome is about 30% or less. In one embodiment, a population of subjects exhibit cytokine release syndrome after administration of an anti-CD20 / anti-CD3 bispecific antibody, and the rate of Grade 2 cytokine release syndrome is about 12% or less. In one embodiment, the rate of subjects exhibiting Grade 3 or higher cytokine release syndrome is about 5% or less. In one embodiment, the rate of subjects exhibiting Grade 3 or higher cytokine release syndrome is about 3% or less. In one embodiment, the rate of subjects exhibiting Grade 3 or higher cytokine release syndrome is about 0% or less. In one embodiment, the grade of CRS is defined by the modified criteria of Lee et al. (Lee et al., Blood, 124:188-195, 2014) and / or the ASTCT consensus grading (criteria of the American Society for Transplantation and Cellular Therapy, 2019; ASTCT; Lee et al., Biol Blood Marrow Transplant, 25(4):625-638, 2019).

[0354] In one embodiment, the stepwise dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein reduces the frequency of Grade 2 or higher CRS compared to the rate of Grade 2 or higher CRS in a patient population treated with a corresponding therapeutic regimen without the stepwise dosing regimen of an anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the frequency of Grade 2 or higher CRS is about 45%, 50%, 55%, or 60% lower compared to the rate of Grade 2 or higher CRS observed in a patient population treated with a corresponding therapeutic regimen without the stepwise dosing regimen of an anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the grade of CRS is defined by the modified criteria of Lee et al. (2014) and / or the ASTCT consensus grading (criteria of the American Society for Transplantation and Cellular Therapy, 2019; ASTCT).

[0355] In one embodiment, a phased dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein results in an objective response rate of at least about 60% in a patient population. In one embodiment, a phased dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein results in an objective response rate of at least about 70% in a patient population. In one embodiment, a phased dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein results in a CRR of at least about 60% in a patient population.

[0356] In one embodiment, a phased dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein results in an objective response rate of at least about 60% in a patient population with aggressive B-NHL (DLBCL, trFL, PMBCL, MCL, Richter's transformation). In one embodiment, a phased dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein results in an objective response rate of at least about 70% in a patient population with aggressive B-NHL (DLBCL, trFL, PMBCL, MCL, Richter's transformation). In one embodiment, a phased dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein results in an objective response rate of at least about 65% in a patient population with grade 1-3A FL.

[0357] In one embodiment, a phased dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein results in a complete remission rate (CRR) of at least about 45% in a patient population with aggressive B-NHL (DLBCL, trFL, PMBCL, MCL, Richter's transformation). In one embodiment, a phased dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein results in a CRR of at least about 50% in a patient population with grade 1-3A FL.

[0358] In one embodiment, CR occurs by Cycle 3. In another embodiment, complete response (CR) occurs at the first or second response assessment (C3 or C6).

[0359] In one embodiment, a phased dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein results in a DOR of at least about 5.5 months in patients with aggressive NHL (DLBCL, trFL, PMBCL, MCL, Richter's transformation).

[0360] In one embodiment, a phased dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein results in a progression-free survival of at least 3 months, hi one embodiment, a phased dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein results in a progression-free survival rate of at least about 30% or about 34% at 6 months.

[0361] In one embodiment, a phased dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein results in a DOR of at least about 10 months in patients with grade 1 to 3A FL. In one embodiment, a phased dosing schedule of an anti-CD20 / anti-CD3 bispecific antibody provided herein results in a progression-free survival of at least about 11 months in patients with grade 1 to 3A FL.

[0362] For certain indications, extended phased dosing has been found to provide a favorable benefit-risk profile. In the extended phased dosing regimen provided herein, an initial low-dose glofitamab is administered on C1D1 and C1D8, followed by an intermediate dose in cycle 2, and the first administration of the target therapeutic dose in cycle 3. Alternatively, the intermediate dose may be administered in cycle 3, and the first target dose in cycle 4. Smaller dose increases at each phase may further improve the clinical benefit / risk of glofitamab by reducing the incidence and severity of CRS in certain indications, such as follicular lymphoma.

[0363] In one embodiment of the present invention, there is provided a method of treating a subject with diffuse large B-cell lymphoma (DLBCL), comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg and C1D2 is 10 mg of the anti-CD20 / anti-CD3 bispecific antibody; b) The second dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1).

[0364] In one embodiment, a single dose of the third dosing cycle (C2D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody.

[0365] In one embodiment, the first dose (C1D1) is administered on day 1 of the first dosing cycle and the second dose (C1D2) is administered on day 8 of the first dosing cycle. In one embodiment, the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle.

[0366] In one embodiment, a method is provided for treating a subject with diffuse large B-cell lymphoma (DLBCL), comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 1 of the first cycle and C1D2 is 10 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 8 of the first dosing cycle; b) The second dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1) administered on day 1 of the second dosing cycle.

[0367] In one embodiment, the method of treating DLBCL comprises 1 to 10 additional dosing cycles (C3D1 through C12D1). In one embodiment, the 1 to 10 additional dosing cycles (C3D1 through C12D1) comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) is administered on day 1 of each additional dosing cycle.

[0368] In one embodiment, a method of treating a subject with diffuse large B-cell lymphoma (DLBCL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising 2 to 12 dosing cycles: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody; b) Subsequent dosing cycles include a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1).

[0369] In one such embodiment, the subsequent dosing cycle comprises a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1). In one embodiment, a method of treating a subject with diffuse large B-cell lymphoma (DLBCL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising 2 to 12 dosing cycles: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 1 of the first cycle and C1D2 is 10 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 8 of the first cycle; b) Subsequent dosing cycles comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1) administered on day 1 of each subsequent cycle.

[0370] In one such embodiment, the subsequent dosing cycle comprises a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1).

[0371] In one embodiment, the method for treating DLBCL described above comprises a total of 12 dosing cycles. In one embodiment, the DLBCL is relapsed or refractory (R / R) DLBCL. In one embodiment, the DLBCL arises from FL, is transformed FL (trFL), or is Richter's transformed. In one embodiment, patients with R / R DLBCL have relapsed after or failed to respond to at least two prior lines of systemic therapy.

[0372] In one embodiment, one or more treatment cycles comprise 14 or 21 days. In one embodiment, one or more treatment cycles comprise 21 days.

[0373] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody c) at least one antigen-binding domain that specifically binds to CD20, comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable region comprising at least one antigen-binding domain that specifically binds to CD20, comprising: d) at least one antigen-binding domain that specifically binds to CD3, comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region comprising (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a light chain variable region comprising at least one antigen-binding domain that specifically binds to CD3, comprising Includes.

[0374] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (i) at least one antigen-binding domain that specifically binds to CD20 comprising a heavy chain variable region sequence of SEQ ID NO: 7 and a light chain variable region sequence of SEQ ID NO: 8; and (ii) at least one antigen-binding domain that specifically binds to CD3, comprising the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16 Includes.

[0375] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises two binding sites for CD20 and one binding site for CD3. In one such embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises HVRs as defined above. In one such embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises VL and VH sequences as defined above.

[0376] In one embodiment of the present invention, there is provided a method of treating a subject with diffuse large B-cell lymphoma (DLBCL), comprising administering glofitamab to the subject in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg and C1D2 is 10 mg of glofitamab; b) The second dosing cycle comprises a single dose of 16 or 30 mg glofitamab (C2D1).

[0377] In one embodiment, a single dose of the second dosing cycle (C2D1) comprises 30 mg of glofitamab.

[0378] In one embodiment, the first dose (C1D1) of glofitamab is administered on day 1 of the first dosing cycle and the second dose (C1D2) of glofitamab is administered on day 8 of the first dosing cycle. In one embodiment, the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle.

[0379] In one embodiment, a method of treating a subject having diffuse large B-cell lymphoma (DLBCL) is provided, comprising administering glofitamab to the subject in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 2.5 mg and is administered on day 1 of the first cycle, and C1D2 is 10 mg of glofitamab and is administered on day 8 of the first dosing cycle; b) The second dosing cycle comprises a single dose of 16 or 30 mg glofitamab (C2D1) administered on day 1 of the second dosing cycle.

[0380] In one embodiment, the method comprises 1 to 10 additional dosing cycles (C3D1 through C12D1). In one embodiment, the 1 to 10 additional dosing cycles (C3D1 through C12D1) comprise a single dose of 16 or 30 mg of glofitamab. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) comprises 30 mg of glofitamab. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) is administered on day 1 of each additional dosing cycle.

[0381] In one embodiment, a method of treating a subject having diffuse large B-cell lymphoma (DLBCL) is provided, comprising administering glofitamab to the subject in a dosing regimen comprising 2 to 12 dosing cycles: a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 2.5 mg and C1D2 is 10 mg of glofitamab; b) Subsequent dosing cycles include a single dose of 16 or 30 mg glofitamab (C2D1 to C12D1).

[0382] In one such embodiment, subsequent dosing cycles comprise a single dose of 30 mg glofitamab (C2D1 to C12D1).

[0383] In one embodiment, a method of treating a subject having diffuse large B-cell lymphoma (DLBCL) is provided, comprising administering glofitamab to the subject in a dosing regimen comprising 2 to 12 dosing cycles: a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 2.5 mg of glofitamab administered on day 1 of the first cycle and C1D2 is 10 mg of glofitamab administered on day 8 of the first cycle; b) Subsequent dosing cycles comprise a single dose of 16 or 30 mg glofitamab (C2D1 through C12D1) administered on day 1 of the second cycle of each subsequent cycle.

[0384] In one such embodiment, subsequent dosing cycles comprise a single dose of 30 mg glofitamab (C2D1 to C12D1).

[0385] In one embodiment of the present invention, there is provided a method of treating a subject with follicular lymphoma (FL), comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg and C1D2 is 10 mg of the anti-CD20 / anti-CD3 bispecific antibody; b) The second dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1).

[0386] In one embodiment, a single dose of the third dosing cycle (C2D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody.

[0387] In one embodiment, the first dose (C1D1) is administered on day 1 of the first dosing cycle and the second dose (C1D2) is administered on day 8 of the first dosing cycle. In one embodiment, the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle.

[0388] In one embodiment, a method of treating a subject with follicular lymphoma (FL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 1 of the first cycle and C1D2 is 10 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 8 of the first dosing cycle; b) The second dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1) administered on day 1 of the second dosing cycle.

[0389] In one embodiment, the method of treating FL comprises 1 to 10 additional dosing cycles (C3D1 through C12D1). In one embodiment, the 1 to 10 additional dosing cycles (C3D1 through C12D1) comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) is administered on day 1 of each additional dosing cycle.

[0390] In one embodiment, a method of treating a subject with follicular lymphoma (FL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising 2 to 12 dosing cycles: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody; b) Subsequent dosing cycles include a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1).

[0391] In one such embodiment, the subsequent dosing cycle comprises a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1).

[0392] In one embodiment, a method of treating a subject with follicular lymphoma (FL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising 2 to 12 dosing cycles: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 1 of the first cycle and C1D2 is 10 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 8 of the first cycle; b) Subsequent dosing cycles comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1) administered on day 1 of each subsequent cycle.

[0393] In one such embodiment, the subsequent dosing cycle comprises a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1).

[0394] In one embodiment, the method for treating FL described above comprises a total of 12 dosing cycles. In one embodiment, the FL is relapsed or refractory (R / R) FL. In one embodiment, the FL is grade 1, 2, or 3a FL. In one embodiment, patients with grade 1-3a FL have relapsed after or failed to respond to at least two prior lines of systemic therapy and have received prior treatment with rituximab and an alkylating agent. In one embodiment, the treated subject has a FLIPI risk score of ≥3.

[0395] In one embodiment, subjects with CLL, Burkitt's lymphoma, and lymphoplasmacytic lymphoma are excluded from the methods of treatment described above.

[0396] In one embodiment, the FL is transformed FL. In one embodiment, a patient with trFL has relapsed after or failed to respond to at least two prior systemic treatment regimens, including at least one prior regimen containing an anthracycline and at least one prior regimen containing an anti-CD20-directed therapy. In one embodiment, the subject: (a) has relapsed after at least two prior therapies or is refractory to at least two prior therapies; (b) relapsed after treatment with a phosphoinositide 3-kinase (PI3K) inhibitor or was resistant to treatment with a phosphoinositide 3-kinase (PI3K) inhibitor; (c) experiencing disease progression within 24 months of frontline treatment; and / or (d) The sum of the products of the lesion diameters is ≥ 3,000 mm 2 have a lesion that is Being a high-risk subject.

[0397] In one embodiment, one or more treatment cycles comprise 14 or 21 days. In one embodiment, one or more treatment cycles comprise 21 days.

[0398] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody e) at least one antigen-binding domain that specifically binds to CD20, comprising: (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region comprising (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable region comprising at least one antigen-binding domain that specifically binds to CD20, comprising: f) at least one antigen-binding domain that specifically binds to CD3, comprising: (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region comprising (vii) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (viii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (ix) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a light chain variable region comprising at least one antigen-binding domain that specifically binds to CD3, comprising Includes.

[0399] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (iii) at least one antigen-binding domain that specifically binds to CD20 comprising the heavy chain variable region sequence of SEQ ID NO: 7 and the light chain variable region sequence of SEQ ID NO: 8; and (iv) at least one antigen-binding domain that specifically binds to CD3, comprising the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16 Includes.

[0400] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises two binding sites for CD20 and one binding site for CD3. In one such embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises HVRs as defined above. In one such embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises VL and VH sequences as defined above.

[0401] In one embodiment of the present invention, there is provided a method of treating a subject with follicular lymphoma (FL), comprising administering glofitamab to the subject in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg and C1D2 is 10 mg of glofitamab; b) The second dosing cycle comprises a single dose of 16 or 30 mg glofitamab (C2D1).

[0402] In one embodiment, a single dose of the second dosing cycle (C2D1) comprises 30 mg of glofitamab.

[0403] In one embodiment, the first dose (C1D1) of glofitamab is administered on day 1 of the first dosing cycle and the second dose (C1D2) of glofitamab is administered on day 8 of the first dosing cycle. In one embodiment, the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle.

[0404] In one embodiment, a method of treating a subject having follicular lymphoma (FL) is provided, comprising administering glofitamab to the subject in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 2.5 mg and is administered on day 1 of the first cycle, and C1D2 is 10 mg of glofitamab and is administered on day 8 of the first dosing cycle; b) The second dosing cycle comprises a single dose of 16 or 30 mg glofitamab (C2D1) administered on day 1 of the second dosing cycle.

[0405] In one embodiment, the method comprises 1 to 10 additional dosing cycles (C3D1 through C12D1). In one embodiment, the 1 to 10 additional dosing cycles (C3D1 through C12D1) comprise a single dose of 16 or 30 mg of glofitamab. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) comprises 30 mg of glofitamab. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) is administered on day 1 of each additional dosing cycle.

[0406] In one embodiment, a method of treating a subject with follicular lymphoma (FL) is provided, comprising administering glofitamab to the subject in a dosing regimen comprising 2 to 12 dosing cycles: a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 2.5 mg and C1D2 is 10 mg of glofitamab; b) Subsequent dosing cycles include a single dose of 16 or 30 mg glofitamab (C2D1 to C12D1).

[0407] In one such embodiment, subsequent dosing cycles comprise a single dose of 30 mg glofitamab (C2D1 to C12D1).

[0408] In one embodiment, a method of treating a subject with follicular lymphoma (FL) is provided, comprising administering glofitamab to the subject in a dosing regimen comprising 2 to 12 dosing cycles: a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 2.5 mg of glofitamab administered on day 1 of the first cycle and C1D2 is 10 mg of glofitamab administered on day 8 of the first cycle; b) Subsequent dosing cycles comprise a single dose of 16 or 30 mg glofitamab (C2D1 through C12D1) administered on day 1 of the second cycle of each subsequent cycle.

[0409] In one such embodiment, subsequent dosing cycles comprise a single dose of 30 mg glofitamab (C2D1 to C12D1).

[0410] In another aspect of the invention, there is provided a method of treating a subject with follicular lymphoma (FL), comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody; b) the second dosing cycle comprises a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1); c) The third dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1).

[0411] In one embodiment, a single dose of the third dosing cycle (C3D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody.

[0412] In one embodiment, the first dose (C1D1) is administered on day 1 of the first dosing cycle and the second dose (C1D2) is administered on day 8 of the first dosing cycle. In one embodiment, the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle. In one embodiment, the single dose of the third dosing cycle (C3D1) is administered on day 1 of the third dosing cycle.

[0413] In one embodiment, a method of treating a subject with follicular lymphoma (FL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg of the anti-CD20 / anti-CD3 bispecific antibody and is administered on day 1 of the first cycle, and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody and is administered on day 8 of the first dosing cycle; b) the second dosing cycle comprises a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1) administered on day 1 of the second dosing cycle; c) The third dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1) administered on day 1 of the third dosing cycle.

[0414] In one embodiment, the method of treating FL comprises 1 to 9 additional dosing cycles (C4D1 through C12D1). In one embodiment, the 1 to 9 additional dosing cycles (C4D1 through C12D1) comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C4D1 through C12D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C4D1 through C12D1) is administered on day 1 of each additional dosing cycle.

[0415] In one embodiment, a method of treating a subject with follicular lymphoma (FL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising 3 to 12 dosing cycles: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody; b) the second dosing cycle comprises a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1); c) Subsequent dosing cycles include a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1 through C12D1).

[0416] In one such embodiment, the subsequent dosing cycle comprises a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1 through C12D1).

[0417] In one embodiment, a method of treating a subject with follicular lymphoma (FL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising 3 to 12 dosing cycles: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 1 of the first cycle and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 8 of the first cycle; b) the second dosing cycle comprises a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1) administered on day 1 of the second cycle; c) Subsequent dosing cycles comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1 through C12D1) administered on day 1 of each subsequent cycle.

[0418] In one such embodiment, the subsequent dosing cycle comprises a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1 through C12D1).

[0419] In one embodiment, the method for treating FL described above comprises a total of 12 dosing cycles. In one embodiment, the FL is relapsed or refractory (R / R) FL. In one embodiment, the FL is grade 1, 2, or 3a FL. In one embodiment, patients with grade 1-3a FL have relapsed after or failed to respond to at least two prior lines of systemic therapy and have received prior treatment with rituximab and an alkylating agent. In one embodiment, the treated subject has a FLIPI risk score of ≥3.

[0420] In one embodiment, subjects with CLL, Burkitt's lymphoma, and lymphoplasmacytic lymphoma are excluded from the methods of treatment described above.

[0421] In one embodiment, the FL is transformed FL. In one embodiment, a patient with trFL has relapsed after or failed to respond to at least two prior systemic treatment regimens, including at least one prior regimen containing an anthracycline and at least one prior regimen containing an anti-CD20-directed therapy.

[0422] In one embodiment, the subject: (a) has relapsed after at least two prior therapies or is refractory to at least two prior therapies; (b) relapsed after treatment with a phosphoinositide 3-kinase (PI3K) inhibitor or was resistant to treatment with a phosphoinositide 3-kinase (PI3K) inhibitor; (c) experiencing disease progression within 24 months of frontline treatment; and / or (d) The sum of the products of the lesion diameters is ≥ 3,000 mm 2 have a lesion that is Being a high-risk subject.

[0423] In one embodiment, one or more treatment cycles comprise 14 or 21 days. In one embodiment, one or more treatment cycles comprise 21 days.

[0424] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody a) at least one antigen-binding domain that specifically binds to CD20, comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable region comprising at least one antigen-binding domain that specifically binds to CD20, comprising: b) at least one antigen-binding domain that specifically binds to CD3, comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a light chain variable region comprising at least one antigen-binding domain that specifically binds to CD3, comprising Includes.

[0425] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (i) at least one antigen-binding domain that specifically binds to CD20 comprising a heavy chain variable region sequence of SEQ ID NO: 7 and a light chain variable region sequence of SEQ ID NO: 8; and (ii) at least one antigen-binding domain that specifically binds to CD3, comprising the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16 Includes.

[0426] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises two binding sites for CD20 and one binding site for CD3. In one such embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises HVRs as defined above. In one such embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises VL and VH sequences as defined above.

[0427] In another aspect of the invention, there is provided a method of treating a subject having follicular lymphoma (FL), comprising administering glofitamab to the subject in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg and C1D2 is 2.5 mg of glofitamab; b) the second dosing cycle comprises a single dose of 10 mg glofitamab (C2D1); c) The third dosing cycle comprises a single dose of 16 or 30 mg glofitamab (C3D1).

[0428] In one embodiment, a single dose of the third dosing cycle (C3D1) comprises 30 mg of glofitamab.

[0429] In one embodiment, the first dose (C1D1) of glofitamab is administered on day 1 of the first dosing cycle and the second dose (C1D2) of glofitamab is administered on day 8 of the first dosing cycle. In one embodiment, the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle. In one embodiment, the single dose of the third dosing cycle (C3D1) is administered on day 1 of the third dosing cycle.

[0430] In one embodiment, a method of treating a subject having follicular lymphoma (FL) is provided, comprising administering glofitamab to the subject in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 0.5 mg and is administered on day 1 of the first cycle, and C1D2 is 2.5 mg of glofitamab and is administered on day 8 of the first dosing cycle; b) the second dosing cycle comprises a single dose of 10 mg glofitamab (C2D1) administered on day 1 of the second dosing cycle; c) The third dosing cycle comprises a single dose of 16 or 30 mg glofitamab (C3D1) administered on day 1 of the third dosing cycle.

[0431] In one embodiment, the method includes 1 to 9 (C4D1 through C12D1) additional dosing cycles. In one embodiment, 1 to 9 additional dosing cycles (C4D1 through C12D1) include a single dose of 16 or 30 mg of glofitamab. In one embodiment, a single dose of the additional dosing cycles (C4D1 through C12D1) includes 30 mg of glofitamab. In one embodiment, a single dose of the additional dosing cycles (C4D1 through C12D1) is administered on day 1 of each additional dosing cycle.

[0432] In one embodiment, a method of treating a subject with follicular lymphoma (FL) is provided, comprising administering glofitamab to the subject in a dosing regimen comprising 3 to 12 dosing cycles: a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 0.5 mg and C1D2 is 2.5 mg of glofitamab; b) the second dosing cycle comprises a single dose of 10 mg glofitamab (C2D1); c) Subsequent dosing cycles include a single dose of 16 or 30 mg glofitamab (C3D1 to C12D1).

[0433] In one such embodiment, subsequent dosing cycles comprise a single dose of 30 mg glofitamab (C3D1 through C12D1). In one embodiment, a method of treating a subject with follicular lymphoma (FL) is provided, comprising administering glofitamab to the subject in a dosing regimen comprising 3 to 12 dosing cycles: a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 0.5 mg of glofitamab administered on day 1 of the first cycle and C1D2 is 2.5 mg of glofitamab administered on day 8 of the first cycle; b) the second dosing cycle comprises a single 10 mg glofitamab administered on day 1 of the second cycle (C2D1); c) Subsequent dosing cycles include a single dose of 16 or 30 mg glofitamab administered on day 1 of each subsequent cycle (C3D1 through C12D1).

[0434] In one such embodiment, subsequent dosing cycles comprise a single dose of 30 mg glofitamab (C3D1 through C12D1).

[0435] In one embodiment, the method for treating FL described above comprises a total of 12 dosing cycles. In one embodiment, the FL is relapsed or refractory (R / R) FL. In one embodiment, the FL is grade 1, 2, or 3a FL. In one embodiment, patients with grade 1-3a FL have relapsed after or failed to respond to at least two prior lines of systemic therapy and have received prior treatment with rituximab and an alkylating agent. In one embodiment, the treated subject has a FLIPI risk score of ≥3.

[0436] In one embodiment, the subject: (a) has relapsed after at least two prior therapies or is refractory to at least two prior therapies; (b) relapsed after treatment with a phosphoinositide 3-kinase (PI3K) inhibitor or was resistant to treatment with a phosphoinositide 3-kinase (PI3K) inhibitor; (c) experiencing disease progression within 24 months of frontline treatment; and / or (d) The sum of the products of the lesion diameters is ≥ 3,000 mm 2 have a lesion that is Being a high-risk subject.

[0437] In one embodiment, subjects with CLL, Burkitt's lymphoma, and lymphoplasmacytic lymphoma are excluded from the methods of treatment described above.

[0438] In one embodiment, the FL is transformed FL. In one embodiment, a patient with trFL has relapsed after or failed to respond to at least two prior systemic treatment regimens, including at least one prior regimen containing an anthracycline and at least one prior regimen containing an anti-CD20-directed therapy.

[0439] In one embodiment, one or more treatment cycles comprise 14 or 21 days. In one embodiment, one or more treatment cycles comprise 21 days.

[0440] Mantle cell lymphoma (MCL) is a relatively rare and incurable B-cell lymphoma where areas of high unmet need include relapsed or refractory (r / r) patients previously treated in the BTK inhibitor setting and previously untreated patients with high-risk disease. To date, relapsed patients have been treated with rituximab-based therapy or CAR-T therapy, with other targeted therapies proving to be of limited use.

[0441] Patients with MCL after BTKi have a poor prognosis due to the aggressive nature of the disease and the lack of curative treatment options. Median survival with available systemic therapies is 6-12 months, with an ORR of ~26%. Therefore, there is currently an urgent unmet medical need for improved methods of treating patients with MCL. Glofitamab monotherapy for MCL offers an off-the-shelf, fixed-duration regimen that is preferable compared to systemic therapies such as rituximab-based and CAR-T therapy.

[0442] In one embodiment of the present invention, there is provided a method of treating a subject with mantle cell lymphoma (MCL), comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg and C1D2 is 10 mg of the anti-CD20 / anti-CD3 bispecific antibody; b) The second dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1).

[0443] In one embodiment, a single dose of the second dosing cycle (C2D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody.

[0444] In one embodiment, the first dose of the anti-CD20 / anti-CD3 bispecific antibody (C1D1) is administered on day 1 of the first dosing cycle, and the second dose of the anti-CD20 / anti-CD3 bispecific antibody (C1D2) is administered on day 8 of the first dosing cycle. In one embodiment, the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle.

[0445] In one embodiment, a method of treating a subject having mantle cell lymphoma (MCL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 1 of the first cycle and C1D2 is 10 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 8 of the first dosing cycle; b) The second dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1) administered on day 1 of the second dosing cycle.

[0446] In one embodiment, the second dosing cycle of b) comprises a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1) administered on day 1 of the second dosing cycle.

[0447] In one embodiment, the method comprises 1 to 10 additional dosing cycles (C3D1 through C12D1). In one embodiment, the 1 to 10 additional dosing cycles (C3D1 through C12D1) comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) is administered on day 1 of each additional dosing cycle.

[0448] In one embodiment, a method of treating a subject having mantle cell lymphoma (MCL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising 2 to 12 dosing cycles: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg and C1D2 is 10 mg of the anti-CD20 / anti-CD3 bispecific antibody; b) Subsequent dosing cycles include a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1).

[0449] In one such embodiment, the subsequent dosing cycle comprises a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1).

[0450] In one embodiment, a method of treating a subject having mantle cell lymphoma (MCL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising 2 to 12 dosing cycles: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 1 of the first cycle and C1D2 is 10 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 8 of the first cycle; b) Subsequent dosing cycles comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1) administered on day 1 of the second cycle of each subsequent cycle.

[0451] In one such embodiment, the subsequent dosing cycle comprises a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1 through C12D1).

[0452] In one embodiment, the method for treating MCL described above comprises a total of 12 dosing cycles. In one embodiment, the method for treating MCL described above comprises a total of 6, 7, 8, 9, or 10 dosing cycles. In one embodiment, the MCL is relapsed or refractory (R / R) MCL. In one embodiment, an R / R MCL patient has relapsed after or failed to respond to at least two prior lines of systemic therapy. In one embodiment, the subject has received at least one prior systemic treatment regimen comprising a Bruton's tyrosine kinase inhibitor (BTKi). In one embodiment, the BTKi comprises ibrutinib, acalabrutinib, or zanubrutinib.

[0453] In one embodiment, one or more treatment cycles comprise 14 or 21 days. In one embodiment, one or more treatment cycles comprise 21 days.

[0454] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody for the method of treating MCL comprises: a) at least one antigen-binding domain that specifically binds to CD20, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable region comprising at least one antigen-binding domain that specifically binds to CD20, comprising: b) at least one antigen-binding domain that specifically binds to CD3, comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a light chain variable region comprising at least one antigen-binding domain that specifically binds to CD3, comprising Includes.

[0455] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody for the method of treating MCL comprises: (i) at least one antigen-binding domain that specifically binds to CD20 comprising a heavy chain variable region sequence of SEQ ID NO: 7 and a light chain variable region sequence of SEQ ID NO: 8; and (ii) at least one antigen-binding domain that specifically binds to CD3, comprising the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16 Includes.

[0456] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises two binding sites for CD20 and one binding site for CD3. In one such embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises HVRs as defined above. In one such embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises VL and VH sequences as defined above.

[0457] In one embodiment of the present invention, a method of treating a subject having mantle cell lymphoma (MCL) is provided, comprising administering glofitamab to the subject in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 2.5 mg and C1D2 is 10 mg of glofitamab; b) The second dosing cycle comprises a single dose of 16 or 30 mg glofitamab (C2D1).

[0458] In one embodiment, a single dose of the second dosing cycle (C2D1) comprises 30 mg of glofitamab.

[0459] In one embodiment, the first dose (C1D1) of glofitamab is administered on day 1 of the first dosing cycle and the second dose (C1D2) of glofitamab is administered on day 8 of the first dosing cycle. In one embodiment, the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle.

[0460] In one embodiment, a method of treating a subject having mantle cell lymphoma (MCL) is provided, comprising administering glofitamab to the subject in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 2.5 mg and is administered on day 1 of the first cycle, and C1D2 is 10 mg of glofitamab and is administered on day 8 of the first dosing cycle; b) The second dosing cycle comprises a single dose of 16 or 30 mg glofitamab (C2D1) administered on day 1 of the second dosing cycle.

[0461] In one embodiment, the method comprises 1 to 10 additional dosing cycles (C3D1 through C12D1). In one embodiment, the 1 to 10 additional dosing cycles (C3D1 through C12D1) comprise a single dose of 16 or 30 mg of glofitamab. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) comprises 30 mg of glofitamab. In one embodiment, the single dose of the additional dosing cycles (C3D1 through C12D1) is administered on day 1 of each additional dosing cycle.

[0462] In one embodiment, a method of treating a subject having mantle cell lymphoma (MCL) is provided, comprising administering glofitamab to the subject in a dosing regimen comprising 2 to 12 dosing cycles: a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 2.5 mg and C1D2 is 10 mg of glofitamab; b) Subsequent dosing cycles include a single dose of 16 or 30 mg glofitamab (C2D1 to C12D1).

[0463] In one such embodiment, subsequent dosing cycles comprise a single dose of 30 mg glofitamab (C2D1 to C12D1).

[0464] In one embodiment, a method of treating a subject having mantle cell lymphoma (MCL) is provided, comprising administering glofitamab to the subject in a dosing regimen comprising 2 to 12 dosing cycles: a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of glofitamab, where C1D1 is 2.5 mg of glofitamab administered on day 1 of the first cycle and C1D2 is 10 mg of glofitamab administered on day 8 of the first cycle; b) Subsequent dosing cycles comprise a single dose of 16 or 30 mg glofitamab (C2D1 through C12D1) administered on day 1 of the second cycle of each subsequent cycle.

[0465] In one such embodiment, subsequent dosing cycles comprise a single dose of 30 mg glofitamab (C2D1 to C12D1).

[0466] In one embodiment, the method for treating MCL described above comprises a total of 12 dosing cycles. In one embodiment, the method for treating MCL described above comprises a total of 6, 7, 8, 9, or 10 dosing cycles.

[0467] In one embodiment, the MCL is relapsed or refractory (R / R) MCL. In one embodiment, patients with R / R MCL have relapsed after or failed to respond to at least two prior lines of systemic therapy. In one embodiment, the subject has received at least one prior systemic treatment regimen comprising a Bruton's tyrosine kinase inhibitor (BTKi). In one embodiment, the BTKi comprises ibrutinib, acalabrutinib, or zanubrutinib.

[0468] In one embodiment, one or more treatment cycles comprise 14 or 21 days. In one embodiment, one or more treatment cycles comprise 21 days.

[0469] In another aspect of the invention, there is provided a method of treating a subject with mantle cell lymphoma (MCL), comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody; b) a second dosing cycle comprising a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1); c) The third dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1).

[0470] In one embodiment, a single dose of the third dosing cycle (C3D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody.

[0471] In one embodiment, the first dose (C1D1) is administered on day 1 of the first dosing cycle and the second dose (C1D2) is administered on day 8 of the first dosing cycle. In one embodiment, the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle. In one embodiment, the single dose of the third dosing cycle (C3D1) is administered on day 1 of the third dosing cycle.

[0472] In one embodiment, a method of treating a subject with mantle cell lymphoma (MCL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg of the anti-CD20 / anti-CD3 bispecific antibody and is administered on day 1 of the first cycle, and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody and is administered on day 8 of the first dosing cycle; b) a second dosing cycle comprising a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1) administered on day 1 of the second dosing cycle; c) The third dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1) administered on day 1 of the third dosing cycle.

[0473] In one embodiment, the method of treating MCL comprises 1 to 9 additional dosing cycles (C4D1 through C12D1). In one embodiment, the 1 to 9 additional dosing cycles (C4D1 through C12D1) comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C4D1 through C12D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody. In one embodiment, the single dose of the additional dosing cycles (C4D1 through C12D1) is administered on day 1 of each additional dosing cycle.

[0474] In one embodiment, a method of treating a subject having mantle cell lymphoma (MCL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising 3 to 12 dosing cycles: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody; b) a second dosing cycle comprising a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1); c) Subsequent dosing cycles include a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1 through C12D1).

[0475] In one such embodiment, the subsequent dosing cycle comprises a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1 through C12D1).

[0476] In one embodiment, a method of treating a subject having mantle cell lymphoma (MCL) is provided, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising 3 to 12 dosing cycles: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 1 of the first cycle and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody administered on day 8 of the first cycle; b) the second dosing cycle comprises a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1) administered on day 1 of the second cycle; c) Subsequent dosing cycles comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1 through C12D1) administered on day 1 of each subsequent cycle.

[0477] In one such embodiment, the subsequent dosing cycle comprises a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1 through C12D1).

[0478] In one embodiment, the method for treating MCL described above comprises a total of 12 dosing cycles. In one embodiment, the MCL is relapsed or refractory (R / R) MCL. In one embodiment, patients with R / R MCL have relapsed after or failed to respond to at least two prior lines of systemic therapy. In one embodiment, the subject has received at least one prior systemic treatment regimen comprising a Bruton's tyrosine kinase inhibitor (BTKi). In one embodiment, the BTKi comprises ibrutinib, acalabrutinib, or zanubrutinib.

[0479] In one embodiment, one or more treatment cycles comprise 14 or 21 days. In one embodiment, one or more treatment cycles comprise 21 days.

[0480] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody a) at least one antigen-binding domain that specifically binds to CD20, (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable region comprising at least one antigen-binding domain that specifically binds to CD20, comprising: b) at least one antigen-binding domain that specifically binds to CD3, comprising: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region comprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a light chain variable region comprising at least one antigen-binding domain that specifically binds to CD3, comprising Includes.

[0481] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody (i) at least one antigen-binding domain that specifically binds to CD20 comprising a heavy chain variable region sequence of SEQ ID NO: 7 and a light chain variable region sequence of SEQ ID NO: 8; and (ii) at least one antigen-binding domain that specifically binds to CD3, comprising the heavy chain variable region sequence of SEQ ID NO: 15 and the light chain variable region sequence of SEQ ID NO: 16 Includes.

[0482] In one embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises two binding sites for CD20 and one binding site for CD3. In one such embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises HVRs as defined above. In one such embodiment, the anti-CD20 / anti-CD3 bispecific antibody comprises VL and VH sequences as defined above.

[0483] In another aspect of the invention, there is provided a method of treating a subject having mantle cell lymphoma (MCL), comprising administering glofitamab to the subject in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: a) a first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, where C1D1 is 0.5 mg and C1D2 is 2.5 mg of glofitamab; b) the second dosing cycle comprises a sing...

Claims

1. 1. A method of treating a subject having a CD20-positive B-cell proliferative disorder, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle, wherein: (a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, wherein C1D1 is 2.5 mg and C1D2 is 10 mg; and (b) the second dosing cycle comprises a single dose (C2D1) of 16 or 30 mg of the anti-CD20 / anti-CD3 bispecific antibody.

2. 10. The method of claim 1, wherein a single dose of the second dosing cycle comprises 30 mg of the anti-CD20 / anti-CD3 bispecific antibody.

3. 3. The method of claim 1 or 2, wherein the first dose (C1D1) is administered on day 1 of the first dosing cycle and the second dose (C1D2) is administered on day 8 of the first dosing cycle.

4. 4. The method of any one of claims 1 to 3, wherein the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle.

5. 5. The method of any one of claims 1 to 4, comprising 1 to 10 (C3D1 to C12D1) additional dosing cycles.

6. 6. The method of claim 5, wherein 1 to 10 additional dosing cycles (C3D1 to C12D1) comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody.

7. 7. The method of claim 5 or 6, wherein a single dose of the additional dosing cycles (C3D1 to C12D1) comprises 30 mg of anti-CD20 / anti-CD3 bispecific antibody.

8. 8. The method of any one of claims 5 to 7, wherein a single dose of each additional dosing cycle (C3D1 through C12D1) is administered on day 1 of each additional dosing cycle.

9. 9. The method of any one of claims 1 to 8, comprising a total of 12 dosing cycles.

10. 10. The method of any one of claims 1 to 9, wherein one treatment cycle comprises 14 or 21 days.

11. 11. The method of claim 10, wherein one treatment cycle comprises 21 days.

12. 12. The method of any one of claims 1 to 11, wherein the CD20-positive B-cell proliferative disorder is non-Hodgkin's lymphoma (NHL).

13. 13. The method of claim 12, wherein the B cell proliferative disorder is relapsed or refractory NHL.

14. 14. The method of claim 12 or 13, wherein the NHL is an indolent NHL (iNHL) or an aggressive NHL (aNHL).

15. 14. The method of claim 12 or 13, wherein the NHL is diffuse large B-cell lymphoma (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal large B-cell lymphoma (PMBCL), or marginal zone lymphoma (MZL).

16. The method of claim 15, wherein the DLBCL is Richter transformed.

17. 14. The method of claim 12 or 13, wherein the NHL is mantle cell lymphoma (MCL).

18. 18. The method of claim 17, wherein the MCL is relapsed and / or refractory (R / R) MCL.

19. 19. The method of claim 17 or 18, wherein the subject has received at least one prior systemic treatment regimen comprising a Bruton's tyrosine kinase inhibitor (BTKi).

20. 20. The method of claim 19, wherein the BTKi comprises ibrutinib, acalabrutinib, or zanubrutinib.

21. The method of claim 12 or 13, wherein the NHL is follicular lymphoma (FL).

22. 22. The method of claim 21, wherein the FL is grade 1, 2, or 3a FL.

23. The method of claim 21 or 22, wherein the FL is a transformed FL.

24. 24. The method of any one of claims 21 to 23, wherein the FL is relapsed or refractory (R / R) FL.

25. If the subject is: (a) has relapsed after at least two prior therapies or is refractory to at least two prior therapies; (b) relapsed after treatment with a phosphoinositide 3-kinase (PI3K) inhibitor or is resistant to treatment with a phosphoinositide 3-kinase (PI3K) inhibitor; (c) experiencing disease progression within 24 months of first-line treatment; and / or (d) The sum of the products of the lesion diameters is ≥ 3,000 mm ２ have a lesion that is 25. The method of any one of claims 21 to 24, in a high-risk subject.

26. 26. The method of any one of claims 1 to 25, wherein a population of subjects with a CD20-positive B-cell proliferative disorder exhibit cytokine release syndrome following administration of the bispecific antibody, and the rate of cytokine release syndrome of Grade 3 or higher (as defined by the American Society for Transplantation and Cellular Therapy, 2019; ASTCT) is about 5% or less.

27. 27. The method of any one of claims 1 to 26, wherein administering the anti-CD20 / anti-CD3 bispecific antibody to a plurality of subjects results in a complete remission rate of at least about 70%.

28. 15. The method of claim 14, wherein administering the anti-CD20 / anti-CD3 bispecific antibody to a plurality of subjects results in a complete remission rate of at least about 70% in subjects afflicted with iNHL.

29. 15. The method of claim 14, wherein administering the anti-CD20 / anti-CD3 bispecific antibody to a plurality of subjects results in a complete remission rate of at least about 70% in subjects afflicted with aNHL.

30. 21. The method of any one of claims 17 to 20, wherein administering the anti-CD20 / anti-CD3 bispecific antibody to a plurality of subjects results in an overall response rate of at least about 80%.

31. 21. The method of any one of claims 17 to 20, wherein administering the anti-CD20 / anti-CD3 bispecific antibody to a plurality of subjects results in a complete remission rate of at least about 65%.

32. 25. The method of any one of claims 21 to 24, wherein administering the anti-CD20 / anti-CD3 bispecific antibody to a plurality of subjects results in an overall response rate of at least about 80%.

33. 26. The method of claim 25, wherein administering the anti-CD20 / anti-CD3 bispecific antibody to a plurality of subjects results in a complete metabolic response rate of at least about 40%.

34. 1. A method of treating a subject with follicular lymphoma (FL), comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: (i) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, wherein C1D1 is 0.5 mg and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody; (ii) the second dosing cycle comprises a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1); (iii) the third dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1); method.

35. 35. The method of claim 34, wherein a single dose (C3D1) of the third dosing cycle comprises 30 mg of the anti-CD20 / anti-CD3 bispecific antibody.

36. 36. The method of claim 34 or 35, wherein the first dose (C1D1) is administered on day 1 of the first dosing cycle and the second dose (C1D2) is administered on day 8 of the first dosing cycle.

37. 37. The method of any one of claims 34 to 36, wherein the single dose of the second dosing cycle (C2D1) is administered on day 1 of the second dosing cycle.

38. 38. The method of any one of claims 34 to 37, wherein the single dose of the third dosing cycle (C3D1) is administered on day 1 of the third dosing cycle.

39. 39. The method of any one of claims 34 to 38, comprising 1 to 9 (C4D1 to C12D1) additional dosing cycles.

40. 40. The method of claim 39, wherein one to nine additional dosing cycles (C4D1 through C12D1) comprise a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody.

41. 41. The method of claim 39 or 40, wherein a single dose of the additional dosing cycles (C4D1 through C12D1) comprises 30 mg of the anti-CD20 / anti-CD3 bispecific antibody.

42. 42. The method of any one of claims 39-41, wherein a single dose of an additional dosing cycle (C4D1 through C12D1) is administered on day 1 of each additional dosing cycle.

43. 43. The method of any one of claims 34 to 42, comprising a total of 12 dosing cycles.

44. 44. The method of any one of claims 34 to 43, wherein one treatment cycle comprises 14 or 21 days.

45. 45. The method of claim 44, wherein one treatment cycle comprises 21 days.

46. 46. ​​The method of any one of claims 34 to 45, wherein the FL is grade 1, 2, or 3a FL.

47. 46. ​​The method of any one of claims 34 to 45, wherein the FL is a transformed FL.

48. 46. ​​The method of any one of claims 34 to 45, wherein the FL is relapsed or refractory (R / R) FL.

49. If the subject is: (a) has relapsed after at least two prior therapies or is refractory to at least two prior therapies; (b) relapsed after treatment with a phosphoinositide 3-kinase (PI3K) inhibitor or is resistant to treatment with a phosphoinositide 3-kinase (PI3K) inhibitor; (c) experiencing disease progression within 24 months of first-line treatment; and / or (d) The sum of the products of the lesion diameters is ≥ 3,000 mm ２ have a lesion that is 49. The method of claim 48, wherein the subject is at high risk.

50. 50. The method of any one of claims 34 to 49, wherein administering the anti-CD20 / anti-CD3 bispecific antibody to a plurality of subjects results in an overall response rate of at least about 80%.

51. 50. The method of claim 49, wherein the subjects are high-risk subjects with R / R FL, and wherein administering the anti-CD20 / anti-CD3 bispecific antibody to a plurality of subjects results in a complete remission rate of at least about 40%.

52. 52. The method of any one of claims 34 to 51, wherein a population of subjects with FL exhibit cytokine release syndrome after administration of the bispecific antibody, and the rate of cytokine release syndrome of Grade 3 or higher (as defined by the American Society for Transplantation and Cellular Therapy, 2019; ASTCT) is about 3%.

53. 53. The method of any one of claims 1 to 52, wherein the method of treatment is combined with administration of obinutuzumab or rituximab.

54. 54. The method of claim 53, wherein obinutuzumab is administered 7 days prior to the first dose (C1D1) of the anti-CD20 / anti-CD3 bispecific antibody.

55. 55. The method of claim 54, wherein obinutuzumab is administered in one single dose of 1000 mg.

56. 55. The method of claim 54, wherein obinutuzumab is administered in first and second doses of 1000 mg each of obinutuzumab.

57. 57. The method of claim 56, wherein the first and second doses of obinutuzumab are administered on the same day.

58. 58. The method of claim 56 or 57, wherein the subject has MCL and has received at least two prior systemic therapies.

59. 59. The method of any one of claims 53 to 58, wherein obinutuzumab or rituximab is administered on day 1 of the second cycle (C2D1) and on day 1 of the subsequent cycle.

60. 60. The method of claim 59, wherein obinutuzumab or rituximab is administered on day 1 of the second cycle (C2D1) and on days 1 of the third cycle (C3D1) through twelfth cycle (C12D1).

61. 60. The method of claim 58 or 59, wherein obinutuzumab is administered at a dose of 1000 mg.

62. 62. The method of any one of claims 1 to 61, wherein the patient is premedicated with corticosteroids prior to the anti-CD20 / anti-CD3 bispecific antibody.

63. 63. The method of claim 62, wherein the corticosteroid premedication comprises prednisolone and methylprednisolone, and / or dexamethasone.

64. 64. The method of claim 62 or 63, wherein the first dose (C1D1) of the anti-CD20 / anti-CD3 bispecific antibody is preceded by premedication with a corticosteroid.

65. 65. The method of any one of claims 1 to 64, wherein treatment is stopped after a total of 12 treatment cycles.

66. 66. The method of claim 65, wherein if relapse occurs and / or the disease progresses, the patient is retreated with the method of any one of claims 1 to 64.

67. A method of treating a subject having a CD20-positive cell proliferative disorder, comprising administering to the subject an anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds to CD20 and CD3.

68. 68. The method of claim 67, wherein administering to a plurality of humans an anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3 results in a complete remission in at least about 60%, at least about 70%, or at least about 80% of the plurality of humans after treatment with the anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3.

69. 69. The method of claim 67 or 68, wherein administering to a plurality of humans an anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3 results in an overall response in at least about 80%, at least about 85%, or at least about 90% of the plurality of humans after treatment with the anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3.

70. 70. The method of any one of claims 67-69, wherein administration of an anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds CD20 and CD3 to a human does not result in CRS of grade 2 or higher.

71. a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: (d) the first dosing cycle comprises an anti-CD20 antibody, cyclophosphamide, doxorubicin, and a first dose of a corticosteroid (C1D1), but no dose of a bispecific antibody; (e) a second dosing cycle comprises a second dose (C2D1) of an anti-CD20 antibody, cyclophosphamide, doxorubicin, and a corticosteroid, and a first dose (C2D8) and a second dose (C2D15) of a bispecific antibody, wherein the C2D8 of the bispecific antibody is about 2.5 mg and the C2D15 is about 10 mg; (f) a third dosing cycle comprises a third dose of an anti-CD20 antibody, cyclophosphamide, doxorubicin, and a corticosteroid (C3D1), and a third dose of a bispecific antibody (C3D8), wherein the bispecific antibody C3D8 is about 30 mg; 71. The method of any one of claims 67 to 70.

72. 72. The method of claim 71, wherein the anti-CD20 antibody, cyclophosphamide, doxorubicin, and corticosteroid are administered on day 1 of each dosing cycle.

73. 73. The method of claim 71 or 72, wherein a first dose (C2D8) of the bispecific antibody is administered on day 8 of the second dosing cycle and a second dose (C2D15) is administered on day 15 of the second dosing cycle.

74. 74. The method of any one of claims 71 to 73, wherein a third dose of the bispecific antibody (C3D8) is administered on day 8 of the third dosing cycle.

75. 75. The method of any one of claims 71 to 74, comprising 1 to 5 (C4 to C8) additional dosing cycles.

76. 76. The method of claim 75, wherein one to five additional dosing cycles (C4 to C8) comprise a single dose of anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a single dose of 30 mg of anti-CD20 / anti-CD3 bispecific antibody.

77. 77. The method of claim 75 or 76, wherein a single dose of anti-CD20 antibody, cyclophosphamide, doxorubicin, and corticosteroid is administered on day 1 and a single dose of anti-CD20 / anti-CD3 bispecific antibody is administered on day 8 of each additional dosing cycle (C4 through C8).

78. 78. The method of any one of claims 67 to 77, wherein the corticosteroid is prednisone and the anti-CD20 antibody is rituximab.

79. a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: (d) the first dosing cycle comprises a first dose (C1D1) of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) without a dose of a bispecific antibody; (e) a second dosing cycle comprises a second dose of R-CHOP (C2D1) and a first dose (C2D8) and a second dose (C2D15) of a bispecific antibody, wherein the C2D8 of the bispecific antibody is about 2.5 mg and the C2D15 is about 10 mg; (f) a third dosing cycle comprises a third dose of R-CHOP (C3D1) and a third dose of the bispecific antibody (C3D8), wherein the bispecific antibody C3D8 is about 30 mg; 79. The method of claim 78.

80. 80. The method of claim 79, wherein R-CHOP is administered on day 1 of each dosing cycle.

81. 81. The method of claim 79 or 80, wherein a first dose (C2D8) of the bispecific antibody is administered on day 8 of the second dosing cycle and a second dose (C2D15) is administered on day 15 of the second dosing cycle.

82. 82. The method of any one of claims 79 to 81, wherein a third dose of the bispecific antibody (C3D8) is administered on day 8 of the third dosing cycle.

83. 83. The method of any one of claims 79 to 82, comprising 1 to 5 (C4 to C8) additional dosing cycles.

84. 84. The method of claim 83, wherein one to five additional dosing cycles (C4 through C8) comprise a single dose of R-CHOP and a single dose of 30 mg of the anti-CD20 / anti-CD3 bispecific antibody.

85. 85. The method of claim 84, wherein a single dose of R-CHOP is administered on day 1 and a single dose of the anti-CD20 / anti-CD3 bispecific antibody is administered on day 8 of each additional dosing cycle (C4 through C8).

86. 86. The method of any one of claims 79-85, wherein rituximab in the first dosing cycle is replaced with obinutuzumab.

87. 87. The method of any one of claims 71 to 86, comprising a total of 6 dosing cycles.

88. 88. The method of any one of claims 71 to 87, wherein one treatment cycle comprises 14 or 21 days.

89. 89. The method of claim 88, wherein one treatment cycle comprises 21 days.

90. 90. The method of any one of claims 67 to 89, wherein the CD20-positive B-cell proliferative disorder is previously untreated DLBCL.

91. 91. The method of claim 90, wherein the subject to be treated has an International Prognostic Index [IPI] of 2 to 5.

92. 92. The method of any one of claims 1 to 91, wherein the anti-CD20 / anti-CD3 bispecific antibody is administered intravenously.

93. 93. The method of any one of claims 1 to 92, wherein the subject is a human.

94. 94. The method of claim 93, wherein the human is a high-risk subject.

95. The anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds to CD20, wherein the antigen-binding domain is (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3 a heavy chain variable region comprising (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5; and (v) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6 a light chain variable region comprising 95. The method of any one of claims 1 to 94, comprising:

96. 96. The method of any one of claims 1 to 95, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds to CD20, and the antigen-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.

97. The anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds to CD3, the antigen-binding domain comprising: (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 9; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 11 a heavy chain variable region comprising (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 13; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 14 a light chain variable region comprising 97. The method of any one of claims 1 to 96, comprising:

98. 98. The method of any one of claims 1 to 97, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises at least one antigen-binding domain that specifically binds to CD3, and the antigen-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.

99. 99. The method of any one of claims 1 to 98, wherein the anti-CD20 / anti-CD3 bispecific antibody is a cross-Fab molecule comprising an antigen-binding domain that specifically binds to CD3 and in which the variable or constant domains of the Fab heavy and light chains have been exchanged.

100. 100. The method of any one of claims 1 to 99, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises an IgG1 Fc domain comprising one or more amino acid substitutions that reduce binding to Fc receptors and / or decrease effector function.

101. 101. The method of any one of claims 1 to 100, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises an IgGl Fc domain comprising the amino acid substitutions L234A, L235A and P329G (numbering according to the Kabat EU index).

102. 102. The method of any one of claims 1 to 101, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises at least one Fab molecule comprising an antigen-binding domain that specifically binds to CD20, wherein in the constant domain CL of the Fab molecule the amino acid at position 124 is substituted by lysine (K) (Kabat numbering) and the amino acid at position 123 is substituted by arginine (R) or lysine (K) (Kabat numbering), and wherein in the constant domain CHI of the Fab molecule the amino acid at position 147 is substituted by glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is substituted by glutamic acid (E) (Kabat EU index numbering).

103. 103. The method of any one of claims 1 to 102, wherein the anti-CD20 / anti-CD3 bispecific antibody comprises two antigen-binding domains that specifically bind to CD20 and one antigen-binding domain that specifically binds to CD3.

104. 104. The method of any one of claims 1 to 103, wherein the anti-CD20 / anti-CD3 bispecific antibody is bivalent for CD20 and monovalent for CD3.

105. The anti-CD20 / anti-CD3 bispecific antibody (i) an antigen-binding domain that specifically binds to CD3 fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain; (ii) a first antigen-binding domain that specifically binds CD20 fused to the N-terminus of the Fab heavy chain of an antigen-binding domain that specifically binds CD3 at the C-terminus of the Fab heavy chain; (iii) a second antigen-binding domain that specifically binds to CD20 fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain; 105. The method of any one of claims 1 to 104, comprising:

106. 106. The method of any one of claims 1 to 105, wherein the anti-CD20 / anti-CD3 bispecific antibody is glofitamab.

107. 1. An anti-CD20 / anti-CD3 bispecific antibody for use in a method of treating a subject having a CD20-positive B-cell proliferative disorder, comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle, wherein: (a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of the anti-CD20 / anti-CD3 bispecific antibody, wherein C1D1 is 2.5 mg and C1D2 is 10 mg; and (b) the second dosing cycle comprises a single dose (C2D1) of 16 or 30 mg of the anti-CD20 / anti-CD3 bispecific antibody. Anti-CD20 / anti-CD3 bispecific antibody.

108. 1. An anti-CD20 / anti-CD3 bispecific antibody for use in a method of treating a subject with follicular lymphoma (FL), comprising administering to the subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: (i) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, wherein C1D1 is 0.5 mg and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody; (ii) the second dosing cycle comprises a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1); (iii) the third dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1); Anti-CD20 / anti-CD3 bispecific antibody.

109. 1. An anti-CD20 / anti-CD3 bispecific antibody for use in a method of treating a subject having a CD20-positive cell proliferative disorder, comprising administering to the subject an anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds to CD20 and CD3.

110. Use of an anti-CD20 / anti-CD3 bispecific antibody in the manufacture of a medicament for the treatment of a CD20-positive cell proliferative disorder, in a method comprising administering to a subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle and a second dosing cycle, wherein: (a) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of the anti-CD20 / anti-CD3 bispecific antibody, wherein C1D1 is 2.5 mg and C1D2 is 10 mg; and (b) the second dosing cycle comprises a single dose (C2D1) of 16 or 30 mg of the anti-CD20 / anti-CD3 bispecific antibody.

111. 1. Use of an anti-CD20 / anti-CD3 bispecific antibody in the manufacture of a medicament for the treatment of a CD20-positive cell proliferative disorder, in a method comprising administering to a subject an anti-CD20 / anti-CD3 bispecific antibody in a dosing regimen comprising at least a first dosing cycle, a second dosing cycle, and a third dosing cycle: (i) the first dosing cycle comprises a first dose (C1D1) and a second dose (C1D2) of an anti-CD20 / anti-CD3 bispecific antibody, wherein C1D1 is 0.5 mg and C1D2 is 2.5 mg of the anti-CD20 / anti-CD3 bispecific antibody; (ii) the second dosing cycle comprises a single dose of 10 mg of anti-CD20 / anti-CD3 bispecific antibody (C2D1); (iii) the third dosing cycle comprises a single dose of 16 or 30 mg of anti-CD20 / anti-CD3 bispecific antibody (C3D1); use.

112. Use of an anti-CD20 / anti-CD3 bispecific antibody in the manufacture of a medicament for the treatment of a CD20-positive cell proliferative disorder in a method comprising administering to a subject an anti-CD20 antibody, cyclophosphamide, doxorubicin, a corticosteroid, and a bispecific antibody that binds to CD20 and CD3.

113. 10. The invention as hereinbefore described.

Images