TWI890657B - Methods of using anti-cd79b immunoconjugates - Google Patents

Abstract

Provided herein are methods of treating B-cell proliferative disorders (such as Diffuse Large B-Cell Lymphoma “DLBCL”) using immunoconjugates comprising anti-CD79b antibodies in combination with an alkylating agent (such as bendamustine) and an anti-CD20 antibody (such as rituximab).

Description

Methods of using anti-CD79B immunoconjugates

The present invention relates to methods for treating B-cell proliferative disorders, such as diffuse large B-cell lymphoma (DLBCL), by administering an immunoconjugate comprising an anti-CD79b antibody in combination with an alkylating agent (e.g., bendamustine) and an anti-CD20 antibody (e.g., rituximab).

Diffuse large B-cell lymphoma (DLBCL) accounts for approximately 25% of all newly diagnosed non-Hodgkin lymphoma cases (Armitage et al., Journal of Clinical Oncology, 16:2780-95, 1998; Swerdlow et al., International Agency for Research on Cancer (IARC), Revised 4th Edition, 2017). 30-40% of patients are refractory to or relapse after treatment with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), the current standard of care (Vitolo et al., Journal of Clinical Oncology, 25:3529-37, 2017; Coiffier et al., New England Journal of Medicine, 346:235-42, 2002). Higher treatment failure rates have been observed in lower-risk subgroups, including activated B-cell-like (ABC) and MYC/BCL2 dual-expressing lymphomas (DEL) (Scott et al., Journal of Clinical Oncology, 33:2848-56, 2015; Johnson et al., Journal of Clinical Oncology, 30:3452-59, 2012). For patients with relapsed/refractory (R/R) disease, platinum salvage therapy followed by high-dose chemotherapy and autologous stem cell transplantation (ASCT) can cure up to 30-40% of patients who are able to undergo this treatment (Gisselbrecht et al., Journal of Clinical Oncology, 28:4184-90, 2010; Crump et al., Blood, 130:1800-08, 2017). However, for most patients with R/R DLBCL who are not candidates for ASCT due to age, comorbidities, or inadequate response to salvage chemotherapy, and for those who relapse after ASCT, the prognosis is poor, with a median overall survival (OS) of approximately 6 months (Czuczman et al., Clinical Cancer Research, 23:4127-37, 2017). There is no standard of care in this setting.

Recently, CD19-directed chimeric antigen receptor (CAR)-T cell therapy was approved for use in the third-line setting or later in the United States and the European Union (Neelapu et al., New England Journal of Medicine, 377:2531-44, 2017; Schuster et al., Blood, 130:577, 2017). Despite its promise, widespread use of CAR-T cell therapy has been limited by a lack of effective bridge therapies, treatment toxicities, and access limitations due to high costs and the need for specialized centers. Consequently, significant unmet medical needs remain for patients with R/R DLBCL who are ineligible for transplantation, including those who have failed ASCT. The present invention addresses this and other needs.

Provided herein are methods and uses of anti-CD79b immunoconjugates for treating B-cell proliferative disorders in individuals (e.g., human individuals) in need thereof. Specifically, these methods and uses are based on data from a randomized Phase II clinical study of polatuxumab vedotin with an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody (e.g., rituximab) in patients with unspecified DLBCL who had received at least one prior therapy for DLBCL. The study demonstrated that treatment with a combination of an anti-CD79b immunoconjugate (e.g., polatuzumab vedotin), an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl), and an anti-CD20 antibody (e.g., rituximab) reduced the risk of disease worsening or death (PFS) compared to treatment with an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody (e.g., rituximab) without an anti-CD79b immunoconjugate. Additionally, patients who received an anti-CD79b immunoconjugate (e.g., polatuzumab vedotin), an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl), and an anti-CD20 antibody (e.g., rituximab) demonstrated a statistically significant improvement in overall survival compared to patients who received only an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody (e.g., rituximab). The safety profile of the combination of an anti-CD79b immunoconjugate (e.g., polatuzumab vedotin), an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl), and an anti-CD20 antibody (e.g., rituximab) appeared consistent with the known safety profiles of the individual agents, and no new safety signals were identified for the combination.

Provided herein are methods for treating diffuse large B-cell lymphoma (DLBCL) in a human in need thereof, comprising administering to the human an effective amount of: (a) an immunoconjugate comprising the formula wherein Ab is an anti-CD79b antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, and wherein p is 1 to 8, b) an alkylating agent, and (c) an anti-CD20 antibody, wherein the treatment prolongs progression-free survival (PFS) in the subject. In some embodiments, the treatment prolongs overall survival (OS) in the subject.

The present invention provides a method for treating diffuse large B-cell lymphoma (DLBCL) in a human in need thereof, comprising administering to the human an effective amount of: (a) an immunoconjugate comprising the formula wherein Ab is an anti-CD79b antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, and wherein p is 1 to 8, (b) an alkylating agent, and (c) an anti-CD20 antibody, wherein the treatment prolongs overall survival (OS) in humans.

In some embodiments, the human achieves a complete response (CR) following treatment with an immunoconjugate, an alkylating agent, and an anti-CD20 antibody. In some embodiments, the anti-CD79 antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 19; and (ii) a VL comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-CD79 antibody comprises (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the immunoconjugate is polatuzumab vedotin. In some embodiments, the alkylating agent is 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid or a salt thereof. In some embodiments, the alkylating agent is bendamustine or a salt thereof. In some embodiments, the alkylating agent is bendamustine-HCl. In some embodiments, the anti-CD20 antibody is rituximab, a humanized B-Ly1 antibody (e.g., atuzumab), ofatumumab, ublituximab, and/or ibritumomab tiuxetan.

In some embodiments, the immunoconjugate is administered at a dose of 1.8 mg/kg, the alkylating agent is administered at a dose of 90 mg/m ² , and the anti-CD20 antibody is administered at a dose of 375 mg/m ² . In some embodiments, the immune conjugate, the alkylating agent, and the anti-CD20 antibody are administered for at least six 21-day cycles, wherein in the 21-day cycle of cycle 1, the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on day 2, the alkylating agent is administered intravenously at a dose of 90 mg/m ² on days 2 and 3, and the anti-CD20 antibody is administered intravenously at a dose of 375 mg/m ² on day 1, and wherein in each 21-day cycle of cycles 2-6, the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on day 1, the alkylating agent is administered intravenously at a dose of 90 mg/m 2 on days 1 and 2. In some embodiments, the immune conjugate is administered intravenously at a dose of ² , and the anti-CD20 antibody is administered intravenously at a dose of 375 mg/m ² on day 1. In some embodiments, the immune conjugate and the alkylating agent are administered sequentially on day 2 of cycle 1. In some embodiments, the immune conjugate is administered before the alkylating agent. In some embodiments, the immune conjugate, the alkylating agent, and the anti-CD20 antibody are administered sequentially on day 1 of cycles 2-6. In some embodiments, the anti-CD20 antibody is administered before the immune conjugate, and the immune conjugate is administered before the alkylating agent on day 1 of cycles 2-6. In some embodiments, the immune conjugate, the alkylating agent, and the anti-CD20 antibody are further administered after cycle 6. In some embodiments, the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on day 1, the alkylating agent is administered intravenously at a dose of 90 mg/m ² on days 1 and 2, and the anti-CD20 antibody is administered intravenously at a dose of 375 mg/m ² on day 1 of each 21-day cycle in each cycle after cycle 6. In some embodiments, the anti-CD20 antibody is administered before the immune conjugate, and wherein the immune conjugate is administered before the alkylating agent on day 1 of each 21-day cycle in each cycle after cycle 6. Other exemplary dosing and administration schedules are provided elsewhere herein.

The present invention provides a method for treating diffuse large B-cell lymphoma in a human in need thereof, comprising administering to the human an effective amount of: (a) an immunoconjugate comprising the formula wherein Ab is an anti-CD79b antibody comprising (i) a heavy chain comprising VH, wherein VH comprises the amino acid sequence of SEQ ID NO: 19, and (ii) a light chain comprising VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 20, wherein p is 2 to 5, (b) bendamustine or a salt thereof, and (c) rituximab, wherein the immunoconjugate is administered at a dose of 1.8 mg/kg, bendamustine or a salt thereof is administered at a dose of 90 mg/m ² , and rituximab is administered at a dose of 375 mg/m 2. ² , wherein the treatment prolongs progression-free survival (PFS) and/or overall survival (OS) in the human, and wherein: i) the DLBCL is activated B-cell-like DLBCL (ABC-DLBCL) or germinal center B-cell-like DLBCL (GCB-BLBCL); iii) the DLBCL is a bi-expressing lymphoma (DEL); iv) the human has received at least two prior therapies for DLBCL; v) the human has received at least three prior therapies for DLBCL; and/or vi) the human has received more than three prior therapies for DLBCL. In some embodiments, p is between 3 and 4 (e.g., 3.5). In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 36, and wherein the light chain comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the bendamustine or a salt thereof is bendamustine-HCl. In some embodiments, the human achieves a complete response (CR) following treatment with the immune conjugate, bendamustine or a salt thereof, and rituximab.

In some embodiments, the immune conjugate, bendamustine or a salt thereof, and rituximab are administered for at least six 21-day cycles, wherein the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on day 2, bendamustine or a salt thereof is administered intravenously at a dose of 90 mg/m ² on days 2 and 3, and rituximab is administered intravenously at a dose of 375 mg/m ² on day 1 of a 21-day cycle in cycle 1, and wherein in each 21-day cycle after cycle 1, the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on day 1, bendamustine or a salt thereof is administered intravenously at a dose of 90 mg/m 2 on days 1 and 2, in each 21-day cycle. In some embodiments, the immune conjugate is administered intravenously at a dose of ² , and rituximab is administered intravenously on day 1 at a dose of 375 mg/m ^2. In some embodiments, the immune conjugate and bendamustine or a salt thereof are administered sequentially on day 2 of cycle 1. In some embodiments, the immune conjugate is administered before bendamustine or a salt or solvate thereof. In some embodiments, the immune conjugate, bendamustine or a salt thereof, and rituximab are administered sequentially on day 1 of cycles 2-6. In some embodiments, rituximab is administered before the immune conjugate, and the immune conjugate is administered before the alkylating agent on day 1 of cycles 2-6. In some embodiments, an immune conjugate, bendamustine or a salt thereof, and rituximab are further administered after cycle 6, and wherein in each cycle after cycle 6, the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on day 1, bendamustine or a salt thereof is administered intravenously at a dose of 90 mg/m ² on days 1 and 2, and rituximab is administered intravenously at a dose of 375 mg/m ² on day 1 of each 21-day cycle. In some embodiments, rituximab is administered before the immune conjugate, and wherein the immune conjugate is administered before the alkylating agent on day 1 of each 21-day cycle of each cycle after cycle 6. Other exemplary dosing and administration schedules are provided elsewhere herein.

In some embodiments, treatment extends PFS in a human by at least about 6, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, or 17 months. In some embodiments, treatment extends PFS by at least 7 months. In some embodiments, treatment extends PFS by at least about 7.6 months. In some embodiments, treatment extends PFS by at least about 8 months. In some embodiments, treatment extends PFS to at least 11 months. In some embodiments, treatment extends PFS to at least 11.1 months.

In some embodiments, treatment extends OS in humans by at least about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, or more than 12.5 months. In some embodiments, treatment extends OS to at least about 12 months. In some embodiments, treatment extends OS by at least about 12.4 months.

In some embodiments, the DLBCL is activated B-cell-like DLBCL (ABC DLBCL). In some embodiments, the DLBCL is germinal center B-cell-like DLBCL (GCB DLBCL). In some embodiments, the DLBCL is DLBCL not otherwise specified (DLBCL-NOS). In some embodiments, the DLBCL is bi-expressing lymphoma (DEL). In some embodiments, the DLBCL is relapsed/refractory DLBCL. In some embodiments, the human does not have grade 3b follicular lymphoma, transformed and slowed non-Hodgkin lymphoma, or CNS lymphoma. In some embodiments, the human has received at least one prior therapy for DLBCL. In some embodiments, the human has received at least two prior therapies for DLBCL. In some embodiments, the human has received at least three prior therapies for DLBCL. In some embodiments, the human has received more than three prior therapies for DLBCL. In some embodiments, the human is not suitable for autologous stem cell transplantation (ASCT). In some embodiments, the ASCT is a first-line ASCT, a second-line ASCT, a third-line ASCT, or beyond a third-line ASCT. In some embodiments, the human has failed a prior autologous stem cell transplant. In some embodiments, the human has received prior treatment with an anti-CD20 agent. In some embodiments, the human has received prior treatment with bendamustine or a salt thereof. In some embodiments, the human is refractory to the most recent prior therapy. In some embodiments, the most recent prior therapy is standard of care therapy. In some embodiments, the individual is refractory to treatment if the individual has demonstrated a partial response, minimal response, or no response to treatment. In some embodiments, the individual is female. In some embodiments, the individual is an adult with DLBCL not otherwise specified (NOS) who has received at least one prior therapy (e.g., for DLBCL).

Also provided are kits comprising an immunoconjugate comprising the formula Wherein Ab is an anti-CD79b antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, and wherein p is 1 to 8, and the antibody is used in combination with an alkylating agent and an anti-CD20 antibody to treat a human with diffuse large B-cell lymphoma (DLBCL) in need thereof according to the methods provided herein. In some embodiments, the anti-CD79b antibody comprises (i) a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 20.

Also provided are kits comprising an immunoconjugate comprising the formula Wherein Ab is an anti-CD79b antibody comprising (i) a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 20, and wherein p is 2 to 5, said antibody is used in combination with bendamustine or a salt thereof and rituximab to treat a human with diffuse large B-cell lymphoma (DLBCL) in need thereof according to the methods provided herein. In some embodiments, p is between 3 and 4 (e.g., 3.5). In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 36, and wherein the light chain comprises the amino acid sequence of SEQ ID NO: 35.

Also provided is an immunoconjugate comprising the formula wherein Ab is an anti-CD79b antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, and wherein p is between 1 and 8, the antibody is for use in a method of treating diffuse large B-cell lymphoma (DLBCL) in a human in need thereof, the method comprising administering to the human an effective amount of an immunoconjugate, an alkylating agent, and an anti-C20 antibody, wherein the treatment prolongs progression-free survival (PFS) and/or overall survival (OS) in the human. In some embodiments, the immunoconjugates are used in the methods provided herein. In some embodiments, the anti-CD79b antibody comprises (i) a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 20.

Also provided is an immunoconjugate comprising the formula wherein Ab is an anti-CD79b antibody comprising (i) a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 20, wherein p is between 2 and 5, and the antibody is for use in a method of treating diffuse large B-cell lymphoma (DLBCL) in a human in need thereof, the method comprising administering to the human an effective amount of (a) an immunoconjugate, (b) bendamustine or a salt thereof, and (c) rituximab, wherein the immunoconjugate is administered at a dose of 1.8 mg/kg, bendamustine or a salt thereof is administered at a dose of 90 mg/m ² , and rituximab is administered at a dose of 375 mg/m 2. ² , and wherein the treatment prolongs progression-free survival (PFS) and/or overall survival (OS) in the human. In some embodiments, the immunoconjugate is used in the methods provided herein. In some embodiments, p is between 3 and 4 (e.g., 3.5). In some embodiments, the anti-CD79 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

Also provided are compositions (e.g., pharmaceutical compositions) comprising an immunoconjugate having the formula wherein Ab is an anti-CD79b antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 26 NO: 26, and wherein p is between 1 and 8, the antibody is for use in a method of treating diffuse large B-cell lymphoma (DLBCL) in a human in need thereof, the method comprising administering to the human an effective amount of a composition, an alkylating agent, and an anti-C20 antibody, wherein the treatment prolongs progression-free survival (PFS) and/or overall survival (OS) in the human. In some embodiments, the composition is used in the methods provided herein. In some embodiments, the anti-CD79b antibody comprises (i) a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 20.

Also provided are compositions (e.g., pharmaceutical compositions) comprising an immunoconjugate having the formula wherein Ab is an anti-CD79b antibody comprising (i) a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 20, wherein p is between 2 and 5, and the antibody is for use in a method of treating diffuse large B-cell lymphoma (DLBCL) in a human in need thereof, the method comprising administering to the human an effective amount of (a) a composition, (b) bendamustine or a salt thereof, and (c) rituximab, wherein the composition is administered to provide a dose of 1.8 mg/kg of the immunoconjugate, bendamustine or a salt thereof is administered at a dose of 90 mg/m ² , and rituximab is administered at a dose of 375 mg/m 2. ² , and wherein the treatment prolongs progression-free survival (PFS) and/or overall survival (OS) in the human. In some embodiments, the composition is used in the methods provided herein. In some embodiments, p is between 3 and 4 (e.g., 3.5). In some embodiments, the anti-CD79 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form further embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description below.

Figure 1A provides a schematic diagram of the study design of the Phase Ib/II clinical trial described in Example 1 .

Figure 2A(i) provides a Kaplan-Meier plot of investigator-assessed progression-free survival (PFS by INV) for patients who received Pola-BR versus those who received BR alone.

Figure 2A(ii) provides a Kaplan-Meier plot of progression-free survival as assessed by an independent review committee (PFS by IRC) for patients who received Pola-BR versus those who received BR alone.

Figure 2B provides a Kaplan-Meier plot of overall survival (OS) for patients who received Pola-BR versus those who received BR alone.

Figure 2C provides a forest plot showing subgroup analysis of overall survival (OS) of patients with various clinical and biological characteristics in the Pola-BR group and the BR group.

Figure 3A provides a forest plot showing subgroup analyses of investigator-assessed progression-free survival (PFS by INV) in patients with various clinical and biological characteristics in the Pola-BR group and the BR group.

Figure 3B provides a forest plot showing subgroup analyses of progression-free survival assessed by an independent review committee (PFS by IRC) in the Pola-BR and BR groups for patients with various clinical and biological characteristics.

Figure 4 provides the results of RNA assessment performed to determine CD79b expression in lymph node biopsies obtained from patients participating in the clinical trial described in Example 1 .

Figure 5 provides the results of experiments performed to assess CD79b protein expression levels in samples obtained from patients participating in the clinical trial described in Example 1 .

Provided herein are methods for treating or delaying the progression of lymphoma (e.g., diffuse large B-cell lymphoma (DLBCL), e.g., relapsed/refractory DLBCL) in an individual (e.g., a human), comprising administering to the individual an effective amount of an anti-CD79b immunoconjugate, an alkylating agent ( e.g. , bendamustine or bendamustine-HCl), and an anti-CD20 agent ( e.g., an anti-CD20 antibody, e.g., rituximab). In some embodiments, treatment with an anti-CD79 immunoconjugate, an alkylating agent, and an anti-CD20 agent prolongs progression-free survival (PFS) and/or overall survival (OS) of the individual. In some embodiments, such treatment prolongs progression-free survival (PFS) and/or overall survival (OS) in a subject, for example, compared to PFS and/or OS in a subject receiving a treatment comprising administration of an alkylating agent (e.g., bendamustine or bendamustine-HCl) and an anti-CD20 agent (e.g., an anti-CD20 antibody) without an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin). In some embodiments, a subject administered an anti-CD79 immunoconjugate, an alkylating agent, and an anti-CD20 agent achieves a complete remission (CR) following administration. A complete remission is also referred to as a "complete response."

In some embodiments, the method comprises treating a subject having diffuse large B-cell lymphoma (DLBCL, e.g., relapsed/refractory DLBCL) by administering to the subject (a) an immunoconjugate comprising the formula wherein Ab is an anti-CD79b antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 26 NO: 26, and wherein p is 1 to 8 (e.g., 2 to 5, or 3 to 4), (b) an alkylating agent (e.g., bendamustine or bendamustine-HCl), and (c) an anti-CD20 agent (e.g., rituximab), wherein the immunoconjugate is administered at a dose of 1.8 mg/kg, the alkylating agent (e.g., bendamustine or bendamustine-HCl) is administered at a dose of 90 mg/m ² , and the anti-CD20 agent (e.g., rituximab) is administered at a dose of 375 mg/m ² , and wherein the treatment prolongs progression-free survival (PFS) and/or overall survival (OS) of the individual. In some embodiments, a subject administered an anti-CD79 immunoconjugate, bendamustine (or bendamustine-HCl), and rituximab achieves a complete remission (CR) following administration. A complete remission is also referred to as a "complete response."

In some embodiments, the individual has activated B-cell-like DLBCL (ABC DLBCL). In some embodiments, the individual has germinal center B-cell-like DLBCL (GCB DLBCL). In some embodiments, the individual has DLBCL not otherwise specified (DLBCL-NOS). In some embodiments, the individual has bi-expressing lymphoma (DEL). In some embodiments, the individual has not responded to initial treatment for DLBCL. In some embodiments, the individual has relapsed/refractory DLBCL. In some embodiments, the individual has received at least one, at least two, or at least three prior therapies for DLBCL. In some embodiments, the individual has received more than three prior therapies for DLBCL. In some embodiments, the individual is not a candidate for autologous stem cell transplant (ASCT) (e.g., first-line ASCT, second-line ASCT, third-line ASCT, or beyond third-line ASCT). In some embodiments, the individual has failed a previous autologous stem cell transplant. In some embodiments, the individual has received prior treatment with an anti-CD20 agent. In some embodiments, the individual has received prior treatment with bendamustine or bendamustine-HCl. In some embodiments, the individual is refractory to the most recent prior therapy.

I. General Technology

Unless otherwise indicated, the practice of the present invention will employ techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. These techniques are fully explained in the literature, for example, in "Molecular Cloning: A Laboratory Manual," 2nd 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); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., ed., 1987 and regularly updated); "PCR: The Polymerase Chain Reaction" (Mullis et al., ed., 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); and "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

II. Definition

Before describing the present invention in detail, it should be understood that the present invention is not limited to specific compositions or biological systems, which may, of course, vary. It should be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

As used in this specification and the accompanying claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" includes combinations of two or more such molecules, and the like.

As used herein, the term "about" refers to the typical error range for the respective values that is readily known to those skilled in the art. References to "about" a value or parameter herein include (and describe) embodiments specific to that value or parameter.

It should be understood that the aspects and embodiments of the present invention described herein include "consisting of aspects and embodiments" and/or "consisting essentially of aspects and embodiments."

Unless otherwise indicated, the term "CD79b" as used herein refers to any native CD79b from any vertebrate source, including mammals, such as primates ( e.g., humans, cynomolgus monkeys ("cyno")), and rodents ( e.g., mice and rats). Human CD79b is also referred to herein as "Igβ,""B29,""DNA225786," or "PRO36249." An exemplary CD79b sequence including a signal sequence is shown in SEQ ID NO: 1. An exemplary CD79b sequence without a signal sequence is shown in SEQ ID NO: 2. The term "CD79b" encompasses "full-length," unprocessed CD79b as well as any form of CD79b resulting from processing in cells. The term also encompasses naturally occurring variants of CD79b, such as splice variants, allelic variants, and isoforms. The CD79b polypeptides described herein can be isolated from a variety of sources, such as from a human tissue type or from another source, or prepared by recombinant or synthetic methods. A "native sequence CD79b polypeptide" comprises a polypeptide having the same amino acid sequence as a corresponding CD79b polypeptide derived from nature. Such native sequence CD79b polypeptides can be isolated from nature or can be produced by recombinant or synthetic methods. The term "native sequence CD79b polypeptide" specifically includes naturally occurring truncated or secreted forms (e.g., extracellular domain sequences) of a particular CD79b polypeptide, naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants of the polypeptide.

As used herein, "CD20" refers to the human B lymphocyte antigen CD20 (also known as CD20, B lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5; the sequence is represented by the SwissProt database entry P11836), a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes. (Valentine, MA et al., J. Biol. Chem. 264(19)(198911282-11287; Tedder, TF et al., Proc. Natl. Acad . Sci. USA 85(1988)208-12; Stamenkovic, I. et al., J. Exp. Med. 167(1988)1975-80; Einfeld, DA et al., EMBO J. 7(1988)711-7; Tedder, TF et al., J. Immunol. 142 (1989) 2560-8). The corresponding human gene is transmembrane 4 domains, subfamily A, member 1, also known as MS4A1. This gene encodes a member of the transmembrane 4A gene family. Members of this family of novel proteins are characterized by shared structural features and similar intron/exon splice boundaries and display distinct expression patterns in hematopoietic cells and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule that plays a role in B cell development and plasmacytogenic differentiation. In clusters of family members, this family member is localized to 11q12. Alternative splicing of this gene produces two transcript variants encoding the same protein.

The terms "CD20" and "CD20 antigen" are used interchangeably herein and include any variants, isoforms, and species homologs of human CD20 that are naturally expressed by cells or expressed on cells transfected with the CD20 gene. Binding of the antibodies of the present invention to the CD20 antigen mediates the killing of cells expressing CD20 ( e.g., tumor cells) by inactivating CD20. Killing of cells expressing CD20 can occur through one or more of the following mechanisms: cell death/apoptosis induction, ADCC, and CDC. As recognized in the art, synonyms for CD20 include B lymphocyte antigen CD20, B lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5.

The term "expression of the CD20 antigen" is intended to refer to the expression of the CD20 antigen at significant levels on cells ( e.g. , T- or B-cells). In one embodiment, patients treated according to the methods of the present invention express significant levels of CD20 on B-cell tumors or cancers. Patients with "CD20-expressing cancers" can be identified using standard assays known in the art. For example , CD20 antigen expression can be measured using immunohistochemistry (IHC) assays, FACS, or PCR-based detection of corresponding mRNA.

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

An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not possess these alterations, which result in improved affinity of the antibody for the antigen.

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

"Antibody fragments" are molecules other than intact antibodies that comprise 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, and F(ab') ₂ ; bifunctional antibodies; linear antibodies; single-chain antibody molecules ( e.g., scFv); and multispecific antibodies formed from antibody fragments.

An "antibody that binds to the same antigenic determinant as a reference antibody" is an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

The term "antigenic determinant" refers to the specific site on an antigen molecule to which an antibody binds.

The term "chimeric" antibody refers to an antibody in which one portion of the heavy and/or light chain is derived from a specific source or species, while the remaining portion of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or region within its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these classes are further divided into subclasses (isotypes), such as _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgA1 , and _IgA2 . The constant domains of the heavy chains corresponding to these different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The terms "anti-CD79b antibody" and "antibody that binds to CD79b" refer to an antibody that is capable of binding to CD79b with an affinity sufficient to render the antibody useful as a diagnostic and/or therapeutic agent when targeting CD79b. Preferably, the anti-CD79b antibody binds to unrelated non-CD79b proteins to an extent that is less than about 10% of the binding of the antibody to CD79b, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to CD79b has 1μM, 100nM, 10nM, 1nM or The dissociation constant (Kd) is 0.1 nM. In certain embodiments, the anti-CD79b antibody binds to an antigenic determinant in CD79b that is conserved among CD79b from different species.

The term "anti-CD20 antibody" according to the present invention refers to an antibody that is capable of binding to CD20 with an affinity sufficient to make the antibody useful as a diagnostic and/or therapeutic agent when targeting CD20. In one embodiment, the extent to which the anti-CD20 antibody binds to an unrelated non-CD20 protein is less than about 10% of the binding of the antibody to CD20, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to CD20 has 1μM, 100nM, 10nM, 1nM or A dissociation constant (Kd) of 0.1 nM. In certain embodiments, the anti-CD20 antibody binds to an epitope in CD20 that is conserved among CD20 from different species.

An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis ( e.g. , SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography ( e.g. , ion exchange or reverse phase HPLC). For a review of methods used to analyze antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007). The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of the antibody and contain the antigen-binding site.

"Isolated nucleic acid encoding an anti-CD79b antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors and such nucleic acid molecules present at one or more locations in a host cell.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and/or bind to the same antigenic determinant, except for possible variant antibodies, for example, those containing naturally occurring mutations or arising during the production of the monoclonal antibody preparation, which variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (antigenic determinants), each individual antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies to be used according to 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. These methods and other exemplary methods for producing monoclonal antibodies are described herein.

A "naked antibody" is an antibody that is not conjugated to a foreign moiety ( e.g. , a cytotoxic moiety) or a radiolabel. Such naked antibodies can be present in pharmaceutical formulations.

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

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in 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.

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

For the purposes of this document, an "acceptor human framework" is a framework that comprises an amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence of a human immunoglobulin framework or a human consensus framework, or it may contain amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the sequence of the VL acceptor human framework is identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody whose structure is substantially similar to that of a native antibody or which has a heavy chain containing an Fc region as defined herein.

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 its progeny, regardless of the number of generations. Progeny may not be completely identical to the parent cell in terms of nucleic acid content and may contain mutations. Mutant progeny that have the same function or biological activity as that screened or selected for in the original transformed cell are included herein.

A "human antibody" is an antibody that has an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by humans or human cells, or that is derived from a non-human source that utilizes a human antibody repertoire or other human antibody-encoding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

A "human consensus framework" is a framework representing the most frequently occurring amino acid residues in a selected human immunoglobulin VL or VH framework sequence. Generally, a human immunoglobulin VL or VH sequence is selected from a subset of variable domain sequences. Generally, the sequence subset is a subset as described in Kabat et al., Sequences of Proteins of Immunological Interest , Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI as described in Kabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup III as described in Kabat et al. (supra).

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

As used herein, the term "hypervariable region" or "HVR" refers to regions of an antibody variable domain whose sequence is highly variable and/or form structurally defined loops ("hypervariable loops"). Generally, a natural four-chain antibody comprises six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically comprise amino acid residues from the hypervariable loops and/or from the complementary determining regions (CDRs), which have the highest sequence variability and/or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are present at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).) With the exception of CDR1 in VH, CDRs typically include the amino acid residues that form the hypervariable loops. CDRs also contain "specificity-determining residues" or "SDRs," which are antigen-contacting residues. SDRs are contained within regions of the CDRs known as abbreviated CDRs or α-CDRs. Exemplary α-CDRs (α-CDR-L1, α-CDR-L2, α-CDR-L3, α-CDR-H1, α-CDR-H2, and α-CDR-H3) are located at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. ( See Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues in variable domains ( e.g. , FR residues) are numbered herein according to Kabat et al., supra.

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

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary 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); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

"CD79b polypeptide variants" refers to CD79b polypeptides, as defined herein, that have at least about 80% amino acid sequence identity to the full-length native sequence CD79b polypeptide sequence disclosed herein, the CD79b polypeptide sequence disclosed herein lacking a signal peptide, the extracellular domain of a CD79b polypeptide as disclosed herein with or without a signal peptide, or any other fragment of the full-length CD79b polypeptide sequence disclosed herein (e.g., those encoded by a nucleic acid that represents only a portion of the complete coding sequence of the full-length CD79b polypeptide), preferably an active CD79b polypeptide. Such CD79b polypeptide variants include, for example, CD79b polypeptides in which one or more amino acid residues are added or deleted at the N- or C-terminus of the full-length native amino acid sequence. Typically, a CD79b polypeptide variant will have at least about 80% amino acid sequence identity, or at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a full-length native sequence CD79b polypeptide sequence disclosed herein, a CD79b polypeptide sequence disclosed herein lacking a signal peptide, an extracellular domain of a CD79b polypeptide as disclosed herein with or without a signal peptide, or any other fragment of a full-length CD79b polypeptide sequence disclosed herein. Typically, the CD79b variant polypeptide is at least about 10 amino acids long, or at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 amino acids in length, or longer. Optionally, the CD79b variant polypeptide will have no more than one conservative amino acid substitution as compared to the native CD79b polypeptide sequence, or no more than about 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions as compared to the native CD79b polypeptide sequence.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the candidate sequences and introducing gaps (if necessary) to achieve maximum percent sequence identity, and not taking into account any conservative substitutions that are part of the sequence identity. Alignment to determine percent amino acid sequence identity can be performed in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. However, for the purposes of this article, the sequence comparison computer program ALIGN-2 is used to generate % amino acid sequence identity values. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code is archived in the U.S. Copyright Office, Washington, D.C., 20559, as user documentation, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available or can be compiled from source code from Genentech, Inc., South San Francisco, California. The ALIGN-2 program should be compiled for UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In cases where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A relative to, with, or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity relative to, with, or against a given amino acid sequence B) is calculated as follows: 100 × score X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity of B relative to A. Unless otherwise specifically stated, all % amino acid sequence identity values used herein were obtained using the ALIGN-2 computer program as described in the previous paragraph.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid molecule to which it is linked. The term includes vectors in the form of autonomously replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents).

In the context of the formulae provided herein, "p" refers to the average number of drug moieties per antibody, which can range, for example, from about 1 to about 20 drug moieties per antibody, and in certain embodiments, from 1 to about 8 drug moieties per antibody. The present invention includes compositions comprising a mixture of antibody-drug compounds of Formula I, wherein the average drug loading per antibody is from about 2 to about 5, or from about 3 to about 4, (e.g., about 3.5).

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes ( e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu); chemotherapeutic agents or drugs ( e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and/or variants thereof; and various antitumor or anticancer agents disclosed below.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, B-cell lymphomas (including low-grade/follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade/follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-dividing cell NHL; large mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with keloids, edema (such as edema associated with brain tumors), and Meig's syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, first-line low-grade NHL, stage III/IV NHL, chemotherapy-resistant NHL, progenitor B-cell lymphoblastic leukemia and/or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma , B-cell prolymphocytic lymphoma, immunocytoma and/or lymphocytic lymphoma, lymphocytic lymphoma, marginal area B-cell lymphoma, splenic marginal area lymphoma, extranodal marginal area-MALT lymphoma, nodular marginal area lymphoma, hairy cell leukemia, plasma cell neoplasm and/or plasma cell myeloma, low grade/follicular lymphoma, intermediate Grade/follicular NHL, mantle cell lymphoma, follicular center lymphoma (follicular), intermediate-grade diffuse NHL, diffuse large B-cell lymphoma (DLBCL), aggressive NHL (including aggressive first-line NHL and aggressive relapsed NHL), NHL that relapses after or is refractory to autologous stem cell transplantation, original Primary longitudinal large B-cell lymphoma, primary exudative lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-mitotic cell NHL, large mass disease NHL, Burkitt's lymphoma, progenitor (peripheral) large granulocytic lymphocytic leukemia, mycosis fungoides and/or Sézary syndrome, cutaneous (cutaneous) lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma.

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

An "effective amount" of a drug (e.g., a pharmaceutical formulation) is an amount effective to achieve the desired therapeutic or prophylactic result at the dosage and for the period of time necessary.

The term "pharmaceutical formulation" means a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective and that contains no additional components that are unacceptably toxic to the subject to which the formulation is to be administered.

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

As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to clinical intervention intended to alter the natural course of a disease in the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desirable therapeutic effects include, but are not limited to, reduction of free light chains, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological consequences of the disease, reduction in the rate of disease progression, amelioration or alleviation of the disease state, and palliation or improvement of prognosis. In some embodiments, the antibodies described herein are used to delay disease onset or slow disease progression.

The term "CD79b-positive cancer" refers to a cancer comprising cells expressing CD79b on their surface. In some embodiments, CD79b expression on the cell surface is determined, for example, using CD79b antibodies in methods such as immunohistochemistry and FACS. Alternatively, CD79b mRNA expression is believed to correlate with CD79b expression on the cell surface and can be determined by a method selected from in situ hybridization and RT-PCR (including quantitative RT-PCR).

As used herein, "in combination with" means administering one therapeutic modality in addition to another therapeutic modality. Thus, "in combination with" means administering one therapeutic modality before, during, or after another therapeutic modality is administered to a subject.

"Chemotherapeutic agents" are chemical compounds used to treat cancer. Examples of chemotherapeutic agents include erlotinib ( ^TARCEVA® , Genentech/OSI Pharm.), bortezomib ( ^VELCADE® , Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant ( ^FASLODEX® , AstraZeneca), sunitinib ( ^SUTENT®, , Pfizer/Sugen), letrozole (FEMARA ^® , Novartis), imatinib mesylate (GLEEVEC ^{® , Novartis), finasuna (VATALANIB ®} , Novartis), oxaliplatin (ELOXATIN ^® , Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus, RAPAMUNE ^® , Wyeth), lapatinib ^{(TYKERB ® ,} GSK572016, Glaxo Smith Kline), lonafamib (SCH 66336), sorafenib (NEXAVAR ^® , Bayer Labs), gefitinib ( ^IRESSA® , AstraZeneca), AG1478, alkylating agents such as thiotepa and ^CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylmelamine, triethylphosphamide, triethylthiophosphamide, and trihydroxymethylmelamine; polyacetogenins (particularly bullatacin and bullatacinone); cinone); camptothecins (including topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycin (specifically prednisone and prednisolone); cyproterone acetate acetate); 5α-reductase (including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat; dolastatin; aldesleukin, talc; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards (such as chlorambucil, chlomaphazine, chloropho sphamide), estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide ), uracil nitrogen mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma and calicheamicin omega (Angew Chem. Intl. Ed. Engl. 1994 ) 33:183-186); dynemicins, including dynemicin A; bisphosphates, such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoproteins (enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, diazoserine (a zaserine), bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-leucine, ADRIAMYCIN ^® (doxorubicin), N-morpholinyl doxorubicin, cyano (N-morpholinyl) doxorubicin, 2-(N-pyrrolyl) doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, everolimus, sotrataurin, idarubicin, marcellomycin, mitomycins (such as mitomycin C), mycophenolic acid acid), nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimethoprim-sulfamethoxazole, dapoxetine ... trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone and dromostanolone propionate propionate), epitiostanol, mepitiostane, testolactone; antiadrenaline drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; ^PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A) A) and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");cyclophosphamide;thiotepa; taxoids, such as TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), ^ABRAXANE® (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE®. ^® (docetaxel/doxetaxel; Sanofi-Aventis); chloranmbucil; GEMZAR ^® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE ^® (vinorelbine); novantrone; teniposide; edatrexate; daunorubicin; aminopterin; capecitabine (XELODA ^® ); ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid acid); and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing; and combinations of two or more of the foregoing, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for the combination therapy of oxaliplatin (ELOXATIN ^™ ) with 5-FU and leucovorin. Additional examples include chemotherapeutic agents including bendamustine (or bendamustine-HCl) (TREANDA®), ibrutinib, lenalidomide, and/or idelaris (GS-1101).

Additional examples of chemotherapeutic agents include antihormones, which are used to modulate, reduce, block, or inhibit the effects of hormones that promote cancer growth, and are usually given as systemic or whole-body treatments. They themselves can be hormones. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene (EVISTA®), droloxifene, 4-hydroxytamoxifen, troloxifene, xifen, LY117018, onapristone, and toremifene (FARESTON®); antiprogestins; estrogen receptor downregulators (ERDs); estrogen receptor antagonists, such as fulvestrant (FASLODEX®); agents used to suppress or shut down the ovaries, such as progesterone-releasing hormone (LHRH) agonists, such as leuprorelin. These include dapoxetine (LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate, and triptorelin; antiandrogens such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors, which inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, nilutamide, megestrol acetate (MEGASE®), exemestane (AROMASIN®), formestane (formestanie), fadrozole, vorozole (RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®). Additionally, this definition of chemotherapeutic agents includes bisphosphonates such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronic acid (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); and troxadronate. Tambine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways associated with abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine.

In some embodiments, the chemotherapeutic agent includes a topoisomerase 1 inhibitor ( e.g. , LURTOTECAN®); an anti-estrogen such as fulvestrant; a Kit inhibitor such as imatinib or EXEL-0862 (a tyrosine kinase inhibitor); an EGFR inhibitor such as erlotinib or cetuximab; an anti-VEGF inhibitor such as bevacizumab; arinotecan; rmRH ( e.g. , ABARELIX®); lapatinib and lapatinib ditosylate (small molecule inhibitors of ErbB-2 and EGFR dual tyrosine kinases, also known as GW572016); 17AAG (a derivative of geldermycin, i.e., a heat shock protein (Hsp) 90 toxin), and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

Chemotherapy agents also include antibodies, such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec), ublituximab, ofatumumab, ibritumomab tiuxetan, pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody-drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Other humanized monoclonal antibodies with therapeutic potential as combination drugs include apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, and pegol), cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin), ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, and pascolizumab. scolizumab), pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab Tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and anti-interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories), a recombinant, specifically human sequence, full-length IgG1 lambda antibody that has been genetically modified to recognize the interleukin-12 p40 protein.

The term "package insert" is used to refer to instructions customarily included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings concerning the use of such therapeutic products.

"Alkyl" refers to a _C1 - _C18 hydrocarbon group containing normal chain carbon atoms, secondary carbon atoms, tertiary carbon atoms, or ring carbon atoms. The alkyl group is methyl (Me, -CH ₃ ), ethyl (Et, -CH ₂ CH ₃ ), 1-propyl (n-Pr, n-propyl, -CH ₂ CH ₂ CH ₃ ), 2-propyl (i-Pr, isopropyl, -CH(CH ₃ ) ₂ ), 1-butyl (n-Bu, n-butyl, -CH ₂ CH _{2 CH 2 CH 3} ), 2-methyl-1-propyl (i-Bu, isobutyl, -CH ₂ CH(CH ₃ ) ₂ ), 2-butyl (s-Bu, sec-butyl, -CH(CH ₃ )CH ₂ CH ₃ ), 2-methyl-2-propyl (t-Bu, tert-butyl, -C(CH ₃ ) ₃ ), 1-pentyl (n-pentyl, -CH ₂ CH ₂ CH ₂ CH ₃ ), 2-pentyl (-CH(CH ₃ )CH 2 CH ₂ CH ₃ ) ₂ CH ₃ ), 3-pentyl (-CH(CH ₂ CH ₃ ) ₂ ), 2-methyl-2-butyl (-C(CH ₃ ) ₂ CH ₂ CH ₃ ), 3-methyl-2-butyl (-CH(CH ₃ )CH(CH ₃ ) ₂ ), 3-methyl-1-butyl (-CH ₂ CH ₂ CH(CH ₃ ) ₂ ), 2-methyl-1-butyl (-CH ₂ CH(CH ₃ )CH ₂ CH ₃ ), 1-hexyl (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-hexyl (-CH(CH ₃ )CH ₂ CH ₂ CH ₂ CH ₃ ), 3-hexyl (-CH(CH ₂ CH ₃ )(CH ₂ CH ₂ CH ₃ )), 2-methyl-2-pentyl (-C(CH ₃ ) ₂ CH ₂ CH ₂ CH ₃ ), 3-methyl-2-pentyl (-CH(CH ₃ )CH(CH ₃ )CH ₂ CH ₃ ), 4-methyl-2-pentyl (-CH(CH ₃ )CH ₂ CH(CH ₃ ) ₂ ), 3-methyl-3-pentyl (-C(CH ₃ )(CH ₂ CH ₃ ) ₂ ), 2-methyl-3-pentyl (-CH(CH ₂ CH ₃ )CH(CH ₃ ) ₂ ), 2,3-dimethyl-2-butyl (-C(CH ₃ ) ₂ CH(CH ₃ ) ₂ ), 3,3-dimethyl-2-butyl (-CH(CH ₃ )C(CH ₃ ) ₃ .

As used herein, the term "C ₁ -C ₈ alkyl" refers to a straight or branched chain saturated or unsaturated hydrocarbon group having 1 to 8 carbon atoms. Representative "C ₁ -C ₈ alkyl groups" include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl, and -n-decyl; and branched chain C ₁ -C ₈ alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C ₁ -C _Alkyl groups include, but are not limited to, vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl. The C ₁ -C ₈ alkyl group may be unsubstituted or substituted with one or more groups including, but not limited to, —C ₁ -C ₈ alkyl, —O—(C ₁ -C ₈ alkyl), —aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH ₂ , —C(O)NHR′, —C(O)N(R′) ₂ —NHC(O)R′, —SO ₃ R′, —S(O) ₂ R′, —S(O)R′, —OH, —halogen, —N ₃ , —NH ₂ , —NH(R′), —N(R′) ₂ , and —CN; wherein each R′ is independently selected from H, —C ₁ -C ₈ alkyl, and aryl.

As used herein, the term "C ₁ -C ₁₂ alkyl" refers to a straight or branched chain saturated or unsaturated hydrocarbon group having 1 to 12 carbon atoms. The _C1 - _C12 alkyl group may be unsubstituted or substituted with one or more groups including, but not limited to, -C1 _{- C8} alkyl, -O-( _C1 - _C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O) _NH2 , -C(O)NHR', -C(O)N(R') _2, -NHC(O)R', -SO3R', -S(O) _2R ', -S(O)R', -OH, -halogen, _-N3 , _-NH2 , -NH(R'), -N(R') ₂ , and -CN; wherein each R' is independently _selected from H, _-C1 - _C8 alkyl, and aryl.

As used herein, the term "C ₁ -C ₆ alkyl" refers to a straight or branched chain saturated or unsaturated hydrocarbon group having 1 to 6 carbon atoms. Representative " _C1 - _C6 alkyl" groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; branched-chain _C1 - _C6 alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl; unsaturated _C1 - _C6 alkyl groups include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, and -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, and 3-hexyl. The C ₁ -C ₆ alkyl group may be unsubstituted or substituted with one or more groups as described above for the C ₁ -C ₈ alkyl group.

As used herein, the term " _C1 - _C4 alkyl" refers to a straight or branched chain, saturated or unsaturated hydrocarbon group having 1 to 4 carbon atoms. Representative " _C1 - _C4 alkyl" groups include, but are not limited to, -methyl, -ethyl, -n-propyl, and -n-butyl; branched chain _C1 - _C4 alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, and -tert-butyl; and unsaturated _C1 - _C4 alkyl groups include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, and -isobutenyl. _C1 - _C4 alkyl groups may be unsubstituted or substituted with one or more groups as described above for C1- _{C8 alkyl} groups.

"Hydroxy" refers to an alkyl group that is single-bonded to an oxygen group. Exemplary alkoxy groups include, but are not limited to, methoxy (-OCH ₃ ) and ethoxy (-OCH ₂ CH ₃ ). "C ₁ -C ₅ alkoxy" refers to an alkoxy group having 1 to 5 carbon atoms. An alkoxy group may be unsubstituted or substituted with one or more groups as described above for an alkyl group.

"Alkenyl" refers to a _C2 - _C18 hydrocarbon group with at least one unsaturated site (i.e., a carbon-carbon sp2 ^double bond) and containing normal, secondary, tertiary, or cyclic _carbon atoms. Examples include, but are _{not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2 ) , cyclopentenyl (-C5H7 )} , and 5-hexenyl ( _{-CH2CH2CH2CH2CH} = _CH2 ) _. " _C2 - _C8 alkenyl" refers to a hydrocarbon group with at least one unsaturated site (i.e., ^a carbon-carbon sp2 double bond) and containing 2 to 8 normal, secondary, tertiary, or cyclic carbon atoms.

"Alkynyl" refers to a _C2 - _C18 hydrocarbon group with at least one unsaturated site (i.e., a carbon-carbon sp bond) and containing normal, secondary, tertiary, or cyclic carbon atoms. Examples include, but are not limited to, ethynyl (-C≡CH) and propargyl ( _-CH2C≡CH ). " _C2 - _C8 alkynyl" refers to a hydrocarbon group with at least one unsaturated site (i.e., a carbon-carbon sp bond) and containing 2 to 8 normal, secondary, tertiary, or cyclic carbon atoms.

"Alkylene" refers to a saturated, branched, straight-chain, or cyclic alkyl group having 1-18 carbon atoms and two monovalent radical centers derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. Typical alkylene groups include, but are not limited to, methylene ( _-CH2- ) _{, 1,2} -ethyl _{( -CH2CH2-} ), 1,3-propyl ( _-CH2CH2CH2- ), 1,4 _- butyl ( _{-CH2CH2CH2CH2-} ) _, and _similar groups.

"C ₁ -C ₁₀ alkylene" is a straight chain saturated alkyl group having the formula -(CH ₂ ) _1-10 -. Examples of C ₁ -C ₁₀ alkylene include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, and decylene.

"Alkenylene" refers to an unsaturated branched, straight, or cyclic alkyl group having 2-18 carbon atoms and two monovalent radical centers derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkene. Typical alkenylene groups include, but are not limited to, 1,2-ethylene (-CH=CH-).

"Alkyne" refers to an unsaturated branched or straight chain or cyclic alkyl group having 2-18 carbon atoms and two monovalent radical centers derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkynyl. Typical alkynyl groups include, but are not limited to, acetylene (-C≡C-), propargyl ( _-CH≡C- ) _, and ₄ -pentynyl (-CH≡CH≡C-) _.

"Aryl" refers to a carbocyclic aromatic group. Examples of aryl include, but are not limited to, phenyl, naphthyl, and anthracenyl. The carbocyclic aromatic group or heterocyclic aromatic group may be unsubstituted or substituted with one or more groups including, but not limited to, -C1 _{- C8} alkyl, -O-( _C1 - _C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O) _NH2 , -C(O)NHR', -C(O)N(R') _2, -NHC(O)R', -S(O) _2R ', -S(O)R', -OH, _-halogen , _-N3 , -NH2, -NH(R'), -N(R') ₂ , and -CN; wherein each R' is independently selected from H, _-C1 - _C8 alkyl, and aryl.

" _C5 - _C20 aryl" refers to an aryl group having 5 to 20 carbon atoms in the carbocyclic aromatic ring. Examples of _C5 - _C20 aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. _C5 - _C20 aryl groups may be substituted or unsubstituted as described above for aryl groups. " _C5 - _C14 aryl" refers to an aryl group having 5 to 14 carbon atoms in the carbocyclic aromatic ring. Examples of _C5 - _C14 aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. _C5 - _C14 aryl groups may be substituted or unsubstituted as described above for aryl groups.

An "arylene" group is an aromatic group having two covalent bonds in an ortho, meta, or para configuration as shown in the following structure: wherein the phenyl group may be unsubstituted or substituted with up to four groups including, but not limited to, _-C1 - _C8 alkyl, -O-( _C1 - _C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O) _NH2 , -C(O)NHR', -C(O)N(R') ₂ , -NHC(O)R', -S(O) _2R ', -S(O)R', -OH, -halogen, _-N3 , _-NH2 , -NH(R'), -N(R') ₂ , and -CN; wherein each R' is independently selected from H, _-C1 - _C8 alkyl, and aryl.

"Arylalkyl" refers to an acyclic alkyl group in which a hydrogen atom bonded to a carbon atom (typically a terminal or ^sp3 carbon atom) is replaced by an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenyleth-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthyleth-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthphenyleth-1-yl, and the like. Arylalkyl groups contain 6 to 20 carbon atoms, for example, the alkyl portion (including alkyl, alkenyl, or alkynyl) of the arylalkyl group has 1 to 6 carbon atoms and the aryl portion has 5 to 14 carbon atoms.

"Heteroarylalkyl" refers to an acyclic alkyl group in which a hydrogen atom bonded to a carbon atom (typically a terminal or ^sp3 carbon atom) is replaced by a heteroaryl group. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, and similar groups. Heteroarylalkyl groups contain 6 to 20 carbon atoms, for example, the alkyl portion (including alkyl, alkenyl, or alkynyl) of the heteroarylalkyl group contains 1 to 6 carbon atoms and the heteroaryl portion contains 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. The heteroaryl portion of the heteroarylalkyl group can be a monocyclic ring having 3 to 7 ring members (2 to 6 carbon atoms) or a bicyclic ring having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S), for example, a bicyclic [4,5], [5,5], [5,6] or [6,6] system.

"Substituted alkyl,""substitutedaryl," and "substituted arylalkyl" mean alkyl, aryl, and arylalkyl, respectively, in which one or more hydrogen atoms are each independently replaced with a substituent. Typical substituents include, but are not limited to, -X, -R, ^-O- , -OR, -SR, ^-S- , _-NR2 , _-NR3 , =NR, _-CX3 , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, _-NO2 , = _N2 , _-N3 , NC(=O)R, -C(=O)R, -C(=O) _NR2 , _-SO3- , ^-SO3H , -S ₍ =O) _2R , -OS(=O) _2OR , -S(=O) _2NR , -S(=O)R, -OP(=O)(OR) ₂ , -P(=O)(OR) ₂ , -PO ^- ₃ , _-PO3H2 , -C(=O ₎ R, -C(=O)X, -C(=S)R, _-CO2R , -CO _2- ^, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR ₂ , -C(=S)NR ₂ , -C(=NR)NR ₂ , wherein each X is independently a halogen: F, Cl, Br, or I; and each R is independently -H, C ₂ -C ₁₈ alkyl, C ₆ -C ₂₀ aryl, C ₃ -C ₁₄ heterocycle, protecting group, or prodrug moiety. The alkylene, alkenylene, and alkynylene groups described above may also be similarly substituted.

"Heteroaryl" and "heterocyclic" refer to ring systems in which one or more ring atoms is a heteroatom (e.g., nitrogen, oxygen, and sulfur). Heterocyclic groups contain 3 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. Heterocyclic groups can be monocyclic with 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) or bicyclic with 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), for example, bicyclic [4,5], [5,5], [5,6], or [6,6] systems.

Exemplary heterocycles are described, for example, in Paquette, Leo A., "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (published by John Wiley & Sons, New York, 1950), particularly Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.

Examples of heterocyclic rings include, for example, but not limited to, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothienyl, tetrahydrothienyl thioxylate, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthyl, indolyl, indolene, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidinyl, 4-piperidinyl, 4-pyrid ... Keto, pyrrolidinyl, 2-pyrrolidinone, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azoctanyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl , isobenzofuranyl, benzofuranyl, benzophenone, benzophenone, phenoxathiol, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, oxazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, octinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbazolyl Phylinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrinyl, phenazinyl, phenathiazinyl, furazanyl, phenoxazinyl, isoquinoline, quinoline, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinidinyl, oxolinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, hydroxyindolyl, benzoxazolinyl and isatinyl.

By way of example and not limitation, the carbon-bonded heterocyclic ring is bonded at the 2, 3, 4, 5, or 6 position of pyridine; the 3, 4, 5, or 6 position of oxazine; the 2, 4, 5, or 6 position of pyrimidine; the 2, 3, 5, or 6 position of pyrazine; the 2, 3, 4, or 5 position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole; the 2, 4, or 5 position of oxazole, imidazole, or thiazole; the 3, 4, or 5 position of isoxazole, pyrazole, or isothiazole; the 2 or 3 position of aziridine; the 2, 3, or 4 position of azacyclobutane; the 2, 3, 4, 5, 6, 7, or 8 position of quinoline; or the 1, 3, 4, 5, 6, 7, or 8 position of isoquinoline. More typically, carbon-bonded heterocyclic rings include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-oxazinyl, 4-oxazinyl, 5-oxazinyl, 6-oxazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen-bonded heterocyclic rings are bonded at the following positions: position 1 of aziridine, azacyclobutane, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazoidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole; position 2 of isoindole or isoindoline; position 4 of oxoline; and position 9 of carbazole or β-carboline. More typically, nitrogen-bonded heterocyclic rings include 1-aziridinyl, 1-azacyclobutanyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

"C ₃ -C ₈ heterocycle" refers to an aromatic or non-aromatic C ₃ -C ₈ carbocyclic ring in which one to four ring carbon atoms are independently replaced by a heteroatom selected from the group consisting of O, S, and N. Representative examples of C ₃ -C ₈ heterocyclic ring include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, thienyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridyl, pyridonyl, pyrazinyl, oxazinyl, isothiazolyl, isoxazolyl, and tetrazolyl. The C ₃ -C ₈ heterocyclic ring may be unsubstituted or substituted with up to seven groups including, but not limited to, -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C(O)N(R') ₂ -NHC(O)R', -S(O) ₂ R', -S(O)R', -OH, -halogen, -N ₃ , -NH ₂ , -NH(R'), -N(R') ₂ , and -CN; wherein each R' is independently selected from H, -C ₁ -C ₈ alkyl, and aryl.

"C ₃ -C ₈ heterocyclic group" refers to a C ₃ -C ₈ heterocyclic group as defined above, wherein one of the hydrogen atoms of the heterocyclic group is replaced by a bond. The C ₃ -C ₈ heterocyclic group may be unsubstituted or substituted with up to six groups including, but not limited to, -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C(O)N(R') ₂ -NHC(O)R', -S(O) ₂ R', -S(O)R', -OH, -halogen, -N ₃ , -NH ₂ , -NH(R'), -N(R') ₂ , and -CN; wherein each R' is independently selected from H, -C ₁ -C ₈ alkyl, and aryl.

"C ₃ -C ₂₀ heterocycle" refers to an aromatic or non-aromatic C ₃ -C ₈ carbon ring in which one to four ring carbon atoms are independently replaced by a heteroatom selected from the group consisting of O, S and N. The C ₃ -C ₂₀ heterocycle may be unsubstituted or substituted with up to seven groups including, but not limited to, -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C(O)N(R') ₂ -NHC(O)R', -S(O) ₂ R', -S(O)R', -OH, -halogen, -N ₃ , -NH ₂ , -NH(R'), -N(R') ₂ , and -CN; wherein each R' is independently selected from H, -C ₁ -C ₈ alkyl, and aryl.

The "C ₃ -C ₂₀ heterocyclic group" refers to a C ₃ -C ₂₀ heterocyclic group as defined above, wherein one hydrogen atom of the heterocyclic group is replaced by a bond.

"Carbocycle" means a saturated or unsaturated ring having 3 to 7 carbon atoms in a monocyclic form or 7 to 12 carbon atoms in a bicyclic form. Monocyclic carbocycles have 3 to 6 ring atoms, more typically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms arranged, for example, in a bicyclic [4,5], [5,5], [5,6], or [6,6] system, or 9 or 10 ring atoms arranged in a bicyclic [5,6] or [6,6] system. Examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl, and cyclooctyl.

" _C3 - _C8 carbocycle" is a 3-, 4-, 5-, 6-, 7-, or 8-membered saturated or unsaturated, non-aromatic carbocycle. Representative _C3 - _C8 carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl. The C ₃ -C ₈ carbocyclic group may be unsubstituted or substituted with one or more groups including, but not limited to, -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C(O)N(R') ₂ -NHC(O)R', -S(O) ₂ R', -S(O)R', -OH, -halogen, -N ₃ , -NH ₂ , -NH(R'), -N(R') ₂ and -CN; wherein each R' is independently selected from H, -C ₁ -C ₈ alkyl and aryl.

"C ₃ -C ₈ carbocyclyl" refers to a C ₃ -C ₈ carbocyclyl group as defined above, wherein one of the hydrogen atoms of the carbocyclyl group is replaced by a bond.

"Linker" refers to a chemical moiety comprising a covalent bond or series of atoms that covalently links the antibody to the drug moiety. In various embodiments, linkers include divalent groups such as alkyldiyls, aryldiyls, heteroaryldiyls, moieties such as -( _CR2 ) _nO ( _CR2 ) _n- , repeating units of alkyloxy groups ( e.g., polyethyleneoxy, PEG, polymethyleneoxy) and alkylamine groups ( e.g., polyethyleneamine, Jeffamine ^™ ); and diacids and amides, including succinate, succinamide, diglycolate, malonate, and hexamethylene. In various embodiments, linkers may comprise one or more amino acid residues such as valine, phenylalanine, lysine, and homolysine.

The term "chiral" refers to molecules that have the property of non-superimposition of their mirror image partners, while the term "non-chiral" refers to molecules that are superimposable on their mirror image partners.

The term "stereoisomers" refers to compounds that have the same chemical composition but differ in the arrangement of their atoms or groups in space.

"Non-mirror stereoisomers" are stereoisomers with two or more chiral centers that are not mirror images of each other. Non-mirror stereoisomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. Mixtures of non-mirror stereoisomers can be separated by high-resolution analytical procedures such as electrophoresis and chromatography.

"Mirror isomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of each other.

Stereochemical definitions and conventions used herein generally follow those of S.P. Parker, ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, New York. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center. The prefixes d and l, or (+) and (-), are used to designate the rotational signature of plane-polarized light through the compound, where (-) or l means that the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. Specific stereoisomers may also be referred to as mirror image isomers, and mixtures of such isomers are often referred to as mirror image mixtures. A 50:50 mixture of mirror image isomers is called a racemic mixture or racemate, which can occur when stereoselectivity or stereospecificity is absent in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two mirror image isomers that lacks optical activity.

A "leaving group" refers to a functional group that can be substituted with another functional group. Certain leaving groups are well known in the art, and examples include, but are not limited to, halogen groups ( e.g. , chloro, bromo, iodo), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), and triflate.

The term "protecting group" refers to a substituent generally used to block or protect a particular functional group while reacting other functional groups on a compound. For example, an "amine-protecting group" is a substituent attached to the amino functional group in a blocking or protecting compound. Suitable amine-protecting groups include, but are not limited to, acetyl, trifluoroacetyl, tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethyleneoxycarbonyl (Fmoc). For a general description of protecting groups and their use, see TW Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991 or later editions.

III. Methods

Provided herein are methods for treating a B-cell proliferative disorder (e.g., diffuse large B-cell lymphoma (DLBCL), e.g., relapsed/refractory DLBCL) in a subject (human subject) in need thereof, comprising administering to the subject an effective amount of (a) an immunoconjugate comprising an antibody that binds to CD79b linked to a cytotoxic agent and (b) at least one additional therapeutic agent, wherein the treatment (e.g., a therapeutic regimen) prolongs progression-free survival (PFS) in the subject. In some embodiments, the treatment (e.g., a therapeutic regimen) prolongs overall survival (OS) in the subject. Also provided herein are methods for treating a B-cell proliferative disorder (e.g., diffuse large B-cell lymphoma (DLBCL), e.g., relapsed/refractory DLBCL) in a subject, comprising administering to the subject an effective amount of (a) an immunoconjugate comprising an antibody that binds to CD79b linked to a cytotoxic agent and (b) at least one additional therapeutic agent, wherein the treatment (e.g., a therapeutic regimen) prolongs the subject's overall survival (OS). In some embodiments, following treatment with the immunoconjugate and at least one additional therapeutic agent, the subject achieves a complete remission (CR), e.g., as described in further detail elsewhere herein. (Additional details regarding CR are provided below.) In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the at least one additional therapeutic agent is a cytotoxic agent.

Provided herein are methods for treating a B cell proliferative disorder in a subject (human subject) in need thereof, comprising administering to the subject an effective amount of (a) an immunoconjugate comprising an anti-CD79b antibody linked to a cytotoxic agent (i.e., an anti-CD79b immunoconjugate and (b) an alkylating agent. In some embodiments, the alkylating agent is 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazole-2- [amino]butyric acid or a salt or solvate thereof. In some embodiments, the alkylating agent is bendamustine or a salt or solvate thereof. In some embodiments, bendamustine or a salt or solvate thereof is bendamustine-HCl. In some embodiments, the anti-CD79b immunoconjugate is huMA79bv28-MC-vc-PAB-MMAE. In some embodiments, the immunoconjugate is polatuzumab vedotin (CAS Reg. No. 1313206-42-6).

Provided herein are methods for treating a B-cell proliferative disorder (e.g., DLBCL, e.g., relapsed/refractory DLBCL) in a subject (human subject) in need thereof, comprising administering to the subject an effective amount of (a) an immunoconjugate comprising an anti-CD79b antibody linked to a cytotoxic agent (i.e., an anti-CD79b immunoconjugate), (b) an alkylating agent, and (c) an anti-CD20 agent (e.g., an anti-CD20 antibody), wherein the treatment (e.g., a therapeutic regimen) prolongs progression-free survival (PFS) in the subject. In some embodiments, the treatment (e.g., a therapeutic regimen) prolongs overall survival (OS) in the subject. Also provided herein are methods for treating a B-cell proliferative disorder (e.g., DLBCL, e.g., relapsed/refractory DLBCL) in a subject, comprising administering to the subject an effective amount of (a) an immunoconjugate comprising an anti-CD79b antibody linked to a cytotoxic agent (i.e., an anti-CD79b immunoconjugate), (b) an alkylating agent, and (c) an anti-CD20 agent (e.g., an anti-CD20 antibody), wherein the treatment (e.g., a treatment regimen) prolongs the subject's overall survival (OS). In some embodiments, the subject achieves a complete remission (CR) following treatment with the immunoconjugate, alkylating agent, and anti-CD20 agent, e.g., as described in further detail elsewhere herein. (Additional details regarding CR are provided below.) In some embodiments, the anti-CD20 agent is an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20 antibody is a humanized B-Ly1 antibody. In some embodiments, the humanized B-Ly1 antibody is atuzumab. In some embodiments, the anti-CD20 antibody is ofatumumab, ublituximab, and/or ibritumomab tiuxetan. In some embodiments, the alkylating agent is 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid, or a salt or solvate thereof. In some embodiments, the alkylating agent is bendamustine, or a salt or solvate thereof. In some embodiments, bendamustine, or a salt or solvate thereof, is bendamustine-HCl. In some embodiments, the anti-CD79b immunoconjugate is huMA79bv28-MC-vc-PAB-MMAE. In some embodiments, the immunoconjugate is polatuzumab vedotin (CAS Reg. No. 1313206-42-6).

In some embodiments, the B-cell proliferative disorder is such as lymphoma (e.g., B-cell non-Hodgkin lymphoma (NHL)) and lymphocytic leukemia. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, b) small non-mitotic cell lymphoma/Burkitt's lymphoma (including endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma, and non-Burkitt's lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B-cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), 4) large cell lymphoma (including B-cell diffuse large cell lymphoma (DLCL), ), diffuse mixed cell lymphoma, immunoblastic lymphoma, primary septal B-cell lymphoma, angiocentric lymphoma-pulmonary B-cell lymphoma), f) hairy cell leukemia, g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia, h) acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasma cell neoplasm, plasma cell myeloma, multiple myeloma, plasma cell neoplasm, and/or j) Hodgkin's disease.

In some embodiments, the B cell proliferative disorder is cancer. In some embodiments, the B cell proliferative disorder is lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed remitting NHL, refractory NHL, refractory remitting NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), or mantle cell lymphoma. In some embodiments, the B cell proliferative disorder is NHL, such as remitting NHL and/or aggressive NHL. In some embodiments, the B-cell proliferative disorder is leukemic follicular lymphoma or diffuse large B-cell lymphoma (DLBCL).

The term "co-administration" or "co-administering" refers to administering an anti-CD79b immunoconjugate and at least one additional therapeutic agent (e.g., an alkylating agent and an anti-CD20 agent) as two (or more) separate formulations (or as a single formulation comprising an anti-CD79b immunoconjugate and at least one additive). When separate formulations are used, co-administration can be performed simultaneously or in any order, preferably with a time period during which all active agents simultaneously exert their biological activities. The anti-CD79b immunoconjugate and at least one additional therapeutic agent ( e.g., an alkylating agent and an anti-CD20 agent) are co-administered simultaneously or sequentially. In some embodiments, when all therapeutic agents are co-administered sequentially, the doses are administered on the same day in two separate administrations, or one agent is administered on day 1 and the other agent is co-administered between days 2 and 7, e.g., between days 2 and 4. In some embodiments, the term "sequentially" means within 7 days after the first dose of the component, e.g., within 4 days after the first dose of the component; the term "concurrently" means at the same time. The term "co-administered" with respect to a maintenance dose of an anti-CD79b immunoconjugate and at least one additional therapeutic agent ( e.g. , an alkylating agent and an anti-CD20 agent) means that the maintenance dose can be co-administered simultaneously if the treatment cycle is appropriate for all agents, e.g., weekly. Alternatively, for example, the anti-CD79b immunoconjugate is administered every one to three days, and at least one additional therapeutic agent ( e.g. , an alkylating agent and an anti-CD20 agent) is administered weekly. Alternatively, the maintenance doses are co-administered sequentially on one or several days.

The anti-CD79b immunoconjugates and additional therapeutic agents provided herein for use in any of the treatment methods described herein ( e.g. , alkylating agents and anti-CD20 agents) will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the reagents, the method of administration, the timing of administration, and other factors known to medical practitioners. The antibody is not required but is optionally formulated with one or more agents currently used to prevent or treat the disorder.

The amount of the anti-CD79b immunoconjugate and the additional therapeutic agent to be co-administered, as well as the timing of co-administration, will depend on the type (species, sex, age, weight, etc.) and condition of the patient being treated, as well as the severity of the disease or condition being treated. The anti-CD79b immunoconjugate and at least one additional therapeutic agent (e.g., an alkylating agent and an anti-CD20 agent) are suitably co-administered to the patient at once or over a series of treatments, for example, on the same day or the following day.

In some embodiments, the dose of the anti-CD79b immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin) is about any one of 1.4-5 mg/kg, 1.8-4 mg/kg, 1.8-3.2 mg/kg, and/or 1.8-2.4 mg/kg. In some embodiments of any of these methods, the dose of the anti-CD79 immunoconjugate is about any one of 1.4, 1.8, 2.0, 2.2, 2.4, 2.8, 3.2, 3.6, 4.0, 4.4, and/or 4.8 mg/kg. In some embodiments, the dose of the anti-CD79b immunoconjugate is about 1.8 mg/kg. In some embodiments, the dose of the anti-CD79b immunoconjugate is about 2.4 mg/kg. In some embodiments, the dose of the anti-CD79b immunoconjugate is about 3.2 mg/kg. In some embodiments, the dose of the anti-CD79b immunoconjugate is about 3.6 mg/kg. In some embodiments of any of these methods, the anti-CD79b immunoconjugate is administered every 3 weeks. In some embodiments, the anti-CD79b immunoconjugate is administered by intravenous infusion. The dose administered by infusion is about 1 μg/m ² to about 10,000 μg/m ² per dose, typically once a week for a total of one, two, three, or four doses. Alternatively, the dosage range is about 1 μg/m ² to about 1000 μg/m 2, about 1 μg/m ² to about 800 μg/m ² , about 1 μg/m 2 to about 600 μg/m 2, about 1 μg/m ² to about 400 μg/m ² , about 10 μg/m 2 to about 500 μg/m 2, about 10 μg/m ² to about 300 μg/m ² , about 10 μg/m 2 to about 200 μg/m 2, and about 1 μg/m ² to about 200 μg/m ^2. The dosage can be administered once a day, once a week, multiple times a week but less than once a day, multiple times a month but less than once a day, multiple times a month but less than once a week, once a month, or intermittently to alleviate or reduce the symptoms of the disease. Administration may be continued at any disclosed interval until the symptoms of the B-cell proliferative disorder or tumor being treated are alleviated. In cases where such alleviation or alleviation is prolonged by continued administration, administration may be continued after alleviation or alleviation of symptoms is achieved.

In some embodiments, the dose of the anti-CD20 agent ( e.g. , anti-CD20 antibody) is about 300-1600 mg/m ² and/or 300-2000 mg. In some embodiments, the dose of the anti-CD20 antibody is about 300, 375, 600, 1000, or 1250 mg/m ² and/or any of 300, 1000, or 2000 mg. In some embodiments, the anti-CD20 antibody is rituximab and the dose administered is 375 mg/m ^2. In some embodiments, the anti-CD20 antibody is atoxizumab and the dose administered is 1000 mg/m ^2. In some embodiments, the anti-CD20 antibody is administered q3w (i.e., every 3 weeks). In some embodiments, the dose of the non-fucosylated anti-CD20 antibody (preferably a non-fucosylated humanized B-Ly1 antibody) can be 800 to 1600 mg (in one embodiment, 800 to 1200 mg) on days 1, 8, and 15 of a 3- to 6-week dosing cycle, followed by a dose of 400 to 1200 mg (in one embodiment, 800 to 1200 mg) on day 1 of up to nine 3- to 4-week dosing cycles. In some embodiments, the dose is a flat dose of 1000 mg over a three-week dosing schedule, with an additional 1000 mg flat dose cycle in the second week.

Exemplary dosing regimens for combination therapy of an anti-CD79b immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin) and other agents include, but are not limited to, an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE) administered at approximately 1.4-5 mg/ ^kg q3w, plus rituximab at 375 mg/ ^m q3w, and bendamustine ( e.g., bendamustine-HCl) at 25-120 mg/m q3w on days 1 and 2 of a 21-day cycle (e.g. , q3w days 1 and 2). In some embodiments, the anti-CD79 immunoconjugate is administered at about 1.8 mg/kg, 2.0 mg/kg, 2.2 mg/kg, 2.4 mg/kg, 3.2 mg/kg, or 4.0 mg/kg. In some embodiments, the anti-CD79b immunoconjugate is administered at about 1.8 mg/kg. In some embodiments, the anti-CD79b immunoconjugate is administered at about 2.4 mg/kg. In some embodiments, bendamustine ( e.g. , bendamustine-HCl) is administered at about 90 mg/m ² .

Another exemplary dosing regimen for combination therapy of an anti-CD79b immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin) and other agents includes, but is not limited to, an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin) administered at about 1.4-5 mg/kg q3w, plus 1000 mg/m ² q3w atozotocin, and 25-120 mg/m ² bendamustine (e.g., bendamustine-HCl) administered on days 1 and 2 of a 21-day cycle (e.g., q3w days 1 and 2). In some embodiments, the anti-CD79 immunoconjugate is administered at about 1.8 mg/kg, 2.0 mg/kg, 2.2 mg/kg, 2.4 mg/kg, 3.2 mg/kg, or 4.0 mg/kg. In some embodiments, the anti-CD79b immunoconjugate is administered at about 1.8 mg/kg. In some embodiments, the anti-CD79b immunoconjugate is administered at about 2.4 mg/kg. In some embodiments, bendamustine (e.g., bendamustine-HCl) is administered at about 90 mg/m ² .

The immunoconjugates provided herein (and any other therapeutic agents, such as alkylating agents and anti-CD20 agents) for use in any of the treatment methods described herein can be administered by any suitable route, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. A variety of dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations over multiple time points, bolus administration, and pulse infusion.

Provided herein are methods for treating diffuse large B-cell lymphoma (DLBCL) in a subject (human subject) in need thereof, comprising administering to the subject an effective amount of: (a) an immunoconjugate comprising the formula wherein Ab is an anti-CD79b antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, and wherein p is 1 to 8; (b) an alkylating agent, and (c) an anti-CD20 antibody, wherein the treatment (e.g., the treatment regimen) prolongs progression-free survival (PFS) of the individual. In some embodiments, the treatment (e.g., the treatment regimen) prolongs overall survival of the individual. Also provided herein are methods for treating diffuse large B-cell lymphoma (DLBCL) in a subject (human subject) in need thereof, comprising administering to the subject an effective amount of: (a) an immunoconjugate comprising the formula wherein Ab is an anti-CD79b antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, and wherein p is 1 to 8; (b) an alkylating agent, and (c) an anti-CD20 antibody, wherein the treatment (e.g., a therapeutic regimen) prolongs the overall survival (OS) of the individual. In some embodiments, the individual achieves a complete remission (CR) after treatment with the anti-CD79b immunoconjugate, the alkylating agent, and the anti-CD20 antibody. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 37 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, p is 2 to 7, 2 to 6, 2 to 5, 3 to 5, or 3 to 4. In some embodiments, p is 3.4. In some embodiments, the anti-CD79b immunoconjugate is huMA79bv28-MC-vc-PAB-MMAE. In some embodiments, the immunoconjugate is polatuzumab vedotin (CAS Reg. No. 1313206-42-6). In some embodiments, the alkylating agent is 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid or a salt thereof. In some embodiments, the alkylating agent is bendamustine or a salt or solvate thereof. In some embodiments, bendamustine or a salt or solvate thereof is bendamustine-HCl. In some embodiments, the anti-CD20 antibody is rituximab, humanized B-Ly1 antibody, atuzumab, ofatumumab, ublituximab, or ibritumomab tiuxetan.

In some embodiments, the anti-CD79b immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin) is administered at a dose of 1.8 mg/kg. Alternatively or additionally, in some embodiments, the alkylating agent (e.g., bendamustine or bendamustine-HCl) is administered at a dose of 90 mg/m ^2. Alternatively or additionally, in some embodiments, the anti-CD20 antibody is administered at a dose of 375 mg/m ^2. In some embodiments, the anti-CD79b immunoconjugate, alkylating agent, and anti-CD20 antibody are administered for at least six 21-day cycles. In some embodiments, for a 21-day cycle in Cycle 1, the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on day 2, the alkylating agent is administered intravenously at a dose of 90 mg/m2 on days 2 and 3, and the anti-CD20 antibody is administered intravenously at a dose of 375 mg/m2 on day 1, and wherein for Cycles 2-6, the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on day 1, the alkylating agent is administered intravenously at a dose of 90 mg/m2 on days 1 and 2, and the anti-CD20 antibody is administered intravenously at a dose of 375 mg/m2 on day 1 in each 21-day cycle. Exemplary dosing and administration schedules are provided in Table A1 below:

In some embodiments, the anti-CD79b immune conjugate and the alkylating agent are administered sequentially on day 2 of cycle 1. In some embodiments, the anti-CD79b immune conjugate is administered before the alkylating agent. In some embodiments, the anti-CD79b immune conjugate, the alkylating agent, and the anti-CD20 antibody are administered sequentially on day 1 of cycles 2-6. In some embodiments, the anti-CD20 antibody is administered before the anti-CD79b immune conjugate, and the anti-CD79b immune conjugate is administered before the alkylating agent. In some embodiments, the anti-CD79b immune conjugate, the alkylating agent, and the anti-CD20 antibody are further administered after cycle 6. In some embodiments, in each cycle after cycle 6, the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on day 1, the alkylating agent is administered intravenously at a dose of 90 mg/m ² on days 1 and 2, and the anti-CD20 antibody is administered intravenously at a dose of 375 mg/m ² on day 1. In some embodiments, the anti-CD20 antibody is administered before the anti-CD79b immune conjugate, and the anti-CD79b immune conjugate is administered before the alkylating agent.

In some embodiments, the anti-CD79b immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin) is administered at a dose of 1.8 mg/kg. Alternatively or additionally, in some embodiments, the alkylating agent (e.g., bendamustine or bendamustine-HCl) is administered at a dose of 90 mg/m ^2. Alternatively or additionally, in some embodiments, the anti-CD20 antibody is administered at a dose of 375 mg/m ^2. In some embodiments, the anti-CD79b immunoconjugate, alkylating agent, and anti-CD20 antibody are administered for at least six 21-day cycles. In some embodiments, the anti-CD79b immunoconjugate, alkylating agent, and anti-CD20 antibody are administered for no more than six 21-day cycles. In some embodiments, for each 21-day cycle of Cycles 1-6, the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on day 1, the alkylating agent is administered intravenously at a dose of 90 mg/m ² on days 1 and 2, and the anti-CD20 antibody is administered intravenously at a dose of 375 mg/m ² on day 1. In some embodiments, in each 21-day cycle after cycle 6, the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on day 1, the alkylating agent is administered intravenously at a dose of 90 mg/m ² on days 1 and 2, and the anti-CD20 antibody is administered intravenously at a dose of 375 mg/m ² on day 1. Another exemplary dosing and administration schedule is provided in Table A2 below:

In some embodiments, an anti-CD79b immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin), an alkylating agent, and an anti-CD20 antibody are administered sequentially on day 1 of each 21-day cycle (e.g., cycles 1-6 and/or cycles beyond cycle 6). In some embodiments, an anti-CD20 antibody is administered before an anti-CD79b immunoconjugate, and an anti-CD79b immunoconjugate is administered before an alkylating agent. In some embodiments, an anti-CD79b immunoconjugate, an alkylating agent, and an anti-CD20 antibody are administered in any order.

Also provided are methods of treating diffuse large B-cell lymphoma (DLBCL) in a subject (human subject) in need thereof, comprising administering to the subject an effective amount of (a) an immunoconjugate comprising the formula wherein Ab is an anti-CD79b antibody comprising (i) a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 20, wherein p is 2 to 5, (b) bendamustine or a salt or solvate thereof, and (c) rituximab, wherein the immunoconjugate is administered at a dose of 1.8 mg/kg, bendamustine or a salt or solvate thereof is administered at a dose of 90 mg/m ² , and rituximab is administered at a dose of 375 mg/m ² , and wherein the treatment prolongs progression-free survival (PFS) and/or overall survival (OS) of the subject. In some embodiments, p is 2 to 4 or 3 to 4. In some embodiments, p is 3.5. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:36 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:35. In some embodiments, the anti-CD79b immunoconjugate is huMA79bv28-MC-vc-PAB-MMAE. In some embodiments, the immunoconjugate is polatuzumab vedotin (CAS Reg. No. 1313206-42-6). In some embodiments, the bendamustine or a salt or solvate thereof is bendamustine-HCl. In some embodiments, the subject achieves a complete remission (CR) following treatment with the anti-CD79b immunoconjugate, bendamustine or a salt or solvate thereof (e.g., bendamustine-HCl), and rituximab.

The anti-CD79b immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin), the alkylating agent (e.g., bendamustine or bendamustine-HCl), and the anti-CD20 antibody (e.g., rituximab) can be administered by the same route of administration or by different routes of administration. In some embodiments, the anti-CD79b immunoconjugate is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraocularly, by implantation, inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraocularly, by implantation, inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the anti-CD20 antibody (e.g., rituximab) is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraocularly, by implant, inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the anti-CD79b immunoconjugate, alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl), and anti-CD20 antibody (e.g., rituximab) are administered by intravenous infusion. An effective amount of the anti-CD79b immunoconjugate, alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl), and anti-CD20 antibody (e.g., rituximab) can be administered for the prevention or treatment of a disease.

In some embodiments, an anti-CD79b immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin), bendamustine (e.g., bendamustine-HCl), and rituximab are administered for at least six 21-day cycles, wherein for the first 21-day cycle, the anti-CD79b immunoconjugate is administered intravenously at a dose of 1.8 mg/kg on day 2, bendamustine (e.g., bendamustine-HCl) is administered intravenously at a dose of 90 mg/m ² on days 2 and 3, and rituximab is administered intravenously at a dose of 375 mg/m 2 on day 1. ² , and wherein in each 21-day cycle following cycle 1, the anti-CD79b immunoconjugate is administered intravenously at a dose of 1.8 mg/kg on day 1, bendamustine (e.g., bendamustine-HCl) is administered intravenously at a dose of 90 mg/m ² on days 1 and 2, and rituximab is administered intravenously at a dose of 375 mg/m ² on day 1 in each 21-day cycle. Exemplary dosing and administration schedules are provided in Table B1 below:

In some embodiments, an anti-CD79b immune conjugate and bendamustine (e.g., bendamustine-HCl) are administered sequentially on day 2 of cycle 1. In some embodiments, the anti-CD79b immune conjugate is administered before bendamustine (e.g., bendamustine-HCl). In some embodiments, the immune conjugate, bendamustine (e.g., bendamustine-HCl), and rituximab are administered sequentially on day 1 of cycles 2-6. In some embodiments, rituximab is administered before the anti-CD79b immune conjugate, and the anti-CD79b immune conjugate is administered before bendamustine (e.g., bendamustine-HCl). In some embodiments, an anti-CD79b immune conjugate, bendamustine (e.g., bendamustine-HCl), and rituximab are further administered after cycle 6. In some embodiments, in each cycle after cycle 6, the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on day 1, bendamustine (e.g., bendamustine-HCl) is administered intravenously at a dose of 90 mg/m ² on days 1 and 2, and rituximab is administered intravenously at a dose of 375 mg/m ² on day 1 in each 21-day cycle. In some embodiments, rituximab is administered before the anti-CD79b immune conjugate, and the anti-CD79b immune conjugate is administered before bendamustine (e.g., bendamustine-HCl).

In some embodiments, the anti-CD79b immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin) is administered at a dose of 1.8 mg/kg. Alternatively or additionally, in some embodiments, bendamustine (e.g., bendamustine-HCl) is administered at a dose of 90 mg/m ^2. Alternatively or additionally, in some embodiments, rituximab is administered at a dose of 375 mg/m ^2. In some embodiments, the anti-CD79b immunoconjugate, bendamustine (e.g., bendamustine-HCl), and rituximab are administered for at least six 21-day cycles. In some embodiments, the anti-CD79b immunoconjugate, bendamustine (e.g., bendamustine-HCl), and rituximab are administered for no more than six 21-day cycles. In some embodiments, for each 21-day cycle of Cycles 1-6, the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on Day 1, bendamustine (e.g., bendamustine-HCl) is administered intravenously at a dose of 90 mg/m ² on Days 1 and 2, and anti-rituximab is administered intravenously at a dose of 375 mg/m ² on Day 1. In some embodiments, in each 21-day cycle after cycle 6, the immune conjugate is administered intravenously at a dose of 1.8 mg/kg on day 1, bendamustine (e.g., bendamustine-HCl) is administered intravenously at a dose of 90 mg/m ² on days 1 and 2, and rituximab is administered intravenously at a dose of 375 mg/m ² on day 1. Another exemplary dosing and administration schedule is provided in Table B2 below:

In some embodiments, an anti-CD79b immune conjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin), bendamustine (e.g., bendamustine-HCl), and rituximab are administered sequentially on day 1 of each 21-day cycle (e.g., cycles 1-6 and/or cycles other than cycle 6). In some embodiments, rituximab is administered before the anti-CD79b immune conjugate, and the anti-CD79b immune conjugate is administered before bendamustine (e.g., bendamustine-HCl). In some embodiments, the anti-CD79b immune conjugate, bendamustine (e.g., bendamustine-HCl), and rituximab are administered in any order.

In any of the above embodiments (e.g., dosing and administration regimens provided herein), the anti-CD20 antibody (e.g., rituximab) is administered for between 6 and 8 cycles. In some embodiments, the anti-CD20 antibody is administered for more than 8 cycles. In any of the above embodiments (e.g., dosing and administration regimens provided herein), wherein the individual has peripheral neuropathy (e.g., before starting treatment) or develops peripheral neuropathy (e.g., during treatment), the dose of the anti-CD79b immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin) in any of the embodiments provided herein is reduced to 1.4 mg/kg. Additionally or alternatively, wherein the individual has neutropenia (e.g., Grade 3-4 neutropenia) or thrombocytopenia (e.g., Grade 3-4 thrombocytopenia), e.g., prior to initiating treatment, or develops neutropenia (e.g., Grade 3-4 neutropenia) or thrombocytopenia (e.g., Grade 3-4 thrombocytopenia), the dose of the alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) is reduced to about 70 mg/m ² or about 50 mg/m ² , e.g., during treatment.

In some embodiments of any of the methods described herein, CR (complete response/complete remission) is a complete radiographic response. In some embodiments, a complete radiographic response is assessed by computed tomography (CT). In some embodiments, a complete radiographic response is characterized by the following criteria: (a) all target nodes or nodal masses in the subject have regressed to a maximum diameter of 1.5 cm as measured by CT, (b) any previously unmeasured lesions in the subject have disappeared, (c) the subject has no extralymphatic sites of disease, and (d) the subject has no organomegaly (i.e., abnormal enlargement of an organ). In some embodiments, a complete radiographic response is further characterized by normal bone marrow morphology. Alternatively or additionally, in some embodiments, CR is a complete metabolic response (CMR). In some embodiments, CMR is assessed by F ¹⁸ -2-fluoro-2-deoxy-d-glucose positron emission tomography-computed tomography (FDG-PET/CT). In some embodiments, CMR is characterized by the following criteria: (a) a score of 1 (FDG uptake no higher than background), 2 (uptake longitudinally), or 3 (uptake > longitudinally but > background) according to the Deauville 5-point scale. liver) with or without residual mass, where residual mass is allowed if the disease is not FDG-avid, and (b) no evidence of FDG-avid lesions in the bone marrow. Further details on clinical staging and response criteria for lymphomas such as DLBCL are provided, for example, in Van Heertum et al. (2017) Drug Des. Devel. Ther. 11:1719-1728; Cheson et al. (2016) Blood. 128:2489-2496; Cheson et al. (2014) J. Clin. Oncol. 32(27):3059-3067; Barrington et al. (2017) J. Clin. Oncol. 32(27):3048-3058; Gallamini et al. (2014) Haematologica. 99(6):1107-1113; Barrinton et al. (2010) Eur. J. Nucl. Med. Mol. Imaging. 37(10):1824-33; Moskwitz (2012) Hematology Am Soc. Hematol. Educ. Program 2012:397-401; and Follows et al. (2014) Br. J. Haematology 166:34-49. The progress of any of the treatment methods provided herein can be monitored by techniques known in the art.

In some embodiments, PFS is measured from the date of the first complete or partial response to treatment (e.g., a documented complete or partial response to treatment) to the date of disease progression, recurrence, or death from any cause. In some embodiments, PFS is measured based on PET-CT or CT alone from the date of the first treatment to the first occurrence of progression, recurrence, or death from any cause. In some embodiments, complete response, partial response, progression, and/or recurrence are assessed according to the modified Lugano criteria, as measured by PET/CT or CT (see, e.g., Cheson et al. (2014) "Recommendations for initial evaluation, staging, and response assessment of hodgkin and non-hodgkin lymphoma: The Lugano Classification." J. Clin. Oncol. 32:3059-67). In some embodiments, PFS is measured as the time from the start of treatment ( e.g. , treatment with an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin), an alkylating agent ( e.g. , bendamustine, such as bendamustine-HCl), and an anti-CD20 antibody ( e.g. , rituximab)) to the time of death. In some embodiments, PFS is the median PFS. In some embodiments, treatment with an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin), an alkylating agent (e.g., bendamustine, such as bendamustine-HCl), and an anti-CD20 antibody (e.g., rituximab)) is the time from the start of treatment to the time of death. In some embodiments, treatment with an anti-CD20 antibody (e.g., rituximab) or a combination of an alkylating agent ( e.g. , bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody ( e.g. , rituximab) prolongs PFS in a subject to at least about any of 6, 6.1, 6.2, 6.3, 6.4, 6.5, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, including any ranges therebetween, or more than 17 months. In some embodiments, treatment with an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin), an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl), and an anti-CD20 antibody (e.g., rituximab) extends PFS in a subject to at least about 7.6 months. In some embodiments, treatment with an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin), an alkylating agent ( e.g. , bendamustine or bendamustine-HCl), and an anti-CD20 antibody ( e.g., rituximab) extends PFS in a subject to at least about 11.1 months. In some embodiments, the treatment (e.g., a treatment regimen) prolongs PFS in the subject by at least any of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 (including any ranges therebetween), or more than 8.5 months compared to an subject with DLBCL (e.g., a subject with DLBCL who did not receive treatment). In some embodiments, the treatment (e.g., treatment regimen) increases the subject's PFS by at least about any of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 (including any ranges therebetween), or more than 8.5 months compared to a subject with DLBCL who received a treatment comprising an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody (e.g., rituximab) without an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin). In some embodiments, the median PFS is extended to at least about 11.1 months ( e.g. , at least about 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11, including any ranges therebetween) compared to individuals with DLBCL who received a therapy comprising an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody (e.g., rituximab) without an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin). In some embodiments, the median PFS with a 95% confidence interval is between 6.2 and 13.9 months. In some embodiments, the median PFS is extended to at least about 7.6 months (e.g., about any one of 4, 4.5, 5, 5.5, 6, 6.5, 7, or 7.5, including any range therebetween) compared to individuals with DLBCL who received a therapy comprising an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody (e.g., rituximab) without an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin). In some embodiments, the median PFS with a 95% confidence interval is between 6.0 and 17.0 months.

In some embodiments, OS is measured as the time from diagnosis to death. In some embodiments, overall survival (OS) is measured as the time from the start of treatment (e.g., with an anti-CD79 immunoconjugate, an alkylating agent (e.g., bendamustine, such as bendamustine-HCl), and an anti-CD20 antibody (e.g., rituximab)) until death (e.g., from any cause). In some embodiments, treatment with an anti-CD79 immunoconjugate, an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl), and an anti-CD20 antibody (e.g., rituximab) extends the subject's OS to at least about any of 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5 (including any ranges therebetween), or more than 12.5 months. In some embodiments, treatment with an anti-CD79 immunoconjugate, an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl), and an anti-CD20 antibody (e.g., rituximab) extends the subject's OS to at least about 12.4 months. In some embodiments, the treatment (e.g., a treatment regimen) extends the subject's OS by at least about any of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 (including any ranges therebetween), or more than 7.5 months compared to a subject with DLBCL (e.g., a subject with DLBCL who did not receive treatment). In some embodiments, the treatment (e.g., a treatment regimen) increases the subject's OS by at least about any of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 (including any ranges therebetween), or more than 7.5 months compared to a subject with DLBCL who received a treatment comprising an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody (e.g., rituximab) without an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin). In some embodiments, the median OS is extended to at least about 12.4 months ( e.g. , at least about 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12, inclusive), with a hazard ratio (HR) of 0.42 or less, compared to individuals with DLBCL who received a therapy comprising an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody (e.g., rituximab) without an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin). In some embodiments, the median OS with a 95% confidence interval is at least 9.0 months.

In some embodiments, the individual is an adult. In some embodiments, the individual has activated B-cell DLBCL (ABC DLBCL). In some embodiments, the individual has germinal center B-cell-like DLBCL (GCB DLBCL). In some embodiments, the individual has DLBCL not otherwise specified (DLBCL-NOS). In some embodiments, DLBCL classification by cell of origin (COO) is performed using the NanoString Research-Only Lymphoma Subtype Test (LST) assay. In some embodiments, the individual has dual-expression lymphoma (DEL). DEL is a DLBCL characterized by overexpression of MYC and BLC2. In some embodiments, DEL is determined by immunohistochemistry (IHC). In some embodiments, IHC assays are performed using monoclonal antibodies against BCL2 (124) and MYC (Y69). In some embodiments, IHC assays are performed on the Ventana Benchmark XT platform. In some embodiments, MYC overexpression is characterized by 40% of tumor cell nuclei stained positively. The characteristic of BCL2 overexpression is 50% of tumor cell nuclei stain positively. In some embodiments, the individual has relapsed/refractory DLBCL (RR-DLBCL). In some embodiments, RR-DLBCL is characterized by (a) the appearance of a new lesion or an increase in size of a previously involved disease site by more than 50% after achieving remission, (b) an increase of more than 50% in the maximum diameter of any previously identified abnormal node with a minor axis greater than 1 cm, or in the sum of the product diameters (SPD) of more than one abnormal node, (c) an increase of more than 50% in the SPD of any previously identified abnormal node from its nadir, and/or (d) the appearance of a new lesion during or at the end of treatment. Further details regarding the criteria for characterizing RR-DLBCL are provided in Cheson et al. (1999) J. Clin. Oncol. 17(4): 1244. In some embodiments, the individual does not have grade 3 follicular lymphoma, transformed and rheumatic non-Hodgkin lymphoma, or central nervous system (CNS) lymphoma. In some embodiments, the individual is not a candidate for autologous stem cell transplantation (ASCT), e.g., first-line ASCT, second-line ASCT, third-line ASCT, or beyond third-line ASCT. In some embodiments, the individual has a T-cell/tissue-rich large B-cell lymphoma. In some embodiments, the individual has a high-grade B-cell lymphoma with a MYC and BCL-2 and/or BCL-6 rearrangement. In some embodiments, the individual has high-grade B-cell lymphoma, not otherwise specified (NOS). In some embodiments, the individual has primary longitudinal (thymic) large B-cell lymphoma. In some embodiments, the individual has Epstein-Barr virus-positive DLBCL, not otherwise specified (NOS). In some embodiments, the individual has at least one two-dimensionally measurable lesion on imaging scan with a maximum dimension of >1.5 cm. In some embodiments, the individual has not received prior treatment for DLBCL (e.g., prior chemotherapy or prior antibody therapy). In some embodiments, the individual has undergone at least one prior treatment for DLBCL. In some embodiments, the individual has undergone at least two prior treatments for DLBCL. In some embodiments, the individual has received at least three prior therapies for DLBCL. In some embodiments, the individual has received more than three prior therapies for DLBCL. In some embodiments, the individual has failed a prior autologous stem cell transplant (ASCT), e.g., relapsed after ASCT or is refractory to ASCT. In some embodiments, the individual has received prior treatment with an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl). In some embodiments, the duration of prior treatment with an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) is 1 year. In some embodiments, the individual has received prior therapy with an anti-CD20 agent, such as an anti-CD-20 antibody. In some embodiments, the individual is refractory to the most recent prior therapy. In some embodiments, the most recent prior therapy is standard of care therapy. In some embodiments, an individual is refractory to treatment if they have demonstrated a partial response, minimal response, or no response to treatment. In some embodiments, the individual is female. In some embodiments, the individual is an adult with DLBCL not otherwise specified (NOS) who has received at least one prior therapy (e.g., for DLBCL).

In some embodiments, the DLBCL is BCL2 positive (e.g., positive for a BCL2 gene rearrangement, t(14;18)(q32;q21)). In some embodiments, the DLBCL is BCL2 negative (e.g., negative for a BCL2 gene rearrangement, t(14;18)(q32;q21)). In some embodiments, the individual has (e.g., further has) one or more of the following characteristics: (a) at least one two-dimensionally measurable lesion on an imaging scan, defined as >1.5 cm in its longest dimension; (b) a life expectancy of at least 24 weeks; (c) an Eastern Cooperative Oncology Group (ECOG) performance status of 0, 1, or 2; (d) adequate hematologic function.

In some embodiments, the individual does not have a history of severe allergic or anaphylactic reactions to humanized or murine monoclonal antibodies (MAbs or recombinant antibody-related fusion proteins); known sensitivity or anaphylaxis to mouse products; or contraindications to bendamustine (e.g., bendamustine-HCl), rituximab, or atezolizumab. In some embodiments, the individual does not have a history of allergy to mannitol. In some embodiments, the individual does not receive corticosteroids in doses greater than 30 mg/day of prednisone or equivalent for purposes other than lymphoma symptom management.

In some embodiments of any of these methods, if administration is intravenous, the initial infusion of the anti-CD79b immunoconjugate or additional therapeutic agent can be longer than subsequent infusions, for example, an initial infusion of about 90 minutes and subsequent infusions of about 30 minutes (if the initial infusion is well tolerated).

Provided herein are methods for improving PFS in an individual (human individual) suffering from DLBCL, comprising administering to the individual an effective amount of: (a) an immunoconjugate comprising the formula wherein Ab is an anti-CD79b antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, and wherein p is 1 to 8; (b) an alkylating agent, and (c) an anti-CD20 antibody according to any embodiment described herein. In some embodiments, improving PFS comprises improving median PFS. In some embodiments, the method improves OS in individuals with DLBCL. In some embodiments, improving OS comprises improving median OS. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 37 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, p is 2 to 7, 2 to 6, 2 to 5, 3 to 5, or 3 to 4. In some embodiments, p is 3.4. In some embodiments, the anti-CD79b immunoconjugate is huMA79bv28-MC-vc-PAB-MMAE. In some embodiments, the immunoconjugate is polatuzumab vedotin (CAS Reg. No. 1313206-42-6). In some embodiments, the alkylating agent is 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid or a salt thereof. In some embodiments, the alkylating agent is bendamustine or a salt or solvate thereof. In some embodiments, bendamustine or a salt or solvate thereof is bendamustine-HCl. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the improvement in PFS is relative to that of individuals with DLBCL treated with an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody without an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin). In some embodiments, the improvement in OS is relative to that of individuals with DLBCL treated with an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody without an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin).

Provided herein are methods for improving OS in a subject (human subject) suffering from DLBCL, comprising administering to the subject an effective amount of: (a) an immunoconjugate comprising the formula wherein Ab is an anti-CD79b antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, and wherein p is 1 to 8; (b) an alkylating agent, and (c) an anti-CD20 antibody according to any embodiment described herein. In some embodiments, improving OS comprises improving median OS. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 19 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 37 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, p is 2 to 7, 2 to 6, 2 to 5, 3 to 5, or 3 to 4. In some embodiments, p is 3.4. In some embodiments, the anti-CD79b immunoconjugate is huMA79bv28-MC-vc-PAB-MMAE. In some embodiments, the immunoconjugate is polatuzumab vedotin (CAS Reg. No. 1313206-42-6). In some embodiments, the alkylating agent is 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid or a salt thereof. In some embodiments, the alkylating agent is bendamustine or a salt or solvate thereof. In some embodiments, bendamustine or a salt or solvate thereof is bendamustine-HCl. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the improvement in OS is compared to individuals with DLBCL treated with an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody without an anti-CD79 immunoconjugate (e.g., huMA79bv28-MC-vc-PAB-MMAE or polatuzumab vedotin).

Also provided are uses of the anti-CD79b immunoconjugates described herein in the preparation or manufacture of a medicament for use in combination with at least one additional therapeutic agent, e.g., an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody (e.g., rituximab), for treating DLBCL in a subject in need thereof (e.g., a human subject having one or more of the characteristics described above), wherein administration prolongs the subject's PFS and/or OS.

Provided is an immunoconjugate comprising the formula wherein Ab is an anti-CD79b antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 26 NO: 26, and wherein p is between 1 and 8, the antibody is for use in a method of treating diffuse large B-cell lymphoma (DLBCL) in a subject (human subject) in need thereof, the method comprising administering to the subject an effective amount of an immunoconjugate, an alkylating agent, and an anti-C20 antibody, wherein the treatment prolongs progression-free survival (PFS) and/or overall survival (OS) of the subject. In some embodiments, the immunoconjugate is used in the methods described herein. In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising (i) a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 20.

Also provided is an immunoconjugate comprising the formula wherein Ab is an anti-CD79b antibody comprising (i) a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 20, wherein p is between 2 and 5, the antibody being for use in a method of treating diffuse large B-cell lymphoma (DLBCL) in a subject (human subject) in need thereof, the method comprising administering to the subject an effective amount of (a) an immunoconjugate, (b) bendamustine (e.g., bendamustine-HCl), and (c) rituximab, wherein the immunoconjugate is administered at a dose of 1.8 mg/kg, the bendamustine (e.g., bendamustine-HCl) is administered at a dose of 90 mg/m ² , and the rituximab is administered at a dose of 375 mg/m 2. ² , and wherein the treatment prolongs the subject's progression-free survival (PFS) and/or overall survival (OS). In some embodiments, the immunoconjugate is used in a use according to the methods described herein. In some embodiments, p is between 3 and 4. In some embodiments, p is 3.5. In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 36, and wherein the light chain comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 37 and a light chain comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 38.

IV. Immunoconjugates Comprising an Anti-CD79b Antibody and a Drug/Cytotoxic Agent ("Anti-CD79b Immunoconjugates")

In some embodiments, an anti-CD79b immunoconjugate comprises an anti-CD79b antibody (Ab) that targets cancer cells (e.g., diffuse large B-cell lymphoma (DLBCL) cells), a drug moiety (D), and a linker moiety (L) that attaches Ab to D. In some embodiments, the anti-CD79b antibody is linked to the linker moiety (L) via one or more amino acid residues (e.g., lysine and/or cysteine). In some formulas Ab-(L-D)p, (a) Ab is an anti-CD79b antibody that binds to CD79b on the surface of cancer cells (e.g., DLBCL cells); (b) L is a linker; (c) D is a cytotoxic agent; and (d) p ranges from 1 to 8.

Exemplary anti-CD79b immunoconjugates comprise Formula I: (I) Ab-(LD) _p wherein p is 1 to about 20 ( e.g. , 1 to 15, 1 to 10, 1 to 8, 2 to 5, or 3 to 4). In some embodiments, the number of drug moieties that can be conjugated to an anti-CD79b antibody is limited by the number of free cysteine residues. In some embodiments, free cysteine residues are introduced into the antibody amino acid sequence by methods described elsewhere herein. Exemplary anti-CD79b immunoconjugates of Formula I include, but are not limited to, anti-CD79b antibodies comprising 1, 2, 3, or 4 engineered cysteine amino acids (Lyon, R. et al. (2012) Methods in Enzym. 502: 123-138). In some embodiments, one or more free cysteine residues are already present in the anti-CD79b antibody without engineering, in which case the existing free cysteine residues can be used to conjugate the anti-CD79b antibody to the drug/cytotoxic agent. In some embodiments, the anti-CD79b antibody is exposed to reducing conditions and then conjugated to the drug/cytotoxic agent to generate the one or more free cysteine residues.

A. Demonstration Connector

A "linker" (L) is a bifunctional or multifunctional moiety that can be used to link one or more drug moieties (D) to an anti-CD79b antibody (Ab) to form an anti-CD79b immunoconjugate of Formula I. In some embodiments, anti-CD79b immunoconjugates can be prepared using a linker having reactive functional groups that are covalently linked to the drug and the anti-CD79b antibody. For example, in some embodiments, a cysteine thiol of an anti-CD79b antibody (Ab) can form a bond with a reactive functional group of a linker or a drug-linker intermediate to prepare an anti-CD79b immunoconjugate.

In one embodiment, the linker has a functional group capable of reacting with a free cysteine present on the anti-CD79b antibody to form a covalent bond. Exemplary reactive functional groups include, but are not limited to, cis-butylenediamide, halogenated acetamide, α-halogenated acetyl, activated esters (such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters), acid anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, for example, Klussman et al. (2004), Bioconjugate Chemistry 15(4):765-773, page 766 for conjugation methods and examples herein.

In some embodiments, the linker has a functional group capable of reacting with an electrophilic group present on the anti-CD79b antibody. Exemplary electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, the heteroatom of the reactive functional group of the linker can react with the electrophilic group on the antibody and form a covalent bond with the antibody unit. Exemplary reactive functional groups include, but are not limited to, hydrazides, oximes, amines, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazides.

In some embodiments, the linker comprises one or more linker components. Exemplary linker components include, for example, 6-cis-butenediimidohexanoyl ("MC"), cis-butenediimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), and 4-(N-cis-butenediimidomethyl)cyclohexane-1-carboxylate ("MCC"). Various linker components are known in the art, some of which are described below.

In some embodiments, the linker is a "cleavable linker" that facilitates drug release. Non-limiting exemplary cleavable linkers include acid-labile linkers ( e.g., comprising a hydrazone), protease-sensitive ( e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); US Pat. No. 5,208,020).

In certain embodiments, the linker (L) has the following formula II: (II) _-Aa - _Ww - _Yy- wherein A is a "Stretcher Unit" and a is an integer from 0 to 1; W is an "Amino Acid Unit" and w is an integer from 0 to 12; Y is a "Spacer Unit" and y is 0, 1, or 2; and Ab, D, and p are as defined above for Formula I. Exemplary embodiments of such linkers are described in U.S. Patent No. 7,498,298, which is expressly incorporated herein by reference.

In some embodiments, the linker component comprises a "stretcher unit" that connects the antibody to another linker component or a drug moiety. Non-limiting exemplary stretcher units are shown below (where the wavy line indicates the site of covalent attachment to the antibody, drug, or other linker component):

In some embodiments, the linker component comprises an amino acid unit. In some of these embodiments, the amino acid unit allows the linker to be cleaved by a protease, thereby facilitating release of the drug/cytotoxic agent from the anti-CD79b immunoconjugate upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe-homolys); and N-methyl-valine-citrulline (me-val-cit). Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid units may contain naturally occurring amino acid residues and/or minor amino acids and/or non-naturally occurring amino acid analogs, such as citrulline. Amino acid units can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

In some embodiments, the linker component comprises a "spacer" unit that links the antibody to the drug moiety, either directly or via a stretcher unit and/or an amino acid unit. The spacer unit can be "self-immolative" or "non-self-immolative." A "non-self-immolative" spacer unit is one in which a portion or all of the spacer unit remains bound to the drug moiety after cleavage of the ADC. Examples of non-self-immolative spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. In some embodiments, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by tumor cell-associated proteases results in the release of the glycine-glycine-drug moiety from the remainder of the ADC. In some of these embodiments, the glycine-glycine-drug moiety undergoes a hydrolysis step in tumor cells, thereby cleaving the glycine-glycine spacer unit from the drug moiety.

The "self-immolative" spacer unit allows for the release of the drug moiety. In certain embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In some of these embodiments, the p-aminobenzyl alcohol is linked to the amino acid unit via an amide bond, and a carbamate, methyl carbamate, or carbonate is formed between the benzyl alcohol and the drug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15: 1087-1103). In some embodiments, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In some embodiments, the anti-CD79b immunoconjugate comprises a self-immolative linker comprising the following structure: wherein Q is -C ₁ -C ₈ alkyl, -O-(C ₁ -C ₈ alkyl), -halogen, -nitro, or -cyano; m is an integer in the range of 0 to 4; and p is in the range of 1 to about 20. In some embodiments, p is in the range of 1 to 10, 1 to 7, 1 to 5, or 1 to 4.

Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (U.S. Patent No. 7,375,078; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and o- or p-aminobenzyl acetal. In some embodiments, spacers that undergo cyclization upon hydrolysis of the amide bond may be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al. (1995) Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al. (1972) J. Amer. Chem. Soc. 94:5815), and 2-aminophenylpropionic acid amide (Amsberry et al. (1990) J. Org. Chem. 55:5867). The linkage of the drug to the α-carbon of a glycine residue is another example of a self-immolative spacer that may be suitable for use in an ADC (Kingsbury et al. (1984) J. Med. Chem. 27:1447).

In some embodiments, Linker L can be a tree-type linker used to covalently attach more than one drug moiety to an antibody via a branched multifunctional linker moiety (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768). Tree-like linkers can increase the drug-to-antibody molar ratio, i.e., loading capacity, which correlates with ADC potency. Thus, when an antibody carries only one reactive cysteine thiol group, multiple drug moieties can be attached via a tree-like linker.

Non-limiting exemplary linkers are shown below in the context of the anti-CD79 immunoconjugates of Formula III, IV, and V:

wherein (Ab) is an anti-CD79b antibody, (D) is a drug/cytotoxic agent, "Val-Cit" is a valeric acid-citrulline dipeptide, MC is 6-cis-butenediimidohexanoyl, PAB is p-aminobenzyloxycarbonyl, and p is 1 to about 20 (e.g., 1 to 15, 1 to 10, 1 to 8, 2 to 5, or 3 to 4).

In some embodiments, the anti-CD79b immunoconjugate comprises the structure of any one of the following Formulas VI-V: Where X is: ,-(CH ₂ ) _n -,-(CH ₂ CH ₂ O) _n -, Y is: Each R is independently H or C ₁ -C ₆ alkyl; and n is 1 to 12.

Typically, peptide linkers can be prepared by forming peptide bonds between two or more amino acids and/or peptide fragments. For example, such peptide bonds can be prepared according to liquid-phase synthesis methods (e.g., E. Schröder and K. Lübke (1965) "The Peptides", Vol. 1, pp. 76-136, Academic Press).

In some embodiments, the linker is substituted with groups that modulate solubility and/or reactivity. As a non-limiting example, charged substituents such as sulfonate (-SO ₃ ^- ) or ammonium can increase the water solubility of the linker reagent and facilitate the coupling reaction between the linker reagent and the antibody and/or drug moiety, or facilitate the coupling reaction between Ab-L (anti-CD79b antibody-linker intermediate) and D, or DL (drug/cytotoxic agent-linker intermediate) and Ab, depending on the synthetic route used to prepare the anti-CD79b immunoconjugate. In some embodiments, one portion of the linker is coupled to the antibody and one portion of the linker is coupled to the drug, and the anti-CD79 Ab, (linker portion) ^a, is then coupled to the drug/cytotoxic agent, (linker portion) ^b , to form an anti-CD79b immunoconjugate of Formula I. In some of these embodiments, the anti-CD79b antibody comprises more than one (linker portion) ^a substituent, allowing for more than one drug/cytotoxic agent to be coupled to the anti-CD79b antibody in the anti-CD79b immunoconjugate of Formula I.

The anti-CD79b immunoconjugates provided herein specifically encompass, but are not limited to, anti-CD79b immunoconjugates prepared with the following linker reagents: bis-cis-butylenediimido-trioxyethylene glycol (BMPEO), N-(β-cis-butylenediimidopropyloxy)-N-hydroxysuccinimide ester (BMPS), N-(ε-cis-butylenediimidohexanoyloxy)succinimide ester (EMCS), N-[γ-cis-butylenediimidobutyryloxy]succinimide ester (GMBS), 1,6-hexane-bis-vinylsulfonate (HBV S), succinimidyl 4-(N-cis-butylenediimidomethyl)cyclohexane-1-carboxyl-(6-amidohexanoate) (LC-SMCC), m-cis-butylenediimidobenzyl-N-hydroxysuccinimidyl ester (MBS), 4-(4-N-cis-butylenediimidophenyl)butyric acid hydrazide (MPBH), succinimidyl 3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA), succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl-3- (2-Pyridyldithio) propionate (SPDP), N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP), succinimidyl-4-(N-cis-butylenediimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl-4-(p-cis-butylenediimidophenyl)butyrate (SMPB), succinimidyl-6-[(β-cis-butylenediimidopropionamido)hexanoate] (SMPH), iminothiocyclopentane (IT), sulfonic acid EMCS, sulfonic acid GMBS, sulfonic acid KMU S, sulfonic acid-MBS, sulfonic acid-SIAB, sulfonic acid-SMCC and sulfonic acid-SMPB and succinimidyl-(4-vinylsulfonate) benzoate (SVSB), and including bis-cis-butylenediimide reagents: disulfide bis-cis-butylenediimide ethane (DTME), 1,4-bis-cis-butylenediimide butane (BMB), 1,4-bis-cis-butylenediimide-2,3-dihydroxybutane (BMDB), bis-cis-butylenediimide hexane (BMH), bis-cis-butylenediimide ethane (BMOE), BM (PEG) ₂ (shown below) and BM(PEG) ₃ (shown below); difunctional derivatives of imino esters (such as dimethyl diimidoadipate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In some embodiments, a bis-cis-butylenediimide reagent allows for the attachment of thiol groups of cysteine residues in antibodies to thiol-containing drug moieties, linkers, or linker-drug intermediates. Other functional groups that react with thiol groups include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfides, pyridyl disulfides, isocyanates, and isothiocyanates.

Certain useful linker reagents can be obtained from various commercial sources, such as Pierce Biotechnology, Inc. (Rockford, IL), Molecular Biosciences, Inc. (Boulder, CO), or synthesized according to methods described in the art; for example, in Toki et al. (2002) J. Org. Chem. 67: 1866-1872; Dubowchik et al. (1997) Tetrahedron Letters , 38: 5257-60; Walker, MA (1995) J. Org. Chem. 60: 5352-5355; Frisch et al. (1996) Bioconjugate Chem. 7: 180-186; US 6214345; WO 02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; and WO 04/032828.

Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See, for example, WO94/11026.

B. Anti-CD79b Antibody

In some embodiments, the immunoconjugate (e.g., an anti-CD79b immunoconjugate) comprises an anti-CD79b antibody comprising at least one, two, three, four, five, or six HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some of these embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising at least one of: (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23, and/or (ii) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising at least one of: (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23, and/or (ii) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23. In another embodiment, the immunoconjugate comprises an anti-CD79b antibody comprising HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23.

In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the immunoconjugate comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22, and (iii) HVR-H3 comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 23; and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising at least one of: (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23, and/or (ii) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24.

In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the immunoconjugate comprises at least one of: HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23, and/or HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the anti-CD79b immunoconjugate comprises a humanized anti-CD79b antibody. In some embodiments, the anti-CD79b antibody comprises the HVRs of any of the embodiments provided herein and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework. In certain embodiments, the human acceptor framework is a human VLκ1 (VL _KI ) framework and/or a VH framework VH _III . In some embodiments, the humanized anti-CD79b antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the humanized anti-CD79b antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the immunoconjugate ( e.g., an anti-CD79b immunoconjugate) comprises an anti-CD79 antibody comprising a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 19 contains substitutions ( e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-CD79b immunoconjugate comprising that sequence retains the ability to bind to CD79b. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 19. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 19. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., within the FRs). In certain embodiments, the immunobinder ( e.g., an anti-CD79b immunobinder) comprises the VH sequence of SEQ ID NO: 19, including post-translational modifications thereof. In certain embodiments, the VH comprises one, two, or three HVRs selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 23.

In some embodiments, the immunoconjugate ( e.g., an anti-CD79b immunoconjugate) comprises an anti-CD79b antibody comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 20 contains substitutions ( e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-CD79b immunoconjugate comprising that sequence retains the ability to bind to CD79b. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 20. In certain embodiments, a total of 1 to 5 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 20. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). In certain embodiments, the anti-CD79b immunoconjugate comprises an anti-CD79b antibody comprising a VL sequence of SEQ ID NO: 20, including post-translational modifications of that sequence. In certain embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the immunoconjugate ( e.g. , an anti-CD79b immunoconjugate) comprises an anti-CD79b antibody comprising a VH as described in any of the embodiments provided herein and a VL as described in any of the embodiments provided herein. In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising the VH and VL sequences of SEQ ID NO: 19 and SEQ ID NO: 20, respectively, including post-translational modifications of those sequences.

In some embodiments, the immunoconjugate ( e.g. , an anti-CD79b immunoconjugate) comprises an anti-CD79b antibody that binds to the same epitope as an anti-CD79b antibody described herein. For example, in some embodiments, the immunoconjugate ( e.g. , an anti-CD79b immunoconjugate) comprises an anti-CD79b antibody that binds to the same epitope as an anti-CD79b antibody comprising the VH sequence of SEQ ID NO: 19 and the VL sequence of SEQ ID NO: 20.

In some embodiments, the immunoconjugate comprises an anti-CD79b antibody that is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the immunoconjugate comprises an antigen-binding fragment of an anti-CD79b antibody described herein, such as an Fv, Fab, Fab', scFv, a mini-bifunctional antibody, or a F(ab') ₂ fragment. In some embodiments, the immunoconjugate comprises a substantially full-length anti-CD79b antibody, such as an IgG1 antibody or other antibody classes or isotypes as described elsewhere herein.

In some embodiments, the immunoconjugate comprises an anti-CD79b antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 36, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 37 and a light chain comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and a light chain comprising the amino acid sequence of SEQ ID NO: 38.

C. Drug cytotoxic agents

Anti-CD79 immunoconjugates comprise an anti-CD79b antibody ( e.g., an anti-CD79b antibody described herein) conjugated to one or more drugs/cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins ( e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes (i.e., radioconjugates). These immunoconjugates are targeted chemotherapeutic molecules that combine the properties of antibodies and cytotoxic drugs by targeting the delivery of potent cytotoxic drugs to cancer cells (e.g., tumor cells) expressing the antigen (Teicher, BA (2009) Current Cancer Drug Targets 9:982-1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, PJ and Senter PD (2008) The Cancer Jour. 14(3):154-169; Chari, RV (2008) Acc. Chem. Res. 41:98-107. That is, anti-CD79 conjugates selectively deliver effective doses of drugs to cancer cells/tissues, thereby achieving higher selectivity (i.e., lower effective doses) and increasing the therapeutic index ("therapeutic window") (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).

The anti-CD79 immunoconjugates used in the methods provided herein include anti-CD79 immunoconjugates with anti-cancer activity. In some embodiments, the anti-CD79 immunoconjugate comprises an anti-CD79b antibody conjugated (i.e., covalently linked) to a drug portion. In some embodiments, the anti-CD79b antibody is covalently linked to the drug portion via a linker. The drug portion (D) of the anti-CD79 immunoconjugate may include any compound, moiety, or group having a cytotoxic or cytostatic effect. The drug portion may impart its cytotoxic and cytostatic effects by mechanisms including, but not limited to, tubulin binding, DNA binding or insertion, and inhibition of RNA polymerase, protein synthesis, and/or topoisomerase. Exemplary drug agents include, but are not limited to, maytansinoids, dolastatins, orestadins, kacinomycins, anthracyclines, duocarmycins, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecins, elinafide, and their stereoisomers, isosteres, analogs, and derivatives having cytotoxic activity.

(i) Maytansine and maytansinoid

In some embodiments, the anti-CD79b immunoconjugate comprises an anti-CD79b antibody bound to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine and are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was originally isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinoids are disclosed in, for example, U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428 No. 4,313,946; No. 4,315,929; No. 4,317,821; No. 4,322,348; No. 4,331,598; No. 4,361,650; No. 4,364,866; No. 4,424,219; No. 4,450,254; No. 4,362,663; and No. 4,371,533.

Maytansinoid drug moieties are attractive drug moieties for antibody-drug conjugates because they are: (i) relatively easy to prepare by fermentation or chemical modification or derivatization of fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation to antibodies via non-disulfide linkers, (iii) stable in plasma, and (iv) active against a variety of tumor cell lines.

Certain maytansinoids suitable for use as maytansinoid drug moieties are known in the art and can be isolated from natural sources according to known methods or produced using genetic engineering techniques ( see, e.g., Yu et al. (2002) PNAS 99:7968-7973). Maytansinoids can also be prepared synthetically according to known methods.

Exemplary maytansinoid drug moieties include, but are not limited to, maytansinoid drug moieties having modified aromatic rings such as: C-19-dechloro (U.S. Patent No. 4,256,746) (prepared, for example, by reduction of ansamitocin P2 with lithium aluminum hydroxide); C-20-hydroxy (or C-20-demethyl) +/- C-19-dechloro (U.S. Patent Nos. 4,361,650 and 4,307,016); No. 4,294,757) (prepared, for example, by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), +/-dechlorination (U.S. Patent No. 4,294,757) (prepared, for example, by acylation using acyl chloride), and those with modifications at other positions on the aromatic ring.

Exemplary maytansinoid drug moieties also include those with modifications such as: C-9-SH (U.S. Patent No. 4,424,219) (e.g., prepared by reacting maytansinol with H ₂ S or P ₂ S ₅ ); C-14-alkoxymethyl (demethoxy/CH ₂ OR) (U.S. Patent No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH ₂ OH or CH ₂ OAc) (U.S. Patent No. 4,450,254) (e.g., prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Patent No. 4,364,866) (e.g., prepared by converting maytansinol with Streptomyces); C-15-methoxy (U.S. Patent Nos. 4,313,946 and 4,315,929) (e.g., prepared from Trewia nudlflora); C-18-N-desmethyl (U.S. Patent Nos. 4,362,663 and 4,322,348) (prepared, for example, by demethylation of maytansinol using Streptomyces cerevisiae); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by reduction of maytansinol with titanium trichloride/LAH).

Many positions on a maytansinoid compound are suitable for bonding. For example, an ester bond can be formed by reaction with a hydroxyl group using conventional coupling techniques. In some embodiments, the reaction can occur at the C-3 position with a hydroxyl group, the C-14 position modified with a hydroxyl group, the C-15 position modified with a hydroxyl group, and the C-20 position with a hydroxyl group. In some embodiments, the bond is formed at the C-3 position of maytansinol or a maytansinol analog.

The maytansinoid drug portion includes those having the following structure: The wavy line indicates the covalent attachment of the sulfur atom of the maytansinoid drug moiety to the linker of the anti-CD79b immunoconjugate. Each R can independently be H or a _C1 - _C6 alkyl group. The alkylene chain connecting the amide group to the sulfur atom can be methane, ethyl, or propyl, i.e., m is 1, 2, or 3 (US 633410; US 5208020; Chari et al. (1992) Cancer Res. 52:127-131; Liu et al. (1996) Proc. Natl. Acad. Sci USA 93:8618-8623).

All stereoisomers of the maytansinoid drug moiety are contemplated for use in the anti-CD79b immunoconjugates provided herein, i.e., any combination of R and S configurations at the chiral carbon ( US 7276497 ; US 6913748 ; US 6441163 ; US 633410 (RE39151 ); US 5208020 ; Widdison et al., (2006) J. Med. Chem. 49: 4392-4408 , which are incorporated herein by reference in their entirety). In some embodiments, the maytansinoid drug moiety has the following stereochemistry:

Exemplary embodiments of the maytansinoid drug moiety include, but are not limited to, DM1; DM3; and DM4 having the following structures:

The wavy line indicates the covalent connection between the sulfur atom of the drug and the linker (L) of the anti-CD79b immunoconjugate.

Other exemplary maytansinoid anti-CD79b immunoconjugates have the following structures and abbreviations (wherein Ab is an anti-CD79b antibody and p is 1 to about 20. In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4):

An exemplary antibody-drug conjugate in which DM1 is linked to the thiol group of the antibody via a BMPEO linker has the following structure and abbreviation: wherein Ab is an anti-CD79b antibody; n is 0, 1, or 2; and p is 1 to about 20. In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4.

Maytansinoid-containing immunoconjugates, their preparation methods, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020 and 5,416,064; US 2005/0276812 A1; and European Patent No. 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference. See also Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996); and Chari et al., Cancer Research 52:127-131 (1992).

In some embodiments, anti-CD79b antibody-maytansinoid conjugates can be prepared by chemically linking an anti-CD79b antibody to a maytansinoid molecule without significantly weakening the biological activity of the antibody or maytansinoid molecule. See, for example, U.S. Patent No. 5,208,020 (the disclosure of which is expressly incorporated herein by reference). In some embodiments, anti-CD79b immunoconjugates with an average of 3-4 maytansinoid molecules bound to each antibody molecule have been shown to be effective in enhancing the cytotoxicity of target cells without negatively affecting the function or solubility of the antibody. In some cases, it is expected that even one toxin/antibody molecule will enhance cytotoxicity more than using naked anti-CD79b antibodies.

Exemplary linking groups for preparing antibody-maytansinoid conjugates include, for example, those described herein and those disclosed in U.S. Patent No. 5,208,020; EP Patent No. 0,425,235 Bl; Chari et al. Cancer Research 52:127-131 (1992); US 2005/0276812 Al; and US 2005/016993 Al, the disclosures of which are expressly incorporated herein by reference.

(2) Orlistatin and Dolastatin

The drug segment includes dolastatin, aurestin, and their analogs and derivatives (US 5635483; US 5780588; US 5767237; US 6124431). Aurestin is a derivative of the marine mollusk compound dolastatin-10. While not intending to be bound by any particular theory, dolastatins and auricularisin have been shown to disrupt microtubule dynamics, GTP hydrolysis, nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer (US 5663149) and antifungal activities (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin/orestatin drug moiety can be linked to the antibody via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02/088172; Doronina et al., (2003) Nature Biotechnology 21(7):778-784; Francisco et al., (2003) Blood 102(4):1458-1465).

Exemplary aurestudin embodiments include the N-terminally linked monomethyl aurestudin drug moieties _DE and _DF disclosed in US Pat. No. 7,498,298 and US Pat. No. 7,659,241, the disclosures of which are expressly incorporated herein by reference in their entirety:

wherein the wavy lines _DE and _DF indicate the sites of covalent attachment to the antibody or antibody-linker component, and independently at each position: ^R2 is selected from H and _C1 - _C8 alkyl; ^R3 is selected from H, _C1 - _C8 alkyl, _C3 - _C8 carbocycle, aryl, _C1 - _C8 alkyl-aryl, _C1 - _C8 alkyl-( _C3 - _C8 carbocycle), _C3 - _C8 heterocycle, and _C1 - _C8 alkyl-( _C3 - _C8 heterocycle); ^R4 is selected from H, _C1 - _C8 alkyl, _C3 - _C8 carbocycle, aryl, _C1 - _C8 alkyl-aryl, C1- _C8 alkyl- _{(C3 - C8} carbocycle), _C3 - _C8 heterocycle, and _C1 -C8 R ⁵ is selected from H and methyl; or R ⁴ and R ⁵ together form a carbocycle and have the formula -(CR ^a R ^b ) _n -, wherein ^Ra and R ^b are independently selected from H, C _{1 -C 8} alkyl and C ₃ -C ₈ carbocycle and n is selected from 2 _, 3 _, 4, 5 and 6; R ⁶ is selected from H and C ₁ -C ₈ alkyl; R ⁷ is selected from H, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle, aryl, C ₁ -C ₈ alkyl-aryl, C ₁ -C ₈ alkyl-(C ₃ -C ₈ carbocycle), C ₃ -C ₈ heterocycle and C ₁ -C ₈ alkyl-(C ₃ -C ₈ heterocycle); each R ⁸ is independently selected from H, OH, C ₁ -C ₈ alkyl, C ₃ -C ₈ carbocycle and O-(C ₁ -C ₈ alkyl); R ⁹ is selected from H and C ₁ -C ₈ alkyl; R ¹⁰ is selected from aryl or C ₃ -C ₈ heterocycle; Z is O, S, NH or NR ¹² , wherein R ¹² is C ₁ -C ₈ alkyl; R ¹¹ is selected from H, C ₁ -C ₂₀ alkyl, aryl, C ₃ -C ₈ heterocycle, -(R ¹³ O) _m -R ¹⁴ or -(R ¹³ O) _m -CH(R ¹⁵ ) ₂ ; m is an integer in the range of 1-1000; R ¹³ is C ₂ -C ₈ alkyl; R ¹⁴ is H or C ₁ -C ₈ alkyl; R ¹⁵ is independently H, COOH, -(CH ₂ ) _n -N(R ¹⁶ ) ₂ , -(CH ₂ ) _n -SO ₃ H, or -(CH ₂ ) _n -SO ₃ -C ₁ -C ₈ alkyl; R ¹⁶ at each occurrence is independently H, C ₁ -C ₈ alkyl, or -(CH ₂ ) _n -COOH; R ¹⁸ is selected from -C(R ⁸ ) ₂ -C(R ⁸ ) ₂ -aryl, -C(R ⁸ ) ₂ -C(R ⁸ ) ₂ -(C ₃ -C ₈ heterocyclic) and -C(R ⁸ ) ₂ -C(R ⁸ ) ₂ -(C ₃ -C ₈ carbocyclic); and n is an integer ranging from 0 to 6.

In one embodiment, R ³ , R ⁴ and R ⁷ are independently isopropyl or t-butyl and R ⁵ is -H or methyl. In an exemplary embodiment, R ³ and R ⁴ are each isopropyl, R ⁵ is -H, and R ⁷ is t-butyl.

In another embodiment, R ² and R ⁶ are each methyl, and R ⁹ is -H.

In another embodiment, each occurrence of R ⁸ is -OCH ₃ .

In an exemplary embodiment, R ³ and R ⁴ are each isopropyl, R ² and R ⁶ are each methyl, R ⁵ is -H, R ⁷ is t-butyl, each occurrence of R ⁸ is -OCH ₃ , and R ⁹ is -H.

In one embodiment, Z is -O- or -NH-.

In one embodiment, R ¹⁰ is aryl.

In an exemplary embodiment, R ¹⁰ is -phenyl.

In an exemplary embodiment, when Z is -O-, R ¹¹ is -H, methyl or t-butyl.

In one embodiment, when Z is -NH, R ¹¹ is -CH(R ¹⁵ ) ₂ , wherein R ¹⁵ is -(CH ₂ ) _n -N(R ¹⁶ ) ₂ , and R ¹⁶ is -C ₁ -C ₈ alkyl or -(CH ₂ ) _n -COOH.

In another embodiment, when Z is -NH, R ¹¹ is -CH(R ¹⁵ ) ₂ , wherein R ¹⁵ is -(CH ₂ ) _n -SO ₃ H.

An exemplary auricular statin embodiment having formula _DE is MMAE, wherein the wavy line indicates the covalent attachment to the linker (L) of the anti-CD79b immunoconjugate:

An exemplary auriculariastatin embodiment having Formula _DF is MMAF, wherein the wavy line indicates the covalent attachment to the linker (L) of the anti-CD79b immunoconjugate:

Other exemplary embodiments include monomethyl valeric acid compounds having a phenylalanine carboxyl modification at the C-terminus of the pentapeptide aurestin drug moiety (WO 2007/008848) and monomethyl valeric acid compounds having a phenylalanine side chain modification at the C-terminus of the pentapeptide aurestin drug moiety (WO 2007/008603).

Non-limiting exemplary embodiments of anti-CD79b immunoconjugates of Formula I comprising MMAE or MMAF and various linker components have the following structures and abbreviations (wherein "Ab" is an anti-CD79b antibody; p is 1 to about 8, "Val-Cit" is a valeric acid-citrulline dipeptide; and "S" is a sulfur atom):

In certain embodiments, the anti-CD79b immunoconjugate comprises the structure of Ab-MC-vc-PAB-MMAE, wherein p is, for example, about 1 to about 8; about 2 to about 7; about 3 to about 5; about 3 to about 4; or about 3.5. In some embodiments, the anti-CD79b immunoconjugate is huMA79bv28-MC-vc-PAB-MMAE, such as an anti-CD79b immunoconjugate comprising the structure of MC-vc-PAB-MMAE, wherein p is, for example, about 1 to about 8; about 2 to about 7; about 3 to about 5; about 3 to about 4; or about 3.5, wherein the anti-CD79 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36, and wherein the light chain comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the anti-CD79b immunoconjugate is polatuzumab vedotin (CAS No. 1313206-42-6).

Non-limiting exemplary embodiments of anti-CD79b immunoconjugates of Formula I comprising MMAF and various linker components further include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Immunoconjugates comprising MMAF linked to the antibody via a non-proteolytically cleavable linker have been shown to have similar activity to immunoconjugates comprising MMAF linked to the antibody via a proteolytically cleavable linker (Doronina et al., (2006) Bioconjugate Chem. 17: 114-124). In some of these embodiments, drug release is believed to be achieved by degradation of the antibody in cells.

Typically, peptide-based drug moieties can be prepared by forming peptide bonds between two or more amino acids and/or peptide fragments. For example, such peptide bonds can be prepared according to liquid phase synthesis methods ( see, e.g., E. Schröder and K. Lübke, "The Peptides", Vol. 1, pp. 76-136, 1965, Academic Press). In some embodiments, the auriculariastatin/dolastatin drug moiety can be prepared according to the methods of: US 7498298; US 5635483; US 5780588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; Pettit, GR et al. Synthesis , 1996, 719-725; Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 15:859-863; and Doronina (2003) Nat. Biotechnol. 21(7):778-784.

In some embodiments, auriculariastatin/dolastatin drug moieties of formula _DE , e.g., MMAE, and _DF , e.g., MMAF, and drug-linker intermediates and derivatives thereof, e.g., MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, can be prepared using the methods described in US 7498298; Doronina et al. (2006) Bioconjugate Chem. 17: 114-124; and Doronina et al. (2003) Nat. Biotech. 21: 778-784 and then conjugated to an antibody of interest.

(3) Kazimycin

In some embodiments, the anti-CD79b immunoconjugate comprises an anti-CD79b antibody bound to one or more calicheamicin molecules. The calicheamicin family of antibiotics and their analogs are capable of inducing double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al., (1993) Concer Research 53:3336-3342; Lode et al., (1998) Cancer Research 58:2925-2928). The calicheamicins have intracellular sites of action but, in some cases, are less able to cross the plasma membrane. Therefore, in some embodiments, cellular uptake of these agents via antibody-mediated internalization can significantly enhance their cytotoxic effects. Non-limiting exemplary methods for preparing anti-CD79b antibody immunoconjugates having a kachyclone drug moiety are described, for example, in US Pat. Nos. 5,712,374, 5,714,586, 5,739,116, and 5,767,285.

(4) Other medicines

In some embodiments, the anti-CD79b immunoconjugate comprises geldermycin (Mandler et al. (2000) J. Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791); and/or enzymatically active toxins and fragments thereof, including but not limited to diphtheria chain A, nonbinding active fragments of diphtheria toxin, exotoxin chain A (from Pseudomonas aeruginosa), ricin chain A, abrin chain A, modeccin chain A, α-sarcin, Aleurites fordii (Aleurites obscurus), ricin chain A, abrin chain A, modeccin chain A, α-sarcin, tung oil (Aleurites obscurus), ricin chain B, ricin chain C, ricin chain D, ricin chain E, ricin chain F ... fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitors, crotins, crotonins, sapaonaria officinalis inhibitors, gelonins, mitogellins, restrictocins, phenomycins, enomycins, and tricothecenes. See, for example, WO 93/21232.

The drug moiety also includes compounds with nucleolytic activity ( eg, ribonucleases or DNA endonucleases).

In certain embodiments, the anti-CD79b immunoconjugate comprises a highly radioactive atom. A variety of radioisotopes can be used to produce radioactive conjugated antibodies. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , and radioisotopes of Lu. In some embodiments, when anti-CD79b immunoconjugates are used for detection, they may include radioactive atoms such as Tc ⁹⁹ or I ¹²³ for scintillation imaging studies, or spin labels such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging). Zirconium-89 can be complexed with various metal chelators and conjugated to antibodies, for example, for PET imaging (WO 2011/056983).

Radiolabels or other labels can be incorporated into anti-CD79b immunoconjugates in known manner. For example, peptides can be biosynthesized or chemically synthesized using suitable amino acid precursors containing, for example, one or more fluorine-19 atoms in place of one or more hydrogens. In some embodiments, labels such as Tc ⁹⁹ , I ¹²³ , Re ¹⁸⁶ , Re ¹⁸⁸ , and In ¹¹¹ can be attached via a cysteine residue in an anti-CD79b antibody. In some embodiments, yttrium-90 can be attached via a lysine residue in an anti-CD79b antibody. In some embodiments, iodine-123 can be incorporated using the IODOGEN method (Fraker et al., (1978) Biochem. Biophys. Res. Commun. 80: 49-57). Some other approaches are described in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

In certain embodiments, anti-CD79b immunoconjugates may comprise an anti-CD79b antibody conjugated to a prodrug-activating enzyme. In some of these embodiments, the prodrug-activating enzyme converts a prodrug ( e.g., a peptidyl chemotherapeutic agent, see WO 81/01145) into an active drug, such as an anticancer drug. In some embodiments, these immunoconjugates are suitable for use in antibody-dependent enzyme-mediated prodrug therapy ("ADEPT"). Enzymes that can be conjugated to anti-CD79b antibodies include, but are not limited to, alkaline phosphatases, which are useful for converting phosphate-containing prodrugs into free drugs; arylsulfatases, which are useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminases, which are useful for converting non-toxic 5-fluorocytosine into the anticancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases, and cathepsins (such as cathepsins B and L), which are useful for converting peptide-containing prodrugs into free drugs. into free drugs; D-alanylcarboxypeptidases, which are suitable for converting prodrugs containing D-amino acid substituents; carbohydrate cleavage enzymes, such as β-galactosidase and neuraminidase, which are suitable for converting glycosylated prodrugs into free drugs; β-lactamases, which are suitable for converting drugs derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase and penicillin G amidase, which are suitable for converting drugs derivatized with phenoxyacetyl or phenylacetyl groups at their amine nitrogens, into free drugs. In some embodiments, the enzyme can be covalently linked to the antibody using recombinant DNA technology well known in the art. See, eg, Neuberger et al., Nature 312: 604-608 (1984).

D. Drug loading

Drug loading is represented by p, which is the average number of drug moieties per anti-CD79b antibody in a molecule of Formula I. Drug loading can range from 1 to 20 drug moieties (D) per antibody. The anti-CD79b immunoconjugates of Formula I include a collection of anti-CD79b antibodies that bind a range (1 to 20) of drug moieties. The average number of drug moieties per anti-CD79b antibody in the anti-CD79b immunoconjugate preparation obtained from the binding reaction can be characterized by conventional methods such as mass spectrometry, ELISA analysis, and HPLC. The quantitative distribution of the anti-CD79b immunoconjugate, represented by p, can also be determined. In some cases, separation, purification, and characterization of homogeneous anti-CD79b immunoconjugates having a p value from anti-CD79b immunoconjugates having other drug loadings can be achieved by methods such as reverse phase HPLC or electrophoresis.

For some anti-CD79b immunoconjugates, p may be limited by the number of attachment sites on the anti-CD79b antibody. For example, when the attachment is a cysteine thiol as in certain exemplary embodiments above, the anti-CD79b antibody may have only one or a few cysteine thiol groups, or may have only one or a few thiol groups with sufficient reactivity for attachment of a linker. In certain embodiments, higher drug loadings (e.g., p>5) may result in aggregation, insolubility, toxicity, or reduced cell permeability of certain anti-CD79b immunoconjugates. In certain embodiments, the average drug loading of the anti-CD79b immunoconjugate ranges from 1 to about 8; about 2 to about 6; about 3 to about 5; or about 3 to about 4. Indeed, it has been shown that for certain antibody-drug conjugates, the optimal ratio of drug moieties per antibody can be less than 8 and can be between about 2 and about 5 (US 7498298). In certain embodiments, the optimal ratio of drug moieties per antibody is between about 3 and about 4. In certain embodiments, the optimal ratio of drug moieties per antibody is about 3.5.

In certain embodiments, less than the theoretical maximum amount of the drug moiety is bound to the anti-CD79b antibody during the binding reaction. The antibody may contain, for example, lysine residues that are unreactive with the drug-linker intermediate or linker reagent, as discussed below. Generally, antibodies do not contain many free and reactive cysteine thiol groups that can be linked to the drug moiety; in fact, most cysteine thiol residues in the antibody exist in the form of disulfide bridges. In certain embodiments, the anti-CD79b antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or full reducing conditions to generate reactive cysteine thiol groups. In certain embodiments, the anti-CD79b antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups, such as lysine or cysteine.

The loading capacity (drug/antibody ratio) of the anti-CD79b immunoconjugate can be controlled in various ways, for example, by: (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the binding reaction time or temperature, and (iii) partially or completely limiting the reducing conditions used for cysteine thiol modification.

It should be understood that when more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent, the resulting product is a mixture of anti-CD79b immunoconjugate compounds having a distribution of one or more drug moieties attached to the anti-CD79b antibody. The average number of drug molecules bound to each antibody can be calculated from the mixture by performing a duplex ELISA assay using antibodies specific for both the antibody and the drug. Individual anti-CD79b immunoconjugate molecules can be identified in a mixture by mass spectrometry and separated by HPLC, e.g., hydrophobic interaction chromatography ( see, e.g., McDonagh et al. (2006) Prot. Engr. Design & Selection 19(7): 299-307; Hamblett et al. (2004) Clin. Cancer Res. 10: 7063-7070; Hamblett, KJ et al. "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate," Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004; Alley, SC et al. "Controlling the location of drug attachment in antibody-drug conjugates," IEEE Trans. Cancer Res., ... "Controlling the location of drug attachment in antibody-drug conjugates," IEEE Trans. Cancer Re conjugates," Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004). In certain embodiments, homogeneous anti-CD79b immunoconjugates having a single loading amount can be separated from the conjugate mixture by electrophoresis or chromatography.

E. Methods for preparing anti-CD79b immunoconjugates

Anti-CD79b immunoconjugates of Formula I can be prepared by a number of routes using organic chemical reactions, conditions, and reagents known to those skilled in the art, including, but not limited to, for example: (1) reacting the nucleophilic group of the anti-CD79b antibody with a bivalent linker reagent to form Ab-L via a covalent bond, followed by reaction with the drug moiety D; and (2) reacting the nucleophilic group of the drug moiety with a bivalent linker reagent to form D-L via a covalent bond, followed by reaction with the nucleophilic group of the anti-CD79b antibody. Exemplary methods for preparing anti-CD79b immunoconjugates of Formula I via the latter route are described in US Pat. No. 7,498,298, which is expressly incorporated herein by reference.

Nucleophilic groups on antibodies include, but are not limited to, (i) an N-terminal amine group, (ii) pendant amine groups such as lysine, (iii) pendant thiol groups such as cysteine, and (iv) sugar hydroxyl groups or amine groups, where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker moieties and linker reagents, including: (i) active esters such as NHS esters, HOBt esters, halogenated formates, and acid halides; (ii) alkyl and benzyl halides such as halogenated acetamides; and (iii) aldehyde, ketone, carboxyl, and cis-butylenediamide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine bridges. The anti-CD79b antibody can be rendered reactive for conjugation to a linker reagent by treating it with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) to fully or partially reduce it. Each cysteine bridge will theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the anti-CD79b antibody by modifying the lysine residue, for example, by reacting the lysine residue with 2-iminothiolane (Traut's reagent), thereby converting the amine into a thiol. Reactive thiol groups can also be introduced into anti-CD79b antibodies by introducing one, two, three, four, or more cysteine residues ( e.g., by preparing variant antibodies containing one or more non-natural cysteine amino acid residues).

The anti-CD79b immunoconjugates described herein can also be produced by the reaction between an electrophilic group (such as an aldehyde or ketone carbonyl) on the anti-CD79b antibody and a nucleophilic group on a linker reagent or drug. Suitable nucleophilic groups on the linker reagent include, but are not limited to, hydrazides, oximes, amines, hydrazines, thiosemicarbohydrazones, hydrazine carboxylates, and arylhydrazides. In one embodiment, the anti-CD79b antibody is modified to introduce an electrophilic moiety capable of reacting with a nucleophilic substituent on the linker reagent or drug. In another embodiment, the sugars of the glycosylated anti-CD79b antibody can be oxidized, for example, with a periodate oxidizing reagent, to form an aldehyde or ketone group that can react with an amine group on the linker reagent or drug moiety. The resulting imine Schiff base group can form a stable linkage or can be reduced, for example, by a borohydride reagent, to form a stable amine linkage. In one embodiment, reaction of the carbohydrate portion of a glycosylated anti-CD79b antibody with galactose oxidase or sodium metaperiodate can generate carbonyl groups (aldehydes and ketones) in the anti-CD79b antibody that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, an anti-CD79b antibody containing an N-terminal serine or threonine residue can be reacted with sodium metaperiodate to generate an aldehyde in place of the first amino acid (Geoghegan and Stroh, (1992) Bioconjugate Chem. 3:138-146; US Pat. No. 5,362,852). This aldehyde can be reacted with a drug moiety or a linker nucleophile.

Exemplary nucleophilic groups on the drug moiety include, but are not limited to, amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups, which are capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including: (i) active esters, such as NHS esters, HOBt esters, halogenated formates, and acid halides; (ii) alkyl and benzyl halides, such as halogenated acetamides; and (iii) aldehyde, ketone, carboxyl, and cis-butylenediamide groups.

Non-limiting exemplary cross-linker reagents that can be used to prepare anti-CD79b immunoconjugates are described herein in the section entitled "Exemplary Linkers." Methods for using such cross-linker reagents to link two moieties, including proteinaceous and chemical moieties, are known in the art. In some embodiments, a fusion protein comprising an anti-CD79b antibody and a cytotoxic agent can be prepared, for example, by recombinant techniques or peptide synthesis. The recombinant DNA molecule can comprise regions encoding the antibody and cytotoxic portions of the conjugate, which regions are adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate. In another embodiment, anti-CD79b antibodies can be conjugated to a "receptor" (such as the antibiotic protein streptavidin) for use in tumor pretargeting, wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and subsequent administration of a "ligand" (such as the antibiotic protein avidin) conjugated to a cytotoxic agent (such as a drug or radionucleotide). Additional details regarding anti-CD79b immunoconjugates are provided in U.S. Patent No. 8,545,850 and WO/2016/049214, the contents of which are expressly incorporated herein by reference in their entireties.

V. Alkylating agents

Alkylating agents are a class of anti-tumor or anti-cancer drugs that work by inhibiting the transcription of DNA into RNA and thereby stopping protein synthesis. Alkylating agents replace hydrogen atoms on DNA with alkyl groups (C _n H _2n+1 ), causing cross-links to form within the DNA chain, which in turn leads to DNA chain breakage, abnormal base pairing, inhibition of cell division, and ultimately cell death. This effect occurs in all cells, but rapidly dividing cells, such as cancer cells, are generally most sensitive to the effects of alkylating agents.

Alkylating agents are generally classified into six categories: (1) nitrogen mustards, including but not limited to methylethylamine, cyclophosphamide, isocyclophosphamide, bendamustine, melphalan, and chlorambucil; (2) ethyleneamine and methyleneamine derivatives, including but not limited to altretamine and thiotepa; (3) alkyl sulfonates, including but not limited to busulfan; (4) nitrosoureas, including but not limited to carmustine and lomustine; (5) triazenes, including but not limited to dacarbazine, procarbazine, and temozolomide; and (6) platinum-containing antitumor agents, including but not limited to cisplatin, carboplatin, and oxaliplatin. Any known alkylating agent (including but not limited to those listed above) can be used in the treatment methods provided herein.

Bendamustine is an exemplary alkylating agent for use in the methods described herein. Bendamustine's chemical name is 4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoic acid. Bendamustine has the following structural formula:

Bendamustine (CAS registration #16506-27-7) has a molecular formula of C ₁₆ H ₂₁ Cl ₂ N ₃ O ₂ and a molecular weight of 358.263 g/mol. Bendamustine is a bifunctional dichloromethyldiethylamine derivative containing a purine-like benzimidazole ring. Bendamustine is available as a solution and a powder for solution preparation.

In some embodiments, the alkylating agent used in the methods described herein is a salt or solvate of bendamustine. In some embodiments, the bendamustine salt is bendamustine-HCl (CAS# 3543-75-7), which has a molecular formula of C ₁₆ H ₂₁ Cl ₂ N ₃ O _{2 ·} HCl and a molecular weight of 394.72 g/mol.

Bendamustine-HCl is available under the trade names BENDEKA, TREANDA, TREAKISYM, RIBOMUSTIN, LEVACT, and MUSTIN.

VI. Anti-CD20 agents

Two types of anti-CD20 antibodies (type I and type II anti-CD20 antibodies) can be distinguished based on their binding properties to the CD20 antigen and their biological activity, according to Cragg, MS et al., Blood 103 (2004) 2738-2743; and Cragg, MS et al., Blood 101 (2003) 1045-1052, see Table C.

Examples of type I anti-CD20 antibodies include, for example, rituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (as disclosed in WO 2005/103081), 2F2 IgG1 (as disclosed in WO 2004/035607 and WO 2005/103081), and 2H7 IgG1 (as disclosed in WO 2004/056312).

In some embodiments, the anti-CD20 antibody used in the treatment methods provided herein is rituximab. In some embodiments, rituximab (reference antibody; an example of a type I anti-CD20 antibody) is a genetically engineered chimeric monoclonal antibody containing a human γ1 murine constitutive domain that is directed against the human CD20 antigen. However, the antibody is not glycosylated and is not non-fucosylated, thus having a fucosylated content of at least 85%. The chimeric antibody contains a human γ1 constitutive domain and is identified by the designation "C2B8" in US Patent No. 5,736,137 (Andersen et al. ), issued April 17, 1998 to IDEC Pharmaceuticals Corporation. Rituximab is approved for the treatment of patients with relapsed or refractory low-grade or follicular CD20-positive B-cell non-Hodgkin's lymphoma. In vitro mechanism of action studies have shown that rituximab has human complement-dependent cytotoxicity (CDC) (Reff, ME et al., Blood 83(2) (1994) 435-445). In addition, it has demonstrated activity in assays measuring antibody-dependent cytotoxicity (ADCC).

In some embodiments, the anti-CD20 antibody used in the treatment methods provided herein is a non-fucosylated anti-CD20 antibody.

Examples of type II anti-CD20 antibodies include, for example, 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. Generally, type II anti-CD20 antibodies of the IgG1 isotype exhibit characteristic CDC properties. Compared to type I antibodies of the IgG1 isotype, type II anti-CD20 antibodies have reduced CDC (if of the IgG1 isotype). In some embodiments, type II anti-CD20 antibodies, such as the GA101 antibody, have increased antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the type II anti-CD20 antibody is more preferably a non-fucosylated humanized B-Ly1 antibody as described in WO 2005/044859 and WO 2007/031875.

In some embodiments, the anti-CD20 antibody used in the treatment methods provided herein is the GA101 antibody. In some embodiments, the GA101 antibody as used herein refers to any of the following antibodies that bind to human CD20: (1) an antibody comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 5, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 6, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 7, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 8, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 10; (2) an antibody comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 11 and a VL domain comprising the amino acid sequence of SEQ ID NO: 12, (3) an antibody comprising an amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14. NO: 14; (4) an antibody called atozumab; or (5) an antibody comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 13 and an antibody comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 14. In one embodiment, the GA101 antibody is an IgG1 isotype antibody.

In some embodiments, the anti-CD20 antibody used in the treatment methods provided herein is a humanized B-Ly1 antibody. In some embodiments, the humanized B-Ly1 antibody refers to the humanized B-Ly1 antibody disclosed in WO 2005/044859 and WO 2007/031875, which is derived from the murine monoclonal anti-CD20 antibody B-Ly1 (mouse heavy chain (VH) variable region: SEQ ID NO: 3; mouse light chain (VL) variable region: SEQ ID NO: 4 - see Poppema, S. and Visser, L., Biotest Bulletin 3 (1987) 131-139) by chimerizing with human constant domains from IgG1 and after humanization (see WO 2005/044859 and WO 2007/031875). Humanized B-Ly1 antibodies are disclosed in detail in WO 2005/044859 and WO 2007/031875.

In some embodiments, the humanized B-Ly1 antibody comprises a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 15-16 and 40-55 (corresponding to B-HH2 to B-HH9 and B-HL8 to B-HL17 of WO 2005/044859 and WO 2007/031875). In some embodiments, the variable domain is selected from the group consisting of SEQ ID NOs: 15, 16, 42, 44, 46, 48, and 50 (corresponding to B-HH2, BHH-3, B-HH6, B-HH8, B-HL8, B-HL11, and B-HL13 of WO 2005/044859 and WO 2007/031875). In some embodiments, the humanized B-Ly1 antibody has a light chain variable region (VL) of SEQ ID NO: 55 (corresponding to B-KV1 in WO 2005/044859 and WO 2007/031875). In some embodiments, the humanized B-Ly1 antibody has a heavy chain variable region (VH) of SEQ ID NO: 42 (corresponding to B-HH6 in WO 2005/044859 and WO 2007/031875) and a light chain variable region (VL) of SEQ ID NO: 55 (corresponding to B-KV1 in WO 2005/044859 and WO 2007/031875). In some embodiments, the humanized B-Ly1 antibody is an IgG1 antibody. These non-fucosylated humanized B-Ly1 antibodies are glycoengineered (GE) in the Fc region according to the procedures described in WO 2005/044859, WO 2004/065540, WO 2007/031875, Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180, and WO 99/154342. In some embodiments, the non-fucosylated glycoengineered humanized B-Ly1 is B-HH6-B-KV1 GE. In some embodiments, the anti-CD20 antibody is atezolizumab (Recommended INN, WHO Drug Information, Vol. 26, No. 4, 2012, p. 453). As used herein, atezolizumab is synonymous with GA101 or RO5072759. This replaces all previous versions (e.g., Volume 25, Issue 1, 2011, pp. 75-76), formerly known as afutuzumab (Recommended INN, WHO Drug Information, Volume 23, Issue 2, 2009, p. 176; Volume 22, Issue 2, 2008, p. 124). In some embodiments, the humanized B-Ly1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 18, or an antigen-binding fragment of such an antibody. In some embodiments, the humanized B-Ly1 antibody comprises a heavy chain variable region comprising three heavy chain CDRs of SEQ ID NO: 17 and a light chain variable region comprising three light chain CDRs of SEQ ID NO: 18.

In some embodiments, the humanized B-Ly1 antibody is a non-fucosylated, glycosylated humanized B-Ly1 antibody. These glycosylated humanized B-Ly1 antibodies have an altered glycosylation pattern in the Fc region, preferably with reduced levels of fucose residues. In some embodiments, the amount of fucose is about 60% or less of the total oligosaccharides at Asn297 (in one embodiment, the amount of fucose is about 40% to about 60%, in another embodiment, the amount of fucose is about 50% or less, and in another embodiment, the amount of fucose is about 30% or less). In some embodiments, the oligosaccharides in the Fc region are bisected. These glycosylated humanized B-Ly1 antibodies have increased ADCC.

As described in Example 2, the anti-CD20 antibody conjugated to Cy5 and rituximab conjugated to Cy5 were used in a FACS array (Becton Dickinson) with Raji cells (ATCC-No. CCL-86) to determine the ratio of the binding ability of the anti-CD20 antibody to that of rituximab to CD20 on Raji cells (ATCC-No. CCL-86) by direct immunofluorescence measurement (measuring the mean fluorescence intensity (MFI)) and calculated as follows: MFI is the mean fluorescence intensity. As used herein, "Cy5 labeling ratio" refers to the number of Cy5-labeled molecules per antibody.

Typically, the type II anti-CD20 antibody has a ratio of the binding ability of the second anti-CD20 antibody to CD20 on Raji cells (ATCC No. CCL-86) compared to rituximab of 0.3 to 0.6, and in one embodiment, 0.35 to 0.55, and in another embodiment, 0.4 to 0.5.

"Antibody having increased antibody-dependent cellular cytotoxicity (ADCC)" means an antibody (as that term is defined herein) having increased ADCC as measured by any suitable method known to those of ordinary skill in the art.

An exemplary acceptable in vitro ADCC assay is described below: 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) PBMCs are isolated using a standard density centrifugation procedure and suspended in RPMI cell culture medium at 5×10 ⁶ cells/ml; ii) target cells are cultured by standard tissue culture methods, harvested from the exponential growth phase at a viability greater than 90%, washed in RPMI cell culture medium, and stained with 100 μCi of ⁵¹ Cr was used to label the cells, washed twice with cell culture medium, and resuspended in cell culture medium at a density of 10 ⁵ cells/ml; iii) 100 μl of the final target cell suspension was transferred to each well of a 96-well microtiter plate; iv) the antibody was serially diluted from 4000 ng/ml to 0.04 ng/ml in cell culture medium, and 50 μl of the resulting antibody solution was added to the target cells in the 96-well microtiter plate, and various antibody concentrations covering the entire concentration range were tested in triplicate; v) for the maximum For spontaneous release (MR) control, three additional wells of the plate containing labeled target cells received 50 μl of 2% (VN) nonionic detergent in water (Nonidet, Sigma, St. Louis) instead of the antibody solution (point iv above); vi) for spontaneous release (SR) control, three additional wells of the plate containing labeled target cells received 50 μl of RPMI cell culture medium instead of the antibody solution (point iv above); vii) the 96-well microtiter plate was then centrifuged at 50 × g for 1 minute and incubated at 4°C for 1 hour; viii) 50 μl of the PBMC suspension (point i above) was added to each well to produce an effector:target cell ratio of 25:1, and the plate was placed in a 5% CO Incubate in a 37°C incubator under _{a CO} atmosphere for 4 hours; ix) Harvest the cell-free supernatant from each well and quantify the experimentally released radioactivity (ER) using a gamma counter; x) Calculate the percentage of specific lysis for each antibody concentration according to the formula (ER-MR)/(MR-SR) × 100, where ER is the mean radioactivity quantified for that antibody concentration (see point ix above) and MR is the mean radioactivity quantified for the MR control (see point v above). 4) "Increased ADCC" is defined as an increase in the maximum percentage of specific lysis observed within the antibody concentration range tested above, and/or a decrease in the antibody concentration required to achieve half the maximum percentage of specific lysis observed within the antibody concentration range tested above. In one embodiment, the increase in ADCC is relative to ADCC measured by the above assay mediated by the same antibody produced by the same type of host cells using the same standard production, purification, preparation, and storage methods known to those skilled in the art, except that the comparator antibody (lacking increased ADCC) is not produced by host cells engineered to overexpress GnTIII and/or engineered to have reduced expression of the fucosyltransferase 8 (FUT8) gene ( e.g. , including, engineered with a FUT8 gene knockout).

In some embodiments, "increased ADCC" can be achieved, for example, by mutation and/or glycosylation modification of the antibodies. In some embodiments, the anti-CD20 antibodies are glycosylated to have a biantennary oligosaccharide linked to the Fc region of the antibody, the oligosaccharide being bisected by GlcNAc. In some embodiments, the anti-CD20 antibodies are glycosylated to lack fucosylation on the carbohydrate linked to the Fc region by expressing the antibodies in host cells lacking protein fucosylation ( e.g. , Lec13 CHO cells or cells lacking the α-1,6-fucosyltransferase gene (FUT8) or knocking down FUT gene expression). In some embodiments, the anti-CD20 antibody sequences have been modified in their Fc region to enhance ADCC. In some embodiments, such engineered anti-CD20 antibody variants comprise an Fc region having one or more amino acid substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, the term "complement-dependent cytotoxicity (CDC)" refers to the lysis of human cancer target cells by an antibody according to the present invention in the presence of complement. CDC can be measured by treating a CD20-expressing cell preparation with an anti-CD20 antibody of the present invention in the presence of complement. CDC is detected if the antibody induces lysis (cell death) of 20% or more tumor cells after 4 hours at a concentration of 100 nM. In some embodiments, the assay is performed using tumor cells labeled with ⁵¹ Cr or Eu, and the released ⁵¹ Cr or Eu is measured. A control includes incubating tumor target cells with complement but without antibody.

In some embodiments, the anti-CD20 antibody is a monoclonal antibody, such as a human antibody. In one embodiment, the anti-CD20 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, minidiabody, or F(ab') ₂ fragment. In another embodiment, the anti-CD20 antibody is a substantially full-length antibody, such as an IgG1 antibody, an IgG2a antibody, or another antibody class or isotype as defined herein.

VII. Antibodies

In some embodiments, the antibodies used in the treatment methods provided herein (e.g., anti-CD79b antibodies or anti-CD20 antibodies) can incorporate any of the features described below, alone or in combination.

A. Antibody affinity

In certain embodiments, the antibodies used in the treatment methods provided herein (e.g., anti-CD79b antibodies or anti-CD20 antibodies) have 1μM, 100nM, 50nM, 10nM, 5nM, 1nM, 0.1nM, 0.01nM, or 0.001nM, and depending on the situation A dissociation constant (Kd) of 10 ⁻¹³ M. (e.g., 10 ⁻⁸ M or less, e.g., ^{10 −8 M} to 10 ⁻¹³ M, e.g., 10 ⁻⁹ M to 10 ⁻¹³ M).

In one embodiment, Kd is measured by performing a radiolabeled antigen binding assay (RIA) using a Fab version of the relevant antibody and its antigen, as described in the following assay. The solution binding affinity of the Fab for the antigen is measured by equilibrating the Fab with a minimal concentration of ( ¹²⁵ I)-labeled antigen in the presence of a titration series of unlabeled antigen, followed by capturing the bound antigen with an anti-Fab antibody-coated plate ( see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, ^MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with 5 μg/ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and subsequently blocked for two to five hours at room temperature (approximately 23°C) with PBS containing 2% (w/v) bovine serum albumin. In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ ¹²⁵ I]-antigen was mixed with serial dilutions of the relevant Fab ( e.g. , as evaluated for anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, incubation can be continued for a longer period (e.g., 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate is washed eight times with PBS containing 0.1% polysorbate 20 (TWEEN- ^20® ). When the plate is dry, 150 μl/well of scintillator (MICROSCINT-20 ^™ ; Packard) is added and the plates are counted for 10 minutes on a TOPCOUNT ^™ gamma counter (Packard). A concentration of each Fab that provides less than or equal to 20% of maximal binding is selected for use in the competitive binding assay.

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

B. Antibody fragments

In certain embodiments, the antibodies used in the treatment methods provided herein ( e.g. , anti-CD79b antibodies or anti-CD20 antibodies) are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al . Nat. Med. 9: 129-134 (2003). For a review of scFv, see Pluckthün, in The Pharmacology of Monoclonal Antibodies , Vol. 113, Rosenburg and Moore, eds. (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab') ₂ fragments comprising a salvage receptor-binding epitope residue and having extended in vivo half-life, see U.S. Patent No. 5,869,046.

Bifunctional antibodies are antibody fragments with two antigen-binding sites and can be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad . Sci. USA 90:6444-6448 (1993). Trifunctional and tetrafunctional antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments that comprise all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).

Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and, as described herein, production by recombinant host cells ( e.g., E. coli ) or bacteriophage.

C. Chimeric and humanized antibodies

In certain embodiments, the antibodies used in the treatment methods provided herein ( e.g. , anti-CD79b antibodies or anti-CD20 antibodies) are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad . Sci. USA, 81: 6851-6855 (1984). In one example, 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 (e.g., monkey)) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

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

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

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 the consensus sequence of human antibodies of a particular subset 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 (somatic cell mutation) framework regions or human germline framework regions ( see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions obtained by screening FR libraries ( see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

D. Human antibodies

In certain embodiments, the antibodies used in the treatment methods provided herein (e.g., anti-CD79b antibodies or anti-CD20 antibodies) are human antibodies. 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).

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

Human antibodies can also be produced using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing 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 using human B cell hybridoma technology are also described in Li et al. , Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other 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 (triple hybridoma 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).

Human antibodies can also be generated by isolating pure Fv variable domain sequences selected from human phage display libraries. These variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

E. Library-derived antibodies

In some embodiments, antibodies for use in the therapeutic methods provided herein (e.g., anti-CD79b antibodies or anti-CD20 antibodies) can be isolated by screening combinatorial libraries for antibodies with desired activity. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and further described, for example, in 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., 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).

In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phage, as described in Winter et al . Ann. Rev. Immunol. , 12:433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries derived from immune sources provide high-affinity antibodies against the immunogen without the need for constructing hybridomas. Alternatively, natural 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 prepared synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences encoding the highly variable CDR3 regions and achieving rearrangement in vitro 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. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are referred to herein as human antibodies or human antibody fragments.

F. Multispecific antibodies

In certain embodiments, the antibodies used in the treatment methods provided herein (e.g., anti-CD79b antibodies or anti-CD20 antibodies) are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for one antigen ( e.g., CD79b or CD20), and the other binding specificity is for any other antigen. In certain embodiments, one binding specificity is for one antigen ( e.g., CD79b or CD20) and the other is for CD3. See, for example, U.S. Patent No. 5,821,337. In certain embodiments, bispecific antibodies can bind to two different antigenic determinants of a single antigen ( e.g. , CD79b or CD20). Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing an antigen ( e.g. , CD79b or CD20). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for preparing multispecific antibodies include, but are not limited to, recombining two immunoglobulin heavy chain-light chain pairs that co-express 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. Patent No. 5,731,168). Multispecific antibodies can also be prepared by the following methods: electrostatic switching effect designed for the preparation of antibody Fc-heterodimer molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments ( see, for example, U.S. Patent No. 4,676,980, and Brennan et al., Science , 229: 81 (1985)); using leucine to produce bispecific antibodies ( see, for example, Kostelny et al., J. Immunol. , 148(5): 1547-1553 (1992)); using "mini-bifunctional antibody" technology to prepare bispecific antibody fragments ( see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA , 90: 6444-6448 (1993)); and using single-chain Fv (sFv) dimers ( see, e.g., Gruber et al., J. Immunol. , 152: 5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. , J. Immunol. 147: 60 (1991).

Also included herein are engineered antibodies with three or more functional antigen-binding sites, including "Octopus antibodies" ( see, e.g., US 2006/0025576A1).

The antibodies or fragments herein also include "dual-acting FAbs" or "DAFs," which contain antigen binding sites that bind to CD79b and another different antigen ( see , e.g., US 2008/0069820).

G. Antibody variants

In certain embodiments, amino acid sequence variants of antibodies ( e.g. , anti-CD79b antibodies or anti-CD20 antibodies) used in the treatment methods provided herein are encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of an anti-CD79b antibody or an anti-CD20 antibody. The amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

(i) Substitution, insertion and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table D under the heading "Preferred Substitutions." More substantial changes are provided in Table D under the heading "Exemplary Substitutions" and as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibodies of interest, and the products can be screened for desired activities such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves replacing one or more hypervariable region residues of a parent antibody ( e.g. , a humanized or human antibody). Generally, the resulting variants selected for further study will have modifications ( e.g. , improvements) in certain biological properties relative to the parent antibody ( e.g. , increased affinity, reduced immunogenicity) and/or will have certain biological properties of the parent antibody that are substantially maintained. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated, for example, using affinity maturation techniques based on phage display (such as those described herein). Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a specific biological activity ( e.g. , binding affinity).

Alterations ( e.g. , substitutions) can be made in HVRs, for example, to improve antibody affinity. These alterations can be made in HVR "hotspots" (i.e., residues encoded by codons that undergo mutation at high frequency during in vivo maturation) ( see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)) and/or SDRs (a-CDRs), and the resulting variant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001). In some embodiments of affinity maturation, diversity is introduced into a variable gene selected for maturation by any of a variety of methods ( e.g. , error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves HVR site-directed approaches, in which several HVR residues ( e.g. , 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often particularly targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes that do not substantially reduce binding affinity ( e.g. , conservative substitutions as provided herein) may be made in the HVRs. Such changes may be outside of HVR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains only one, two, or three amino acid substitutions.

A suitable method for identifying residues or regions in antibodies that can be targeted for mutation induction is called "alanine scanning mutation induction", as described by Cunningham and Wells (1989) Science , 244: 1081-1085. In this method, a residue or a group of target residues ( e.g. , charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids ( e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitutions. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. These contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include N- or C-terminal fusions of the antibody to enzymes ( e.g. , against ADEPT) or polypeptides that increase the serum half-life of the antibody.

(ii) Glycosylation variants

In certain embodiments, the antibodies used in the treatment methods provided herein ( e.g. , anti-CD79b antibodies or anti-CD20 antibodies) are modified to increase or decrease the degree of glycosylation of the antibody. Adding glycosylation sites to the antibody or deleting glycosylation sites from the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

Where the antibody comprises an Fc region, the carbohydrates attached thereto may be varied. Native antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides that are generally linked by an N-link to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides may include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention may be made to generate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided that lack a carbohydrate structure of trehalose attached (directly or indirectly) to the Fc region. For example, the amount of trehalose in the antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of trehalose is determined by calculating the average amount of trehalose at Asn 297 within the sugar chain relative to the sum of all carbohydrate structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008/077546. Asn297 refers to the asparagine residue located at approximately position 297 (Eu numbering of Fc region residues) in the Fc region. However, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Publications US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to "de-trehalosylated" or "trehalose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al. , especially Example 11) and gene-knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 gene-knockout CHO cells ( see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. ,94(4):680-688(2006);and WO2003/085107).

Antibody variants further have bisecting oligosaccharides, for example, wherein a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al .). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

(iii) Fc variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody ( e.g. , an anti-CD79b antibody or an anti-CD20 antibody) used in the treatment methods provided herein to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence ( e.g. , a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification ( e.g. , substitution) at one or more amino acid positions.

In certain embodiments, the present invention encompasses antibody variants that possess some, but not all, effector functions that make them candidates for applications where in vivo half-life of the antibody is important but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity) but retains FcRn binding ability. Primary NK cells used to mediate ADCC express only Fc(RIII), whereas monocytes express Fc(RI, Fc(RII), and Fc(RIII). FcR expression on hematopoietic cells is summarized in Table 3 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991), p. 464. Non-limiting examples of in vitro assays for evaluating ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362 ( see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)). 82: 1499-1502 (1985); 5,821,337 ( see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays can be used ( see , for example, the ACTI ^™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnogy, Inc., Mountain View, CA); and the CytoTox ^96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Suitable effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be determined, for example, in animal models, such as Clynes et al., Proc. Nat'l Acad. Sci. USA In vivo assessment can be performed in animal models as disclosed in WO 2006/029879 (1996 ) ; Cragg, MS et al. , Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 102:1060-1080 (2003). FcRn binding and in vivo clearance/half-life can also be determined using methods known in the art ( see, for example , Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

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

In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

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

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

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

(iv) Cysteine-modified antibody variants

In certain embodiments, it may be desirable to generate cysteine-modified antibodies, such as "thioMAbs," in which one or more residues of the anti-CD79b or anti-CD20 antibodies used in the therapeutic methods provided herein are substituted with cysteine residues. In specific embodiments, the substituted residues occur at accessible sites on the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby positioned at accessible sites on the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541.

(v) Antibody derivatives

In certain embodiments, the antibodies used in the treatment methods provided herein ( e.g. , anti-CD79b antibodies or anti-CD20 antibodies) can be further modified to include other non-protein moieties that are known in the art and readily available. Suitable moieties for derivatization of 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, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polyoxypropylene/ethylene oxide copolymers, polyoxyethylated polyols ( e.g. , glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may offer advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and/or type of polymers used for derivatization can be determined based on a variety of considerations, including, but not limited to, the specific properties or functions of the antibody to be modified, whether the antibody derivative will be used therapeutically under specified conditions, etc.

In another embodiment, an antibody is conjugated to a non-protein moiety that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad . Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to wavelengths that do not harm normal cells but heat the non-protein moiety to a temperature that kills cells adjacent to the antibody-non-protein moiety.

H. Recombination Methods and Compositions

Antibodies can be produced using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acids encoding the antibodies described herein are provided. Such nucleic acids may encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody ( e.g., the light chain and/or heavy chain of the antibody). In another embodiment, one or more vectors ( e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises ( e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell ( e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method for producing an antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of antibodies, nucleic acids encoding antibodies, such as those described above, are isolated and inserted into one or more vectors for further propagation and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using known procedures ( e.g. , by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not desired. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. ( See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) Following expression, the antibodies can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable hosts for the cloning or expression of antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" so that the antibodies produced have a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).

Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous bacilliform virus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES ^™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be suitable. Other examples of useful mammalian host cell lines are monkey kidney CV1 transformed by SV40 (COS-7); human embryonic kidney strains (such as 293 or 293 cells described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); hamster kidney cells (BHK); mouse Segmental cells (such as TM4 cells described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL); 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

I. Inspection

Antibodies (e.g., anti-CD79b antibodies or anti-CD20 antibodies) used in the treatment methods provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities using various assays known in the art.

In one aspect, the antibodies used in the treatment methods provided herein (e.g., anti-CD79b antibodies or anti-CD20 antibodies) are tested for antigen binding activity, for example, by known methods such as ELISA, ^BIACore® , FACS, or Western blotting.

In another aspect, competition assays can be used to identify antibodies that compete with any of the antibodies described herein for binding to a target antigen. In certain embodiments, such competing antibodies bind to the same epitope ( e.g., a linear or conformational epitope) as the antibodies described herein. Detailed exemplary methods for mapping epitopes bound by antibodies are provided in Morris (1996), "Epitope Mapping Protocols," in Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competitive assay, the immobilized antigen is incubated in a solution comprising a first labeled antibody ( e.g., any of the antibodies described herein) that binds to the antigen and a second, unlabeled antibody whose ability to compete with the first antibody for antigen binding is tested. The second antibody can be present in the hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution comprising the first labeled antibody but without the second, unlabeled antibody. Following incubation under conditions permissive for binding of the first antibody to the antigen, excess unbound antibody is removed, and the amount of label associated with the immobilized antigen is measured. If the amount of label associated with the immobilized antigen is substantially reduced in the test sample relative to the control sample, this indicates that the second antibody is competing with the first antibody for antigen binding. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

VIII. Pharmaceutical preparations

Pharmaceutical formulations of any of the agents described herein ( e.g. , anti-CD79b immunoconjugates, anti-CD20 agents, and alkylating agents) for use in any of the methods described herein are prepared by mixing the agent having the desired purity with one or more optional pharmaceutically acceptable carriers ( Remington's Pharmaceutical Sciences , 16th edition, Osol, A., ed. (1980)) in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexadecyl quaternary ammonium chloride; benzathonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides. proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersions, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 ( ^HYLENEX® , Baxter International). Certain exemplary sHASEGPs (including rHuPH20) and methods of use are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGPs are combined with one or more additional mucopolysaccharidases (such as chondroitinases).

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

The formulations herein may also contain more than one active ingredient as necessary for the specific indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other.

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

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, such matrices being in the form of shaped articles such as films or microcapsules.

Formulations intended for intravenous administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filter membranes.

Further details regarding pharmaceutical formulations comprising anti-CD79 immunoconjugates are provided in WO 2009/099728, the contents of which are expressly incorporated herein by reference in their entirety.

IX. Manufacturing Objects and Kits

In another embodiment, an article of manufacture or kit is provided comprising an anti-CD79b immunoconjugate (as described herein) and at least one additional agent. In some embodiments, the at least one additional agent is an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody (e.g., rituximab). In some embodiments, the article of manufacture or kit further comprises a package insert comprising instructions for using the anti-CD79b immunoconjugate with at least one additional agent, such as an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody (e.g., rituximab), for treating or delaying the progression of a B-cell proliferative disorder (e.g., DLBCL) in a subject. Any anti-CD79b immunoconjugate and anticancer agent known in the art may be included in the article of manufacture or kit. In some embodiments, the kit comprises an immunoconjugate having the formula wherein Ab is an anti-CD79b antibody comprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 22, (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 23, (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 24, (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and (vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 26; and wherein p is in the range of 1 to 8. In some embodiments, the kit comprises an immunoconjugate comprising the formula Wherein Ab is an anti-CD79b antibody comprising (i) a heavy chain comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and (ii) a light chain comprising a VL comprising the amino acid sequence of SEQ ID NO: 20, wherein p is between 2 and 5. In some embodiments, p is between 3 and 4, e.g., 3.5. In some embodiments, the immunoconjugate comprises an anti-CD79 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 36, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 35. In certain embodiments, the anti-CD79b immunoconjugate comprises the structure Ab-MC-vc-PAB-MMAE. In some embodiments, the anti-CD79b immunoconjugate is polatuzumab vedotin (CAS No. 1313206-42-6). In some embodiments, the at least one additional agent is an alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl) and an anti-CD20 antibody (e.g., rituximab).

In some embodiments, the kit is used to treat DLBCL in a subject (e.g., a subject having one or more of the characteristics described herein) according to the methods provided herein.

In some embodiments, the anti-CD79 immunoconjugate, the alkylating agent (e.g., bendamustine, e.g., bendamustine-HCl), and the anti-CD20 antibody (e.g., rituximab) are in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The container can be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloy (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and a label on or associated with the container may indicate instructions for use. The article or kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more other pharmaceutical agents (e.g., chemotherapeutic agents and anti-tumor agents). Suitable containers for the one or more pharmaceutical agents include, for example, bottles, vials, bags, and syringes.

The foregoing written description is believed to be sufficient to enable one skilled in the art to practice the present invention. Various modifications of the present invention, in addition to those shown and described herein, will become apparent to one skilled in the art from the foregoing description and are within the scope of the appended patent applications. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

Example

The following are examples of methods and compositions of the present invention. It should be understood that many other embodiments can be implemented given the general description provided above.

Case 1: Combination of an anti-CD79b immunoconjugate (Polatuzumab Vedotin) with an anti-CD20 antibody (rituximab) and an alkylating agent (Bendamustine) for relapsed or refractory diffuse large B-cell lymphoma (DLBCL)

CD79b is a signaling component of a B cell receptor located on normal B cells and most mature B cell malignancies, including >95% of DLBCL (Dornan et al., Blood, 114: 2721-9, 2009; Pfeifer et al., Leukemia, 29: 1578-86, 2015). Polatuzumab vedotin (CAS No. 1313206-42-6) has shown encouraging activity in R/R DLBCL as a monotherapy (Palanca-Wessels et al., Lancet Oncology, 16:704-15, 2015) and in combination with an anti-CD20 monoclonal antibody (Morschhauser et al., Journal of Clinical Oncology, 32:15-suppl, 8519, 2014), resulting in overall response rates (ORRs) ranging from 13-56%. However, complete response (CR) rates are low (0-15%), prompting its use in combination with other agents. Bendamustine and rituximab (BR) are commonly used in transplant-ineligible R/R DLBCL, with a reported median progression-free survival (PFS) of 3.6-6.7 months (Ohmachi et al., Journal of Clinical Oncology 31:2103-9, 2013; Vacirca et al., Annals of Hematology, 93:403-9, 2014). Described below are results from a phase Ib/II trial evaluating the combination of polatuzumab vedotin with bendamustine and atezolizumab (Pola-BG) and the combination of polatuzumab vedotin with BR (Pola-BR) compared with BR alone in transplant-ineligible R/R DLBCL, including patients who had failed prior autologous stem cell transplantation (ASCT).

patient

Patients aged 18 years or above who Patients with biopsy-confirmed relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) after 1 prior therapy, Eastern Cooperative Oncology Group (ECOG) performance status of 0-2, and peripheral neuropathy (PN) Patients with a history of autologous stem cell transplantation (SCT) were eligible if their treating physician deemed them unsuitable for autologous stem cell transplantation (SCT) or if they had previously failed SCT. Patients with a history of SCT were eligible if the SCT occurred >100 days before Day 1 of Cycle 1.

Patients with grade 3b follicular lymphoma, transformed lymphoma, transformed and rheumatic non-Hodgkin lymphoma (NHL), and central nervous system (CNS) lymphoma were excluded. Patients who were suitable for SCT were excluded. Patients who had received a prior allogeneic stem cell transplant were excluded.

Experimental design

The Phase Ib safety trial included six patients treated with polatuzumab vedotin in combination with bendamustine and rituximab ("Pola-BR") and six patients treated with polatuzumab vedotin in combination with bendamustine and atuzumab ("Pola-BG"). See Figure 1A. The Phase II portion included an expansion cohort evaluating Pola-BG (20 patients) and a randomized cohort comparing Pola-BR to BR alone (80 patients, 40 patients in each treatment group), stratified by duration of response (DOR) to last treatment ( 12 months vs. >12 months). See Figure 1A.

All patients received bendamustine ("B") 90 mg/ ^m² intravenously on Days 2 and 3 of Cycle 1, then on Days 1 and 2 of subsequent cycles. They also received rituximab ("R") 375 mg/ ^m² intravenously on Day 1 of each cycle or atezolizumab ("G") 1000 mg intravenously on Days 1, 8, and 15 of Cycle 1 and on Day 1 of subsequent cycles. Those treated with polatuzumab vedotin ("Pola") received 1.8 mg/kg intravenously (IV) on Day 2 of Cycle 1 and on Day 1 of subsequent cycles. See Tables 1A-1C below . Patients received treatment for up to six 21-day cycles.

On days scheduled for bendamustine and rituximab, rituximab should be administered before bendamustine. On days scheduled for polatuzumab vedotin, bendamustine, and rituximab, rituximab should be administered before polatuzumab vedotin, and polatuzumab vedotin should be administered before bendamustine. On days scheduled for polatuzumab vedotin, bendamustine, and atezolizumab, atezolizumab should be administered before polatuzumab vedotin, and polatuzumab vedotin should be administered before bendamustine.

Evaluation and Endpoints

The primary endpoint of the Phase Ib study was safety and tolerability. The primary endpoint of the Phase II study was the complete response (CR) rate with Pola-BR compared to BR, as measured by 18F-fluorodeoxyglucose-positron emission tomography-computed tomography (PET-CT) at end of treatment (EOT, ^6-8 weeks after Day 1 of Cycle 6 or the last dose of study treatment) by an independent review committee (IRC) using modified Lugano response criteria (see Cheson et al. (2014) "Recommendations for initial evaluation, staging, and response assessment of hodgkin and non-hodgkin lymphoma: The Lugano Classification." J. Clin. Oncol. 32:3059-67). The Lugano classification was modified as follows: 1) For patients with bone marrow involvement or unknown status at baseline, a CR assessment based on imaging alone without confirmatory bone marrow testing was classified as a partial response (PR); 2) A partial response (by IRC alone) required a partial metabolic response on fluorodeoxyglucose-PET and a complete or partial response on CT; otherwise, the response according to the modified Lugano criteria (see above) was classified as stable disease.

Secondary endpoints included overall response rate (ORR) at end-of-life (EOT), best overall response (BOR), duration of response (DOR), and progression-free survival (PFS) by IRC. Exploratory endpoints included biomarker assessment of efficacy in cell of origin (COO) as determined by the Nanostring Lymphoma Subtyping Test (LST) or Hans algorithm criteria, immunohistochemical staining for dual-expressing lymphoma (DEL), investigator-assessed (INV) DOR and PFS, and overall survival (OS).

Response was assessed after three cycles (mid-term) and at the end of treatment (end of treatment evaluation) by computed tomography (CT), PET-CT, and bone marrow examination (if necessary to confirm CR). Follow-up CT scans were performed every six months for two years or until progressive disease (PD) or patient withdrawal.

The National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.03) was used to assess and score all adverse events (AEs) throughout the study. All AEs, including serious adverse events (SAEs), were reported from Day 1 of Cycle 1 through Day 90 after the last dose of study drug, regardless of their relationship to the study drug. Thereafter, all SAEs continued to be reported indefinitely.

biomarkers

The following method was used for exploratory biomarker assessment of CD79b expression, cell of origin (COO), and dual-expression lymphoma (DEL), i.e., dual expression of MYC and BCL2.

CD79b

CD79b tumor cell protein expression was assessed by immunohistochemistry (IHC) in a central laboratory using the AT 107-2 antibody (Serotec) and the Ventana Benchmark XT platform. Expression was scored using staining intensity (0-3+). Furthermore, the range of expression was assessed with greater granularity by assessing the H-score, a continuous measurement that considers the percentage of tumor cells with staining intensities of 0, 1, 2, or 3+ and ranges from 0 to 300. The H-score of tumor cell staining was calculated using the following formula: H-score = (% at 0) × 0 + (% at 1+) × 1 + (% at 2+) × 2 + (% at 3+) × 3. Therefore, the score generates a continuous variable ranging from 0 to 300 (Pfeifer et al. (2015) "Anti-C22 and anti-CD79B antibody drug conjugates are active in different molecular diffuse large B-cell lymphoma subtypes." Leukemia. 29:1578-86). Cells with an H-score staining greater than "0" are considered positive.

Cell of Origin (COO)

Samples were sent to Labcorp for NanoString Research-Only Lymphoma Subtype Test (LST) analysis. If COO classification was not possible by Nanostring LST ( e.g. , due to tissue availability), COO classification was performed by central pathology review (Histogenex) and IHC using the Hans algorithm (Hans et al. (2004) "Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray." Blood. 103:275-282) using local pathology reports. In this analysis, Hans non-GCB (i.e., non-germinal center B cells) were counted as ABC (i.e., activated B cells).

Dual-expressing lymphoma (DEL)

IHC was performed at Ventana (Santa Clara, CA) on the Ventana Benchmark XT platform using the research-use-only BCL2 (124) mAb and MYC (Y69) IHC assay. MYC IHC overexpression was defined as 40% of tumor cell nuclei were positively stained, and BCL2 overexpression was defined as 50% of tumor cells have 2+ cytoplasmic staining intensity.

Statistical analysis

The sample size for Phase Ib was determined by a 3+3 design ( see Storer BE. (1989) “Design and analysis of phase I clinical trials.” Biometrics. 45:925-37). The sample size for the Phase II randomized cohort was determined based on the assumption that the difference in CR rate between Pola-BR and BR was 25%, which allowed the exclusion of zero as the lower limit of the 95% confidence interval (CI, 3.8 to 46.2%). For the safety assessment of the Phase II portion, the sample sizes of 20 patients in the expansion cohort and 40 patients in each randomized cohort provided the observed true incidence of 10% and 5%, respectively. 1 AE 85% probability.

All accepted Patients receiving any study treatment at dose 1 were included in the safety analysis (safety assessment). Efficacy analyses were performed in the intention-to-treat population.

result

One hundred and thirteen patients with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) who were ineligible for transplant were enrolled. The demographic and disease characteristics of all patients are shown in Table 2 below. For the randomized phase II cohort, patients received a median of two prior lines of therapy, with patients in the Pola-BR group receiving 1 to 7 prior lines of therapy and those in the BR group receiving 1 to 5 prior lines of therapy.

High-dose chemotherapy with autologous stem cell transplantation (ASCT) was counted as 1 therapy. DLBCL, NOS — diffuse large B-cell lymphoma, not otherwise specified; ABC — activated B-cell-like; GCB — germinal center B-cell-like; ECOG PS — Eastern Cooperative Oncology Group performance status.

^* No response or progressive disease within 6 months of the last dose of treatment

The safety trial included 6 patients receiving Pola-BR and 6 patients receiving Pola-BG. The Phase II Pola-BG cohort enrolled 21 patients and treated 20 patients. For the Phase II randomized cohort, 40 patients were enrolled in each arm and 39 patients were treated in each arm. See Figure 1B . Baseline characteristics of the randomized patients were generally balanced, with patients receiving an average of 2 prior lines of therapy. Seventy-five percent of Pola-BR and 85% of BR patients were refractory to their last therapy ( e.g. , no response or progressive disease within 65 months of the last dose of therapy).

effect

The EOT response rate and mean event time endpoint are shown in Table 3 .

NE = Not Estimable

*HR (95% CI) = Not applicable

HR(95% CI)=0.40(0.16,1.01); p=0.0462

HR(95% CI)=0.44(0.2,0.95); p=0.0321

§HR(95% CI)=0.36(0.21,0.63); p=0.002

§§HR(95% CI)=0.34(0.2,0.57); p<0.0001

**HR(95% CI)=0.42(0.24,0.75); p=0.0023

HR and p-values based on stratified analysis.

In the Phase Ib Pola-BR arm, the CR rate was 50% (3/6) as assessed by end-of-life IRC, and all three patients remained in remission at a median follow-up of 37.6 months (DOR: 28.9-38.2 months). One nonresponder received subsequent treatment and remains alive, and two patients died from PD. In the combined Phase Ib/II Pola-BG arm, the CR rate was 29.6% (8/27) as assessed by end-of-life IRC. At a median follow-up of 26.9 months, median PFS (IRC) and OS were 6.3 and 10.8 months, respectively. Two patients went on to undergo consolidation SCT (one autologous and one allogeneic). Four patients (15%) documented responses lasting at least 20 months (range, 20.7-22.5 months) without requiring further treatment. At the final follow-up, eight patients remained alive, 17 died (12 due to PD, five due to AEs), and two discontinued the study (one by physician decision, one due to an AE).

The primary analysis of the randomized phase II cohort showed that the CR rate at EOT was significantly higher in the Pola-BR group compared with BR (40.0% vs. 17.5%; P = 0.026; Table 3 ). A higher proportion of patients were considered ineligible for EOT response by IRC compared with investigator assessment because patients with evidence of clinical progression but who did not undergo follow-up PET-CT were not evaluable by IRC. However, this did not affect the assessment of CR rate, in which the agreement between IRC and investigator assessment was > 90%. BOR and best CR rate were also higher in the Pola-BR group compared with BR. See Table 3. Duration of response without further treatment was 6 patients (15%) 20 months.

After a median follow-up of 22.3 months, PFS and OS were significantly improved with Pola-BR compared with BR, as was DOR (see Figures 2A and 2B) . Consistent risk reduction benefits were observed in IRC- and investigator-assessed DOR (IRC: HR 0.40 [95% CI, 0.16 to 1.01]; INV: HR 0.44 [95% CI, 0.20 to 0.95]) and PFS (IRC: HR 0.36 [95% CI, 0.21 to 0.63]; INV: 0.34 [95% CI, 0.20 to 0.57]). IRC assessments of DOR and PFS are slightly longer than INV assessments, primarily due to the lag time between the receipt or non-receipt of the follow-up confirmatory scans required for IRC assessments following clinical progression determined by INV.

The Pola-BR group showed a significant improvement in overall survival (OS), with a 58% reduction in the risk of death (HR, 0.42; 95% CI, 0.24 to 0.75). The median OS was longer with Pola-BR (12.4 months [95% CI, 9.0 to not evaluable [NE]]) compared with BR alone (4.7 months [95% CI, 3.7 to 8.3]). See Figure 2B .

During the follow-up period, 11 patients in the Pola-BR group and 4 patients in the BR group were still alive.

Post hoc subgroup analyses demonstrated a consistent survival benefit across all clinical and biologic subgroups examined (see Figures 2C , 3A , and 3B ). The benefit was similar regardless of the patient's refractory status or the number of prior therapies they had received.

In addition, 7 (18%) Pola-BR patients had sustained DOR The patients were followed for 20 months (range, 20.0-22.5 months) and remained in complete remission at the time of the last follow-up. One patient underwent consolidation of allogeneic SCT; the other six received no additional treatment. Only two patients with BR (5%) continued to follow-up without progression; both received consolidation therapy (one underwent allogeneic SCT and the other received radiation therapy).

Security

In the phase Ib Pola-BR and phase Ib/II Pola-BG arms, treatment delivery and adverse events (AEs) were similar to those in the phase II randomized Pola-BR arm.

(i) Phase Ib Pola-BR

Of the 6 patients treated in the Phase Ib Pola-BR arm, The most common adverse events (AEs) in one patient were decreased appetite, weight loss, diarrhea, hypocalcemic pneumonia, fever, thrombocytopenia (all in 33.3%), hypokalemia and nausea (both in 50%), and fatigue (66.7%). One patient experienced the following Grade 3-4 AEs: febrile neutropenia, pneumonia, and thrombocytopenia. No Grade 5 AEs occurred.

(ii) Phase Ib/II Pola-BG

In the combined Phase Ib/II Pola-BG arm, patients received a median of four cycles, with 42.3% completing all treatment cycles. Overall, this was similar to Pola-BR. Across all arms, the median dose intensity for dose modifications and dose delays was approximately 99-100%. No patients underwent dose reductions for polatuzumab vedotin. Bendamustine dose reductions occurred in 26.9% (7/26) of patients. The most common reasons for bendamustine dose reductions were neutropenia (15.4%) and fatigue/asthenia (7.7%). One patient underwent a dose reduction due to both neutropenia and fatigue (during the same cycle). Twelve patients (46.2%) experienced treatment delays. The most common reasons for treatment delays were cytopenias (neutropenia or thrombocytopenia [23.1%]) and infection (15.4%). Treatment delays occurred in two patients due to elevated transaminases, and in one patient due to peripheral neuropathy (PN).

The most common AEs occurring in at least 20% of patients were diarrhea (61.5%), fatigue (53.8%), nausea (53.8%), constipation (42.3%), decreased appetite (42.3%), pyrexia (42.3%), thrombocytopenia (30.8%), neutropenia (26.9%), anemia (19.2%), vomiting (34.6%), and hypokalemia (23.1%). The most common Grade 3-4 adverse events reported in at least 10% of patients were neutropenia (26.9%), thrombocytopenia (23.1%), febrile neutropenia (11.5%), anemia (11.5%), nausea (11.5%), and fatigue (11.5%). The incidence of grade 3-4 infection was 23.1%.

Peripheral neuropathy (PN) of all grades occurred in 38.5% of patients and 15.4% of patients Grade 2. Two patients reported Grade 3 muscle weakness, but in one case, this was consistent with disease progression. Two patients withdrew from all study treatment: one due to Grade 2 PN and the other due to Grade 3 muscle weakness.

There were five fatal AEs. Three of the fatal AEs were infections (pneumonia, fungal pneumonia, and sepsis). The other two were myelodysplastic syndrome (occurring 2 years after subsequent autologous transplantation) and general health deterioration.

(iii) Fatal AEs in Pola-BR and BR

Three fatal AEs in Pola -BR (pneumonia, hemoptysis, and pulmonary edema) and four in BR (cerebrovascular accident, sepsis[2], and pneumonia) occurred within 30 days of treatment.

Fatal AEs occurring during the follow-up period (including those with PD) were: Pola-BR (distributive shock [PD], pneumonia [PD], renal failure [PD], intracranial hemorrhage [PD], herpes encephalitis, and sepsis); BR (multiple organ dysfunction [2 cases, both with PD], cerebral hemorrhage [PD], leukoencephalopathy [PD], sepsis [PD], heart failure, and unexplained death).

(iv) Phase II Pola-BR and BR

Among randomized patients, the treatment completion rate was higher in the Pola-BR group than in the BR group (46.2% vs. 23.1%), as was the median number of completed cycles (5 vs. 3), primarily due to a higher rate of disease progression in the BR group. Progressive disease led to treatment discontinuation in 53.8% of patients treated with BR and 15.4% of patients treated with Pola-BR. AEs were the most common reason for discontinuation of Pola-BR (33.3%; Supplementary Table 1). In both groups, the most common reason for bendamustine dose reduction was cytopenia (4 Pola-BR, 3 BR). The most common all-grade and grade 3-4 AEs are shown in Table 4 below. Although the Pola-BR group had a higher rate of grade 3-4 anemia and thrombocytopenia, transfusion rates were similar between the Pola-BR and BR groups (red blood cell: 25.6% vs. 20.5%; platelet: 15.4% vs. 15.4%). Grade 3-4 neutropenia was more frequent with the Pola-BR group (46.2% vs. 33.3%), but grade 3-4 infections were similar between the two groups (23.1% Pola-BR and 20.5% BR). The overall incidence of peripheral neuropathy (PN) in Pola-BR patients was 41.0% (16/39) (11 grade 1, 5 grade 2), with 10 patients having resolved and 1 patient having improved at clinical cutoff. PN was the only reason for polatuzumab vedotin dose reduction, which occurred in 2 (7.7%) patients (both with grade 2 PN) and resolved in both cases.

Fatal AEs occurred in 9 patients receiving Pola-BR and 11 patients receiving BR, with infection being the most common cause (4 Pola-BR, 5 BR).

*Show Adverse events of all grades occurred in 20% of patients. Grade 3-4 AEs (safety assessment) occurred in 10% of patients. Preferred terms are shown for each system organ class, except for peripheral neuropathy.

†Includes: peripheral motor neuropathy, peripheral sensory neuropathy, decreased vibration sensation, hypoesthesia, paresthesia

Biomarkers: CD79b, cell of origin (COO), and double-expressing lymphoma (DEL)

Of 83 stained patient samples, 80 (96.4%) had detectable CD79b (immunohistochemistry [IHC] H-score 1-300 or 1 ⁺ -3 ⁺ ). RNA assessment showed measurable expression of CD79b in all samples, including 3 samples that were negative by IHC. See Figure 4. In the three samples where CD79b was undetectable by IHC, parallel RNA assessment showed measurable expression significantly above background levels that was inconsistent with the IHC data. Each dot represents a single sample or negative control probe. In the NanostringQCPro Bioconductor R-package, gene expression levels were normalized to the median by default. Response to Pola-based therapy was not correlated with CD79b levels by IHC or gene expression. As shown in Figure 5 , there was no significant difference in performance between responders and non-responders (p-value = 0.24, Wilcoxon rank-sum test with continuity correction).

COO was assessed in 107 patient samples, of which 97 were evaluable. The COO distribution was 46.4% activated B cell (ABC), 47.4% germinal center B cell-like (GCB), and 6.2% unclassifiable. In the randomized cohort, improvements achieved with Pola-BR were observed in both the ABC and GCB subgroups. See Tables 5 and 6 .

ABC - activated B-cell-like; GCB - germinal center B-cell-like; n - number; Pola-BR - polatuzumab vedotin-BR; BR - bendamustine plus rituximab; CR - complete response; PR - partial response; SD - stable disease; PD - progressive disease; NE - not estimable

PFS - progression-free survival; OS - overall survival; COO - cell of origin; ABC - activated B-cell-like; GCB - germinal center B-cell-like; Pola-BR - polatuzumab vedotin-BR; BR - bendamustine plus rituximab; NE - not estimable.

*Patients classified as non-GCB by the Hans algorithm were grouped together with ABC patients.

HR: 0.20 (95% CI, 0.09-0.45)

HR: 0.49 (95% CI, 0.23-1.05)

§HR: 0.21 (95% CI, 0.09-0.51)

§§HR: 0.57 (95% CI, 0.25-1.31)

Of 62 patient samples assessed for DEL status, 41.9% were identified as DEL, defined as dual expression of MYC and BCL2. In the randomized cohort, improvements achieved with Pola-BR were observed in both DEL and non-DEL patients. See Tables 7 and 8 .

DEL - dual-expressed lymphoma; Pola-BR - polatuzumab vedotin-BR; BR - bendamustine plus rituximab; n - number; CR - complete response; PR - partial response; SD - stable disease; PD - progressive disease; NE - not estimable.

PFS - progression-free survival; OS - overall survival; Pola-BR - polatuzumab vedotin-BR; BR - bendamustine plus rituximab; DEL - bi-expressed lymphoma; non-DEL - bi-expressed lymphoma; NE - not estimable;

HR: 0.35 (95% CI, 0.12-1.11)

HR: 0.53 (95% CI, 0.23-1.25)

§HR: 0.39 (95% CI, 0.12-1.22)

§§HR: 0.58 (95% CI, 0.24-1.40)

Patients with R/R DLBCL who are not transplant candidates, including those who have failed autologous SCT, face frustrating outcomes due to limited treatment options. In this randomized comparison, treatment with Pola-BR resulted in significant improvements in CR rates, PFS, and OS compared with BR alone across all COO and DEL subgroups. Although 13 patients received additional therapies after progression, those treated with BR fared poorly, highlighting the limitations of currently available agents. This is the first randomized trial to demonstrate an OS benefit in patients with R/R DLBCL who are not transplant candidates.

Compared with BR alone, patients who received Pola-BR had significantly longer OS (median 12.4 months vs. 4.7 months, HR 0.42; 95% CI 0.24, 0.75). Benefit appeared to be present in all subgroups examined, including refractory patients and those who had received at least one, at least two, at least three, or more prior lines of therapy. Furthermore, biomarker studies indicated that Pola-BR appeared to benefit patients regardless of COO or DEL status. CD79b expression was ubiquitous, and no correlation was found between CD79b expression levels and response. Although the independent contribution of bendamustine to the overall efficacy could not be measured, the 40% CR rate observed with Pola-BR was significantly higher than the 15% previously reported with the combination of polatuzumab vedotin and an anti-CD20 monoclonal antibody (Morschhauser et al. (2014) “Preliminary results of a phase II randomized study (ROMULUS) of polatuzumab vedotin (PoV) or pinatuzumab vedotin (PiV) plus rituximab (RTX) in patients (Pts) with relapse/refractory (R/R) non-Hodgkin lymphoma (NHL).” J. Clin. Oncol. 32:15suppl,8519). Achievement of a CR is associated with improved outcomes in DLBCL, and the high CR rate observed may partially explain the durable responses seen in a subset of patients receiving Pola-BR, many of whom remain disease-free without additional treatment. Pola-BR can be used as a stand-alone therapy or as a bridge to consolidation therapy.

Peripheral neuropathy (PN) is a well-known toxicity associated with monomethyl auristatin E (MMAE)-based antibody-drug conjugates and was closely monitored during the study. Despite prior exposure to vincristine or platinum in many patients, the majority of PN observed was low-grade and reversible, with relatively few patients requiring dose reductions or delays. A higher rate of grade 3-4 cytopenias was observed with Pola-BR compared to BR, but this did not result in a higher risk of infection or the need for transfusions.

The significant and profound PFS and OS benefits observed with Pola-BR made a randomized phase III trial impractical. Although this study examined Pola-BR as a stand-alone treatment, given the higher CR rates and prolonged disease control observed, Pola-BR may offer a valuable treatment option that can be readily offered to a broader patient population.

Polatuzumab vedotin combined with BG or BR has a tolerable safety profile. After a median follow-up of 26.9 months, the CR rate in patients receiving Pola-BG was 29.6%, and the median OS was 10.8 months. 80 patients were randomly assigned (40 in each group) to Pola-BR or BR. After a median follow-up of 22.3 months, patients receiving Pola-BR had a significantly higher CR rate (40% vs. 17.5%, P=0.026), longer PFS and OS (median OS 12.4 vs. 4.7 months, HR 0.42; 95% CI, 0.24 to 0.75). Compared with BR, patients receiving pola-BR had a higher incidence of grade 3-4 neutropenia, anemia, and thrombocytopenia, but similar rates of grade 3-4 infections and transfusions. Peripheral neuropathy associated with polatuzumab vedotin was primarily low-grade and resolved in the majority of patients.

Compared with BR, treatment with Pola-BR more than doubled overall survival. Treatment with Pola-BR resulted in a 66% reduction in the risk of disease progression or death (as measured by investigator-assessed progression-free survival; PFS; HR = 0.34; 95% CI 0.2-0.570; p < 0.0001). Complete remission (CR) was achieved in 40% (16/40) of patients receiving Pola-BR, compared with approximately 18% (7/40) of patients receiving BR (primary endpoint, CR rate assessed by positron emission tomography (PET) and assessed by an independent review committee; p = 0.026). Furthermore, patients treated with Pola-BR achieved higher CR rates and longer PFS and OS compared with BR in all subgroups tested, including those from the cell-of-origin group, germinal center B-cell-like, and activated B-cell-like, which are associated with a worse prognosis in DLBCL.

Approximately 40% of patients with diffuse large B-cell lymphoma do not respond to initial treatment or relapse after initial treatment. This disease trajectory is associated with a poor prognosis. Polatuzumab vedotin has demonstrated sustained clinical benefit and may improve survival in this population. These study results demonstrate a survival benefit for patients with relapsed/refractory DLBCL who are ineligible for hematopoietic stem cell transplantation.

For the purpose of clarity of understanding, the present invention is described in detail through illustrations and examples, but such descriptions and examples should not be construed as limiting the scope of the present invention. The disclosures of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims (50)

Use of polatuzumab vedotin for the preparation of a pharmaceutical for treating diffuse large B-cell lymphoma (DLBCL) in a human in need thereof, wherein the pharmaceutical is for use in combination with (a) bendamustine or a salt thereof, and (b) rituximab, wherein the treatment prolongs progression-free survival (PFS) in the human. The use of claim 1, wherein the treatment prolongs the overall survival (OS) of the person. Use of polatuzumab vedotin for the preparation of a pharmaceutical for treating diffuse large B-cell lymphoma (DLBCL) in a human in need thereof, wherein the pharmaceutical is used in combination with (a) bendamustine or a salt thereof, and (b) rituximab, wherein the treatment prolongs the human's overall survival (OS). The use of any one of claims 1 to 3, wherein the human achieves a complete response (CR) after treatment with the vetinib-pollotuzumab, the bendamustine or a salt thereof, and the rituximab. The use according to any one of claims 1 to 3, wherein the bendamustine or a salt thereof is bendamustine-HCl. The use of any one of claims 1 to 3, wherein the vetin-pollotuzumab is administered at a dose of 1.8 mg/kg, the bendamustine or a salt thereof is administered at a dose of 90 mg/m ² , and the rituximab is administered at a dose of 375 mg/m ² . The use of any one of claims 1 to 3, wherein the vetin-pollotuzumab, the bendamustine or a salt thereof, and the rituximab are administered for at least six 21-day cycles, wherein in the 21-day cycle of the first cycle, the vetin-pollotuzumab is administered intravenously at a dose of 1.8 mg/kg on day 2, the bendamustine or a salt thereof is administered intravenously at a dose of 90 mg/m ² on days 2 and 3, and the rituximab is administered intravenously at a dose of 375 mg/m 2 on day 1. ² , and wherein in each 21-day cycle of cycles 2 to 6, the vetin-pollotuzumab is administered intravenously at a dose of 1.8 mg/kg on day 1, the bendamustine or a salt thereof is administered intravenously at a dose of 90 mg/m ² on days 1 and 2, and the rituximab is administered intravenously at a dose of 375 mg/m ² on day 1. The use of claim 7, wherein the vetin-pollotuzumab and the bendamustine or a salt thereof are administered sequentially on the second day of the first cycle. The use of claim 8, wherein the vetin-polotuzumab is administered before the bendamustine or a salt thereof. The use of claim 7, wherein the vetin-pollotuzumab, the bendamustine or a salt thereof, and the rituximab are administered sequentially on day 1 of cycles 2 to 6. The use of claim 10, wherein on day 1 of cycles 2-6, the rituximab is administered before the vetin-pollotuzumab, and wherein the vetin-pollotuzumab is administered before the bendamustine or a salt thereof. The use of claim 7, wherein the vetin-pollotuzumab, the bendamustine or a salt thereof, and the rituximab are further administered after the sixth cycle. The use of claim 12, wherein in each cycle after the 6th cycle, in each 21-day cycle, the vetin-pollotuzumab is administered intravenously at a dose of 1.8 mg/kg on day 1, the bendamustine or a salt thereof is administered intravenously at a dose of 90 mg/m ² on days 1 and 2, and the rituximab is administered intravenously at a dose of 375 mg/m ² on day 1. The use of claim 12, wherein in each cycle after the 6th cycle, on day 1 of each 21-day cycle, the rituximab is administered before the vetin-pollotuzumab, and wherein the vetin-pollotuzumab is administered before the bendamustine or a salt thereof. Use of polatuzumab vedotin for the preparation of a medicament for treating diffuse large B-cell lymphoma (DLBCL) in a patient in need thereof, wherein the medicament is for use in combination with (a) bendamustine or a salt thereof, and (b) rituximab, wherein the polatuzumab vedotin is administered at a dose of 1.8 mg/kg, the bendamustine or a salt thereof is administered at a dose of 90 mg/m ² , and the rituximab is administered at a dose of 375 mg/m 2. ² , wherein the treatment prolongs progression-free survival (PFS) and/or overall survival (OS) in the human, and wherein: i) the DLBCL is activated B-cell-like DLBCL (ABC-DLBCL) or germinal center B-cell-like DLBCL (GCB-DLBCL); ii) the DLBCL is a bi-expressing lymphoma (DEL); iii) the human has received at least two prior therapies for DLBCL; iv) the human has received at least three prior therapies for DLBCL; and/or v) the human has received more than three prior therapies for DLBCL. The use of claim 15, wherein the bendamustine or its salt is bendamustine-HCl. The use of claim 15, wherein the human achieves a complete response (CR) after treatment with the vetinib-pollotuzumab, the bendamustine or a salt thereof, and the rituximab. The use of claim 15, wherein the vetin-pollotuzumab, the bendamustine or a salt thereof, and the rituximab are administered for at least six 21-day cycles, wherein in the 21-day cycle of the first cycle, the vetin-pollotuzumab is administered intravenously at a dose of 1.8 mg/kg on day 2, the bendamustine or a salt thereof is administered intravenously at a dose of 90 mg/m ² on days 2 and 3, and the rituximab is administered intravenously at a dose of 375 mg/m 2 on day 1. ² , and in each 21-day cycle after the first cycle, the vetin-pollotuzumab is administered intravenously at a dose of 1.8 mg/kg on day 1, the bendamustine or a salt thereof is administered intravenously at a dose of 90 mg/m ² on days 1 and 2, and the rituximab is administered intravenously at a dose of 375 mg/m ² on day 1 in each 21-day cycle. The use of claim 18, wherein the vetin-polotuzumab and the bendamustine or a salt thereof are administered sequentially on the second day of the first cycle. The use of claim 19, wherein the vetin-polotuzumab is administered before the bendamustine or a salt thereof. The use of claim 18, wherein the vetin-pollotuzumab, the bendamustine or a salt thereof, and the rituximab are administered sequentially on day 1 of cycles 2 to 6. The use of claim 21, wherein on day 1 of cycles 2-6, the rituximab is administered before the vetin-pollotuzumab, and wherein the vetin-pollotuzumab is administered before the bendamustine or a salt thereof. The use of claim 18, wherein the vetin-pollotuzumab, the bendamustine or a salt thereof, and the rituximab are further administered after the 6th cycle, and wherein in each cycle after the 6th cycle, the vetin-pollotuzumab is administered intravenously at a dose of 1.8 mg/kg on day 1, the bendamustine or a salt thereof is administered intravenously at a dose of 90 mg/m ² on days 1 and 2, and the rituximab is administered intravenously at a dose of 375 mg/m ² on day 1 in each 21-day cycle. The use of claim 23, wherein on day 1 of each 21-day cycle after cycle 6, the rituximab is administered before the vetin-pollotuzumab, and wherein the vetin-pollotuzumab is administered before the bendamustine or a salt thereof. The use of any of claims 1, 2, and 15, wherein the treatment prolongs PFS to at least about 6 months. The use of any of claims 1, 2, and 15, wherein the treatment prolongs PFS to at least 7 months. The use of claim 26, wherein the treatment prolongs PFS to at least approximately 7.6 months. The use of claim 27, wherein the treatment prolongs PFS to at least about 8 months. The use of any of claims 1, 2, and 15, wherein the treatment prolongs PFS to at least 11 months. For the use of claim 29, wherein the treatment prolongs PFS to at least 11.1 months. The use of any of claims 2, 3, and 15, wherein the treatment prolongs OS to at least about 11 months. The use of any of claims 2, 3, and 15, wherein the treatment prolongs OS to at least about 12 months. The use of claim 32, wherein the treatment prolongs OS to at least about 12.4 months. The use of any one of claims 1-3 and 15, wherein the DLBCL is activated B cell-like DLBCL (ABC DLBCL). The use of any one of claims 1-3 and 15, wherein the DLBCL is germinal center B cell-like DLBCL (GCB DLBCL). The use of any of claims 1-3 and 15, wherein the DLBCL is not otherwise specified (DLBCL-NOS). The use of any one of claims 1-3, and 15, wherein the DLBCL is a double-expression lymphoma (DEL). The use of any of claims 1-3 and 15, wherein the DLBCL is relapsed/refractory DLBCL. The use of any of claims 1-3 and 15, wherein the human does not have grade 3b follicular lymphoma, transformed slowed non-Hodgkin's lymphoma, or CNS lymphoma. The use of any of claims 1-3 and 15, wherein the person has received at least one prior therapy for DLBCL. The use of claim 40, wherein the person has received at least two prior therapies for DLBCL. The use of claim 41, wherein the person has received at least three prior therapies for DLBCL. The use as claimed in claim 42, wherein the person has received more than three prior therapies for DLBCL. The use of any of claims 1-3 and 15, wherein the person is not suitable for autologous stem cell transplantation (ASCT). The use of claim 44, wherein the ASCT is a first-line ASCT, a second-line ASCT, a third-line ASCT, or an ASCT beyond the third line. For the use as claimed in claim 40, wherein the person has previously failed autologous stem cell transplantation. The use of claim 40, wherein the person has received prior treatment with an anti-CD20 agent. For the use described in claim 40, wherein the person has previously received treatment with bendamustine or a salt thereof. For the use described in item 40, where the person is refractory to the most recent prior treatment. A kit comprising polatuzumab vedotin and a package insert containing instructions for use, for use in combination with bendamustine or a salt thereof and rituximab, for treating a human with diffuse large B-cell lymphoma (DLBCL) in need thereof, according to the treatment as described in any of claims 1-3 and 15.